OBSERVATION AND RESULT

7. Observation and Result

7.1 Behavioral Parameters

Values are expressed MEAN±SEM, n = 6, ** = P<0.01, *** = P<0.001 when compared to normal control group, b = ns when compared to normal control group, a***=P<0.001 when compared to negative compared to negative control group, c = ns when compared to standard group. Standard: – Diazepam (5mg/kg)

Fig. 7.1 Effect of Semecarpus Anacardium on Behavioral Parameters on Stress Induced Anxiety in Mice.

7.2 Biochemical Estimation

Values are expressed MEAN±SEM, n = 6, ** = P<0.01, *** = P<0.001 when compared to normal control group, b = ns when compared to normal control group, a***=P<0.001 when compared to negative compared to negative control group, c = ns when compared to standard group. Standard: – Diazepam (5mg/kg)

Fig. 7.2 Effect of Semecarpus Anacardium on different Biochemical Parameters in Stress Induced Anxiety in Mice.

Fig. 7.3 Effect of Semecarpus Anacardium on glutathione reductase activity in Stress Induced Anxiety in Mice.

8. Discussion

Behavioral parameters are the primary evidence to confirm anxiety as well as anti-anxiety effect of treatments. All the parameters are based on pathophysiology of anxiety because anxiety or fear is evaluated through stress or immobilization of animal like mice and rats.

Elevated Plus Maze (EPM)

After immobilization of animals for 3hr, the drug treatment was started for all groups except negative control. Time spent in open arm and closed arm were observed. Time spent in open arm were significantly increased (P>0.001) after administration of Semecarpus anacardium at dose of 200 mg/kg 175±2.2046 sec. as compared with negative control (258±3.2018 sec.). In fear, animal is more favorable to dark area which was shows in negative control.

Force Swim Test (FST)

Time cycle in seconds was count in all groups. Time cycle per five minute were significantly increased (P>0.001) in Semecarpus anacardium at dose of 200 mg/kg (20±4.2044) compared with negative control (25±2.5421).

Light and Dark Test

After immobilization of animals for 3 hr, the drug treatment was started for all groups except negative control. Time spent in light and dark area was observed. Time spent in light area were significantly increased (P>0.001) after administration of Semecarpus anacardium at dose of 200mg/kg (178±3.5041 sec.) as compared with negative control (58±2.1245 sec.). In fear, animal is more favorable to dark area which was shows in negative control.

Open Field Test (OFT)

OFT is the test to evaluate anti- anxiety effect as well as to compare the statistics with actophotometer because each squire in OFT is 10 ×10 cm and each electrode’s difference in actophotometer is 6 cm so the reading should be double in OFT. Animal in control group were shows significant walk fullness in OFT (45±2.2405 sec.). After administration of Semecarpus anacardium at dose of 200mg/kg, the animal was shows significant effect (P>0.001). 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 (P>0.001) in 38±4.0510 sec. compared with negative control (18±2.5402 sec.).

The gaseous messenger molecule nitric oxide (NO) is synthesized from its precursor L-arginine by a family of three NO Synthases (NOS), designated as “neuronal” NOS-I, “inducible” NOS-II and “endothelial” NOS-III. In the adult brain, the inducible iso form NOS-II is present only at very low levels in microglia and immune cells, while “endothelial” NOS-III is expressed predominantly in the vasculature. Whether or not this isoform is also expressed in neural cells, is still a matter of debate but data arguing for this are only sparse. The quantitatively major source 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 pathway have been implicated in almost every major neuropsychiatric disorder including Schizophrenia, affective disorders, Alcoholism, Alzheimer’s dementia, Parkinson and Huntington’s disease. For some of these disorders, NOS-I has also been identified as a risk gene in human case-control association 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.

Plasma nitrates level was significantly decreased (P>0.001) after administration of Semecarpus anacardium at dose of 200 mg/kg (52.23±2.1401sec.) as compared with negative control (74.24±2.2406). In fear or anxiety, animal were showed increased level of plasma nitrates which was shows in negative control.

iNOS level was significantly increased (P>0.001) after administration of Semecarpus anacardium at dose of 200mg/kg (78.37±3.2131sec.) as compared with negative control (26.23±2.5470 sec.).

In addition to its role in cholinergic transmission, substantial evidence has accumulated over the last two decades which suggests a non- cholinergic neuromodulatory function for AChE. Few studies have demonstrated that the expression of AChE during early development correlate closely with the major phase of neurite outgrowth. Layer et al. have showed that AChE inhibitors have been shown to retard neuritic outgrowth in a dose dependent manner in retinal ganglion cells, dorsal root ganglion and sympathetic ganglion neurons. There is a growing body of evidence supporting the morphogenic effects of AChE in both in vivo and in vitro systems. AChE is known to regulate the neuritic outgrowth and survival 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. Furthermore, 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 hippocampus, suggest that chronic drug administration induces AChE activity which in turn might modulate dendritic branching pattern in specific brain regions.

Ach level was significantly decreased (P>0.001) after administration of Semecarpus anacardium at dose of 200mg/kg (53.26±2.0987 sec.) as compared with negative control (81.23±3.0245 sec.).

