Phenolic compounds and antifungal activity of ethyl acetate extract and methanolic extract from Capsicum chinense Jacq. ripe fruit

Abstract Food loss due to contamination caused by fungi has much impact on agriculture and leads to significant economic losses. Synthetic and natural fungicides have been used for avoiding losses of several food products due to fungal contamination. As a result, species of the genus Capsicum have been used for preserving food because of their chemical compounds with antifungal activity. Therefore, this study aimed at identifying some phenolic compounds found in both ethyl acetate extract (EAE) and methanolic extract (ME) from habanero pepper (C. chinense) ripe fruit by liquid chromatography tandem mass spectrometry with electrospray ionization (LC-ESI-MS/MS) and at evaluating their antifungal activities against fungi Sclerotinia sclerotiorum, Rhizopus stolonifer and Colletotrichum gloeosporioides. Extracts resulted from a sequential process of maceration. Antifungal activity was evaluated by the disk diffusion method (DDM) at the following doses of both diluted extracts: 25 µL, 50 µL, 100 µL and 200 µL. The chemical analysis showed that there were protocatechuic acid, gentisic acid, vanillic acid, kaempferol-3-O-robinobiosideo and naringenin in both extracts. EAE showed high inhibition of mycelial growth at both doses 100µL and 200µL against the three fungi while methanolic exhibited weak activity even at the highest dose under investigation. However, further in-depth studies are needed to reinforce their uses and practical applications to the agricultural field.

Resumo As perdas de alimentos por contaminação causada por fungos são de grande impacto negativo para a agricultura, gerando altos prejuízos econômicos. Para evitar as perdas de diversos produtos alimentícios pela contaminação fúngica são utilizados fungicidas sintéticos e naturais. As espécies do gênero Capsicum são usadas há muitos anos para auxiliar na conservação de alimentos por possuírem substâncias químicas com ação antifúngica entre outras. Neste contexto, o objetivo deste estudo foi identificar alguns compostos fenólicos por cromatografia líquida de alta eficiência acoplada à espectrometria de massas sequencial (LC-ESI-MS/MS) presentes nos extratos acetato de etila (EAE) e metanólico (ME) dos frutos maduros da pimenta biquinho (C. chinense) e avaliar atividade antifúngica de EAE e ME contra os fungos Sclerotinia sclerotiorum, Rhizopus stolonifer e Colletotrichum gloeosporioides. Os extratos foram obtidos de forma sequencial, utilizando o procedimento de maceração. A atividade antifúngica foi avaliada seguindo a metodologia de difusão em disco, nas doses de 25 µL, 50 µL, 100 µL e 200 µL de cada extrato diluído. A análise química evidenciou a presença de ácido protocatequico, ácido gentisico, ácido vanílico, kaempferol-3-O-robinobiosídeo e naringenina em ambos os extratos. EAE revelou maior poder de inibição do crescimento micelial nas doses de 100µL e 200µL contra os três fungos testados, enquanto ME exibiu fraca atividade inclusive na maior dose investigada. Entretanto, estudos mais aprofundados ainda são necessários para consolidar seu uso e aplicação prática na área agronômica.

Phenolic compounds and antifungal activity of ethyl acetate extract and methanolic extract from Capsicum chinense Jacq. ripe fruit / Compostos fenólicos e atividade antifúngica dos extratos acetato de etila e metanólico dos frutos maduros de Capsicum chinense Jacq

Food loss due to contamination caused by fungi has much impact on agriculture and leads to significant economic losses. Synthetic and natural fungicides have been used for avoiding losses of several food products due to fungal contamination. As a result, species of the genus Capsicum have been used for preserving food because of their chemical compounds with antifungal activity. Therefore, this study aimed at identifying some phenolic compounds found in both ethyl acetate extract (EAE) and methanolic extract (ME) from habanero pepper (C. chinense) ripe fruit by liquid chromatography tandem mass spectrometry with electrospray ionization (LC-ESI-MS/MS) and at evaluating their antifungal activities against fungi Sclerotinia sclerotiorum, Rhizopus stolonifer and Colletotrichum gloeosporioides. Extracts resulted from a sequential process of maceration. Antifungal activity was evaluated by the disk diffusion method (DDM) at the following doses of both diluted extracts: 25 µL, 50 µL, 100 µL and 200 µL. The chemical analysis showed that there were protocatechuic acid, gentisic acid, vanillic acid, kaempferol-3-O-robinobiosideo and naringenin in both extracts. EAE showed high inhibition of mycelial growth at both doses 100µL and 200µL against the three fungi while methanolic exhibited weak activity even at the highest dose under investigation. However, further in-depth studies are needed to reinforce their uses and practical applications to the agricultural field.

