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Evaluation of the antioxidant potential of NR-ANX-C (a polyherbal formulation) and its individual constituents in reversing haloperidol-induced catalepsy in mice Albina Arjuman, Vinod Nair*, H.N. Gopalakrishna*, M. Nandini evaluate the possible role of the antioxidant activity of the polyherbal formulation, NR-ANX-C and its individual components in reversing haloperidol-induced catalepsy in Swiss albino mice.
Materials and Methods: Materials and Methods: Catalepsy was induced with haloperidol (1 mg/kg i.p) in 13 Departments of Biochemistry and groups of male albino mice (n = 6 / group). Three groups received NR-ANX-C (10, 25, *Pharmacology, Kasturba Medical 50 mg/kg), three groups received Withania somnifera (1.7, 4.25, 8.5 mg/kg), another three College, Mangalore, Karnataka, groups Ocimum sanctum (1.7, 4.25, 8.5 mg/kg), three other groups Camellia sinensis (3.4, 8.5, 17 mg/kg) and one group received the vehicle (1% Gum acacia) orally, 30 minutes prior to haloperidol administration, for a duration of seven days. Animals were sacrificed Received: 22.10.2006 on the seventh day and superoxide dismutase (SOD) activity was estimated in the brain.
Results: A significant (P < 0.01) reduction in the cataleptic scores was observed in all the Accepted: 10.5.2007 drug-treated groups as compared to the control, with maximum reduction in the NR-ANX-C Correspondence to: Correspondence to: 25 mg/kg group. Similarly, a reduction in SOD activity was observed in the NR-ANX-C-, O. sanctum- and the W. somnifera-treated groups. An increase in SOD activity was observed in the C. sinensis-treated groups.
Conclusion: With the exception of C. sinensis, the antioxidant potential of NR-ANX-C and its individual constituents has contributed to the reduction in the oxidative stress and the catalepsy induced by haloperidol administration. WORDS: Camellia sinensis, catalepsy, haloperidol, Ocimum sanctum, oxidative stress, Withania somnifera medknow the amounts of peroxidative products or the rates of enzyme- This PDF is available for free download from Haloperidol is an antipsychotic drug, which is used in the catalysed reactions neutralizing free radical intermediates such treatment of schizophrenia and other affective disorders. It as those of superoxide dismutase (SOD). SOD is a primary, blocks the dopaminer a site hosted by Medknow Publications (www gic action in the nigro-strial pathway natural, free radical-scavenging, antioxidant enzyme in the leading to high frequency of extrapyramidal motor side effects.[1] body. The estimation of the activity of such antioxidant enzymes In animal models, haloperidol induces a behavioural state such as superoxide dismutase, catalase, glutathione peroxidase known as catalepsy in which the animals are unable to correct etc, can be used to assess the therapeutic effects of different externally imposed postures.[2] The use of haloperidol has been antioxidant agents.
associated with an increased level of oxidative stress in the The administration of any exogenous agent is associated brain.[3] This evidence suggests a possible role for antioxidants with the development of cellular oxidative stress as a result in the treatment of haloperidol-induced catalepsy.
of its metabolism in the body. The ability of the compound to The brain is made up of 70% lipid and any kind of stress is counteract the oxidative stress induced by its metabolism is usually manifested by lipid peroxidative damage.[4] The extent known as its antioxidant potential. Recently, the evaluation of this damage can be used to evaluate the degree of cellular of the extent of cellular oxidative stress has been applied to harm. In practical terms, this stress-induced lipid peroxidative determine the therapeutic / toxic doses of synthetic and herbal damage in the brain can be quantified by either determining drugs. A step ahead of this approach would be to evaluate Indian J Pharmacol June 2007 Vol 39 Issue 3 151-154 Arjuman, et al.: Effect of NR-ANX-C on catalepsy
the best therapeutic dose of a natural or synthetic compound 20 seconds, it was said to be cataleptic and given one point. in a specific disease state, giving better or at least similar For every additional 20 seconds that the animal continued to therapeutic action on a tissue but with the least amount of maintain the cataleptic posture, one extra point was given. The damage to the same, thus ensuring mammalian well being and animals were then sacrificed by cervical dislocation and the avoiding supplementary tissue toxicity.
