|Year : 2016 | Volume
| Issue : 2 | Page : 41-47
Study of anxiolytic effect of ethanolic extract of drumstick tree leaves on albino mice in a basic neuropharmacology laboratory of a postgraduate teaching institute
Ayon Bhattacharya1, Soumya Santra2, Soumyadeep Mahapatra2, Pratap Kumar Sahu3, Divya Agrawal4, Sanjay Kumar2
1 Department of Pharmacology, KPC Medical College and Hospital, WBUHS, Kolkata, West Bengal, India
2 Department of Pharmacology, IMS and SUM Hospital, SOA University, Odisha, India
3 Department of Pharmacology, SPS, SOA University, Bhubaneswar, Odisha, India
4 Department of Anatomy, Mahaveer Institute of Medical Sciences and Research, MPMSU, Jabalpur, Madhya Pradesh, India
|Date of Web Publication||17-Jun-2016|
Department of Pharmacology, KPC Medical College andHospital, IF, Raja Subodh Chandra Mullick Road, Jadavpur, Kolkata - 700 032, West Bengal
Source of Support: None, Conflict of Interest: None
Objectives: Anxiety is a serious condition, leading to depression. The statistics of anxiety is alarming and needs an effective, low-cost remedy. Drumstick tree is known as Moringa oleifera belonging to the Moringaceae family. In previous studies, drumstick leaves revealed a storehouse of active ingredients possessing anxiolytic property. In this study, the anxiolytic effect of drumstick leaves was studied using elevated plus maze (EPM) and staircase models at four doses 50, 100, 200, and 400 mg/kg. Materials and Methods: It is a randomized control study. Albino mice were used in this study, a total of 72, divided into six groups, each group consisting of 6 mice. Group I: Control (normal saline orally at 2 ml/kg body weight); Group II: Standard (diazepam 10 mg/kg orally); Group III, IV, V, and VI (ethanolic extract of Moringa oleifera [EMO] 50, 100, 200, and 400 mg/kg, respectively). EMO leaves were administered at 50, 100, 200, and 400 mg/kg doses orally 1 h before the experiments and their effects observed. Results: In the EPM method, the time spent in open arm and closed arm before and after drug administration was analyzed by one-way ANOVA followed by Bonferroni's test. In the staircase test, the comparison of steps climbed and rears before and after drug administration were done by nonparametric tests, Kruskal-Wallis test followed by Mann-Whitney's test. In both models, the extract showed a significant response at 200 and 400 mg/kg. Conclusion: Thus, EMO leaves exhibited anxiolytic activity in both models at higher doses.
Keywords: Anxiolytic, drumstick, elevated plus maze test, staircase test
|How to cite this article:|
Bhattacharya A, Santra S, Mahapatra S, Sahu PK, Agrawal D, Kumar S. Study of anxiolytic effect of ethanolic extract of drumstick tree leaves on albino mice in a basic neuropharmacology laboratory of a postgraduate teaching institute. J Health Res Rev 2016;3:41-7
|How to cite this URL:|
Bhattacharya A, Santra S, Mahapatra S, Sahu PK, Agrawal D, Kumar S. Study of anxiolytic effect of ethanolic extract of drumstick tree leaves on albino mice in a basic neuropharmacology laboratory of a postgraduate teaching institute. J Health Res Rev [serial online] 2016 [cited 2018 Apr 22];3:41-7. Available from: http://www.jhrr.org/text.asp?2016/3/2/41/184228
| Introduction|| |
Anxiety is a state of excessive fear accompanied by motor tension, apprehension, sympathetic over-activity and vigilance syndrome eventually causing impairment of memory, intelligence, and psychological function.  The spectrum of anxiety disorder consists of panic disorder, generalized anxiety disorder, posttraumatic stress disorder, phobias, and separation anxiety disorder.  In the world scenario, anxiety occupies one-eighth of the population.  The spectrum of anxiety diseases is estimated to be lateralized more to the female population compared to males.  5.7-28.8% of community adolescents are a victim to anxiety.  The dangerous triad of depression, anxiety, and drug use has a major impact on health of youth in India.  Recent data have shown that in India one in every four Indians that is about 25% of the population are affected by anxiety disorders and among them 10% suffer from depression. 
Modern day allopathic system is still in its infancy in developing an effective remedy for anxiety. Benzodiazepines, azapirones are the classes of drugs commonly used in anxiety. However, the major limitations to the use of these drugs are due to unfavorable kinetics, withdrawal symptoms on sudden stoppage of the drugs, cognitive, and psychomotor impairment and gastrointestinal symptoms. Moreover, as anxiety sometimes persists for a long time, it poses a financial burden to the patient. The various approaches in the treatment of anxiety have also confounded the modern day doctors as to inability in deciding which patient shall be benefitted to which therapy. Thus, this stream of never-ending problems has motivated us to look for safer alternatives. 
