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Cardiovascular Effects Of Boophone DistichaAqueous Ethanolic Extract On Early Mater-nally Separated Balb/C Mice
Pote William, Tagwireyi Dexter, ChinyangaHerbertM. , Musara Collin, Nyandoro George,Chifamba Jephat, Nkomozepi Pilani
PII: S0378-8741(13)00142-6DOI: http://dx.doi.org/10.1016/j.jep.2013.03.001Reference: JEP7964
To appear in: Journal of Ethnopharmacology
Received date: 11 December 2012Revised date: 12 February 2013Accepted date: 1 March 2013
Cite this article as: Pote William, Tagwireyi Dexter, Chinyanga HerbertM. ,Musara Collin, Nyandoro George, Chifamba Jephat, Nkomozepi Pilani,Cardiovascular Effects Of Boophone Disticha Aqueous Ethanolic Extract OnEarly Maternally Separated Balb/C Mice, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2013.03.001
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1
CARDIOVASCULAR EFFECTS OF BOOPHONE DISTICHA AQUEOUS ETHANOLIC
EXTRACT ON EARLY MATERNALLY SEPARATED BALB/c MICE.
a,*Pote William., bTagwireyi Dexter., cChinyanga Herbert. M., aMusara Collin., aNyandoro
George., cChifamba Jephat., and dNkomozepi Pilani.
aDepartment of Preclinical Veterinary Studies; bDrug and Toxicology Information Service-
School of Pharmacy and c Department of Physiology, University of Zimbabwe P O Box MP167,
Mount Pleasant Harare Zimbabwe and dDepartment of Neuroscience, University of
Witwatersrand, South Africa.
*Corresponding author. Tel.: +263773234621/+263736566176 Fax: +2634333678
E-mail address [email protected]/[email protected] (W. Pote)
Abstract
Ethnopharmacological relevance: There are a number of reports from traditional medical
practice in Zimbabwe and neighboring countries and few in-vitro studies suggesting an effect
with extracts of Boophone disticha in some forms of anxiety disorders.
Aim of the study: In order to validate the use of B. disticha in treatment of anxiety, this study was
set to determine the effects of the plant extracts on blood pressure (BP) and heart rate (HR) in
adult BALB/c mice subjected to repeated early maternal separation (MS) stress.
Materials and Methods: To test whether early life stress increases anxiety in mice, non-invasive
tail cuff method was used to examine the autonomic nervous system activity by assessing
cardiovascular reactivity and response to acute mixing stress (AMS) and restraint stress (RS) in
adult mice subjected to early postnatal stress as compared to control. AMS-induced
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cardiovascular response was then evaluated in adult MS mice treated with B. disticha as
compared to vehicle and diazepam.
Results: Comparisons of the BP and HR measurements indicated that MS significantly reduced
AMS-induced HR responses in BALB/c mice when compared with control. B. disticha treatment
significantly reduced AMS-induced BP response in BALB/c MS as compared to vehicle and
diazepam treatments.
Conclusions: Our findings demonstrate for the first time that postnatal stress can induce short-
term changes in the sensitivity of the cardiovascular system to subsequent stress which can be
reduced by treatment with a frieze dried aqueous ethanolic extract of B. disticha.
Keywords: Boophone disticha; maternal separation, anxiety disorders, blood pressure, heart rate.
Abbreviations: AMS, acute mixing stress; ANS, autonomic nervous system; BP, blood pressure;
DBP, diastolic BP; HR, heart rate; MAP, mean arterial pressure; MS, maternal separation; PND,
postnatal day; RS, restraint stress; SSRI, selective serotonin reuptake inhibitor; SBP, Systolic
BP; TLC, thin layer chromatography.
1. Introduction
Anxiety disorders are the most common psychiatric illnesses encountered in clinical practice.
Often, they are chronic conditions, associated with substantial cardiovascular-related morbidity
and mortality for sufferers, as well as considerable costs to society (Schwartz and Nihalani,
2006). Research has shown that individuals displaying symptoms of anxiety or depression are at
higher risk for cardiovascular-related morbidity and mortality (Larsen and Christenfeld, 2009).
However, treatment of anxiety disorders is still limited since current drugs are effective only in a
3
certain proportion of patients hence the need to develop effective treatment options (Schwartz
and Nihalani, 2006). Nonetheless, efforts are continuously being made to identify and develop
new and improved herbal remedies and lead compounds for drug discovery.
One of the psychoactive plants that have been widely used in herbal medicine is Boophone
disticha (L.f.) Herb. belonging to the Amaryllidaceae family. B. disticha, commonly known as
munzepete in Shona or Ingcoto in Isindebele, has been used systemically in herbal medical
practice in Zimbabwe and neighbouring countries for the management of various central nervous
system conditions including anxiety and depression (de Smet, 1996; du Plooy et al. 2001;
Gadaga et al. 2011; Gelfand et al. 1985; Perderson et al. 2008; Stafford et al. 2008). There is,
however, limited literature, most of it is unpublished, that have demonstrated in vitro and in vivo
effects in managing depression and anxiety, (Chingombe et al. 2010, Chuma et al. 2010;
Mungadzi et al. 2008; Musarira et al. 2011; Neergaard et al. 2009; Pederson et al. 2008;
Sandager et al. 2005). Crude extracts of the leaves have affinity to the selective serotonin
reuptake inhibitor (SSRI) site on the serotonin transporter in a radio-ligand binding assay
(Nielsen et al. 2004; Sandager et al. 2005). Several alkaloids from this plant have been isolated
and identified (Adewusi et al. 2012; Cheesman et al. 2012; de Smet, 1996; Hautch and
Stauffacher, 1961; Neergaard et al. 2009; Steenkamp, 2005).
Maternal separation (MS) is one animal model that has been studied extensively to characterize
the long-term effects of early life experience on subsequent behavior in adulthood and the
development of psychiatric disorders such as anxiety and depression (Daniels et al. 2009; Faure
et al. 2006; Plotsky and Meaney, 1993; Pryce and Feldon, 2003). Repeated early maternal
4
separation stress have demonstrated an altered cardiovascular response which may be mediated
by changes in the adjustment of the tone in the parasympathetic and sympathetic nervous system
and glucocorticoid effects in limbic structures in rodents (Grillison et al. 2007; Igosheva et al.
2004; Volkmar et al. 2005). Previous studies have focused on the short-term and long-term
effects of maternal separation, including changes in neurobehavior and neurophysiology but
lacked evidence of possible interventions to curb or reverse associated dysfunctions.
