Evaluation of Punica granatum fruit juice for anti-anxiety activity

Background and Objectives: In traditional system of Indian medicine, Punica granatum L. fruits are widely used for treatment of brain diseases, fever, heart diseases, diarrhea, dysentery, piles, inflammation and bronchitis. However, there is no authentic scientific data reported regarding anxiolytic activity of Punica granatum (Linn) fruit juice. To evaluate the anxiolytic activities of Punica granatum L. fruit juice in various validated animal models of anxiety in mice. Methods: The preliminary phytochemical investigation was carried out with the fruit juice of Punica granatum for qualitative identification of phytoconstituents. Animals were administered with orally 0.1, 0.2, 0.4 and 0.8 ml of Punica granatum fruit juice and observed for its mortality upto 48 hours study period (short term toxicity) to determine LD50. From the LD50 dose, 1/20, 1/10 & 1/5 doses were selected and considered as low, medium and high dose respectively. For assessing the anxiolytic activity, models like Elevated Plus Maze, Hole-Board were used Diazepam was used as a standard reference for anxiolytic. Results: Preliminary phytochemical investigation of the PGFJ (Punica granatum L. fruit juice) revealed the presence of flavonoids, saponins, tannins, sterols, polyphenols, alkaloids, carbohydrates and proteins. LD50 (acute oral toxicity) of Punica granatum fruit juice was determined in mice with a dose limit of 2000 mg/kg as per OECD guidelines no.425 and even upto 2000 mg/kg dose no mortality was recorded. Hence the experimental doses selected were 1/20, 1/10 and 1/5 of the LD50 value and considered as low, medium and high doses of Punica granatum fruit juice 100, 200 and 400 mg/kg respectively. In Elevated Plus Maze model, low, medium and high doses PGFJ and Diazepam (2 mg/kg) had significantly increased number of entries, time spent in open arms and decreased the number of entries and time spent in closed arms. In Holeboard model, Diazepam (2 mg/kg), medium and high doses (200 and 400mg/kg) but not the low dose (100mg/kg) of PGFJ had significantly increased the number of head dips, latency of first head dip and number of rearings. Conclusion: The present study ascertains that the plant Punica granatum L. fruit juice possesses significant antianxiety activity in mice and study concludes the beneficial effects of Punica granatum fruit juice in treatment of anxiety signifies the rational basis for its traditional use.


Introduction
In recent years, anxiolytic drugs have been among the front-runners in terms of the number of prescription written in medical practice. This may be due to the tense life style imposed on man by the competitive atmosphere. Some degree of anxiety is a part of normal life, but treatment needed when it is disproportionate to the situation and excessive. [1] Anxiety disorders may be due to life experiences, heredity and brain chemistry. Chemical messenger's (neurotransmitters) in the brain regulate thought and feeling. Sometimes has a problem with brain messages sent out because of a chemical imbalance. Approximately 4-6% of the population suffers from anxiety so severely, that it disrupts routine life functions. [2] In today's world of stressful life style with the individuals, anxiety is one of the common symptoms associated, affecting almost 1/8 th of the total population worldwide and has became a very important area of research interest in psychopharmacology during this decade and is also obvious component of many psychiatric and medical conditions. [3] Currently, the most widely prescribed medications for anxiety disorders are the Benzodiazepines. However, the clinical uses of Benzodiazepines are limited by their side effects such as psychomotor impairment, potentiation of other central depressant drugs and dependence liability. Therefore, the development of new herbal medications possessing anxiolytic effect without the complication of BZD would be of great importance in the treatment of anxiety-related disorders. In recent years, interest is growing in alternative medicines that include plant derived medications, polyherbal formulations that affect the mind. During last few years there has been increase in usage of alternative medicines by the patients for IJBAR (2016) 07 (09) www.ssjournals.com such ailments, Therefore many herbal medicines have been accepted in our country for treating anxiety disorders as Bacopa monniera (Brahmi) [4] and Withania somnifera (Ashwagandha). [5] Punica granatum is one such medicinal plant that has been extensively used in the traditional system of medicine. In ayurveda, this plant is used to treat brain disorders, heart disease, anthelmentic, diarrhea, dysentery, piles, colic, inflammation, aphrodisiac diuretic and bronchitis. [6] Ingesting healthy Punica granatum fresh fruit juice has also been known to alter anxiety, insomnia and improves circulation of the heart.[7] No major investigation reports were found pertaining to its CNS activity. Hence, the present study is proposed to evaluate the anxiolytic activity of Punica granatum fruit juice by using different validated animal models for anxiety based on exploratory behaviour in mice.

