Long-term cardiovascular autonomic responses to aqueous ethanolic extract of Boophone disticha bulb in early maternally separated BALB/c mice

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South African Journal of Botany 94 (2014) 33–39

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South African Journal of Botany

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Long-term cardiovascular autonomic responses to aqueous ethanolicextract of Boophone disticha bulb in early maternally separatedBALB/c mice

William Pote a,⁎, Dexter Tagwireyi b, Herbert M. Chinyanga c, Colin Musara a, Davies M. Pfukenyi d,Pilani Nkomozepi e, Louis L. Gadaga b, George Nyandoro a, Jephat Chifamba c

a Preclinical Veterinary Studies Department, Faculty of Veterinary Science, University of Zimbabwe, Zimbabweb Drug and Toxicology Information Service, School of Pharmacy, College of Health Sciences, University of Zimbabwe, Zimbabwec Department of Physiology, College of Health Sciences, University of Zimbabwe, Zimbabwed Clinical Veterinary Studies, Faculty of Veterinary Science, University of Zimbabwe, Zimbabwee Division of Neuroscience, University of Witwatersrand, South Africa

Abbreviations: AMS, acute mixing stress; BP, blood preiability; BW, bodyweight; DBP, diastolic blood pressure; DB. disticha; HR, heart rate; HRV, heart rate variability; LDBMAP, mean arterial pressure; MDBD, medium dose B. disPND, postnatal day; PTD, post-treatment day; SBP, systoli⁎ Corresponding author at: PO Box MP167, Mount

Tel.: +263 773234621, +263 736566176; fax: +263E-mail addresses: [email protected], potewilliam@gm

[email protected] (W. Pote).

http://dx.doi.org/10.1016/j.sajb.2014.04.0120254-6299/© 2014 SAAB. Published by Elsevier B.V. All rig

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 10 December 2013Received in revised form 16 April 2014Accepted 22 April 2014Available online xxxx

Edited by JJ Nair

Keywords:B. distichaAntihypertensive activityAutonomic responseCardiovascular comorbid disordersAnxiety disorders

Background: Boophone disticha is commonly used in southern Africa for the management of mental-relatedillnesses. Recently, it was shown to reduce blood pressure (BP) in maternally separated mice immediatelyafter withdrawal of treatment. However, the long-term cardiovascular effects and the underlying mechanismsare still illusive. Based on the reputed traditional use of the plant for anxiety and stress-related disorders,the aqueous-ethanolic extract of B. disticha was screened for its long-term effects on the cardiovascular andautonomic responses to repeated acute stressors in adult early maternally separated BALB/c mice.Methods: Five groups (n= 6 each) of adult BALB/c mice subjected to early maternal separation (MS) were givensix daily oral doses of vehicle (normal saline); low, medium and high doses of B. disticha (10, 25 and 40 mg/kgbody weight, respectively); and 1 mg/kg body weight diazepam during adulthood. The control (un-separated)group (n = 6) received vehicle treatment. Cardiovascular parameters (BP and heart rate (HR)) were recordedusing non-invasive tail-cuff methods on post-treatment days (PTDs) 9 and 30 to compare short-term andlong-term effects of the plant extract, respectively. Autonomic responses were measured by estimating BPvariability (BPV) and HR variability (HRV).

Results: Early maternal separation significantly increased systolic BP (SBP), and decreased HR on PTD9 whileraising BPV on PTD30when compared to control un-separatedmice (p b 0.05). B. disticha at low dose significant-ly reduced short-termSBP andmean arterial pressure (MAP), whilemediumdose reduced long-termdiastolic BP(DBP) and MAP in maternally separated mice when compared to vehicle and diazepam (p b 0.05). High dosesignificantly decreased SBP and MAP at both occasions (p b 0.05).Conclusions: The current results have led to the identification of long-term antihypertensive-like activity of theaqueous ethanolic extract of B. distichawhich was found to last for several weeks after withdrawal of treatment.

© 2014 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction

Boophone disticha (L.f.) Herb. (Amaryllidaceae) known as ‘sore-eyeflower’ (‘munzepete’ in Shona and ‘ingcotho’ in IsiNdebele), is used formedicinal purposes among the indigenous people of southern Africa.

ssure; BPV, blood pressure var-ZP, diazepam; HDBD, high doseD, low dose Boophone disticha;ticha; MS, maternal separation;c blood pressure; Veh, vehicle.Pleasant, Harare, Zimbabwe.4333678.ail.com,

hts reserved.

It is used for the treatment of several mental-related conditions suchas epilepsy, seizures, psychosis, anxiety, depression and cognitive disor-ders (De Smet, 1996; Gelfand and Mitchell, 1952; Gelfand et al., 1985;Gomes et al., 2009; Hutchings et al., 1996; Nair and Van Staden, 2014;Stafford et al., 2008; Steenkamp, 2005). Although, the mechanism un-derlying the effects of the plant is largely unknown, in vivo studiesdone on the crude extract showed that the plant has potential antide-pressant (Chingombe et al., 2010; Pedersen et al., 2008), anxiolytic(Chuma et al., 2010; Musarira et al., 2011), nootropic (Gadaga, 2012)and antihypertensive (Pote et al., 2013) activities in animal models.

