




































_____________________________________________________________________________________________________ 
 
*Corresponding author: E-mail: evbuomwanlucky1@gmail.com; 
 
 
 

Asian Journal of Immunology 
 
1(1): 36-43, 2018; Article no.AJI.46581 
 

 
 

 

 

Antifungal and Immunomodulatory Activity of 
Bryophyllum pinnatum Leaf Extracts 

 
Jacinta Edesiri Okpoho1, Lucky Evbuomwan1* and Fortune Itojie Ebiala1 

 
1
Department of Microbiology, Faculty of Life Sciences, University of Benin, Benin City, P.M.B. 1154, 

Nigeria. 
 

Authors’ contributions  
 

This work was carried out in collaboration between all authors. Author JEO designed the study, 
performed the statistical analysis, wrote the protocol, and wrote the first draft of the manuscript. 

Authors LE and FIE managed the analyses and literature searches of the study. All authors read and 
approved the final manuscript. 

 
Article Information 

 
DOI: 10.9734/AJI/2018/v1i130092 

Editor(s): 
(1) Dr. Jaffu Othniel Chilongola, Department of Biochemistry and Molecular Biology, Kilimanjaro Christian Medical University 

College, Tumaini University, Tanzania. 
(2) Dr. Darko Nozic, Professor, Higher Medical School in Belgrade, University of Belgrade, Serbia. 

Reviewers: 
(1) Muhammad Shahzad Aslam, Xiamen University, China. 

(2) Mahendran Sekar, Universiti Kuala Lumpur, Malaysia. 
(3) Vivek Kumar Singh, Public Health and Infectious Disease Research Center (PHIDReC), Nepal. 

Complete Peer review History: http://www.sdiarticle3.com/review-history/46581 

 
 
 

Received 26
th

 October 2018  
Accepted 30

th
 January 2019 

Published 26
th

 February 2019 

 
 

ABSTRACT 
 

This study was carried out to investigate the antifungal and immunomodulatory activities of 
Bryophylum pinnatum. Both aqueous and ethanol solvents were used for extraction. Five fungal 
species including Aspergillus niger, Aspergillus flavus, Cladosporium herbarium, Candida albicans 
and Penicillium italicum were obtained from the University of Benin Teaching Hospital and they 
were preliminarily identified using standard microbiological methods. Wistar rat models for the 
study were purchased and acclimatized for a period of two weeks. The rats were divided into five 
groups and orally administered with the ethanol extract of the plant while one group served as 
control. Antimicrobial and hematological parameters including packed cell volume (PCV), 
hemoglobin, white blood cell counts, platelets and CD4 count were assayed using standard 
methods. The only fungus sensitive to aqueous extract was Aspergillus niger, with zone of 
inhibition ranging from 6.00±0.58-11.33±0.33 mm at concentration range of 50-100 mg/ml. Mean 
zones of inhibition of ethanolic extract ranged from 11.67±0.67-20.33±0.33 mm against 

Original Research Article 



 
 
 
 

Okpoho et al.; AJI, 1(1): 36-43, 2018; Article no.AJI.46581 
 

 

 
37 

 

Cladosporium herbarium at concentration range of 6.25-100 mg/ml. Minimum inhibitory 
concentrations (MIC) of ethanol extract ranged from 6.25- 50 mg/ml against fungal isolates. While 
MIC of aqueous extract was 50 mg/ml against susceptible fungal isolate. Minimum fungicidal 
concentrations of ethanol and aqueous were 25- 100 mg/ml and 100 mg/ml respectively. Significant 
difference was observed between the treatment and control groups in platelet counts (range: 
95.40±1.86-126.20±5.40% and control: 108.60±4.19%), PCV (range: 39.00±0.71-44.20±0.58%; 
control: 39.00±0.71%) and hemoglobin (range:12.94±0.21-14.62±0.24 g/dl; control: 12.94±0.21 
g/dl). There was no significant difference between the treatment and control groups in CD4 counts 
(75.40±19.32-99.00±6.33cells/ml and control 75.4±19.32 cells/ml). Bryophylum pinnatum has been 
shown in this work to possess both antimicrobial and hematological properties. 
 

 
Keywords: Fungicidal; immunomodulatory; phytochemical; extract, inhibition. 
 