As perdas de alimentos por contaminação causada por fungos são de grande impacto negativo para a agricultura, gerando altos prejuízos econômicos. Para evitar as perdas de diversos produtos alimentícios pela contaminação fúngica são utilizados fungicidas sintéticos e naturais. As espécies do gênero Capsicum são usadas há muitos anos para auxiliar na conservação de alimentos por possuírem substâncias químicas com ação antifúngica entre outras. Neste contexto, o objetivo deste estudo foi identificar alguns compostos fenólicos por cromatografia líquida de alta eficiência acoplada à espectrometria de massas sequencial (LC-ESI-MS/MS) presentes nos extratos acetato de etila (EAE) e metanólico (ME) dos frutos maduros da pimenta biquinho (C. chinense) e avaliar atividade antifúngica de EAE e ME contra os fungos Sclerotinia sclerotiorum, Rhizopus stolonifer e Colletotrichum gloeosporioides. Os extratos foram obtidos de forma sequencial, utilizando o procedimento de maceração. A atividade antifúngica foi avaliada seguindo a metodologia de difusão em disco, nas doses de 25 µL, 50 µL, 100 µL e 200 µL de cada extrato diluído. A análise química evidenciou a presença de ácido protocatequico, ácido gentisico, ácido vanílico, kaempferol-3-O-robinobiosídeo e naringenina em ambos os extratos. EAE revelou maior poder de inibição do crescimento micelial nas doses de 100µL e 200µL contra os três fungos testados, enquanto ME exibiu fraca atividade inclusive na maior dose investigada. Entretanto, estudos mais aprofundados ainda são necessários para consolidar seu uso e aplicação prática na área agronômica.

Phenolic compounds and antifungal activity of ethyl acetate extract and methanolic extract from Capsicum chinense Jacq. ripe fruit.

Food loss due to contamination caused by fungi has much impact on agriculture and leads to significant economic losses. Synthetic and natural fungicides have been used for avoiding losses of several food products due to fungal contamination. As a result, species of the genus Capsicum have been used for preserving food because of their chemical compounds with antifungal activity. Therefore, this study aimed at identifying some phenolic compounds found in both ethyl acetate extract (EAE) and methanolic extract (ME) from habanero pepper (C. chinense) ripe fruit by liquid chromatography tandem mass spectrometry with electrospray ionization (LC-ESI-MS/MS) and at evaluating their antifungal activities against fungi Sclerotinia sclerotiorum, Rhizopus stolonifer and Colletotrichum gloeosporioides. Extracts resulted from a sequential process of maceration. Antifungal activity was evaluated by the disk diffusion method (DDM) at the following doses of both diluted extracts: 25 µL, 50 µL, 100 µL and 200 µL. The chemical analysis showed that there were protocatechuic acid, gentisic acid, vanillic acid, kaempferol-3-O-robinobiosideo and naringenin in both extracts. EAE showed high inhibition of mycelial growth at both doses 100µL and 200µL against the three fungi while methanolic exhibited weak activity even at the highest dose under investigation. However, further in-depth studies are needed to reinforce their uses and practical applications to the agricultural field.

Risk assessment of in vitro cytotoxicity, antioxidant and antimicrobial activities of Mentha piperita L. essential oil.

The objective of this study was to determine the chemical composition as well as antioxidant, antibacterial, and cytotoxic properties of the essential oil of Mentha piperita L. (peppermint). Fifteen chemical constituents were identified in the essential oil, for a total of 99.99% of the compounds. The essential oil exhibited antimicrobial activity against two Gram-positive bacteria Staphylococcus aureus and Listeria monocytogenes. The minimum inhibitory concentration (MIC) of essential oil of Mentha piperita L. for Staphylococcus aureus and Listeria monocytogenes was 1.84 µg/ml, whereas the minimum bactericidal concentration (MBC) values were 3.7 and 7.43 µg/ml, respectively. The oil displayed potent antioxidant activity inhibiting up to approximately73% of 2,2'-azinothiobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals. In the cytotoxicity assay, the highest essential oil concentration (100 µg/ml) resulted in viability of approximately 90% human epidermal keratinocyte (HaCaT) cells. With respect to antitumor activity in C6 rat glioma cells, there was significant reduction in cell viability: 56-74% in 24 hr, and 71-77% in 48 hr. Data suggest that in presence of the essential oil of Mentha piperita L. antioxidant, antibacterial, antitumor and non-cytotoxic properties were noted.

Valerian treatment during the postpartum period alters breast milk composition and impairs long-term memory in female rat offspring.

Maternal anxiety symptoms in the perinatal period might have long-term health effects on both the mother and the developing child. Valerian is a phytotherapeutic agent that is widely used for the treatment of anxiety. This study investigated the effects of valerian treatment in postpartum rats on maternal care, toxicity, and milk composition. Postnatal development, memory, and anxiety behavior in the offspring were also assessed. Postpartum Wistar rats received the valerian (500, 1000, or 2000 mg·kg-1·day-1) by oral gavage. Clinical and biochemical toxicity was evaluated with commercial kits. Maternal behavior was observed daily. Milk composition was analyzed by colorimetric methods. Physical and neuromotor tests were used to analyze postnatal development. Anxiolytic activity was assessed by the elevated plus maze, and memory was evaluated by the step-down inhibitory avoidance task. Maternal toxicity and care behavior were not altered by the treatment, while only the highest dose promoted a significant increase of lactose, and the doses 1000 and 2000 mg·kg-1·day-1 promoted a reduction of protein contents in milk. Postnatal development was similar in all offspring. Adult offspring did not display altered anxiety behavior, while long-term memory was impaired in the female adult offspring by maternal treatment with 1000 mg·kg-1·day-1. These results suggested that high doses of valerian had significant effects on important maternal milk components and can cause long-term alterations of offspring memory; thus, treatment with high doses of valerian is not safe for breastfeeding Wistar rat mothers.

