Istanbul J Pharm 52 (3): 271-280
DOI: 10.26650/IstanbulJPharm.2022.1007696
Original Article
Protective effects of Brenania brieyi (De Wild)
E.M.A.Petit root bark fractions against inflammatorymediated hemolysis and dyslipidemia in rats
Ifeoma Felicia Chukwuma1 , Victor Onukwube Apeh2 , Florence Nkechi Nworah1 ,
Felix Ifeanyi Nwafor3 , Lawrence Uchenna Sunday Ezeanyika1 , Victor Nwadiogo Ogugua1
University of Nigeria, Nsukka, Department of Biochemistry, Nigeria
Federal College of Dental Technology and Therapy, Enugu, Department of Applied Sciences, Nigeria
3
University of Nigeria, Nsukka, Department of Pharmacognosy and Environmental Medicine, Nigeria
1
2
ORCID IDs of the authors: I.F.C. 0000-0001-9629-213; V.O.A 0000-0003-2987-4046; F.N.N. 0000-0002-7724-9846;
F.I.N. 0000-0003-1889-6311; L.U.S.E. 0000-0002-3124-066X; V.N.O. 0000-0001-6302-7137
Cite this article as: Chukwuma, I.F., Apeh, V.O., Nworah, F.N., Nwafor, F.I., Ezeanyika, L.U.S., & Ogugua, N. (2022). Protective effects of Brenania brieyi (De Wild) E.M.A.petit root bark fractions against inflammatory -mediated hemolysis and dyslipidemia in
rats. Istanbul Journal of Pharmacy,52(3), 271-280. DOI: 10.26650/IstanbulJPharm.2022.1007696
ABSTRACT
Background and Aims: The inflammatory response, though protective, is the major cause of debilitating diseases when provoked excessively or if left unresolved. Brenania brieyi (De Wild) E.M.A.Petit is widely used in folk medicine for the treatment
of inflammatory-related diseases. This study investigated the protective effects of methanol and chloroform root bark fractions of Brenania brieyi on inflammation-induced hemolysis and dyslipidemia.
Methods: Anti-inflammatory activity was investigated by inserting 20 mg of autoclaved cotton pellets into forty-five rats randomly distributed into nine groups (n=5), this excluded group 1 (baseline). The extent of hemolysis and dyslipidemia in the
inflamed rats was ascertained from hematological parameters, lipid profile, and lipidemic index, while the possible underlying mechanisms of inflammation were determined using standard procedures.
Results: Treatment with varying doses of the root bark fractions of B. brieyi elicited a significant (p< 0.05) decrease in granuloma tissue and an increase (p<0.05) in hemoglobin, red and white blood cell count, packed cell volume, and platelets compared
with the untreated group 2. A significant (p<0.05) decrease in cholesterol, triacylglycerols, and low-density lipoprotein, and a
non-significant (p>0.05) increase in high-density lipoprotein were observed in almost all the test groups compared with group
2. There was a significant restoration of atherogenic and dyslipidemia indices and inhibition of acetic acid-induced vascular permeability, membrane hemolysis, and platelet aggregation in the fraction-treated groups compared with the control.
Conclusion: The findings from this study suggest that B. brieyi inhibits exudation and proliferation of granuloma-forming
cells and also has the potential to restore the hematological parameters and lipid anomalies to their physiologic state under
chronic inflammation. The possible mechanisms of its action could be inhibition of vascular permeability, stabilization of the
membrane, or inhibition of platelet aggregation. This justifies the use of the plant in traditional medicine and also demonstrates its potential as a target for the discovery of new anti-inflammatory agents.
Keywords: Acute toxicity, Anti-inflammatory activity, Brenania brieyi, Chronic inflammation, Hematological parameters,
Platelet aggregation, Lipid profile
Address for Correspondence:
Ifeoma Felicia CHUKWUMA, e-mail: chukwuma.ifeoma@unn.edu.ng
This work is licensed under a Creative Commons Attribution 4.0 International License.