Anxiety and depression disorders are themost prevalent psychiatricconditions. These conditions are commonly undertreated because theyoccur along with other mental or physical illnesses, mostly comorbidcardiovascular disorders, that mask their symptoms causing socio-

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34 W. Pote et al. / South African Journal of Botany 94 (2014) 33–39

economic burden and personal distress (Belzung and Griebel, 2001;Sarris and Kavanagh, 2009). Current treatments are less than satisfacto-ry; for example, a considerable proportion of patients are non-responsive to first-line treatment, the onset of action is delayed andthe drugs can induce side effects which significantly impair compliance(Sarris and Kavanagh, 2009). In addition, these medications are benefi-cial to only 65% of the patients; hence, most people with severe depres-sion and anxiety attacks use complementary and alternative medicineto treat these conditions (Sarris and Kavanagh, 2009). Since anxietysignificantly increases morbidity and mortality in heart diseases(Belzung and Griebel, 2001), successful treatment for psychiatric disor-ders should aim to correct the abnormalities of central cardiovascularautonomic regulation (Cohen and Benjamin, 2006; Depino and Gross,2007; Igosheva et al., 2004; Volkmar et al., 2005).

Most recently, a study done by Pote et al. (2013) found thatB. disticha extract (10 to 40 mg/kg body weight (BW)), significantlyreduced systolic BP (SBP), diastolic BP (DBP) andmean arterial pressure(MAP) inmaternally separated (MS)mice immediately afterwithdraw-al of treatment. B. disticha may therefore be utilized to develop a drugfor treatment of comorbid cardiovascular disorders that are frequentlyassociated with anxiety and mood disorders. However, the low re-sponse rate to treatment makes it very difficult to prove on short-termbasis that the plant has anxiolytic, antidepressant or antihypertensiveefficacy. Therefore, the present study was set to explore the long-termpharmacological effects of the aqueous ethanolic extract of B. distichaon blood pressure (BP), heart rate (HR), BP variability (BPV) and HRvariability (HRV) in a maternal separation animal model of anxietydisorder.

2. Materials and methods

The experimental protocols, care and handling of animals used inthis study were in accordance with international guidelines (EuropeanCommunity guidelines, EEC Directive of 1986; 86/609/EEC) on the useand care of laboratory animals and were approved by the Division ofVeterinary Services, Zimbabwe (Pote et al., 2013).

2.1. Plant materials, extraction and qualitative analysis of alkaloids

Bulbs of B. distichawere harvested, authenticated, and dried and theaqueous ethanolic (70% v/v) extract was prepared as described previ-ously (Gadaga et al., 2011; Pote et al., 2013). Chromatographic analysis(Zulu et al., 2011) confirmed the presence of isoquinoline alkaloidsreported in previous studies (Adewusi et al., 2012; Cheesman et al.,2012; Hauth and Stauffacher, 1961; Neergaard et al., 2009; Sandageret al., 2005; Steenkamp, 2005).

2.2. Animals and housing conditions

Pregnant BALB/cmice, obtained from the Central Veterinary Labora-tory Breeding Unit in The Ministry of Agriculture (Zimbabwe), werehoused in the Animal Holding facilities at the University of Zimbabwe(UZ) under conditions previously described by Pote et al. (2013).

2.3. Experimental protocols

Control pups (un-separated group) were not separated from theirdams during the deprivation period while the treatment pups werematernally separated. The procedures for maternal separation (MS)were as described earlier by Pote et al. (2013). The study animalswere weaned, caged, grouped and given treatments from post-natalday (PND) 71–76 as reported previously (Pote et al., 2013). Briefly, thecontrol group (Group A) comprised of six mice (three males and threefemales) randomly selected from an un-separated litter. For thetreatment groups, 30 mice (15 males and 15 females) were selectedrandomly from a maternally separated colony. These were randomly

assigned to five treatment groups: B, C, D, E, and F (n = 6 each; with3 males and 3 females housed in separate cages, Fig. 1). Groups A(control + Veh) and B (MS + Veh) were given a vehicle (normal sa-line). Groups C (MS+ LDBD), D (MS+ MDBD) and E (MS+ HDBD) re-ceived low (10 mg/kg body weight (BW)), medium (25 mg/kg BW)and high (40 mg/kg BW) doses of B. disticha, respectively; whileGroup F (MS + DZP) received 1 mg/kg BW diazepam (Fig. 1).

2.3.1. Cardiovascular responses to acute mixing stressThe present study repeated the procedures of acute mixing stress

(AMS) and restraint stress described recently by Pote et al. (2013) onpost-treatment day 9 (PTD9) and PTD30 (i.e. PND85 and PND106, re-spectively). Blood pressure (systolic blood pressure (SBP), diastolic BP(DBP), mean arterial pressure (MAP)) and heart rate (HR) were record-ed for 5min between 09.00 and 15.00 h on PTD9 (short-term response)and PTD30 (long-term response). A non-invasive tail-cuff system(BIOPAC® System Inc., CA) was used to monitor all the cardiovascularparameters (Pote et al., 2013).

