Dilip K. Ganguly

Thearubigin, the major polyphenol of black tea, ameliorates mucosal injury in trinitrobenzene sulfonic acid-induced colitis

Inflammatory bowel disease is characterized by oxidative and nitrosative stress, leukocyte infiltration and upregulation of proinflammatory cytokines. The aim of the present study was to examine the protective effects of thearubigin, an anti-inflammatory and anti-oxidant beverage derivative, on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice, a model for inflammatory bowel disease. Intestinal lesions (judged by macroscopic and histological score) were associated with neutrophil infiltration (measured as increase in myeloperoxidase activity in the mucosa), increased serine protease activity (may be involved in the degradation of colonic tissue) and high levels of malondialdehyde (an indicator of lipid peroxidation). Both nitric oxide (NO) and O(2)(-) were increased with concomitant upregulation in the mRNA expression of proinflammatory cytokine response and inducible NO synthase (iNOS). Dose-response studies revealed that pretreatment of mice with thearubigin (40 mg kg(-1) day(-1), i.g. for 10 days) significantly ameliorated the appearance of diarrhoea and the disruption of colonic architecture. Higher dose (100 mg kg(-1)) had comparable effects. This was associated with a significant reduction in the degree of both neutrophil infiltration and lipid peroxidation in the inflamed colon as well as decreased serine protease activity. Thearubigin also reduced the levels of NO and O(2)(-) associated with the favourable expression of T-helper 1 cytokines and iNOS. Consistent with these observations, nuclear factor kappa B (NF-kappa B) activation in colonic mucosa was suppressed in thearubigin-treated mice. The results of this study suggest that thearubigin, the most predominant polyphenol of black tea, exerts beneficial effects in experimental colitis and may, therefore, be useful in the treatment of inflammatory bowel disease.

Triterpenoid saponins from the roots of tea plant (Camellia sinensis var. assamica).

Three olean-12-ene type triterpenoid saponins, named TR-saponins A, B and C, were isolated as methyl esters from tea roots (Camellia sinesis var. assamica) after treatment with diazomethane. Their structures were established as the methyl esters of 3-O-alpha-L-arabinopyranosyl (1-->3)-beta-D-glucuronopyranosyl-21, 22-di-O-angeloyl-R1-barrigenol-23-oic acid, 3-O-alpha-L-arabinopyranosyl (1-->3)-beta-D-glucuronopyranosyl-21-O-angeloyl-22-O-2-me thylbutanoyl-R1- barrigenol-23-oic acid and 3-O-alpha-L-arabinopyranosyl (1-->3)-beta-D-glucuronopyranosyl-16 alpha-O-acetyl-21-O-angeloyl-22-O-2-methylbutanoyl-R1-bar rigenol-23-oic acid, by extensive 1D and 2D-NMR as well as FABMS and HR-MS analyses.

Effect of prostaglandin E2 on acetylcholine release from some peripheral cholinergic nerve terminals.

The effect of prostaglandin E2 (PGE2) on the acetylcholine (ACh) release evoked from rat phrenic nerve terminals and from Auerbachs plexus of the guinea-pig ileum was investigated. PGE2 enhanced the evoked release of ACh from phrenic nerve terminals and from Auerbachs plexus in a concentration-dependent manner. Preincubation with 7-oxa-13-prostynoic acid, the PGE receptor blocker, and indomethacin inhibited the PGE2-induced increase of evoked release of ACh while atropine failed to do so. Whereas a single administration of either 7-oxa-13-prostynoic acid or indomethacin significantly inhibited the control evoked release of ACh from the Auerbachs plexus, they failed to alter the control evoked release of ACh from the phrenic nerve terminals. The study indicates that the PGE2-induced increase in release of ACh from cholinergic nerve terminals is accomplished through activation of prostaglandin receptors and that PGE2 may play a physiological role in ACh liberation from the cholinergic autonomic nerve terminals but not from motor nerve terminals.

