INTRODUCTION:

Stress, a state of metabolic and behavioral responses occurs in response to chemical, physical, biological and emotional fluctuations and helps in establishment of the organism¹. If the stress is exciting, the homeostatic function of the organism develops deficit and the existence of the organism is endangered. Stress activates a wide range of the body changes called General Adaptation Syndrome (GAS) and persuade a marked increase in the brain stages of biogenic amines such as adrenaline and nor-adrenaline which backing the organisms to cope with stress².

Glucocorticoids secretion mediates the endocrine response, though, augmented and lengthy severe stress is accountable for lowered mood, fatigue, reduced stamina, and in the etiopathogenesis of diversity of diseases like anxiety, depression, endocrine disorders, immunosuppression, male impotency, peptic ulcer, cognitive dysfunction, hypertension and ulcerative colitis³.

Stroke is one of the foremost causes of mortality worldwide⁴. Shortage of blood in a part, usually due to practical constriction or actual block of blood vessel is called ischemia. Ischemia plays to the pathophysiology of many situations faced by anesthesiologists, including peripheral vascular insufficiency, myocardial infarction, stroke, and hypovolemic shock. Though the restoration of blood flow to an ischemic organ is indispensable to prevent irreparable cellular injury, reperfusion may supplement tissue injury in addition to that shaped by ischemia alone. For example, the histological variations of injury after 3 h of feline intestinal ischemia, shadowed by 1 h of reperfusion are far worse than the variations observed after 4 h of ischemia alone⁵.

Sarcostemma acidum is an Indian traditional therapeutic herb. It has been considered as aspirant of Soma plants by numerous authors. It was said that Soma (Somlata) was used to make ‘Som ras’ (Rejuvenating drink) by Aryans. The original source of 'Soma' plant is a mystery that has been discussed by the vedic and botanical researchers for more than two and a half centuries⁶. It is available wildly in India, Pakistan and Europe etc. It is spread in numerous parts of India. It is originating in dry rocky places in Bihar, Konkan, Tamil Nadu, Bengal, Deccan, Madhya Pradesh, Maharashtra, and Kerala. The plant is unpleasant, acrid, chilling, alternate, sedative, emetic, antiviral and revitalizing. Several chemical constituents present in the plant like succinic acid, malic acid, reducing sugar-sucrose, traces of tannin, alkaloids, phytosterols, alpha and beta amyrins, lupeol and lupeol acetate, beta sitosterol. Antifertility, anti-microbial and in vitro anti-inflammatory actions have been described on this plant^7,8.

Many useful medications to relieve stress such as benzodiazepines, certain CNS stimulants such as amphetamines and caffeine as well as some anabolic steroids are routinely prescribed to combat stress. The incidence of toxicity and dependence has limited the therapeutic usefulness of these drugs. The potential utility of safer and cheaper herbal medicines against stress has been reported as they can withstand the stress without altering the physiological functions of the body. Sarcostemma acidum traditionally using as rejuvenating drink, has wound healing property, anti-microbial and in vitro anti-inflammatory actions. The whole plant extractives were reported to have a number of psychopharmacological effects including antipsychotic, anxiolytic and CNS inhibitory activity⁹ and it was claimed to have antistress activity by folklore.

So, the present study was designed to study antioxidant, anti-stress and cerebroprotective activities of Sarcostemma acidum in experimental animal models.

MATERIALS AND METHODS:

Chemicals and drugs

Withania Somnifera tablets (Himalaya, Kadapa). Nicotinamide adenine dinucleotide (NADH), Bradford reagent, Griess reagent, phenazine methosulphate, potassium dihydrogen phosphate, sodium pyrophosphate, 1,1-diphenyl-2-picrylhydrazyl (DPPH), disodium hydrogen phosphate and sodium dihydrogen phosphate were bought from Sigma Aldrich life sciences, Bangalore, India. Nitro blue tetrazolium (NBT), sodium lauryl sulfate and trichloroacetic acid (TCA) were bought from Molychem, Mumbai, India. 5, 5¹-dithio-bis-2-nitrobenzoic acid was bought from Kemphosol, Bombay, India. Thiobarbituric acid (TBA) was bought from Hi Media Laboratories Pvt. Ltd, Mumbai, India. All other substances used were of analytical rank with high purity.

