Effect of Semecarpus Anacardium on Plasma Nitrates

encumbrance of Semecarpus Anacardium on job plasma NitratesOBSERVATION AND RESULT7. Observation and import7.1 Behavioral ParametersValues ar show MEANSEM, n = 6, ** = PFig. 7.1 gear up of Semecarpus Anacardium on Behavioral Parameters on Stress bring forth misgiving in Mice.7.2 Biochemical EstimationValues ar explicit MEANSEM, n = 6, ** = PFig. 7.2 Effect of Semecarpus Anacardium on opposite Biochemical Parameters in Stress Induced Anxiety in Mice.Fig. 7.3 Effect of Semecarpus Anacardium on glutathione blood-reductase activity in Stress Induced Anxiety in Mice.8. DiscussionBehavioral parameters are the primary evidence to subscribe anxiety as surface as anti-anxiety g all overnment issue of treatments. only the parameters are based on pathophysiology of anxiety because anxiety or dismay is rated with stress or immobilization of zoology like mice and rats.Elevated overconfident Maze (EPM) laterwards immobilization of animals for 3hr, the drug treatment was star ted for all chemical groups neglect prohibit cut back. eon spent in open leg and shut arm were ascertained. Time spent in open arm were importantly alteration magnitude (P0.001) later on judgeship of Semecarpus genus Anacardium at venereal disease of cc mg/kg 1752.2046 sec. as canvassd with forbid view (2583.2018 sec.). In fear, animal is more favorable to dark playing area which was shows in prohibit control.Force Swim Test (FST)Time cycle in bits was find knocked out(p) in all groups. Time cycle per five here and now were importantly increased (P0.001) in Semecarpus anacardium at dit of two hundred mg/kg (204.2044) compared with negative control (252.5421).Light and Dark Testafter immobilization of animals for 3 hr, the drug treatment was started for all groups except negative control. Time spent in depress and dark area was observed. Time spent in light area were significantly increased (P0.001) after politics of Semecarpus anacardium at dose of 200mg/kg (1783.5041 sec.) as compared with negative control (582.1245 sec.). In fear, animal is more favorable to dark area which was shows in negative control.Open Field Test (OFT)OFT is the analyze to evaluate anti- anxiety set as well as to compare the statistics with actophotometer because each squire in OFT is 10 10 cm and each electrodes difference in actophotometer is 6 cm so the reading should be double in OFT. creature in control group were shows significant walk fullness in OFT (452.2405 sec.). aft(preno arcminuteal) organisation of Semecarpus anacardium at dose of 200mg/kg, the animal was shows significant effect (P0.001). Rearing is the parameter in OFT which shows alertness of animals. After administration of Semecarpus anacardium at dose of 200mg/kg the animal was shows significant effect (P0.001) in 384.0510 sec. compared with negative control (182.5402 sec.).The gaseous messenger pinpoint nitric oxide (NO) is synthesized from its precursor L-arginine by a family of thr ee NO Synthases (NOS), designated as neuronal NOS-I, inducible NOS-II and endothelial NOS-III. In the adult mindset, the inducible iso form NOS-II is present wholly at very low takes in microglia and immune cells, while endothelial NOS-III is verbalized predominantly in the vasculature. Wh divinyl ether or not this isoform is also show in neural cells, is still a matter of debate only data arguing for this are only sparse. The quantitatively major commencement for NO in the CNS thus is the neuronal isoform NOS-I present in approximately 1% of all neurons. Nitrinergic transmission is especially important in limbic structures, in the basal ganglia where NO regulates striatal output and in the cerebellum. NO exerts multiple action in the CNS and from animal studies, it has been suggested that it is involved in behavioral processes such as learning and memory formation. Pathologies of the NO passage take hold been implicated in almost every major neuropsychiatric disorder inclu ding Schizophrenia, affective disorders, Alcoholism, Alzheimers dementia, Parkinson and Huntingtons disease. For some of these disorders, NOS-I has also been determine as a risk gene in human case-control linkup studies. The role of NO in the regulation of normal human brain functioning however is still unclear, although first genetic studies argue for a function of NOS-I in the regulation of impulsive behaviors. In a