SOD activity in brain tissue was estimated.
NR-ANX-C is a polyherbal formulation containing the Assay of SOD aqueous extract of W. somnifera (17% withanolides, 2.1% The assay of SOD was carried out based on the SOD- w/w), 70% ethanolic extract of O. sanctum (17% Ursolic acid, mediated inhibition of the reduction of nitroblue tetrazolium to 2.9% w/w) and 70% ethanolic extract of C. sinensis (34%, total blue formazan by superoxide anions as described by Beauchamp polyphenols 60.1% w/w). These agents have been used in the and Fridovich.[14] Ayurvedic system of medicine for over 3000 years. W. somnifera The total protein present in the homogenate was estimated (Ashwagandha) has been used as a liver tonic, anti-inflammatory following the method described by Lowry et al.[15] Units of SOD agent and antioxidant, in the treatment of bronchial asthma, activity determined were expressed in terms of milligrams of insomnia, as a nervine tonic in the treatment of senile dementia, total protein (TP).
cognitive and neurological disorders and Parkinson's disease.[5-8] Preparation of homogenate O. sanctum (Tulsi, India's Holy Basil) has been shown to have The brains isolated from the individual groups of mice were hypoglycemic, hypolipidemic, adaptogenic, antidepressant, homogenized (20% w/v) in 10 mM Phosphate buffer, pH 7.8. The hepatoprotective, anticancer, antioxidant, analgesic and anti- homogenate was subjected to centrifugation. 0.1 ml aliquot of inflammatory properties.[9-11] C. sinensis (Green tea) has been the homogenate (1:10 dilution) was used for the assay.
used in the treatment of cancer, rheumatoid arthritis, high cholesterol levels, cardiovascular disease, infection and other impaired immune functions. It has also been shown to have Statistical analysis was done using ANOVA followed by Dunnett's multiple comparison.
The objective of our study was to evaluate the efficacy of NR-ANX-C and its individual components by virtue of their antioxidant potential in mouse brain using haloperidol-induced The cataleptic scores of the present study are given in Table catalepsy as our model of investigation.
1. Significant reversal in haloperidol-induced catalepsy (P < 0.01) was observed with NR-ANX-C at a dose of 25 mg/kg. Materials and Methods
This dose was the most effective of the three tested doses. Of Prior approval was obtained from the Institutional Animal the individual constituents of NR-ANX-C, W. somnifera and O. Ethics Committee, Kasturba Medical College, Mangalore, for sanctum at a dose of 8.5 mg/kg showed a significant reversal in conducting this study.
the catalepsy (P < 0.01). The result observed with C. sinensis though significant when compared to the control, is not as Study design pronounced as with NR-ANX-C, W. somnifera or O. sanctum. Adult male Swiss albino mice (25-30 g) were allocated to The SOD activity in the brain of the mice treated with thirteen groups (n = 6/ group) and housed in polypropylene haloperidol and the test drugs are also presented in Table 1. C with 12 h:12 h light-dark cycle. The animals The SOD activity in the brain was found to be elevated in the were allowed free access to food (standard pellet feed) and haloperidol-treated group. The observed increase was about water. They were allowed an acclimation period of seven days 66% above the normal. The test drug formulation, NR-ANX-C before the study.
