Effects of Compound troxerutin and poreine cerebroside injection on cytochrome P450 enzyme in vivo and in vitro / 中国药房

OBJECTIVE To investigate the effects of Compound troxerutin and poreine cerebroside injection on the activity of cytochrome P450 （CYP450） enzyme in vivo and in vitro. METHODS Human liver microsomes were incubated with Compound troxerutin and poreine cerebroside injection （volume fraction 0.05%-10%） and the specific probe substrates of CYP1A2， CYP2B6， CYP2C8， CYP2C9， CYP2C19， CYP2D6 and CYP3A4 for 30 min. The production of corresponding metabolites was detected by ultra-high performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）， and the half inhibitory concentration （IC50） was calculated. The relative mRNA expression （i.e. induction multiple） of CYP450 enzyme was determined by real-time fluorescence quantitative PCR after human primary hepatocytes were incubated with Compound troxerutin and poreine cerebroside injection （volume fraction 0.05%-10%） or 3 positive inducers of CYP1A2， CYP2B6， CYP3A4 for 48 hours. Male SD rats were randomly divided into control group （normal saline+probe substrates of CYP1A2， CYP2B6， CYP2C8， CYP2C9， CYP2C19， CYP2D6， CYP3A4 8， 2， 1， 1， 10， 10， 8 mg/kg） and experimental group （Compound troxerutin and poreine cerebroside injection 0.9 mL/kg+probe substrates of CYP1A2， CYP2B6， CYP2C8， CYP2C9， CYP2C19， CYP2D6， CYP3A4 8， 2， 1， 1， 10，10， 8 mg/kg）， with 6 rats in each group. The pharmacokinetic parameters of probe substrates were detected by UPLC-MS/MS and Cocktail probe drug method. RESULTS After the lzqpharm@126.com treatment of 0.05%-10% Compound troxerutin and poreine cerebroside injection， the activities of CYP2B6， CYP2C8 and CYP2C19 in human liver microsomes had no significant change， and IC50 could not be fitted； IC50 of CYP1A2， CYP2C9， CYP2D6 and CYP3A4 were 419.90%， 97.78%， 176.00%， 19.42%， respectively. After the treatment of 0.05%-10% Compound troxerutin and poreine cerebroside injection， the average induction multiple of CYP3A4 mRNA in human primary hepatocytes （No. MHK） was 4.88 （and the average induction multiples of 2 concentration points were higher than 2）. After the treatment of Compound troxerutin and poreine cerebroside injection， AUC0－t and AUC0－∞ of CYP2C8， CYP2C9 and CYP2C19 substrates were increased significantly， CL of CYP2C8 and CYP2C19 substrates were decreased significantly， while t1/2 of CYP2C9 substrate was prolonged significantly （P＜0.05）. CONCLUSIONS Compound troxerutin and poreine cerebroside injection has no obvious inhibitory effect on CYP1A2， CYP2B6， CYP2C8， CYP2C9， CYP2C19， CYP2D6 and CYP3A4 in human liver microsomes in vitro， but can induce the mRNA expression of CYP3A4 in human primary hepatocytes in vitro， and can inhibit the activities of CYP2C8， CYP2C9 and CYP2C19 in rats in vivo.

Strategies to increase cellulase production with submerged fermentation using fungi isolated from the Brazilian biome / Estratégias para produção de celulases através de fermentação submersa utilizando fungos isolados do bioma brasileiro

Studies on new microbial sources of cellulase and accurate assessment of the steps that increase cellulase production are essential strategies to reduce costs of various processes using such enzymes. This study aimed at the selection of cellulase-producing filamentous fungi, and at the research of parameters involving cellulase production by submerged fermentation. The first test consisted of selecting the best cellulase-producing microorganisms (FPase) in Erlenmeyer flasks containing 200 mL of specific growth medium. The next test was designed to further investigate the enzyme production in fermentation with four types of soluble sugars: glucose, lactose, sucrose and xylose. In bioreactor tests, three different inoculation strategies were analyzed. The best FPase activity was presented by the strain Trichoderma sp. CMIAT 041 (49.9 FPU L-1) and CMCase by the fungus Lasiodiplodia theobromae CMIAT 096 (350.0 U L-1). Sucrose proved to be the best option among the soluble sugars tested, with higher rates of FPase activity (49.9 FPU L-1) and CMCase (119.7 U L-1). The best inoculation strategy for the bioreactor was a spore suspension obtained from a semi-solid state fermentation of wheat bran for 72h.