Animals:

Male Wistar albino rats (200 ± 10 g) and male swiss albino mice (25 ± 5 g) were picked for the study. The animals were retained in clean polypropylene cages under sterile and regular environmental circumstances at 22°C ± 2°C, 12:12 h light: dark cycle and 60 ± 5% relative humidity with free access to regular laboratory food and water ad libitum (Sai Durga Feeds and Foods, Bangalore). Mice were familiarized to laboratory settings for 1 week before the test. All the experiments were permitted out during the light period (08:00-16:00) and conducted in agreement with the guidelines given by the committee for the purpose of control and supervision of experiments on animals (CPCSEA), New Delhi (India) and the Institutional Animal Ethics Committee (1220/a/08/CPCSEA/ ANCP-08/15) permitted the experimental protocol.

Plant material and preparation of extract¹⁰

The stem of Sarcostemma acidum was collected from the Tirumala forests, Tirupati, A.P, India in the month of Feb 2016 and was authenticated by Dr. K. Madhava Chetty, Professor and Head, Department of Botany, S. V. University, Tirupati and the voucher sample number was lodged (plant specimen no: 1102) and conserved in the herbarium, which was reserved in our lab for future reference.

The ethanolic extract was prepared by using the shade dried and crushed stem of Sarcostemma acidum (500 g) by using a Soxhlet extractor in presence of 70% ethanol for 72 h. The obtained ethanolic extract was concentrated using rota evaporator at 70 degree Celsius (Sigma Scientific Glass Pvt. Ltd. India). The yield of the extract was found to be 12.3 %.

Preliminary phytochemical screening:

Ethanolic extract of Sarcostemma acidum was tested for the existence of various phytoconstituents¹¹.

High performance thin layer chromatography:

The EESA was then subjected to high performance thin layer chromatography (HPTLC) for identification of specific phytoconstituents¹¹. The extract was dissolved in HPTLC grade chloroform: methanol (6:4), which was used for sample application on precoated silica gel GF 254 aluminum sheets. The samples (5 μL-10 mg/5 ml) were spotted in the form of bands of width 8 mm with a 100 μL sample using a Hamilton syringe on silica gel, which was precoated on aluminum plate GF 254 plates (5 × 10 cm-E. MERCK KGaA, Bangalore) with the help of Linomat 5 applicator attached to CAMAG HPTLC system, which was programmed through WIN CATS software (CAMAG, USA). The developed plate was kept in photo-documentation chamber (CAMAG REPROSTAR 3) and captured the images under ultraviolet (UV) light at 254 and 366 nm, respectively (Aetron, Mumbai, India). The Rf values and finger print data were recorded by WIN CATS software.

Estimation of Flavonoids¹²

An aliquot of the sample was pipetted out in a test tube and the volume was made up to 0.5 ml with distilled water. Sodium nitrite (5%; 0.03 ml) was added to the tube and incubated for 5 min. at room temperature. Aluminum chloride solution (10%; 0.06 ml) was added and incubated for 5 min. at room temperature. Sodium hydroxide solution (1 M; 0.2 ml) was added and the total volume was made up to 1 ml with distilled water. Absorbance was measured at 510 nm against a reagent blank. Standard curve using different concentrations of rutin was prepared. From the standard curve, the concentration of flavonoids in the test sample was determined and expressed as mg of rutin equivalent.

In vitro antioxidant activity:

Different parameters like reducing power¹³,2,2-diphenyl-1-picrylhydrazyl assay¹⁴, Peroxide radical scavenging activity¹⁵, Nitric oxide radical scavenging activity¹⁶ and Superoxide radical scavenging activity¹⁷were studied.

Acute oral toxicity study:

The acute oral toxicity study was performed according to the method described by Lorke¹⁸. EESA up to a dose of 2000 mg/kg did not produce any signs of toxicity and mortality. Based on this the doses of EESA for further experimental study were selected.

Anti-stress activity

Swimming endurance test

The mice were randomly divided into five groups of six animals each and the animals were treated for 7-days same as in the above model. On day 7, 1 h after the administration of extracts, all the animals were subjected to swimming stress by keeping them in a polypropylene vessel of dimension 40 × 40 × 30 cm with a water level of 20 cm and the immobility time of each mouse was recorded for 30 min¹⁹.

