|Year : 2012 | Volume
| Issue : 4 | Page : 489-492
Evaluation of Anxiolytic effect of Erythrina mysorensis Gamb. in mice
Thandaga S Nagaraja1, Riaz Mahmood2, V Krishna2, BS Thippeswamy3, VP Veerapur3
1 Department of Pharmaceutics, SJM College of Pharmacy, Chitradurga, India
2 Department of Biotechnology and Bioinformatics, Kuvempu University, Shankarghatta, Shimoga, India
3 Department of Pharmacology, Siddaganga College of Pharmacy, Tumkur, India
|Date of Submission||10-Jun-2011|
|Date of Decision||12-Oct-2011|
|Date of Acceptance||30-Apr-2012|
|Date of Web Publication||3-Aug-2012|
Thandaga S Nagaraja
Department of Pharmaceutics, SJM College of Pharmacy, Chitradurga
Source of Support: None, Conflict of Interest: None
Aim and Objectives: To evaluate anxiolytic effect of stem bark ethanol and chloroform extracts of Erythrina mysorensis in mice.
Materials and Methods: The anxiolytic activity was examined by using the elevated plus maze (EPM) and open field test (OFT), and motor coordination by rotarod test (RRT). Twenty four Swiss albino male mice were divided into four groups of six mice each. Group 1 received vehicle (normal saline); group 2 received diazepam (1 mg/kg); groups 3 and 4 received ethanolic and chloroform extract of Erythrina mysorensis, 200 and 400 mg/kg p.o., respectively.
Results: Mice treated with diazepam (1 mg/kg, p.o.) showed significant ( P < 0.001) increase ini the percentage of open arms entries and time spent whereas, in closed arm the number of entries and time spent were significantly ( P < 0.05) decreased. Oral administration of chloroform and ethanol extract of E. mysorensis exhibited significant ( P < 0.05) increase in the number of open arm entries and time spent with significant ( P < 0.05) reduction in number of entries and time spent in the closed arm as compared to group 1. Chloroform and ethanol extracts treated mice also produced significant increase in the number of rearings ( P < 0.05), assisted rearings and number of squares crossed ( P < 0.01). Rotarod test showed significant ( P < 0.01) reduction in motor activity at 45 min with diazepam and E. mysorensis extracts (400 mg/kg) as compared to groups 3 and 1.
Conclusion: Erythrina mysorensis possess significant anxiolytic activity in the mice. It can be a promising anxiolytic agent.
Keywords: Elevated plus maze test, Erythrina mysorensis, open field test, Rotarod test
|How to cite this article:|
Nagaraja TS, Mahmood R, Krishna V, Thippeswamy B S, Veerapur V P. Evaluation of Anxiolytic effect of Erythrina mysorensis Gamb. in mice. Indian J Pharmacol 2012;44:489-92
|How to cite this URL:|
Nagaraja TS, Mahmood R, Krishna V, Thippeswamy B S, Veerapur V P. Evaluation of Anxiolytic effect of Erythrina mysorensis Gamb. in mice. Indian J Pharmacol [serial online] 2012 [cited 2022 Jun 25];44:489-92. Available from: https://www.ijp-online.com/text.asp?2012/44/4/489/99316
| » Introduction|| |
Benzodiazepines are the major class of compounds commonly prescribed for treating anxiety. However, their use is associated with side effects like psychomotor impairment, potentiating of other central depressant drugs and addiction liability.  A number of plants are being investigated in complementary alternative medicines for anxiety. , Research has been conducted in the search of an alternate, more specific and cost-effective therapy. 
Erythrina mysorensis belongs to family Leguminosae, is a small tree with few or no prickles, rarely distributed in the eastern Himalaya and deciduous forest of the Western Ghats. , Traditionally, plants belonging to genus Erythrina are used for biliousness, fever, rheumatism, itch, asthma, leprosy and epilepsy.  Total alkaloid fraction from bark of E. indica, E. variegata and E. americana seeds extract reportedly possesses central nervous system (CNS) depressant and anticonvulsant property and may be clinically useful as muscle relaxants and as sedatives. ,,, In view of these reports, the present study was taken to evaluate the anti-anxiety activity of stem bark of Erythrina mysorensis.
| » Material and Methods|| |
The stem bark of Erythrina mysorensis. Gamb was collected from the regions of Shimoga District, Karnataka, India, and authenticated by Prof. V. Krishna, Department of Biotechnology and Bioinformatics, Kuvempu University, Shankarghatta, Shimoga, India.
Preparation of Extracts
Coarse powder was extracted successively with petroleum ether 60-80, chloroform and alcohol in a Soxhlet apparatus, finally with chloroform water by maceration. All the extracts were concentrated in vacuum using rotary flash evaporator. They were further concentrated and dried in desiccators. The yield of ethanolic and chloroform extract of Erythrina mysorensis. Gamb was found to be 4.62% and 1.8% w/w, respectively. The extracts were stored in a refrigerator and used for the pharmacological investigations.