Valerian treatment during the postpartum period alters breast milk composition and impairs long-term memory in female rat offspring

Maternal anxiety symptoms in the perinatal period might have long-term health effects on both the mother and the developing child. Valerian is a phytotherapeutic agent that is widely used for the treatment of anxiety. This study investigated the effects of valerian treatment in postpartum rats on maternal care, toxicity, and milk composition. Postnatal development, memory, and anxiety behavior in the offspring were also assessed. Postpartum Wistar rats received the valerian (500, 1000, or 2000 mg·kg-1·day-1) by oral gavage. Clinical and biochemical toxicity was evaluated with commercial kits. Maternal behavior was observed daily. Milk composition was analyzed by colorimetric methods. Physical and neuromotor tests were used to analyze postnatal development. Anxiolytic activity was assessed by the elevated plus maze, and memory was evaluated by the step-down inhibitory avoidance task. Maternal toxicity and care behavior were not altered by the treatment, while only the highest dose promoted a significant increase of lactose, and the doses 1000 and 2000 mg·kg-1·day-1 promoted a reduction of protein contents in milk. Postnatal development was similar in all offspring. Adult offspring did not display altered anxiety behavior, while long-term memory was impaired in the female adult offspring by maternal treatment with 1000 mg·kg-1·day-1. These results suggested that high doses of valerian had significant effects on important maternal milk components and can cause long-term alterations of offspring memory; thus, treatment with high doses of valerian is not safe for breastfeeding Wistar rat mothers.

Chemical composition and in vitro inhibitory effects of essential oils from fruit peel of three Citrus species and limonene on mycelial growth of Sclerotinia sclerotiorum / Composição química e efeito inibitório in vitro dos óleos essenciais das cascas dos frutos de três espécies de Citrus e do limoneno sobre o crescimento micelial de Sclerotinia sclerotiorum

Abstract Essential oils (EO) from aromatic and medicinal plants generally perform a diverse range of biological activities because they have several active constituents that work in different mechanisms of action. EO from Citrus peel have an impressive range of food and medicinal uses, besides other applications. EO from Citrus reticulata, C. sinensis and C. deliciosa were extracted from fruit peel and analyzed by GC-MS. The major constituent of EO under evaluation was limonene, whose concentrations were 98.54%, 91.65% and 91.27% for C. sinensis, C. reticulata and C. deliciosa, respectively. The highest potential of inhibition of mycelial growth was observed when the oil dose was 300 μL. Citrus oils inhibited fungus growth in 82.91% (C. deliciosa), 65.82% (C. sinensis) and 63.46% (C. reticulata). Anti-Sclerotinia sclerotiorum activity of 90% pure limonene and at different doses (20, 50, 100, 200 and 300 μL) was also investigated. This monoterpene showed to be highly active by inhibiting 100% fungus growth even at 200 and 300 μL doses. This is the first report of the in vitro inhibitory effect of natural products from these three Citrus species and its results show that there is good prospect of using them experimentally to control S. sclerotiorum, in both greenhouse and field conditions.

Resumo Óleos essenciais de plantas aromáticas e medicinais geralmente exibem uma gama diversificada de atividades biológicas, pois possuem vários constituintes ativos que atuam por meio de vários mecanismos de ação. Os óleos essenciais das cascas de Citrus têm uma variedade impressionante de usos em alimentos, medicamentos entre várias outras aplicações. Os óleos essenciais (OE) de Citrus reticulata, C. sinenses e C. deliciosa foram extraídos das cascas dos frutos e analisados por CG-EM. O limoneno foi o constituinte majoritário encontrado nos óleos essenciais avaliados, nas concentrações de 98,54%, 91,65% e 91,27% para C. sinensis, C. reticulata e C. deliciosa, respectivamente. Os maiores potenciais de inibição do crescimento micelial foi observado na dose de 300 µL dos óleos. Os óleos de Citrus inibiram em 82,91% (C. deliciosa), 65,82% (C. sinensis) e 63,46% (C. reticulata) o crescimento do fungo. A atividade anti-Sclerotinia sclerotiorum do limoneno 90% puro e em diferentes doses (20, 50, 100, 200 e 300 µL) foi também investigada e este monoterpeno demonstrou-se altamente ativo inibindo 100% o crescimento do fungo inclusive nas doses de 200 e 300 µL. Este é o primeiro relato sobre o efeito inibitório in vitro dos óleos essenciais destas três espécies de Citrus e os resultados deste estudo mostram que existe uma boa perspectiva de uso destes produtos naturais experimentalmente para controlar o S. sclerotiorum tanto em condições de estufa como em condições de campo.

Chemical composition and in vitro inhibitory effects of essential oils from fruit peel of three Citrus species and limonene on mycelial growth of Sclerotinia sclerotiorum.

Essential oils (EO) from aromatic and medicinal plants generally perform a diverse range of biological activities because they have several active constituents that work in different mechanisms of action. EO from Citrus peel have an impressive range of food and medicinal uses, besides other applications. EO from Citrus reticulata, C. sinensis and C. deliciosa were extracted from fruit peel and analyzed by GC-MS. The major constituent of EO under evaluation was limonene, whose concentrations were 98.54%, 91.65% and 91.27% for C. sinensis, C. reticulata and C. deliciosa, respectively. The highest potential of inhibition of mycelial growth was observed when the oil dose was 300 µL. Citrus oils inhibited fungus growth in 82.91% (C. deliciosa), 65.82% (C. sinensis) and 63.46% (C. reticulata). Anti-Sclerotinia sclerotiorum activity of 90% pure limonene and at different doses (20, 50, 100, 200 and 300 µL) was also investigated. This monoterpene showed to be highly active by inhibiting 100% fungus growth even at 200 and 300 µL doses. This is the first report of the in vitro inhibitory effect of natural products from these three Citrus species and its results show that there is good prospect of using them experimentally to control S. sclerotiorum, in both greenhouse and field conditions.

