Adongo et al. 
Natural Products and Bioprospecting           (2023) 13:33  Natural Products and 
https://doi.org/10.1007/s13659-023-00399-8 Bioprospecting
ORIGINAL ARTICLE Open Access
Anxiolytic-like effects of Pseudospondias 
microcarpa hydroethanolic leaf extract 
in zebrafish: Possible involvement of GABAergic 
and serotonergic pathways
Donatus Wewura Adongo1*  , Charles Kwaku Benneh1, Augustine Tandoh1, Robert Peter Biney2, 
Kennedy Kwami Edem Kukuia3, Priscilla Kolibea Mante4, Benjamin Kingsley Harley5, David Oteng1, 
Emmanuel Aduboffour Appiah1, Ernest Cudjoe Anorbor1 and Eric Woode1 
Abstract 
Pseudospondias microcarpa is used in ethnomedicine to manage central nervous system diseases. The hydroethanolic 
extract (PME) from the leaves of the plant has shown anxiolytic-like properties in mice anxiety models. However, its 
effects in chronic anxiety models and possible mechanism(s) of action were not studied. Therefore, the current study 
evaluated the anxiolytic-like mechanisms of PME in zebrafish models of anxiety. The zebrafish light dark test (LDT) 
and novel tank test (NTT) were employed to assess the anxiolytic-like effects of PME (0.1, 0.3, 1.0 mg m L−1), fluox-
etine (3 ×  10−5 mg m L−1) and diazepam (1.5 ×  10−7 mg  mL−1). The chronic unpredictable stress (CUS) test was used 
to further evaluate the extract’s anxiolytic-like properties. The potential mechanisms of anxiolytic action of the extract 
was evaluated after pre-treated with flumazenil, granisetron, methysergide, or pizotifen, all at 1 ×  10−3 mg  mL−1. The 
extract significantly decreased anxiety behaviours in the NT and LD tests. These observed effects of the extract were 
however counteracted by flumazenil, granisetron, methysergide and pizotifen pre-treatment. In addition, PME treat-
ment significantly reversed CUS-induced anxiety behaviours in zebrafish. Results show that PME possesses anxiolytic-
like effects possibly through interaction with serotonergic and gamma-aminobutyric acid mediated pathways.
Keywords Anxiety disorders, Pseudospondias microcarpa, Zebrafish, Novel tank, Benzodiazepines
*Correspondence:
Donatus Wewura Adongo
dadongo@uhas.edu.gh
Full list of author information is available at the end of the article
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Adongo et al. Natural Products and Bioprospecting           (2023) 13:33 Page 2 of 13
Graphical abstract
1 Introduction Despite significant advancements, many people with 
Anxiety disorders typically depict physiological, psycho- anxiety disorders do not respond to pharmacological 
logical, and behavioural changes brought on by an actual therapies in a satisfactory way [3]. This makes it necessary 
or perceived threat to survival or well-being in humans or to identify and develop medications that are free of these 
animals, and is often marked by an increase in nervous- tolerance and efficacy limitations [7]. In clinical studies, a 
ness, anticipation, hormonal and autonomic stimulation, number of medicinal plants including Kava kava, Valeri-
as well as particular behavioural changes like feeding and ana officinalis, Passiflora incarnata, Withania somnifera, 
exploration to escape [1]. The most prevalent psychiatric and Hypericum perforatum have revealed encouraging 
diseases globally are anxiety disorders, which also have a results in treating anxiety disorders [8]. Thus, research 
huge disease burden [2]. into medicinal plants may help in the identification and 
Antidepressants and benzodiazepines are two classes subsequent development of new agents for managing 
of medications that are frequently used to manage anx- anxiety disorders.
iety-related disorders. Although antidepressants includ- In various regions of Africa, the plant Pseudospondias 
ing selective serotonin reuptake inhibitors (SSRIs) and microcarpa is frequently used to treat diseases, includ-
serotonin norepinephrine reuptake inhibitors (SNRIs) ing conditions of the central nervous system (CNS). 
are the preferred medications owing to their favourable The plant is alleged to sedate individuals who sleep or 
benefit/risk ratio [2, 3], their use nevertheless results in sit underneath it, hence local folks of the Akan tribe in 
sexual dysfunction and delayed anxiolytic effects [4–6]. Ghana popularly refer to it as katawani “close your eyes”. 