Given the extensive illicit and medicinal use of B. disticha as well as its potential as a source of
psychoactive pharmacological therapies, it is important to accurately validate its possible
anxiolytic short term and long term pharmacological effects. B. disticha extract may be a useful
addition to currently available drugs for anxiety; however, the data from limited in vitro studies
remain to be confirmed in controlled in vivo preclinical and clinical investigations. Since there is
little scientific evidence on the in vivo effects of B. disticha in treatment of anxiety disorders in
animals or humans the present study was set to validate the use of B. disticha in treatment of
anxiety and cardiovascular comorbid disorders. It was hypothesized that mice subjected to
maternal separation develop cardiovascular changes which may be reversed by treating the
underlying anxiety disorders with a freeze dried aqueous ethanolic extract of B. disticha.
2. Materials and methods
The experimental protocols, care and handling of animals used in this study were in accordance
with international guidelines and Zimbabwean legislation on the use and care of laboratory
animals and were approved by the Division of Veterinary Services, Zimbabwe.
2.1.Plant materials and extraction
5
2.1.1. Plant material and preparation of the crude extract
Bulbs of Boophone disticha were harvested at Melford plains close to Marondera in December
2010. The plant sample was authenticated by a taxonomist from the Botanical Gardens and
National Herbarium and a voucher specimen (reference number D.BUZPP4P 07-2011) was
retained and refrigerated in the department of Clinical Pharmacology, College of Health Sciences
University of Zimbabwe. The crude aqueous ethanolic (70% v/v) extract was prepared as
previously described by Gadaga et al. (2011).
2.1.2. Qualitative analysis of alkaloids
The data and results on extraction, purification and chromatographic analysis (thin layer
chromatograph (TLC) and column chromatograph) of alkaloids in the freeze dried aqueous
ethanolic extract of B. disticha obtained above have been described elsewhere by Zulu et al.
(2011) and therefore the detailed procedure and results were not included in this report.
2.2.Animals and housing conditions
Twenty-two pregnant dams of BALB/c mice were purchased from the Central Veterinary
Laboratory Breeding Unit in The Ministry of Agriculture, Lands and Irrigation Development,
Zimbabwe. Study animals were kept in the Animal House facility at University of Zimbabwe
throughout the experiments. Each pregnant dam was separated and kept in an individual cage
under standard conditions. Close to delivery, pregnant mice were carefully monitored to
determine the exact day of birth. Birth was designated as postnatal day 0 (PND0). At birth, dams
were assigned randomly into two groups: Unseparated control group (Group A) and repeated
early maternal separation (MS) group (Group B) and enriched with 5-6 pups each to standardize
conditions and ensure an adequate milk supply until weaning age. Plexiglas cages with wood
6
shavings as bedding were used. Temperature was maintained between 20 and 24°C; light/dark
cycle was constant (12/12, light on from 06.00 h to 18.00 h); humidity was maintained between
52 and 60%; and, food and water were available ad libitum for the full duration of the
experiment.
2.3.Experimental protocols
2.3.1. Postnatal stress
The protocol for maternal separation was previously described in detail by Faure et al. (2005)
and Loria et al. (2010). Briefly, the offsprings were left undisturbed on the day of birth (PND0)
and day 1 after birth (PND1). Maternal separation occurred daily from PND2 until PND14 for
180 minutes between 0930 hours and 1500 hours (Faure et al. 2006). Maternal separation
involved removal of the dam from the home cage containing the pups. This was done in a
dedicated room in which the room temperature was maintained between 30�C and 33oC. The
dam was returned to the colony housing room during the deprivation period. After the 3 h
maternal deprivation period, the dam was placed back with the pups and returned to the colony
housing room. Control pups were not subjected to maternal deprivation between postnatal days 2
and 14. Pups were weaned at PND 21 by removing the dams from test cage and placing them in
separate cages. Male and female offsprings were separated on PND28, housed with 3-4 pups per
cage at 5 weeks and 2–3 mice per cage at 8 weeks of age.
2.3.2. Grouping and drug administration
On PND60, six mice (3males and 3 females) were randomly selected from un-separated litter,
Group A (control) and thirty (15 males and 15 females) were randomly selected from offs MS
litter, Group B; and were randomly assigned to five treatment groups, Group B, C, D, E, and F,
7
(n=6 each; 3males and 3 females). Mice with body weight between 20 and 30g were included in
this study. Males and females were housed in separate cages and handled daily for one week
before starting the experimental protocol. From PND71 to PND 76 all mice were given the
following doses by oral gavage. Group A (Control+NS) received normal saline; Group B
(MS+NS) received normal saline; Group C (MS+LDBD) received 10mg/kg body weight (BW)
low dose of B. disticha; Group D (MS+MDBD) received 25mg/kg BW medium dose of B.
disticha; Group E (MS+HDBD) received 40mg/kg BW high dose of B. disticha; Group F
(MS+DZP) received 1mg/kg BW diazepam (positive control).
2.3.3. Acute mixing stress and cardiovascular parameters
A model of acute mixing stress (AMS) that combines mixing with unfamiliar cage mates of the
same sex followed subsequently by restraint stress was developed. On PND 78 (day 2 post-
treatment), test mice were transferred to the testing room 1 hour before testing for
acclimatization and then subjected to adulthood acute stress in the form of 30 minutes of mixing,
acute mixing stress (AMS). Briefly, a single mouse from each cage (Group A, B, C, D, E and F)
was tail marked for identification and mixed in a single cage with five other unfamiliar mice
from different cages of the same sex for 30 minutes (AMS). AMS was followed by placing the
mixed mice in a heating chamber for thirty minutes to warm them to 32°C and a 10 minutes
restraint stress (RS) during blood pressure (BP) and heart rate (HR) recording. All mice were
acclimatized to the restrainer tube prior to actual experimentation to minimize trauma associated
with entry into the tubular plexiglas restrainers.
Hemodynamic parameters; blood pressure and heart rate, were monitored by non-invasive tail
cuff methods using BIOPAC NIBP200A system following manufacture’s guidelines (BIOPAC
8
System Inc, CA). Systolic blood pressure (SBP), diastolic BP (DBP), mean arterial pressure
(MAP), and heart rate (HR), were recorded for 10 minutes between 09.00 and 15.00 h. using the
NIBP200A MP36 Acquisition program (BIOPAC Systems, Inc., CA). Animals were returned to
the UZ Animal holding unit after the experiments.
