- Email: [email protected]
c mice
Author’s Accepted Manuscript Effects of a hydroethanolic extract of Boophone disticha bulb on anxiety-related behaviour in naive BALB/c mice William Pote, Shamiso Musarira, Donald Chuma, Louis L. Gadaga, Ellen Mwandiringana, Dexter Tagwireyi www.elsevier.com/locate/jep
PII: DOI: Reference:
S0378-8741(17)32435-2 https://doi.org/10.1016/j.jep.2017.12.001 JEP11132
To appear in: Journal of Ethnopharmacology Received date: 28 June 2017 Revised date: 23 November 2017 Accepted date: 3 December 2017 Cite this article as: William Pote, Shamiso Musarira, Donald Chuma, Louis L. Gadaga, Ellen Mwandiringana and Dexter Tagwireyi, Effects of a hydroethanolic extract of Boophone disticha bulb on anxiety-related behaviour in naive BALB/c mice, Journal of Ethnopharmacology, https://doi.org/10.1016/j.jep.2017.12.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effects of a hydroethanolic extract of Boophone disticha bulb on anxiety-related behaviour in naive BALB/c mice
William Pote
a,b,c*
, Shamiso Musarira a, Donald Chuma a, Louis L. Gadaga a, Ellen
Mwandiringana b, Dexter Tagwireyi a. a
Drug and Toxicology Information Service (DaTIS), School of Pharmacy, College of
Health Sciences, University of Zimbabwe, P.O. Box A178, Avondale, Harare, Zimbabwe. b
Department of Preclinical Veterinary Studies, Faculty of Veterinary Science,
University of Zimbabwe, P O Box MP167, Mount Pleasant, Harare, Zimbabwe. c
Department of Physiology, Faculty of Medicine, Midlands State University, Private
Bag, 9055, Senga, Gweru, Zimbabwe.
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Shamiso Musarira: [email protected] Donald Chuma: [email protected] Louis L. Gadaga: [email protected] Ellen Mwandiringana: [email protected] Dexter Tagwireyi: [email protected] email: [email protected]
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*Corresponding Author is Pote William: Department of Preclinical Veterinary Studies, Faculty of Veterinary Science, University of Zimbabwe, P O Box MP167, Mount Pleasant, Harare, Zimbabwe. Phone: +263773234621
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Abstract Ethnopharmacological relevance Boophone disticha is one of the most important medicinal bulbs of Southern Africa. Previous in vitro studies have shown that it’s crude ethanolic extracts and some alkaloidal phytoconstituents possesses high affinity for the serotonin transporter protein (SERT) and serotonin receptor 1a (5HT1a) which are both implicated in the pathogenesis and treatment of anxiety disorders. However, there are no in vivo studies that validates the anxiolytic actions of the plant.
Aim of the study This study was therefore set to determine the anxiolytic-like activity of an orally administered hydroethanolic extract of B. disticha bulbs in naive mice using the behavioural tests of anxiety.
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Materials and Methods Naïve adult male BALB/c mice were randomly placed into five treatment groups (n = 6-10): vehicle control (10ml/kg 0.9% NaCl), positive control (1mg/kg diazepam) and the hydroethanolic extract of B. disticha (10, 25 and 40mg/kg p.o.). Souk test, elevared plus maze and open field tests were used to evaluate the anxiolytic-like activity of the B. disticha extract.
Results Diazepam-treated mice exhibited higher number of sector visits and line crossings in the ST, rearings in the OF and head dips in the EPM than the control (p<0.05). B. disticha extract treated groups expressed higher sector visits at 10mg/kg, and, unprotected head dips at 25mg/kg in the ST, as well as, open arm time entries at 10mg/kg dose, and unprotected head dips at all doses in the EPM than the control group (p<0.05). The 25mg/kg B. disticha dose group exhibited highest anxiolytic-like activity in both the ST and OF, while the 10mg/kg was most active in the EPM.
Conclusion The extract of B. disticha exerted good anxiolytic-like activity in both the ST and OF at medium dose (25mg/kg), while the low dose (10mg/kg) showed prominent anxiolytic-like activity in the EPM.
Graphical abstract
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Experiment 1
Suok Test
Anxioly c Tests Open field
BALB/c mice
Boophone dis cha extract
Experiment 2
Elevated plus maze
Key words: Boophone disticha, anxiolytic-like activity, BALB/c mice, Souk test; open field, elevated plus maze.
1.
Introduction
Anxiety disorders constitute a considerable proportion of the global burden of disease with more than 20% of the adult population suffering from the disorder at some time during their life and with projections that anxiety disorders will form the second most common cause of disability by 2020 (Buller and Legrand, 2001). Until now, the mainstay for treating anxiety has been benzodiazepines, drugs only effective for acute anxiety cases (Kumar et al. 2011). However, benzodiazepine use is often negated by side effects related to increased GABAergic neurotransmission. Clinical signs of this side effect include, ataxia, amnesia, muscle relaxation and sedation. Furthermore, there exists a potential for abuse and dependence associated with long-term use of these medicines (Basile et al. 2004; Cryan and Sweeney, 2011). Antidepressants like
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selective serotonin reuptake inhibitors (SSRI) and tricyclic acids (TCAs) are used to treat anxiety disorder, yet, they also produce many systemic side effects associated with their chronic use (Cryan and Sweeney, 2011). Thus, the search for newer anxiolytic medicines continues to be a priority in drug discovery.
In light of the therapeutic limitations presented by most conventional medicines, different approaches to treatment and management of anxiety disorders are being considered. New approaches at the moment are in favour of herbal medicines with the same or more therapeutic efficacy as conventional medicine but fewer side effects. A number of various herbal options are available and documented as having medicinal or other effects on the central nervous system (CNS) hence posing as potentially affective in chronic conditions such as anxiety, depression, headaches or epilepsy known to be partially non responsive to conventional medicines (Phillipson, 2001). Herbal medicines are in use to combat mental disorders, however, in most instances there is no scientific evidence to support their use (Eisenberg, 1997; Talalay, 2001). An example of such herbs is Boophone disticha (L.f.) Herb. (Amaryllidaceae), known as ‘sore-eyeflower’ or ‘munzepete’ in Shona and ‘ingcotho’ in IsiNdebele, which is one of the most important medicinal bulbs of Southern Africa and has been used traditionally in the management of various neuropsychiatric illnesses including anxiety (Pote et al. 2013 and 2014; Sandager et al. 2005; Stafford et al. 2008; Steenkamp, 2005 ;De Smet, 1996; Gelfand et al. 1985; Hutchings et al. 1996;). However, little is known about its efficacy or mechanism of action for the purported anxiolytic effects.
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Previous in vitro studies done by Nielsen and colleagues (2004) have shown that the aqueous and ethanolic extracts of the leaves and bulbs of B. disticha possessed high affinity for serotonin reuptake transporter protein (SERT), which is important in the etiology of depression and anxiety-related disorders. Two alkaloids found in the plant, i.e., buphanamine and buphanindrine were subsequently isolated and found to have high affinity for the SERT and low affinity for serotonin receptor (5-HT1a) (Sandager et al. 2005). Thus, the crude extract or these bioactive constituents may elicit an anxiolytic response in the brain (Kent et al. 2002). In recent studies, Pote and colleagues (2013, 2014) reported that a hydroethanolic extract of the bulb of B. disticha marginally decreased blood pressure, immediately and several weeks after withdrawal of repeated oral treatments, and significantly reduced heart rate variability several days post treatment in maternally separated mice. Amongst other things, the authors attributed these effects to possible anxiolytic activity of the plant related to modulating the autonomic nervous system (Pote et al. 2014). Although the results of all studies cited above point to potential anxiolytic effects of extracts of B. disticha, none of there is no single in vivo study that was designed specifically to examine this using a scientifically validated animal models of anxiety. Therefore in view of this, and given the potential of B. disticha as an anxiolytic agent, the present study was set to determine if oral administration of hydroethanolic extracts of the plant in vivo, would have anxiolytic-like effects in naive BALB/c mice using three behavioural anxiety: Suok test (ST) (Hart et al. 2010; Kalueff and Tuohimaa, 2005; Kalueff et al. 2007), open field (OF) (Holmes et al. 2001, 2003; Mathis et al. 1994; Miyakawa et al. 2001) and elevated plus maze (EPM) (Pellow et al. 1985; Grundmann et al. 2007).
2.