Drugs and Chemicals
Diazepam tablets (Calmpose 5 mg, Ranbaxy Laboratories Limited Navi-Mumbai, India B-No. 1700593) were used as a standard drug. Diazepam was suspended in 0.5% of carboxymethyl cellulose in distilled water. Accurately weighed quantity of ethanolic and chloroform extracts of E. mysorensis separately suspended in distilled water using 1% (w/v) of gum acacia as suspending agent and administered to experimental animals.
Swiss albino mice (22-25 g) bred in animal house of Sree Siddaganga College of Pharmacy, Tumkur, Karnataka was used. The animals were housed under standard laboratory conditions and had free access to food and water. The experimental protocols were approved by the Institutional Animal Ethics Committee (SSCPT/IAEC/88/2010-11, date 27/07/2010) and conducted according to CPCSEA guidelines, Govt. of India.
Acute Oral Toxicity Studies
Acute oral toxicity test was performed according to Organization for Economic Co-operation and Development (OECD) guideline test, ANNEX-423. Ethanolic and chloroform extract of E. mysorensis at 5, 50, 150, 300, 500, 1000 and 2000 mg/kg b.w. were administered orally to separate set of three female mice for each dose. The animals were observed for signs of toxicity such as hyperactivity, grooming, convulsions, sedation, and hypothermia continuously for 2 h, and for mortality up to 24 h, after administration of the doses.
The anxiolytic activity was examined by using the elevated plus maze (EPM) and open field test (OFT) and rotarod test for motor coordination assessment. A total of 24 male mice were randomly divided into four groups of 6 each and treated as follows: Group 1 received vehicle (normal saline); group 2 received diazepam (1 mg/kg, p.o.); groups 3 and 4 received ethanolic and chloroform extract of Erythrina mysorensis extract (200 and 400 mg/kg, p.o., respectively).
Elevated Plus-Maze Test
After 1 h of oral administration of vehicle, diazepam and ethanolic/chloroform extract of E. mysorensis animals were assessed for anxiolytic behavior using elevated plus-maze test. The elevated plus-maze apparatus consists of two opposite open arms (20 cm × 7 cm), crossed with two closed arms of same dimensions with 30 cm high wall. The arms are connected with central square (7 cm × 7 cm). The walls and floor, of the apparatus colored with black paint and the apparatus was kept in sound proof and 200 lux light illuminated room. To assess plus maze test, the mouse was individually placed on the central platform facing towards open arm. The percentage of time spent (duration) in open arms and frequency of open arm entries were counted for a period of 5 min. All precaution was taken to ensure that no external stimuli, other than the height of plus maze could invoke anxiety in the animals. Arm entry was defined as all four paws having crossed the dividing line between an arm and the central area. The percentage of time spent in the open arms and number of open arm entries were calculated using the formulas [100 × open/ (open + enclosed)] and (100 × open / total entries), respectively. 
This test was used to detect angiogenic and anxiolytic activity under identical situations. Various types of Open field apparatus have been used to test the mice.  The apparatus consisted of a wooden box (60 × 60 × 60 cm). The arena of the open field was divided into 16 squares (15 × 15 cm), the four inner squares in the center and 12 squares in the periphery along the walls. The experimental room was dark and sound attenuated. The open field arena was illuminated with a 40-W lamp, focusing on the field from a height of about 75-100 cm. After 60 min of oral treatment with vehicle, diazepam and ethanolic or chloroform extract of E. mysorensis extract, animals were placed individually in one of the corner squares and number of rearings, assisted rearings and number of squares crossed were observed for the period of 5 min. 
Motor Co-Ordination Test by Rotarod
The effect on motor co-ordination test was assessed using rotarod apparatus rotating at a speed of 32 rpm. The apparatus consisted of a base platform, an iron rod of 3 cm in diameter and 30 cm long with non-slippery surface. The rod was divided into three equal sections by two disks, thus enabling three mice to walk on the rod at the same time and speed. Intervals between the mounting of the animal on the rod and falling off were recorded as the performance time. The training of mice on rotarod test was given 20 times at 5-15 min intervals. Thereafter, three mice were randomly selected to determine the retention of the walking technique. Animals that performed on the rod for more than 10 s were included for the study. After the administration of the standard or test drug, the performance time was measured at 15 min time intervals for 90 min,  for each dose with six trained mice.
| » Results|| |
Acute Oral Toxicity Studies
Acute toxicity studies revealed that ethanolic and chloroform extract of E. mysorensis was safe at a maximum oral dose of 2000 mg/kg b.w. in mice. No lethal or toxic reactions were observed in 24-h period. However, the animals showed slight sedation after 1 h of both extract administration. Based on this data, 1/10 th and 1/5 th of maximum safe oral dose (2000 mg) i.e. 200 mg/kg and 400 mg/kg, respectively, were selected for further study.