Plant Material
Fresh fruits of the plant Punica granatum were collected around the fields of Agricultural University, Raichur and authenticated by Prof. Vedavyas, Department of Botany, L.V.D College Raichur.

Preparation of fruit juice of Punica granatum:
The freshly collected fruit was taken and peel was removed then, arils/seeds were taken from the fruit then squeezed in muslin cloth. The obtained juice was collected and filtered in whatmann filter paper. The juice was kept in refrigerator at 0-8 o C until used.

Preliminary phytochemical screening
The preliminary phytochemical investigation was carried out with the fruit juice of Punica granatum for qualitative identification of phytoconstituents and to detect carbohydrates, proteins, amino acids, flavonoids, saponins, alkaloids, glycosides, phytosterols, tannins and phenolics.

Experimental animals:
Swiss albino mice of either sex weighing between 20-25g were procured from Shri Venkateshwara Enterprises, Bangalore for experimental purpose. The animals were fasted overnight and during the experiment. All experiments were carried out during the light period. The approval of the Institutional Animal Ethical Committee (IAEC) of N.E.T Pharmacy College, Raichur (Karnataka) was taken prior to the experimentation. All the protocols and the experiments were conducted in strict compliance according to ethical principles and guidelines provided by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), with registration number 576/02/bc/CPCSEA.

Acute toxicity studies:
The oral acute toxicity of Punica granatum was determined by using albino mice of either sex (20-25 g), maintained under standard conditions. The animals were fasted for 3h prior to the experiment. Animals were administered with single dose of Punica granatum fruit juice (PGFJ) and observed for its mortality up to 48 h study period (short term toxicity). Based on the short-term toxicity profile, the next dose was determined as per OECD guidelines No 425. From the LD 50 dose 1/20, 1/10 and 1/5 th doses were selected and considered as low, medium and high dose respectively. The PGFJ was administered orally at the doses of 50, 100, 200, 400, 800, 1000 and 2000 mg/kg to different groups of mice. No mortality was observed for 48hr study period. PGFJ was found to be safe at the given doses.

Experimental design:
In the investigation, Albino mice of either sex weighing between 20-25g were used for the study. The animals were divided into five groups, each group containing six mice. ` The Elevated plus-maze apparatus comprises of two open arms (16×5cm) and two closed arms (16×5×12cm) that extend from a common central platform (5×5cm). The entire maze is elevated to a height of 25cms above the floor level. During a five minutes test period, the following parameters were recorded using Sony handy camera: The apparatus used in this model consists of wooden chamber (40x40x25 cm) with 16 holes (diameter 3 cm) on the floor, elevated from the ground so that the rats could peep through the holes. During a five minutes test period the following parameters were recorded using Sony handy camera.  Latency to the first head dips  The number of head dips through the holes  Number of rearings

Statistical Analysis
The data were expressed as mean ± S.E.M from 6 animals [n=6]. The results were subjected to statistical analysis by using Unpaired t-test to calculate the significance difference if any among the groups. P<0.05 was considered as statistical significance using Graph Pad Prism Software.