2.3.2. Autonomic reactivityThe standard deviation of theMAP (SDMAP) calculated for every seg-

ment (24 s) of the recording period was used as a measure of bloodpressure variability (BPV) (Igosheva et al., 2004). Standard deviationof heart rate (SDHR) calculated from every heart beat, recorded on theinterval between the SBP and DBP, was used as a measure of heartrate variability (HRV).

2.4. Statistical analysis

Data was initially captured with Excel where it was sorted and thenexported to statistical package software (SPSS® version 16.0) for furtheranalysis. Comparisons were done on treatment groups using multivari-ate multiple comparison analysis and independent Student's t-test.Changes in cardiovascular parameters were calculated by subtractingvalues recorded on PTD9 from PTD30 readings and the mean changeswere analyzed using independent t-test and one-wayANOVAwithmul-tiple comparisons across all test groups. The normal mice (Group A;Control + Veh) was first compared to Group B (MS + Veh; Fig. 1) onPTD9 and PTD30 to determine how habituation to repeated acutemixing stress would affect the cardiovascular and autonomic reactivity.MS mice treated with three doses of B. disticha extract (low, mediumand high doses: Groups C, D and E), were compared to the vehicle(Group B) and diazepam (Group F; Fig. 1). Comparisons were alsomade on normal un-separated mice that received vehicle (Group A)and all MS treated mice Groups C–F (Fig. 1) to find which treat-ment would retain and maintain the animal health towards thenormal range on PTD9 and PTD30. All data were expressed as meandifference ± standard error (S.E.) with a significance level of p b 0.05.

3. Results

MS mice had a significantly higher SBP when compared to controlmice on PTD9 with a mean difference of 4.6 ± 1.8 millimeter mercury(mm Hg, p b 0.02) but this effect was absent on PTD30 (p N 0.05).DBP, MAP and changes in BP recorded over the entire test period werecomparable between the two groups (p N 0.05) (Fig. 2). When earlymaternally separated mice were compared to the control, initially onPTD9 they exhibited lower HR (mean difference 53.7 ± 16.5 beats perminute (bpm); p b 0.001), but the difference disappeared on PTD30(mean difference 11.8 ± 13.9 bpm; p N 0.05). The change in HRover the testing period was non-significantly higher in the formergroup (p = 0.091).

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Fig. 1.A schematic diagram summarizing the study design, protocols and procedures used in this study. PTD, post-treatment day; PND, post-natal day;MS,maternal separation; Veh, vehicle.

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3.1. Long-term cardiovascular response to B. disticha treatment in adult MSmice

3.1.1. Systolic blood pressure (SBP)At PTD9 (short-term observations), MS mice treated with low and

high doses of B. disticha had significantly lower SBP when comparedto vehicle treated MS mice (mean differences; 6.28 ± 1.83 mm Hg,p b 0.03 and 3.80 ± 1.83 mm Hg, p b 0.05, respectively). However,onlyMS animals treatedwith the lowdose of B. distichahad a significant-ly lower SBP on PTD9 when compared to the group treated with diaze-pam (mean difference of 4.63 ± 1.83 mm Hg, p b 0.02). At PTD30(long-term observations), only the group treated with a high dose ofB. disticha had a significantly reduced mean SBP when compared to thevehicle treatment in MS mice (mean difference of 4.84 ± 1.78 mm Hg,p b 0.02). In contrast, when compared to diazepam-treated MS mice,medium and high doses of B. disticha-treated MS mice had significantlylower SBP on PTD30 with mean differences of 3.85 ± 1.78, p b 0.05and 5.46 ± 1.78 mm Hg, p b 0.01, respectively. MS mice treated withlow dose of B. disticha had a significantly smaller change in SBP thanthe vehicle (p b 0.03), medium (p b 0.04) and high dose (p b 0.01)(Fig. 2). However, all the MS groups given vehicle, B. disticha extract ordiazepam treatments had comparable changes in SBP to control, non-separated group (Fig. 2). Lastly, dose-dependent effect of B. distichatreatment in MS mice on SBP was evident on PTD30 i.e. increasing dosefrom low through medium to high dose proportionally increased themagnitude of SBP differences when compared to vehicle (0.86 ± 1.78,3.23 ± 1.78 and 4.84 ± 1.78 mm Hg, respectively) or to diazepam(0.616± 1.78, 1.48± 1.78, 3.85±1.78 and 5.46±1.78mmHg, respec-tively) for vehicle, low, medium and high doses of B. disticha, respective-ly. This trend was confirmed when the magnitude changes in SBPbetween PTD9 and 30 were compared among the three doses ofB. disticha (Fig. 2).

