Biotransformation of bromhexine by Cunninghamella elegans, C. echinulata and C. blakesleeana

Abstract Fungi is a well-known model used to study drug metabolism and its production in in vitro condition. We aim to screen the most efficient strain of Cunninghamella sp. among C. elegans, C. echinulata and C. blakesleeana for bromhexine metabolites production. We characterized the metabolites produced using various analytical tools and compared them with mammalian metabolites in Rat liver microsomes (RLM). The metabolites were collected by two-stage fermentation of bromhexine with different strains of Cunninghamella sp. followed by extraction. Analysis was done by thin layer chromatography, high performance thin layer chromatography, Fourier transform infrared spectroscopy, high performance liquid chromatography and Liquid chromatography–mass spectrometry. The role of Cytochrome P3A4 (CYP3A4) enzymes in bromhexine metabolism was studied. Fungal incubates were spiked with reference standard – clarithromycin to confirm the role of CYP3A4 enzyme in bromhexine metabolism. Three metabolites appeared at 4.7, 5.5 and 6.4 min retention time in HPLC. Metabolites produced by C. elegans and RLM were concluded to be similar based on their retention time, peak area and peak response of 30.05%, 21.06%, 1.34%, and 47.66% of three metabolites and bromhexine in HPLC. The role of CYP3A4 enzyme in metabolism of bromhexine and the presence of these enzymes in Cunninghamella species was confirmed due to absence of peaks at 4.7, 5.4 and 6.7 min when RLM were incubated with a CYP3A4 enzyme inhibitor – clarithromycin.

Anti-ulcer & antioxidant activities of Hedranthera barteri {(Hook F.) Pichon} with possible involvement of H+, K+ ATPase inhibitory activity.

Background & objective: Hedranthera barteri (HB) is used in folk medicine as a vermifuge, laxative and an anti-inflammatory agent. The aim of this study was to evaluate the anti-ulcer and antioxidant properties of the dichloromethane fraction of HB root (DMHBR). Methods: Anti-ulcerogenic activity was assessed in cold-restraint (CRU), aspirin (ASP), alcohol (AL), pyloric ligation (PL) induced gastric ulcer models in rats and histamine-induced duodenal ulcer (HST) in guinea pigs. The effect of DMHBR (100 mg/kg) on gastric juice for free and total acidity, peptic activity and mucin secretion, using the pylorus ligated model, were evaluated. The H+, K+-ATPase activity was assayed in gastric microsomes, spectrophotometrically. The in vitro anti-oxidant assays were explored through DPPH, nitric oxide, hydroxyl radical, superoxide anion scavenging assays. Results: DMHBR reduced the incidence of ulcers in CRU (63.3%), PL (58.5%), ASP (52.7%), HST (75.0%) and AL (53.87%). Also, reductions were observed in the free acidity (49.4%), total acidity (45.8%) and peptic activity (32.9%) with increase in the mucin secretion by 81.6 per cent. DMHBR (60-100 μg/ml) inhibited the H+,K+-ATPase activity with IC50 of 89.64 μg/ml compared with omeprazole (10-50 μg/ml ) with IC50 of 32.26 μg/ml. DMHBR showed antioxidant activity with IC50 values of DPPH (397.69 μg/ml), nitric oxide (475.88 μg/ml), hydroxyl radical (244.22 μg/ml) and superoxide anion radical (285.20 μg/ml). Interpretation & conclusion: DMHBR showed anti-ulcer activity against experimentally-induced peptic ulcer models and exhibited both cytoprotective and anti-secretory property. It exhibited a proton pump inhibition activity and its anti-ulcer properties may be partly ascribed to its antioxidant activities.

Effect of metformin on renal microsomal proteins, lipid peroxidation and antioxidant status in dexamethasone-induced type-2 diabetic mice.