2.4.Statistical analysis
All data are expressed as mean ± standard error of mean (S.E.M) with a significance level of p<
0.05. Hemodynamic parameters were analysed using SPSS version 16.0 and Minitab version
15.0. Unpaired t-test and 2-way ANOVA was performed for each of the cardiovascular
parameters and comparisons of the means of SBP, DBP, MAP and HR, between groups.
3. Results
3.1.The effects of Boophone disticha on hemodynamic response to MS in adulthood
To study the effects of repeated early maternal separation on the cardiovascular parameters in
response to acute stressors during adulthood, the means of blood pressure (BP) and heart rate
(HR) of MS mice(Group B) was compared to control mice (Group A). The dose dependent
effects of a aqueous ethanolic extract of B. disticha on mean BP and HR of MS mice were then
determined following six daily oral administration of low (LDBD, Group C), medium (MDBD,
Group D) and high (HDBD, Group E) doses (10, 25 and 40mg/kgBW/day) and the results were
compared to vehicle (0.9% normal saline, Group B) and diazepam (1mg/kgBW/day, group F) on
PND 78. Figure 1 to Figure 4 below shows the measurements of blood pressure (BP) and heart
rate (HR) across experimental groups described above.
3.1.1. Systolic blood pressure in response to acute mixing stress and in control, MS and MS
treated BALB/c mice
9
Maternal separation significantly increased SBP as compared to control response in adulthood
(133.8±1.9 and 129.51±1.2mmHg respectively, p<0.05). Mice treated with low dose B. disticha
B. (LDBD; Group C) exhibited significantly decreased SBP (127.9±1.4mmHg) as compared to
both vehicle and diazepam (133.8±1.9 and 132.3±0.3mmHg respectively, p<0.05; Figure 1).
Although medium and high doses of B. disticha did not affect acute SBP stress responses, both
treatments seams to decrease SBP two day post treatment recovery period when challenged with
acute stressors (Figure 1).
Figure 1. Graph illustrating how Boophone disticha treatment in BALB/c mice after maternal
separation affected systolic blood pressure (SBP) as compared to control vehicle and diazepam.
3.1.2. Diastolic blood pressure responses to acute stress and recovery in control and
MS+treatment BALB/c mice
Changes of DBP in control and MS+treatment mice following mixing stress recorded two days
post treatment recovery period are presented in Figure 2. MS stress had no significant effect on
124
126
128
130
132
134
136
Systolic Blood
Pressure (m
mHg)
Experimental Groups
10
the DBP response. Medium (MDBD) and high (HDBD) doses of B. disticha had similar DBP
results (94.3±0.51 and 93.2±1.8mmHg respectively) when compared to the vehicle (95.6±3.0,
but, significantly lower when compared to diazepam (97.7±0.5mmHg, p<0.05). No significant
effect was observed on DBP of MS mice treated with either B. disticha or diazepam when
compared to vehicle. A dose dependent trend was observed on DBP with increasing doses of B.
disticha significantly decreasing DBP. Low and medium doses of B. disticha had significantly
lower DBP than high dose B. disticha (p<0.05; Figure 2).
Figure 2. Graph illustrating how Boophone disticha treatment in BALB/c mice after maternal
separation affected diastolic blood pressure (DBP) as compared to control vehicle and diazepam.
3.1.3. Mean arterial pressure (MAP) responses in control and MS treated BALB/c mice
Figure 3 shows changes in mean arterial pressure during restraint stress and following acute
mixing stress on PND78, two days post-treatment recovery period, in control and MS and MS +
treated (with B. disticha and diazepam) mice. MAP was neither affected by repeated early life
stress (MS) nor with six daily treatments with B. disticha or diazepam when compared to
84
86
88
90
92
94
96
98
100
102
104
Diastolic Blood
Pressure (m
mHg)
Experimental groups
11
vehicle. However, low and high doses of B. disticha were found to significantly decrease MAP
(109.7±1.3 and 111.3±0.3mmHg, respectively) as compared to diazepam (113.8±0.35, p<0.05
and 0.001 respectively; Figure 3).
Figure 3. Changes in mean arterial pressure (MAP) in control and postnatally repeated MS
stressed BALB/c mice. The graph also shows how Boophone disticha treatment affected Mean
arterial pressure measurements in BALB/c mice after maternal separation as compared to control
vehicle and diazepam.
3.1.4. Heart rate responses to acute mixing and restraint stress and recovery in control and
MS mice receiving vehicle, B. disticha and diazepam
The mean HR response to acute mixing stress and restraint stress on post-treatment day 2 is
shown in Figure 4. HR results were significantly lower in MS Group B, 480.5±16, as compared
to control, Group A, 568.9±27; (p<0.03). HR response was similar in magnitude in MS mice
treated with B. disticha, diazepam and vehicle. ANOVAs for HR stress values also revealed no
differences between doses of B. disticha.
102
104
106
108
110
112
114
116
118
120
Mean arterial Pressure (m
mHg)
Experimental groups
12
Figure 4. Diagram showing Changes in heart rate (HR) in control and repeated MS stressed
BALB/c mice. Also shown are effects of Boophone disticha treatment after maternal separation
as compared to control vehicle and diazepam.
4. Discussion
4.1.The cardiovascular response to repeated early maternal separation in adulthood
Blood pressure and heart rate response to B. disticha treatment was tested in a mouse model of
early life stress during adulthood. The present results indicate that maternal separation
significantly raised systolic blood pressure and markedly lowered heart rate when MS and
control mice were compared following acute stress (Figure 1 and 4). However, mean BP (DBP
and MAP parameters) was similar in MS and control animals (Figure 2 and 3). There is large
body of epidemiological evidence in humans showing that early social experiences can promote
phenotypic changes that persist in adult life (Loria et al. 2010). In humans, child abuse is a risk
factor for subsequent onset of mental disorders such as depression and post-traumatic stress
470480490500510520530540550560
Heart rate (beats per m
inute)
Experimental groups
13
disorder whilst in animals, different forms of early life stress resulted in altered behavior
(Daniels et al. 2004 and 2009; Faure et al. 2006; Marais et al. 2008).
As in previous studies, a maternal separation paradigm was used as a model for studying the
consequences of early adversity (Daniels et al. 2004 and 2009; Faure et al. 2007; Ladd et al.
2000; Slotten et al. 2006). This protocol was chosen because it has been used in many previous
studies and shown to significantly affect neuro-endocrine and neuro-behavioural stress reactivity
in offspring producing permanent alterations in brain neurotransmitter systems which are
involved in cardiovascular response to stress (Daniels et al. 2009; Faure et al. 2006; Ladd et al.
