INTRODUCTION:

Homeostasis is the term refers to maintenance of our internal medium constant in face of changing environment.

The things which menace the homeostasis is known as stress. Stress represents a reaction of the body to a stimulus that tends to alter its regular physiological equilibrium or homeostasis and has been described as a nonspecific response of the physique to any demand imposed on it.¹Stress alters the normal physiological conditions and spring in state of endangered homeostasis. This has been result into the etiopathogenesis of disorders such as GI disorders, psychiatric disorders, immunosuppression, and endocrine disorders including diabetes mellitus, male sexual dysfunction and cognitive dysfunctions.^2,3 The increased level of free radicals results into oxidative stress, oxidative stress has been involved in several diseases includes cancer, atherosclerosis and neurodegenerative diseases.⁴Several drugs have been developed for the management of the stress disorders which includes, flurazepam, diazepam and chlordiazepoxide used to treat the anxiety and tricyclic antidepressant used for treating the depression. However, the resulting side-effects associated with these agents have limited their use.^5,6 Therefore, there is a need for an effective herbal antioxidant, antistress and nootropic agents in the treatment of stress and stress induced disorders. Herbal drugs have been used to overcome the stress with the aid of bettering the nonspecific resistance of organisms towards a range of stressors and represent a unique category of compound regarded as adaptogen.⁷ A plant adaptogen is defined as smooth pro‑stressors which reduces reactivity of host defense systems and the increased level of mediators involved in the stress response will decrease the damaging effects of various stressors.⁸A number of plants have been investigated and reported for their antistress activity, a few salient ones are Ocimum sanctum, Withania somnifera, Eleuthrococcus senticosus, and Panax ginseng, Lagerstroemia speciosa, Bacopa monniere, Zizyphus jujuba, Morinda citrifolia, Punica granatum, Shisandrae chinensis and Lycium barbarum.^9-11The Pomegranate (Punica granatum) fruit is an ancient and rich source of bioactive compounds and also it has been used in folkcore medicine for centuries.¹² Pomegranate bark and the rind of the fruit contain tannin and the root contains punico-tannic acid, mannite, sugar, gum, pectin and an active liquid alkaloid "pelletierine" and oil liquid "isopelletierine" and rich in polyphenols.¹³Punica Granatum Linn family Lythraceae is well known plant in traditional Indian system of medicine Ayurveda. This tree is wildly found in Persia, Arabia, Afghanistan and Baluchistan, and cultivated nearly all over India.The pomegranate juice constitute of 85.4% water, 10.6% total sugars, 1.4% pectin, 0.2-1.0% polyphenols. Other reported minor compounds include fatty acids, amino and organic acids, indoleamines, sterols, triterpenoids and α-tocopherol. Anthocyanins, potent antioxidant flavonoids, provide pomegranate juice with its brilliant color, which increases in intensity during ripening and declines after pressing.^13,14 The plant has reported for various pharmacological activities includes, antioxidant, antiulcerogenic, antidiarrheal, antibacterial and antifungal, antimutagenic, preventive effects on Alzheimer’s disease, Antierectile dysfunction, improved learning, antiatherosclerotic, antihypertensive, and inhibits prostate cancer cell growth.^15-20 It is commonly used as a febrifuge, during malaria and seasonal fevers, and in all bilious complaints as a cooling drink to ameliorate the action of bile. Previous studies reported antioxidant, antidiarrheal, antibacterial and antifungal activity, antimutagenic effects, antioxidant and antihemolytic activity.^16,21In order to validate the traditional usage of drug as Punica granatum the aim of present study was undertaken to investigate the antioxidant, antistress and memory enhancing activity of the fruit extract of Punica granatum Linn.