Drumstick tree is known as Moringa oleifera belonging to the Moringaceae family could be one such alternative to low-cost therapy.  It is soft wooded perennial tree. Moringa oleifera is known by many names such as the "miracle tree," "tree of life," "mother's best friend," "God's Gift to Man," and "Savior of the Poor." , The advantages of this "miracle tree" are that it is widely cultivated, every part of the plant is edible, multitude of medicinal uses and other uses, fast growing, and being drought resistant. , The leaves are bipinnate or more commonly tripinnate, slender about 45 cm long, and a storehouse of nutrients such as amino acids, potassium, Vitamin E, zinc, and natural antioxidants. ,
Phytochemical ingredients in leaves showed the presence of flavonoids, tannins, saponins, terpenoids, phenolic acid which possess antioxidant, and anxiolytic activity. 
The findings of phytochemical analysis were further fortified by GC-MS analysis of the ethanolic extract of Moringa oleifera leaves revealing 35 compounds with 15 major compounds.  L galactose 6 deoxy, n-hexadecanoic acid, tetradecanoic acid, beta-l-rhamnofuranoside 5-O-acetyl-thio-ocyl, Vitamin E, and gamma sitosterol are some of compounds having antioxidant activity isolated in GC-MS profiling which can contribute to the antianxiety effect.  The leaves also have a wide array of central nervous system (CNS) activities such as analgesic, anti-inflammatory, antipyretic, neuroprotective in Alzheimer's disease, and antidepressant action. , Thus, on the basis of these findings, the present study is undertaken to evaluate the antianxiety effect of drumstick leaves at four doses (50, 100, 200, and 400 mg/kg).
| Materials and methods|| |
The extract was obtained from Vidya herbs, 30-33 main, J.P. Nagar, 6 th Phase, Bengaluru - 560 078.
Diazepam (Calmpose, Ranbaxy Laboratories Ltd., Haryana, India) and other solvent chemicals used were of analytical grade.
Wistar strain of albino mice of either sex (20-30 g) was randomly selected from the central animal house. The animals were kept at a temperature of 22 ± 1°C, with 12 h light and dark cycle. Food and water was given ad libitum. Animals were trained and acclimatized to laboratory conditions for 7 days prior to subjecting them for experimentation. The study was approved by the Institutional Animal Ethical Committee (IAEC) of Siksha O Anusandhan University (NAAC "A" Grade), Bhubaneswar, under the approval number 22/12/IAEC/SPS/SOA. All experiments and animal care strictly adherent to the CPCSEA and Good Laboratory Practice Guidelines. No animals were sacrificed at the end of the study.
It is a randomized control study. The animals were randomly divided into six groups with 6 mice in each group; Group I: Control (normal saline given orally at 2 ml/kg body weight); Group II: Standard (diazepam 10 mg/kg orally); Group III, IV, V, and VI (extract of Moringa oleifera [EMO] 50, 100, 200, and 400 mg/kg, respectively). The total number of mice used in each experiment was 36, so a total of 72 mice were used in this study.
In this method, there are 5 identical steps. Step climbing shows locomotor activity while rearing behavior is an index of anxiety state. EMO and the standard (diazepam 10 mg/kg) drug were administered 1hr before the test. The number of steps climbed and the number of rears were counted over the 5 min period. The steps are counted only when all the four paws are on the step.  When the mice are anxious then the number of steps climbed will reduce, and the number of rears will increase while drugs which relieve tension will decrease the rears and increase the number of steps climbed. Statistical evaluation was done using SPSS version 16, SPSS Inc. 2007, Chicago, IL using nonparametric tests, the Kruskal-Wallis test followed by Mann-Whitney's test.
Elevated plus maze test
Here, the maze is elevated to a height of 70 cm. It consists of two arms open and two enclosed arms. EMO and the standard (diazepam 10 mg/kg) were administered 1 h before the test. In a period of 5 min, the number of entries and time spent in open and enclosed arms were recorded. The natural habitat of rats and mice is in burrows, holes, and crevices where there is darkness, so naturally it will seek shelter in those places. The mice when in tension will run to hide in the place where there is darkness like here the closed arms of the apparatus. The reduction in an entry in the open arms, time spent, rearing in open arms, ratio of open arm to total arm entries, and increased defecation are the indications of the high level of fear or anxiety.  Drugs which reduce anxiety will increase the open arm exploratory time while in anxiety state, the opposite thing will happen. Here, parametric tests were used in the study; one-way ANOVA followed by post hoc Bonferroni's test using SPSS version 16.