Neonatal treatment with 5-HT antiserum alters 5-HT metabolism and function in adult rats

Adult rats were studied for serotonin and its metabolite in basal ganglia, nociceptive responses to electric current and tremor induced by 5-methoxy-N,N-dimethyltryptamine (5-MeODMT) to assess the effects of a single systemic injection of serotonin antiserum given at 1 day old. Antiserum-treated animals showed no overt behavioural abnormalities, but tremor induced by 5-MeODMT was augmented. Basal nociceptive thresholds for tail withdrawal and vocalization were unaffected, whereas vocalization after-discharge was significantly reduced. Significantly decreased serotonin levels and increased turnover were found in nucleus caudatus putamen, nucleus accumbens and tuberculum olfactorium of adult rats treated as neonates with serotonin antiserum. These results demonstrate long-lasting functional alterations and neurochemical suppression of the central serotoninergic system following neonatal administration of serotonin antiserum.

Thermodynamics of the interaction of d-tubocurarine with nicotinic receptors of mammalian skeletal muscle in vitro

Thermodynamic study is important for defining drug receptor interactions, and denervated rat hemidiaphragm is a unique preparation for such a study on nicotinic receptors. As a continuation of our earlier study with acetylthiocholine on the same preparation, we now report on the characteristics of temperature-dependent binding of d-tubocurarine, a reversible antagonist. The O. Arunlakshana and H.O. Schild (1959, Br. J. Pharmacol. 14, 48) equation, as improved by D.R. Woud and R.B. Parker (1971, J. Pharmacol. Exp. Ther. 177, 13), was used to calculate the dissociation constant of d-tubocurarine at various temperatures (10-37 degrees C) from the parallel shift of the acetylcholine dose-response curve to the right by effective doses of d-tubocurarine. It was observed that the values of the dissociation constant increased with a decrease in temperature. Both the enthalpy (delta H degree) and entropy (delta S degree) changes as evaluated from the vant Hoff plot (In Kd vs. 1/T) were found to be positive and their relative value (delta H degree - T delta S degree) produced a negative free energy change which characterises the binding of d-tubocurarine as an entropy-controlled process. This finding is in agreement with the neurotoxin binding reported earlier. The present finding and earlier observations with acetylthiocholine reveal that agonist and antagonist binding to the nicotinic receptor may differ depending on the experimental conditions.

Thermodynamic studies with acetylthiocholine on nicotinic receptors of mammalian skeletal muscle in vitro

The temperature dependency of binding of acetylthiocholine, a specific nicotinic agonist, to the nicotinic receptor of mammalian skeletal muscle was studied using isotonic contractions of the rat denervated diaphragm preparation in vitro. The dissociation constants at different temperatures (22-39 degrees) were determined by the Furchgott method using alpha-bungarotoxin as an irreversible antagonist. Both free energy of association (delta G zero = -22.93 kJ/mol at 37 degrees) and enthalpy of binding (delta H zero = -58.35 kJ/mol) calculated from Kd (dissociation constant) and slope of lnKd versus 1/T (vant Hoff plot) respectively were found to be negative. The negative entropy value (delta S zero = -0.113 kJ/mol/deg) obtained from the intercept of this vant Hoff plot differs from the large positive value obtained earlier employing radioligand binding studies of the nicotinic receptor of Electrophorus electricus.