second series of experiments, we investigated whether NOS-I knockdown animals have cognitive deficits.plasm nitrates take was significantly change magnitude (P0.001) after administration of Semecarpus anacardium at dose of 200 mg/kg (52.232.1401sec.) as compared with negative control (74.242.2406). In fear or anxiety, animal were showed increased level of plasma nitrates which was shows in negative control.iNOS level was significantly increased (P0.001) after administration of Semecarpus anacardium at dose of 200mg/kg (78.373.2131sec.) as compared with negative con trol (26.232.5470 sec.).In auxiliary to its role in cholinergic transmission, substantial evidence has accumulated over the last two decades which suggests a non- cholinergic neuromodulatory function for torment. Few studies have demonstrated that the materialisation of AChE during early development correlate tight with the major phase of neurite outgrowth. Layer et al. have showed that AChE inhibitors have been shown to stop neuritic outgrowth in a dose dependent manner in retinal ganglion cells, dorsal root ganglion and sympathetic ganglion neurons. There is a ripening body of evidence supporting the morphogenic effects of AChE in twain in vivo and in vitro systems. AChE is known to regulate the neuritic outgrowth and selection of cultured neurons and also has morphogenic and axogenic role in the developing nervous system. In addition, AChE has a role in cell growth and survival. These functions are considered to be the non-classical roles of this classical enzyme. Further more, ACh is also known to enhance the neuritic outgrowth and in turning of the nerve growth cones. These studies, together with the present demonstration of increased dendritic arborization in the genus Hippocampus, suggest that chronic drug administration induces AChE activity which in turn might modulate dendritic fork pattern in specific brain regions.Ach level was significantly decreased (P0.001) after administration of Semecarpus anacardium at dose of 200mg/kg (53.262.0987 sec.) as compared with negative control (81.233.0245 sec.).The efficacy of this plant pick toward the transmitters was significant. MAO regulates metabolic degradation of catecholamine, serotonin and another(prenominal) endogenous amines in CNS. Inhibition of this enzyme causes decline of metabolism of these transmitters and subsequent increase of these biogenic amines. MAO-A level was significantly decreased (P0.001) after administration of Semecarpus anacardium at dose of 200mg/kg (56.63.3245 sec.) a s compared with negative control (86.12.3024 sec.). MAO-B level was significantly decreased (P0.001) after administration of Semecarpus anacardium at dose of 200 mg/kg (44.83.2431 sec.) as compared with negative control (73.42.2061 sec.).Glutathione reductase level was significantly decreased (P0.001) after administration of Semecarpus anacardium at dose of 200mg/kg (1478.53.2436 sec.) as compared with negative control (16342.2102 sec.). All values are expressed in U/I. Glutathione reductase level was decreased after administration of extract of Semecarpus anacardium at dose 200 mg/kg in mice. Glutathione reductase is the enzyme which increases in anxiety and depression. This enzyme secretes from hippocampus region of brain. The level of this enzyme was significantly reduced in mice compared with vehicle handle control group.On the bases of behavioral as well as biochemical inclination, study concludes that Semecarpus anacardium shows significant effect in plasma nitrates and othe r chemical messenger in anxiety at dose of 200mg/kg compared with negative control.9. SUMMARY AND CONCLUSIONThe present study is designed to evaluate Effect of Semecarpus anacardium on plasma nitrates on stress bring on anxiety in mice.Behavioral parameters show following result After administration of Semecarpus anacardium Time spent in open arm in Elevated Plus Maze, Time cycle per five mi cranke in Force Swim Test, Time spent in light area in Light and Dark Test, no(prenominal) of Squire intersection in Open Field Test was significantly increased after administration of Semecarpus anacardium at dose of 200 mg/kg as compared with negative control.Biochemical Estimations show following result blood plasma nitrates level, Ach level, MAO-A level, MAO-B level, Glutathione reductase level was significantly decreased after administration of