An SDS-PAGE analysis of renal microsomal fraction of albino mice was performed to study the involvement of proteins in dexamethasone-induced type-2 diabetes mellitus (DM) and their alterations by metformin, a widely accepted oral antidiabetic drug. In addition, changes in renal lipid peroxidation (LPO), activities of superoxide dismutase (SOD) and catalase (CAT), reduced glutathione (GSH) content, as well as renal somatic index (RSI) and daily rate of water consumption were also investigated. While dexamethasone administration (1.0 mg/kg for 21 days) expressed two renal proteins (43 kDa and 63.23 kDa), in addition to the increased fasting serum levels of glucose and insulin, renal LPO, RSI and daily rate of water consumption, a parallel decrease in renal SOD, CAT and GSH was also observed. Treatment with metformin normalized these alterations including the renal proteins and LPO, confirming its efficacy in ameliorating dexamethasone-induced type-2 DM and also the association of two proteins with type-2 DM.

Cytochrome P450 enzyme isoforms and their therapeutic implications: an update.

Clinicians should be cognizant of potential drug drug interactions and become familiar with the substrates, inhibitors and inducers of the common enzymatic pathways responsible for drug metabolism. Our knowledge of and ability to predict drug interactions have improved with growing understanding of substrates, inhibitors and inducers of cytochrome P450 (CYP-450) isoenzymes. These isoenzymes are a major determinant of the pharmacokinetic behavior of numerous drugs. In addition to inhibition and induction, microsomal drug metabolism is affected by genetic polymorphisms, age, nutrition, hepatic disease and endogenous chemicals. Prescribing physicians by understanding the unique characteristics of these isoenzymes may better anticipate and manage drug drug interactions.

Inhibition of rat microsomal lipid peroxidation by the oral administration of D002

The effect of D002, a defined mixture of higher primary alcohols purified from bee wax, on in vivo and in vitro lipid peroxidation was studied. The extent of lipid peroxidation was measured on the basis of the levels of thiobarbituric acid reactive substances (TBARS). When D002 (5-100 mg/kg body weight) was administered orally to rats for two weeks, a partial inhibition of the in vitro enzymatic and non-enzymatic lipid peroxidation was observed in liver and brain microsomes. Maximal protection (46 percent) occurred at a dose of 25 mg/kg. D002 behaved differently depending on both the presence of NADPH and the integrity of liver microsomes, which suggests that under conditions where microsomal metabolism was favored the protective effect of D002 was increased. D002 (25 mg/kg) also completely inhibited carbon tetrachloride- and toluene-induced in vivo lipid peroxidation in liver and brain. Also, D002 significantly lowered in a dose-dependent manner the basal level of TBARS in liver (19-40 percent) and brain (28-44 percent) microsomes. We conclude that the oral administration of D002 (5, 25 and 100 mg/kg) for two weeks protected rat liver and brain microsomes against microsomal lipid peroxidation in vitro and in vivo. Thus, D002 could be useful as a dietary natural antioxidant supplement. More studies are required before these data can be extrapolated to the recommendation for the use of D002 as a dietary antioxidant supplement for humans

A microtiter plate assay for superoxide using MTT reduction method.

A simple, rapid and sensitive microtiter plate assay for superoxide using the reduction of tetrazolium dye MTT to its coloured formazan has been developed. The colour formed can be measured using a microtiter plate reader and the extent of reduction of MTT indicates the amount of superoxide generation. A comparison of the sensitivities of different procedures for the quantitation of superoxide generated by X-XO system has been made. The MTT reduction due to superoxide was confirmed by inhibiting the reduction using purified superoxide dismutase. Using this method superoxide generation by mitochondria and microsomes was demonstrated and this procedure is suitable for detection of intracellularly generated superoxide. The proposed method is inexpensive and is suitable for a routine analysis of large number of samples.

Involvement of a protease in tert-butylhydroperoxide-mediated activation of Ca2+ ATPase in microsomes of pulmonary smooth muscle.