MATERIALS AND METHODS:

Aqueous extracts of Punica granatum was obtained as a gift samples from M/s Laila Implex, Vijayawada and Kisalaya Herbals Ltd. Ratlam Koti, Indore, 452001. Ethylenediaminetetraacetic acid (EDTA), sodium cyanide (NaCN), nitro blue tetrazolium, deoxy-D-ribose, ferric chloride, sodium dodecyl sulphate, ascorbic acid, Tris HCl buffer, sodium nitro prusside, vanillyl mandelic acid (VMA), homovanillic acid (HVA) and 5-hydroxy indole acetic acid (5-HIAA) were purchased from Sigma Chemicals Co., U.S.A. Acetonitrile (HPLC grade) was obtained from Qualigens, methanol (HPLC grade) from SRL, hydrochloric acid, ortho-phosphoric acid, potassium dihydrogen phosphate all from Merck. All other chemicals and reagents used were of analytical grade.

Preparation of extract and preliminary phytochemical screening:

Punica granatum was obtained from the Laila Implex Laboratory (batch number: L242134, product code: C/AU/PILO-03) and was authenticated. Aqueous extract was qualitatively analyzed for the presence of various phytochemical constituents. The aqueous extract of the Punica granatum wastested for the presence of carbohydrates, proteins, alkaloids, flavonoids, glycosides, saponins, tannins, and essential oilsusing the standard procedures.²²

Superoxide, hydroxyl radical scavenging in-vitro antioxidant activity:

Super oxide scavenging activity of the extracts was determined by the McCord and Fridovich methodwhich looks at light induced superoxide generation by riboflavin and the corresponding reduction of nitro blue tetrazolium.²³

0.2ml of deoxyribose (2.8mM), 0.2 ml of each of ferric chloride (0.1mM), EDTA (0.1mM), H₂O₂ (1mM), were added to the mixture 0.2ml of different dilutions of extract and ascorbic acid and 1.2ml of phosphate buffer (20mM, pH 7.4) and kept in the incubator at 37°C for 1 h. The reaction mixture (1ml) was treated with sodium dodecyl sulfate (0.2ml, 8.1%), thiobarbituric acid (1.5 ml, 0.8%) and acetic acid (1.5ml, 20%, pH 3.5). The percentage inhibition of the extracts on hydroxyl radicals was determined by comparing the absorbance values of the control, and experimental tubes as in the case of superoxide assay.^24-25

Animals:

Healthy adult albino wistar rats (180-200g) were used for the forced swimming stressand Y-mazetest. The animals were housed in polypropylene cage at an ambient temperature of 27°C±2°C with 65±10% relative humidity and a 12 h light/dark cycle. All animals were fed with standard pellet diet and water ad libitum supplied by Pranav Agro Industries Ltd, Sangli, India. The study protocol was approved (CPCSEA/IAEC/341) by the Institutional Animal Ethics Committee (IAEC) and conducted according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

Forced swim stress test for antistress activity:

Albino wistar rats were randomly divided into four groups of four animals each of either sex. The rats were segregated into groups in such a way that the total weight of each group was approximately the same. The animals were maintained on constant diet with 12 h dark and light cycle at 25°C. The treatment groups were pretreated with Punica granatum (100mg/kg, 300 mg/kg, p.o.) for four days. The control group was pretreated with normal saline (1ml/kg, p.o.), while the reference standard group received Bacopa monniere (100mg/kg, p.o.) for four days. The swimming test was carried out on the fourth day, after one hour of oral administration of the drug, using a polypropylene vessel (45×40×30cm) with a water level of 30cm at room temperature (28°C). After forced swimming stress, 24h urine sample from each group were collected into two different beakers, one containing 5ml of 10% oxalic acid for the determination of ascorbic acid by spectrophotometric method at 550nm and the other containing 0.5ml of 6N hydrochloric acid for the determination of metabolites of noradrenaline (VMA), 5-hydroxytryptamine or serotonin (5HIAA) and dopamine (HVA) by UPLC. Recovery from stress was assessed on the fourth day by collecting 24h urine samples and analyzed.

