Sheelendra Pratap Singh

Reversal of cadmium induced oxidative stress by chelating agent, antioxidant or their combination in rat

The influence of an antioxidant agent such as N-acetyl cysteine (NAC) or mannitol on the cadmium chelating ability of monoisoamyl 2,3-dimercaptosuccinate (MiADMS) was investigated in cadmium pre-exposed rats. This ester of 2,3-dimercaptosuccinic acid (DMSA), an accepted drug for lead poisoning, being lipophilic in nature was expected to be an efficient cadmium chelator. The treatment of cadmium intoxicated animals with MiADMS reversed cadmium induced increase in blood catalase, superoxide dismutase (SOD) and malondialdehyde (MDA), liver MDA and brain SOD and MDA levels but not the decrease in blood, liver brain reduced glutathione (GSH) and increase in oxidized glutathione (GSSG) levels, consistent with the lowering of tissue cadmium burden. The administration of NAC or mannitol reversed the cadmium induced alterations in blood and liver GSH, GSSG, blood catalase, SOD, MDA, liver SOD, MDA and brain MDA levels without lowering blood and tissue cadmium contents. However, treatments with the combination of MiADMS and NAC or MiADMS and mannitol reversed these alterations as well as reduced blood and tissue cadmium concentrations. The combined treatment with MiADMS and mannitol was better than that with MiADMS and NAC, and was significantly more effective in normalizing blood, liver GSH, GSSG, brain GSSG, and their GSH/GSSG ratios than that by either of them alone. The combined treatments also improved liver and brain endogenous zinc levels, which were decreased due to cadmium toxicity. The results suggest that the administration of an antioxidant during chelation of cadmium may provide beneficial effects by reducing oxidative stress without its cadmium removing ability.

Effect of donepezil and tacrine on oxidative stress in intracerebral streptozotocin-induced model of dementia in mice.

Oxidative stress is a major factor implicated in the degeneration of cholinergic neurons in Alzheimers disease. Presently, cholinesterase inhibitors are the mainstay of therapy for Alzheimers disease. However, the potential of cholinesterase inhibitors as antioxidants, an important aspect for neuroprotection, has not been properly investigated. Therefore, the present study was designed to investigate the influence of antidementia drugs, tacrine and donepezil, on biochemical markers of oxidative stress, glutathione (GSH) and malondialdehyde (MDA), and acetylcholinesterase activity in the brain in a streptozotocin-induced experimental model of dementia in mice. Intracerebral (i.c.) injection of streptozotocin at a dose of 0.5 mg/kg on 1st and 3rd days caused significant deficits in memory function, as evaluated in a passive avoidance test and Morris Water Maze (spatial memory) test 14 days after the 1st dose. Mice were treated with tacrine and donepezil at a dose of 5 mg/kg orally in separate groups. Both tacrine- and donepezil-treated mice showed a significant improvement of the streptozotocin (i.c.)-induced memory impairment. Streptozotocin (i.c.) administration caused a significant decrease in GSH and increase in MDA as compared to control, indicating a state of oxidative stress in the brain of streptozotocin (i.c.) amnesic mice. Treatment of streptozotocin (i.c.) amnesic mice with tacrine or donepezil did not cause significant changes in GSH and MDA levels in the brain as compared to control. Streptozotocin amnesic mice had raised acetylcholinesterase activity in the brain while there was a significant decrease in brain acetylcholinesterase activity in tacrine- and donepezil-treated streptozotocin (i.c.) mice. Thus, results indicate that tacrine and donepezil, beside inhibition of acetylcholinesterase, may also suppress oxidative stress.