Additionally, adverse effects could be more severe during As a result, it is utilized in Ghana as a sedative and to 
the first 2 weeks. Initial jitters or an increase in symptoms treat common CNS diseases [9]. A previous investigation 
regarding anxiety could happen, which could negatively showed PME to have anxiolytic-like properties in rodent 
affect patient adherence to their treatment regimen [2]. models of anxiety [10]. A different study also reported 
Benzodiazepines, unlike antidepressants, do not initially that PME exhibited similar effects to those of antide-
cause increased jitteriness and inability to sleep. How- pressants probably through the 5-hydroxytryptamine 
ever, they may cause CNS depression, leading to fatigue, (5-HT) pathway [11]. Additionally, the extract produced 
drowsiness, slowed reaction times, declined cognitive a fast-onset and long-lasting antidepressant-like activity 
function, dependence, and tolerance [2, 3]. in chronic animal models depicting human depression, 
A dongo et al. Natural Products and Bioprospecting           (2023) 13:33 Page 3 of 13
improving cognitive function and reversing depression- chronic anxiety states. In addition, the possible anxiolytic 
induced anxiogenic behaviour [12, 13]. mechanism(s) were investigated.
Although rodent models depicting human neuropsy-
chiatric diseases have long been employed in the search 2  Results
for novel therapies, inefficient experimentation and 2.1 A nalysis of PME with Fourier‑transform infrared 
likely high expenses remain barriers [14]. The zebrafish, spectroscopy (FT‑IR)
an inexpensive, marine vertebrate species that shares a Over an IR band of 400–4000  c m−1, different func-
great deal of human genetic and physiological makeup, tional groups were identified using FT-IR spectroscopy. 
has in the last decade been recognized as a potent ani- In order to compare extracts afterwards, characteristic 
mal for modelling several CNS disorders in humans [15– spectra in the region were employed as the fingerprint 
18]. Additionally, approximately 82% of genes linked to spectra. Additional file 1: Figure S1 and Table S1 show IR 
human diseases have orthologues in the fully sequenced spectra and peak values respectively.
zebrafish genome [19].
According to a recent review, the zebrafish has been 2.2  Acute anxiolytic effects
used in a number of research as an effective tool for find- 2.2.1 N ovel tank test (NTT)
ing natural therapies with possible anxiolytic benefits The effects of acute treatment of PME, diazepam or 
[20]. Although the anxiolytic effects of PME have been fluoxetine on zebrafish behaviours in the NTT are 
established in rodent models, its effects in chronic anxi- shown in Fig. 1. The duration of fish in upper 2/3 com-
ety models and possible mechanism(s) of action are yet to partments of the tank significantly increased following 
be studied. Thus, this study explored the anxiolytic-like acute treatment with PME (F5,20 = 4.025, P = 0.0109). A 
effects of PME in zebrafish models depicting acute and post hoc analysis as seen in Fig.  1a showed significance 
Fig. 1 Effects of acute administration of PME, fluoxetine, and diazepam on time spent in upper 2/3 (a), number of entries to upper 2/3 (b) 
and latency to upper 2/3 (c) in the novel tank test. Data are expressed as group mean ± SEM (n = 5). Significantly different from control: *P < 0.05, 
**P < 0.01 (one-way ANOVA followed by Newman–Keuls post hoc test)
Adongo et al. Natural Products and Bioprospecting           (2023) 13:33 Page 4 of 13
at 0.3  mg   mL−1 (P < 0.05) and 1  mg   mL−1 (P < 0.01) for in Fig.  2b. However, treatment with 1  mg   mL−1 PME 
PME, and (P < 0.05) for both fluoxetine and diazepam. showed significance (P < 0.05).