1996; Loria et al. 2010; Plotsky and Meaney, 1993; Pryce and Feldon, 2003).
Alterations in cardiovascular function of MS mice were evident when the animals were
challenged by acute mixing and subsequent restraint stress. The changes in HR observed in the
present study in MS mice may be similar to behavioral changes observed in previous
investigations in animals and humans which have shown that early adversity results in altered
behavior and neurophysiology (Daniels et al. 2004 and 2009; Faure et al. 2007; Ladd et al. 2000;
Slotten et al. 2006). This study has also shown that exposure to stress early in development can
have long-term effects on cardiovascular stress responses in adulthood. Reduced HR MS mice
values in response to acute stress as compared to control may indicate changes in autonomic
activity associated with abnormalities in heart rate variability (Cohen and Benjamin, 2006) and
anxiety in anxiety-like behavior (Loria et al. 2010). On the other hand, raised systolic BP may be
indicative of abnormal blood pressure variability (Cohen and Benjamin, 2006) and anxiety-like
disorders observed in previously studies (Faure et al. 2006). Furthermore, various cardiovascular
14
diseases have been shown to be associated with alterations in ANS function (Cohen and
Benjamin, 2006). Maternal separation has been observed previously that it leads to anxious-like
behavior and hyperactivity (Daniels et al. 2004 and 2009; Slotten et al. 2006). It was suggested
previously that elevated corticosterone levels are indicative of the disturbed function of the HPA
axis in separated animals (Daniels et al. 2009; Ladd et al. 2000; Kalinichev et al. 2002). These
findings and ours therefore support the hypothesis that stress leads to allostatic load and in order
to counter this, heart rate is decreased and blood pressure elevated. Decreased HR and raised BP
in MS mice may also be an indication of compensatory mechanisms that attempt to maintain
cardiac output and to combat the long-term deleterious effects of maternal separation associated
with increased neurotrophin levels and corticosterone release (Daniels et al. 2009; Faure et al.
2006).
4.2.The effects of Boophone disticha on hemodynamic response to MS in adulthood
The major hypothesis of this study that maternally separated mice treated with B. disticha would
show reduced autonomic reactivity and cardiovascular response shortly after acute mixing stress
during adulthood, was confirmed for the first time in the present mouse model. Our results
demonstrated that low dose B. disticha treated MS mice had the lowest SBP (Figure 1) but
showed no effect on DBP responses. On the other hand, high dose had the lowest DBP and MAP
(Figure 2). These changes may be indicative of increased recovery from anxiety in these animals
associated with Boophone treatment. The current results confirm findings from previous studies
in our laboratory with various mouse models of anxiety developed using open field test, elevated
plus maze and souk test that showed that the aquous ethanolic extract of B. disticha had
anxiolytic-like activity in these specific tests for anxiety (Chuma et al. 2010; Musarira et al. 2011
Unpublished results). These findings and current observation in an early life stress model that B.
15
disticha decrease the raised systolic and diastolic BP of MS mice with anxiety-like disorder at
when compared to a vehicle or diazepam may indicate the anxiolytic-like activity of the plant
extract through reduction of autonomic reactivity to acute stress. The current study, therefore,
demonstrated that blood pressure changes following early adverse events in the form of repeated
maternal separation are reversed or reduced by a freeze dried aqueous ethanolic extract of B.
disticha (Figure 1, 2 and 3). Neither B. disticha nor diazepam treatment had a significant effect
on mean HR (Figure 4) in MS subjected to adulthood acute stressors when compared to the
vehicle. Furthermore, diazepam did not show an effect on all BP parameters measured (SBP,
DBP and MAP) when compared to the vehicle (Figure1, 2 and 3).
The hypotensive-like effects of B. disticha treatment on the cardiovascular function in adulthood
may be due to alkaloids which are present in the crude extract. B. disticha extract was therefore
found to significantly decrease systolic BP and diastolic BP at 10 and 40 mg/kg respectively
which is likely to be a therapeutic anxiolytic-like activity of the extract investigated. These
effects are in contrast to previously reported hallucinogenic effects of the bulb in a poisoning
case that presented with tachycardia, raised BP, dilated pupil, pyrexia and laboured respiration
(du Plooy, 2001). The contradicting effects might be due to doses used in this study which were
below toxic levels of 50mg/kg BW found in previous study (Gadaga, et al. 2011). The present
findings effects may be direct effects on the heart or indirect effects to the autonomic nervous
system regulation of the cardiovascular system. Some studies in our laboratory have also shown
that the same extract would exert anxiolitic-like activity in a mouse model of anxiety of open
field test, elevated plus maze and souk test (Musarira et al. and Chuma et al. unpublished
results). Similar studies done by Pederson et al. 2008 on in a mouse and rat model of depression
16
of tail suspension test and forced swim stress used higher doses than ours and found
antidepressant-like dose dependent effects of B. disticha ethanolic extract at doses of 250 and
500mg/kg body which were higher than doses used in the present study. The Amaryllidaceae
alkaloids represent a still expanding group of isoquinoline alkaloids, which are found exclusively
in plants belonging to this family (Viladomat et al. 1997). Some alkaloids of this series, such as
Dubiusine, homolycorine, 8-O-demethylhomolycorine, lycorenine, Tazettine, haemanthamine or
papyramine may have a hypotensive effects which may be mediated by stimulation of β-
adrenergic receptors. For instance, lycorine shows antiarrhythmic action, appreciable inhibitory
activity against acetylcholinesterase, relaxant effects on arteries and positive ionotropic and
chronotropic effects on the heart. On the other hand, lycorenine has a vasodepressor action by
blocking α-adrenergic receptors, and produces bradycardia by modifying vagal activity (Bastida
et al. 2011).
