Depression is a significant contributor to the total economic and health burden of every country [
Plants have served as a rich source of new molecules with pharmacological properties that fill an essential gap in the search for superior therapeutic agents. Local remedies, over the years, have served as a relatively cheap source of therapy and have been employed in the management of disorders such as anxiety, schizophrenia, and epilepsy. The therapeutic claims of preparations from local herbs have over the years provided valuable clues for the direction of pharmacological investigations [
Despite the plants popular use, there is sparse scientific evidence supporting its purported CNS activity. Hence, it is important to investigate the potential of
The spectral region between 400 and 1400 cm−1 is usually considered as the unique region for every compound/compound mixtures and hence can be used for identification and quality control. Hence, triplicate FT-IR (PerkinElmer® UATR Two) spectra were subsequently generated for the extract.
Imipramine; pentylenetetrazole; caffeine; sodium pentobarbitone; Tween 80 (Sigma-Aldrich Inc., St. Louis, MO, USA), fluoxetine (Eli Lilly and Co., Indianapolis, IN, USA), diazepam (INTAS, Gujarat, India) were used. Caffeine, pentobarbitone, diazepam, and pentylenetetrazole were dissolved in distilled water before oral or intraperitoneal administration. To avoid temperature-induced breakdown of pentylenetetrazole, the solution was constantly kept on ice throughout the experimental duration.
Male ICR mice (20-25 g) were obtained from the vivarium of the Department of Pharmacology, KNUST, Kumasi, Ghana. They were housed, in groups of 5, in stainless steel cages (34 × 47 × 18 cm3) with soft wood shavings as bedding and housing conditions as follows: temperature maintained at 23-25°C, relative humidity 60-70 %, and 12 h light-dark cycle. All mice had free access to water and pellet diet (GAFCO, Tema, Ghana). All experiments were compliant with NIH Guidelines for the Care and Use of Laboratory Animals. Ethical approval was obtained from the Department of Pharmacology, Animal Ethics Committee, KNUST..
Irwin’s test is a battery of tests that assesses the behaviour and autonomic response of mice/rats after pretreatment with a compound. The test measures individual behavioural and autonomic parameters of each mouse in a regularly spaced time interval. In addition to the parameters assessed the test provided data on the lethality of the extract or compound in a 24 h period.
Mice were randomly distributed into seven groups (n=5) and left to acclimate to the experimental room for 24 h. Mice were fasted during this period but had free access to glucose solution. The animals were treated with six (6) oral doses of MAE (30, 100, 300, 1000, 3000, and 5000 mg kg−1
The effect of MAE on spontaneous locomotor activity was evaluated using an activity cage (Ugo Basile model 7401, Comerio, VA, Italy). Mice (20-25 g) were randomly divided into eleven groups (n=6-8) and treated with MAE (30, 100, 300, 1000 mg kg−1
Male mice randomly were assigned to six (6) groups and received either MAE (30, 100, 300, 1000 mg kg−1
The effect of MAE on pentobarbitone-induced sleeping time was investigated. Male mice were assigned randomly to seven groups (n=7) and received either MAE (30, 100, 3000, and 1000 mg kg−1
The regular Suok test was carried out as described by Kalueff et al., 2008, with slight modifications [
Forty-two (42) mice were allowed to acclimatize for 24 h in a dimly lit experimental room for an hour before drug treatment and testing. Mice were the randomly selected and distributed into seven groups of six (6) animals each. Animals received either MAE (30, 100, and 300 mg kg−1,
The elevated plus-maze test was performed according methods described by to Pawlak et al. [
The forced swim test was carried out according to the method described by Porsolt et al. [
The tail suspension test was carried out according to the method previously described by Steru et al., 1985. ICR mice were randomly assigned to ten groups of seven animals each. After acclimatization, mice were dosed with either MAE (100, 300, and 1000 mg kg−1,
Behaviours assessed included the following: (1) immobility, a mouse was judged to be immobile when it hung by its tail without engaging in any active behaviour; (2) swinging, a mouse was judged to be swinging when it continuously moved its paws in the vertical position while keeping its body straight and/or it moved its body from side to side; (3) curling, a mouse was judged to be curling when it engaged in active twisting movements of the entire body; (4) pedaling was defined as when the animal moved its paws continuously without moving its body. Mice that climbed on their tail were gently pulled down and the test continued.