Submitted: 12.10.2021
Revision Requested: 04.01.2022
Last Revision Received: 09.09.2022
Accepted: 22.09.2022
Published Online: 00.00.0000
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Istanbul J Pharm 52 (3): 271-280
INTRODUCTION
An inflammatory response is a defense mechanism which
eradicates noxious stimuli as well as initiating the tissue repair
process (Kuum et al., 2018; Naher, Aziz, Akter, Rahman, & Sajon,
2019). In a bid to eliminate the injurious agent and restore tissue injury in the body, a network of mediators, cells, and pathways that send chemical signaling cascades are stimulated
(Chen et al., 2018; Altan et al., 2020). Paradoxically, when this
response is not tightly controlled, regulated, or provoked excessively, it leads to inflammatory-induced diseases (Patil & Patil, 2017). Notably, the major predisposing factors to these diseases are not only the excessive generation of reactive oxygen
and nitrogen species (Hwang & Thi, 2020), but more importantly, the provoked perturbation of blood homeostasis (Mosser, Hamidzadeh & Goncalves, 2021), and increased hydrolysis
of polyunsaturated fatty acids, causing an increase in plasma
lipids leading to dyslipidemia (Ogbe, Aghese &Abu, 2020). The
increase in plasma lipids, especially triacylglycerols (TAG) and
total cholesterol (TC), has been reported as a biomarker for the
onset of cardiovascular diseases (CVD) (Aladaileh et al., 2019).
Due to the complex etiology of inflammation, the identification
of effective therapeutic options has been a great challenge.
Currently, in clinical settings, synthetic anti-inflammatory
drugs such as steroidal and non-steroidal drugs are prescribed
for the management of inflammatory-related diseases (Kuum
et al., 2018; Alabi et al., 2019; Khan et al., 2021). Although these
drugs transiently suppress symptoms and ameliorate inflammation, their chronic usage has severe adverse effects (Patil et
al., 2019). Interestingly, recent studies have shown that a good
percentage of the world’s population relies solely on botanical preparations as medicine to meet their health needs (Fernandez-Moriano, Gomez-Serranillos & Crespo, 2016; Oloyede,
Lukman & Salamu, 2020) in the management and treatment of
numerous diseases, including inflammation (Majouli, Hamdi &
Hlila., 2017; Antonisamy et al., 2019; Majumder, Ghosh & Bhattacharya, 2020). So, in the milieu of the discovery of newer anti-inflammatory drug targets, natural products have remained
of great interest (Rhetso, Seshadri, Ramnath, & Venkataramegowda, 2021) due to their better safety profile and lower cost
in comparison to the increasing side effects and high cost of
their synthetic counterparts (Kumar, Gupta & Singh, 2016; VasudhaUdupa et al., 2021).
272
A wide array of extensive studies have investigated the antiinflammatory and anti-hemolytic effects of several plants on
animal models (Dragomanova, Tancheva, Georgieva, & Klisurov, 2019; Patil et al., 2019), especially those with known
folk remedies, but there is still a paucity of information about
the potential of B. brieyi in the management of chronic inflammation-induced hemolysis and dyslipidemia. Brieyi, a member
of the Rubiaceae family of flowering plants, is a herbal plant
employed as a folk remedy in the management of several diseases, including swelling, infection, and endocrine disorders
(Chukwuma, Nkwocha, Ezeanyika, & Ogugua, 2020a). In addition, a high abundance of phytoconstituents with reported
anti-inflammatory activity such as squalene, hexadecenoic
acids, 9-octadecanoic acids, eicosanoic acids, and pentadeca-
noic acids were identified in B. brieyi root bark (Odo, Ezeanyika,
Ogugua, Joshua, & Okagu, 2017). Hence, this research was carried out to determine the protective effects of methanol and
chloroform fractions of the root bark of B. brieyi against chronic
inflammation in rats subjected to cotton pellet-induced inflammation, a model that could represent the proliferation of
macrophages, fibroblasts, and neutrophils in human beings.