Effects of supplementation with ruminally protected choline on performance of multiparous Holstein cows did not depend upon prepartum caloric intake.

Objectives were to evaluate the effect of prepartum energy intake on performance of dairy cows supplemented with or without ruminally protected choline (RPC; 0 or 17.3 g/d of choline chloride; 0 or 60 g/d of ReaShure, Balchem Corp., New Hampton, NY). At 47 ± 6 d before the expected calving date, 93 multiparous Holstein cows were assigned randomly to 1 of 4 dietary treatments in a 2 × 2 factorial arrangement. Cows were fed energy to excess [EXE; 1.63 Mcal of net energy for lactation/kg of dry matter (DM)] or to maintenance (MNE; 1.40 Mcal of net energy for lactation/kg of DM) in ad libitum amounts throughout the nonlactating period. The RPC was top-dressed for 17 ± 4.6 d prepartum through 21 d postpartum (PP). After calving, cows were fed the same methionine-balanced diet, apart from RPC supplementation, through 15 wk PP. Liver was biopsied at -14, 7, 14, and 21 d relative to parturition. Cows fed EXE or MNE diets, respectively, consumed 40 or 10% more Mcal/d than required at 15 d before parturition. Cows fed the MNE compared with the EXE diet prepartum consumed 1.2 kg/d more DM postpartum but did not produce more milk (41.6 vs. 43.1 kg/d). Thus, PP cows fed the EXE diet prepartum were in greater mean negative energy balance, tended to have greater mean concentrations of circulating insulin, fatty acids, and ß-hydroxybutyrate, and had greater triacylglycerol in liver tissue (8.3 vs. 10.7% of DM) compared with cows fed the MNE diet prepartum. Cows fed RPC in transition tended to produce more milk (43.5 vs. 41.3 kg/d) and energy-corrected milk (44.2 vs. 42.0 kg/d) without increasing DM intake (23.8 vs. 23.2 kg/d) during the first 15 wk PP, and tended to produce more milk over the first 40 wk PP (37.1 vs. 35.0 kg/d). Energy balance of cows fed RPC was more negative at wk 2, 3, and 6 PP, but mean circulating concentrations of fatty acids and ß-hydroxybutyrate did not differ from those of cows not fed RPC. Despite differences in energy balance at 2 and 3 wk PP, mean concentration of hepatic triacylglycerol did not differ between RPC treatments. Feeding RPC reduced the daily prevalence of subclinical hypocalcemia from 25.5 to 10.5%, as defined by concentrations of total Ca of <8.0 mg/dL in serum in the first 7 d PP. Pregnancy at first artificial insemination tended to be greater for cows fed RPC (41.3 vs. 23.6%), but the proportion of pregnant cows did not differ by 40 wk PP. Heifers born from singleton calvings from cows fed RPC tended to experience greater daily gain between birth and 50 wk of age than heifers from cows not supplemented with RPC. Feeding RPC for approximately 38 d during the transition period tended to increase yield of milk for 40 wk regardless of amount of energy consumed during the pregnant, nonlactating period.

Effect of supplemental yeast culture and dietary starch content on rumen fermentation and digestion in dairy cows.

The objectives of this experiment were to evaluate the effect of feeding a culture of Saccharomyces cerevisiae on rumen metabolism and digestibility when cows are fed diets varying in starch content. Four lactating Holstein cows were assigned to a 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments. Treatments were low starch (LS; 23% of diet DM) and no yeast culture (YC; LS-control), LS and 15 g of YC/d (LS-YC), high starch (HS; 29% of diet DM) and no YC (HS-control), and HS and 15 g of YC/d (HS-YC). Periods lasted 28 d, with the last 9 d for data collection. Days 20 to 24 were used to determine production, nutrient flow, and digestibility. On d 25, 3 kg of corn grain DM was placed in the rumen 1 h before the morning feeding, and yields of milk and milk components were measured after the challenge. Blood was sampled -1, 3, 7, and 11 h relative to the morning feeding on d 24 and 25. Rumen pH was measured continuously on d 24 and 25. Rumen papillae were collected on d 24 and 28 to quantify mRNA expression of select genes. Supplementing YC increased yields of milk (26.3 vs. 29.6 kg/d), energy-corrected milk (ECM; 26.5 vs. 30.3 kg/d), fat (0.94 vs. 1.08 kg/d), true protein (0.84 vs. 0.96 kg/d), and ECM/dry matter intake (1.15 vs. 1.30) compared with the control but did not affect dry matter intake (22.6 vs. 22.9 kg/d). Cows fed HS had increased milk true protein percentage (3.18 vs. 3.31%) and yield (0.87 vs. 0.94 kg/d) compared with cows fed LS. Feeding HS-YC increased the proportion of dietary N incorporated into milk true protein from 24.9% in the other 3 treatments to 29.6%. Feeding HS increased the concentration of propionate (21.7 vs. 32.3 mM) and reduced that of NH3-N (8.3 vs. 6.7 mg/dL) in rumen fluid compared with the control, and combining HS with YC in HS-YC tended to increase microbial N synthesis compared with LS-YC (275 vs. 322 g/d). Supplementing YC to cows fed HS reduced plasma haptoglobin and rumen lactate concentrations, increased mean rumen pH, reduced the time with pH <6.0, and prevented the decrease in rumen neutral detergent fiber digestion caused by HS. Cows fed HS had less total-tract digestion of organic matter (73.9 vs. 72.4%) because of reduced acid detergent fiber (57.6 vs. 51.7%) and neutral detergent fiber (60.9 vs. 56.7%) digestibility. Production performance after the challenge was similar to that before the challenge, and YC improved yield of ECM. After the challenge, supplementing YC tended to reduce rumen lactate concentration compared with the control and reduced haptoglobin in cows fed HS. Feeding HS but not YC increased expression in rumen papillae of genes for receptors (FFAR2 and FFAR3) and transporter (SLC16A3) of short-chain fatty acids but did not affect genes involved in transport of Na+/H+ or water or in inflammatory response. Supplementing YC to dairy cows improved lactation performance in diets containing low or high starch, and mechanisms might be partially attributed to improvements in rumen pH, digestion of fiber, microbial N synthesis, and reduction in acute phase response.