Gugulipid, an extract of Commiphora whighitii with lipid-lowering properties, has protective effects against streptozotocin-induced memory deficits in mice

Gugulipid, an ethyl acetate extract of the resin of plant Commiphora whighitii is an established hypolipidemic agent in clinical practice. The major constituent of gugulipid is guggulsterone [4, 17 (20)-pregnadiene-3, 16-dione]. It has been observed recently that patients receiving lipid-lowering drugs like statins have a reduced risk of dementia. Therefore, the present study was planned to explore the potential of gugulipid as cognitive enhancer. Gugulipid (12.5, 25 and 50 mg/kg, p.o.) showed dose dependent improvement in scopolamine-induced deficits in passive avoidance test. The maximal effective dose of gugulipid i.e. 50 mg/kg, p.o. was used for further studies on streptozotocin (STZ) model of dementia in mice. Gugulipid was investigated for its effect on learning and memory, parameters of oxidative stress (GSH and MDA) and acetylcholinesterase (AChE) activity in the STZ (ic)-treated mice. Intracerebral (ic) injections of STZ (0.5 mg/kg) on 1st and 3rd day caused significant deficit in memory in passive avoidance and Morris water maze test after the 14th day of first dose. In passive avoidance, transfer latency time (TLT) was not increased on retention trials in STZ (ic) group while gugulipid treatment resulted in significant increase in TLT on retention trials in STZ (ic)-treated mice. In Morris water maze test the latency time to reach platform in STZ (ic)-treated mice was significantly higher than control and vehicle (artificial CSF). Pre-treatment of gugulipid (50 mg/kg, p.o.) daily for 14 days started with the first dose of STZ (ic), significantly prevented STZ (ic)-induced memory deficit. Post-treatment i.e. after 14 days of first dose of STZ (ic) of gugulipid (50 mg/kg, p.o.) significantly decreased the latency time indicating anti-dementia activity. Effect of gugulipid and STZ in visible platform test was similar to those seen with hidden platform. Gugulipid and STZ-treated mice did not cause significant change in locomotor activity. Furthermore, STZ (ic) resulted into increase in AChE activity, low level of GSH and high concentration of MDA in brain on 21st day as compared to control. Gugulipid treatment caused significant decrease in AChE activity, low level of MDA and high concentration of GSH in brain following STZ (ic) as compared to vehicle administration in STZ (ic)-treated mice. The study demonstrated that gugulipid has significant protective affect against streptozotocin-induced memory deficits model of dementia that can be attributed to anti-oxidant and anti-AChE activity of gugulipid. These observations suggest gugulipid as a potential anti-dementia drug (CDRI, Lucknow has obtained US patent No. 6896901 for use of gugulipid as cognitive enhancer).

Hepatic and renal metallothionein induction by an oral equimolar dose of zinc, cadmium or mercury in mice

The hepatic and the renal subcellular distribution of zinc, cadmium or mercury and induction of tissue metallothionein (MT) at 24, 48 and 72 h following an oral equimolar dose (15 micro;mol metal/kg) of zinc (II) chloride, cadmium (II) chloride or mercury (II) chloride in male albino mice were investigated. There was a moderate increase in hepatic and renal zinc levels mainly in their nuclear mitochondrial fraction (NMF) 24 h post zinc chloride administration. Subsequently, the hepatic zinc increased and the renal zinc declined with time. The zinc-induced hepatic MT level was maximum at 48 h, which decreased slightly thereafter, while there was no marked increase in renal MT level at any time interval. The cadmium was equally distributed in liver and kidney more in their supernatant cytosol fraction (SCF) than in their NMF at 24 h after a dose of cadmium chloride. The cadmium levels showed a decreasing trend in hepatic fractions and an increasing trend in renal fractions with time. The cadmium-induced hepatic and renal MT were substantial at 24 h post cadmium administration, the former decreased thereafter while the latter enhanced at 48 h before declining. The accumulation of mercury in kidney was 1.5 times that in liver, which was localised more in their SCF than in their NMF at 24 h in response to a dose of mercuric chloride. The mercury levels of hepatic and renal subcellular fractions started declining after 24 h and at 72 h they were significantly lower. The induction of hepatic and renal MT was maximum at 24 h after mercuric chloride administration, which declined thereafter concomitant with the decrease in their mercury levels. However, the MT levels in both the organs remained considerably higher than in normal animals at 72 h post exposure. The results show that the accumulation of metal in liver and kidney follows the order: Hg > Cd > Zn and the induction of MT follows Hg > Cd > Zn in liver and Cd > Hg > Zn in kidney. The alterations in zinc and copper homeostasis were more marked in liver than in kidney and follows the order: Hg > Cd > Zn.