Estudos sobre novas fontes microbianas e análises mais acuradas das etapas que compõem a produção de celulases são essenciais como estratégias para diminuir os custos gerados pelo uso de celulases nos processos de obtenção de açúcares fermentescíveis. O trabalho teve como objetivo a seleção de fungos filamentosos produtores de celulases e a investigação de parâmetros que envolvem a produção enzimática em fermentação submersa. O primeiro teste consistiu em selecionar os melhores fungos produtores de celulases totais em frascos Erlenmeyer contendo 200 mL de meio de cultura específico. O teste subsequente teve o intuito de investigar a produção enzimática com quatro tipos de açúcares solúveis: glicose, lactose, sacarose e xilose. Nos testes em biorreator foram analisados três diferentes estratégias de inoculação. Na etapa de seleção a melhor atividade de FPase foi apresentada por Trichoderma sp. CMIAT 041 (49,9 FPU L-1) e CMCase pelo fungo Lasiodiplodia theobromae CMIAT 096 (350,0 U L-1). O uso de sacarose mostrou-se ser a melhor opção dentre os açúcares solúveis testados, apresentando os maiores valores de atividade de FPase (49,9 FPU L-1) e CMCase (119,7 U L-1). A melhor estratégia de inoculação foi a suspensão de esporos obtidos a partir de fermentação em farelo de trigo, no tempo 72h.

Inhibitory and inductive effects of Polygonum capitatum water extract on CYP450 / 中国药理学通报

Aim To evaluate the inhibitive and induc-tive effects of Polygonum capitatum water extract on main cytochrome P450 isoforms in human and liver mi-crosomes of mouse in vitro for predicting the herb-drug interactions in clinical application. Methods The in vitro inhibitory effect was evaluated by incubating Po-lygonum capitatum water extract with the probe sub-strates of main phase I metabolic enzymes in human liver microsomes, including CYP1A2, CYP2E1, CYP2C9,CYP2C19 and CYP3A4. Mice were adminis-tered with Polygonum capitatum water extract at dosage of 0 . 58 g · kg-1 and 1 . 16 g · kg-1 by gastric lavage for successive 7 days and 14 days, then the cocktail-LC-MS/MS method was applied to assess the inductive effect of main CYP450 isoforms in mouse liver micro-somes. Results The IC50 values of Polygonum capita-tum water extract on main CYP450 isoforms in human liver microsomes were from 849 . 6 mg · L-1 to 2 287 mg·L-1 . Compared with the blank control group, the activites of CYP2C9 and CYP3A4 in 1. 16 g·kg-1 7 d group were about 49 . 9 % and 21. 1 % higher ( P <0. 01, P < 0. 05 ) respectively, the activities of CYP2C9 and CYP3A4 in 0. 58 g·kg-1 7 d group were 27. 6 % and 15. 5 % higher ( P <0. 01 , P <0. 05 ) respectively, the activities of CYP2C9 and CYP3A4 in 1. 16 g·kg-1 14 d group were 67. 5 % and 32. 1 %higher (P<0. 01) respectively, while the activities of CYP1 A2 , CYP2 E1 and CYP2 C19 were not increased significantly in Polygonum capitatum treatment group. Conclusions Polygonum capitatum water extract do not show the inhibitory effect on main CYP450 in hu-man liver microsomes. There is induction on CYP2C9 and CYP3 A4 in mouse liver microsomes by Polygonum capitatum water extract.