The present study was to investigate the anxiolitic-like activity of B. disticha on the autonomic
response of the cardiovascular system by assessing BP and HR changes known to be associated
with anxiety-like disorders in animal models of anxiety. It was found that in the maternal
separation model, systolic BP was raised and HR decreased significantly. Previous studies had
shown that maternal separation is associated with increased anxiety-like behaviour in an animal
model. Several other studies done both in humans and animals have shown that early maternal
deprivation is associated with increased BP and decrease heart rate variability. These findings
may be indicative of increased risk to anxiety disorders in adulthood in face of acute social stress
like acute mixing and restraint stress administered to the current model. Such a pattern of
hemodynamic responses to stress following B. disticha treatment would have significant
17
consequences for adult cardiovascular health since in clinical studies, delayed blood pressure
(Floras and Senn, 1991) and heart rate (Cole et al. 1999; Watanabe et al. 2001) recovery to basal
levels after stress has been shown to correlate highly with increased cardiovascular morbidity
and mortality. Furthermore, augmented blood pressure variability is found in patients with
hypertension as well as anxiety disorders and it may play a pathophysiololgical role in the
development of end-organ damage (Mancia and Parati, 2003). Given that restraint stress has
been reported to increase blood pressure largely due to sympathoadrenal system activation (Chen
and Herbert, 1995) and the stress-induced increase in blood pressure variability is mediated by
sympathetic excitation (Blanc et al. 1991; Gaudet et.al. 1996), it is possible that the decreased BP
responses associated with Boophone treatment seen in MS mice under stressful conditions is due
to reduced activation of the sympathetic nervous system. It seems likely that B. disticha may
alter the sensitivity and/or density of cardiac adrenergic receptors rather than vascular adrenergic
receptors in MS mice, since the magnitude of HR stress responses differ between MS and control
animals (Igosheva et al. 2004). The findings might also indicate a decreased peripheral
resistance, a resetting of the spontaneous baroreflex or altered baroreflex sensitivity following B.
disticha treatment.
A second major mechanism by which B. disticha treatment of postnatally stressed mice would
exert their effects may be through programmed subsequent enhanced blood pressure
responsiveness to stress by causing direct alteration in the HPA axis (Igosheva et al. 2004). The
present results suggest that B. disticha may have possible associated anxiolytic-like activity
which may alter pathophysiological changes in behavior and endocrine system associated with
repeated early MS previously described as increased plasma corticosterone release and elevated
18
NGF levels in the hippocampus which can affect the development and maturation of specific
organs related to blood pressure control and maintenance, such as heart, vasculature, kidney and
brain (Daniels et al. 2009; Igosheva et al. 2004). Glucocorticoids can directly regulate blood
pressure, increasing sodium and calcium uptake by vascular smooth muscle (Kornel, 1993) and
increasing vascular responsiveness to angiotensin II (Provencher et al. 1995) and noradrenaline
(Walker and Williams, 1992). These glucocorticoid-induced changes in both central and effector
sites of the cardiovascular control may be reflected in enhanced cardiovascular responses to
acute stress seen here in MS mice and seem to be decreased by treatment with B. disticha freeze
dried aqueous ethanolic crude extract. However, further investigations are required for
clarification of changes in the central or local control of the cardiovascular system to understand
the mechanisms mediating hypotensive-like activity underlying B. disticha treatment.
5. Conclusions
The present study, confirmed that maternal separation during early life can lead to changes in
hemodynamic parameters. The disruptions in maternal care during the separation period
significantly increase systolic blood pressure, whilst heart rate was markedly decreased in
adulthood. MS mice treated with low dose Boophone disticha had a lowest systolic blood
pressure and MS mice treated with high dose B. disticha had the lowest diastolic blood pressure
and mean arterial pressure following acute stress in adulthood than control and diazepam treated
MS animals. Future studies should focus on cardiovascular effects of B. disticha and also
simultaneously investigate autonomic and behavioural anxiolytic-like effects of B. disticha
extract.
6. Acknowledgments
19
This study was supported by IFS and the University of Zimbabwe (UZ) Research Grants. Many
thanks to the UZ Preclinical Veterinary Studies and Physiology Department, UZ School of
Pharmacy and Animal House (UZ) staff for advice, technical support, animal facilities and
equipment. We are also very grateful to Mr Louis L. Gadaga and Mr Zulu Daniel for solvent
extraction and characterisation of the freeze dried aqueous ethanolic B. disticha extract used in
this study and Mr Harmony Kwitiri for animal care.
7. References
1. Adewusi, A.E., Fouche, G., Steenkamp, V., 2012. Cytotoxicity and acetylcholinesterase
inhibitory activity of anisolated crinine alkaloid from Boophane disticha (Amaryllidaceae).
Journal of Ethnopharmacology http://dx.doi.org/10.1016/j.jep.2012.07.011.
2. Bastida, J., Berkov, S., Torras, L., Pigni N.B., de Andrade, J.P., Martínez, V., Codina, C.,
Viladomat, F., 2011. Chemical and biological aspects of Amaryllidaceae alkaloids. Recent
Advances in Pharmaceutical Sciences, 2011: 65-100.
3. Bian, X.P., Seidler, F.J., Slotkin, T.A., 1992. Promotional role for glucocorticoids in the
development of intracellular signalling: enhanced cardiac and renal adenylate cyclase reactivity
to beta-adrenergic and non-adrenergic stimuli after low-dose fetal dexamethasone exposure.
Journal of Developmental Physiology 17, 289–297.
4. Blanc, J. Grichois, M.L., Elghozi, J.L., 1991. Effects of clonidine on blood pressure and heart
rate responses to an emotional stress in the rat: a spectral study. Clinical and Experimental
Pharmacology and Physiology 18, 711–717.
5. Botha, E.W., Kahler, C.P., du Plooy, W.J., du Plooy, S.H., Mathibe, L., 2005. Effects of
Boophone disticha on human neutrophils. Journal of Ethnopharmacology 96, 385–388.
6. Buchholz, J., Duckles, S.P., 2001. Chronic hypoxia alters prejunctional alpha2-receptor function
20
in vascular adrenergic nerves of adult and fetal sheep. American Journal of Physiology.
Regulatory, Integrative and Comparative Physiology 281, R926–R934.
7. Cheesman, L., Nair, J.J., Van Staden, J., 2011. Pronounced seasonal variation effects in alkaloid
metabolite production in Boophone disticha (unpublished results). In Cheesman, L., Nair, J.J.,
Van Staden, J., 2012. Antibacterial activity of crinane alkaloids from Boophone disticha
(Amaryllidaceae). Journal of Ethnopharmacology 140, 405– 408.
8. Cheesman, L., Nair, J.J., Van Staden, J., 2012. Antibacterial activity of crinane alkaloids from
Boophone disticha (Amaryllidaceae). Journal of Ethnopharmacology 140, 405– 408
9. Chen, X., Herbert, J., 1995. Regional changes in c-fos expression in the basal forebrain and
brainstem during adaptation to repeated stress: correlations with cardiovascular, hypothermic and
endocrine responses. Neuroscience 64, 675–685.