Effects of supplementing yeast culture to diets differing in starch content on performance and feeding behavior of dairy cows.

The objectives were to evaluate the effects of a culture of Saccharomyces cerevisiae (YC) on lactation performance of cows fed diets differing in starch content. Fifty-six Holstein cows at 42 d postpartum were blocked by parity and milk production and randomly assigned to 1 of 4 treatments, low starch (23% diet DM) and no YC (LS-control), low starch and 15 g/d of YC (LS-YC), high starch (29% diet DM) and no YC (HS-control), and high starch and 15 g/d of YC (HS-YC). The experiment lasted 14 wk. Blood was sampled twice weekly during the first 5 wk in the experiment. Feeding behavior was evaluated in 2 consecutive days when cows were 33 d in the experiment. On d 92 in the experiment, cows were challenged with 3 kg of corn grain DM immediately before the morning feeding. Blood was sampled in the first 12 h after the challenge. Rumen fluid was collected 5 h after the challenge, and pH, ammonia N, short-chain fatty acids, and lactate concentrations were quantified. Lactation performance was measured daily before and after the challenge. Supplementation with YC increased yields of 3.5% fat-corrected milk and energy-corrected milk by 2.2 and 2.0 kg/d, and the increments were observed in both low- and high-starch diets. Feeding HS tended to decrease milk fat content (LS = 3.88 vs. HS = 3.73%), but increased concentration (LS = 2.87 vs. HS = 3.00%) and yield (LS = 1.11 vs. HS = 1.20 kg/d) of milk true protein. Feeding YC increased yields of fat and true protein in milk by 100 and 60 g/d. Energy balance, body weight, and feed efficiency did not differ with treatments. Feeding HS reduced eating time (LS = 177 vs. HS = 159 min/12 h) and intermeal interval (LS = 103 vs. HS = 82 min), but tended to increase eating rate (LS = 139 vs. HS = 150 g/min). Interactions were detected between level of starch and YC for ruminating time, meal duration, and meal size because within LS, feeding YC increased ruminating time 23 min/12 h, but reduced meal duration 6 min/meal and meal size 0.7 kg/meal. Concentrations of glucose in plasma increased (LS = 62.1 vs. HS = 63.8 mg/dL), whereas those of urea N decreased (LS = 10.1 vs. HS = 9.4 mg/dL) with feeding HS compared with LS in the first 5 wk in the experiment, and the same responses were observed after the challenge with corn grain. After the challenge, rumen pH was less and short-chain fatty acid concentrations were greater in cows fed HS compared with those fed LS; however, supplementing YC to high-starch diets increased rumen pH (HS-control = 5.72 vs. HS-YC = 6.12) and reduced concentrations of lactate in rumen fluid (HS-control = 7.72 vs. HS-YC = 1.33 mM) and haptoglobin in plasma 28%. Feeding YC improved lactation performance irrespective of the level of dietary starch and reduced the risk of subacute rumen acidosis induced by a grain challenge when cows were fed a high-starch ration.

Hemoencephalography self-regulation training and its impact on cognition: A study with schizophrenia and healthy participants.

BACKGROUND: Cognitive impairments in schizophrenia are strongly correlated to functional outcome and recovery rates, with no pharmacological agent approved for its treatment. Neurofeedback has emerged as a non-pharmacological approach to enhance neuroplasticity, which consists in inducing voluntary control of brain responses through operant conditioning. METHOD: The effects of hemoencephalography neurofeedback (HEG-NFBK) in 4 brain sites (F7, Fp1, Fp2 and F8) was studied in 8 patients with schizophrenia (SCH, mean age 36.5±9.98) and 12 health controls (mean age 32.17±5.6). We analyzed groups' performance (10 sessions) and cognitive differences in 3 time points (baseline, after training and follow-up) with generalized estimated equations. For SCH we also evaluate the impact on psychopathology. RESULTS: We found a group∗time interaction for HEG-NFBK performance in the left hemisphere sites (F7 an Fp1) and a near-to-significant in the right frontotemporal region (F8), with no group differences and a significant time effect. Most of cognitive domains improved after intervention, including information processing speed, attention processing, working memory, executive functioning, verbal and visual learning. No group∗time interaction was found. Results suggest that both groups benefit from HEG-NFBK training regardless of cognitive differences at baseline. No significant time effects were found for Calgary and PANSS total scale and subscales (positive, negative neither general). CONCLUSION: To our knowledge, this is the first controlled trial showing effects of NFBK on cognitive performance improvement in schizophrenia. Further research investigating the effects of HEG-NFBK training in schizophrenia should be performed.