Materials and Methods
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The study protocols, animal care and handling procedures, were conducted in accordance with the international guidelines on the use and care of experimental animals (European Community guidelines, EEC Directive of 1986) and were approved by The Joint Parirenyatwa Group of Hospitals - College of Health Sciences, University of Zimbabwe Research Ethical Committee (JREC) and the Division of Livestock and Veterinary Services, Ministry of Agriculture, Mechanisation and Irrigation Development, Zimbabwe.
2.1.
Plant material and extraction
2.1.1. Plant material Bulbs of B. disticha were harvested in Mashonaland East province of Zimbabwe and were authenticated by a taxonomist (botanist) from the Botanical Gardens and National Herbarium. A voucher specimen (reference number D.BUZPP4P 07-2011) was refrigerated in the School of Pharmacy, College of Health Sciences, University of Zimbabwe (Pote et al. 2013). 2.1.2.
Preparation of the crude extract
The crude extract was prepared as previously described by Gadaga et al. (2011) and Pote et al. (2013 and 2014) with few modifications. Briefly, the bulbs were sliced, sun dried for a week, oven dried at a temperature of about 50˚C and ground into a powder using a two-speed blender. Four samples of the powdered B. disticha bulb weighing approximately 100g each, constituting equal proportions of powdered dried inner scales and powdered dried outer scales were each added to 600 ml of hydroethanolic solvent (70% v/v). The four mixtures were left to stand overnight, refluxed the following morning for 1 hour at 70˚C on a heating mantle, cooled and left overnight again. The following day, the acquired hydroethanolic extract solution was decanted
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into a beaker. Pressing the plant material with a spatula pushed out remaining extract. Extracts obtained from all four samples were mixed and placed in one volumetric flask. The extract mixture was then vacuum with Whatman filter paper No. 1, to get rid of residual fine particulate plant material. The filtrate obtained (1800 ml) was rotary evaporated at 90 revolutions per minute at 80˚C with a Heidolph 4000 Rotavapor (Heidolph, Germany) to remove solvent to a volume of 100 ml, which was then freeze-dried overnight at temperatures between -40˚C and -20˚C. A semi-solid crude extract (32.06g) was obtained and kept in an airtight container placed in a desiccator at room temperature.
2.1.3. Qualitative analysis of alkaloids Thin layer chromatography, viewed under UV light of 365nm wavelength, revealed that isoquinoline alkaloids were present in the crude extract of B. disticha used in this study. Most of the alkaloids were detected under strongly basic conditions whilst few others were detected under the neutral to weakly basic conditions (Zulu, 2011).
2.2.
Animal Husbandry
Forty-five adult male BALB/c mice (8-12 weeks old) weighing between 20 and 35 g were purchased from the Animal House Unit, University of Zimbabwe (UZ) and transported to the animal holding facility at the Clinical Pharmacology Department, College of Health Sciences (CHS), UZ. The animals were housed in standard Plexiglas cages (4-5 mice per cage) and allowed to acclimatise for two weeks to the holding room. Temperature in the animal holding facility was 22 ± 20C with a humidity of 60 ± 5% and lighting was maintained using the natural cycle of the day, light phase was at 5 am and dark phase at 7 pm. Wood shavings were used as
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bedding. Food and water was available ad libitum throughout the study except when the animals were fasted by removing both water and feed for 18 hours before the behavioural testing. All behavioural experiments were carried out, between 8am and 4pm during the light phase, in a sound proof room located at the College of Health Sciences, University of Zimbabwe. Soon after each test, the animal was placed back into its home cage. All test apparatus were cleaned thoroughly with 70% (v/v) aqueous ethanol and dried before putting another animal to preclude the possible cueing effects of odours left by previous subjects. After the neurobehavioural experiments, the mice were returned to the Animal House Unit (UZ) and humanely sacrificed by chloroform euthanasia.
2.3.
Treatment groups and dosage administration
Adult male mice were randomly assigned to five groups for the first experiment and tested in the Suok test (n = 6-9 mice in each group with a total of 36 animals) followed by open field (n = 8-10 mice in each group with a total 45 animals). The other cohort of mice (n=40) were also randomly assigned to five similar treatment groups (n = 8 each) for the second experiment and tested in the elevated plus maze. The five treatment groups included the vehicle control, three B. disticha treatment groups and a positive control group. The vehicle control group received normal saline while the positive control group received 1mg/kg of diazepam. Each of the B. disticha groups received 10, 25 or 40mg/kg of the hydroethanolic extract of the plant suspended in normal saline. All treatments were given as a single dose by oral gavage at a volume of 10 ml/kg one hour before behavioural testing was conducted.
2.4.
Experiment 1
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In this experiment, forty-five mice were randomly assigned to five treatments as described above (n = 8-10 each) and were placed in cages with 4 or 5 animals each. Cage number and tail markings were used to identify the animals. Before the testing day, the mice were fasted by removing both water and feed for 18 hours. On the testing day, all the animals were moved to the temporary holding room in holding cages on a trolley and allowed to rest for one hour to acclimatise to the test room. Each animal was weighed and was orally gavaged 30 minutes prior to testing with the respective treatment described above. Each animal was first tested using the Suok test (ST) for ten minutes (Kalueff and Tuohimaa, 2005; Kalueff et al. 2007, 2008a), rested for another ten minutes, placed in the open field (OF) and then observed for five minutes (Mathis et al. 1994; Holmes et al. 2001, 2003; Miyakawa et al. 2001). The anxiolytic activity was assessed based on four behavioural domains: exploration, displacement, risk assessment and motor co-ordination.
2.4.1.
Suok test (ST)
The Suok test (ST) apparatus consisted of a 2 m aluminium rod, with a diameter of 3 cm, covered with masking tape to improve grip. The rod was marked into 20 sectors, each measuring 10 cm, by line drawings. There was a 20 cm virtual two-sector central zone about the placement point at the centre of the rod. This rod was also elevated to a height of 20 cm from the floor and fixed to the central groove of two wooden end walls which measured 40 cm (height) x 40 cm (width) x 2 cm (thick). Paper towels were cushioned underneath the rod to avoid any harm to the animals which fell from the rod. The experimental room was dimly lit with a 40 watt light bulb fixed two meters above the apparatus. A digital camera was positioned about 1 metre away from
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the apparatus to record all movements done by the mouse. A mouse was placed on the central zone of the rod and left to explore the apparatus for a period of 10 minutes. The following anxiety-related behaviours were then scored: exploration (horizontal activity [number of sectors visited and number of line crossings] and vertical activity [number of rears and wall leanings, head dips and number of stops]); displacement: (number of grooming behaviour); risk assessment behaviour (number of times of stretch attends postures); and motor coordination (number of missteps) (Hart et al. 2010; Kalueff and Tuohimaa, 2005; Kalueff et al. 2008a). When number of sectors visited, rearings and head dips are high, it means the mouse is not anxious, but, if the number of stops is high, it shows that the animal is anxious (Kalueff et al. 2008a).
2.4.2. The open field (OF) The open field (OF) apparatus consisted of a rectangular orange plastic box of 42 cm (length) x 30 cm (width) x 18 cm (height). The apparatus was placed on a table with a height of 62 cm from the ground. The floor of the box was divided by lines into 12 sectors measuring 10 cm x 7 cm. The experimental room was dimly lit with a 40 W light bulb fixed about two meters above the apparatus. A digital camera was positioned about 75 cm above the apparatus to record all movements done by the mouse. A mouse was placed on the central square and allowed to explore the apparatus for 5 minutes. The following anxiety-like behaviours were scored: exploration (number of line crossing, number of vertical rears, number of centre square entries and number of stops); displacement (number of grooming behaviour); and risk assessment behaviour (number of stretch attends posture) (Hart et al. 2010; Holmes et al. 2001, 2003; Miyakawa et al. 2001).
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2.5.
Experiment 2
Forty mice included in this experiment were randomly assigned to five treatment groups, housed four animals per cage, tail marked, fasted, weighed and given respective treatments as described in experiment 1. Thirty minutes after gavaging, each animal was then placed on the elevated plus maze and observed for 5 minutes (Pellow et al. 1985).
2.5.1.