10. Chingombe, P., Tagwireyi D., Gadaga L.L., 2010. Investigating the antidepressant-like activity
of Boophone disticha after repeated daily dosing in a mouse model. University of Zimbabwe
Library. (Unpublished results).
11. Chuma., Tagwireyi D., Gadaga L.L., 2010. Investigation of the anxiolytic-like activity of
Boophane disticha extract in mice. University of Zimbabwe Library. (Unpublished results)
12. Cohen, H., Benjamin, J., 2006. Power spectrum analysis and cardiovascular morbidity in anxiety
disorders. Autonomic Neuroscience: Basic and Clinical 128, 1-8.
13. Cole, C.R., Blackstone, E.H., Pashkow, F.G., Snader, C.E., Lauer, M.S., 1999. Heart rate
recovery immediately after exercise as a predictor of mortality. New England Journal of
Medicine 342, 1351–1357.
14. Dampney, R.A., 1994. Functional organization of central pathways regulating the cardiovascular
system. Physiological Reviews 74, 323–364.
21
15. Daniels, W.M., Pietersen, C.Y., Carstens, M.E., Stein, D.J., 2004. Maternal Separation in Rats
Leads to Anxiety-Like Behavior and a Blunted ACTH Response and Altered Neurotransmitter
Levels in Response to a Subsequent Stressor. Metabolic Brain Disease 19, 3–14.
16. Daniels, W.M.U., Fairbairn, L.R., van Tilburg, G., McEvoy, C.R.E., Zigmond, M.J., Russell,
V.A., Stein, D.J., 2009. Maternal separation alters nerve growth factor and corticosterone levels
but not the DNA methylation status of the exon 17 glucocorticoid receptor promoter region.
Metabolic Brain Disease 24, 615–627.
17. De Smet, P.A.G.M., 1996. Some ethnopharmacological notes on African hallucinogens. Journal
of Ethnopharmacology 50, 141–146.
18. Du Plooy, W.J., Swart, L. Van Huysteen, G.W., 2001. Poisoning with Boophane disticha: a
forensic case. Human and Experimental Toxicology 20, 277–278.
19. Ecklund, M.B., Arborelius, L., 2006. Twice daily long maternal separations in Wistar rats
decreases anxiety-like behaviour in females but does not affect males. Behavioural Brain
Research, 172, 278–85.
20. Faure, J., Uys, J.D.K., Marais, L., Stein, D.J., Daniels, D.M.U., 2006. Early maternal separation
followed by later stressors leads to dysregulation of the HPA-axis and increases in hippocampal
NGF and NT-3 levels in a rat model. Metabolic Brain Disease, 21(2-3), 181-88.
21. Faure, J., Uys, J.D.K., Marais, L., Stein, D.J., Daniels, W.M.U., 2007. Early maternal separation
alters the response to traumatization resulting in increased levels of hippocampal neurotrophic
factors. Metabolic Brain Disease 22(2), 183–195.
22. Floras, J.S., Senn, B.L., 1991. Absence of post exercise hypotension and sympathoinhibition in
normal subject: additional evidence for increased sympathetic outflow in borderline
hypertension. Canadian Journal of Cardiology 72, 53–258.
22
23. Gadaga, L.L., Tagwireyi, D., Dzangare, J., and Nhachi, C.F.B., 2011. Acute oral toxicity and
neurobehavioural toxicological effects of a hydroethanolic extract of Boophone disticha in rats.
Human and Experimental Toxicololgy 30(8), 972–980.
24. Gaudet, E., Blanc, J., Elghozi, J.L., 1996. Role of angiotensin II and catecholamines in blood
pressure variability responses to stress in SHR. American Journal of Physiology 270, R1265–
R1272.
25. Grillison, C., Duncko, R., Covington, M.F., Kopperman, L., Kling, M.A., 2007. Acute stress
potentiates anxiety in humans. Biological Psychiatry. 62, 1183–1186.
26. Hautch, H., Stauffacher, D., 1961. Die alkaloide von Buphane disticha (L.f.) Herb. Helvetica
Chimica Acta 44, 491–502.
27. Hayashi, A., Nagaoka, M., Yamada, K., Ichitani, Y., Miake, Y., Okado, N., 1998. Maternal
stress induces synaptic loss and developmental disabilities of offspring. International Journal of
Developmental Neuroscience16, 209–216.
28. Holst, S., Uvnas, K., Petersson, M., 2002. Postnatal oxytocin treatment and postnatal stroking of
rats reduce blood pressure in adulthood. Autonomic Neuroscience 99, 85–90.
29. Huff, R.A., Seidler, F.J., Slotkin, T.A., 1991. Glucocorticoids regulate the ontogenetic transition
of adrenergic receptor subtypes in rat liver. Life Sciences 48, 1059–1065.
30. Igosheva, N., Klimova, O., Anishchenko, T., Glover, V., 2004. Prenatal stress alters
cardiovascular responses in adult rats. Journal of Physiology 557, 273–285.
31. Kalinichev, M., Easterling, K.W., Plotsky, P.M., Holtzman, S.G., 2002. Long-lasting changes in
stress-induced corticosterone responses and anxiety-like behaviors as a consequence of neonatal
maternal separation in Long-Evans rats. Pharmacology, Biochemistry and Behavior 73(1), 131–
40.
23
32. Kornel, L., 1993.The role of vascular steroid receptors in the control of vascular contractility and
peripheral vascular resistance. The Journal of Steroid Biochemistry and Molecular Biology 45,
195–203.
33. Ladd, C.O., Huot, R.L., Thrivikraman, K.V., Nemeroff, C.B., Meany, M.J., Plotsky, P.M., 2000.
Long-term behavioural and neuroendocrine adaptations to adverse early experience. Progress in
Brain Research 122, 81–103.
34. Ladd, C.O., Owens, M.J., Nemeroff, C.B., 1996. Persistent changes in corticotropin-releasing
factor neuronal systems induced by maternal deprivation. Endocrinology 137, 1212–218.
35. Loria, A.S., D'Angelo, G., Pollock, D.M., Pollock, J.S., 2010. Early life stress down regulates
endothelin receptor expression and enhances acute stress-mediated blood pressure responses in
adult rats. American Journal of Physiology: Regulation Integration Comparative Physiology.
299(1), R185–R191.
36. Larsen B.A., and Christenfeld N.J.S., 2009. Cardiovascular Disease and Psychiatric
Comorbidity: The Potential Role of Perseverative Cognition. Cardiovascular, Psychiatry and
Neurology. Volume, page 1-8.