Tail suspension test²⁰

The mice were randomly divided into five groups of six animals each. The treatment protocol was, group 1 received 2% tween 80 orally and served as vehicle control, group 2 received 2% tween 80 orally and stress only, served as stress control, group 3 and 4 received 200 mg/kg and 400 mg/kg orally of EESA respectively and group 5 received Withania somnifera 100 mg/kg orally. On the 7^th day, 1 hr after the extract treatment all the animals were suspended individually by end of tail with adhesive tape with the head 50 cm from the bottom. Mice were suspended for a total of 10 min during the final 5 min interval of the test, duration of immobility was recorded. Mice were considered immobile only when they hung passively and completely motionless.

Cerebral ischemia model:

The animals are divided into 4 groups (n=6 per group). Group –I received 2% tween 80 (10 ml/kg p.o) and served as normal control, Group-II received iron and served as disease control, Group-III received the EESA 400 mg/kg p.o. Group–IV received the Withania somnifera 100 mg/kg p.o for 28 days. Iron dextrose injection (0.5 mg/kg i.p) was administered 24 hrs prior to the carotid artery occlusion then the animals were subjected for carotid artery occlusion²¹ for 15 min followed by reperfusion 30 min prior to scarification. Then the animals were sacrificed, and the homogenates obtained were used for the estimation of TBA reactive substance (TBARS)²², reduced glutathione (GSH)²³, superoxide dismutase (SOD)²⁴, catalase²⁵ and nitrate²⁶.

Estimation of Monoamine Oxidase A²⁷

Rat brain mitochondrial fraction was prepared, and the protein concentration was adjusted to 1 mg/ml. To the 250 µl of the homogenate add 250 µl of serotonin and 250 µl of buffer. The reaction tube was placed at 37°C for 20 minutes and the reaction was arrested by the addition of 200 µl of 1M HCl. The reaction product was extracted with 5 ml of butyl acetate. The organic phase was separated and measured at 280 nm using a spectrophotometer. Blank samples were prepared by adding 1M HCl (200 µl) prior to reaction and the reaction was carried out. The MAO-A is expressed in nmoles/ mg protein.

Estimation of serum corticosterone²⁸

Serum corticosterone (CORT) level was determined quantitatively using rat ELISA kits.

Measurement of infracted area:

After scarification, rat brains were isolated and infract sizes caused by cerebral ischemia were visualized by TTC staining. After 24 h brains were taken out from the dye and observed the infracted area by naked eye where the normal cells will receive stain and visualized in red colour while the infracted cells will not receive the stain and visualized in pale colour.

Histopathology:

The brains were isolated and washed immediately with ice cold saline and fixed in 10% buffered formalin. The brains stored in 10% buffered formalin, were embedded in paraffin, section cut at 5µm and stained with hematoxylin and eosin. These secreations were then examined under a light microscope for histological changes.

Statistical analysis:

Results were expressed as mean ± standard deviation and analyzed using Graph Pad Prism version 5.1 Graph Pad Software, Inc using one-way analysis of variance followed by Dunnett's post-test. P < 0.05 was considered to be significant.

RESULTS:

Preliminary phytochemical screening

The preliminary phytochemical analysis revealed the presence of alkaloids, carbohydrates, saponins, phenolic compounds, and flavonoids.

High performance thin layer chromatography finger print analysis of EESA

High performance thin layer chromatography finger printing analysis of EESA revealed several peaks and were recorded. HPTLC profile under UV 366 and 254 nm was recorded in the [Figure 1]. The Rf values of spots observed were 0.12, 0.81 and 0.93. Appearance of blue and orange color under UV examination from the chromatogram after derivatization confirmed the presence of flavone and flavonoid components in the test extract (T) and standard (S).

Figure 1. HPTLC (Chromatogram) with peaks test (T) and standard (S) of ethanolic leaf extract of Sarcostemma acidum at 366 nm and 254 nm.

Flavonoid content of EESA

Flavonoid content of EESA was determined and the results indicated that EESA contains 13.6 mg/g of rutin equivalent flavonoid.

In-vitro antioxidant activity of EESA

The plant extract was potently active and has good free radical scavenging activity. DPPH radical, reducing power and nitric oxide reactive free radical scavenging activity of EESA was lower than ascorbic acid but comparable to ascorbic acid.

Antistress activity: Swimming endurance test

Oral administration of EESA in two different doses significantly (P<0.001) reduced the immobility time in mice during swimming endurance test. The percentage inhibition at high dose of EESA (54.3%) was incomparable to reference standard Diazepam (61.64%). The results were present in figure 2.