The efficacy of this plant extract toward the transmitters was significant. MAO regulates metabolic degradation of catecholamine, serotonin and other endogenous amines in CNS. Inhibition of this enzyme causes reduction of metabolism of these transmitters and subsequent increase of these biogenic amines. MAO-A level was significantly decreased (P>0.001) after administration of Semecarpus anacardium at dose of 200mg/kg (56.6±3.3245 sec.) as compared with negative control (86.1±2.3024 sec.). MAO-B level was significantly decreased (P>0.001) after administration of Semecarpus anacardium at dose of 200 mg/kg (44.8±3.2431 sec.) as compared with negative control (73.4±2.2061 sec.).

Glutathione reductase level was significantly decreased (P>0.001) after administration of Semecarpus anacardium at dose of 200mg/kg (1478.5±3.2436 sec.) as compared with negative control (1634±2.2102 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 treated control group.

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.

9. SUMMARY AND CONCLUSION

The present study is designed to evaluate “Effect of Semecarpus anacardium on plasma nitrates on stress induced 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 minute in Force Swim Test, Time spent in light area in Light and Dark Test, No. of Squire Cross 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: 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 Semecarpus 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 & Methods

6.1 Materials

2. Collection & Authentication

The 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.

3. Extraction procedure

6.3.1 Petroleum ether extract: The whole 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 Rotary evaporator at 60 ⁰C for 20 min at a speed 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 about 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 vacuum distillation and kept in refrigerator for further use. ⁽⁵¹⁾

4. Phytochemical screening

6.4.1 Tests for Alkaloids

Mayer’s Test: Extract treated with Mayer’s reagent (Potassium Mercuric Iodide). Formation of a yellow coloured precipitate indicated the presence of alkaloids.

Wagner’s Test: Extract treated with Wagner’s reagent (Iodine in Potassium Iodide). Formation of brown/reddish precipitate indicated the presence of alkaloids.

Dragendroff’s Test: Extract treated with Dragendroff’s reagent (solution of Potassium Bismuth Iodide). Formation of red precipitate indicated the presence of alkaloids.

Hager’s Test: Extract treated with Hager’s reagent (saturated picric acid solution). Presence of alkaloids confirm by the formation of yellow coloured precipitate.

Tannic acid test: Extract treated with 10% Tannic acid solution. Alkaloids gave buff colour precipitate. ⁽⁵²⁾

6.4.2 Detection of Phenols

Bromine water test: Test solution treated with few milliliters of bromine water. Formation of yellow precipitate indicated presence of Phenols.

Ferric chloride test: Test solution gave blue green colour with ferric chloride. ⁽⁵³⁾

6.4.3 Detection of Saponins

Emulsion test: 1 ml of the extract filtrate added to few drops of olive oil. The mixture added to another two drops of olive. The mixture shakes and observed for the formation of emulsion.

Frothing test: 1 ml of the extract filtrate diluted with 4 ml of distilled water. The mixture was shake vigorously and then observed on standing for a stable froth.

6.4.4 Detection Steroids and Triterepenoids

Libermann- 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 junction of two layers and the upper layer turns green which shows the presence of Steroids and formation of deep 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 Tannins

Lead sub-acetate test: 1 ml of the filtrate added to 3 drops of the lead sub-acetate solution. A cream gelatinous precipitate indicated the presence of tannins.

Ferric chloride test: 1 ml of the filtrate diluted with distilled water and added with 2 drops of ferric chloride. A transient greenish to black colour indicated the presence of tannins.

6.4.6 Detection of Flavonoids

Shinoda test (Magnesium Hydrochloride reduction 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 test: A quantity (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 Anthraquinones

1. 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 parts of Fehling’s 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.

5. Pharmacological Screening

6.5.1 Animal: Mice required as Animal model

• Body weight: 25 gms.
• Floor area per animal: 15 in².
• Cage height: 5 inch.
• Temperature: 64° to 79°F (18° to 26°C).
• Relative Humidity: 40% to 70%.
• Number of air 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 Estimation

6.5.3.1 Plasma Nitrate estimation:

• Plasma nitrate were measured by spectrophotomeric assay 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 estimation:

Sample 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, anticoagulated with 1.5% EDTA centrifuged at 12,000 rpm for 10 minutes, and then the supernatant was collected. All these samples stored at −80°C for further analysis.

RNA extraction and reverse transcription Total RNA extracted from the brain tissue using Trizol reagent. Total mRNA (1 μg) was transcribed using Quant script cDNA RT Kits according to the manufacturer’s manual. Briefly, RNA (1 μg) 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 μl at 37°C 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 Applied Rotor-Gene 3000 under the following conditions: iNOS and GAPDH for 40 cycles at 94°C for 30 s, 63°C for 60 s, and 72°C for 90 s. Relative quantitative measurements of target gene levels was performed using the ΔCt 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 Student’s Statistical analysis

The statistical analysis carried out as per standard method. All result expressed as MEAN±SEM. Groups of data were compared with the analysis of variance (ANOVA) followed by dunnett’s t-test values for statistical significance.

Sagar Institute of Pharmaceutical Sciences, Sagar M.P. Page 1