Influence of Punica granatum on urinary levels of VMA, 5-HIAA, HVA and AA:

In the first part, estimation of levels of VMA, 5HIAA, HVA and ascorbic acid were carried out in 24h urine sample in normal rats. In the second part, the animals in each group except the first group were subjected to fresh water swimming stress individually and estimation of VMA, 5HIAA, HVA and ascorbic acid levels were done in 24h urine sample in stress induced rats. The recovery from stress was assessed on the fourth day by collecting 24 hurine samples and analyzed. In the third part of the experiment, the animals were treated bydrug as forced swimming stress test and observed influence of Punica granatum on levels of VMA, 5HIAA, HVA and ascorbic acid in stress induced rats. In the final part, aqueous extracts of Bacopa monniere and Punica granatum were administered as mentioned in the third part. After 30 minutes the animals were subjected to stress as earlier and 24h urine samples were collected to study the influence of selected herbal extracts on stress induced biochemical changes. The analysis was done on the same day and on fourth day for recovery. The 24h urine sample from each rat was collected in two different beakers using an inverted 'Y' tube fixed at the bottom of each metabolic cage. The urine from one end of tube was collected into a beaker containing 5 ml of 10% oxalic acid for determination of ascorbic acid at 550nm using UPLC (UPLC-Shimadzu, Model–SIL-20 ACHT) method. Ascorbic acid in urine was determined by the modified method developed by Roe JH.²⁶

Nootropic activity:

The spontaneous alteration task paradigms is the simplest version of Y-maze task used to measure spatial working memory (short term memory) in rats and mice.²⁷Albino wistar rats were divided into four groups each containing four animals. Group I served as control, group II served as Bacopa monniere (reference standard), Groups III and IV received orally Punica granatum 100 and 300mg per kg respectively for 7 days. Y-maze was used to study the effect of Bacopa monniere (reference standard) and Punica granatum on spatial memory in stress induced amnesia model. Enclosing each goal box is a guillotine door, similar to the start door, located 30cm from the end of the goal zone. A metal cup (0.5cm in radius) is fixed 0.5cm from the end of the each goal arm. In the second trail rats were allowed to explore the three arms; the number and order of arm entries for total 6 min duration was recorded. Total number of arm entries indicates the locomotor activity and successive entries into the three arms on overlapping triplet sets (SSD, SDS, and DSS) were used to calculate the spontaneous alteration behavior. The step first includes the rats were trained to find the safer zone two days before the experiment and the next day assessed for the nootropic activity. In the second steps, stress was induced before subjecting the animal for nootropic activity, and third step,the animals were treated after the recovery from stress as; group I has kept as control and 1ml of normal saline was administered orally, group II, III and IV was administered orally Bacopa monniere 100mg/kg dose (standard), Punica granatum 100mg/kg and Punica granatum 300mg/kg dose. After 30min the animals were assessed for nootropic activity. In the final step, after 3 days washout period, aqueous extracts of Bacopa monniere and Punica granatum were administered respectively to the groups of animals as mentioned in the third step.

Statistical Analysis:

All values are expressed as mean±SEM. Comparison between the stress control and drug-treated groups were made by one way ANOVA followed by the tukey test, <0.01 were considered to be significant

RESULTS:

The phytochemical analysis of extract revealed the presence of alkaloids, tannins, phenols and flavonoids in the fruits of Punica granatum Linn. The extract of Punica granatum was found to scavenge the superoxides generated by photo reduction of riboflavin (Table 1). The quantity of the extract of Punica granatum was needed for 50% scavenging of superoxides was found to be 179.2µg. The quantity needed for the same effect by the known antioxidant, ascorbic acid was 114.00µg. This indicates that the aqueous extract of Punica granatum possess antioxidant activity. Degradation of deoxyribose mediated by hydroxyl radicals generated by Fe³⁺/EDTA/H₂O₂ system was found to be inhibited by the extracts. The quantity of extract of Punica granatum was needed for 50% scavenging of hydroxyl radicals 266.2 and the same effect by the known antioxidant, ascorbic acid was 140.7µg. The quantity of the extract of Punica granatum was needed for 50% scavenging of lipid peroxides was found to be 278.7µg. The quantity needed for the same effect by the known antioxidant, ascorbic acid was 196.5µg. Punica granatum at quantities of 50, 100, 150, 200, 300µg produced dose dependent inhibition of hydroxyl radicals and, superoxide radicals. The extracts were found to be safe at a dose of 2000mg/kg since non mortality was observed. Signs and symptoms of intoxication were not observed 24h post-treatment and as dose did not produce any significant changes in behavioral pattern and failed to elicit any clinical abnormality.