1. INTRODUCTION 
 
Bryophyllum pinnatum (Kalanchoe pinnata; 
Lamarch Crassulaceae) is an erect, succulent, 
perennial shrub that grows about 1.5m tall and 
reproduces through seeds and also vegetatively 
from leaf bubils. It has a tall hollow stem, freshly 
dark green leaves that are distinctively scalloped 
and trimmed in red and dark bell-like pendulous 
flowers [1]. Bryophyllum pinnatum can easily be 
propagated through stems or leaf cutting. It is an 
introduced ornamental plant that is now growing 
as a weed around plantation crop and widely 
used in traditional medicines [2]. Nigeria is richly 
endowed with indigenous plants which are used 
in herbal medicine to cure diseases and heal 
injuries. Some of the plants are used as food or 
medicine. These plants exhibit a wide range of 
biological and pharmacological activities such as 
anti-cancer, anti-inflammatory, diuretic, oxytocic, 
laxative, antispasmodic, antihypertensive, anti-
diabetic, and anti-microbial and 
immunomodulatory functions [3]. The secondary 
metabolites of plants provides humans with 
numerous biological active products which have 
been used extensively as drugs, foods, additives, 
flavours, insecticides, colorants, fragrances and 
chemicals. These secondary metabolites include 
several classes such as saponins, terpenoids, 
phenolic compounds, steroids, alkaloids and 
flavonoids [4]. In recent years, researches into 
new sources of chemotherapeutic agents have 
intensified due to failure or non-effectiveness of 
conventional drugs to which many pathogens 
have developed resistance. Many compounds 
with potential biological activities have been 
isolated from many plants.  In this regard, 
Bryophyllum pinnatum is used in ethnomedicine 
for the treatment of earache, burns, abscesses, 
ulcers, insect bites, whitlow, diarrhoea and 
cithiasis [5]. In South eastern Nigeria, this herb is 
used to facilitate the dropping of the placenta of 
new born baby [6]. The lightly roasted leaves are 

used externally for skin fungus and 
inflammations. The leaf infusions are an internal 
remedy for fever [4]. Different naturally occurring 
flavonoids have been described and 
subcategorized into flavones, falvans, 
flavanones, isoflavonoids, chalcones, aurones 
and anthocyanidines. These flavonoids exhibit 
remarkable biological activities including 
inhibitory effects on enzymes, modulatory effects 
on some cell types, protection against allergies, 
antiviral, anti-malarial, antioxidant, anti-
inflammatory and anti-carcinogenic properties 
[7]. 
 
Phytochemical screenings of Bryophyllum 
pinnatum have yielded Alkaloids, Triterpenes, 
Glycosides, Flavonoids, Steroids, 
Butadienolides, Lipids, and organic acids, Phenol 
and Tannis, free amino acid and Terpenoids. 
Arachidic acid, Astragalin, Behenic acid, beta 
Amyrin, Benzenoids, Bersaldegenin, beta-
Sitosterol, Bryophollenone, Bryophollone, 
Bryophyllin, Caffeic acid, Ferulic acid, Quercetin, 
Steroids, Taraxerol have also been found from 
extracts of Brophyllum pinnatum [8]. 
 
Two novel Flavanoids; 5 Methyl 4,5,7 trihydroxyl 
flavones and 4,3,5,7 Tetrahydroxy 5 methyl 5 
propenamine anthocyanidines from this plant 
have shown potential antimicrobial activities 
against Pseudomonas aeruginosa, Klebsiella 
pneumoniae, E. coli, Staphylococcus aureus, 
Candida albicans and Aspergillus niger. When 
60% methanolic extract of Bryophyllum pinnatum 
leaf was used to inhibit the growth of bacteria, at 
a concentration of 25 mg/ml it showed good 
antibacterial effects. Further the Plant is effective 
in the treatment of typhoid fever and other 
bacterial infections, particularly those caused by 
S. aureus, E. coli, B. subtilis, P. aeruginosa, K. 
aerogenes, K. pneumonia and S. typhi due to the 
presence of phenolic compounds [9]. The anti-
inflammatory potential of Bryophyllum pinnatum 



 
 
 
 

Okpoho et al.; AJI, 1(1): 36-43, 2018; Article no.AJI.46581 
 

 

 
38 

 

was investigated by [10]. However, the antifungal 
and immunomodulating activity of the plant has 
not been well studied. Therefore, this work was 
carried out to determine the immunomodulatory 
properties and antifungal activities of 
Bryophyllum pinnatum leaf extract. 
 