| Results|| |
In the elevated plus maze (EPM) method, the time spent in open and closed arms before and after drug administration have been analyzed through one-way ANOVA followed by post hoc Bonferroni's test. As the data confirms to the test of normality, the basic assumption of ANOVA was put to use. The diazepam treatment group has the maximum anxiolytic effect, in comparison to normal saline and the EMO at different doses (P < 0.05). Mean time spent in open arm in the diazepam group at 10 mg/kg increased from 50.5 to 123.0 s after the drug administration [Table 1]. In the closed arm, the mean time spent decreased from 205.6 to 106.33 s after drug administration [Table 1]. The effect of test drug at 50 mg/kg is not different from the normal saline group, but the test drug at increased doses exhibited a significant anxiolytic effect in comparison to control. In [Table 2]a-f, post hoc Bonferroni's test reveals significant differences among the groups except for EMO 50 mg/kg and normal saline. [Figure 1] and [Figure 2] clearly illustrates the dose dependent responses of EMO, where with the increase in dose the time spent in open arm increases and in closed arm decreases.
|Figure 1: Bar diagram showing the effect of ethanolic extract of Moringa oleifera in elevated plus maze test in the mean time spent in open arm before and after drug administration with error bars|
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|Figure 2: Bar Diagram showing the effect of ethanolic extract of Moringa oleifera in elevated plus maze test in the mean time spent in enclosed arm before and after drug administration with error bars|
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|Table 1: Comparison among different groups using one-way ANOVA showing the effect of ethanolic extract of Moringa oleifera on mean time spent by mice in the open and closed arms before and after drug administration (mean±standard error)|
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In the staircase test for the comparison of steps climbed and rears before and after drug administration among the groups were done through nonparametric test, the Kruskal-Wallis test. The pairwise comparison was done through Mann-Whitney's U-test. The nonparametric test was followed because the assumption of normality was not fulfilled for the parametric test. After the administration of the test drug, there was a significant difference in the steps climbed as well as the rears among the groups (P < 0.05). Multiple comparisons exhibited diazepam having the most significant anxiolytic effect. Mean steps climbed increased from 2.17 before drug administration to 4.17 after drug administration. Similarly, mean rears decreased from 4.50 to 0.67 after drug administration with diazepam. The EMO at 50 mg/kg was not having any significant impact in comparison to control; however, the EMO at 400 mg/kg the mean steps climbed increased from 2.67 to 3.5 and the mean rears decreased from 3.5 to 1 [Table 3] and [Table 4]. Mann-Whitney's U-test reveals no significant difference between EMO 400 mg/kg and diazepam [Table 5].
|Table 3: Comparison among the groups in the staircase test showing the mean ranks in the steps climbed by mice using Kruskal - Wallis test|
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|Table 4: Comparison among the groups in the staircase test showing the mean ranks in the rears by mice using Kruskal - Wallis test|
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|Table 5: Comparison by Mann-Whitney's U - test between the diazepam group and ethanolic extract of Moringa oleifera at 400 mg/kg|
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| Discussion|| |
Anxiety is caused by many mechanisms, and the neurobiological basis is still obscure. Research has shown that it could be due to the involvement of neurotransmitters and transporters of noradrenergic, serotonergic, glutaminergic, and GABAergic neurons and hormones (neuropeptide Y, CCK).  Low level of GABA in CNS plays a vital role in causing anxiety.  In addition to the GABA release, 5-HT also plays a major role in the development of anxiety.  Free radical-induced damage to the noradrenergic and serotonergic nervous systems is another contributing factor. In this study, the mechanism of action could be related to GABA-mediated facilitation or by either modifying the hormones, transmitters, or by multiple mechanisms. However, the specific mechanism of the extract remains to be unveiled and needs further study.
Rats exhibit neophobia and thus in EPM, the psychomotor performance and emotional aspects of rodents were evaluated .  Anxiolytic agents increase the frequency of entries and the time spent in open arms of the EPM. 
In the staircase test, we have observed that the number of rears has reduced with no significant reduction in the number of steps climbed, and thus the anxiolytic activity of the extract was unveiled by the test. 
Previous phytochemical analysis of the plant leaf extract showed the presence of flavonoids. tannins, saponins, terpenoids which have antioxidant property. Flavonoids are natural active compounds that tend to bind to benzodiazepine GABA A receptors, and they act pharmacologically as partial agonists. Some semi-synthetic flavone derivatives are much more potent than diazepam in vivo. ,,
Free radical mediated damage could be tackled by the presence of compounds such as L-galactose 6 deoxy, n-hexadecanoic acid, tetradecanoic acid, beta-l-rhamnofuranoside 5-O-acetyl-thio-ocyl, Vitamin E, gamma sitosterol, isolated during GC-MS analysis the extract. 
| Conclusion|| |
The EMO leaves showed a significant anxiolytic effect at 100, 200, and 400 mg/kg. Due to complex neurobiological actions of anxiety, we are still in infancy to find out the exact mechanism of action of the extract and thus needs to be explored in details before making a final statement.
I would sincerely like to thank Dr. Manas Naik, Tutor, Department of Pharmacology, VSS Medical College, Burla and Dr. B. B Nanda, M.Sc (Stat), Ph.d (Stat), former biostatistician, SCB Medical College, Cuttack.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]