LD₅₀was considered as more than 2000mg/kg. The entire two test groups of the aqueous extract of Punica granatum showed dose-dependent decrease in immobility time when compared against control as well as against Bacopa monnieri. The treatment with the selected plant extract found to increase the duration of time to swim until get exhausted was 2.3 to 3.4 min. A wide variety of methods have been reported for the measurement of VMA. 5-HIAA, HVA in urine of rats and humans.^28,29 The urinary values of VMA, 5 HIAA, HVA and AA are shown in Table 2. With administration of Punica granatum extract 1 hr prior to the induction of stress, the VMA and 5HIAA levels were significantly (p<0.01) decreased and HVA, AA were significantly (p<0.01) increased compared to stress alone.

Table 2: Effect of stress and Punica granatum extract on the total amount of metabolites (μg) excreted in 24-h rat urine

Level	Rats	Control	Stress	Recovery	Control + Extract	Recovery	Stress + Extract	Recovery
VMA	Group I	41.94±1.3	40.78±1.86	40.59±1.87	38.17±1.93	40.9±1.49	44.1±1.96	41.62±1.27
5HIAA	Group I	77.40± 1.18	75.43± 0.45	74.86±1.5	72.00± 1.36	68.16±1.78	68.74±1.62	65.49±0.25
HVA	Group I	217.44±1.38	194.16± 2.82	194.02±1.87	184.81 ±1.3	176.06±1.4	176.81± 17.50	169.30± 1.8
AA	Group I	23.16± 0.89	23.59±1.3	23.73±0.23	21.78±0.08	23.88±0.28	23.68±0.65**	24.03±1.66
VMA	Group II	43.59±1.49	142.17±2.56**	43.47±1.88	41.27±1.6	50.94±0.51	92.03± 1.51**	46.94±1.26
5HIAA	Group II	61.78±1.59	205.71± 2.7**	54.96± 1.51	52.56± 0.47	52.75± 1.18	76.86±1.00**	53.31±1.34
HVA	Group II	233.80±1.86	30.9± 1.30**	225.52±1.52	215.16± 1.6	219.85±1.2	133.39±1.60**	196.92±2.13
AA	Group II	23.79±1.1	8.45±0.64**	23.88±0.92	25.1±0.52	24±0.78	24.34±0.07**	25.1±0.48
VMA	Group III	40.67±1.6	149.79±1.21**	40.41±0.71	37.51±0.83	40.18±1.01	87.94± 1.71**	41.43±0.66
5HIAA	Group III	73.98±4.03	202.68±1.06**	65.05±1.23	61.64±1.99	66.6± 1.57	76.57±1.28**	65.53±1.23
HVA	Group III	253.42±1.92	32.53± 0.82**	229.95 ±2.88	218.8±1.69	236.29±1.82	114.57±1.21**	233.25±1.8
AA	Group III	25.40±0.52	8.34±0.09**	22.71±0.77	24.46±1.26	24.06±0.31	24.63±0.65**	23.52±0.34
VMA	Group IV	37.37±2.08	155.3 ±2.50**	37.90±1.27	35.41 ±1.49	37.80±1.34	83.91±1.80**	38.63±1.17
5HIAA	Group IV	66.97± 0.79	222.12±2.13**	67.01±0.96	63.91± 1.75	67.72± 1.88	70.82±0.77**	67.24±1.03
HVA	Group IV	228.26±1.29	29.04± 1.84**	222.58 ±1.78	211.9 ±1.24	226.47±1.49	118.25±0.70**	224.51±1.7
AA	Group IV	25.33±0.36	9.89±1.06**	22.90±0.69	24.76±0.68	24.09±0.59	24.74±0.31**	23.7±0.85