Data are presented as mean ± SEM. Data were analyzed using one-way analysis of variance (ANOVA). When ANOVA was significant, multiple comparisons between treatments were done using Sidak
Dose-response curves are constructed using iterative curve fitting with the following nonlinear regression (three-parameter logistic) equation:
The characteristic spectra (Figure
Baseline corrected infrared spectra of the petroleum ether/ethyl acetate fraction of
Oral administration of MAE produced analgesia and reduced fear response at all tested doses. The onset of action of these responses was observed to be shorter at higher doses (Table
Effects of
| | |
| ||
0 | No effect (0-180 min) | 0/5 |
100 | Analgesia, reduced fear response (60-120 min) | 0/5 |
300 | Analgesia, reduced fear response (30-120 min) | 0/5 |
1000 | Analgesia, reduced fear and touch response (30-120 min) | 0/5 |
3000 | Analgesia, reduced fear, touch response & activity (30-120 min) | 0/5 |
5000 | Analgesia, reduced fear, touch response and activity (30-120 min) Increased jumps (15-30 min). | 0/5 |
D= number of deaths recorded T= number of animals treated
Effects of MAE (30-1000 mg kg−1,
MAE (1000 mg kg−1
Effect of MAE (100- 1000 mg kg−1
A Kaplan-Meier estimate of overall survival of animals treated with (a) MAE (100 -1000 mg kg−1
Acute administration of MAE (30 -1000 mg kg−1
Effects of acute MAE (30-1000 mg kg−1 p.o.), diazepam (8 mg kg−1
Effects of acute MAE (30-1000 mg kg−1,
Behaviours assessed include the following: duration and number of freezing bouts and leg slips. Although the duration of freezing (MAE=
Effects of MAE (30-300 mg kg−1) and diazepam (0.1-1.0 mg kg−1) on duration of immobility (a & b) and number of freezing bouts (c & d) over a 5-min test period in the regular Suok test. Data are expressed as group mean ± SEM. Significant difference:
Effects of MAE (30-300 mg kg−1) and diazepam (0.1-1.0 mg kg−1) on the number of leg slips over a 5-min test period in the regular Suok test. Data are expressed as group mean ± SEM. Significant difference:
Testing in the elevated plus maze after acute MAE administration significantly favoured anxiolytic parameters (Figures
Effect of MAE (30-1000 mg kg−1) and diazepam (0.1-1.0 mg kg−1) on mice behaviour in the EPM test, over a 5-min test period. (a) Percentage open arm entries. (b) Percentage of time spent in the open arms of the EPM. Data are presented as group mean ± SEM. Significantly different from control:
Effect of MAE (30-1000 mg kg−1) and diazepam (0.1-0.3 mg kg−1) on mice behaviour in the EPM test, over a 5-min test period. (a) Percentage duration of protected head dips. (b) Percentage of a number of protected head dips. Data are presented as group mean ± SEM. Significantly different from control:
Effect of MAE (30-1000 mg kg-1) and diazepam (0.1-0.3 mg kg-1) on mice behaviour in the EPM test, over a 5-min test period. (a) Percentage duration of protected stretch attend postures (SAPs). (b) Percentage of number of protected SAPs. Data are presented as group mean ± SEM. Significantly different from control:
Figure
Performance of mice in the FST: behavioural assessment including immobility and swimming duration (a, d, g), climbing duration (b, e, h), and latency to immobility (c, f, i) after acute treatment with MAE (100-1000 mg kg−1), imipramine (10-100 mg kg−1), and fluoxetine (3-30 mg kg−1). Data are expressed as group mean ± SEM. Significantly different from control:
From Figure
Dose-response curves for MAE (100-1000 mg kg−1), fluoxetine (3-30 mg kg−1), and imipramine (10-100 mg kg−1) with respect to (a) decrease in immobility and (b) increase in swimming time in the forced swim test. Each point represents the mean ± SEM (n=7).
A two-way ANOVA analysis revealed that 1000 mg kg−1 of MAE significantly decreased the immobility time and increased swimming time (
Similar to MAE, ANOVA analysis revealed that fluoxetine at all tested doses significantly increased swimming time (
Imipramine, similar to the above treatment, increased the swimming time duration at all doses and decreased the immobility duration at 100 and 1000 mg kg−1 (
Figure
Performance of mice in the TST: behavioural assessment including duration of immobility (a, b, c), pedaling (d, e, f), curling (g, h, i), and swinging (j, k and l) after acute treatment of mice with MAE (100-1000 mg kg−1), imipramine (10-100 mg kg−1), and fluoxetine (3-30 mg kg−1). Significantly different from control:
Administration of fluoxetine, MAE, and imipramine reduced immobility time in a dose-dependent manner by a maximum (
Dose-response curves for MAE (100-1000 mg kg−1), fluoxetine (3-30 mg kg−1), and imipramine (10-100 mg kg−1) with respect to % decrease in immobility in the tail suspension test in mice. Each point represents the mean ± SEM (n=7).