Evaluación de la harina de plátano (Musa paradisíaca L.) en ratones (M. musculus) fenilcetonúricos / Evaluation of platain (Musa paradisíaca L.) meal in phenylketonurics mice (M. musculus)

Los errores innatos del metabolismo se definen como trastornos genéticos que ocurren en la estructura y/o función de las moléculas proteicas. Uno de estos trastornos, es la fenilcetonuria (PKU, por sus siglas en inglés), la cual se presenta debido a la alteración en la reacción enzimática de la hidroxilación de fenilalanina (F). El tratamiento de la PKU es básicamente nutricional, por lo cual, en Venezuela, se están evaluando nuevas materias primas con bajo contenido de F, de manera que puedan ser incluidas en dietas de niños fenilcetonúricos. Estas materias primas, deben ser validadas a través de pruebas biológicas in vivo usando animales experimentales, antes de suministralos a los humanos. En el presente estudio se evaluó la harina de plátano Musa paradisíaca (como ingrediente único) como coadyudante en el manejo nutricional de ratones fenilcetonuricos inducidos experimentalmente utilizando un bloqueante de la enzima a-metilfenilalanina a una dosis de 24 µM/10 g peso vivo (PV) y el aminoácido D-fenilalanina a una dosis de 52µM/10 g p.v. Se observó que los ratones presentaron solo una elevación de los niveles de F (P≤0,05) alcanzando una hiperfenilalaninemia leve (4-10mg/dL de sangre) durante la etapa predestete. Sin embargo, al ser sometidos a una alimentación postdestete con la harina de plátano, como única fuente nutritiva, se pudo observar una diferencia en el desarrollo del animal (P≤0,05), evidenciándose un marcado deterioro y desnutrición. Se recomienda realizar estudios adicionales, que incorporen junto con la harina de plátano, un glicomacropéptido que supla los requerimientos mínimos nutricionales de los ratones en crecimiento y mantenga bajo los niveles de F.

Inborn errors of metabolism are defined as genetic disturbances that occur in the structure and/or function of protein molecules. Phenylketonuria (PKU) is a good example of this type of disturbance, which is due to an alteration in the enzymatic reaction responsible for phenylalanine (Phe) hydroxylation. Treatment of this disease is basically nutritional. In Venezuela, new raw materials with a low content of Phe are being tested, so that they can be included in the diets of children suffering from PKU. Previous administration to humans, these raw materials should be validated by in vivo biological tests, using laboratory animals. Therefore, the purpose of this study was to assess the plantain (Musa paradisíaca) meal, as the only ingredient, in the nutritional management of experimentally induced PKU in laboratory mice, using a-methyphenylalanine, as the agent responsible for blocking phenylalanine hydroxylase, at a dose of 24 µM/10 of body weight (BW). To help blockade, a dose of D-phenylalanine (52 µM/10g of BW), was also administered. The results of the investigation showed that during the pre-weaning stage, mice exhibited a slight elevation in Phe levels that was statistically significant (P≤0.05), reaching a hyperphenylalalinemia level of 4-10 mg/dL of blood. In contrast, when mice were subjected to a post-weaning diet with plantain meal as the only nutritional source, a difference (P≤0.05) in their development could be observed, which evidenced marked deterioration and malnutrition. It is recommended that additional studies be performed that include a glycomacropeptid along with the plantain meal, so that minimum nutritional requirements can be supplied, keeping low levels of Phe in PKU patients.

Evaluation of the induction of lipolytic enzymes froma Pseudomona aeruginosa isolated from african palm fruit (Eleasis guimeensis) / Evaluación de la inducción de enzimas lipolíticas a partir de una Pseudomona aeruginosa aislada del fruto de palma africana (Eleasis guineensis)