After acute treatment with PME, fluoxetine, or diazepam, 
latency to the upper 2/3 region (Fig. 2c) of the novel tank 
was significantly decreased (F5,20 = 4.866, P = 0.0045). 2.3  CUS
However, neither PME nor the standard drugs had any 2.3.1  NTT
statistically significant effects on entries (F5,20 = 0.249, In comparison with the non-stressed group, zebrafish 
P > 0.05) into the upper 2/3 region (Fig. 1b). exposed to the CUS schedule displayed anxiety behav-
iours by showing increased latency to enter upper 2/3 
2.2.2  Light dark test (LDT) region (P < 0.05) of the NT and spending less time 
Total time spent in the light region of the LD appara- (P < 0.05) in the same region (Fig.  3). With regards 
tus increased significantly after treatment with PME to the number of entries into the upper 2/3 region 
(F5,20 = 4.44, P = 0.0070). A post hoc analysis (Fig.  2a) by stressed fish, a significant decrease (P < 0.05) was 
showed significance at 0.3  mg  m L−1 (P < 0.05) and observed compared to the non-stressed group, indi-
1  mg   mL−1 (P < 0.01) for PME, and (P < 0.05) for both cating decreased locomotor activity. Treatment with 
fluoxetine and diazepam. Latency to the light region PME or fluoxetine however significantly reversed 
(Fig.  2c) was also significantly reduced after acute these effects in the upper 2/3 regions as observed for 
administration of PME, fluoxetine, or diazepam time spent (F5,20 = 5.14, P = 0.0034; Fig. 3a), number of 
(F5,20 = 4.625, P = 0.0058). Treatment with PME or the entries (F5,20 = 6.26, P = 0.0012; Fig.  3b), and latency 
standard drugs did not affect the number of entries (F5,20 = 4.18, P = 0.0092; Fig. 3c).
into the light region (F5,20 = 2.534, P = 0.0623), as shown 
Fig. 2 Effects of acute administration of PME, fluoxetine, and diazepam on time spent in light region (a), number of entries to light region (b) 
and latency to light region (c) in the novel tank test. Data are expressed as group mean ± SEM (n = 5). Significantly different from control: *P < 0.05, 
**P < 0.01 (one-way ANOVA followed by Newman–Keuls post hoc test)
A dongo et al. Natural Products and Bioprospecting           (2023) 13:33 Page 5 of 13
Fig. 3 Effects of acute administration of PME and fluoxetine on time spent in light region (a), number of entries to light region (b) and latency 
to light region (c) in the novel tank test after the CUS procedure. Data are expressed as group mean ± SEM (n = 5). Significantly different from control: 
*P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA followed by Newman–Keuls post hoc test)
2.3.2  LDT was observed, indicating an anxiety state resulting from 
Chronic exposure of zebrafish to the CUS protocol the CUS paradigm. However, acute treatment with the 
resulted in reduced entries to the  light compartment extract or fluoxetine decreased shoal cohesion duration 
(P < 0.05) and decreased time spent in the same com- (F5,20 = 9.37, P = 0.0141; Fig.  5a) and increased latency 
partment (P < 0.05), while increasing the latency to entry to shoal cohesion (F5,20 = 7.01, P = 0.0047; Fig.  5b) in 
(Fig.  4). However, PME or fluoxetine treatment dem- stressed fish, indicating anxiolytic-like effect.
onstrated effects similar to anxiolytics by significantly 
increasing the time stressed zebrafish spent in the light 2.4  Assessment of possible anxiolytic mechanisms
region (F5,20 = 6.54, P = 0.0009; Fig.  4a) and decreas- 2.4.1 I nvolvement of the GABAergic system
ing latency to the light region (F5,20 = 5.06, P = 0.0037; As shown in Fig.  6, time spent in the upper sections 
Fig.  4c). The number of entries into the light compart- of the NT and light compartment of the LD equip-
ment also increased significantly (F5,20 = 4.76, P = 0.0050; ment were not significantly altered after immersion in 
Fig.  4b), with PME at 1  mg   mL−1 showing significance 1 × 1 0−3  mg  m L−1 flumazenil alone. Treatment with 
(P < 0.001). 1 mg  mL−1 PME produced an anxiolytic-like effect similar 
to 1.5 × 1 0−7  mg  m L−1 DZP, demonstrated by increased 
2.3.3  Shoal cohesion time spent in the upper 2/3 and light compartments in 
Stressed fish displayed noticeably altered shoal cohe- the NT and LD tests, respectively (P < 0.001 for PME and 
sion (Fig. 5). The duration of shoal cohesion in stressed P < 0.01 for DZP in both tests). However, the observed 
fish increased considerably (P < 0.05), when compared anxiolytic-like effects of the extract in both experiments 
to the naïve group. Additionally, a decrease in time was reversed significantly with flumazenil pre-treatment 
taken to shoal cohesion in the CUS zebrafish (P < 0.05) (all at P < 0.01). Diazepam had similar effects as well.