The elevated plus maze (EPM)
The elevated plus maze (EPM) consisted of two open arms (25 × 5 cm) crossed with two closed arms (25 × 5 × 20 cm) made from wood. The maze was elevated to a height of 40 cm, while, a 40 W light bulb was fixed 80 cm above the maze and an overhead digital camera was fixed 110 cm above the maze. Mice were taken from the home cage by the base of the tail and placed individually on the EPM facing an open arm. Mice were observed for five minutes and number of entries and time spent in open and closed arms was recorded. The percentage time spent on the open arms was calculated (100 × open arm time / 300s) for each animal (Grundmann et al. 2007). Arm entries were defined as entry of all four paws into an arm (Pellow et al. 1985). Percentage open arm entries were calculated for each animal as well (100 x number of open arm entries/(total entries into open arm and closed arms). Other anxiety-like behaviours scored were, number of head dips (HD), stretch attend posture (SAP) and rearings. For SAP and HD a distinction was made between ‘protected’ (if the behaviours are displayed from the central platform or closed arm) and ‘unprotected’ (if it occurs while being in an open arm). Percentage of unprotected head dips was calculated for each animal (100 x unprotected head dips / total number of head dips) or SAP (100 x unprotected SAP /total number of SAP). Anxiety-like behaviours were
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classified as exploration (open arm time, open arm entries, rearing and head dip); and risk assessment (number of stretch attends posture) (Hart et al. 2010; Mathis et al. 1994; Holmes et al. 2001, 2003; Miyakawa et al. 2001). The EPM was cleaned with 70% v/v ethanol solution and tissue paper to remove any residual scent.
2.6.
Statistical analysis
Statistical analysis was performed using Graph Pad Prism® 7.0 software. One-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was used to compare groups. Data was presented as mean ± standard error of mean (SEM) and significant level was considered at p<0.05.
3.
Results
3.1.
Suok test
Treatments had significant effect on number of sectors visited and lines crossed (F4,31 = 2.962, p = 0.0350; Figure 1A and F4,31 = 3.795, p =0.0126; Figure 1B, respectively).
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Figure 1. Effects of B. disticha on anxiety-related explorative behavior measured by the Souk Test (n= 6-9 in each group). Number of: A. sectors visited, B. line crossings, C. rearings, D. head dips and E. stops. BD, B. disticha; DZP, diazepam.*p < 0.05 vs. vehicle control, ## p < 0.01 vs. diazepam.
However, treatment of mice with either B. disticha or diazepam had no effect on the number of rearings, head dips, stops, groomings, SAP and missteps (F4,31 = 1.673, p = 0.1814 (Figure 1C); F4,31 = 2.123, p = 0.1016 (Figure 1D), F4,31 = 1.134, p = 0.3587 (Figure 1E), F4,30 = 1.42, p = 0.2513; Figure 2A), F4,31 = 2.062, p = 0.1099, Figure 2B), and F4,31 = 1.767, p = 0.1607, Figure 2C), respectively). Groups treated with either B. disticha at 25mg/kg or diazepam, visited significantly more sectors than the vehicle control group (p<0.05, Figure 1A). Animals given diazepam significantly crossed more lines than either the vehicle control or the group treated with B. disticha at 40mg/kg (p<0.05 and p<0.01, respectively, Figure 1B). And, mice treated with
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25mg/kg B. disticha expressed significantly more head dip behaviours when compared to the vehicle control group (p<0.05).
Figure 2. Effects of an ethanol extract of B. disticha on risk assessment behavior and motor activity in the Souk Test (n= 6-9 in each group). Number of: A. grooming behaviour; B. number of stretch attend postures; and C. missteps. BD, B. disticha; DZP, diazepam.
3.2.
Open Field
Treatments had significant effect on the number of rearings in the open field (F4,40 = 2.87, p = 0.0351; Table 1). However, treatments did not affect the number of stops, line crossings, centre square entries, grooming and stretch attend posture in the open field (F4,40 = 1.386, p = 0.2560, F4,40 = 2.13, p = 0.0949, F (4, 40) = 0.5178, p = 0.7231, F4,40 = 1.374, p = 0.2603 and F4,40 = 1.244, p = 0.3079, respectively; Table 1). Diazepam-treated mice expressed significantly higher rearing and line crossings scores than the control and the 40mg/kg B. disticha treated groups, respectively (p<0.05, Table 1).
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Table 1. Effects of B. disticha hydroethanolic extract anxiety-like behavior in the open field.§ Treatment Group
Parameter
Control
BD10mg/kg
BD25mg/kg
BD 40mg/kg
DZP 1mg/kg
Line crossings
65.75±11.95
56.70±6.32
63.67±4.33
47.30±5.83##
75.63±7.07
Centre Square entries
4.50±0.96
7.13±1.04*
4.88±0.69
4.86±0.74
5.14±0.86
Rearings
17.33±2.99
16.13±2.88
20.13±1.52
19.14±1.50
81.57±4.41*
Stops
1.33±0.615
0.375±0.183
0.875±0.350
0.286±0.184
0.571±0.429
Groomings
4.83±0.95
4.63±0.71
5.13±0.83
5.43±0.84
3.57±0.78
SAP
5.67±0.61
7.875±1.246
7.000±1.309
7.43±2.29
10.57±2.79
§Each group (n= 8-10); BD, B. disticha; DZP, diazepam; SAP, stretch attend posture. *p<0.05 vs. Control; ##p<0.01 vs. DZP.
3.3.
Elevated plus maze
Animals treated with B. disticha extract, at 10mg/kg dose, spent significantly more time in open arms and scored significantly higher number of open arm entries than the vehicle control group (p<0.05, Table 2). It was also observed that mice treated with ether diazepam or B. disticha at 10, 25 and 40mg/kg had significantly higher number of unprotected head dips than the vehicle control group (p<0.05, p<0.05, p<0.005 and p<0.01, respectively; Table 2). And, mice treated with either diazepam or B. disticha at doses of 10 and 25mg/kg, had significantly higher scores of stretch attend postures
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than the control (p<0.01, p<0.05 and p<0.05, respectively Table 2). However, animals treated with either B. disticha or diazepam expressed same scores of rearing behaviour as the control group (p > 0.05) (Table 2).
Table 2. Effects of B. disticha extract on anxiety-like behaviours in the elevated plus maze. §
Treatment Group
Control
BD10mg/kg
BD25mg/kg
BD 40mg/kg
DZP 1mg/kg
Open arm entries (%)
35.36±3.99
49.08±3.91*
45.53±2.75
46.14±3.45
46.74±1.50
Time spent in Open arms (%)
17.83±4.22
45.24±9.46*
34.76±4.82
31.42±6.55
25.44±6.85
Unprotected Head dips (%)
46.83±9.97
86.11±3.17*
84.64±4.79***
73.77±7.45**
78.15±8.97*
Unprotected SAP (%)
26.49±11.25
66.17±7.69**
60.83±6.11*
42.51±6.19
57.02±7.47*
Rearings
19.17±4.02
13.43±1.80
17.00±1.35
13.75±1.22
15.83±2.47
Parameter
§Each group (n = 8 in); BD, B. disticha; DZP, diazepam; SAP, stretch attend posture. *p<0.05, **p<0.01, ***p<0.001 vs. control.
4.
Discussion
The present study was set to investigate the effects of B. disticha hydroethanolic extract on anxiety-like behaviours in mice. Diazepam was used as the reference drug (positive control) and was used to determine the predictive validatity of the paradigm employed in this study. This study reported that the diazepam-treated group expressed higher number of sectors visited and line crossings in the ST than the control, which
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indicated that diazepam increased horizontal explorative activity (Kalueff et al. 2008a and 2008b) and decreased anxiety in mice. Meanwhile, the former group also expressed higher scores of rearings than the latter group in the OF which suggested that diazepam increased vertical exploratory activity and reduced anxiety. In addition the former group scored more number of head dips in the EPM than the control indicating that diazepam reduced fear and anxiety in these animals. These results supported findings reported in previous studies demonstrating that a standard anxiolytic drug like diazepam elicits anxiolytic-like behaviours in the ST (Kalueff et al. 2008a), OF and EPM (Cryan and Sweeney, 2011). These results confirmed that the tests selected in this study (ST, OF and EPM) and the study design were valid for screening substances with potential anxiolytic activity (Brown et al. 1999). The present findings, therefore, established that diazepam effectively reduced anxiety-like behaviours and showed that the behavioural paradigm employed in this study was pharmacologically valid (Cryan and Sweeney, 2011).