Background: Extracellular lipases are found in the culture broth when the fermentation is at the end of the exponential phase. Lipases can be induced easily since they are produced by the presence of oily sources or other materials as surfactants, fatty acids, some esters, glycerol and biliary salts. Objective: The aim of this work is to study the effect of carbon source concentration and the use of inductors on biomass production, and the lipolytic activity of a bacterium isolated from mature palm oil fruits. Methods: The yield biomass/substrate was evaluated with glucose as carbon source at different concentrations (3, 5, 7, 10, 15 y 20 g/L) by dry weight and OD (600 nm). Lipolytic activity was evaluated by spectrophotometric assay using p-nitrofenilpalmitate at 37°C for 15 min. Results: Gram negative microorganisms with lipolytic activity isolated from palm fruit were identified as Pseudomona aeruginosa. The growth of the bacteria was inhibited when glucose was used at concentrations greater than 5%. The production of lipase was induced by using three inducers (Palm oil, Tween 20 and palm oil:Tween 20 mixture), at three different induction times (0, 11 and 18 hours of fermentation). The highest activity (3,81 µmoles/mL*min) was observed when the palm oil:Tween 20 mixture was added at 11 hours of fermentation. The kinetic of p-nitrophenylpalmitate hydrolysis using the supernatant of a culture induced with palm oil:Tween 20 mixture at 11 hours showed the production of p-nitrophenol beyond 300 minutes, with the greatest hydrolysis rate during the first 7 minutes. Conclusions: The growth of P. aeruginosa was not affected by using glucose as carbon source at concentrations of 3% and 5%. There was a basal level of lipase production without inducer, and greater lipolytic activity was achieved with the addition of inducers.

Upregulation of extracellular matrix metalloproteinase inducer (EMMPRIN) and gelatinases in human atherosclerosis infected with Chlamydia pneumoniae: The potential role of Chlamydia pneumoniae infection in the progression of atherosclerosis

Chlamydia pneumoniae infection implicated as an important etiologic factor of atherosclerosis, especially in coronary artery disease (CAD), was found in vitro to be associated with the induction of matrix metalloproteinases (MMPs). An extracellular matrix metalloproteinase inducer (EMMPRIN)/membrane-type 1 matrix metalloproteinase (MT1-MMP) system which induces and activates MMPs,is suggested to be functional and were upregulated in the failing myocardium. However, the upstream regulation of MMPs by C. pneumoniae within atheroma itself remains unclear. We evaluated the seroepidemiologic study of C. pneumoniae infection in CAD patients (n = 391) and controls (n = 97) and performed histopathological and in vitro analysis in atherosclerotic vascular tissues obtained from patients with seropositive to C. pneumoniae (n = 20), by using immunochemistry for C. pneumoniae, EMMPRIN/MT1-MMP, MMP-2, and MMP-9. The seropositive rates of both anti-C. pneumoniae IgG and IgA were 56.7% in CAD group and 43.3% in control group (P =0.033). Seropositive rate was increased in subgroups of CAD patients without conventional coronary risk factors compared to those with conventional risk factors. Immunoreactivities of EMMPRIN, MT1-MMP, MMP-2, and MMP-9 were increased in the atheromatous plaque itself, predominantly in immunoreactive macrophages/mononuclear cells to C. pneumoniae. Furthermore, Western blot analysis showed that EMMPRIN and MMP-2 were detected more prominently in atherosclerotic tissues infected with C. pneumoniae compared to control tissues. Zymographic analysis revealed that activities of MMP-2 and MMP-9 were more increased in atherosclerotic tissues infected with C. pneumoniae compared to control tissues. The present study demonstrated upstream regulation of MMPs can be induced by C. pneumoniae within atheromatous plaque itself. These findings help to understand the potential role of C. pneumoniae in the progression of atherosclerosis.

Arylamine N-methyltransferase and thiol methyltransferase activities in cholestatic rat liver induced by common bile duct ligation