Adongo et al. Natural Products and Bioprospecting           (2023) 13:33 Page 6 of 13
Fig. 4 Effects of acute administration of PME and fluoxetine on time spent in light region (a), number of entries to light region (b), and latency 
to light region (c) in the light–dark test after the CUS procedure. Data are expressed as group mean ± SEM (n = 5). Significantly different from control: 
*P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA followed by Newman–Keuls post hoc test)
Fig. 5 Effects of acute administration of PME and fluoxetine on shoaling cohesion duration region (a) and latency to shoal cohesion (b) 
after the CUS procedure. Data are expressed as group mean ± SEM (n = 3). Significantly different from control: *P < 0.05, **P < 0.01 (one-way ANOVA 
followed by Newman–Keuls post hoc test)
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Fig. 6 Effects of acute administration of PME (1 mg  mL−1) and diazepam (1.5 ×  10−7 mg m L−1) after pre-treatment with flumazenil 
(1 × 1 0−3 mg  mL−1) on the time spent in upper 2/3 and light region of the novel tank test (a) and light–dark test (b) respectively. Data are expressed 
as group mean ± SEM (n = 5). Significantly different from control: **P < 0.01, ***P < 0.001 compared to control group; ##P < 0.01, ###P < 0.001 compared 
to group pre-treated with antagonist (one-way ANOVA followed by Newman–Keuls post hoc test)
2.4.2  Involvement of the serotonergic system Most zebrafish behavioural models of anxiety were 
Figure 7 shows the effects of PME, fluoxetine or various developed from rodent models, as these fish are often 
serotonergic antagonists on fish behaviour in the NT and exposed to various stressors such as utilizing lit or 
LD tests. In comparison to the control group, adminis- dark areas, unfamiliar settings, and models of poten-
tration of granisetron, pizotifen, or methysergide (all at tial predators. Clinically effective anxiolytic drugs are 
1 ×  10−3 mg   mL−1) had no significant alteration on time used to validate these adjustments [20, 21]. This enables 
spent in the upper 2/3 region of the NT. Similarly, the the investigation of possible anxiolytic effects of natural 
time spent in the light region did not alter significantly products.
in the LDT. The zebrafish instinctively seeks protection when 
Administration of PME or fluoxetine demonstrated placed in a novel environment, and the NTT is built on 
anxiolytic-like effects by increasing the time spent in this behaviour. Zebrafish prefer to remain on the tank’s 
light region of the LD apparatus. However, the extract’s bottom until they feel safe enough to explore the whole 
observed effect was blocked by pre-treatment with tank [20, 22]. In order to evaluate anxiety in adult fish, the 
granisetron (P < 0.01; Fig. 7a), pizotifen (P < 0.05; Fig. 7c) NTT generally measures a number of metrics including 
or methysergide (P < 0.05; Fig.  7e). Fluoxetine showed latency to explore the top, duration in the upper regions, 
comparable results: [granisetron (P < 0.001), pizotifen entries to top, frequency of freezing episodes, and fre-
(P < 0.05), or methysergide (P < 0.01)]. quency of erratic engagements [23]. In this paradigm, 
Similar to effects in the NTT, administration of PME or increased irregular movements and freezing, together 
fluoxetine increased significantly the upper 2/3 duration with a major reduction in exploration (increased latency 
in the NTT. This was however reversed by pre-treatment to upper regions, decreased duration in upper regions, 
with the various serotonergic antagonists: PME [grani- and fewer entries), are signs of elevated anxiety state and 
setron (P < 0.01; Fig.  7b), pizotifen (P < 0.01; Fig.  7d) or stress [24]. Drugs with anxiolytic effects, including ben-
methysergide (P < 0.001; Fig.  7f )], and fluoxetine [grani- zodiazepines, antidepressants, and buspirone decrease 
setron (P < 0.05), pizotifen (P < 0.001) or methysergide the latency and increase exploratory behaviour in the 
(P < 0.001)]. upper regions [22, 25, 26]. The NTT was therefore used 
to evaluate acute anxiolytic effects of PME. The extract 
significantly decreased latency to enter the upper regions 
3  Discussion and conclusion of the tank while increasing duration, indicating anxio-
In the zebrafish models of anxiety used in this investiga- lytic effects. Similar results were obtained for the anxio-
tion, administration of PME demonstrated anxiolytic-like lytic drugs fluoxetine and diazepam. However, the total 
activity comparable to that of fluoxetine and diazepam. number of transitions to the upper regions of the NT 
In addition, the extract reversed anxiety state induced by did not significantly change with the doses of the extract 