Differential effects of formononetin and cladrin on osteoblast function, peak bone mass achievement and bioavailability in rats

Dietary soy isoflavones including genistein and daidzein have been shown to have favorable effects during estrogen deficiency in experimental animals and humans. We have evaluated osteogenic effect of cladrin and formononetin, two structurally related methoxydaidzeins found in soy food and other natural sources. Cladrin, at as low as 10 nM, maximally stimulated both osteoblast proliferation and differentiation by activating MEK-Erk pathway. On the other hand, formononetin maximally stimulated osteoblast differentiation at 100 nM that involved p38 MAPK pathway but had no effect on osteoblast proliferation. Unlike daidzein, these two compounds neither activated estrogen receptor in osteoblast nor had any effect on osteoclast differentiation. Daily oral administration of each of these compounds at 10.0 mg kg(-1) day(-1) dose to recently weaned female Sprague-Dawley rats for 30 consecutive days, increased bone mineral density at various anatomic positions studied. By dynamic histomorphometry of bone, we observed that rats treated with cladrin exhibited increased mineral apposition and bone formation rates compared with control, while formononetin had no effect. Cladrin had much better plasma bioavailability compared with formononetin. None of these compounds exhibited estrogen agonistic effect in uteri. Our data suggest that cladrin is more potent among the two in promoting parameters of peak bone mass achievement, which could be attributed to its stimulatory effect on osteoblast proliferation and better bioavailability. To the best of our knowledge, this is the first attempt to elucidate structure-activity relationship between the methoxylated forms of daidzein and their osteogenic effects.

Determination of lumefantrine in rat plasma by liquid-liquid extraction using LC-MS/MS with electrospray ionization: assay development, validation and application to a pharmacokinetic study.

A simple, sensitive and rapid method for the analysis of lumefantrine in rat plasma using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed. Detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. The method included a chromatographic run of 5 min using a C(18) analytical column and the calibration curve was linear over the concentration range of 2-500 ng/mL with a correlation coefficient (r) of 0.996 or better. The intra- and inter-day assay precision ranged from 1.5 to 7.5% and 5.5 to 7.7%, respectively, and intra- and inter-day assay accuracy was between 91.3-109.7% and 97.0-104.7%, respectively. The method was successfully applied for the pharmacokinetic study in rats.

Chelation in metal intoxication. XVIII. Combined effects of thiamine and calcium disodium versenate on lead toxicity

Calcium disodium ethylenediaminetetraacetate (Ca-Na2EDTA; Versenate) was more effective than thiamine (vitamin B1) in enhancing the urinary excretion of lead, reducing tissue lead and restoring lead induced biochemical alterations in rats. However, the combination of CaNa2EDTA and vitamin B1 enhanced the beneficial effect of CaNa2EDTA in lead intoxication and was particularly effective in reducing the brain concentration of lead.

Permeability determination and pharmacokinetic study of nobiletin in rat plasma and brain by validated high-performance liquid chromatography method

In the present study, we are reporting permeability and pharmacokinetics of nobiletin in rat plasma and brain, using a validated reverse phase high performance liquid chromatographic method. Protein precipitation method was used for the extraction of nobiletin and coumarin (IS) from rat plasma and brain tissue. The system was run in isocratic mode with mobile phase consisting of potassium dihydrogen ortho-phosphate (pH 4.5; 0.04 mM) and acetonitrile in ratio of 50:50, v/v. The total chromatographic run time was 9.0 min. The method was proved to be accurate and precise at linearity range of 0.05-10 μg/mL with a correlation coefficient (r) of ≥ 0.994 in rat plasma and ≥ 0.995 in rat brain. The intra- and inter-day precision and accuracy values are found to be within the assay variability limits as per the FDA guidelines. Nobiletin was found stable in the battery of stability studies viz., bench-top, auto-sampler, freeze/thaw cycles and long term storage in a freezer at -70±10°C. Maximum concentrations of nobiletin in both plasma and brain were observed at 1h after single oral dosing (50 mg/kg). The maximum concentration in plasma and brain were 1.78 and 4.20 μg/mL, respectively. The AUC(0-t) in plasma and brain were 7.49 and 20.66 μg·h/mL, respectively. The mean elimination half life (t(½) in plasma and brain were 1.80 and 11.42 h, respectively. The Parallel Artificial Membrane Permeability Assay (PAMPA) permeability of nobiletin was found to be high at both pH 4.0 and 7.0.