Microsomes isolated from bovine pulmonary artery smooth muscle tissue treated with the oxidant t-buOOH stimulated Ca2+ ATPase activity dose-dependently as also protease activity when tested with a synthetic substrate N-benzoyl-DL-arginine p-nitroanilide. At 300 microM, t-buOOH optimally stimulated these activities. Treatment of the microsomes with t-buOOH stimulated ATP dependent Ca2+ uptake while Na+ dependent Ca2+ uptake was inhibited by t-buOOH. Pretreatment of the microsomes with vitamin E (1 mM) and aprotinin (1 mg/ml) prevented t-buOOH caused stimulation of protease activity and Ca2+ ATPase activity, and also stimulation of ATP dependent Ca2+ uptake while t-buOOH caused inhibition of Na+ dependent Ca2+ uptake was reversed by vitamin E and aprotinin. Treatment of the microsomes with trypsin (1 microgram/ml) stimulated Ca2+ ATPase and ATP dependent Ca2+ uptake while Na+ dependent Ca2+ uptake was inhibited. Pretreatment of the microsomes with aprotinin prevented trypsin caused stimulation of Ca2+ ATPase and ATP dependent Ca2+ uptake, while trypsin caused inhibition of Na+ dependent Ca2+ uptake was reversed by aprotinin.

Fast and slow gating types of SR ryanodine receptor/channel purified from canine latissimus dorsi muscle

The ryanodine receptor/channel (RyR) mediates the release of calcium from the sarcoplasmic reticulum (SR) in both skeletal and cardiac muscle cells. There are three isoforms of the RyR: RyR1, RyR2, and RyR3. RyR1 is specifically expressed in skeletal muscles and RyR2 in cardiac muscles. RyR3 is yet another isoform found in non-muscle cells such as neuronal cells. Single channel recordings of RyR1 and RyR2 reconstituted in artificial lipid bilayer show that the characteristics of two isoforms are very distinct. RyR1 has a shorter mean open time and is activated at a higher concentration of Ca2+ than RyR2. In this study, we isolated the heavy SR membranes from canine latissimus dorsi muscles and investigated the single channel activities from the heavy SR membrane fraction using Cs+ as a charge carrier. Two different types of activities were observed. The fast-gating type (FG) with the mean open time of 0.9 ms was more frequently recorded (n = 12) than the slow-gating type (SG) with the mean open time of 269.2 ms. From the I-V relation, the slope conductance of the FG was calculated to be 514.7 pS and the SG, to 625.6 pS. The activity of the fast gating type increased by raising the concentration of Ca2+ in the cis-solution up to 100 microM. The appearance of the SG in the canine heavy SR membrane fraction suggests a possibility that two types of RyR isoform are co-expressed in mammalian skeletal muscle as well as in avian, amphibian and piscine fast twitch muscles.

Toxicity of endosulfan on kidney of male rats in relation to drug metabolizing enzymes and microsomal lipid peroxidation.

Endosulfan administration (po, 15 and 30 days at 7.5 and 10 mg/kg body wt respectively) inhibited the activity of microsomal mixed function oxidases in kidney tissue of male rats. Microsomal and cytosolic protein contents of kidney were significantly increased following 30 days endosulfan exposures. Profound induction in the activity profiles of alcohol dehydrogenase and cytosolic glutathione s-transferase was noticed, however, no such change was apparent in the activity of aldehyde dehydrogenase. Microsomal preparations from treated animals showed a dose and duration dependent increase in spontaneous lipid peroxidation. The observed biochemical changes persisted even after 7 days normalcy allowance provided after the endosulfan (10 mg/kg body wt) withdrawl. The results suggest a substantial renal toxicity of endosulfan to male rats in relation to microsomal mixed function oxidases and associated functions which possibly resulted from lipid peroxidative damage of microsomal membrane in treated animals.

Changes in lipid composition and some biologically important enzyme activities in the microsomal membranes of developing toad ovary in different seasons.