UV 366 nm

Table 1. In-vitro antioxidant activity of EESA

Concentration µg/mL^-1	Reducing Power (OD)		DPPH assay (%)		Superoxide radical scavenging assay (%)		H₂O₂ radical scavenging assay (%)		Nitric oxide inhibition assay (%)
Concentration µg/mL^-1	EESA	AA	EESA	AA	EESA	AA	EESA	AA	EESA	AA
10	0.132±0.01	0.167±0.02	29±1.03	64±0.66	34±2.86	57±0.21	18±5.29	26±1.56	23±23.23	31±1.45
50	0.169±0.05	0.192±0.01	37±2.47	79±2.68	56±0.82	61±3.39	37±5.95	42±1.17	29±3.87	58±1.20
100	0.218±0.01	0.247±0.05	56±1.29	85±1.40	68±2.21	72±0.14	59±2.37	67±1.11	48±3.21	61±0.21
200	0.237±0.02	0.276±0.01	72±2.41	96±0.77	89±0.77	92±2.47	72±6.40	86±0.86	76±2.85	83±0.07
500	0.285±0.03	0.324±0.02	94±1.97	99±0.71	100±0.11	100±0.2	91±1.21	100±0.15	88±1.91	100±0.21
IC₅₀	---	---	89±1.12	7.8±0.92	44.6±4.74	8.8±4.70	85±3.51	75±0.80	104±2.40	44±2.36

EEAS - Ethanolic extract of stem of Sarcostemma acidum; AA – Ascorbic acid

All values expressed as mean±SEM. One way ANOVA followed by Dunnet’s post-test. Values are signiﬁcantly different at P<0.05

Figure 2. Effect of EESA on swimming endurance test

All values expressed as mean±SEM. One way ANOVA followed by Dunnet’s post-test. ^a P<0.001 vs Stress control

Tail suspension test:

In tail suspension test, seven-day pre-treatment with two doses (200 mg/kg, 400 mg/kg) of EESA significantly reduced the immobility time as compared to the stress control group. Withania somnifera (100 mg/kg) showed significant reduction in immobility time as compared to stress control group. The results were shown in Figure 3.

Figure 3. Effect of EESA on Tail suspension test

All values expressed as mean±SEM. One way ANOVA followed by Dunnet’s post-test. ^a P<0.001 vs Stress control

There were no significant changes in body weight and tissue weight in stress control and EESA treated mice. So, the data not presented.

Results of tissue antioxidant parameters in cerebral ischemia model rats (Table 2)

Level of thiobarbituric acid reactive substances (TBARS):

There was significant (p˂0.001) increase in the level of TBARS in disease control when compared with normal control. There was less significant (p˂0.001) decrease in level of TBARS in groups treated with EESA and standard drug when compared with disease control.

Level of reduced glutathione:

There was significant (p˂0.001) decrease in level of GSH in disease control when compared with normal control. There was less significant (p˂0.001) increase in level of GSH in group treated with EESA and standard drug when compared with disease control.

Level of superoxide dismutase:

There was significant (P˂0.001) decrease in level of enzyme catalase in disease control when compared with normal control. There was less significant increase (P˂0.001) in level of catalase in groups treated with EESA and standard drug when compared with disease control.

Level of Catalase:

Nitrates level:

There was significant (p˂0.001) increase in the level of nitrates in disease control when compared to normal control. There was significant (P˂0.001, P˂0.01) decrease in level of nitrates in group treated with EESA and standard drug when compared with disease control.

Level of monoamine oxidase A in brain tissue (Table 3)

There was significant (p˂0.001) increase in the level of MAO-A between disease control when compared with normal control. There was less significant (p˂0.001) decrease in level of MAO-A in groups treated with plant extract and standard drug.

Level of CORT in serum (Table 3):

There was significant (p˂0.001) increase in the level of CORT between disease control when compared with normal control. There was less significant (p˂0.001) decrease in level of CORT in groups treated with plant extract and standard drug.

Table 3. Level of monoamine oxidase A in brain tissue and CORT in serum

Group	MAO-A activity is expressed in nmoles/ mg protein	CORT ng/ml
Control	47.2±0.1	38.4±1.8
Disease control	74.6±1.4^#	68.9±1.2^#
EESA 400 mg/kg	54.1±0.2***	45.1±1.6***
WS 100mg/kg	50.18±1.2***	40.6±0.8***

All values expressed as mean±SEM. One way ANOVA followed by Dunnet’s post-test.