The current findings also demonstrated that B. disticha extract increased horizontal activity (number of sectors visited) at 10mg/kg, vertical explorative activity (unprotected head dips) at 25mg/kg, and directed explorative activity (unprotected head dips) at all doses in the ST. Furthermore, the extract increased horizontal exploratory activity and risk-taking behaviour (open arm time and entries), and vertical explorative activity (unprotected head dips) in the EPM. These consistent observations that animals treated with B. disticha expressed higher exploratory activity in both the ST and EPM tests than the vehicle control suggest that the extract effectively reduced anxiety and fear in mice (Brown et al. 1999, Heinrichs et al. 1992; Rodgers and Cole, 1993; Holmes et al. 2001, 2003; Miyakawa et al. 2001). Thus B.
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disticha exhibit an anxiolytic-like activity at doses of 10 and 25mg/kg (Mathis et al. 1994; Holmes et al. 2001, 2003).
In a contrary observation, the present study found that mice given B. disticha extract at doses of 10 and 25mg/kg exhibited higher scores of unprotected SAP in the EPM indicating increased risk assessment activity, which is suggestive of increased fear and anxiety. This paradoxical finding might have been due to the fact that the hydroethanolic extract may affect exploration and risk assessment behaviour in different ways (Bourin et al. 2007; Navarro et al. 2006). Risk assessment is a behavioural measure that indicates that the animal is hesitant to move from its present location to a new position and thus a high frequency of these postures indicates a higher level of anxiety (Brown et al. 1999).
The failure of B. disticha extract to affect other anxiety-like behaviours in the ST, OF and EPM, could be due to several reasons. Missteps can reflect vestibular, motor coordination or neuromuscular deficits or stress-evoked sensori-motor disintegration (SSD) (Kalueff et al. 2008a). Hence, current findings showing no difference between groups on missteps suggest that B. disticha extract does not cause motor incoordination and hallucinations in mice at doses between 10 and 40mg/kg. This is consistent with previous studies, which reported that at doses less than 50mg/kg do not elicit SSD and hallucinations seen in higher doses (Gadaga et al. 2011; Gadaga, 2012; Ganga et al. 2017; Mutseura et al. 2013). Furthermore, the lack of effects on grooming behaviour was not surprising since this measure is not a reliable indicator when screening potential anxiolytic drugs (Kalueff et al. 2008b). Grooming is a behaviour that shows displacement activity seen when the mouse, while stationary,
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licks, or scratches using front or back paws, displayed in a novel environment showing that the mice is anxious (Brown et al. 1999).
A combined agonism/antagonism occurring at different receptors by various compounds in the crude extract might also account for the absence of effects on such behaviours (Grundmann et al. 2007). Lastly, it has been shown that drugs that act on the serotonin system do not show significant difference in the number of open arm entries in the EPM (Dawson and Tricklebank, 1995).
The anxiolytic activity demonstrated by the plant extract in this study may be due to the active alkaloidal phytoconstituents reported by previous studies (Bastida et al. 2011; Botha et al. 2005; Cheesman et al. 2012; Nair and Van Staden, 2014; Nair et al. 2013; Neergaard et al. 2009; Nielsen et al. 2004; Viladomat et al. 1997; Gadaga et al. 2011). The alkaloids may act directly or indirectly through their active metabolites released into the blood from the liver and/or other tissues, or by the brain tissue into the surrounding tissue and/or cranium blood supply. It may be likely that the extract modified various neurotransmitter systems because numerous neural pathways are involved in the pathophysiology of anxiety disorders (Kent et al. 2002). These pathways include the monoamines: 5-hydroxytryptamine (5-HT), noradrenaline and the γ amino butyric acid (GABA)-benzodiazepine receptor complex and a number of unrelated compounds that are known to provoke anxiety and/or panic in humans (Sandford, 2000). The alkaloids or their metabolites may exert their effects by binding on the serotonergic receptors (5-HT1a receptors) or the serotonin transporter protein (SERT) in the central nervous system (Nair and Van Staden, 2014; Neergaard et al. 2009; Nielsen et al. 2004; Sandager et al. 2005). The absence of activity in some
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behavioral parameters in the current results could explain and also further support the theory that B. disticha extract’s anxiolytic actions work through the serotonin system. However, the behavioural tests employed in this study do not allow discerning of the underlying mechanism of action of B. disticha extract. Hence, more neurochemistryrelated work would need to be done to investigate these hypotheses. Further studies also need to ascertain the activity of the extract in pathological animal models of anxiety disorders and to identify the bioactive compounds responsible for the anxiolytic-like activity of the plant extract.
5.
Conclusion
The current study demonstrated for the first time that a hydroethanolic extract of B. disticha has anxiolytic-like activity in naïve BALB/c mice. The extract of B. disticha exerted good anxiolytic-like activity in both the souk test and open field at medium dose (25mg/kg), while the low dose (10mg/kg) showed prominent anxiolytic-like activity in the elevated plus maze. The identification that the activity of the plant is similar to the well-known conventional anxiolytic diazepam supports its safe use as a natural health product. However, future studies are required to elucidate concisely the mechanism underlying anxiolytic activity of the plant extract.
Acknowledgements: We are very grateful to Mr Murambiwa and staff at the of Clinical Pharmacology Department, Food Science Department, School of Pharmacy, and Animal House Unit at University of Zimbabwe. Funding: This work was supported by IFS (grant number F/4187-1). Conflicts of interest: none
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Holmes, A., Hollon, T.R., Liu, Z., Sibley, D.R., Dreiling, J., Gleason, T.C., et al., 2001. Dopamine D5 receptor null mutant mice show attenuated behavioral responses to a dopamine agonist. Behavioral Neuroscience 115, 1129–1144. Holmes, A., Kinney, J.W., Wrenn, C.C., Li, Q., Yang, R.J., Ma, L. et al. 2003. Galanin GAL-R1 receptor null mutant mice display increased anxiety-like behavior specific to the elevated plus-maze test. Neuropsychopharmacology 28, 1031–1044. Hutchings, A., Scott, A.H., Lewis, G., Cunningham, A.B., 1996. Zulu Medicinal Plants: An Inventory. University of Natal Press, Pietermaritzburg. Kalueff, A.V., Tuohimaa, P., 2005. The Suok (ropewalking) murine test of anxiety. Brain Research Protocols 14, 87-89 Kalueff, A.V., Keisala, T., Minasyan, A., Tuohimma, P., 2007. Pharmacological modulation of anxiety related behaviors in the murine Suok test. Brain Research Bulletin 74: 45-50. Kalueff, A.V., Keisala, T., Minasyan, A., Kumar, S.R., LaPorte, J.L., Murphy, D.L., Tuohimaa, P., 2008a. The regular and light-dark Suok tests of anxiety and sensorimotor integration: utility for behavioral characterization in laboratory rodents. Nature Protocols 3(1), 129–36. Kalueff, A.V., La Porte, J., Smolinsky, A., 2008b. Measuring Behavior: Understanding Brain affective states by measuring animal grooming patterns. 6th International Conference on Methods and Techniques in Behavioral Research pp 113. Kent J.M., Mathew S.J., and Gorman M.J., 2002. Molecular Targets in the Treatment of Anxiety. Biological Psychiatry 52, 1008-1030. 26
Kumar, R., Muruganathan, R., Nandakumar, K., 2011. Isolation of anxiolytic principle from ethanolic root extract of Cardiospermum halicacabum. Phytomedicine 18(2-3), 219-23. Mathis, C., Paul, S.M., Crawley, J.N., 1994. Characterization of benzodiazepinesensitive behaviors in the A/J and C57BL/6J inbred strains of mice. Behavioral Genetics 4(2), 171-80. Mutseura, M., Tagwireyi, D., Gadaga, L. L. 2013. Pre-treatment of BALB/c mice with a centrally acting serotonin antagonist (cyproheptadine) reduces mortality from Boophone disticha poisoning. Clinical Toxicology (Philadelphia, Pa.), 51(1), 16–22. https://doi.org/10.3109/15563650.2012.748194 Miyakawa, T., Yamada, M., Duttaroy, A., Wess, J., 2001. Hyperactivity and intact hippocampus-dependent learning in mice lacking the M1 muscarinic acetylcholine receptor. Journal of Neuroscience 21, 5239–5250. Nair, J.J., Bastida, J., Codina, C., Viladomat, F., van Staden, J., 2013. Alkaloids of the South African Amaryllidaceae: a review. Natural Products Communications 8(9):1335-50 Nair, J.J., Van Staden, J., 2014. Traditional usage, phytochemistry and pharmacology of the South African medicinal plant Boophone disticha (L.f.) Herb. (Amaryllidaceae). Journal of Ethnopharmacology 151, 12–26. Navarro, J.F., Buron, E., and Lopez, M.M., 2006. Anxiolytic-like activity of SB205384 in the elevated plus maze test in mice. Psicothema 18 (1), 100-104.