2. MATERIALS AND METHODS 
 
2.1 Plant Material 
 
Bryophylum pinnatum leaves were obtained from 
Adolor Street in Benin City and identified at the 
Herbarium, Department of Plant Biology and 
Biotechnology, University of Benin, Benin City, 
Edo State. The leaves were air-dried, ground 
using sterilized laboratory blender. The 
powdered leaf was kept in a sterile bottle 
container until required. 
 

2.2 Preparation of Crude Extracts 
 
Fifty grams (50 g) of the grinded Bryophylum 
pinnatum leaves was soaked in 250 ml each of 
distilled water and ethanol for 24 hrs. The extract 
was filtered through a sieve with pore size of 
250µm to remove debris. The filtrate was then 
filtered through membrane filter paper. The final 
filtrate was evaporated in a water bath at 40°C to 
get the crude extract. The crude aqueous and 
ethanol extracts were stored at 4°C until required 
[11].  
 

2.3 Preparation of Concentration of Plant 
Extract 

 
One gram (1g) each of both ethanol and 
aqueous extract was added to 10ml of ethanol 
and distilled water respectively to give a 
concentration of 100mg/ml. Other concentrations 
of 50, 25 and 12.5 and 6.25mg/ml were prepared 
by double dilution method. In this procedure, 1ml 
of content in the stock concentration (containing 
100mg/ml) was added to 1ml each of distilled 
water and ethanol in test tubes to give 50mg/ml 
concentration. 1ml of this 50mg/ml is added to 
another test tube of 1ml distilled water and 
ethanol to give 25mg/ml and so on [12]. 
 

2.4 Test Microorganisms  
 
Five fungal isolates, Aspergillus niger, 
Aspergillus flavus, Cladosporium herbarium, 
Candida albicans and Penicillium italicum were 
used in this study. The fungi were obtained from 

the Microbiology Laboratory stocks in University 
of Benin Teaching Hospital. They were then 
identified based on their cultural and microscopic 
characteristic before further work on them were 
carried out. 
 

2.5 Fungal Inoculum Preparation 

 
The fungi inocula were prepared by inoculating 
the test organisms on potato dextrose broth and 
kept at room temperature for 24hr. 0.2 ml of the 
different fungal broth culture was used for the 
antifungal study [12]. 
 

2.6 Agar Well Diffusion Technique 
 
The ability of the various extracts to inhibit the 
growth of the clinical test organisms was 
determined using the agar well technique. The 
inoculated potato dextrose agar plates were 
allowed to dry. After which, wells were bored on 
the surface of inoculated agar plates using 4mm 
cork borer. Zero point one millilitres (0.1ml) of the 
different concentration of each extracts was 
transferred into the well using Pasteur pipette. 
The wells were sufficiently spaced to prevent the 
resulting zones of inhibition from overlapping. 
The plates were kept at room temperature for 
24hr. The experiment was performed in triplicate 
and the resulting zones of inhibition were 
recorded as mean ± standard error [13]. 
 

2.7 Determination of Minimum Inhibitory 
Concentration (MIC) and Minimum 
Fungicidal Concentration (MFC) 

 
The minimum inhibitory concentration (MIC) of 
the extracts was determined for each of the test 
organisms at varying concentrations of 100, 50, 
25, 12.5 and 6.25mg/ml. 1 ml of potato dextrose 
broth and 1 ml each of the extracts were added 
to test tubes and a loopful of fungi isolates was 
introduced. A tube containing Potato dextrose 
broth only was seeded with the fungi isolates to 
serve as control and all tubes were kept at room 
temperature for 24hr to check for growth. The 
minimum fungicidal concentration of the plant 
extract was carried out according to [14]. Briefly, 
1 ml fungal culture was pipetted from the mixture 
obtained in the determination of MIC tubes which 
did not show any growth and subcultured on to 
potato dextrose agar. The agar plates were left at 
room temperature. After incubation the 
concentration at which there was no single 
growth of fungi was taken as MFC. 