Methylation catalyzed by methyltransferases is a major metabolic pathway for an inactivation of some catecholamines, niacinamide as well as aliphatic sulfhydryl drugs and toxic hydrogen sulfides. To investigate the effects of obstructive jaundice in an animal model, common bile duct ligation (CBDL) was performed in the rat and enzyme activities of S-adenosyl-L-methionine-dependent arylamine N-methyltransferase and thiol methyltransferase were examined in liver cell fractions and serum for a period of 42 d after CBDL. Both mitochondrial and microsomal arylamine N-methyltransferase showed significant increases in their activities between the 1st through the 7th day (P < or = 0.05 to 0.001), and between the 1st through the 28th day (P X or = 0.01 to 0.001) post-ligation, although the cytosolic arylamine N-methyltransferase activity did not show a significant change compared to the activities from the sham-operated control. The mitochondrial as well as microsomal thiol methyltransferase showed significant increases in their activities between the 1st through the 28th day (P < or = 0.05 to 0.01 and P < or = 0.01 to 0.001, respectively) post-ligation, although the cytosolic thiol methyltransferase activity did not show a significant change compared to the activities from the sham-operated control. Arylamine N-methyltransferase and thiol methyltransferase in the serum from cholestatic rats also showed significant increases in their activities between the 1st through 28th day (P < or = 0.01 to 0.001), and between the 0.5th through the 42nd day (P < or = 0.05 to 0.001) post-ligation compared to the sham-operated control, respectively. Enzyme kinetic parameters (Km and Vmax) of hepatic membrane-bound arylamine N-methyltransferase and thiol methyltransferase were analyzed with the preparation from the 7th day post-ligation, using tryptamine or 4-chlorothiophenol as substrates and S-Adenosyl-L-[methyl-3H]methionine as co-substrate. The results indicate that although the Km values were about the same as the sham-operated control, the Vmax values of both enzymes increased significantly (P < or = 0.01 and 0.001, respectively). These results suggest that the biosynthesis of arylamine N-methyltransferase and thiol methyltransferase have been induced in response to obstructive jaundice.

Antiproliferation and induction differentiation of rosiglitazone in human gastric carcinoma transplanted into nude mice / 肿瘤研究与临床

Objective To investigate the antiproliferation and induction differentiation of human gas- tric carcinoma which human gastric lower-differentiation mucinous carcinoma MGC-803 cells transplanted in- to nude mice by using rosiglitazone(ROS),and to preliminarily explore the mechanism of differentiation. Methods The mice were randomly divided into five groups:model,ATRA,ROS 25 mg?kg~(-1),ROS 50 mg/kg, ROS 100 mg/kg.After that the volumes were measured and inhibition rates were calculated.The cell cycle was detected by FCM.The protein expression level of Mucin SAC was detected by immunohistochemistry. Results The volume of tumor decreased significantly in ROS treatment groups,the differences had statistical significance compared with model group(P0.05).The xenograft tumors of ROS groups demonstrated the characteristics of differentiation.Xenograft tumor cells were arrested in G_0/G_1 phase,and the cells in S phase decreased significantly,and up-regulated Mucin SAC gene expression.Conclusion ROS could inhibit the growth of tumor,and the effect were dose-dependent with ROS.ROS could induce the differentiation of Xenograft tumor cells of gastric cancer.Its mechanism might be related to the inhibit of transition from G_1 to S phase,degrade the activity of proliferation,regulate the expres- sion of Mucin 5AC.

The influence of Herba Artemisiae capillaris on the UDPGT activity in rat's hepat / 中成药

Objective:In order to investigate the influence of Herba Artiemisiae Capilloais on the UDPGT activity in rat's hepat.Methods: The UDPGT activities induced by Herba Artemisae capillaris were determined by the Black's assay.Results: Herba Artemisiae capillaris increased the activity of UDPGT in rats' hepat and also increased the ratio of liver weight/body weight. Conclusions: Herba Artemisiae capillaris was able to induce the acitvity of UDPGT in rats' hepat.

Mechanism of action of aflatoxin B1.

The inhibitory effects of aflatoxin B1 were found to be related to the gram character in procaryotes, used in this study. Ethylene diamine tetra chloroacetic acid (0·05 % w/v) or Tween-80 (0·05 % v/v) addition accentuated the aflatoxin B1 growth inhibition in Salmonella typhi and Escherichia coli at different pH values. The inhibition of lipase production was only 5–20 % in Pseudomonas fluorescence ca. 25–48% in Staphylococcus aureus and Bacillus cereus at different aflatoxin B1 concentrations (4–16 μg/ml).However, inhibition of α-amylase induction was complete in Bacillus megaterium whereas the inhibition was partial in Pseudomonas fluorescence (27–40%) at 32 μg aflatoxin B1 concentration. An increase in leakage of cell contents and decreased inulin uptake were observed in toxin incubated sheep red blood cell suspension (1 %) with increased aflatoxin B1 concentration.