Holm-Sidak
The duration of pedaling was significantly altered after MAE (300 mg kg−1) (
The current study demonstrates the anxiolytic and acute antidepressant effects of the petroleum/ethyl acetate extract of
The Irwin test evaluates the qualitative effects of test substances on the behaviour and autonomic and the physiological function of a test animal [
Continuous observation for 48 hours after the test revealed no physical signs of toxicity or lethality at all tested doses. This suggests that the LD50 in mice is beyond 5000 mg kg−1. The onset of action was increased with dose increments, with the fastest onset observed at 30 min. Based on onset and duration of the effects registered in the Irwin test, further tests were carried out 60 minutes after oral administration since lower doses (below 1000 mg kg−1) were adopted for subsequent tests.
The activity meter test was then employed to assess, quantitatively, the spontaneous behaviour with respect to locomotion after oral administration of MAE. There was significant reduction in locomotor activity after 1000 mg kg−1 MAE administration. Locomotor activity can be reduced significantly after dosing test animals with a sedative dose of CNS depressants. Also, a reduction in locomotor activity could also be due to motor impairment induced by the test compound. Consequently, the effects observed after MAE administration can be attributed to the sedative or locomotor impairment potential of the extract. Caffeine, a CNS stimulant, on the other hand, increased whilst diazepam, a CNS depressant, decreased the locomotor activity in this test.
In the Irwin test, test compounds that possess seizure induction potential can be identified by observing physical signs such as tonic and or clonic convulsions during the test. However, the test is not sensitive at identifying proconvulsant effects of compounds. Instead, the proconvulsant potential (seizure liability) is uncovered after pretreatment with chemoconvulsants such as pentylenetetrazole. Such treatments can additionally be used to screen potential anticonvulsants since compounds with anticonvulsant properties being known to reduce seizure parameters induced by these agents. In the convulsive threshold test in mice, only the highest dose of the MAE increased the latency to clonic convulsion and survival compared to the saline group. A Kaplan-Meier analysis of survival revealed no significant protection compared to vehicle control. This indicates that the extract was not effective in delaying and preventing lethality induced by pentylenetetrazole (85 mg kg−1, s.c.).
Barbiturates are general CNS depressants that induce a state of calm, sedation, and hypnosis at high doses [
Anxiety studies were performed in the mice models. The amelioration of innate anxiety induced by novel environment was explored in the elevated plus maze test and the regular Suok test. The elevated plus maze and Suok tests assess the behaviour of mice in a conflict situation. The elevated plus maze assesses the aversion to height and open spaces [
In the EPM test, vehicle-treated mice exposed to the maze made fewer entries and spent less time in the open unprotected arms compared to the closed arms, a behaviour which is consistent with literature that mice generally avoid open unprotected arenas [
Over the past 25 years, several workers have developed protocols that allow a comprehensive profiling of behaviour of mice in the elevated-plus maze based on the defensive behaviours exhibited in the test. While the open arm entries as well as % open arm entries have been found to be a consistent measure of anxiety in rodents, the total head-dips (HD) is a measure of exploration. Additionally, the total SAP provides an additional measure of risk assessment of the test organism. Behaviours such as freezing, stretch attend postures, and head-dips are some of the ethological parameters that can give an indication of the anxiety and behavioural state of the test mouse [
Mice in general tend to move freely in the closed arms with an increased tendency to freeze in the open arm of the EPM. Anxiolytics reduce this freezing behaviour while a converse occurs after giving an appropriate dose of an anxiogenic agent. Based on the above facts, it was realized that a single ethological parameter could increase or decrease depending on whether it occurs in the open or closed arms of the EPM. Hence the designation, “protected” and “unprotected”, is ascribed to an ethological parameter occurring in the closed or opened arms, respectively [