37. Mancia, G., Parati, G., 2003. The role of blood pressure variability in end-organ damage. Journal
of Hypertension 6 (Suppliment), S17–S23.
38. Marais. L., Van Rensburg, S.J., Van Zyl, J.M., Stein, D.J., Daniels, W.M.U., 2008. Maternal
separation of rat pups increases the risk of developing depressive-like behavior after subsequent
chronic stress by altering corticosteorne and neurotrophin levels in the hippocampus.
Neurochemical Research 61(1), 106–12.
39. McMillen, I.C., Adams, M.B., Ross, J.T., Coulter, C.L., Simonetta, G., Owens, J.A., Robinson,
J.S., Edwards, L.J., 2001. Fetal growth restriction: adaptations and consequences. Reproduction
24
122,195–204.
40. Muneoka, K., Mikuni, M., Ogawa, T., Kitera, K., Kamei, K., Takigawa, M., Takahashi, K.,
1997. Prenatal dexamethasone exposure alters brain monoamine metabolism and adrenocortical
response in rat offspring. American Journal of Physiology 273, R1669–R1675.
41. Mungadzi, E., Tagwireyi D., and Gadaga L.L., 2008. Investigation of anxiolytic and memory
enhancing properties of Boophone disticha in a rat model. University of Zimbabwe Library.
(Unpublished results)
42. Musarira, S., Tagwireyi D., and Gadaga L.L., 2011. Effects of a hydroethanolic extract of
Boophone disticha bulb in behavioral models of anxiety in mice. University of Zimbabwe.
(Unpublished results)
43. Neergaard, J.S., Andersen, J., Pedersen, M.E., Stafford, G.I., Van Staden, J., Jäger, A.K., 2009.
Alkaloids from Boophone disticha with affinity to the serotonin transporter. South African
Journal of Botany 75, 371–374.
44. Nielsen, N.D., Sandager, M., Stafford, G.I., Van Staden, J., Jäger, A.K., 2004. Screening of
indigenous plants from South Africa for affinity to the serotonin reuptake transport protein.
Journal of Ethnopharmacology 94, 159–163.
45. Pedersen, M.E., Vestergaard, H.T., Stafford, G.I., van Staden, J., Jäger, A.K. 2008. The effect of
extracts of Searsia species on epileptiform activity in slices of the mouse cerebral cortex. Journal
of Ethnopharmacology 119, 538–541.
46. Pedersen, M.E., Szewczyk, B., Stachowicz, K., Wieronska, J., Andersen, J., Stafford, G.I., van
Staden, J., Pilc, A., Jäger, A.K. 2008. Effects of South African traditional medicine in animal
models for depression. Journal of Ethnopharmacology 119, 542–548.
47. Peters, D.A., 1982. Prenatal stress: effects on brain biogenic amine and plasma corticosterone
25
levels. Pharmacology, Biochemistry and Behavior 17, 721–725.
48. Peyronnet, J., Dalmaz, Y., Ehrstrom, M., Mamet, J., Roux, J.C., Pequignot, J.M., Thoren, H.P.,
Lagercrantz, H., 2002. Long-lasting adverse effects of prenatal hypoxia on developing
autonomic nervous system and cardiovascular parameters in rats. Pflugers Arch 443, 858–865.
49. Plotsky, P.M., Meany, M.J., 1993. Early, postnatal experience alters hypothalamic
corticotrophin-releasing factor (CRF) mRNA, median eminence CRF content and stress-induced
release in adult rats. Molecular Brain Research18, 195–200.
50. Provencher, P.H., Saltis, J., Funder J.W., 1998. Glucocorticoids but not mineralocorticoids
modulate endothelin-1 and angiotensin II binding in SHR vascular smooth muscle cells. The
Journal of Steroid biochemistry and molecular biology 52(3), 219-25.
51. Pryce, C.R., Feldon, J., 2003. Long-term neurobehavioral impact of the postnatal environment in
rats: manipulations, effects, and mediating mechanisms. Neuroscience Biobehavioural Review
27, 57–71.
52. Raffauf, R.F., 1970. A Handbook of Alkaloids and Alkaloid-Containing Plants. Wiley-
Interscience, New York.
53. Sandager, M., Nielsen, N.D., Stafford, G.I., van Staden, J., Jäger, A.K., 2005. Alkaloids from
Boophane disticha with affinity to the serotonin transporter in rat brain. Journal of
Ethnopharmacology 98, 367–370.
54. Slotten, H.A., Kalinichev, M., Hagen, J.J., Marsden, C.A., Fona, K.C., 2006. Long-lasting
changes in behavioural and neuroendocrine indices in the rat following neonatal maternal
separation: gender dependent effects. Brain Research, 1097, 123–132.
55. Stafford, G.I., Pedersen, M.E., Van Staden, J., Jäger, A.K., 2008. Review on plants with CNS-
effects used in traditional South African medicine against mental diseases. Journal of
26
Ethnopharmacology 119, 513–537.
56. Schwartz T.L., and Nihalani N., 2006. Tiagabine in anxiety disorders. Expert Opinion in
Pharmacotherapy 7(14), 1977-1987.
57. Takahashi, L.K., Turner, J.G., Kalin, N.H., 1992. Prenatal stress alters brain catecholaminergic
activity and potentiates stress-induced behavior in adult rats. Brain Research 574, 131–137.
58. Tonkiss, J., Trzcinska, M., Galler, J.R., Ruiz-Opazo, N., Herrera, V.L., 1998. Prenatal
malnutrition-induced changes in blood pressure: dissociation of stress and nonstress responses
using radiotelemetry. Hypertension 32, 108–114.
59. 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.
60. Volkmar, G., Tank, J., Obst, M., Plehm, R., Blumer, K.J., Diedrich, A., Jordan, J., Luft, F.C.,
2005. Autonomic nervous system and blood pressure in RGS2-deficient mice. American Journal
of Physiology- Regulatory, intergrative and comparative Physiology. 228, R1132-R1142.
61. Wagner, H., Bladt, S., 1996. Plant Drug Analysis—A Thin Layer Chromatography Atlas.
Springer-Verlag, Berlin Heidelberg.
62. Walker, B.R., Williams, B.C., 1992. Corticosteroids and vascular tone: mapping the messenger
maze. Clinical Science (London) 82, 597–605.
63. Watanabe, J., Thamilarasan, M., Blackstone, E.H., Thomas, J.D., Lauer, M.S., 2001. Heart rate
recovery immediately after treadmill exercise and left ventricular systolic dysfunction as
predictors of mortality: the case of stress echocardiography. Circulation 104, 1911–1916.