showed an overall reduction in SOD activity at all the three A pilot study was carried out and suitable doses were doses with a maximum reduction observed in the group treated selected. The individual doses used were suspended in 1% with 25 mg/kg. In the W. somnifera-treated groups, there was a This PDF is available for free download from gum acacia and administered orally to the different groups. dose-dependent decrease in the brain SOD activity. Significant One group received the vehicle (1% gum acacia solution) and (P < 0.05) reduction was observed at a dose of 8.5 mg/kg with a site hosted by Medknow Publications (www ved as the control. Three groups received NR-ANX-C (10, W. somnifera. In the O. sanctum-treated groups, there was a 25 and 50 mg/kg), three groups received W. somnifera (1.7, decrease in the SOD activity at the lower dose with maximum 4.25 and 8.5 mg/kg), three groups received O. sanctum (1.7, reduction seen at a dose of 4.25 mg/kg. However, with the 4.25 and 8.5 mg/kg) and three groups received C. sinensis (3.4, higher dose (8.5 mg/kg), there was a slight increase in the SOD 8.5 and 17 mg/kg). Thirty minutes after the administration of activity. Unlike the groups treated with the other constituents of these drugs, catalepsy was induced by the i.p. administration of NR-ANX-C, all the C. sinensis-treated groups showed an increase haloperidol at a dose of 1 mg/kg body weight, in the form of an in the SOD activity compared to the control. injectable solution constituted in normal saline. This procedure was repeated for seven days. On the seventh day, catalepsy was measured 240 minutes after haloperidol administration Induction of free radicals in mammals by haloperidol is in a manner similar to the method described by Ahtee and well established. Previous studies have shown that dopamine Buncombe.[13] All observations were made between 14:00 and receptors in the striatum are involved in neuroleptic-induced 16.00 hours. The animals were placed on a flat surface and their catalepsy. It has been demonstrated that cataleptic effects of front paws were elevated and placed on a 4 cm-high wooden haloperidol are apparently mediated by dopamine receptors bar. If the animal maintained the imposed posture for at least localized postsynaptically on strial neurons.[2] It is also well Indian J Pharmacol June 2007 Vol 39 Issue 3 151-154 Arjuman, et al.: Effect of NR-ANX-C on catalepsy
Effect of test drugs on haloperidol-induced catalepsy and brain superoxide dismutase activity
SOD (U/mg TP)
% decrease
% increase from
(n = 6)
(n = 3)
Normal (no haloperidol) W. somnifera O. sanctum C. sinensis All values are Mean ± SE; Statistical analysis by One-way ANOVA followed by Dunnett's Multiple Comparison Test; *P < 0.05 **P < 0.01 established that administration of haloperidol leads to an can be attributed to its role in reversing haloperidol-induced increase in the oxidative stress in the brain tissue.[3] The increase in SOD observed in the present study supports the Of its individual constituents, W. somnifera appears to above concept.
be the major contributor of antioxidant activity leading to Superoxide formation is a major factor in oxygen toxicity the reduction of SOD activity. The antioxidant activity of W. and the superoxide dismutase enzyme constitutes an essential somnifera could be attributed to the direct scavenging of free defence against it. Under normal conditions, decreased activity radicals by polyphenols present in it.
of antioxidant enzymes, viz. SOD, glutathione peroxidase (GSH- The antioxidant activity of NR-ANX-C could be possibly PX) and catalase in the brain leads to accumulation of oxidative due to the direct scavenging of the superoxide radicals by free radicals resulting in degenerative effects.[6] An increase the polyphenols[9,12,16] or the flavanoids known to be present in these enzymes under normal conditions would represent in its individual constituents which have been established to increased antioxidant activity and a protective mechanism have an antioxidant potential. From the present study, it can in neuronal tissue, thus, constituting the first line of defence be concluded that the polyherbal formulation, NR-ANX-C and against oxidative stress in our body. However, in the presence of its individual components can be beneficial adjuvants in the a free radical-quenching agent, the induction of the antio treatment of drug-induced extrapyramidal side effects and enzymes is minimized. So, an overall decrease in cataleptic related disorders. Clinical trials in humans suffering from scores and also SOD activity in the NR-ANX-C-treated groups Parkinson's disease may provide us with greater evidence for indicates the ability of NR-ANX-C to combat oxidative stress the neuroprotective activity of this formulation.
in brain tissue and reduce the severity of haloperidol-induced This PDF is available for free download from ith regard to the individual constituents, W. somnifera We are grateful to Natural Remedies Pvt. Ltd. Bangalore for providing us with the test drugs. We also appreciate the technical assistance offered to us by P. Dorababu, and O. sanctum[10] showed a dose-dependent decrease in the a site hosted by Medknow Publications (www Lovelyn Joseph, Rose Merin Chacko, Rejeesh E. P. and Anjali Varghese (Postgradu- cataleptic scores. Of the two, W. somnifera showed a dose- ates, Dept. of Pharmacology, Kasturba Medical College, Mangalore, Karnataka).
dependent decrease in SOD activity while O. sanctum showed decrease in SOD activity only at the two lower doses (1.7 and 4.25 mg/kg).
