Effect of (-)-∆9-tetrahydrocannabinoid on the hepatic redox state of mice

(-)-∆9-Tetrahydrocannabinol (∆9-THC), a psychoactive component of marijuana, has been reported to induce oxidative damage in vivo and in vitro. In this study, we administered ∆9-THC to healthy C57BL/6J mice aged 15 weeks in order to determine its effect on hepatic redox state. Mice were divided into 3 groups: ∆9-THC (N = 10), treated with 10 mg/kg body weight ∆9-THC daily; VCtrl (N = 10), treated with vehicle [1:1:18, cremophor EL® (polyoxyl 35 castor oil)/ethanol/saline]; Ctrl (N = 10), treated with saline. Animals were injected ip twice a day with 5 mg/kg body weight for 10 days. Lipid peroxidation, protein carbonylation and DNA oxidation were used as biomarkers of oxidative stress. The endogenous antioxidant defenses analyzed were glutathione (GSH) levels as well as enzyme activities of superoxide dismutase, catalase, glutathione S-transferase, glutathione reductase, and glutathione peroxidase (GPx) in liver homogenates. The levels of mRNA of the cannabinoid receptors CB1 and CB2 were also monitored. Treatment with ∆9-THC did not produce significant changes in oxidative stress markers or in mRNA levels of CB1 and CB2 receptors in the liver of mice, but attenuated the increase in the selenium-dependent GPx activity (Δ9-THC: 8%; VCtrl: 23% increase) and the GSH/oxidized GSH ratio (Δ9-THC: 61%; VCtrl: 96% increase), caused by treatment with the vehicle. Δ9-THC administration did not show any harmful effects on lipid peroxidation, protein carboxylation or DNA oxidation in the healthy liver of mice but attenuated unexpected effects produced by the vehicle containing ethanol/cremophor EL®.

Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithelial cells, while endothelin-3 was added to stimulate their growth. By adding endothelin-3, the achievement ratio (viable cell cultures/total cultures) was enhanced to 60% of a total of 10 cultures (initiated from 8 distinct fetal small intestines), allowing the generation of viable epithelial cell cultures. Western blot, real-time PCR and immunofluorescent staining showed that cytokeratins 8, 18 and mouse intestinal mucosa-1/39 had high expression levels in human intestinal epithelial cells. Differentiated markers such as sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV also showed high expression levels in human intestinal epithelial cells. Differentiated human intestinal epithelial cells, with the expression of surface markers (cytokeratins 8, 18 and mouse intestinal mucosa-1/39) and secretion of cytokines (sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV), may be cultured by the thermolysin and endothelin-3 method and maintained for at least 20 passages. This is relatively simple, requiring no sophisticated techniques or instruments, and may have a number of varied applications.

The chronic blockade of angiotensin I-converting enzyme eliminates the sex differences of serum cytokine levels of spontaneously hypertensive rats

Sex hormones modulate the action of both cytokines and the renin-angiotensin system. However, the effects of angiotensin I-converting enzyme (ACE) on the proinflammatory and anti-inflammatory cytokine levels in male and female spontaneously hypertensive rats (SHR) are unclear. We determined the relationship between ACE activity, cytokine levels and sex differences in SHR. Female (F) and male (M) SHR were divided into 4 experimental groups each (n = 7): sham + vehicle (SV), sham + enalapril (10 mg/kg body weight by gavage), castrated + vehicle, and castrated + enalapril. Treatment began 21 days after castration and continued for 30 days. Serum cytokine levels (ELISA) and ACE activity (fluorimetry) were measured. Male rats exhibited a higher serum ACE activity than female rats. Castration reduced serum ACE in males but did not affect it in females. Enalapril reduced serum ACE in all groups. IL-10 (FSV = 16.4 ± 1.1 pg/mL; MSV = 12.8 ± 1.2 pg/mL), TNF-α (FSV = 16.6 ± 1.2 pg/mL; MSV = 12.8 ± 1 pg/mL) and IL-6 (FSV = 10.3 ± 0.2 pg/mL; MSV = 7.2 ± 0.2 pg/mL) levels were higher in females than in males. Ovariectomy reduced all cytokine levels and orchiectomy reduced IL-6 but increased IL-10 concentrations in males. Castration eliminated the differences in all inflammatory cytokine levels (IL-6 and TNF-α) between males and females. Enalapril increased IL-10 in all groups and reduced IL-6 in SV rats. In conclusion, serum ACE inhibition by enalapril eliminated the sexual dimorphisms of cytokine levels in SV animals, which suggests that enalapril exerts systemic anti-inflammatory and anti-hypertensive effects.