Chemical composition and in vitro inhibitory effects of essential oils from fruit peel of three Citrus species and limonene on mycelial growth of Sclerotinia sclerotiorum

Abstract Essential oils (EO) from aromatic and medicinal plants generally perform a diverse range of biological activities because they have several active constituents that work in different mechanisms of action. EO from Citrus peel have an impressive range of food and medicinal uses, besides other applications. EO from Citrus reticulata, C. sinensis and C. deliciosa were extracted from fruit peel and analyzed by GC-MS. The major constituent of EO under evaluation was limonene, whose concentrations were 98.54%, 91.65% and 91.27% for C. sinensis, C. reticulata and C. deliciosa, respectively. The highest potential of inhibition of mycelial growth was observed when the oil dose was 300 L. Citrus oils inhibited fungus growth in 82.91% (C. deliciosa), 65.82% (C. sinensis) and 63.46% (C. reticulata). Anti-Sclerotinia sclerotiorum activity of 90% pure limonene and at different doses (20, 50, 100, 200 and 300 L) was also investigated. This monoterpene showed to be highly active by inhibiting 100% fungus growth even at 200 and 300 L doses. This is the first report of the in vitro inhibitory effect of natural products from these three Citrus species and its results show that there is good prospect of using them experimentally to control S. sclerotiorum, in both greenhouse and field conditions.

Resumo Óleos essenciais de plantas aromáticas e medicinais geralmente exibem uma gama diversificada de atividades biológicas, pois possuem vários constituintes ativos que atuam por meio de vários mecanismos de ação. Os óleos essenciais das cascas de Citrus têm uma variedade impressionante de usos em alimentos, medicamentos entre várias outras aplicações. Os óleos essenciais (OE) de Citrus reticulata, C. sinenses e C. deliciosa foram extraídos das cascas dos frutos e analisados por CG-EM. O limoneno foi o constituinte majoritário encontrado nos óleos essenciais avaliados, nas concentrações de 98,54%, 91,65% e 91,27% para C. sinensis, C. reticulata e C. deliciosa, respectivamente. Os maiores potenciais de inibição do crescimento micelial foi observado na dose de 300 µL dos óleos. Os óleos de Citrus inibiram em 82,91% (C. deliciosa), 65,82% (C. sinensis) e 63,46% (C. reticulata) o crescimento do fungo. A atividade anti-Sclerotinia sclerotiorum do limoneno 90% puro e em diferentes doses (20, 50, 100, 200 e 300 µL) foi também investigada e este monoterpeno demonstrou-se altamente ativo inibindo 100% o crescimento do fungo inclusive nas doses de 200 e 300 µL. Este é o primeiro relato sobre o efeito inibitório in vitro dos óleos essenciais destas três espécies de Citrus e os resultados deste estudo mostram que existe uma boa perspectiva de uso destes produtos naturais experimentalmente para controlar o S. sclerotiorum tanto em condições de estufa como em condições de campo.

The diversity and abundance of phytase genes (ß-propeller phytases) in bacterial communities of the maize rhizosphere.

UNLABELLED: The ecology of microbial communities associated with organic phosphorus (P) mineralization in soils is still understudied. Here, we assessed the abundance and diversity of bacteria harbouring genes encoding ß-propeller phytases (BPP) in the rhizosphere of traditional and transgenic maize cultivated in two Brazilian soils. We found a soil-dependent effect towards a higher abundance of phytase genes in the rhizosphere, and an absence of any impact of plant genotype. Phylogenetic analyses indicated members of the genera Pseudomonas, Caulobacter, Idiomarina and Maricaulis, close to 'uncultured bacteria', to constitute the dominant bacteria hosting this gene. The results obtained validate a methodology to target bacteria that are involved in the organic P cycle, and depict the responsiveness of such bacteria to the rhizosphere, albeit in dependency of the soil in which maize is cultivated. The data also identified the major bacterial groups that are associated with the organic P mineralization function. SIGNIFICANCE AND IMPACT OF THE STUDY: Micro-organisms play a key role in nutrient balance in soil ecosystems that are essential to life on the planet. However, some processes such as organic phosphorus mineralization, an important source of phosphorus supply in soil, is poorly studied mainly due the absence of an efficient methodology to assess the phytase-producing micro-organisms. In this study, a method to assess beta-propeller phytase (BPP)-carrying bacteria in soil was validated. This method may contribute to the knowledge of how these micro-organisms behave in the environment and contribute for plant growth promotion.

Antimutagenic action of the triterpene betulinic acid isolated from Scoparia dulcis (Scrophulariaceae).

The mutagenic and antimutagenic activities of triterpene betulinic acid {3b-3-hydroxy-lup-20(29)-en-28-oic} isolated from the roots of Scoparia dulcis (Scrophulariaceae) were analyzed using the somatic mutation and recombination test (SMART) in the wings of Drosophila melanogaster. The mutagenic potential of betulinic acid was evaluated at 3 different concentrations (1.64, 3.28, and 6.57 mM). Antimutagenic activity evaluation was performed by co-treatment trials in which the flies received betulinic acid at 3 different concentrations in addition to 10 mM pro-mutagenic urethane. The results demonstrated that betulinic acid was not capable of causing DNA damage. However, the frequency of small single spots, large spots, and twin spots was significantly reduced. In the high bioactivation cross, betulinic acid was significantly active and exerted enhanced antimutagenic activity, possibly as a desmutagen.

Tuberculosis screening in patients receiving biological therapy.