Data was expressed as Mean±SEM ,**significance at P<0.01,*significance at P<0.05 compared with group I followed by tukey test

The urinary levels of VMA, 5HIAA, HVA and AA with 100mg/kg b.w. of standard Bacopa monnieri were found to be within 92.03±2.51 - 24.34±0.07. The percentage reduction in VMA and 5HIAA was 54.42% and 166.0%. The percentage increase in HVA and ascorbic acid was found to be 76.3% and 65.4% of 100mg/kg/b.w of Bacopa monnieri compared to stress control. The percentage increase in HVA and ascorbic acid were found to be 71.4% and 66.1% with 100mg and 300mg/kg b.w. of Punica granatum compared to stress control (figure 1).

Figure 1: The percentage level of VMA, 5-HIAA, HVA and AA in stress and stress-extract in 24 h rat urine

The number of entries in safer zones obtained by each rat were counted and percent mean entries dataare given in figure 2. The normal (basal) values of number entries into safer zone and danger zone were expressed in percentage, the percentage of entries into safer zone were seen to be 59% and in danger zone were 41% respectively. In the stress induced animals the percentage entry of rats into safer zone was 33.3%, the percentage entry into safer zone was found to be decreased as to compared basal values, in danger zone were 66.8%, the percentage entry in danger zone was found to be increased as compared to normal values this indicates that the stress induced loss of memory. The aqueous extracts of Bacopa monniere (standard) and Punica granatum were administered to rats (groups II-IV) and group I treated with normal saline. The percentage entries into safer zone (58.5%) and danger zone (41.5%) were found to similar to normal, it indicates that the extracts were does not show the effect on normal condition of rats. The aqueous extract of Bacopa monniere was shown 50% of positive entries and 50% of negative entries but the positive entries relatively increased as compared to stress group. The aqueous extract of Punica granatum at 100 and 300 mg/kg body weight were shown 35% and 50% of positive entries respectively which indicates that, the effect was shown dose dependently.

Figure 2: Nootropic effect of Punica granatum on spontaneous alteration behavior and locomotor in induced memory impairment in a Y-maze test for percentage mean entry of rat in safe zone (+ve)

DISCUSSION:

The selected plant, Punica granatum has been used in folkcore medicine for centuries, the basic phenols of pomegranate fruit are gallic acid, chlorogenic acid, caffeic acid, coumaric acid and catechin. The literature suggests polyphenols; flavonoids and tannins are involved in antioxidant property.^30-34Stress induces free radicals, and produces biochemical modifications in all tissues, leading to different diseases and disorders. The IC₅₀ values of superoxide, hydroxyl radical, nitric oxide scavenging effect of Punica granatum were comparable to those of ascorbic acid. The hydroxyl radicals scavenging activity is measured as IC₅₀ of ascorbic acid and aqueous extract of Punica granatum for inhibition of hydroxyl radicals generated in fenton reaction mixture by studying the competition between deoxyribose and the extract for hydrogen radicals generated from Fe³⁺/ascorbate/EDTA/H₂O₂ systems.³⁴ The hydroxyl radicals attack deoxyribose which eventually results in thiobarbituric acid reactive substances (TBARS), from present study it was seen that the extract of aqueous extract of Punica granatum shows potential hydroxyl radical scavenging activity in comparison to ascorbic acid. The inhibition could be caused by absence of ferryl-perferryl complex or scavenging the hydroxyl radical or the superoxide radical or changing the Fe³⁺/Fe²⁺ or by chelating the iron itself.³²The acute toxicity studies for the selected plant extract in mice showed that there was no sign of toxicity upto 2000mg/kg body weight, orally. Stress alters the normal functioning of the body, in spatial contrivance, when an animal forced to swim, becomes immobile after an initial period of vigorous activity. Stress response is a complex system with involvement of HPA axis, which affected both peripheral and central components of the HPA axis. The metabolites of noradrenaline, adrenaline, dopamine and 5-HT are used to estimate as biomarkers which are altered in stress. Similarly 24h urinary level of ascorbic acid was used as another parameter, since it is involved in the synthesis of catecholamines and as endogenous substance for scavenging free radicals. The treatment with the selected plant extracts did not alter the normal levels of VMA, 5HIAA, HVA and ascorbic acid. The 24h urinary levels of VMA and 5HIAA were increased and HVA and ascorbic acid were decreased in rats subjected to forced swim stress. On withdrawal of stress on 4^th day the above changes were reverted back to near normal level. Prolonged stress as a result of the mental/emotional upset or due to physical factors such as malnutrition, surgery, chemical exposures, excessive exercise, sleep deprivation and environmental factors, that results in systemic effects.³⁵ The various screening models of stress includes shaker stress, immobilization stress, travel stress, noise and light stress and foot shock, 1-methyl, 4-phenyl, 1,2,3,6-tetrahydropyridine induced oxidative stress, Electric shock and restraint stress in rats, acute stress.^36-38The changes in urinary levels of the metabolites of catecholamines VMA, 5HIAA and HVA can be taken as an index of induction of stress.^39,40 In the present study, stress was applied through fresh water swimming which represents both physical stress and psychological stress, since the animal cannot escape from the swimming representing central activity, The VMA, 5HIAA and HVA were used as stress index. The change in the stress hormone level may be due to actual stress induction or due to sample collection or both. But in the presence study used noninvasive technique was adopted which avoid additional stress during collection sample and change in biochemical parameters where due to forced swim induced stress only, hence more advantageous than other methods. Evidence supporting a role for noradrenaline and serotonin in stress was change in extracellular levels of in different brain areas, including hypothalamus, amygdala, frontal cortex and raphe nuclei after exposure to several stressors.^41-42The treatment with aqueous extract of Punica granatum shown to decrease the elevated levels of VMA during stress indicating that, the decreased synthesis of noradrenaline. Treatment with aqueous extract of Punica granatum shown to decrease elevated levels of 5-HIAA, indicating that the extract had decreased synthesis of serotonin. Treatment with aqueous extract of Punica granatum shown to normalize the altered levels of HVA as compared to stress control; it might be due to the inhibition of synthesis of noradrenaline from dopamine. The antistress effect could be responsible, the presence of water soluble compounds (polyphenolics) in selected plant extracts that possess antioxidant activity as mentioned earlier, suggested that oxidative changes lead to stress, which in turn involves oxidative changes in the release of catecholamines. The potency of selected plant extracts in abolishing stress induced changes correlated well with their free radical scavenging (superoxide, hydroxyl, and nitric oxide) and inhibition of lipid peroxidation seen in in-vitro studies indicating that the in-vivo method was dependable to project the in-vitro antioxidant activity. Since the aqueous extracts of Punica granatum was at 100mg and 300mg/kg body weight did not change the levels of metabolites in normal conditions and blocked only stress induced changes, they appear to be good adaptogenic agents in comparatively Bacopa monniere (100mg/kg body weight). Agents like tacrine, physostigmine are evaluated to improve acetylcholine activity, which is one of the neuronal mediators, concerned with memory. The reduced level of ascorbic acid in stress induced animals was found to be raised to normal with treatment of aqueous extract of Punica granatum indicates the selected plant extracts might be inhibiting utilization of ascorbic acid in the synthesis of noradrenaline and reduces the generation of free radicals. Since the aqueous extracts of Punica granatum did not alter the levels of metabolite in normal conditions and ceased an only stress induced change that shows, they are good antistress agents. The memory loss associated with increased oxidative stress during stress and its reversal by antioxidant. The herbal drug Bacopa monniere is known to produce nootropic activity. Y-maze spontaneous alternation is a behavioral test for measuring the willingness of rodents to explore new environments. The number of entries of rat into safer zone was used as indicator of spatial working memory. The number of entries of the animal into safer zone with groups I, II, III and IV were found to be variable. The percentage of entry of animal into safer zone, in stress induced rats was found to be significantly reduced as compared to normal. The results indicated that stress created confusion in the animals. The results are in associated with stress reduces memory. The ability of animal to identify the safer zone involves recall of the task and may implicate memory. The stress significantly decrease the percentage of entry of animal into safer zone when compared with the control, while Bacopa monniere (100mg/kg body weight) shown reduced effect of stress, while the Punica granatum were shown the considerable increase in the percentage of entry of animal into safer zone as compared to stress induced group respectively. The study also indicates the percentage of entry of animal into safer zone without stress but, treated with extract was does not shown any considerable difference in the percentage entry of animal into safer zone as compared to control. This indicates the effect of extracts on memory which altered in stress. Stressful condition can leads to anxiety and depression, which further leads to excessive production free radicals. Application of stressincreased the VMA and 5HIlevels and decreased HVA and ascorbic acid levels, which returned back to normal level on cessation of the stress. Studies indicated that acute stress deflates memory. Hence, the Punica granatum extracts, being good antistress agent is considered to possess nootropic activity. Nootropic effect of Punica granatum extracts might be due to effect on dopamine, norephinephrine and serotonin synthesis in acute stress induced memory loss. Thus the study revealed the antioxidant and antistress properties of Punica granatum extract was might be responsible and correlated for their nootropic effect. Finally, we reported that urinary biomarkers levels were decreased and memory loss is absent in the stress exposed to aqueous extract of Punica granatum, suggesting that itpossess dose dependent antistress and nootropic activity.