Maternal protein restriction affects gene expression and enzyme activity of intestinal disaccharidases in adult rat offspring

This study investigated the consequences of intrauterine protein restriction on the gastrointestinal tract and particularly on the gene expression and activity of intestinal disaccharidases in the adult offspring. Wistar rat dams were fed isocaloric diets containing 6% protein (restricted, n = 8) or 17% protein (control, n = 8) throughout gestation. Male offspring (n = 5-8 in each group) were evaluated at 3 or 16 weeks of age. Maternal protein restriction during pregnancy produced offspring with growth restriction from birth (5.7 ± 0.1 vs 6.3 ± 0.1 g; mean ± SE) to weaning (42.4 ± 1.3 vs 49.1 ± 1.6 g), although at 16 weeks of age their body weight was similar to control (421.7 ± 8.9 and 428.5 ± 8.5 g). Maternal protein restriction also increased lactase activity in the proximal (0.23 ± 0.02vs 0.15 ± 0.02), medial (0.30 ± 0.06vs 0.14 ± 0.01) and distal (0.43 ± 0.07vs 0.07 ± 0.02 U·g-1·min-1) small intestine, and mRNA lactase abundance in the proximal intestine (7.96 ± 1.11vs 2.38 ± 0.47 relative units) of 3-week-old offspring rats. In addition, maternal protein restriction increased sucrase activity (1.20 ± 0.02 vs 0.91 ± 0.02 U·g-1·min-1) and sucrase mRNA abundance (4.48 ± 0.51 vs 1.95 ± 0.17 relative units) in the duodenum of 16-week-old rats. In conclusion, the present study shows for the first time that intrauterine protein restriction affects gene expression of intestinal enzymes in offspring.

Activation of endogenous angiotensin converting enzyme 2 prevents early injuries induced by hyperglycemia in rat retina

Diabetic retinopathy (DR) is a serious complication of diabetes mellitus that may result in blindness. We evaluated the effects of activation of endogenous angiotensin converting enzyme (ACE) 2 on the early stages of DR. Rats were administered an intravenous injection of streptozotocin to induce hyperglycemia. The ACE2 activator 1-[[2-(dimethylamino) ethyl] amino]-4-(hydroxymethyl)-7-[[(4-methylphenyl) sulfonyl] oxy]-9H-xanthone 9 (XNT) was administered by daily gavage. The death of retinal ganglion cells (RGC) was evaluated in histological sections, and retinal ACE2, caspase-3, and vascular endothelial growth factor (VEGF) expressions were analyzed by immunohistochemistry. XNT treatment increased ACE2 expression in retinas of hyperglycemic (HG) rats (control: 13.81±2.71 area%; HG: 14.29±4.30 area%; HG+XNT: 26.87±1.86 area%; P<0.05). Importantly, ACE2 activation significantly increased the RCG number in comparison with HG animals (control: 553.5±14.29; HG: 530.8±10.3 cells; HG+XNT: 575.3±16.5 cells; P<0.05). This effect was accompanied by a reduction in the expression of caspase-3 in RGC of the HG+XNT group when compared with untreated HG rats (control: 18.74±1.59; HG: 38.39±3.39 area%; HG+XNT: 27.83±2.80 area%; P<0.05). Treatment with XNT did not alter the VEGF expression in HG animals (P>0.05). Altogether, these findings indicate that activation of ACE2 reduced the death of retinal ganglion cells by apoptosis in HG rats.

EFEITO DO TRATAMENTO TÉRMICO SOBRE A ATIVIDADE DE PEROXIDASE (POD) E POLIFENOLOXIDASE (PPO) EM MAÇÃ (Mallus comunis)

O extrato enzimático foi preparado a partir da polpa e casca da maçã de cultivares Fuji e Gala utilizando tampão fosfato de sódio 100mM, pH 5,0 como solução extratora. Dentre as análises determinou-se a concentração de proteína nos extratos enzimáticos concentrados de polpa e casca, sendo que o cultivar Fuji apresentou teores mais elevados em comparação ao cultivar Gala. Os tratamentos térmicos foram realizados nas temperaturas de 60, 65, 70 e 75°C por períodos que variaram de 1 a 10 minutos, sendo observado diminuição da atividade de POD e PPO com o aumento da temperatura e tempo; no entanto a POD não chegou a ser inativada em nenhum dos tratamentos realizados. A PPO foi inativada totalmente após 10 minutos de tratamento a 75°C. A eletroforese mostrou uma composição diferente de isoenzimas aniônicas e catiônicas da peroxidase.