^#P<0.001 vs Control; *** P<0.001 vs Disease control

Measurement of Infracted Area:

The brain tissue showed less infracted area in EESA and WS when compared with disease control which contained unstained infracted cells.

Histopathological Evaluation:

Photomicrograph of rat brain showed normal architecture with regular morphology of brain cell membrane and well-preserved cytoplasm in control group (A). The disease control group (B) showed disruption of epidermal cell integrity, and damage to the neuronal cells and glial cells. Rats pretreated with EESA (C), WS (D) have shown no damage to the neuronal and glial cells with normal epidermal cell integrity similar to that of normal control group. The results were presented in Figure 4.

Figure 4. Histopathological results of brain tissue

Note: Arrows indicate the status of epidermal cell integrity, cytoplasm, neuronal cells and glial cells in rat brain.

DISCUSSION:

Stress is major problem globally which arise through several factors to affect human health and organ system. Short duration of stress does not cause any harm to body but longer duration of stress cause development of abnormal conditions like anxiety, behavioural depression, cognitive dysfunction, and increase in serum corticosterone level, increase in blood glucose, immune suppression, gastric ulcers and increased oxidative stress.²⁹ Many medicinal plants shows effectiveness against the developed stress which causes harm to the body.

Plants and plant products have been used medicinally for numerous sicknesses for years. Sarcostemma acidum is demanded to be having antistress activity by folklore which is offered plentifully in several places of India. The roots and leaves of the plant are used for viral infection, mental diseases, allergic rhinitis, and sinusitis. Latex applied superficially to cure wounds and burns. So the current study was undertaken to evaluate the antistress and cerebroprotective effect of EESA on mice and rat experimental model.

In the swimming endurance model conducted, a significant reduction in immobility time was observed at 400 mg/kg of EESA administered. This increase endurance could be due to normalization of blood catecholamine and monoamine oxidase levels, a decline in muscle glycogenolysis, or a decline in muscle concentrations of two toxic by-products of muscular efforts; lactic acid and ammonia.³⁰ It may also be attributed to increase in the utilization of the Adenosine Triphosphate (ATP) pathway or the anti-oxidant effect of EESA which prevents free radical-induced damage to vital organs. These extracts showed dose dependent antistress activity.

The tail suspension test and swimming endurance test exhibit acute compassion and specificity for antistress and antidepressants and also these tests are easy to achieve and afford reliability. Both animal tests offer a stressful environment, where mice cannot drip and after an initial period of struggling, the animals become motionless, which mimics a depressed and desperate behavior. Also, the tail suspension test has been recognized as having great importance due to its major sensitivity in detecting selective serotonin reuptake inhibitor (SSRI) type antidepressants.³⁰ Since both the doses of EESA successfully presented antidepressant-like behavior in the tail suspension test, we suggest that these drugs may have similar actions to that class of drugs.³¹

In cerebral ischemia model, the cerebral ischemic control group animals showed decreased amount of antioxidant defense enzymes (SOD, Catalase, GSH) and increased amount of lipid peroxidation (TBARS) in brain because of induction of ischemia whereas the group of animals that were treated with EESA, and WS had shown increased levels of SOD, Catalase, GSH enzymes and reduced the total nitrite (NO) and decreased TBARS which is the marker of lipid peroxidation suggesting that the antioxidant potential of EESA may have reduced the formation of reactive oxygen species (ROS) and subsequently prevented the disastrous chain reaction. This may be due to the antioxidant principles in brain regions that are associated with motor and sensory such as flavanoids and phenolic compounds present in the plant extract. This finding is supported by the previous studies where curcumin, resveratrol, silymarin, green tea extract containing antioxidant potential have counteracted the ROS generation during ischemia-reperfusion induced brain injury.³²

Corticosterone (CORT) is an significant stress hormone in animals and has been convoluted in major depressive disorder. CORT is an endocrinological diagnostic marker which is frequently used based on its raise in chronic stress in animal models.³³ Two isoforms of monoamine oxidase (MAO-A and MAO-B) exist, with complex substrate preference of MAO-A isoform for serotonin; and it is the main target for the antidepressant MAO inhibitors. The metabolic deprivation of serotonin and catecholamines in noradrenaline and serotonin neurons is structured by MAO-A. This pathway is supposed to be the site of therapeutic exploit in stress.³⁴ In the present study, cerebral ischemic rats showed significant increase in serum level of CORT and brain activity of MAO-A relative to control rats. This effect was nearly restored by oral administration of EESA and standard Withania somnifera (WS) relative to cerebral ischemic rats, which reflected the antistress-like potential.