3.1.2. Diastolic blood pressure (DBP)MS mice treated with either the high or the medium dose of

B. disticha had significantly reduced DBP at PTD9 and PTD30, whencompared to the vehicle-treated group (mean difference of 8.8 ±4.08 mm Hg and 5.6 ± mm Hg, respectively; p b 0.05). However,when compared to MS mice treated with diazepam, only the mediumdose of B. disticha-treated group had significantly reduced DBP atPTD9 (mean difference of 6.45 ± 4.08 mm Hg, p b 0.03). The dose-dependent effect on DBP was exhibited at PTD9 by B. disticha-treatedgroups which increased response with an increase in dosage whenlow, medium and high dose treatment groups were compared tovehicle-treated MS animals (mean differences of 2.48 ± 4.08, 5.70 ±4.08 and 8.80 ± 4.08 mm Hg, respectively). Although this responsewas absent at PTD30, it was observed that the medium dose group sig-nificantly reduced DBP when compared to the low dose group ofB. disticha treatment (mean difference: 6.2 ± 2.69 mm Hg, p b 0.4,Fig. 2). Generally, DBP decreased in all groups over time, but, the changein DBP during the recording periods was comparable among all testgroups (Fig. 2).

3.1.3. Mean arterial pressure (MAP)MS mice treated with a low dose of B. disticha had a significantly

reduced MAP when compared to the vehicle group (mean differenceof 8.5 ± 3.4 mm Hg. p b 0.02) at PTD9. However, this response patternwas not evident at PTD30 (p N 0.05). On the other hand, MSmice treat-ed with a medium dose of B. disticha had a significantly higher MAPwhen compared to those treated with diazepam at PTD30 (mean differ-ence of 5.5 ± 2.4 mmHg, p b 0.04) but comparable to vehicle group (pN 0.05). The change in MAP for the low dose B. disticha-treated groupwas significantly smaller when compared to the vehicle or themediumdose of B. disticha-treated animals (p b 0.05) (Fig. 2). There was nodose-dependent effect of B. disticha on MAP.

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Fig. 2. The diagram illustrates the cardiovascular response (change in blood pressure (BP)) to repeated exposure to acute mixing (AMS) and restraint stress in BALB/c mice. Changes insystolic BP (SBP), diastolic BP (DBP) andmean arterial pressure (MAP)were given by subtracting recordingsmade on PTD9 fromPTD30 recordings. Control (un-separated, andmaternallyseparated (MS))mice were gavagedwith vehicle (Groups A and B respectively)while four otherMS groupswere given B. disticha aqueous ethanolic extract at low (LDBD, 10 mg/kg bodyweight (BW)), medium (MDBD, 25 mg/kg BW) and high doses (40 mg/kg BW) for Groups C, D and E, respectively and diazepam (DZP) at 1 mg/kg BW (Group F). *p b 0.05 (significantdifferenceswhen treatmentwith B. disticha (Groups C, D and E)was compared to vehicle (GroupB) inMSmice) and #p b 0.05 (significant dose response effect ofB. distichawhen lowdose(Group C) is compared to either medium or high dose (Group D or E respectively).

36 W. Pote et al. / South African Journal of Botany 94 (2014) 33–39

3.1.4. Heart rate (HR)The changes in HRwere comparable among all the groups (p N 0.05)

(Fig. 2). However, it was noticed that MS mice treated with B. distichahad a significantly lower HR when compared to the vehicle group atPTD30 (p b 0.05).

3.2. Long-term autonomic reactivity in response to B. disticha treatment inMS mice

3.2.1. Blood pressure variability (BPV)Blood pressure variability (BPV) was estimated by measuring the

standard deviation of the MAP (SDMAP) of the six experimental groups(Igosheva et al., 2004). Comparisons across all groups at PTD9 showedthat BPV was comparable. However, it was observed that, at PTD30, allMS mice treatment groups, given vehicle; low, medium and highdoses of B. disticha; or diazepam, had a significantly higher BPV whencompared to control (un-separated) mice treated with vehicle (meandifferences of 3.44 ± 1.48, 3.27 ± 1.48, 4.00 ± 1.48, 3.14 ± 1.48 and3.26 ± 1.48 mm Hg respectively, p b 0.05). BPV improved over timeacross all groups. However, neither B. disticha group nor diazepamgroup displayed significant differences on BPV when compared tovehicle in MS mice (p N 0.05). The changes in BPV were comparableacross all treatment groups (p N 0.05) (Fig. 3).

3.2.2. Heart rate variability (HRV)HRV was estimated by calculating the mean standard deviation of

HR (SDHR) for all treatment groups. The HRV of MS mice was compara-ble to that of the control un-separated group, during the entire studype-riod, despite being challenged by repeated acute stressors at PTD9 andPTD30. However, it was noticed that MS mice given low and medium

doses of B. disticha or diazepam had higher HRV when compared tothose given vehicle at PTD9 (mean differences of 1.85 ± 0.83, 1.83 ±0.83 and 1.77 ± 0.83 bpm, respectively, p b 0.05). This effect disap-peared one month post-treatment. Furthermore, the magnitude ofchange in HRV observed in MS mice treated with either a low or medi-umdose of B. disticha, or diazepamwas significantly smaller than that inMS mice given vehicle (p b 0.05) (Fig. 3).