AIM: Biological therapies are a risk factor for tuberculosis (TB). Portuguese recommendations recommend universal baseline screening for TB before starting biologics (2006) and annually thereafter if screened negative (2012 update). The gain with re-screening remains unknown. We aimed to i)identify the risk of latent TB infection at baseline screening among patients candidates to initiate biologics ii)present follow-up results for patients receiving different biological therapies and analyse intolerance or toxicity related to preventive therapy, conversions of immunodiagnostic tests under biological therapy and development of active TB. METHODS: Patients screened for TB at a reference centre before starting biological therapy between 2008-2012 were identified. Medical files were retrospectively reviewed. Demographic data, screening and follow-up results and information on biological therapy were collected. EXCLUSION CRITERIA: unavailable information on initiation of biological therapy. RESULTS: 183 patients were included in the study, with 115 starting biological therapy. The baseline screening was positive in 52(45,2%) patients - 50(96,2%) were proposed for preventive treatment (2 had abnormal liver enzymes). Mild hepatotoxicity occurred in 4(8%) patients without need to interrupt TB prophylaxis. No cases of active TB occurred during follow-up in patients with positive baseline screening. Among the 63(54,8%) patients who screened negative, 2(3,2%) developed active TB (under infliximab and adalimumab) more than one year after initiation of biologics. 26(41,3%) patients were re-screened at the TB centre. 5(19,2%) had tuberculin skin test (TST) conversion and one concomitantly undetermined IGRA. No IGRA conversions were observed. The follow-up period was 4,0 years. TB baseline screening's negative predictive value (NPV) was 96,8% (95%CI: 89,0% to 99.5%). A low rate of re-screening was observed. CONCLUSION: The rate of latent TB at baseline screening was higher than expected. Preventive treatment was well tolerated. No patients with positive baseline screening developed active TB. Efforts should be made to raise awareness concerning the risk of TB exposure, specially considering that the active TB cases were compatible with new infection. The rate of re-screening suggests a low awareness regarding current recommendations Nation-wide studies are necessary to evaluate the efficacy of the re-screening strategy and to clarify what risk groups most benefit from it.

Fenóis totais, atividade antioxidante e inibição da enzima tirosinase de extratos de Myracrodruon urundeuva Fr. All. (Anacardiaceae) / Total phenolics, antioxidant activity and tyrosinase enzyme inhibition by Myracrodruon urundeuva Fr. All. (Anacardiaceae) extracts

RESUMOOs radicais livres e outros oxidantes demonstram ser parcialmente responsáveis pelo envelhecimento e pelas doenças degenerativas associadas à produção de espécies reativas de oxigênio. Além disto, os compostos fenólicos são reconhecidamente detentores de elevada atividade antioxidante, que geralmente estão envolvidos em tratamentos de problemas de pigmentação da pele, que resultam em hiperpigmentações. Sendo assim este trabalho foi realizado para avaliar o conteúdo de fenóis totais, atividade antioxidante e a capacidade de inibição da tirosinase dos extratos das folhas e cascas do caule da espécie Myracrodruonurundeuva Fr. All. Para determinação do conteúdo de fenóis totais dos extratos orgânicos de M. urundeuva utilizou-se o reativo Folin-Ciocalteau, na avaliação da atividade antioxidante empregando o radical livre DPPH, enquanto que os testes de inibição da enzima tirosinase foram realizados utilizando L-tirosina. A concentração de fenóis totais foi de 77 mg EAG g-1 e 194 mg EAG g-1 nos extratos hexânico e metanólico das folhas e de 45 mg EAG g-1 e 193 mg EAG g-1 nos extratos hexânico e metanólico das cascas do caule. O potencial antioxidante dos extratos indicaram que o extrato metanólico das cascas do caule (10,9 ± 0,5 µg mL-1), em comparação ao hexânico (12,9 ± 0,2 µg mL-1) e ao BHT (220 ± 7,0 µg mL-1), possui atividade antioxidante levemente mais acentuada. No ensaio de inibição da tirosinase, o extrato metanólico das cascas do caule demonstrou a inibição da enzima em 42% após uma hora.

ABSTRACTFree radicals and other oxidants had demonstrated to be partially responsible for aging and for degenerative diseases associated with the production of reactive oxygen species. In addition, the phenolic compounds are recognized as holders of high antioxidant activity, which usually are involved in the treatment of pigmentation problems, resulting in hyper pigmentation. So this study was carried out in order to evaluate the content of total phenols, antioxidant activity and capacity inhibition of tyrosinase in extracts from leaves and stem barks of the species Myracrodruon urundeuva Fr. All. In order to determine the content of total phenols from organic extracts of M. urundeuva, a Folin-Ciocalteau reagent was employed in the evaluation of antioxidant activity,using the DPPH free radical, while the enzyme tyrosinase inhibition tests were performed using L-tyrosine. The amount of total phenols was 77 mg EAG g-1 and 194 in the hexane and methanolic extracts from the leaves and 45 mg EAG g-1 and 193 mg EAG g-1in the hexane and methanolic extracts of the stem barks. The antioxidant potential of extracts indicated that the methanolic extract of stem bark (10.9 ± 0.5 µg mL-1), when compared to the hexane (12.9 ± 0.2 µg mL-1) and BHT (220 ± 7.0 µg mL-1), presented a slightly higher antioxidant activity. For the tyrosinase inhibitiontrial , the methanolic extract ofstem barks indicated anenzyme inhibition of 42% after an hour.

Protective effects of the antileishmanial extract of Tephrosia cinerea (L.) Pers. (Fabaceae) against cyclophosphamide-induced damage.

