INTRODUCTION:

According to the World Health report¹ approximately 450 million people suffer from mental or behavioral disorders, and only small minorities of them receive even the most basic treatment. In the search for new therapeutic products for the treatment of neurological disorders, medicinal plant research, worldwide, has progressed constantly, demonstrating the pharmacological effectiveness of different plant species in a variety of animal models².

Embelia ribes Burm f. (Myrsinaceae), commonly known as Vidanga, is a large woody climbing shrub and is sparsely distributed in the moist deciduous forests of India, Sri Lanka, Malaysia and South China³. It is highly esteemed in Ayurveda as a powerful anthelmintic⁴. The whole plant is used in the treatment of inflammation and in rheumatism and fever⁵. The fruits of Embelia ribes are used in folk medicine for treating mental disorders, nervous debility, psychopathy and as a brain tonic^{6, 7}.

Fruits contain a quinine derivative embelin, an alkaloid christembine⁸, volatile oil vilangin, flavonoids, tannin and saponins⁹.

ER is reported to possess anti-spermatogenic¹⁰, antidiabetic¹¹, antidyslipidemic¹², antioxidant, cardioprotective activities¹³. It also possesses neuroprotective¹⁴, antioxidant activity in rat brain¹⁵ and in-vitro acetyl-cholinesterase enzyme inhibitory activity¹⁶.

Hence, taking into consideration the traditional claims and reported pharmacological activities of Embelia ribes, the need was felt to assess the therapeutic efficacy of this plant as a useful candidate in treating anxiety.

MATERIALS AND METHODS:

Plant material and extract preparation:

The dried fruits of Embelia ribes were purchased from local market, Pune, India, in the month of February 2009. Plant material was authenticated by Dr. Rajesh Dabur, Director, Regional Research Institute, Pune, India, where a sample specimen has been deposited (Voucher number: 1338).

The dried and coarsely powdered fruits of Embelia ribes were subjected to maceration using methanol as solvent. The filtrate was evaporated and brown mass residue obtained was stored at 4 C for further use. The yield obtained was 5.14 % w/w. The methanolic extract was used throughout the study.

Animals:

Swiss Albino mice weighing 20-30 g were used in the present study. The above animals of either sex were purchased from National Toxicology Center, Pune. The animals were housed under standard laboratory conditions of temperature (25 ± 2°C) and humidity 54% was maintained and 12/12 hr light/dark cycle. Animals had free access to standard pellet diet (Amrut laboratory animal feed, Sangli-Maharashtra) and water ad libitum. The protocol of the study was approved by the Institutional Animal Ethics Committee (IAEC). The care of animals was taken as per the guidelines of CPCSEA, Department of Animal Welfare, and Government of India. 1% v/v tween 80 was used as vehicle. Doses of ER were prepared by suspending in the vehicle in such concentration as to administer these to mice in a volume ranging 30-300 mg/kg per oral route. Diazepam (DIA) was used as standard drug.

Acute toxicity study:

Acute toxicity study was carried out according to OECD guideline AOT 425. Mice were fasted overnight prior to drug administration. Each animal received a single dose of ER (2000 mg/kg, p.o.). The animals were observed for mortality for 24 hrs. ER was found safe upto 2000 mg/kg.

Anti-anxiety effect:

Elevated plus-maze test:

The apparatus consisted of two open arms (25 cm X 5 cm), facing each other, and two closed arms (25 cm X 5 cm X 15 cm) with an open roof and walls having 40 cm in height. The number of entries into open arm and total time spent in open arm (sec) was recorded by an independent observer during a 5 min test period after the mice had been placed in the centre of the maze.

Hole board test:

Mice were individually placed in the centre of a perforated board and the numbers of head-dips were registered during a 3 min trial. The hole board test was made by using a wooden floorboard; (40 cm X 40 cm X 25 cm) with 16 holes equally spaced in the floor. The holes provide a measure of the number of head-dips.

Mirrored chamber apparatus:

The mirrored chamber apparatus consisted of a mirrored cube (30 cm) open on one side that was placed inside a square wooden box (40 x 40 x 30.5 cm). The mirrored cube was constructed of five pieces of mirrored glass. The mirrors used were mirrored on one surface only (back surface of it was painted dark brown). The three mirrored side panes, a top pane, and the floor pane faced the interior of the cube. The mirrored cube was placed in the centre of the wooden container to form a 5 cm corridor that completely surrounded the mirrored chamber. A mirrored was also placed on the container wall so that it faced the single open side of the mirrored chamber. The other three walls of the container were painted dark brown. Sixty min after administration of the test drug or standard, the mice were placed individually in the chamber of mirrors at a fixed corner. During 5-min test period, the following parameters were noted: (a) latency to enter the chamber (sec), i.e., the time spent in seconds for the first entry into the chamber of mirrors and (b) no. of entries and (c) total time spent in the chamber (sec) during 5 min. period.

Estimation of GABA concentration in the mice brain:

The brain concentration of GABA was measured, in mice, 60 min after treatment with 1% v/v tween 80, diazepam and ER; by fluorimetric method¹⁷.

Statistical analysis:

The results were expressed as mean ± SEM and statistically analyzed by one-way analysis of variance (ANOVA) followed by Dunnet’s test, with level of significance set at P<0.05.

RESULTS:

Elevated plus-maze test:

ER (100 and 300 mg/kg, p.o.) significantly increased (P<0.01) the number of entries in open arm and time spent in open arm. Diazepam (2 mg/kg, p.o.) significantly increased (P<0.01) the number of entries in open arm and time spent in open arm, indicating anxiolytic activity.