the CUS paradigm confirming anxiolytic-like effects. used, eliminating any potential influence of locomotor 
Adongo et al. Natural Products and Bioprospecting           (2023) 13:33 Page 8 of 13
Fig. 7 Effects of acute administration of granisetron (a, b), pizotifen (c, d) and methysergide (e, f) given alone or in combination with PME 
(1 mg  mL−1) or FLX (3 ×  10−4 mg  mL−1) on the time spent in the upper 2/3 of the novel tank test and the light region of LDT. Data are expressed 
as group mean ± SEM (n = 5). Significant difference: *P < 0.05, **P < 0.01, ***P < 0.001 compared to control group; #P < 0.05, ##P < 0.01, ###P < 0.001 
compared to group pre-treated with antagonist (one-way ANOVA followed by Newman–Keuls post hoc test)
activity on its anxiolytic effects. Comparable effects on The light/dark test, which uses zebrafish’s natural aver-
locomotor function was observed for the standard anxio- sion to highly lit places and their spontaneous explora-
lytics used. tory behaviour in unfamiliar situations as an anxiety 
A dongo et al. Natural Products and Bioprospecting           (2023) 13:33 Page 9 of 13
index, is another frequently employed behaviourally-val- shoal cohesion indicate that after experiencing chronic 
idated test to evaluate anxiety in zebrafish [27]. Increased unpredictable stress, zebrafish developed a pheno-
time spent in the dark by zebrafish (scototaxis) is a sign type associated with anxiety and other mood disorders. 
of an anxiety behaviour that is affected by both anxi- These behaviours were however reversed by PME and 
ogenic and anxiolytic drugs [28]. The extract significantly fluoxetine, further suggesting anxiolytic-like effects and 
reversed scototaxis by increasing duration in the light confirming the observed effects in the acute anxiolytic 
compartment. Latency to light region was also decreased studies. These results are quite similar to an earlier study 
suggesting anxiolytic-like effects. The effects of the stand- we conducted where the extract reversed chronic unpre-
ard anxiolytics used in this test are consistent with previ- dictable mild stress-induced anxiety in mice [13].
ous reports where benzodiazepines and antidepressants The neurotransmitter gamma-aminobutyric acid 
produced anxiolytic effects [26, 29]. Locomotor activ- (GABA) is an important regulator of anxiety [35, 36], and 
ity in this test is measured by the frequency of crossings zebrafish have shown to have a well-described GABAe-
between the light and dark regions [29]. This parameter rgic system [24, 37]. Similar to this, agents such as pen-
wasn’t decreased by acute treatment with the extract or tylenetetrazole that interfere with the GABAergic system 
standard drugs, suggestive of normal locomotor activity. in zebrafish induce convulsions. On the contrary, drugs 
The anxiolytic-like effects of the extract observed in the that enhance GABAergic transmission such as diazepam 
two models are quite consistent with previous studies in attenuates convulsions [38]. Many natural compounds 
rodent models of anxiety [10, 13]. have  GABAA receptor-modulating activities because of 
Numerous biological markers for central nervous sys- the structural variety of G ABAA receptors [39]. There-
tem (CNS) drug testing have been discovered through fore, we used flumazenil to assess if the GABAergic sys-
the use of chronic stress-induced neuropsychiatric tem may be contributing to the anxiolytic-like effects 
models in rodents in order to produce more effective of the extract. Flumazenil is a selective antagonist at 
therapies. However, using rodent models for CNS drug the  GABAA receptor complex that has been shown to 
development is highly expensive [30]. Zebrafish are sim- antagonize the sedative, anxiolytic, and anticonvulsant 
ple to handle and cost-effective for screening compounds effects of benzodiazepines, making it a valuable agent for 
as potential agents for treating CNS diseases, hence G ABAA receptor investigations [26, 40]. Pre-treatment 
chronic models for anxiety and associated mood disor- with flumazenil reserved the extract’s anxiolytic-like 
ders have been designed. One of such is the CUS model effects, indicating a potential role of the G ABAA recep-
which appears to be particularly effective in causing a tor complex. This finding is consistent with an earlier 
pattern of behaviour of anxiety and other affective disor- study which suggested that the GABAergic system may 
ders in zebrafish [30, 31]. We therefore assessed behav- be implicated in the anticonvulsant activity of the extract 
ioural effects of the extract including shoal cohesion in [41].