Behavioral and neurochemical alterations following intracerebroventricular administration of anti-serotonin antibodies in adult Balb/c mice

The effects of intracerebroventricular injections of serotonin (5-HT) antibodies were studied for changes in 5-HT, dopamine (DA), their metabolites and norepinephrine (NE) as well as 5-HT mediated behavior in adult mice. While nociceptive thresholds (tail-flick latency) were inhibited in antibody treated animals, tremor response to 5-methoxy-N,N-dimethyl tryptamine administration was increased. 5-HT and DA in the nucleus raphe dorsalis (NRD), substantia nigra (SN), nucleus caudatus putamen (NCP) and in the substantia grisea centralis, and NE in the former two nuclei were significantly decreased in these animals. 5-Hydroxyindoleacetic acid was unaffected in all nuclei except NRD, where it was inhibited. Homovanillic acid and 3,4-dihydroxyphenylacetic acid were inhibited in all nuclei except in NCP. The brunt of insult was more evident in NRD and SN where all neurotransmitters were inhibited for a longer period. 5-HT turnover was increased in all the nuclei, however only SN showed increased DA turnover. It may be assumed that the observed neurochemical and behavioral changes were the consequence of the antibodies binding to 5-HT, which in turn influenced the anatomically and functionally connected neurotransmitters. While the study contributes to the existing understanding of central neurotransmitter control on behavior, it fails to delineate the underlying mechanism. The possibility of developing a useful, drug-free 5-HT deficient animal model for studying clinical disorders, as well as for solving some of the basic questions related to the physiological functions of 5-HT in adult animals are envisaged from the study.

Interactions of some cholinolytic anti-Parkinson drugs with nicotine and oxotremorine on rat diaphragm

Abstract Interactions between the cholinolytic anti-Parkinson drugs cycrimine, benztropine, and biperiden, and the tremorogenic agents oxotremorine and nicotine were examined employing isolated phrenic nerve-diaphragm preparations in rats. Preincubation with the anti-Parkinson drugs prevented the impairment of neuromuscular transmission caused by oxotremorine and nicotine. The results indicate that the skeletal myoneural site may play a role in chemically induced Parkinson-like features in experimental animals, as well as in its prevention by the anti-Parkinson drugs.

Abrus (A. precatorius L.) Leaf Extract as a Novel Antitumor Agent

The 50% ethanolic extract of the leaf of Abrus (AE) has been used to observe antitumor properties against the human leukemic cell lines ML-1, ML-2, U-937, K-562, MOLT-16 (in vitro), and against mouse tumor, Ehrlich ascites carcinoma (EAC) cells (in vivo). Significant cell growth inhibitions were observed both in vitro and in vivo using 20μg/ml or 25mg/kg of AE, respectively. Depletion of “S”-phase cells was observed with accumulation of “G0-G1”-phase cells in the human leukemic cell line ML-2 using flow cytometry. Significant inhibitions of DNA, RNA, and protein synthesis were observed after 24h in treated cells. Morphological observation in treated leukemic cells showed shrinkage and fragmentation. However, the treated cells did not show nitroblue tetrazolium (NBT)-reducing activity, indicating no differentiation. In the in vivo system, increased peritoneal exudate cells in AE (25mg/kg)-treated mice may have been partially responsible for EAC cell growth inhibition. Glycerrhizin is at least one of the constituents of the extract which may have been responsible for such activity.

α2-antagonistic effect of N-carbamoyl-2-(2,6-dichlorophenyl) acetamidine hydrochloride (LON-954)

The effect of N-carbamoyl-2-(2,6-dichlorophenyl) acetamidine hydrochloride (LON-954) on alpha 2-adrenoceptors was studied in in vivo and in vitro preparations. Like yohimbine, LON-954 antagonised the clonidine-induced inhibition of twitch responses in the Auerbachs plexus of guinea pig ileum and rat vas deferens preparations. It also reversed the anti-convulsant effect of clonidine on pentylenetetrazol (PTZ)-induced convulsions in the rat, a property shared by yohimbine. However LON-954 failed to prevent the hypothermic response to clonidine in mice. The dissimilarity in action of LON-954 and yohimbine on clonidine-induced hypothermia could be due to the fact that the anticlonidine effect of yohimbine on hypothermia is mediated through its antiserotonin action. The results indicate that LON-954 acts as an alpha 2-antagonist both centrally and peripherally.