The microsomal membranes isolated by sucrose density gradient centrifugation from developing toad ovary have been found to differ significantly in lipid composition and various enzyme activities in different seasons. All the enzymes studied, viz. Na+, K(+)-ATPase, delta 5-3 beta-hydroxysteroid dehydrogenase (delta 5-3 beta HSD) and prostaglandin synthetase, exhibited maximum activity during the breeding season (July-September) at all stages of development (a,b,c & d). The activities of Na+, K(+)-ATPase and delta 5-3 beta HSD increased with development while that of prostaglandin synthetase followed the reverse order. The total phospholipid, cholesterol and fatty acid contents also varied with season and development. The increase in Na+, K(+)-ATPase and delta 5-3 beta HSD activities in the microsomal membranes of toad ovary at breeding season is accompanied with concomitant increase in phospholipid and unsaturated fatty acid contents at different stages in this season, thereby suggesting some correlation between them.

Effect of high carbohydrate and high protein diets on microsomal fatty acid composition, fluidity and delta 6 desaturation activity in kidney and lung

La administración dietas hiperglucídicas e hiperproteicas suministradas a ratas durante 3 días produce respectivamente una disminución y un aumento en el cociente araquidonato/linoleato en los lípidos totales de microsomas de pulmón, riñon e hígado. En el hígado y el riñon este efecto está correlacionado con un significativo descenso de la actividad de la delta6 desaturasa para el caso de la dieta hiperflucídica y con un aumento de la misma actividad enzimática en la dieta hiperproteica. La actividad de la delta6 desaturasa, medida a través de la conversión del ácido 1-14**C linoleico a ácido alfa-linolénico, no se detectó en los microsomas de pulmón debido probablemente a la poca capacidad de este tejido para producir el éster de CoA del sustrato usado, y a que el cociente 20:4/18:2 en este tejido fue similar al del hígado bajo las condiciones dietéticas analizadas. La anisotropía de fluorescencia (r) del definilhexatrieno mostró diferencias significativas entre los tres tejidos analizados, efecto que se correlacionó con sus respectivos cocientes colesterol/fosfolípidos. Ambos parámetros fueron inferiores en los microsomas hepáticos que en los de los otros tejidos y permanecieron sin modificarse bajo los diferentes regímenes estudiados. Los resultados indican que el efecto de las dietas hiperhidrocarbonada e hiperproteica sobre la delta6 desaturasa no conduce a alteraciones aparentes en las propiedades físicas de las membranas microsomales

Effect of dietary columbinic acid on the fatty acid composition and physical membrane properties of different tissues of EFA- deficient rats

Se estudió el efecto del agregado de ácido columbínico (5 trans, 9 cis, 12 cis octadeca-trienoico) a una dieta libre de grasas sobre la composición de ácidos grasos de distintos tejidos de rata, y estos datos se correlacionaron con las propiedades físicas de dichos tejidos. La ausencia de lípidos en la dieta produjo cambios en la composición de ácidos grasos que son característicos de la deficiencia de ácidos grasos esenciales (AGE). Se observó un incremento significativo del porcentaje relativo de ácidos grasos monoenoicos acompañado de una disminución de los ácidos linoleico y araquidónico y un aumento del ácido eicosa-5,8,11-trienoico en los homogenatos de hígado riñon, pulmón y bazo. El ácido columbínico agregado a una dieta libre de grasas durante 24 ó 48 horas se incorporó en los distintos tejidos, elongándose parcialmente al ácido eicosa-7 trans 11 cis, 14 cis-trienoico, pero sin ser desaturado. El ácido columbínico modificó el perfil de composición de ácidos grasos de los lípidos en los distintos tejidos, de manera tal que su porcentaje de distribución fue similar al observado en los animales no deficientes en AGE, excepto por el descenso del ácido linoleico. La ausencia de lípidos en la dieta produjo un incremento en la anisotropía de fluorescencia determinada con excitación continua (rs) del 1,6-difenil-1,3,5-hexatrieno (DPH) en microsomas hepáticos, que se corrigió con la administración de ácido columbínico durante 24 hr. Se concluye que el ácido columbínico produjo un efecto favorable de corto alcance sobre las propiedades físicas de la membrana microsomal hepática (rs) atribuible a las modificaciones en la composición de ácidos grasos. El ácido columbínico, por lo tanto, induciría también un efecto favorable a corto plazo sobre la producción de eicosanos, pero no así a largo plazo