Utility of noninvasive biomatrices in pharmacokinetic studies

Blood and plasma are the biomatrices traditionally used for drug monitoring and their pharmacokinetic profiling. Blood is the circulating fluid in contact with all organs and tissues of body and thus is the most representative fluid for measuring systemic drug levels. However, venipuncture suffers from the caveat of being an invasive technique which often makes people reluctant to participate in clinical studies. Thus, there is a need for noninvasive bio-fluids that are ethically appropriate, cost-efficient and toxicologically relevant. These alternate bio-fluids may prove clinically useful as alternatives to plasma/serum in therapeutic drug monitoring, pharmacokinetic and toxicokinetic studies, doping control in sports medicine and to monitor local adverse effects. These may be of particular interest in the case of special population groups such as neonates, children, the elderly, terminally ill patients and pregnant or lactating women, and offer the advantage of circumvention of the demand for specialized personnel for sample collection. This review describes such noninvasive bio-fluids (saliva, sweat, tears and milk) that have been considered for pharmacokinetic drug analysis, emphasizing their sample preparation, its associated difficulties and their correlation with plasma.

A novel flavonoid, 6-C-beta-d-glucopyranosyl-(2S,3S)-(+)-3',4',5,7-tetrahydroxyflavanone, isolated from Ulmus wallichiana Planchon mitigates ovariectomy-induced osteoporosis in rats.

Objective: The aim of this study was to determine the skeletal effect of 6-C-&bgr;-d-glucopyranosyl-(2S,3S)-(+)-3&vprime;,4&vprime;,5,7-tetrahydroxyflavanone (GTDF)/Ulmoside A, a new compound isolated from the extract of Ulmus wallichiana in a rat model of postmenopausal bone loss. Methods: GTDF (1.0 and 5.0 mg kg−1 d−1) was given orally to ovariectomized (OVx) rats (180-200 g) for 12 weeks. Sham operated + vehicle, ovariectomy + 17&bgr;-estradiol (2.5 &mgr;g kg−1 d−1), and ovariectomy + vehicle groups served as various controls. Bone mineral density (BMD), trabecular microarchitecture, bone biomechanical strength, levels of bone turnover/resorption markers, uterotropic effect, and plasma pharmacokinetics were studied. One-way analysis of variance was used to test significance of effects. Results: OVx rats treated with both doses of GTDF exhibited significantly higher BMD in the trabecular (distal femur, proximal tibia, and vertebrae) and cortical (femur shaft) regions compared with the ovariectomy + vehicle group. Micro-CT demonstrated that OVx rats treated with 5.0 mg kg−1 day−1 of GTDF had better bone microarchitectural parameters compared with the ovariectomy + vehicle group. Serum osteocalcin and urinary C-terminal teleopeptide of Type I collagen levels in OVx rats treated with GTDF (at both doses) were significantly lower than those in the ovariectomy + vehicle group. At neither of the two doses did GTDF exhibit uterine estrogenicity. A pharmacokinetic study revealed that GTDF achieved maximum plasma concentration (40.67 ng mL−1) at ∼1 hour, indicating its slow absorption. Its absolute bioavailability was found to be 1.04% with a plasma elimination half-life of ∼5 hours. Conclusions: GTDF, a novel compound isolated from U wallichiana extract, improves bone biomechanical quality through positive modifications of BMD and trabecular microarchitecture without a hyperplastic effect on the uterus.