Semecarpus anacardium at dose of 200 mg/kg as compared with negative control. iNOS level was significantly increased after administration of Seme carpus anacardium at dose of 200mg/kg as compared with negative control.On the bases of behavioral as well as biochemical estimation, study concludes that Semecarpus anacardium shows significant effect in plasma nitrates and other chemical messenger in anxiety at dose of 200mg/kg compared with negative control.6. Materials Methods6.1 MaterialsCollection Au indeedticationThe plant Semecarpus anacardium has been taken from local market authenticated from Department of Botany Dr. H.S. Gour University, Sagar M.P. Herbarium No. Bot./her/A/1124. condenseion procedure6.3.1 Petroleum ether extract The totally plant nuts was cleaned and shaded dried for 10-15 days. The dried nuts were pulverized by an electrical blender and nut paste obtained. About 30-40 g of the nut paste was subject for extraction with 400 ml of Petroleum ether solvent by Soxhlet apparatus for 24 hrs. Constant heats of 50 60 0C provided by Mantox heater of Soxhlet for recycling the solvent. The extract was concentrate using orbitual evaporator at 60 0C for 20 min at a travel rapidly of 5m/s. The concentrated extract kept in refrigerator at 4 0C for further use. (50)6.3.2 Ethanol extract The nuts were shed dried for to the highest degree 20 days and then subsequent to reduce coarse drug particle into fine powder using pestle and mortar. The extraction was carrying out by ethanol solvent Soxhlet extraction techniques. Solvent used consecutively with gradient polarity. The extract evaporated to complete dryness by using emptiness distillation and kept in refrigerator for further use. (51)Phytochemical screening6.4.1 Tests for AlkaloidsMayers Test Extract hard- grinded with Mayers reagent (Potassium Mercuric Iodide). Formation of a sensationalistic food coloured precipitate indicated the movement of alkaloids.Wagners Test Extract treated with Wagners reagent (Iodine in Potassium Iodide). Formation of brown/ violent precipitate indicated the social movement of alkaloids.Dragendroffs Test Ext ract treated with Dragendroffs reagent ( resultant role of Potassium Bismuth Iodide). Formation of red precipitate indicated the presence of alkaloids.Hagers Test Extract treated with Hagers reagent (saturated picric mordant dissolving agent). Presence of alkaloids actualize by the formation of yellowish coloured precipitate.Tannic acrid seek Extract treated with 10% Tannic acid solution. Alkaloids gave buff colour precipitate. (52)6.4.2 Detection of PhenolsBromine water supply seek Test solution treated with few milliliters of bromine water. Formation of yellow precipitate indicated presence of Phenols.ferrous chloride streamlet Test solution gave rich viridity colour with ferric chloride. (53)6.4.3 Detection of SaponinsEmulsion test 1 ml of the extract deform added to few sacks of olive oil. The intermixture added to another two drops of olive. The mixture shakes and observed for the formation of emulsion.Frothing test 1 ml of the extract strive diluted with 4 ml o f distilled water. The mixture was shake vigorously and then observed on standing for a stable froth.6.4.4 Detection Steroids and TriterepenoidsLibermann- Buchard test Extract treated with few drops of acetic anhydride, boil and cool, conc. Sulphuric acid added from the sides of the test tube. Formation of a brown ring at the juncture of two layers and the upper layer turns green which shows the presence of Steroids and formation of belatedly red colour indicated the presence of Triterepenoids.Salkowski test Treated extract in Chloroform with few drops of cone. Sulphuric acid, shaked well and allowed standing for some time, red colour appeared at the lower layer indicates the presence of Steroids and formation of yellow coloured lower layer indicated the presence of Triterepenoids.6.4.5 Detection of TanninsLead sub-acetate test 1 ml of the filtrate added to 3 drops of the lead sub-acetate solution. A baste gelatinous precipitate indicated the presence of tannins.Ferric chloride t est 1 ml of the filtrate diluted with distilled water and added with 2 drops of ferric chloride. A fugitive greenish to black colour indicated the presence of tannins.6.4.6 Detection of FlavonoidsShinoda test (Magnesium Hydrochloride decline test) To the test Solution, added few fragments of