Características da atividade da peroxidase de abacaxis (Ananas comosus (L.) Merrill) da cultivar IAC Gomo-de-mel e do clone IAC-1

No presente trabalho foram estudadas as características bioquímicas das peroxidases de novos abacaxis, cultivar IAC Gomo-demel e clone IAC-1. As peroxidases dos sucos destes abacaxis apresentaram atividade ótima entre 45ºC e 50°C e entre 50°C e 55°C, respectivamente. Estas peroxidases apresentaram atividade ótima em pH 4,5 e mostraram-se estáveis na faixa de pH 4,0 a 9,0, retendo, após 24 horas de incubação a 50ºC, mais de 80% da atividade inicial. Foi observada regeneração parcial da atividade da peroxidase, após tratamento a 75ºC por 10 minutos. As peroxidases dos sucos dos abacaxis estudados foram inativadas, após tratamento a 90ºC por 2 minutos.

Peroxidase (POD) e polifenoloxidase (PPO) em polpa de goiaba (Psidium guajava R.)

A peroxidase E.C. 1.11.1.7 (POD) e a polifenoloxidase E.C. 1.10.3.1 (PPO) foram extraídas da polpa de goiaba. Os extratos foram preparados utilizando-se a polpa da goiaba e solução tampão fosfato de sódio 100mM com pH variando de 6,0 a 7,0 em intervalos de 0,1. Foi determinada a atividade enzimática da peroxidase e da polifenoloxidase desses extratos, a fim de se observar o melhor pH para a extração de cada enzima. O pH 6,3 foi considerado o melhor para a extração da POD da polpa de goiaba, enquanto que para PPO, o pH foi 6,8. Os extratos brutos de POD e PPO foram submetidos a temperaturas de 60 °C, 65 °C, 70 °C, 75 °C e 80 °C por um período de 0 a 10 min. Os resultados demonstraram um decréscimo da atividade enzimática nos extratos com o aumento da temperatura e do tempo. No entanto, a total inativação não foi atingida o que sugere a presença de isoenzimas termoresistentes.

Obtenção de nova fonte de peroxidase de folha de Copaifera langsdorffii Desf. com alta atividade

Objetivou-se neste trabalho extrair peroxidase de folha de Copaifera langsdorffii (COP), medir sua atividade, compará-la com a peroxidase de raiz forte (Horseradish peroxidase - HRP) e determinar o pH ótimo, a melhor solução extratora e o efeito de aditivos sobre a atividade da COP. Os resultados mostraram que a COP atingiu 81,6% da atividade de HRP e a faixa de pH ótimo foi de 5,5 a 6,0. A melhor solução extratora da enzima foi o tampão fosfato de sódio 50 mM, pH 6,0 e o melhor aditivo foi o PVPP. Concluindo, a COP apresenta atividade mais alta que outras peroxidases de diferentes fontes citadas na literatura.

Extração e caracterização parcial de peroxidase de folhas de Copaifera langsdorffii Desf.

Em recentes publicações têm sido descritos vários processos para obtenção de peroxidases. O propósito deste trabalho foi extrair peroxidase de folhas de Copaifera langsdorffii e caracterizar parcialmente a enzima usando planejamento experimental e teste univariado, para confirmação dos resultados obtidos por planejamento experimental. A atividade da peroxidase foi medida usando sistema guaiacol: peróxido de hidrogênio. A peroxidase isolada apresentou 81,6% da atividade da horseradish peroxidase e é de fácil obtenção, a partir de folhas de uma árvore abundante em todo o país. A peroxidase semi-purificada (COP) foi obtida pela precipitação do extrato bruto com acetona 65% (v.v-1), produzindo o pó cetônico. A COP apresentou atividade ótima na faixa de pH 5,0 a 7,0 e temperatura de 5 a 45 °C, com atividade máxima em pH 6,0 e 35 °C. A enzima mostrou-se estável em temperaturas inferiores a 50 °C e pH entre 4,5 e 9,0, por até 24 horas. A peroxidase foi inativada após 4 horas a 80 °C e após 3 minutos a 96 °C. Esta enzima demonstra possibilidade para ser usada como reagente para diagnósticos, construção de biossensores e outros métodos analíticos em vários campos da ciência.

Atividades das enzimas peroxidase (POD) e polifenoloxidase (PPO) nas uvas das cultivares benitaka e rubi e em seus sucos e geléias

A peroxidase e a polifenoloxidase estão relacionadas com o escurecimento de frutas, por isso o controle das atividades destas enzimas é de grande importância para a tecnologia de alimentos. Neste trabalho estudaram-se as atividades dessas enzimas nas uvas frescas das cultivares Benitaka e Rubi, bem como as suas termoestabilidades e as suas atividades enzimáticas residuais no suco e nas geléias (extra e light). Foi também avaliada a qualidade microbiológica dos produtos elaborados. As atividades da enzima POD, tanto da fração solúvel quanto da ionicamente ligada, foram semelhantes nas uvas das duas variedades, Benitaka e Rubi. A atividade da enzima polifenoloxidase foi maior na variedade Rubi. As operações de cocção e pasteurização foram mais eficientes para baixar as atividades enzimáticas residuais da POD e PPO quando aplicadas às geléias de uva, em comparação com o suco. Embora não foram suficientes para a total inativação enzimática, essas operações reduziram-nas consideravelmente, e foram eficientes para garantir seguridade microbiológica dos produtos, geléias e suco.