the CUS paradigm. The therapeutic benefits of anxiolytic medications in 
The behavioural studies demonstrated the effectiveness zebrafish have also been linked to the serotonergic sys-
of the CUS paradigm in making fish extremely anxious, tem. [42]. According to studies, pharmacologically acti-
as shown by the decreased duration in light compart- vating serotonin receptors reduces anxiety-like behaviour 
ment, increased latency to light compartment and in zebrafish [20] and these days, such drugs are now the 
decreased transitions to light compartment in the LDT. recommended first-line medications for treating anxi-
Similar to the LDT, chronic exposure of fish to the CUS ety disorders. In the present investigation, pre-treatment 
procedure also resulted in an anxiety state in the NTT. with the 5-HT receptor antagonists pizotifen, methy-
All these behaviours in the CUS fish reflect an anxiety sergide, and granisetron reversed the extract’s anxiolytic-
state and are quite consistent with previous studies [26, like effects, suggesting the possible involvement of the 
30, 32–34]. Numerous fish species have shown to engage serotonergic pathway. This could be as a result of the ser-
in shoaling, a social and adaptive behaviour. Shoal cohe- otonin transporter being blocked, consequently increas-
sion, which is a pronounced propensity to form groups ing the concentration of 5-HT downstream and may 
or shoals in zebrafish, is associated with feeding, preda- possibly activate the 5-HT1–3 receptors directly or indi-
tor defence, mating, and fear response [31]. Some studies rectly [26]. This is consistent with a prior study that dem-
have revealed a link between shoal cohesion and anxiety onstrated the extract’s antidepressant effects involved the 
behaviour in zebrafish [30, 31]. In this study, exposure of serotonergic system [11]. Following pre-treatment with 
fish to the CUS paradigm increased shoaling behaviour the antagonists, similar effects were seen for fluoxetine.
as observed by an increased shoal cohesion duration Overall, our results show that the hydroethanolic leave 
and decreased time to shoal formation, which is consist- extract of P. microcarpa possess anxiolytic-like effects in 
ent with the study by [30]. Therefore, findings from the acute and chronic zebrafish anxiety models and that this 
Adongo et al. Natural Products and Bioprospecting           (2023) 13:33 Page 10 of 13
effect may be mediated via GABAergic and serotonergic their natural habitat. Fish were maintained under a 14 h 
systems. light/10 h dark cycle with lights switched on at 9:00 a.m. 
Commercial fish flakes and high-protein pellets were 
4  General experimental procedures alternately fed to adult fish twice daily. Before the inves-
4.1  Plant extraction tigations, fish spent 15 days acclimating to the laboratory 
Leaves of P. microcarpa were harvested from the environment.
Kwame Nkrumah University of Science and Technol-
ogy (KNUST) campus in Kumasi, Ghana (6° 40.626′ N, 4.5  Acute anxiolytic effects
1° 34.041′ W), and confirmed by Dr. George Sam of the 4.5.1 N ovel tank test (NTT)
Department of Herbal Medicine, KNUST. A voucher The method as outlined by Benneh et  al. was uti-
specimen with number KNUST/HM1/2013/L005 was lized [26]. The behavioural apparatus was a glass tank 
then kept at the Faculty’s herbarium. After air-drying (15  cm × 10  cm × 25  cm) divided into three horizontal 
for a week, the leaves were pulverized into fine powder segments of equal dimensions by lines on the exterior of 
and cold macerated with 70% ethanol for three (3) days. the tank, and filled with water to 18 cm. Zebrafish were 
A rotary evaporator with temperature set at 60  °C and given an acute treatment by immersion with PME (0.1, 
under reduced pressure, was used to condense the filtrate 0.3, 1.0  mg   mL−1), fluoxetine (3 × 1 0−5  mg   mL−1), diaz-
into a brown syrupy substance. After a week of additional epam (1.5 ×  10−7 mg m L−1) or distilled water (control) for 
drying in a hot air oven at 50 °C, a yield (w/w) of 20.5% 20 min prior to the experiment. Following a gentle intro-
was obtained. The crude extract was labelled as PME. duction into the test tank, each zebrafish’s behaviour was 
recorded with a camcorder for 5 min. Video outputs were 
4.2 F ourier‑transform infrared spectroscopy (FT‑IR) analysed with the public domain software JWatcher™ 
The spectrum two FT-IR spectrometer (PerkinElmer for time spent  in upper 2/3, number of entries to upper 
UATR Two) was used to conduct the FT-IR analysis in 2/3 and latency to enter upper 2/3. Increased anxiety is 
order to identify any possible functional groups that indicated by an inclination for lowest section of the tank 
could possibly be present in the extract. The analysis and decreased exploration of the upper levels. A longer 
was done over a range of 400–4000   cm−1 as this spec- delay to enter the upper 2/3 is also suggestive of anxiety 
tral region is unique for every compound or compound behaviour.