Magnesium ribbon and added concentrate Hydrochloric acid drop wise, pink scarlet, crimson red or occasionally green to blue colour appeared after few minutes.Alkaline reagent test To the test solution added few drops of sodium hydroxide solution formation of an intense yellow colour, which turned to Colourless on addition of few drops of dil. acid, indicated presence of Flavonoids. ammonium ion test A touchstone (4 ml) each of the filtrates was shaking with 1 ml of dilute ammonia solution (1%). The layers allowed to separating. A yellow coloration at the ammonia layer indicates the presence of Flavonoids.Aluminium chloride test A quantity (4 ml) each of the filtrates was shake with 1 ml of 1% aluminium chloride solution and observed for light yellow coloration. A yellow precipitate indicated the presence of Flavonoids.6.4.7 Detection of Anthraquinones1. Dilute sulphuric acid (5 ml) added to 0.1 g of the test extract in a test tube and boil for 15 min in a water bath. It was then cool and neutralize with 20% potassium hydroxide solution. A mixture, 10 ml of equal move of Fehlings solution A and B will add and boil for 5 min. A more dense red precipitate indicated the presence of glycoside.2. About 0.5 ml of extract taken and subject to the following tests.1 ml of glacial acetic acid containing traces of ferric chloride and 1ml of concentrate sulphuric acid added to the extract and observed for the formation of the reddish brown colouration at the junction of two layers and the upper layer turned bluish green showed presence of Glycosides.Pharmacological Screening6.5.1 Animal Mice required as Animal modelBody weight 25 gms.Floor area per animal 15 in2.Cage height 5 inch. Temperature 64 to 79F (18 to 26C). telling Humidity 40% to 70%.Number of air out changes per hour 10 15.Light levels 30 foot-candles.Duration of Light 12 -14 hours.Duration of Darkness 10 12 hours.6.5.3 Biochemical Estimation6.5.3.1 Plasma Nitrate estimationPlasma nitrate were measured by spectrophotomeric chip based on Griess reaction.Blood were withdrawn from tail vein of mice and plasma were using cooling centrifuge at 2500 rpm for 10 min.Plasma were mixed with equal volumes of Griess reagent (1% Sulphanilamide+ 0.1% naphthylelediamine dihydrochloride+ 2.5 % phosphoric acid) and incubated at room temp for 10 min. to yield a chromophore.Absorbance was read at 543 nm spectophotometrically.(59)6.5.3.2 i NOS estimationSample collection After the behavioral tests, three mice from each group was deeply anesthetized and perfuse with 4% paraformaldehyde for subsequent Nissl staining. The other animals were anesthetized and kill blood was collected and brains were removed. Blood, anti coagulated with 1.5% EDTA centrifuged at 12,000 rpm for 10 minutes, and then the supernatant was collected. All these samples stored at 80C for further analysis.RNA extraction and sneak transcription fare RNA extracted from the brain tissue using Trizol reagent. Total template RNA (1 g) was transcribed using Quant script cDNA RT Kits according to the manufacturers manual. Briefly, RNA (1 g) pretreated with DNA-free DNase treatment and removal reagents. RNA samples incubated with a mixture consisting of containing dNTPs, random primers, 10 RT mix, Quant Reverse Transcriptase, a reverse transcriptase and RNase-free water to a final volume of 10 l at 37C for 1 h.Real-time RT-PCR cDNA l used for quantification of mRNA by real-time RT-PCR. Real-time RT-PCR will perform on an employ Rotor-Gene 3000 under the following conditions iNOS and GAPDH for 40 cycles at 94C for 30 s, 63C for 60 s, and 72C for 90 s. Relative quantitative measurements of target gene levels was performed using th e Ct method, where Ct is the threshold concentration. GAPDH used as endogenous control to normalize gene expression data, and an RQ value calculated for each sample. RQ values was presented as fold change in gene expression relative to the control group, which normalized to 1. (60)The activity was expressed as m moles hydrolyzed per min per gram of tissue. AChE activity was statistically analyzed by Students Statistical analysisThe statistical analysis carried out as per standard method. All result expressed as MEANSEM. Groups of data were compared with the analysis of variance (ANOVA) followed by dunnetts t-test values for statistical significance.Sagar Institute of Pharmaceutical Sciences, Sagar M.P. Page 1