 



 
 
 
 

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39 

 

2.8 Experimental Animals 
 
Eight weeks old Wistar rats weighing between 
200-270 g were used in this experiment. The rats 
were housed at the Department of Microbiology 
Animal House and were fed with pelleted rat food 
and water daily. After acclimatization for two 
weeks, rats were divided into six groups, with five 
rats in each group. Temperature ranged from 
30°C to 37°C during the period of experiment. 
The groups were labelled A to F. Group F served 
as the control group and did not receive 
administration of extract. Groups A to E received 
different dosage of the ethanolic plant extract. 
Group A received 300mg/kg, group B-250mg/kg, 
group C – 200mg/kg, group D – 150mg/kg and 
group E -100mg/kg body weight. On Day 15, 
2.5ml of blood sample was collected from each 
rat into EDTA anticoagulant container and was 
used to estimate the counts of CD4, neutrophils, 
lymphocytes, eosinophils and total leukocytes of 
each group [15]. 
 

2.9 FULL Blood Count (FBC) 
 
This was carried out using Symex21N Japan 
model auto-analyzer. Automated cell counters 
was used to quantify and classify the different 
cell populations according to [16]. 
 

2.10 Enumeration of CD4Count  
 
This was carried out using Partec CyFlow 
Counter (CY-S-3022 model). Briefly, 20µl whole 
blood was added to a Partec test tube and 20µl 
of CD4 mAb PE was added. This was gently 
mixed and incubated for 15min at room 
temperature in the dark. After incubation, 800µl 
of no lyse buffer was added and shaken gently. 
The sample was then analyzed on the Partec 
device [16]. 
 

2.11Statistical Analysis 
 
Data were analysed using statistical package for 
social sciences (SPSS) version 23.0. 
Descriptive statistic was used to present values 
in means and standard errors. One way ANOVA 
was used to find significant difference among 
different parameters [17].   

 
3. RESULTS 
 
The zone of inhibition of aqueous extract of 
Brophyllum pinnatum against funga isolates is 
shown in Table 1. Many fungi species were 

resistant to all concentrations of aqueous extract 
of B. pinnatum except the fungus Aspergillus 
niger with zones of inhibition ranging from 
6.0±0.58 – 11.33±0.33 mm at concentration 
range of 50-100mg/ml. 
 
A comparatively higher antifungal activity for 
ethanolic extract was observed with 
Cladosporium herbarium, Candida albicans and 
Aspergillus niger as shown in Table 2. The order 
of antifungal activity was Aspergillus niger (50-
100mg/ml) ˂ Candida albicans (25-100mg/ml) ˂ 
Cladosporium herbarium (6.25-100mg/ml). 
 
Minimum antifungal activity of aqueous extract 
was also observed against fungal isolates when 
compared with ethanolic extract and higher 
zones of inhibition were observed at higher 
concentrations in susceptible fungi species. 
 

The minimum inhibitory concentration of 
ethanolic extract against fungi species as shown 
in Table 3 ranged from 6.25-50mg/ml while that 
of aqueous extract was 50mg/ml. The minimum 
fungicidal concentration of ethanolic extract 
ranged from 25-50mg/ml while that of aqueous 
extract was 100mg/ml against susceptible fungal 
isolates. 
 

Effects of ethanolic extract of B. pinnatum on 
haematological parameters of wistar rat models 
is shown in Table 4. The PCV values ranged 
from 39.00±0.71- 44.20±0.58% with the control 
being 39.00±0.71%. Haemoglobin values ranged 
from 12.94±0.21-14.62±0.24g/dl, while the 
control was 12.94±0.21g/dl. 
 

Effect of ethanolic extract of B. pinnatum on CD4 
count as shown in Table 5 has values ranging 
from 81.60±2.84- 99.00±6.33 cells/ml while the 
control was 75.40±19.32 cells/ml. There was no 
significant difference between treated and control 
groups. 
 

4. DISCUSSION 
 

Plants have been reported to be vast repertoire 
of bioactive phytochemical compound. These 
compounds which include flavonoids, alkaloids, 
tannins etc., are usually responsible for the 
various biologic properties of the plant, including 
antimicrobial and other medicinal properties. It 
has been reported that organic solvent such as 
ethanol, will usually extract more of the bioactive 
phytochemical component of the plant compared 
to aqueous solvent, hence the reason for higher 
antibacterial activity in the ethanolic fraction of 
the leaf extract [18]. 