4. Discussion

The present results show that adult mice exposed to early maternalseparation (MS) and subjected to acute stressors displayed elevatedblood pressure (BP) and decreased heart rate (HR) over the onemonth observation period. It is interesting that the changes in cardio-vascular response were restricted only to the initial testing period. Spe-cifically, MS mice exhibited increased SBP and decreased HR at PTD9 aswas observed earlier after immediate withdrawal of treatments onPTD2 (Pote et al., 2013). However, this response to acute stress in MSmice was absent on PTD30 suggesting that maternal separation is aweak model of anxiety as reported previously (Faure et al., 2006;Liebsch et al., 1998). This may also be caused by habituation of MSmice to repeated exposure of the same stressor. Therefore, futurefollow-up studies need to vary stressors in the MS model in orderto continue simulating the underlying autonomic disturbances. Thisstudy also showed that blood pressure variability (BPV) was increasedin MS mice. BPV is positively related to end-organ damage (Cohen andBenjamin, 2006; Igosheva et al., 2004; Mancia and Parati, 2003) hencematernal separation may cause increased cardiac damage in mice.

Repeated maternal separation may augment cardiovascular reactiv-ity and increase long-term anxiety-like and depression-related

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Fig. 3.A graph showing the autonomic reactivity to repeated exposure to acutemixing (AMS) and restraint stress in BALB/cmice. Changes in blood pressure variability (BPV) and heart ratevariability (HRV) were given by subtracting recordings made on PTD9 from PTD30 recordings. Control (un-separated, and maternally separated (MS)) mice were gavaged with vehicle(Groups A and B respectively) while four other MS groups were given B. disticha aqueous ethanolic extract at low (LDBD, 10 mg/kg body weight (BW)), medium (MDBD, 25 mg/kgBW) and high doses (40 mg/kg BW) for Groups C, D and E, respectively and diazepam (DZP) at 1 mg/kg BW (Group F). * p b 0.05 (significant differences when treatment withB. disticha (Groups C, D and E) was compared to vehicle (Group B) in MS mice).

37W. Pote et al. / South African Journal of Botany 94 (2014) 33–39

behaviors in response to acute stressors in adults (Caldji et al., 2000;Daniels et al., 2004; Faure et al., 2007; Huot et al., 2001; Kalinichevet al., 2002; Lippmann et al., 2007; McIntosh et al., 1999; Pote et al.,2013; Wigger and Neumann, 1999). Earlier studies demonstrated thathigh anxiety breeds of rats are more sensitive to the anxiolytic effectof diazepam than low anxiety breeds (Liebsch et al., 1998). In the pres-ent study, MS mice treated with diazepam had comparable BP and HRto vehicle-treated MS group. The results suggest that diazepam is inef-fective in treatment of cardiovascular disturbances seen in maternallyseparated mice.

The increased BP in MS mice in response to AMS, observed in thisstudy, may be mediated through exaggerated behavioral and neuroen-docrine stress responses, increased secretion of catecholamines tomobilize energy tomeet raisedmetabolic requirements, increased sym-pathetic tone to optimize cardiovascular and respiratory functions, anda slightly delayed increase in glucocorticoid secretion to suppress theacute response (Daniels et al., 2004, 2009; Cohen and Benjamin, 2006;Faure et al., 2006; Øverli, 2001). These disturbances may be mediatedby dysfunction of central neurotransmitter pathways (monoaminereceptors and production); intracellular transduction messenger sys-temmalfunction (Gproteins, cyclic adenosinemonophosphate); hyper-activity of the hypothalamic–pituitary–adrenal (HPA)-axis or alteredneurotrophin levels (Daniels et al., 2004, 2009; Faure et al., 2006,2007; Head and Kelly, 2009; Øverli, 2001; Sarris and Kavanagh, 2009).

4.1. Long-term cardiovascular response to B. disticha treatment in adult MSmice

Maternally separated mice treated with B. disticha showed signifi-cantly reduced cardiovascular parameters when compared to thosegiven vehicle or diazepam. MS mice treated with a low dose ofB. disticha had significantly low SBP, DBP and MAP on PTD9 suggesting

short-term antihypertensive activity at 10 mg/kg BW. Those gavagedwith medium dose had significantly decreased SBP and DBP on PTD30pointing to long-term effects of B. disticha at a dose of 25 mg/kg BW.However, the group treatedwith a high dose had a significantly reducedSBP, DBP andMAP thus exhibiting short-term and long-term antihyper-tensive effects of B. disticha at 40 mg/kg BW.

These results indicate that low doses of B. disticha are only effectivefor about oneweek and do not persist longer while the effects of higherdoses of B. disticha on SBP start one week and persist for more than amonth. The cardiovascular effects of B. disticha in maternally separatedmice on SBP were also found to be dose-dependent. These effects ofB. disticha extract can be attributed to several alkaloids present in theplant thatmediate their biological activities through the cholinergic, ad-renergic and/or serotonergic systems (Bastida et al., 2011; Cheesmanet al., 2012; Nair et al., 2013; Nair and Van Staden, 2014; Neergaardet al., 2009; Nielsen et al., 2004; Sandager et al., 2005; Viladomat et al.,1997).