Fig. 1. Effect of ER on number of entries into open arm

Values are presented as mean ± SEM, (n=5). **P<0.01 compared to control group (ANOVA followed by Dunnet’s test).

Fig. 2. Effect of ER on time spent in open arm

Values are presented as mean ± SEM, (n=5). **P<0.01 compared to control group (ANOVA followed by Dunnet’s test).

Hole board test:

ER (100 and 300 mg/kg, p.o.) significantly increased (P<0.01) the number of head dips. Diazepam (2 mg/kg, p.o.) treated group showed significant increase (P<0.01) in the exploratory activity, indicating anxiolytic activity.

Fig. 3. Effect of ER on number of head dips

Values are presented as mean ± SEM, (n=5). **P<0.01 compared to control group (ANOVA followed by Dunnet’s test).

Mirrored chamber apparatus:

ER showed significant and dose dependent increase in the number of entries and time spent in mirrored chamber. There was significant and dose dependent decrease in the latency to enter the mirrored chamber when compared with control group. Diazepam (2 mg/kg, p.o.) treated group also showed significant increase (P<0.01) in the number of entries and time spent in mirrored chamber, along with significant decrease (P<0.01) in the latency to enter the mirrored chamber when compared with control group, indicating anxiolytic activity.

Table 1. Effect of ER on anxiety induced in mice using mirrored chamber apparatus

Groups	Latency to enter the chamber (sec)	No. of entries	Time spent in chamber (sec)
NC	186.00 ± 11.54	1.40 ± 0.24	10.00 ± 1.41
DIA	24.00 ± 4.30**	17.00 ± 1.87**	144.80 ± 7.59**
ER 30	145.00 ± 9.21*	3.60 ± 0.40*	31.40 ± 2.80*
ER 100	116.00 ± 9.13 **	5.00 ± 0.70*	65.80 ± 4.48**
ER 300	81.60 ± 5.63**	8.00 ± 0.44**	78.40 ± 4.64**

Values are presented as mean ± SEM, (n=5). *P<0.05, **P<0.01 compared to control group (ANOVA followed by Dunnet’s test).

Estimation of GABA concentration in the mice brain:

The brain level of GABA was significantly (P<0.01) increased by diazepam. Treatment with ER also significantly (P<0.05) increased the brain GABA level.

Table 2. Effect of ER on the brain level of GABA in mice

Groups	Treatment	GABA (µg/g)
1	1% v/v tween 80 (10 ml/kg, p.o.)	15.23 ± 1.35
2	DIA (2 mg/kg, p.o.)	34.20 ± 3.65**
3	ER 300 (300 mg/kg, p.o.)	29.33 ± 1.78*

Values are presented as mean ± SEM, (n=3). *P<0.05, **P<0.01 compared to control group (ANOVA followed by Dunnet’s test).

DISCUSSION:

The results obtained in the present investigation indicate that ER possesses significant anxiolytic activity. ER significantly increased the number of entries in open arm and time spent in open arm in the EPM. Diazepam also significantly increased the above parameters, when compared with NC, indicating anxiolytic activity. As EPM model is useful for modeling anxiety, and it has been developed for predicting the potency of clinically used compounds for treating this disease¹⁸. Exposure of the animals to novel maze alley evokes an approach-avoidance conflict which is stronger in open arm as compared to enclosed arm. Rodents have an aversion for high and open space and prefer enclosed arm and, therefore, spend greater amount of time in enclosed arm. When animal enters the open arm, they freeze, become immobile and show fear-like movements¹⁹.

The anxiolytic effect was also evidenced in the hole board test and mirrored chamber apparatus. In the hole board test, ER showed significant and dose dependent increase in number of head dips. In general, the high levels of head-dipping are interpreted as indicative of neophilia, while low levels are assumed to result from a lack of neophilia or are assumed to reflect a high anxiety like state in the animal²⁰. In the mirrored chamber apparatus, ER showed significant and dose dependent increase in the number of entries and time spent in mirrored chamber, along with significant decrease in the latency to enter the mirrored chamber. Many animal species exhibit approach-avoidance response upon placement of a mirror within their environment. In order to identify new behavioral measures with qualitatively different responses, a mirrored-chamber apparatus was developed for which mice show an extended latency to enter. Novel stimulation evokes both exploration and anxiety, and thereby generates an approach-avoidance conflict behavior. The response to an apparent animal reflected in the mirror might also be a source of anxiety¹⁹.

Benzodiazepines (BZD), widely used for the treatment of anxiety disorders, produce pharmacological actions via specific high affinity binding sites on a supramolecular complex composed of GABA-A and a BZD receptor coupled with a chloride ion channel. At the cellular level, stimulation of the GABA-A receptor results in an increased chloride conductance and usually a concomitant hyperpolarization. In intact animals, activation of GABA-A receptor is known to be associated with anti-anxiety action¹⁹. ER showed significant increase in the brain GABA level.

Phytochemical tests of ER revealed the presence of tannins, flavonoids, alkaloids, saponins. Flavonoids, as a class of naturally occurring compounds found in most vascular plants, have been demonstrated by a number of groups to be centrally active, possessing efficacies for a number of receptor systems in the CNS^{2, 21}. The anxiolytic activity of ER may be due to the presence of flavonoids. Finally, the result of this study shows that the ER possesses anxiolytic activity, possibly due to its ability to increase the brain GABA levels in the experimental animals.

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Received on 15.04.2010 Modified on 13.05.2010

Research J. Pharm. and Tech.3 (4): Oct.-Dec.2010; Page 1136-1139