mixture.
4.5.2 L ight dark test (LDT)
4.3  Chemicals and drugs The preference for a brightly lit or dark environment 
Diazepam (DZP) was obtained from Sigma-Aldrich, was evaluated using the light–dark apparatus based on 
USA; fluoxetine (FLX) from Eli Lilly and Company Ltd., the procedure as previously described [26]. The device 
England; methysergide (Met) and pizotifen (Piz) were measured 50 cm × 10 cm × 10 cm with its length divided 
acquired from Novartis Pharmaceutical cooperation, into two equal halves, with either black or white back-
Switzerland; granisetron (Gstn) from Corepharma LLC, grounds. Before the experiment, zebrafish were treated 
England; and flumazenil (Fmz) from Roche Pharmaceu- by immersion in PME (0.1, 0.3, 1.0 mg  mL−1), fluoxetine 
tical Ltd., UK. Preparation of drug solutions was done (3 ×  10−5  mg   mL−1), diazepam (1.5 ×  10−7  mg   mL−1) or 
with distilled water, and test compounds administered distilled water for 20  min. Following a gentle introduc-
by immersing fish in 250 mL of the solution for 20 min. tion into the test tank, each zebrafish’s behaviour was 
Based on preliminary tests and other studies, the doses recorded for 5 min and analysed for the following param-
of the agents employed in this investigation were selected eters in the light region; total time spent, latency, and 
[5, 26]. During the experiment, the extract concentrations number of entries. Anxiolytic effects are indicated by 
used had no lethal or sedative effects on the zebrafish. a greater inclination for the light region, and evaluated 
by a longer stay there and more entry into the region. 
4.4 Z ebrafish Decreased latency to light region is also considered as an 
Aquarium Marshals Limited located in Accra supplied anxiolytic behaviour.
us with adult wild type zebrafish that were 3–5 cm long 
and 3  months old. Acclimatization of fish was done in 4.6  Chronic unpredictable stress (CUS)
20 L glass tanks filled with dechlorinated water kept at 4.6.1 S tressor pattern
23–25  °C and a pH of 7–8. To reduce cross contamina- The CUS procedure was performed as previously 
tion, each tank had a separate water inlet and outlet. Each reported [26, 34]. Thirty (30) zebrafish were exposed 
housing tank was planted with Cabomba aquatica and to the stressors listed in Table 1 twice daily for 14 days. 
covered with gravel to a height of about 2 cm to simulate Repeated tank change (RTC) involved transferring fish 
A dongo et al. Natural Products and Bioprospecting           (2023) 13:33 Page 11 of 13
Table 1 Chronic unpredictable stress (CUS) stressor pattern
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
Week 1
 Morning RTC OC RS SI DBE RS CS
 Evening DBE C HS CS C HS OC
Week 2
 Morning C OC SI HS DBE CS HS
 Evening RTC DBE RS RTC C SI OC
RTC repeated tank change; DBE dorsal body exposure; RS restrain stress; SI social isolation; OC overcrowding; HS heat stress; CS cold stress; C chasing stress
quickly between tanks six times; dorsal body exposure PME. Based on results from earlier studies, doses of the 
(DBE) involved reducing the water level in the home tank different antagonists were selected [26].