 
 
 
 

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40 

 

Table 1. Zone of inhibition of aqueous extract of Bryophyllum pinnatum (mm) against fungal 
isolates 

 
Test organism Concentrations (mg/ml) 

100 50 25 12.5 6.25 

Penicillium italicum 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 
Cladosporium herbarium 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 
Candida albicans 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 
Aspergillus flavus 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 
Aspergillus niger 11.33±0.33 6.0±0.58 0.0±0.0 0.0±0.0 0.0±0.0 

 
Table 2. Zone of inhibition of ethanolic extract of Bryophyllum pinnatum (mm) against fungal 

isolates 
 

Test organisms Concentration (mg/ml) 

100 50 25 12.5 6.25 

Penicillium italicum 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 
Cladosporium herbarium 20.33±0.33 15.67±0.67 12.33±0.33 12.0±0.58 11.67±0.67 
Candida albicans 19.0±0.58 18.0±0.58 14.67±0.33 0.0±0.0 0.0±0.0 
Aspergillus flavus 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 
Aspergillus niger 14.0±0.58 9.33±0.33 0.0±0.0 0.0±0.0 0.0±0.0 

 
Table 3. Minimum inhibitory concentration and minimum fungicidal concentrations of 

ethanolic and aqueous extracts of B. pinnatum 
 

Test organisms MIC(mg/ml) MFC (mg/ml) 

Ethanol Aqueous Ethanol Aqueous 

Penicillium italicum ND ND ND ND 
Cladosporium herbarium 6.25 ND 25 ND 
Candida albicans 25 ND 50 ND 
Aspergillus flavus ND ND ND ND 
Aspergillus niger 50 50 100 100 

ND- Not determined 
 

Table 4. Effect of ethanol extract of B. pinnatum on haematological parameters of rat 
 

Groups Parameters  

 PCV(%) Hb(g/dl) 

A 
b
42.6 ± 0.93 

b
14.16±0.31 

B 
b
42.2±1.16 

b
14.0±0.39 

C 
b
43.0±1.70 

b
14.3±0.53 

D 
b
44.2±0.58

 b
14.62±0.24 

E 
b
42.8± 0.86 

b
142.24± 0.29 

F 
a
39.0± 0.71 

a
12.94±0.21 

Letters A-E stands for the groups of organism administered 300, 250, 200, 150 and 100 mg/kg of the ethanolic extract 
respectively while group F is the control. Groups with similar superscript as control has no significance while groups with 

different superscript describes the significance at P <0.05 

 
Table 5. Immuno-modulatory effects of ethanol extract of B. pinnatum on white blood cells 

proliferation 
 

Parameters Groups 

 A B C D E F 

WBC(×10
3
) 6.04±0.12

b 
5.16±0.25

a 
5.52±0.24

a 
4.90±0.25

a 
4.88±0.21

a 
4.96±0.10

a 

Neut(%) 44.8±3.69
a 

36.2±1.53
a 

36.4±9.18
a 

43.4±1.86
a 

33.3±1.48
a 

39.2±4.40
a 

Lymp(%) 49.2±3.15
a 

59.2±1.24
a 

57.4±10.78
a 

52.2±1.88
a 

62.8±2.67
a 

53.2±3.02
a 

Platelet(×10
3
) 105.8±4.10

a 
105.8±4.60

a 
126.2±5.40

c 
103.0±3.38

a 
95.4±1.86

a 
108.6±4.19

b 

CD4(cells/ml) 81.6±2.84
a 

95.8±2.08
a 

99.0±6.33
a 

86.2±4.59
a 

91.6±4.07
a 

75.4±19.32
a 

Letters A-E stands for the groups of organism administered 300, 250, 200, 150 and 100 mg/kg of the ethanolic extract 
respectively while group F is the control. Groups with similar superscript as control has no significance while groups with 

different superscript describes the significance at P <0.05 



 
 
 
 

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41 

 

Many of the fungi species were resistant to the 
all concentrations of the aqueous plant extract. 
The only sensitive fungus was Aspergillus niger. 
This resistance may have resulted from the 
inability of the plant extract to penetrate the cell 
wall of the fungal isolates. Comparatively higher 
antifungal activities were observed in the ethanol 
extract. Only Penicillium italicum and Aspergillus 
flavus were completely resistant to all 
concentrations of the plant extract. Minimum 
inhibitory concentrations of ethanol extract was 
lower than that of the aqueous extract indicating 
a higher efficacy of the ethanol extract. Similarly, 
Minimum fungicidal concentrations of ethanol 
extract was also lower than that of aqueous 
extract. Zakharchenko et al. [19] isolated the 
antimicrobial peptide cecropin P1 from 
Bryophyllum pinnatum and reported its fungicidal 
activity against dermatomycoses and even in 
treatment of wound infected with fungi. In their 
work, the antifungal activity observed against 
Candida albicans agrees with findings from this 
work as the ethanol extract of the plant was 
active against Candida albicans in this work. 
However, there was no in vivo study in this work 
compared to their work. 