In this study infract size as measured with TTC staining was used as the primary experimental measure of the outcome of cerebral artery occlusion. EESA and WS extracts significantly lowered the infract size and the extent of edema in ischemic rats as compared to the control rats. Similar effect was observed in the histopathological report also.

The HPTLC and flavonoids estimation proved that the EESA extracts enriched with flavonoid type compounds which might be responsible for the above said activities.

CONCLUSION:

The present study concluded that, EESA possess significant antioxidant, anti-stress and cerebroprotective activities due to presence of flavonoids as major phyto constituents.

ACKNOWLEDGEMENT:

We are thankful to management of Annamacharya College of Pharmacy for the infrastructure and all other essential facilities provided during the project work.

CONFLICTS OF INTEREST:

The authors declare no conflicts of interest.

REFERENCES:

1. Bharathi KN, Sivaramaiah N, Nagarjuna CG, Gupta AVSSS. Evaluation of antistress, anxiolytic and hypnotic activity of vedic calm, a polyherbal formulation. Pharmacog. Mag 2009; 5(19):124-130.

2. Subarnas A, Tadano T, Nakahata N, Arai Y, Kinemuchi H, Oshima Y et al. A possible mechanism of antidepressant activity of beta-amyrin palmitate isolated from Lobelia inflate leaves in the forced swimming test. Life. Sci 1993; 52:289-296.

3. Esch T, Stefano GB, Fricchione GL, Benson H. The role of stress in neurodegenerative diseases and mental disorders. Neuroendocrinol. Lett 2002; 23:199-208.

4. Rensink M, Schuurmans M, Lindeman E, Hafsteinsdottir T. Task-oriented training in rehabilitation after stroke: Systematic review. J Adv Nurs 2009; 65:737-754.

5. Parks DA, Granger DN. Contributions of ischemia and reperfusion to mucosal lesion formation. Am J Physiol 1986; 250:749-753.

6. Shah NC. The Mystery and Botanical identity of Soma plant: An Appraisal. Herbal Tech Industry 2011; 8:19-28.

7. Anyonyya M, Surendar A, Sruthi G, Khuddus GA. Antimicrobial activity of Sarcostemma acidum Voigt (Apocynaceae) stem. Int J Biol Pharma Res 2012; 3(6):752-757.

8. Verma PK, Sharma A, Mathur A, et al. Effect of Sarcostemma acidum stem extract on spermatogenesis in male albino rats. Asian J Androl 2002; 4(1):43-47.

9. Ittiyavirah P, Rahees T. Evaluation of psychopharmacological activity of ethyl acetate extract of Sarcostemma acidum (Roxb).Voigt. The Journal of Phytopharmacology 2013; 2(5):1-7.

10. Killedar SG, More HN. Screening of Antimicrobial Potential and Phytoconsituents for Different Extracts of Memecylon umbellatum Burm Inflorescences. Asian J. Pharm. Res 2011;1(4):14-118.

11. Harbone JB, Baxter HH. Phytochemical Dictionary: A Hand Book of Bioactive Compound from Plants. Washington: Taylor and Francis; 1993;P.237-240.

12. Wadkar KA, Magdum CS. Evaluation of Total Phenolic Content, Flavonoid Content and Antioxidant Activity of Stem Bark of Careya arborea Roxb. Research J. Pharmacognosy and Phytochemistry 2010; 2(1):49-51.

13. Elias J, Rajesh MG, Anish NP, Deepa P, Jayan N. In Vitro Antioxidant Activity of the Methanolic Extract of Simaruba glauca DC. Asian J. Research Chem 2010;3(2):312-315.

14. Burits M, Bucar F. Antioxidant activity of Nigella sativa essential oil. Phytother Res 2000; 14:323-328.

15. Pagare MS, Joshi H, Patil L, Kadam VJ. In Vitro Antioxidant Activity of Fruit of Benincasa hispida Cogn. Research J. Pharm. and Tech 2011;4(7):1082-1085.

16. Marcocci L, Maguire JJ, Droy-Lefaix MT, Packer L. The nitric oxide-scavenging properties of Ginkgo biloba extract EGb 761. Biochem Biophys Res Commun 1994; 201:748-755.