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Neergaard, J. S., Andersen, J., Pedersen, M. E., Stafford, G. I., Staden, J. Van, and Jäger, A.K., 2009. Alkaloids from Boophone disticha with affinity to the serotonin transporter. South African Journal of Botany 75(2), 371–374. Nielsen, N.D., Sandager, M., Stafford, G.I., Jaeger, A.K., van Staden, J., 2004. Screening of indigenous plants from South Africa for affinity to the serotonin reuptake transport protein. Journal of Ethnopharmacology 94, 159–163. Pellow S., Chopin P., File S.E., and Briley M., 1985. Validation of open: closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. Journal of Neuroscience Methods 14: 149-167. Phillipson, J.D., 2001. Phytochemistry and medicinal plants. Phytochemistry 56, 237–243. Pote, W., Tagwireyi, D., Chinyanga, H. M., Musara, C., Nyandoro, G., Chifamba, J., Nkomozepi, P., 2013. Cardiovascular effects of Boophone disticha aqueous ethanolic extract on early maternally separated BALB/C mice. Journal of Ethnopharmacology 148(2), 379–85. Pote, W., Tagwireyi, D., Chinyanga, H. M., Musara, C., Pfukenyi D.M., Nkomozepi, P., Gadaga L.L., Nyandoro, G., Chifamba, J., 2014. Long-term cardiovascular autonomic responses to aqueous ethanolic extract of Boophone disticha bulb in early maternally separated BALB/c mice. South African Journal of Botany 94 (1), 33-39. Rodgers R.J., Cole J.C., 1993. Anxiety enhancement in the murine elevated plus maze by immediate prior exposure to social stressors. Physiology and Behaviour 53, 383388.
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Sandager, M., Neilsen, N.D., Stafford, G.I., van Staden J., Jaeger, A.K., 2005. Alkaloids from B. disticha with affinity to the serotonin transporter in the rat brain. Journal of Ethnopharmacology 98, 367-370. Sandford, J.J., Argyropoulos, S.V., Nutt, D.J., 2000. The psychobiology of anxiolytic drugs. Part 1: basic neurobiology. Pharmacology and Therapeutics 88 (3), 197–212. Stafford, G.I., Jager, A.K., van Staden, J., 2008. African Psychoactive Plants. African Natural Plant Products: New discoveries and Challenges in Chemistry and Quality. Chapter 18, pp 323-346. Steenkamp, P.A., 2005. Chemical Analysis of Medicinal and Poisonous Plants of Forensic Importance in South Africa. (Philosophiae Doctor Thesis) University of Johannesburg, South Africa. Talalay, P., 2001. The importance of using Scientific Principles in the development of Medicinal Agents from Plants. Academic Medicine 73 (3): 238. Viladomat, F., Bastida, J., Codina, C., Nair, J.J., Campbell, W.E., 1997. Alkaloids of the South African Amaryllidaceae, recent research development. Phytochemistry 1, 131–171. Zulu, D., 2011. Solvent extraction and chromatographic characterization of isoquinoline alkaloids from the bulb of Boophone disticha. Bachelor of Pharmacy Thesis, University of Zimbabwe Library (Unpublished results).
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PII: DOI: Reference:
S0378-8741(17)32435-2 https://doi.org/10.1016/j.jep.2017.12.001 JEP11132
To appear in: Journal of Ethnopharmacology Received date: 28 June 2017 Revised date: 23 November 2017 Accepted date: 3 December 2017 Cite this article as: William Pote, Shamiso Musarira, Donald Chuma, Louis L. Gadaga, Ellen Mwandiringana and Dexter Tagwireyi, Effects of a hydroethanolic extract of Boophone disticha bulb on anxiety-related behaviour in naive BALB/c mice, Journal of Ethnopharmacology, https://doi.org/10.1016/j.jep.2017.12.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effects of a hydroethanolic extract of Boophone disticha bulb on anxiety-related behaviour in naive BALB/c mice
William Pote
a,b,c*
, Shamiso Musarira a, Donald Chuma a, Louis L. Gadaga a, Ellen
Mwandiringana b, Dexter Tagwireyi a. a
Drug and Toxicology Information Service (DaTIS), School of Pharmacy, College of
Health Sciences, University of Zimbabwe, P.O. Box A178, Avondale, Harare, Zimbabwe. b
Department of Preclinical Veterinary Studies, Faculty of Veterinary Science,
University of Zimbabwe, P O Box MP167, Mount Pleasant, Harare, Zimbabwe. c
Department of Physiology, Faculty of Medicine, Midlands State University, Private
Bag, 9055, Senga, Gweru, Zimbabwe.
1
Shamiso Musarira: [email protected] Donald Chuma: [email protected] Louis L. Gadaga: [email protected] Ellen Mwandiringana: [email protected] Dexter Tagwireyi: [email protected] email: [email protected]
2
*Corresponding Author is Pote William: Department of Preclinical Veterinary Studies, Faculty of Veterinary Science, University of Zimbabwe, P O Box MP167, Mount Pleasant, Harare, Zimbabwe. Phone: +263773234621
3
Abstract Ethnopharmacological relevance Boophone disticha is one of the most important medicinal bulbs of Southern Africa. Previous in vitro studies have shown that it’s crude ethanolic extracts and some alkaloidal phytoconstituents possesses high affinity for the serotonin transporter protein (SERT) and serotonin receptor 1a (5HT1a) which are both implicated in the pathogenesis and treatment of anxiety disorders. However, there are no in vivo studies that validates the anxiolytic actions of the plant.
Aim of the study This study was therefore set to determine the anxiolytic-like activity of an orally administered hydroethanolic extract of B. disticha bulbs in naive mice using the behavioural tests of anxiety.
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Materials and Methods Naïve adult male BALB/c mice were randomly placed into five treatment groups (n = 6-10): vehicle control (10ml/kg 0.9% NaCl), positive control (1mg/kg diazepam) and the hydroethanolic extract of B. disticha (10, 25 and 40mg/kg p.o.). Souk test, elevared plus maze and open field tests were used to evaluate the anxiolytic-like activity of the B. disticha extract.
Results Diazepam-treated mice exhibited higher number of sector visits and line crossings in the ST, rearings in the OF and head dips in the EPM than the control (p<0.05). B. disticha extract treated groups expressed higher sector visits at 10mg/kg, and, unprotected head dips at 25mg/kg in the ST, as well as, open arm time entries at 10mg/kg dose, and unprotected head dips at all doses in the EPM than the control group (p<0.05). The 25mg/kg B. disticha dose group exhibited highest anxiolytic-like activity in both the ST and OF, while the 10mg/kg was most active in the EPM.
Conclusion The extract of B. disticha exerted good anxiolytic-like activity in both the ST and OF at medium dose (25mg/kg), while the low dose (10mg/kg) showed prominent anxiolytic-like activity in the EPM.
Graphical abstract
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Experiment 1
Suok Test
Anxioly c Tests Open field
BALB/c mice
Boophone dis cha extract
Experiment 2
Elevated plus maze
Key words: Boophone disticha, anxiolytic-like activity, BALB/c mice, Souk test; open field, elevated plus maze.
1.
Introduction
Anxiety disorders constitute a considerable proportion of the global burden of disease with more than 20% of the adult population suffering from the disorder at some time during their life and with projections that anxiety disorders will form the second most common cause of disability by 2020 (Buller and Legrand, 2001). Until now, the mainstay for treating anxiety has been benzodiazepines, drugs only effective for acute anxiety cases (Kumar et al. 2011). However, benzodiazepine use is often negated by side effects related to increased GABAergic neurotransmission. Clinical signs of this side effect include, ataxia, amnesia, muscle relaxation and sedation. Furthermore, there exists a potential for abuse and dependence associated with long-term use of these medicines (Basile et al. 2004; Cryan and Sweeney, 2011). Antidepressants like
6
selective serotonin reuptake inhibitors (SSRI) and tricyclic acids (TCAs) are used to treat anxiety disorder, yet, they also produce many systemic side effects associated with their chronic use (Cryan and Sweeney, 2011). Thus, the search for newer anxiolytic medicines continues to be a priority in drug discovery.