B. disticha extractmay directly or indirectly reduce physical patterns,blood pressure or respiratory rate, heightened in animals subjected toboth maternal separation and acute mixing stress through the centralmonoamine neurotransmitters serotonin (5-hydroxytryptamin, 5-HT),dopamine (DA) and norepinephrine (NE) (Cohen and Benjamin, 2006;Øverli, 2001). The phyto-constituents of the plant extract or their me-tabolites may reach the central nervous system (Gadaga et al., 2011;Nyazema and Ndiweni, 1986) and effect their actions mainly by affect-ing these monoamine neurotransmitter systems in maternally separat-ed animals. Studies done by Nielsen et al. (2004) and Sandager et al.(2005) have shown that a crude extract of B. disticha has high affinityfor the binding site on the serotonin transporter protein (SERT)in vitro. Furthermore, similar studies found that the plant alkaloidshave inhibitory activity on serotonin transporter protein (SERT) andsome affinity for serotonin receptors (5-HT1A) (Neergaard et al., 2009;

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38 W. Pote et al. / South African Journal of Botany 94 (2014) 33–39

Sandager et al., 2005). This is believed to be the main mechanism bywhich this plant exerts its anxiolytic and antidepressant activitiesin vivo since blocking the serotonin receptors with cyproheptadine,decreased toxicity of B. disticha (Mutseura et al., 2013). The presentfindingsmay therefore suggest long-lasting beneficial autonomic effectsof B. disticha in cardiovascular comorbid disorders present in anxietydisorders explaining why the plant is used frequently in herbal medi-cine in southern Africa (Nair and Van Staden, 2014; Stafford et al.,2008).

4.2. Long-term autonomic responses to B. disticha extract in adult MS mice

Neither diazepam nor B. disticha had an effect on BPV and/or theunderlying cardiac damage in MS mice. Hence, B. disticha may exert itseffects on the heart indirectly by affecting the autonomic regulation ofthe cardiovascular system. The present study also found that MS micetreated with diazepam or B. disticha (low and medium doses) hadsignificantly elevated heart rate variability (HRV) on PTD9. This maybe indicative of short-term anxiolytic activity of B. disticha since HRVis decreased in anxiety disorders and anxiolytic drugs partly correctthis abnormality by acting on the autonomic nervous system (Cohenand Benjamin, 2006; Depino and Gross, 2007; Igosheva et al., 2004;Volkmar et al., 2005). These results are consistent with previous resultsin our laboratory which found that B. disticha exhibited anxiolytic-likeactivity in open field, souk, elevated plus maze and marble bearingtests (Chuma et al., 2010; Gadaga, 2012; Musarira et al., 2011;Punungwe et al., 2013; Tsigo et al., 2013).

5. Conclusion

Current findings indicate that B. disticha aqueous ethanolic extracthas a long-term antihypertensive effect that persists for at least onemonth after withdrawal of the treatment in maternally separatedBALB/c mice. Furthermore, the plant extract increased HRV showingpositive anxiolytic activity. The present results form an important base-line for further studies on drug development from the plant extract.However, future studies are needed to elucidate the underlyingmecha-nism of action of B. disticha extract in a similar animal model.

Acknowledgment

We are very grateful to the University of Zimbabwe Research Boardfor financial assistance (Grant Number: RB/108/11) and the Depart-ments and staff in Faculties of Veterinary Science, Science and Collegeof Health Sciences, University of Zimbabwe for their unwavering sup-port, facilities and equipment used in this project. Many thanks go toMr Zulu Daniel for helping us during plant collection, extraction andcharacterization of the extract used in this study and to Mr. KwitiriHarmony for taking care of our study animals throughout the project.

References

Adewusi, A.E., Fouche, G., Steencamp, V., 2012. Cytotoxicity and acetylcholinesterase inhib-itory activity of an isolated crinine alkaloids from Boophone disticha (Amaryllidaceae).Journal of Ethnopharmacology 143 (2), 572–578.

Bastida, J., Berko, V., Torras, L., Pigni, N.B., de Andrade, J.P., Martinez, V., Codina, C.,Viladomat, F., 2011. Chemical and biological aspects of Amaryllidaceae alkaloids.Recent Advances in Pharmaceutical Sciences 65–100.

Belzung, C., Griebel, G., 2001. Measuring normal and pathological anxiety-like behaviourin mice: a review. Behavioural Brain Research 125, 141–149.

Caldji, C., Francis, D., Sharma, S., Plotsky, P.M., Meaney, M.J., 2000. The effects of early rear-ing environment on the development of GABAA and central benzodiazepine receptorlevels and novelty-induced fearfulness in the rat. Neuropsychopharmacology 22,219–229.

Cheesman, L., Nair, J.J., Van Staden, J., 2012. Antibacterial activity of crinane alkaloids fromBoophone disticha (Amaryllidaceae). Journal of Ethnopharmacology 140, 405–408.

Chingombe, P., Tagwireyi, D., Gadaga, L.L., 2010. Investigating the antidepressant-likeactivity of Boophone disticha after repeated daily dosing in a mouse model. Bachelorof Pharmacy Thesis, University of Zimbabwe Library (Unpublished results).