Elevated Plus-Maze Test
The vehicle-treated mice (10 ml/kg, p. o. normal saline) spent more time in closed arm and showed less entries in open arm compared to closed arm of the maze at 5 min. Animal treated with diazepam (1 mg/kg, p. o.) showed significant (P < 0.001) increase in the percentage of open arms entries as well as time spent in open arm whereas, in closed arm number of entries and time spent were significantly (P < 0.001) decreased. Oral administration of chloroform and ethanol extract of E. mysorensis (200 and 400 mg/kg, respectively) exhibited significant (P < 0.01) increase in the percentage of number of open arm entries and time spent in open arm whereas, in the closed arm number of entries and time spent was significantly (P < 0.01) reduced as compared to vehicle-treated group [Table 1].
|Table 1: Comparison of ethanolic and chloroform extract of Erythrina mysorensis on behavior of mice in elevated plus maze test (n = 6)|
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Open Field Test
In the open field test (OFT), diazepam-treated mice showed significant increase (P < 0.05) in the number of rearings, number of squares crossed and assisted rearings during 5-min interval of test as compared to vehicle-treated groups. Chloroform and ethanol extracts treated mice (200 and 400 mg/kg) also produced significant increase in the number of rearings (P < 0.05), assisted rearings and squares crossed (P < 0.01) [Table 2].
|Table 2: Comparison of ethanolic and chloroform extract of Erythrina mysorensis on behavior of mice in open field test (n = 6)|
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At 45-min interval, diazepam and E. mysorensis extract (400 mg/kg) significantly ( P < 0.01) reduced motor activity of mice whereas E. mysorensis extract (200 mg/kg) failed to produce effect on motor coordination. At 60-min time interval both the doses of E. mysorensis extract and diazepam significantly (P < 0.01) reduced motor activity of mice as compared to vehicle-treated group whereas, at 75 and 90 min time intervals, both the doses of E. mysorensis extracts induced significantly (P < 0.001) comparable results with diazepam [Table 3].
|Table 3: Comparison of ethanolic and chloroform extract of Erythrina mysorensis on rotarod performance in mice (n = 6)|
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| » Discussion|| |
Anxiety, like all emotions, has cognitive, neurobiological and behavioral components. It is a negative emotion that occurs in response to perceived threats that can come from internal or external sources and can be real or imagined.  The incidence of anxiety in the community is very high and associated with lot of morbidity.  Ethnomedical and pharmacological knowledge about the plant under study would allow us to evaluate central nervous system activity, which could be used to treat anxiety type of disorders. The present work has proved the ethno medical claim of this plant Erythrina mysorensis by exhibiting, chloroform and ethanol extracts anxiolytic effect in mice.
The EPMT is used to evaluate psychomotor performance and emotional aspects of rodents. Results obtained on the elevated plus maze after treatment with chloroform and ethanol extracts of E. mysorensis (200 and 400 mg/kg) revealed anxiolytic activity, since increases in open arm entry parameters are the most representative indices of anxiolytic activity.  Time spent on the central platform appears to be related to decision making and/or risk assessment, and the total arm entries is a measure reflecting changes in anxiety or in general activity. 
The OFT is used to evaluate the animal emotional state. The open field model examines anxiety-related behavior characterized by the normal aversion of the animal to an open, brightly lit area. Thus, animals removed from their acclimatized cage and placed in environment express anxiety and fear, by showing alteration in all or some parameters. Anxiolytic treatments reduce such fearful behavior of animals in open field.  Statistical analysis of the data obtained from these experiments supported anxiolytic-like activity of chloroform and ethanol extracts at both the doses (200 and 400 mg/kg) as its effect shows significant increase in the number of rearings, number of assisted rearings and number of squares crossed, as compared to the vehicle-treated group, which indicates its anxiolytic-like effect. RRT was first introduced to screen the assay of neurotoxicity of anticonvulsants and later was reported to predict motor dysfunction produced by centrally acting drugs to determine possible alterations in the motor coordination ability of the animal, often caused by the use of sedative and antipsychotic drugs. In this test, the difference in the fall of time from the rotating rod between the vehicle and extract treated groups is taken as an index of muscle relaxation. The skeletal muscle relaxation together with taming or calming effect also reduces anxiety and tension. Thus, in this study, both the doses of chloroform and ethanol extracts of E. mysorensis (200 and 400 mg/kg) and diazepam significantly reduced the fall of time of the mice from the rotating rod, indicating the skeletal muscle relaxant activity.
Earlier reports on the chemical constituents of plants and their pharmacology suggest that plants containing flavonoids, alkaloids, phenolic acids, essential oil, saponins and tannins possess activity against many CNS disorders.  Investigations on the phytochemical screening of Erythrina mysorensis stem bark extracts revealed the presence of alkaloids, glycosides, steroids, saponins, tannins, proteins, phenolic compounds and flavonoids. It is possible that the mechanism of anxiolytic action of E. mysorensis could be mediated by synergistic action of these phytochemicals. The results obtained in this study suggest that the E. mysorensis possesses anxiolytic and muscle relaxant properties. Thus, E. mysorensis has potential clinical applications in the management of anxiety and muscle tension disorders. Further investigations are warranted for elucidating the exact mechanism and bioactive compounds.
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[Table 1], [Table 2], [Table 3]
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