 
There were changes in haematology parameters 
in experimental animals administered with 
ethanol extract of Bryophyllum pinnatum, 
compared to the control group. Observable 
changes between experimental and control 
animals are indication that ethanol extract of the 
plant has modulatory propensity on 
haematological parameters. Ethanolic extract of 
Brophyllum pinnatum has been shown in this 
work to increase the PCV and haemoglobin 
levels. No significant variation was observed 
between white blood cells proliferation of 
experimental animals and control group. 
Increase in haemoglobin enhances the oxygen 
transporting properties of the red blood cells as a 
result of increased number of red cell. The 
observed increase in the PCV recorded in this 
study on administration of extract of Bryophyllum 
pinnatum may have been due to effect on bone 
marrow stem cell by improving its proliferative 
activity [20]. There was a significant difference in 
platelet numbers between treated and control 
groups. The reduction in platelet counts suggests 
that continuous intake of this extract needs to be 
checked. There was no significant difference in 
white blood cell (WBC) parameters between 
treated and control group except for group A that 
received highest concentration of the extract 
(300mg/kg body weight). This means that the 
ethanolic extract of B. pinnatum may increase 

the WBC at higher concentrations. No significant 
difference was observed in the CD4 counts of 
treated and control groups. This could also be 
due to the concentration of the extract used as 
higher concentration may register significant 
increase.  Results showed that ethanolic extract 
of leaf caused increased haematological 
parameters that help in primary and secondary 
clearance of invading pathogens. This is in line 
with the work of [18] who reported that crude 
methanolic leaf extract of B. pinnatum has 
properties that increase the haemoglobin, 
packed cell volume and total white blood cells 
while decreasing the platelets. However, in 
contrast to their findings, the amount of platelet 
was found to increase at extract concentration 
200mg/kg of experimental animals. In a research 
findings by [21], aqueous extract of Bryophyllum 
pinnatum elevated white blood cell count, 
reduced neutrophil count without affecting 
lymphocyte count and packed cell volume, when 
compared to control, thus agreeing with the 
findings from this study that the extract 
possesses immunomodulating potentials. 
 
The phytochemical constituents of the leaf 
extract of B. pinnatum may have stimulatory 
effect on the bone marrow for leucocyte 
proliferation and haemoglobin production. This 
may be as a result of tannin, ascorbic acid [6] 
and phenolic content. Other phytochemicals that 
may have affected the haematological 
parameters in this study include flavonoids, zinc, 
riboflavin and niacin [22].  
   

5. CONCLUSION 
 
This work has shown that Bryophyllum pinnatum 
ethanol and aqueous extracts have moderate 
antifungal activity. However, the ethanol extract 
was more active compared to the aqueous 
extract. Furthermore, moderate immune-
modulatory activity was observed in Wistar rat 
models with an increase in platelet count, 
haemoglobin and PCV. B. pinnatum has been 
shown to possess hematological properties in 
this study. 
 

ETHICAL APPROVAL 
 
Ethical approval for the experimental protocol 
was obtained from the University of Benin Ethics 
Committee and care of animals was taken as per 
guidelines of Committee for the Purpose of 
Control and Supervision on Experiments on 
Animals (CPCSEA) (with reference number: 
FLS/17/101). 

http://www.sciencedomain.org/journal/86/authors-instruction#SDI-Ethical-approval


 
 
 
 

Okpoho et al.; AJI, 1(1): 36-43, 2018; Article no.AJI.46581 
 

 

 
42 

 

COMPETING INTERESTS 
 
Authors have declared that no competing 
interests exist. 

 

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Okpoho et al.; AJI, 1(1): 36-43, 2018; Article no.AJI.46581 
 

 

 
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© 2018 Okpoho et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License 
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