Tephrosia cinerea L. (Pers.) is a tropical species that exhibits antileishmanial activity in Leishmania amazonensis promastigote cultures and is commonly used to treat infections, inflammations, ulcers, nervous conditions, and diarrhea. However, no studies have investigated its effects on genetic material. Therefore, we evaluated the genotoxic potential, antigenotoxic potential, and cytotoxic effects of hydroalcoholic extracts of T. cinerea leaves. In an in vitro genotoxicity study, human peripheral blood leukocytes were treated for 3, 24 (comet assay), or 48 h (cell death assay) with 22, 44, or 88 µg/mL plant extract. In the in vivo assay, Swiss mice were treated with 500, 1000, or 2000 mg extract/kg body weight by intraperitoneal injection and were evaluated 24 h later. Antigenotoxicity was investigated in pre- and post-treatment assays in which the animals received the plant extract (2000 mg/kg) 24 h before or after receiving cyclophosphamide (50 mg/kg), respectively. The extract had no genotoxic effects in the in vitro or in vivo assays. However, the extract reduced apoptotic cell death and induced necrotic cell death at concentrations that presented leishmanicidal activity in vitro. The extract also had an antigenotoxic effect, reducing the levels of genomic damage that were caused by cyclophosphamide in Swiss mice by more than 80%.

In situ evaluation of low-fluoride toothpastes associated to calcium glycerophosphate on enamel remineralization.

OBJECTIVES: This study aimed to evaluate the effect of low-fluoride toothpastes with calcium glycerophosphate (CaGP) on enamel remineralization in situ. METHODS: Volunteers (n=10) wore palatal devices holding four bovine enamel blocks. The treatments involved 5 experimental phases of 3 days each according to the following toothpastes: placebo, 500 ppm F (500 NaF), 500 ppm F with 0.25% CaGP (500 NaF CaGP), 500 ppm F with 0.25% CaGP (500 MFP CaGP) and 1100 ppm F (1100; positive control). After this experimental period, the fluoride, calcium, and phosphorus ion concentrations from enamel were determined. Surface and cross-sectional hardness were also performed. Data were analysed by 1-way ANOVA, Student-Newman-Keuls' test and by Pearson's correlation. RESULTS: The addition of 0.25% CaGP improved the remineralization potential of low-fluoride toothpastes and the NaF as source of fluoride yielded the best results (p<0.001) as evidenced by the hardness analysis. The 1100 ppm F toothpaste provided higher presence of fluoride in the enamel after remineralization (p<0.001). The addition of CaGP to the NaF and MFP toothpastes led to similar calcium concentration in the enamel as the observed with the positive control (p=0.054). CONCLUSIONS: Toothpastes with 500 ppm F (NaF or MFP) and CaGP showed similar remineralization potential than 1100 ppm F toothpaste. CLINICAL SIGNIFICANCE: Toothpastes containing 500 ppm F associated to CaGP, with both fluoride source (NaF or MFP), showed a potential of remineralization similar to commercial toothpaste. Although there is a need for confirmation in the clinical setting, these results point to an alternative for improving the risk-benefit relationship between fluorosis and dental caries in small children.

Avaliação da absorção colostral em neonatos ovinos da raça Bergamácia / Evaluation of colostral absorption in neonates of Bergamacia breed

Objetivou-se determinar o período de absorção das macromoléculas colostrais e a transferência de imunidade passiva em cordeiros da raça Bergamácia. Avaliou-se o proteinograma sérico dos cordeiros antes da ingestão de colostro até 48 horas de vida e das frações colostrais ao nascimento e 12 horas pós-parto. Foi avaliada a concentração de proteína total no soro por refratometria e sua relação com a densidade e quantidade de gamaglobulinas presentes no colostro. A concentração sérica de gamaglobulina nos cordeiros variou de 0,111±0,07g/dL antes da ingestão de colostro a 1,609±0,72g/dL às 48 horas. Nas amostras de colostro, a concentração variou de 3,125±1,27g/dL, imediatamente após o parto, para 1,378±0,82g/dL, 12 horas após. A concentração de proteína sérica total teve acréscimo de 4,46±0,58g/dL para 5,61±0,75g/dL entre o nascimento e após 48 horas, apresentando correlação positiva com a densidade e a proteína total colostral. A absorção colostral pelo cordeiro foi ascendente até 24 horas subsequentes ao parto, quando, então, iniciou-se sua estabilização. A quantificação da proteína sérica, com uso de refratômetro nos cordeiros, pode ser usada como método para avaliar a transferência de imunidade passiva, pois está diretamente relacionada com a absorção de gamaglobulina colostral...

The period of absorption of colostrum macromolecules and the passive immunity transfer of Bergamacia lambs was determined. The serum proteinogram of lambs before the intake of colostrum up 48 hours of life and colostrum fractions between delivery and twelve hours after birth were measured. The total protein concentration in serum was evaluated by refractometry and also its relationship with the density and amount in colostrum. The serum concentration of gamma globulin in lambs from 0.111±0.07g/dL before the intake of colostrum was 1.609±0.72g/dL at 48 hours. In the colostrum samples, the concentration was 3.125±1.27g/dL immediately after delivery and 1.378±0.82g/dL twelve hours after birth. Serum total protein concentration increased from 4.46±0.58g/dL to 5.61±0.75g/dL between birth and after 48 hours, there was positive correlation with the density and total protein colostrum. The lamb had ascendant colostrum absorption subsequent to delivery, for twelve hours and then began its stabilization. The quantification of serum protein with the use of the refractometer in lambs can be used as a method to evaluate the transfer of passive immunity because it is directly related to the absorption of colostral gammaglobulin...