for 10  min to expose the dorsal body surface; restrain 
stress (RS) required gently placing fish for 10  min in a 
10 mL test tube half-filled with system water; social isola- 4.7.1 I nvolvement of the GABAergic system
tion (SI) entailed housing individuals in 100 mL beakers This experiment evaluated the extract’s anxiolytic-like 
for 60 min; for overcrowding (OC), 10 fish were crammed effects on the  GABAA receptor. Briefly, zebrafish (5 per 
into a 250 mL beaker that was only halfway full of system group) were given one of the following treatments for 
water for an hour; heat stress (HS) involved raising the 20  min: system water, diazepam (1.5 × 1 0−7  mg   mL−1), 
tank’s water temperature to 33 °C for 30 min; cold stress PME (1 mg  mL−1), or flumazenil (1 ×  10−3 mg m L−1), fol-
(CS) entailed lowering the water’s temperature to 23  °C lowed by behavioural assessment in the NT and LD tests. 
for 30  min; and chasing stress (C) defined as groups of Parameters measured were time spent in light and upper 
zebrafish continually chased with a capture net for 5 min. 2/3 compartments in the LDT and NTT respectively.
In order to prevent habituation to stressors, the time and In a separate experiment done on the same day, fish 
order of stressors were changed every day throughout the were immersed in 1 mg  mL−1 PME or 1.5 × 1 0−7 mg  mL−1 
entire duration of the stressor schedule. With the excep- diazepam for 20  min after being exposed to 
tion of heating and cooling stressors, temperature and 1 ×  10−3  mg   mL−1 flumazenil for 20  min. Immediately 
aeration were regulated throughout the execution of each after treatments, zebrafish were individually placed in the 
stressor. The non-stressed group was designated naive NT and the LD equipment for behavioural assessments.
control and kept in the same laboratory during the stress 
period.
4.7.2 I nvolvement of the serotonergic system
4.6.2 B ehavioural testing and analysis Serotonergic antagonists for the following receptors; 
The NT, LD, and shoal cohesion tests were carried out 5-HT1 and 5-HT2A/2C (pizotifen), 5-HT2C/2B (methy-
simultaneously to analyse the behaviours of the naive and sergide) and 5-HT3A/3B (granisetron) were utilized to 
stressed groups 24 h after the CUS procedure. Stressed evaluate the possible participation of the 5-HT sys-
fish were randomly grouped (n = 5/group) as follows: tem in the extract’s anxiolytic-like effects. Briefly, 
control, PME (0.1, 0.3, 1.0  mg   mL−1), and fluoxetine zebrafish (5 per group) were given one of the follow-
(3 ×  10−5 mg  mL−1). Zebrafish were dosed by immersing ing treatments for 20  min: 3 ×  10−4  mg  m L−1 fluox-
them in the drug solutions for 20  min before testing in etine, 1 mg   mL−1 PME, 1 × 1 0−3 mg m L−1 granisetron, 
the LD and NT tests described above. 1 × 1 0−3  mg  m L−1 pizotifen, 1 ×  10−3  mg   mL−1 methy-
Using the procedure outlined by Chakravarty et al. [30], sergide or distilled water, followed by behavioural 
the shoaling response was also evaluated. Videos were assessment in the NT and LD tests. Parameters meas-
captured for 5 min after zebrafish from each group (n = 3) ured were time spent in light and upper 2/3 compart-
were placed into the NT. Shoal cohesion was measured ments in the LDT and NTT respectively.
as the time all three zebrafish swam together in the same In another experiment, fish were immersed in 
quadrant. Latency to shoal cohesion was also measured. 1  mg   mL−1 PME or 3 ×  10−4  mg   mL−1 fluoxetine for 
20  min after being exposed to 5-HT antagonists (all at 
4.7 A ssessment of possible anxiolytic mechanisms 1 × 1 0−3 mg  mL−1) for 20 min. Immediately after the vari-
The NT and LD tests described above were also employed ous treatments, zebrafish were placed individually in the 
to evaluate the possible contribution of the GABAergic NT and the LD equipment for behavioural assessments.
and serotonergic systems in the anxiolytic-like effect of 
Adongo et al. Natural Products and Bioprospecting           (2023) 13:33 Page 12 of 13
4.8  Statistical analysis Received: 11 July 2023   Accepted: 13 September 2023
All data are displayed as mean ± SEM or box and 
whisker plots. The boxes’ lower and upper borders cor-
respond to the 25th and 75th percentiles, and their 
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