In light of the therapeutic limitations presented by most conventional medicines, different approaches to treatment and management of anxiety disorders are being considered. New approaches at the moment are in favour of herbal medicines with the same or more therapeutic efficacy as conventional medicine but fewer side effects. A number of various herbal options are available and documented as having medicinal or other effects on the central nervous system (CNS) hence posing as potentially affective in chronic conditions such as anxiety, depression, headaches or epilepsy known to be partially non responsive to conventional medicines (Phillipson, 2001). Herbal medicines are in use to combat mental disorders, however, in most instances there is no scientific evidence to support their use (Eisenberg, 1997; Talalay, 2001). An example of such herbs is Boophone disticha (L.f.) Herb. (Amaryllidaceae), known as ‘sore-eyeflower’ or ‘munzepete’ in Shona and ‘ingcotho’ in IsiNdebele, which is one of the most important medicinal bulbs of Southern Africa and has been used traditionally in the management of various neuropsychiatric illnesses including anxiety (Pote et al. 2013 and 2014; Sandager et al. 2005; Stafford et al. 2008; Steenkamp, 2005 ;De Smet, 1996; Gelfand et al. 1985; Hutchings et al. 1996;). However, little is known about its efficacy or mechanism of action for the purported anxiolytic effects.
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Previous in vitro studies done by Nielsen and colleagues (2004) have shown that the aqueous and ethanolic extracts of the leaves and bulbs of B. disticha possessed high affinity for serotonin reuptake transporter protein (SERT), which is important in the etiology of depression and anxiety-related disorders. Two alkaloids found in the plant, i.e., buphanamine and buphanindrine were subsequently isolated and found to have high affinity for the SERT and low affinity for serotonin receptor (5-HT1a) (Sandager et al. 2005). Thus, the crude extract or these bioactive constituents may elicit an anxiolytic response in the brain (Kent et al. 2002). In recent studies, Pote and colleagues (2013, 2014) reported that a hydroethanolic extract of the bulb of B. disticha marginally decreased blood pressure, immediately and several weeks after withdrawal of repeated oral treatments, and significantly reduced heart rate variability several days post treatment in maternally separated mice. Amongst other things, the authors attributed these effects to possible anxiolytic activity of the plant related to modulating the autonomic nervous system (Pote et al. 2014). Although the results of all studies cited above point to potential anxiolytic effects of extracts of B. disticha, none of there is no single in vivo study that was designed specifically to examine this using a scientifically validated animal models of anxiety. Therefore in view of this, and given the potential of B. disticha as an anxiolytic agent, the present study was set to determine if oral administration of hydroethanolic extracts of the plant in vivo, would have anxiolytic-like effects in naive BALB/c mice using three behavioural anxiety: Suok test (ST) (Hart et al. 2010; Kalueff and Tuohimaa, 2005; Kalueff et al. 2007), open field (OF) (Holmes et al. 2001, 2003; Mathis et al. 1994; Miyakawa et al. 2001) and elevated plus maze (EPM) (Pellow et al. 1985; Grundmann et al. 2007).
2.
Materials and Methods
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The study protocols, animal care and handling procedures, were conducted in accordance with the international guidelines on the use and care of experimental animals (European Community guidelines, EEC Directive of 1986) and were approved by The Joint Parirenyatwa Group of Hospitals - College of Health Sciences, University of Zimbabwe Research Ethical Committee (JREC) and the Division of Livestock and Veterinary Services, Ministry of Agriculture, Mechanisation and Irrigation Development, Zimbabwe.
2.1.
Plant material and extraction
2.1.1. Plant material Bulbs of B. disticha were harvested in Mashonaland East province of Zimbabwe and were authenticated by a taxonomist (botanist) from the Botanical Gardens and National Herbarium. A voucher specimen (reference number D.BUZPP4P 07-2011) was refrigerated in the School of Pharmacy, College of Health Sciences, University of Zimbabwe (Pote et al. 2013). 2.1.2.
Preparation of the crude extract
The crude extract was prepared as previously described by Gadaga et al. (2011) and Pote et al. (2013 and 2014) with few modifications. Briefly, the bulbs were sliced, sun dried for a week, oven dried at a temperature of about 50˚C and ground into a powder using a two-speed blender. Four samples of the powdered B. disticha bulb weighing approximately 100g each, constituting equal proportions of powdered dried inner scales and powdered dried outer scales were each added to 600 ml of hydroethanolic solvent (70% v/v). The four mixtures were left to stand overnight, refluxed the following morning for 1 hour at 70˚C on a heating mantle, cooled and left overnight again. The following day, the acquired hydroethanolic extract solution was decanted
9
into a beaker. Pressing the plant material with a spatula pushed out remaining extract. Extracts obtained from all four samples were mixed and placed in one volumetric flask. The extract mixture was then vacuum with Whatman filter paper No. 1, to get rid of residual fine particulate plant material. The filtrate obtained (1800 ml) was rotary evaporated at 90 revolutions per minute at 80˚C with a Heidolph 4000 Rotavapor (Heidolph, Germany) to remove solvent to a volume of 100 ml, which was then freeze-dried overnight at temperatures between -40˚C and -20˚C. A semi-solid crude extract (32.06g) was obtained and kept in an airtight container placed in a desiccator at room temperature.
2.1.3. Qualitative analysis of alkaloids Thin layer chromatography, viewed under UV light of 365nm wavelength, revealed that isoquinoline alkaloids were present in the crude extract of B. disticha used in this study. Most of the alkaloids were detected under strongly basic conditions whilst few others were detected under the neutral to weakly basic conditions (Zulu, 2011).
2.2.
Animal Husbandry
Forty-five adult male BALB/c mice (8-12 weeks old) weighing between 20 and 35 g were purchased from the Animal House Unit, University of Zimbabwe (UZ) and transported to the animal holding facility at the Clinical Pharmacology Department, College of Health Sciences (CHS), UZ. The animals were housed in standard Plexiglas cages (4-5 mice per cage) and allowed to acclimatise for two weeks to the holding room. Temperature in the animal holding facility was 22 ± 20C with a humidity of 60 ± 5% and lighting was maintained using the natural cycle of the day, light phase was at 5 am and dark phase at 7 pm. Wood shavings were used as
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bedding. Food and water was available ad libitum throughout the study except when the animals were fasted by removing both water and feed for 18 hours before the behavioural testing. All behavioural experiments were carried out, between 8am and 4pm during the light phase, in a sound proof room located at the College of Health Sciences, University of Zimbabwe. Soon after each test, the animal was placed back into its home cage. All test apparatus were cleaned thoroughly with 70% (v/v) aqueous ethanol and dried before putting another animal to preclude the possible cueing effects of odours left by previous subjects. After the neurobehavioural experiments, the mice were returned to the Animal House Unit (UZ) and humanely sacrificed by chloroform euthanasia.
2.3.
Treatment groups and dosage administration
Adult male mice were randomly assigned to five groups for the first experiment and tested in the Suok test (n = 6-9 mice in each group with a total of 36 animals) followed by open field (n = 8-10 mice in each group with a total 45 animals). The other cohort of mice (n=40) were also randomly assigned to five similar treatment groups (n = 8 each) for the second experiment and tested in the elevated plus maze. The five treatment groups included the vehicle control, three B. disticha treatment groups and a positive control group. The vehicle control group received normal saline while the positive control group received 1mg/kg of diazepam. Each of the B. disticha groups received 10, 25 or 40mg/kg of the hydroethanolic extract of the plant suspended in normal saline. All treatments were given as a single dose by oral gavage at a volume of 10 ml/kg one hour before behavioural testing was conducted.
2.4.
Experiment 1
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In this experiment, forty-five mice were randomly assigned to five treatments as described above (n = 8-10 each) and were placed in cages with 4 or 5 animals each. Cage number and tail markings were used to identify the animals. Before the testing day, the mice were fasted by removing both water and feed for 18 hours. On the testing day, all the animals were moved to the temporary holding room in holding cages on a trolley and allowed to rest for one hour to acclimatise to the test room. Each animal was weighed and was orally gavaged 30 minutes prior to testing with the respective treatment described above. Each animal was first tested using the Suok test (ST) for ten minutes (Kalueff and Tuohimaa, 2005; Kalueff et al. 2007, 2008a), rested for another ten minutes, placed in the open field (OF) and then observed for five minutes (Mathis et al. 1994; Holmes et al. 2001, 2003; Miyakawa et al. 2001). The anxiolytic activity was assessed based on four behavioural domains: exploration, displacement, risk assessment and motor co-ordination.