CONCLUSIONS:

Urinary level of VMA, 5HIAA, HVA and ascorbic acid could be used as reliable indices to indicate stress induced changes and antistress activity of substances. This study provided evidence for correlation of antioxidant, antistress and nootropic activity of Punica granatum aqueous extract. Treatment with Punica granatum increased the movements of rats into safe zones indicating that extract improved nootropic activity. Antioxidant and antistress effect provides the mechanistic basis in relieving stress and memory by way of combating stress in both the models.

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENTS:

The authors are very thankful to Rajiv Memorial Education Society's College of Pharmacy Gulbarga, Karnataka for providing facilities to carry out the research work.The authors gratefully acknowledge M/s Laila Implex, Vijayawada and Kisalaya Herbals Ltd. Ratlam Koti, Indore, India for supported by providing gift samples of aqueous extracts of Punica granatum.

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Received on 04.08.2022 Modified on 06.10.2022

Research J. Pharm. and Tech 2023; 16(10):4751-4758.

DOI: 10.52711/0974-360X.2023.00771

Extracts/AA (µg)	Superoxide radical scavenging activity		Hydroxyl radical scavenging activity
Extracts/AA (µg)	*Ascorbic acid (AA)*	*Punica granatum*	Ascorbic acid (AA)	*Punica granatum*
50	40.95±0.6	30.3±0.14	36.3±0.25	32.9±6.05
100	48.4±0.4	38.3±1.00	45.1±0.60	39.9±0.10
150	54.6±2.65	46.8±0.25	54±0.40	45.2±.10
200	62.7±4.25	54.9±0.45	60.3±0.10	53±1.2
300	74.4±1.15	66.4±0.00	66.6±0.25	57±.60
IC₅₀(μg ml^–1)	114	179.2	140.7	266.2