64. Weinstock M. Does prenatal stress impair coping and regulation of hypothalamic-pituitary-
adrenal axis? Neuroscience Biobehavioural Review 1997, 21:1–10.
65. Young, J.B., 2002. Programming of sympathoadrenal function. Trends in Endocrinology and
27
Metabolism 13, 381–385.
66. Zulu, D., Tagwireyi, D., Gadaga, L.L., 2011. Solvent extraction and chromatographic
characterization of isoquinoline alkaloids from the bulb of Boophone disticha. University of
Zimbabwe Library. (Unpublished results).
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CARDIOVASCULAR EFFECTS OF BOOPHONE DISTICHA HYDROETHANOLIC EXTRACT ON EARLY MATERNALLY SEPARATED BALB/c MICE. a,*Pote William., bTagwireyi Dexter., cChinyanga Herbert. M., aMusara Collin., aNyandoro George., cChifamba Jephat., and dNkomozepi Pilani. aDepartment of Preclinical Veterinary Studies; bDrug and Toxicology Information Service-School of Pharmacy and c Department of Physiology, University of Zimbabwe P O Box MP167, Mount Pleasant Harare Zimbabwe and dDepartment of Neuroscience, University of Witwatersrand, South Africa.
Aim of the study: In order to validate the use of Boophone disticha in treatment of anxiety, this study was set to determine the effects of the plant extracts on blood pressure (BP) and heart rate (HR) in adult BALB/c mice subjected to repeated early maternal separation (MS) stress. Results: Comparisons of the BP and HR measurements indicated that MS significantly reduced the acute AMS-induced HR responses and increased SBP response in BALB/c mice when compared with control. B. disticha treatment significantly reduced the AMS-induced BP response in BALB/c MS as compared to vehicle and diazepam treatments (Table 1). Conclusions: Our findings demonstrate for the first time that postnatal stress can induce short-term, changes in the sensitivity of the cardiovascular system to subsequent stress which can be reduced by treatment with a frieze dried hydroethanolic extract of B. disticha. Table 1. Effect of Boophone disticha hydroethanolic extract on blood pressure and heart rate measurements in BALB/c mice after maternal separation as compared to control vehicle and diazepam. Group SBP DBP MAP HR A 129.51±1.2 a 99.5±0.7 113.34±0.64 568.9±27 ª B 133.84±1.9 a �
� 95.62±3 115.39±3.1 � 480.5±16 ª, �
C 127.85±1.4 � � �
95.62±3 109.69±1.3 � 514.5±10 �
D 130.93±0.5 � � 94.3±0.51 � 111.317±0.3 � 504.9±4.9 E 130.76±1.1 93.16±1.8 � 108±2.7 � � 516.9±9.4 � F 132.291±0.25 � 97.72±0.5 � 113.8±0.35 � � 500±9.9
NB: ª show significant difference of the Group B (MS+NS) as compared to control Group A p<0.05.
� Significant difference between group C as compared to group B p<0.05
� significant difference between Groups C, D, or E as compared to Group F p<0.05
� indicates that there in insignificant tendency of difference between group A and B 0.05<p<0.1
� Insignificant tendency of a difference occurring between Groups C,D, or E; and Group B or
� Group F (0.05<p<0.1)
29
Figures
Figure 1.Diagram showing changes in systolic blood pressure (SBP) on PND 78, following 30
min period of mixing followed by 30 min warming period and subsequent restraint during
recording period in control, and MS mice treated with six oral doses of vehicle, Boophone or
diazepam.
124
126
128
130
132
134
136
Systolic Blood
Pressure (m
mHg)
Experimental Groups
30
Figure 2. Graph illustrating how Boophonedisticha treatment in BALB/c mice after maternal
separation affected diastolic blood pressure (DBP) as compared to control vehicle and diazepam.
84
86
88
90
92
94
96
98
100
102
104Diastolic Blood
Pressure (m
mHg)
Experimental groups
102
104
106
108
110
112
114
116
118
120
Mean arterial Pressure (m
mHg)
Experimental groups
31
Figure 3.Changes in mean arterial pressure (MAP) in control and postnatally repeated MS
stressed BALB/c mice. The graph also shows how Boophonedisticha treatment affected Mean
arterial pressure measurements in BALB/c mice after maternal separation as compared to control
vehicle and diazepam.
Figure 4.Diagram showing Changes in heart rate (HR) in control and repeated MS stressed
BALB/c mice. Also shown are effects of Boophonedisticha treatment after maternal separation
as compared to control vehicle and diazepam.
470480490500510520530540550560
Heart rate (beats per m
inute)
Experimental groups
CARDIOVASCULAR EFFECTS OF BOOPHONE DISTICHA AQUEOUS ETHANOLIC EXTRACT ON EARLY MATERNALLY SEPARATED BALB/c MICE a,*Pote William., bTagwireyi Dexter., cChinyanga Herbert. M., aMusara Collin., aNyandoro George., cChifamba Jephat., and dNkomozepi Pilani. aDepartment of Preclinical Veterinary Studies; bDrug and Toxicology Information Service-School of Pharmacy and c Department of Physiology, University of Zimbabwe P O Box MP167, Mount Pleasant Harare Zimbabwe and dDepartment of Neuroscience, University of Witwatersrand, South Africa.
Figure 1: Diagram showing changes in a) systolic blood pressure (SBP), b) diastolic blood pressure (DBP), c) mean arterial pressure (MAP) and d) heart rate (HR) in BALB/c mice after maternal separation Group B (MS+NS) affected as compared to control Group A (C+NS) two days (postnatal day 78) after receiving six daily doses of normal saline (NS, vehicle). Maternally separated mice were also treated with either Boophone disticha (BD) aqueous ethanolic extract Group C (MS + LDBD), D MS+MDBD) and E (HDBD) receiving low, medium and high doses (10, 25 and 40mg/kg body weight (BW) respectively or 1mg/kg BW diazepam (Group F (MS+DZP)).
124
126
128
130
132
134
136
Systolic Blood
Pressure
(mmHg)
Experimental Groupsa)
8486889092949698
100102104
Diastolic Blood
Pressure
(mmHg)
Experimental groupsb)
102104106108110112114116118120
Mean arterial Pressure (m
mHg)
Experimental groupsc)
470480490500510520530540550560
Heart rate (beats per m
inute)
Experimental groupsd)