Polyphenoloxidase and peroxidase in avocado pulp (Persea americana Mill.)

The aim of the present investigation was to evaluate the enzymatic activity of polyphenoloxidase and peroxidase in avocado pulps, from the Northwest area of Paraná-Brazil, in order to compare the varieties on their enzymatic activity for both, minimum and industrial processing. Enzymatic extracts were prepared from avocado pulp of Choquete, Fortuna and Quintal varieties, in green and ripe maturation stage. Thermal treatment was applied with temperatures 60, 65, 70, 75 and 80 °C. The enzymatic activities were determined by using spectrophotometer. A decline of polyphenoloxidase activity was observed in all of the varieties when both, temperature and time increased. Total inactivation of enzymes was not observed in the largest temperature. Fortuna and Choquete variety showed the lowest polyphenoloxidase activity in the ripe stage. Soluble peroxidase showed activity in the green stage, whereas, ionically bound peroxidase activity increased with the change from green to ripe maturation stage in Choquete variety.

Thermal inactivation of polyphenoloxidase and peroxidase in Jubileu clingstone peach and yeast isolated from its spoiled puree

The thermal inactivation of yeast isolated from spoiled Jubileu peach puree and that of polyphenoloxidase (PPO) and peroxidase (POD) in cv. Jubileu, which is widely cultivated in southern Rio Grande do Sul state, Brazil, were studied. PPO and POD were extracted using the protein powder method and submitted to partial purification by precipitation followed by dialysis. The enzymatic activity was determined measuring the increase in absorbance at 420 nm for PPO and 470 nm for POD. The yeast used in this investigation was isolated from spoiled Jubileu peach puree at 22 °Brix, with total initial microbial count of 22 × 10² UFCmL- 1. Stock cultures were maintained on potato dextrose agar (PDA) slants at 4 °C and pH 5 for later use for microbial growth. In all cases, kinetic analysis of the results suggests that the thermal inactivation was well described by a first-order kinetic model, and the temperature dependence was significantly represented by the Arrhenius law. Both enzymes were affected by heat denaturation, and PPO was more thermostable. PPO was also more thermosTable than the yeast isolated from peach puree. The D60-values were 1.53 and 1.87 min for PPO and yeast isolated from spoiled Jubileu peach puree, respectively.

Peroxidase activity and sensory quality of ready to cook mixed vegetables for soup: combined effect of biopreservatives and refrigerated storage

Enzymatic senescence processes and browning of fresh cut vegetables negatively affect their sensory properties and nutritional value and finally result in the rejection of affected products by consumers. In order to prevent quality decay, the combined effects of natural antioxidants and storage temperature on peroxidase activity and sensory attributes (overall visual quality, browning and odor) of individual and mixed vegetables for soup (butternut squash, leek and celery) were evaluated. Fresh cut vegetables were treated with antioxidant solutions as tea tree essential oil (15 μl/mL), propolis extract (15 μl/mL) and gallic acid (2 mg/mL) and stored at optimal (5 °C) and abusive (15 °C) temperature for a maximum of 14 days. The application of natural preservatives, plus optimal storage conditions, exerted significant inhibitory effects in peroxidase activity of squash, celery and mixed vegetables throughout the storage. Furthermore, propolis treatment applied on mixed vegetables retarded browning appearance and preserved the visual quality for a longer period when compared to untreated product.

Influence of the degree of hydrolysis and type of enzyme on antioxidant activity of okara protein hydrolysates

Abstract The objective of this work was to evaluate the antioxidant activity of protein hydrolysates obtained by the enzymatic hydrolysis of okara using an endopeptidase (Alcalase) and exopeptidase (Flavourzyme). The reaction was monitored by the pH-stat procedure in which five aliquots were collected during the hydrolysis by each enzyme, corresponding to different degrees of hydrolysis (DH). The antioxidant activities of the aliquots were evaluated by the ABTS, DPPH and FRAP methods. For the hydrolysates obtained using Alcalase, the antioxidant activities increased from: 68.6 to 99.5% (ABTS), 14.5 to 17.7% (DPPH) and 222.6 to 684.9 µM Trolox (FRAP), when the DH varied from 0 to 33.6%. With respect to Flavourzyme, the results were: 67.2 to 88.2% (ABTS), 9.5 to 18.5% (DPPH) and 168.0 to 360.3 µM Trolox (FRAP), when the DH increased up to 5.8%. The results showed that the protein hydrolysates had antioxidant capacities, which were influenced by the degree of hydrolysis and the type of enzyme.