2.4.1.
Suok test (ST)
The Suok test (ST) apparatus consisted of a 2 m aluminium rod, with a diameter of 3 cm, covered with masking tape to improve grip. The rod was marked into 20 sectors, each measuring 10 cm, by line drawings. There was a 20 cm virtual two-sector central zone about the placement point at the centre of the rod. This rod was also elevated to a height of 20 cm from the floor and fixed to the central groove of two wooden end walls which measured 40 cm (height) x 40 cm (width) x 2 cm (thick). Paper towels were cushioned underneath the rod to avoid any harm to the animals which fell from the rod. The experimental room was dimly lit with a 40 watt light bulb fixed two meters above the apparatus. A digital camera was positioned about 1 metre away from
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the apparatus to record all movements done by the mouse. A mouse was placed on the central zone of the rod and left to explore the apparatus for a period of 10 minutes. The following anxiety-related behaviours were then scored: exploration (horizontal activity [number of sectors visited and number of line crossings] and vertical activity [number of rears and wall leanings, head dips and number of stops]); displacement: (number of grooming behaviour); risk assessment behaviour (number of times of stretch attends postures); and motor coordination (number of missteps) (Hart et al. 2010; Kalueff and Tuohimaa, 2005; Kalueff et al. 2008a). When number of sectors visited, rearings and head dips are high, it means the mouse is not anxious, but, if the number of stops is high, it shows that the animal is anxious (Kalueff et al. 2008a).
2.4.2. The open field (OF) The open field (OF) apparatus consisted of a rectangular orange plastic box of 42 cm (length) x 30 cm (width) x 18 cm (height). The apparatus was placed on a table with a height of 62 cm from the ground. The floor of the box was divided by lines into 12 sectors measuring 10 cm x 7 cm. The experimental room was dimly lit with a 40 W light bulb fixed about two meters above the apparatus. A digital camera was positioned about 75 cm above the apparatus to record all movements done by the mouse. A mouse was placed on the central square and allowed to explore the apparatus for 5 minutes. The following anxiety-like behaviours were scored: exploration (number of line crossing, number of vertical rears, number of centre square entries and number of stops); displacement (number of grooming behaviour); and risk assessment behaviour (number of stretch attends posture) (Hart et al. 2010; Holmes et al. 2001, 2003; Miyakawa et al. 2001).
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2.5.
Experiment 2
Forty mice included in this experiment were randomly assigned to five treatment groups, housed four animals per cage, tail marked, fasted, weighed and given respective treatments as described in experiment 1. Thirty minutes after gavaging, each animal was then placed on the elevated plus maze and observed for 5 minutes (Pellow et al. 1985).
2.5.1.
The elevated plus maze (EPM)
The elevated plus maze (EPM) consisted of two open arms (25 × 5 cm) crossed with two closed arms (25 × 5 × 20 cm) made from wood. The maze was elevated to a height of 40 cm, while, a 40 W light bulb was fixed 80 cm above the maze and an overhead digital camera was fixed 110 cm above the maze. Mice were taken from the home cage by the base of the tail and placed individually on the EPM facing an open arm. Mice were observed for five minutes and number of entries and time spent in open and closed arms was recorded. The percentage time spent on the open arms was calculated (100 × open arm time / 300s) for each animal (Grundmann et al. 2007). Arm entries were defined as entry of all four paws into an arm (Pellow et al. 1985). Percentage open arm entries were calculated for each animal as well (100 x number of open arm entries/(total entries into open arm and closed arms). Other anxiety-like behaviours scored were, number of head dips (HD), stretch attend posture (SAP) and rearings. For SAP and HD a distinction was made between ‘protected’ (if the behaviours are displayed from the central platform or closed arm) and ‘unprotected’ (if it occurs while being in an open arm). Percentage of unprotected head dips was calculated for each animal (100 x unprotected head dips / total number of head dips) or SAP (100 x unprotected SAP /total number of SAP). Anxiety-like behaviours were
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classified as exploration (open arm time, open arm entries, rearing and head dip); and risk assessment (number of stretch attends posture) (Hart et al. 2010; Mathis et al. 1994; Holmes et al. 2001, 2003; Miyakawa et al. 2001). The EPM was cleaned with 70% v/v ethanol solution and tissue paper to remove any residual scent.
2.6.
Statistical analysis
Statistical analysis was performed using Graph Pad Prism® 7.0 software. One-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was used to compare groups. Data was presented as mean ± standard error of mean (SEM) and significant level was considered at p<0.05.
3.
Results
3.1.
Suok test
Treatments had significant effect on number of sectors visited and lines crossed (F4,31 = 2.962, p = 0.0350; Figure 1A and F4,31 = 3.795, p =0.0126; Figure 1B, respectively).
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Figure 1. Effects of B. disticha on anxiety-related explorative behavior measured by the Souk Test (n= 6-9 in each group). Number of: A. sectors visited, B. line crossings, C. rearings, D. head dips and E. stops. BD, B. disticha; DZP, diazepam.*p < 0.05 vs. vehicle control, ## p < 0.01 vs. diazepam.
However, treatment of mice with either B. disticha or diazepam had no effect on the number of rearings, head dips, stops, groomings, SAP and missteps (F4,31 = 1.673, p = 0.1814 (Figure 1C); F4,31 = 2.123, p = 0.1016 (Figure 1D), F4,31 = 1.134, p = 0.3587 (Figure 1E), F4,30 = 1.42, p = 0.2513; Figure 2A), F4,31 = 2.062, p = 0.1099, Figure 2B), and F4,31 = 1.767, p = 0.1607, Figure 2C), respectively). Groups treated with either B. disticha at 25mg/kg or diazepam, visited significantly more sectors than the vehicle control group (p<0.05, Figure 1A). Animals given diazepam significantly crossed more lines than either the vehicle control or the group treated with B. disticha at 40mg/kg (p<0.05 and p<0.01, respectively, Figure 1B). And, mice treated with
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25mg/kg B. disticha expressed significantly more head dip behaviours when compared to the vehicle control group (p<0.05).
Figure 2. Effects of an ethanol extract of B. disticha on risk assessment behavior and motor activity in the Souk Test (n= 6-9 in each group). Number of: A. grooming behaviour; B. number of stretch attend postures; and C. missteps. BD, B. disticha; DZP, diazepam.
3.2.
Open Field
Treatments had significant effect on the number of rearings in the open field (F4,40 = 2.87, p = 0.0351; Table 1). However, treatments did not affect the number of stops, line crossings, centre square entries, grooming and stretch attend posture in the open field (F4,40 = 1.386, p = 0.2560, F4,40 = 2.13, p = 0.0949, F (4, 40) = 0.5178, p = 0.7231, F4,40 = 1.374, p = 0.2603 and F4,40 = 1.244, p = 0.3079, respectively; Table 1). Diazepam-treated mice expressed significantly higher rearing and line crossings scores than the control and the 40mg/kg B. disticha treated groups, respectively (p<0.05, Table 1).
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Table 1. Effects of B. disticha hydroethanolic extract anxiety-like behavior in the open field.§ Treatment Group
Parameter
Control
BD10mg/kg
BD25mg/kg
BD 40mg/kg
DZP 1mg/kg
Line crossings
65.75±11.95
56.70±6.32
63.67±4.33
47.30±5.83##
75.63±7.07
Centre Square entries
4.50±0.96
7.13±1.04*
4.88±0.69
4.86±0.74
5.14±0.86
Rearings
17.33±2.99
16.13±2.88
20.13±1.52
19.14±1.50
81.57±4.41*
Stops
1.33±0.615
0.375±0.183
0.875±0.350
0.286±0.184
0.571±0.429
Groomings
4.83±0.95
4.63±0.71
5.13±0.83
5.43±0.84
3.57±0.78
SAP
5.67±0.61
7.875±1.246
7.000±1.309
7.43±2.29
10.57±2.79
§Each group (n= 8-10); BD, B. disticha; DZP, diazepam; SAP, stretch attend posture. *p<0.05 vs. Control; ##p<0.01 vs. DZP.
3.3.