1. Farde L, Nordström AL, Wiesel FA, Pauli S, Halldin C, Sedvall G. Positron emis- The increase in SOD activity in C. sinensis-treated groups sion tomographic analysis of central D1 and D2 dopamine receptor occupancy is contrary to the effects of the test drug or its individual in patients treated with classical neuroleptics and clozapine: Relation to extra-pyramidal side effects. Arch Gen Psychiatry 1992;49:538-44.
components namely, W. somnifera and O. sanctum on SOD 2. Sanberg PR. Haloperidol-induced catalepsy is mediated by postsynaptic dopa- activity. The reduced SOD activity seen in the test drug NR-ANX- mine receptors. Nature 1980;284:472-3.
C treated groups could be possibly due to the phytochemicals 3. Sagara Y. Induction of reactive oxygen species in neurons by haloperidol. J present in it which have been shown to possess antioxidant 4. Kedar NP. Can we prevent Parkinson's and Alzheimer's disease? J Postgrad Med 2003;49:236-45.
From these observations, it can be inferred that the test 5. Monograph. Withania somnifera. Altern Med Rev 2004;9:211-4.
drug NR-ANX-C shows significant antioxidant activity, which 6. Naidu PS, Singh A, Kulkarni SK. Effect of Withania somnifera root extract on Indian J Pharmacol June 2007 Vol 39 Issue 3 151-154 Arjuman, et al.: Effect of NR-ANX-C on catalepsy
haloperidol induced orofacial dyskinesia: Possible mechanisms of action. J Med Food 2003;6:107-14.
14. Beauchamp C, Fridovich I. Superoxide dismutase: Improved assays and an assay 7. Mishra LC, Singh BB. ScientiÞ c Basis for the therapeutic use of Withania somnifera applicable to acrylamide gels. Anal Biochem 1971;44:276-87.
(Ashwagandha): A review. Altern Med Rev 2000;5:334-46.
15. Peterson GL. Review of the folin phenol protein quantiÞ cation method of Lowry, 8. Bhattacharya A, Ghosal S, Bhattacharya SK. Antioxidant effect of Withania Rosenbrough, Farr and Randall. Anal Biochem 1979;100:201-20.
somnifera glycowithanolides in chronic footshock stress-induced perturbations 16. Parihar MS, Hemnani T. Phenolic antioxidants attenuate hippocampal neuronal of oxidative free radical scavenging enzymes and lipid peroxidation in rat frontal cell damage against kainic acid induced excitotoxicity. J Biosci 2003;28:121-8.
cortex and striatum. J Ethnopharmacol 2001;74:1-6.
9. Gupta SK, Prakash J, Srivastava S. Validation of traditional claim of tulsi, Ocimum sanctum Linn as a medicinal plant. Indian J Exp Biol 2002;40:765-73.
10. Uma Devi P. Radioprotective, anticarcinogenic and antioxidant properties of the Indian holy basil, Ocimum sanctum (Tulsi). Indian J Exp Biol 2001;39:185-90.
Presentation at a meeting: part of the work was presented
11. Bhargava KP, Singh N. Anti-stress activity of Ocimum sanctum Linn. Indian J Organization: 38th Annual Conference of the Indian
Med Res 1981;73:443-51.
12. Tewari S, Gupta V, Bhattacharya S. Comparative study of antioxidant potential of Pharmacological Society tea with and without additives. Indian J Physiol Pharmacol 2000;44:215-9.
13. Ahtee L, Buncombe G. Metoclopramide induces catalepsy and increases Date: December 2005.
striatal homovanillic acid content in mice. Acta Pharmacol Toxicol (Copenh) This PDF is available for free download from a site hosted by Medknow Publications (www Author Help: Choosing an appropriate category of article for faster publication
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Indian J Pharmacol June 2007 Vol 39 Issue 3 151-154


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