Chuma, D., Tagwireyi, D., Gadaga, L.L., 2010. Investigation of the anxiolytic-like activity ofBoophane disticha extract in mice. Bachelor of Pharmacy Thesis, University ofZimbabwe Library (Unpublished results).

Cohen, H., Benjamin, J., 2006. Power spectrum analysis and cardiovascular morbidity inanxiety disorders. Autonomic Neuroscience: Basic and Clinical 128, 1–8.

Daniels, W.M., Pietersen, C.Y., Carstens, M.E., Stein, D.J., 2004. Maternal separation in ratsleads to anxiety-like behavior and a blunted ACTH response and altered neurotrans-mitter levels in response to a subsequent stressor. Metabolic Brain Disorders 19, 3–14.

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 corticosteronelevels but not the DNA methylation status of the exon 17 glucocorticoid receptorpromoter region. Metabolic Brain Disorders 24, 615–627.

De Smet, P.A.G.M., 1996. Some ethnopharmacological notes on African hallucinogens.Journal of Ethnopharmacology 50, 141–146.

Depino, A.M., Gross, C., 2007. Simultaneous assessment of autonomic function andanxiety-related behaviour in BALB/c mice and c57BL/6 mice. Behavioural BrainResearch 177, 254–269.

Faure, J., Uys, J.D.K., Marais, L., Stein, D.J., Daniels, W.M.U., 2006. Early maternal separationfollowed by later stressors leads to dysregulation of the HPA-axis and increases inhippocampal NGF and NT-3 levels in a rat model. Metabolic Brain Disorders 21,181–188.

Faure, J., Uys, J.D.K., Marais, L., Stein, D.J., Daniels, W.M.U., 2007. Early maternal separationalters the response to traumatization resulting in increased levels of hippocampalneurotrophic factors. Metabolic Brain Disorders 22 (2), 183–195.

Gadaga, L.L., 2012. Investigation of the toxicological and pharmacological activity of ahydroethanolic extract of Boophone disticha bulb. Masters of Philosophy Thesis,University of Zimbabwe Library (Unpublished results).

Gadaga, L.L., Tagwireyi, D., Dzangare, J., Nhachi, C.F.B., 2011. Acute oral toxicity andneurobehavioral toxicological effects of a hydroethanolic extract of Boophone distichain rats. Human and Experimental Toxicology 30 (8), 972–980.

Gelfand, M., Mitchell, C.S., 1952. Buphanine poisoning in man. South African MedicalJournal 26, 573–574.

Gelfand, M., Mavi, S., Drummond, R.B., Ndemera, B., 1985. The Traditional Medical Practi-tioner in Zimbabwe. Mambo Press, Gweru p. 296.

Gomes, N.G.M., Campos, M.G., Orfão, J.M.C., Ribeiro, C.a F., 2009. Plants with neurobiolog-ical activity as potential targets for drug discovery. Progress in Neuro-Psychopharmacology & Biological Psychiatry 33 (8), 1372–1389.

Hauth, H., Stauffacher, D., 1961. Die alkaloide von Buphane disticha (L.f.) Herb. HelveticaChimica Acta 44, 491–502.

Head, K.A., Kelly, S.G., 2009. Nutrients and botanicals for treatment of stress: adrenalfatigue, neurotransmitter imbalance, anxiety and restless sleep. Alternative MedicineReview 2 (2), 114–140.

Huot, R.L., Thrivikraman, K.V., Meaney, M.J., Plotsky, P.M., 2001. Development of adultethanol preference and anxiety as a consequence of neonatal maternal separationin Long Evans rats and reversal with antidepressant treatment. Psychopharmacology158, 366–373.

Hutchings, A., Scott, A.H., Lewis, G., Cunningham, A.B., 1996. Zulu Medicinal Plants: AnInventory. University of Natal Press, Pietermaritzburg.

Igosheva, N., Klimova, O., Anishchenko, T., Glover, V., 2004. Prenatal stress alterscardiovascular responses in adult rats. Journal of Physiology 557, 273–285.

Kalinichev, M., Easterling, K.W., Plotsky, P.M., Holtzman, S.G., 2002. Long-lasting changesin stress-induced corticosterone response and anxiety-like behaviors as a conse-quence of neonatal maternal separation in Long-Evans rats. Pharmacology, Biochem-istry and Behavior 73, 131–140.

Liebsch, G., Linthorst, A.C.E., Neumann, I.D., Reul, J.M.H.M., Holsboer, F., Landgraf, R., 1998.Behavioural, physiological, and neuroendocrine stress responses and differentialsensitivity to diazepam in two Wistar rat lines selectively bred for high- and low-anxiety-related behaviour. Neuropsychopharmacology 19, 381–396.

Lippmann, M., Bress, A., Nemeroff, C.B., Plotsky, P.M., Monteggia, L.M., 2007. Long-termbehavioural and molecular alterations associated with maternal separation in rats.European Journal of Neuroscience 25, 3091–3098.

Mancia, G., Parati, G., 2003. The role of blood pressure variability in end-organ damage.Journal of Hypertension S17–S23 (Supplement).