Elevated plus maze
Animals treated with B. disticha extract, at 10mg/kg dose, spent significantly more time in open arms and scored significantly higher number of open arm entries than the vehicle control group (p<0.05, Table 2). It was also observed that mice treated with ether diazepam or B. disticha at 10, 25 and 40mg/kg had significantly higher number of unprotected head dips than the vehicle control group (p<0.05, p<0.05, p<0.005 and p<0.01, respectively; Table 2). And, mice treated with either diazepam or B. disticha at doses of 10 and 25mg/kg, had significantly higher scores of stretch attend postures
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than the control (p<0.01, p<0.05 and p<0.05, respectively Table 2). However, animals treated with either B. disticha or diazepam expressed same scores of rearing behaviour as the control group (p > 0.05) (Table 2).
Table 2. Effects of B. disticha extract on anxiety-like behaviours in the elevated plus maze. §
Treatment Group
Control
BD10mg/kg
BD25mg/kg
BD 40mg/kg
DZP 1mg/kg
Open arm entries (%)
35.36±3.99
49.08±3.91*
45.53±2.75
46.14±3.45
46.74±1.50
Time spent in Open arms (%)
17.83±4.22
45.24±9.46*
34.76±4.82
31.42±6.55
25.44±6.85
Unprotected Head dips (%)
46.83±9.97
86.11±3.17*
84.64±4.79***
73.77±7.45**
78.15±8.97*
Unprotected SAP (%)
26.49±11.25
66.17±7.69**
60.83±6.11*
42.51±6.19
57.02±7.47*
Rearings
19.17±4.02
13.43±1.80
17.00±1.35
13.75±1.22
15.83±2.47
Parameter
§Each group (n = 8 in); BD, B. disticha; DZP, diazepam; SAP, stretch attend posture. *p<0.05, **p<0.01, ***p<0.001 vs. control.
4.
Discussion
The present study was set to investigate the effects of B. disticha hydroethanolic extract on anxiety-like behaviours in mice. Diazepam was used as the reference drug (positive control) and was used to determine the predictive validatity of the paradigm employed in this study. This study reported that the diazepam-treated group expressed higher number of sectors visited and line crossings in the ST than the control, which
19
indicated that diazepam increased horizontal explorative activity (Kalueff et al. 2008a and 2008b) and decreased anxiety in mice. Meanwhile, the former group also expressed higher scores of rearings than the latter group in the OF which suggested that diazepam increased vertical exploratory activity and reduced anxiety. In addition the former group scored more number of head dips in the EPM than the control indicating that diazepam reduced fear and anxiety in these animals. These results supported findings reported in previous studies demonstrating that a standard anxiolytic drug like diazepam elicits anxiolytic-like behaviours in the ST (Kalueff et al. 2008a), OF and EPM (Cryan and Sweeney, 2011). These results confirmed that the tests selected in this study (ST, OF and EPM) and the study design were valid for screening substances with potential anxiolytic activity (Brown et al. 1999). The present findings, therefore, established that diazepam effectively reduced anxiety-like behaviours and showed that the behavioural paradigm employed in this study was pharmacologically valid (Cryan and Sweeney, 2011).
The current findings also demonstrated that B. disticha extract increased horizontal activity (number of sectors visited) at 10mg/kg, vertical explorative activity (unprotected head dips) at 25mg/kg, and directed explorative activity (unprotected head dips) at all doses in the ST. Furthermore, the extract increased horizontal exploratory activity and risk-taking behaviour (open arm time and entries), and vertical explorative activity (unprotected head dips) in the EPM. These consistent observations that animals treated with B. disticha expressed higher exploratory activity in both the ST and EPM tests than the vehicle control suggest that the extract effectively reduced anxiety and fear in mice (Brown et al. 1999, Heinrichs et al. 1992; Rodgers and Cole, 1993; Holmes et al. 2001, 2003; Miyakawa et al. 2001). Thus B.
20
disticha exhibit an anxiolytic-like activity at doses of 10 and 25mg/kg (Mathis et al. 1994; Holmes et al. 2001, 2003).
In a contrary observation, the present study found that mice given B. disticha extract at doses of 10 and 25mg/kg exhibited higher scores of unprotected SAP in the EPM indicating increased risk assessment activity, which is suggestive of increased fear and anxiety. This paradoxical finding might have been due to the fact that the hydroethanolic extract may affect exploration and risk assessment behaviour in different ways (Bourin et al. 2007; Navarro et al. 2006). Risk assessment is a behavioural measure that indicates that the animal is hesitant to move from its present location to a new position and thus a high frequency of these postures indicates a higher level of anxiety (Brown et al. 1999).
The failure of B. disticha extract to affect other anxiety-like behaviours in the ST, OF and EPM, could be due to several reasons. Missteps can reflect vestibular, motor coordination or neuromuscular deficits or stress-evoked sensori-motor disintegration (SSD) (Kalueff et al. 2008a). Hence, current findings showing no difference between groups on missteps suggest that B. disticha extract does not cause motor incoordination and hallucinations in mice at doses between 10 and 40mg/kg. This is consistent with previous studies, which reported that at doses less than 50mg/kg do not elicit SSD and hallucinations seen in higher doses (Gadaga et al. 2011; Gadaga, 2012; Ganga et al. 2017; Mutseura et al. 2013). Furthermore, the lack of effects on grooming behaviour was not surprising since this measure is not a reliable indicator when screening potential anxiolytic drugs (Kalueff et al. 2008b). Grooming is a behaviour that shows displacement activity seen when the mouse, while stationary,
21
licks, or scratches using front or back paws, displayed in a novel environment showing that the mice is anxious (Brown et al. 1999).
A combined agonism/antagonism occurring at different receptors by various compounds in the crude extract might also account for the absence of effects on such behaviours (Grundmann et al. 2007). Lastly, it has been shown that drugs that act on the serotonin system do not show significant difference in the number of open arm entries in the EPM (Dawson and Tricklebank, 1995).
The anxiolytic activity demonstrated by the plant extract in this study may be due to the active alkaloidal phytoconstituents reported by previous studies (Bastida et al. 2011; Botha et al. 2005; Cheesman et al. 2012; Nair and Van Staden, 2014; Nair et al. 2013; Neergaard et al. 2009; Nielsen et al. 2004; Viladomat et al. 1997; Gadaga et al. 2011). The alkaloids may act directly or indirectly through their active metabolites released into the blood from the liver and/or other tissues, or by the brain tissue into the surrounding tissue and/or cranium blood supply. It may be likely that the extract modified various neurotransmitter systems because numerous neural pathways are involved in the pathophysiology of anxiety disorders (Kent et al. 2002). These pathways include the monoamines: 5-hydroxytryptamine (5-HT), noradrenaline and the γ amino butyric acid (GABA)-benzodiazepine receptor complex and a number of unrelated compounds that are known to provoke anxiety and/or panic in humans (Sandford, 2000). The alkaloids or their metabolites may exert their effects by binding on the serotonergic receptors (5-HT1a receptors) or the serotonin transporter protein (SERT) in the central nervous system (Nair and Van Staden, 2014; Neergaard et al. 2009; Nielsen et al. 2004; Sandager et al. 2005). The absence of activity in some
22
behavioral parameters in the current results could explain and also further support the theory that B. disticha extract’s anxiolytic actions work through the serotonin system. However, the behavioural tests employed in this study do not allow discerning of the underlying mechanism of action of B. disticha extract. Hence, more neurochemistryrelated work would need to be done to investigate these hypotheses. Further studies also need to ascertain the activity of the extract in pathological animal models of anxiety disorders and to identify the bioactive compounds responsible for the anxiolytic-like activity of the plant extract.
5.
Conclusion
The current study demonstrated for the first time that a hydroethanolic extract of B. disticha has anxiolytic-like activity in naïve BALB/c mice. The extract of B. disticha exerted good anxiolytic-like activity in both the souk test and open field at medium dose (25mg/kg), while the low dose (10mg/kg) showed prominent anxiolytic-like activity in the elevated plus maze. The identification that the activity of the plant is similar to the well-known conventional anxiolytic diazepam supports its safe use as a natural health product. However, future studies are required to elucidate concisely the mechanism underlying anxiolytic activity of the plant extract.
Acknowledgements: We are very grateful to Mr Murambiwa and staff at the of Clinical Pharmacology Department, Food Science Department, School of Pharmacy, and Animal House Unit at University of Zimbabwe. Funding: This work was supported by IFS (grant number F/4187-1). Conflicts of interest: none
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