Hole Board Model:
Each mice was placed individually in the Hole board apparatus and the parameters like the number of head dips, duration of head dips, latency to first head dips and no of rearings were recorded.
PGFJ at the dose of 100 mg/kg p.o. produced no significant increase in number of head dips and no of rearings but, significant increase (P<0.05) in the latency to first head dips were noted when compared with control group. PGFJ at the dose of 200 mg/kg p.o had significantly (P<0.01 and P <0.001) increased the number of head dips, number of rearings and latency to first head dips when compared with control group. PGFJ at the doses of 400 mg/kg p.o had significantly (P<0.001 and P<0.01) increased latency to first head dips, number of rearings and the number of head dips when compared with control group.
The reference standard Diazepam (2mg/kg) p.o treated group showed highly significant (P<0.001) increase in the number of head dips, duration of head dips, latency to first head dips and no of rearings as compared with control group. .31*** n = 6 in each group. Values are expressed as mean±SEM. Statistical analysis was carried out by using Unpaired 't' test. *P<0.05, **P<0.01 and ***P<0.001 statistically significant as compared to control group.

Discussion
The significance of anxiety in health and disease is well recognised, its underlying neurobiological mechanisms are not well understood. [2] Anxiety disorders may be due to life experiences, heredity and brain chemistry.
Benzodiazepines, the widely prescribed and clinically effective anxiolytics, are thought to produce their pharmacological actions via specific high affinity binding sites on a supramolecular complex composed of GABA-A and BZD receptor coupled with a chloride ion channel. At the cellular level, stimulation of GABA-A receptor results in an IJBAR (2016) 07 (09) www.ssjournals.com increased chloride conductance and usually hyperpolarisation. In intact animals activation of this receptor is known to be associated with antianxiety and anticonvulsant actions. [2] Animal behavioural models of anxiety have played an important role in the assessment of anxiolytics. Choice of the animal models for anxiety studies and to search for new classes of anxiolytic compounds with potential therapeutic applications is an important parameter in drug development research. [2] Punica granatum is a medicinal plant that has been extensively used in the traditional system of medicine. In ayurveda, this plant is used to treat brain disorders, heart disease, diarrhea, dysentery, piles, colic, inflammation and bronchitis. [6] There are few reports available for its scientific evaluation of the pharmacological effects on CNS activity. [9] The present work was aimed to demonstrate the anxiolytic effects of Punca granatum L. fruit juice in mice in several behavioural models like EPM, Hole-board, Open field and Light-Dark paradigms.
EPM is one of the most popular animal test for research on behavioral pharmacology of anxiety. In EPM, naive mice will normally prefer to spend much of their allotted time in the closed arm. This preference appears to reflect an aversion towards open arms that is generated by fear of open spaces. Drugs that increase open arm exploration are considered as anxiolytics. [10] In our study, we observed that PGFJ (100, 200 and 400 mg/kg) and Diazepam (2 mg/kg) induced significant increase in both the number of entries and time spent in the open arms. The number of entries and time spent in the closed arms were reduced in the PGFJ treated groups as compared to the control group. These observations clearly show statistically significant anxiolytic activity in EPM model in mice.
Hole-board model indicates that head-dipping behaviour is sensitive to changes in the emotional state of the animal and suggests, the expression of an anxiolytic state in animals may be reflected by significant increase in headdipping behaviour. The PGFJ medium and high doses (200 & 400mg/kg) shows increase in number, latency of head dipping and the number of rearing in the hole board test. But the effect of low dose (100mg/kg) was found to be nonsignificant when compared with control. Diazepam (2 mg/kg) had significantly increased the number, latency of head dipping and the number of rearing. Therefore the increased number of head dippings and rearings are representation of anxiolytic effect.
The present work demonstrated that the fruit juice of Punca granatum L. has anxiolytic effects in mice. This action of PGFJ represents the functional similarity to BZD's which are widely used as anxiolytic agents. The benzodiazepines are known to act through the BZD-GABA receptors. The role of GABA in anxiety is well established. The mechanism of anxiolytic action of PGFJ may involve an action on GABAergic transmission; [11] however, further studies are needed to ascertain this.
Earlier reports on the chemical constituents of plants and their pharmacology suggest that plant containing flavonoids, saponins and tannins possesses activity against many CNS disorders. [12] Phytochemical tests of PGFJ revealed the presence of flavonoids, saponins and tannins. It is possible that the mechanism of anxiolytic action of PGFJ could be due to the binding of any of these Phytochemicals to the GABA-A -BZD complex.