Anxiogenic agents increase the duration and frequency of protected behaviours (head dips), with a corresponding decrease in unprotected ethological behaviours. The converse is true for agents that possess anxiolytic properties in mice. Similar to diazepam, MAE (1000 mg/kg) administration increased the number of head dips. An increase in the number of head dips is an indication of low anxiety states while a decrease indicates high anxiety states [
To assess the effect on sensorimotor coordination and further establish the anxiolytic potential of MAE, the regular Suok test was employed. This test combines aspects of the EPM, OFT, and beam walk tests. Behaviours such as head-dips, side-looks, and frequency and duration of freezing bouts are used as endpoints for assessing of the anxiety state of mice whilst the number of falls and missteps are known to predict the degree of impairment of sensorimotor coordination. Diazepam is known to exhibit anxiolytic effects at lower doses in several test paradigms including the Suok test [
Anxiety and depression are intimately linked and usually appear as comorbid states and treatment of both states positively affect the outcome of therapy [
In the TST, significant decrease in immobility duration was achieved after MAE (1000 mg/kg), imipramine (100 mg/kg), and fluoxetine (3-30 mg/kg) treatment suggesting antidepressant activity. Antidepressants that inhibit serotonin and/or NA reuptake decrease immobility and increase swinging behaviour of mice in the TST, a behaviour that was not significantly altered in MAE-treated mice. Opioids are known to decrease immobility whilst increasing curling behaviour [
Similarly, MAE (300 mg/kg) exhibited antidepressant activity comparable to fluoxetine (10 and 30 mg/kg) and imipramine (100 mg/kg) in the FST (Figure
Results from this study indicate that the petroleum ether/ethyl acetate fraction of
See Table
Peak tables for IR spectra of the petroleum ether/ethyl acetate fraction of
Peak | X (cm-1) | Y (%T) | Peak | X (cm-1) | Y (%T) | Peak | X (cm-1) | Y (%T) | Peak | X (cm-1) | Y (%T) |
---|---|---|---|---|---|---|---|---|---|---|---|
1 | 3226.52 | 82.82 | 2 | 2980.94 | 78.51 | 3 | 2100.15 | 98.53 | 4 | 1585.85 | 64.02 |
| |||||||||||
5 | 1515.39 | 78.99 | 6 | 1454.94 | 77.64 | 7 | 1386.52 | 68.83 | 8 | 1310.96 | 75.08 |
| |||||||||||
9 | 1258.88 | 70.16 | 10 | 1150.77 | 67.59 | 11 | 1120.87 | 61.74 | 12 | 1076.32 | 59.83 |
| |||||||||||
13 | 1023.17 | 52.81 | 14 | 869.99 | 74.68 | 15 | 755.57 | 66.71 | 16 | 523.8 | 56.6 |
| |||||||||||
17 | 430.93 | 53.96 |
Peak | X (cm-1) (cm-1) | Y (%T) (%T) | Peak | X (cm-1) (cm-1) | Y (%T) (%T) | Peak | X (cm-1) (cm-1) | Y (%T) (%T) | Peak | X (cm-1) (cm-1) | Y (%T) (%T) |
---|---|---|---|---|---|---|---|---|---|---|---|
1 | 3224.35 | 86.36 | 2 | 2980.96 | 84.61 | 3 | 2099.5 | 98.76 | 4 | 1585.79 | 69.48 |
| |||||||||||
5 | 1515.51 | 82.17 | 6 | 1454.78 | 81.33 | 7 | 1386.55 | 74.32 | 8 | 1310.69 | 78.91 |
| |||||||||||
9 | 1258.95 | 74.98 | 10 | 1150.65 | 73.01 | 11 | 1120.85 | 67.62 | 12 | 1076.39 | 66 |
| |||||||||||
13 | 1022.95 | 59.51 | 14 | 869.99 | 78.29 | 15 | 755.56 | 71.36 | 16 | 523.2 | 62.18 |
| |||||||||||
17 | 430.37 | 59.83 |
Peak | X (cm-1) (cm-1) (cm-1) | Y (%T) | Peak | X (cm-1) (cm-1) (cm-1) | Y (%T) | Peak | X (cm-1) (cm-1) (cm-1) | Y (%T) | Peak | X (cm-1) (cm-1) (cm-1) | Y (%T) |
---|---|---|---|---|---|---|---|---|---|---|---|
1 | 3227.3 | 80.11 | 2 | 2980.92 | 73.02 | 3 | 2103.82 | 97.56 | 4 | 1585.89 | 60.85 |
| |||||||||||
5 | 1515.37 | 76.86 | 6 | 1455 | 75.17 | 7 | 1386.39 | 65.15 | 8 | 1311.06 | 72.64 |
| |||||||||||
9 | 1258.82 | 67.03 | 10 | 1150.79 | 63.95 | 11 | 1120.87 | 58.23 | 12 | 1076.26 | 56.09 |
| |||||||||||
13 | 1023.43 | 49.11 | 14 | 869.93 | 72.48 | 15 | 755.56 | 64.08 | 16 | 523.45 | 53.54 |
| |||||||||||
17 | 427.16 | 50.8 | 18 | 407.14 | 51.61 |
Original spectra of the petroleum ether/ethyl acetate fraction of
Analysis of variance
Central nervous system
Diazepam
Elevated-plus maze
Fourier transform infrared spectroscopy
Forced swim test
Head dips
Intraperitoneal
Infrared spectroscopy
Kwame Nkrumah University of Science and Technology
Open field test
Pentylenetetrazole
Protected head-dips
Stretch attend posture
Subcutaneous
Standard error of mean
Tail suspension test.
The data used to support the findings of this study are available from the corresponding author upon request.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
The authors are very grateful to the laboratory technicians of the Department of Pharmacology, KNUST, Kumasi, Ghana, for their contribution to this study.