17. Misra HP, Fridovich I. The univalent reduction of oxygen by reduced flavins and quinones. J Biol Chem 1972; 247:188-192.

18. Lorke D. A new approach to practical acute toxicity testing. Arch Toxicol 1983; 54:275-287.

19. Pasha S, Khaleel M, Som S. Evaluation of Adaptogenic Activity of Moringa oleifera Lam. Research J. Pharmacology and Pharmacodynamics 2010;2(3) 243-247.

20. Selvi PT, Kumar MS, Kathiravan T, Rajesh R, Megala J, Sravani S. Antistress activity of aqueous extract of leaves of Centella asiatica. Linn by in vivo methods. Asian J. Res. Pharm. Sci 2012;2(3):91-94.

21. Traystman RJ. Animal models of focal and global cerebral ischemia. ILAR J 2003; 44:85-95.

22. Okhawa H, Ohishi N, Yagi K. Assay of lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979; 95(2):351-358.

23. Ellman GL. Tissue sulphydryl groups. Arch Biochem Biophys 1959; 82(1): 70-77.

24. Kakkar P, Das B, Vishwanathan PN. A modified spectrophotometric assay of superoxide dismutase. Ind J Biochem Biophys 1984; 21(2):130-132.

25. Krishnaveni J, Ananthi T. Hepatoprotective Effect of Moringa oleifera in Isoniazid Induced Rats. Research J. Pharm. and Tech 2011;4(12):1901-1903.

26. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite [15 N] and nitrate in biological fluids. Anal Biochem 1982; 126(1):131-138.

27. Habibur R, Eswaraiah MC. Simple spectroscopic Methods for estimating Brain Neurotransmitters, Antioxidant Enzymes of Laboratory animals like Mice: A review. Pharmatutor Art 2008;1244.

28. Dhingra D, Bhankher A. Behavioral and biochemical evidences for antidepressant-like activity of palmatine in mice subjected to chronic unpredictable mild stress. Pharmacological Reports 2014; 66(1):1-9.

29. Dhingra D, Bhankher A. Behavioral and biochemical evidences for antidepressant-like activity of palmatine in mice subjected to chronic unpredictable mild stress. Pharmacol Rep. 2014;66(1):1-9.

30. Parente MSR, Custodio FR, Cardoso NA, Lima MJA, Melo TS, Linhares MI. Antidepressant-Like Effect of Lippia sidoides CHAM (Verbenaceae) Essential Oil and Its Major Compound Thymol in Mice. Sci. Pharm. 2018;86(3):27.

31. Ahirwar D, Ahirwar B. Antidepressant effect of Nigella sativa in stress-induced depression. Research J. Pharm. and Tech. 2020; 13(4):1611-1614.

32. Elizabeth TO, Adegbuyi OA, Olusegun AA, Abayomi MA. Protective Effects of Morin Hydrate on Acute Stress-Induced Behavioral and Biochemical Alterations in Mice. Basic Clin Neurosci. 2018;9(3):195-208.

33. Azmathulla S, Hule A, Naik SR. Evaluation of adaptogenic activity profile of herbal preparation (AVM). Indian J Exp Biol. 2006;44(7):574-9.

34. Xu J, Xu H, Liu Y, He H, Li G. Vanillin-induced amelioration of depression-like behaviors in rats by modulating monoamine neurotransmitters in the brain. Psychiat Res. 2015;225(3):509-14.

Received on 21.10.2020 Modified on 19.11.2020

Research J. Pharm. and Tech 2021; 14(11):5965-5972.

DOI: 10.52711/0974-360X.2021.01036

Treatment group	TBARS (nmol/g wet wt)	GSH (𝜇g/g wet wt)	SOD (IU/mg Protein)	Catalase (IU/mg protein)	NO (µg/dl)
Vehicle Control	23.13±1.34	21.18±0.12	24.15±1.387	0.290 ±0.14	0.41±0.017
Stress control	38.08±2.31^a	18.16±1.11^a	8.25±0.41^a	0.021±0.031^a	0.864±0.025^a
EESA 400 mg/kg	21.45±0.56^c	23.45+0.93^d	16.71+1.2^c	0.173+0.01^c	0.50+0.019^d
WS 100 mg/kg	22.61±1.005^c	22.35+0.489^b	15.62+0.121^c	0.183+0.001^c	0.498+0.047^c