McIntosh, J., Anisman, H., Merali, Z., 1999. Short- and long-periods of neonatal maternalseparation differentially affect anxiety and feeding in adult rats: gender-dependenteffects. Brain Research. Developmental Brain Research 113, 97–106.

Musarira, S., Tagwireyi, D., Gadaga, L.L., 2011. Effects of a hydroethanolic extract ofBoophone disticha bulb in behavioural models of anxiety inmice. Bachelor of Pharma-cy Thesis, University of Zimbabwe Library (Unpublished results).

Mutseura, M., Tagwireyi, D., Gadaga, L.L., 2013. Pre-treatment of BALB/c mice with a cen-trally acting serotonin antagonist (cyproheptadine) reduces mortality from Boophonedisticha poisoning. Clinical Toxicology (Philadelphia, Pa.) 51 (1), 16–22.

Nair, J.J., Van Staden, J., 2014. Traditional usage, phytochemistry and pharmacology of theSouth Africanmedicinal plant Boophone disticha (L.f.) Herb. (Amaryllidaceae). Journalof Ethnopharmacology 151, 12–26.

Nair, J.J., Bastida, J., Codina, C., Viladomat, F., Van Staden, J., 2013. Alkaloids of the SouthAfrican Amaryllidaceae: a review. Natural Product Commununications 8, 1335–1350.

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. SouthAfrican Journal of Botany 75, 371–374.

Nielsen, N.D., Sandager, M., Stafford, G.I., Van Staden, J., Jäger, A.K., 2004. Screening ofindigenous plants from South Africa for affinity to the serotonin reuptake transportprotein. Journal of Ethnopharmacology 94, 159–163.

Nyazema, N., Ndiweni, D., 1986. Effects of Boophone disticha on the central nervoussystem of rats, guinea pig ileum and rabbits' eyes. Bachelor of Pharmacy Thesis,University of Zimbabwe Library (Unpublished results).

Page 7

39W. Pote et al. / South African Journal of Botany 94 (2014) 33–39

Øverli, Ø., 2001. Behavioural and Neuroendocrine Effects of Stress in Salmonid Fish.Comprehensive Summaries of Uppsala PhD DissertationFaculty of Science andTechnology, Uppsala University.

Pedersen, M.E., Szewczyk, B., Stachowicz, K., Wieronska, J., Andersen, J., Stafford, G.I., VanStaden, J., Pilc, A., Jäger, A.K., 2008. Effects of South African traditional medicine inanimal models for depression. Journal of Ethnopharmacology 119, 542–548.

Pote, W., Tagwireyi, D., Chinyanga, H.M., Musara, C., Nyandoro, G., Chifamba, J.,Nkomozepi, P., 2013. Cardiovascular effects of Boophone disticha aqueous ethanolicextract on early maternally separated BALB/c mice. Journal of Ethnopharmacology148, 379–385.

Punungwe, A., Pote, W., Gadaga, L.L., Tagwireyi, D., 2013. Investigation on the possible ac-tivity of a hydroethanolic extract of Boophone disticha in a mouse model of obsessivecompulsive disorder. Bachelor of Pharmacy Thesis, University of Zimbabwe Library(Unpublished results).

Sandager, M., Nielsen, N.D., Stafford, G.I., Van Staden, J., Jäger, A.K., 2005. Alkaloids fromBoophane disticha with affinity to the serotonin transporter in rat brain. Journal ofEthnopharmacology 98, 367–370.

Sarris, J., Kavanagh, D.J., 2009. Kava St. John's Wort: current evidence for use in mood andanxiety disorders. The Journal of Alternative and Complementary Medicine 15 (8),827–836.

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 ofEthnopharmacology 119, 513–537.

Steenkamp, P.A., 2005. Chemical Analysis of Medicinal and Poisonous Plants of ForensicImportance in South Africa. (Philosophiae Doctor Thesis) University of Johannesburg,South Africa.

Tsigo, V., Chikeri, BT., Pote, W., Mwandiringana, E., Dhyiwayo, S., 2013. Investigationof the anxiolytic-like activity of repeated daily dosing of a hydro-ethanolicextract of Boophone disticha in transportation induced anxiety in rats. Bachelorof Veterinary Science Thesis, University of Zimbabwe Library (Unpublishedresults).

Viladomat, F., Bastida, J., Codina, C., Nair, J.J., Cambell, W.E., 1997. Alkaloids of theSouth African Amaryllidaceae, recent research developments. Phytochemistry 1,131–171.

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. AmericanJournal of Physiology. Regulatory, Integrative and Comparative Physiology 228,R1132–R1142.

Wigger, A., Neumann, I.D., 1999. Periodic maternal deprivation induces gender-dependent alterations in behavioral and neuroendocrine responses to emotionalstress in adult rats. Physiology and Behavior 66, 293–302.

Zulu, D., Tagwireyi, D., Gadaga, L.L., 2011. Solvent extraction and chromatographic charac-terization of isoquinoline alkaloids from the bulb of Boophone disticha. Bachelor ofPharmacy Thesis, University of Zimbabwe Library (Unpublished results).