The effects of the inflammatory cascade on hematological and
lipid parameters, which reflect the extent of membrane stability, were ascertained. Additionally, the mechanisms underlying
the fractions’ actions were further investigated using acetic acid-induced permeability and membrane hemolysis inhibitory
effects, as well as anti-platelet aggregatory tests.
MATERIAL AND METHODS
Collection and authentication of plant material
The B. brieyi root bark used for this study were collected from
Njikoka, Anambra State, and identified by Mr. Felix Nwafor, a
plant taxonomist in the Department of Pharmacognosy and
Environmental Medicine. Voucher specimens with identification numbers PCG/UNN/0327 were deposited in his department’s herbarium.
The procedure for extraction
The root barks of B. brieyi were dried at room temperature, pulverized, and extracted with chloroform and methanol in a ratio
of 2:1 for 48 hours under cold maceration. It was filtered using
filter paper (Whatman No. 4). The filtrate was later separated
into two fractions by shaking it in 0.2 mL of distilled water. Using a separating funnel, the fractions were immediately separated into a methanol fraction of B. brieyi root bark (MFBB, upper layer) and a chloroform fraction of B. brieyi root bark (CFBB,
lower layer). The MFBB and CFBB were then evaporated using
a rotary evaporator at 45 °C. Both fractions were stored in a
refrigerator at 4 °C.
Animals
Apparently healthy Swiss mice weighing 16.20 g ± 0.04 g and
adult Wistar albino rats with an average weight of 120.11 ±
0.03 g, bought from the Animal House of the Faculty of Pharmaceutical Sciences, were used in this study. The animals were
kept in a stainless steel cages in a 12 h light and dark cycle, 25
±1 °C temperature, and given clean water and rodents’ feed for
at least two weeks before the procedure to acclimatize them
to the environment. All the animals used were of different sexes, within a small age range, sourced from the same source,
placed under the same environmental conditions, and fed the
same rodent meal to eliminate confounding factors that might
influence the results. This research work was done in conformity with all international and national approved guidelines
on the care and use of laboratory animals as stated by the National Institute of Health Guide for Care and Use of Laboratory
Animals (Pub No. 85-23, 1985). Ethical approval for the study
was obtained from the Faculty of Biological Sciences Ethics
and Biosafety Committee (Ref no: UNN/FBS/EC/1049).
Acute toxicity (LD50)
Acute toxicity and lethality studies of the fractions were determined using the method of Lorke (1983) using 36 mice distrib-
Chukwuma et al. Protective effects of B. brieyi against hemolysis and dyslipidemia
uted into twelve groups (six groups for each fraction) of three
mice each being used for the first and second phases of the
experiment. In the first phase, 3 sets of mice were orally administered 10, 100, and 1,000 mg/kg body weight of MFBB respectively via a cannula. Lethality and behavioral changes such as
dizziness, irritation, jerking, and convulsion were observed for
24 h. This was followed by the administration of 1,600, 2,900,
and 5,000 mg/kg b. w. of the same fraction for the other 3
sets in the second phase. Death and behavioral changes were
also observed for 24 h after the administration of the test substance. The same procedure was also used to determine acute
toxicity for CFBB.
LD50 of each extract was calculated using this formula:
Where Do= highest dose that gave no mortality
D100= lowest dose that produced mortality.
Cotton pellet-induced chronic inflammatory model
A total of 45 male Wistar rats were randomly grouped into nine
groups of five rats each and were implanted with 20 mg of autoclaved cotton pellets according to the method of Mosquera
et al. (2011) with the exception of group 1, which served as the
baseline. Group 2 was administered normal saline, group 3 was
treated with indomethacin (10 mg/kg body weight), groups
4-6, and groups 7-9 received 50, 100, and 200 mg/kg b. w. of
MFBB and CFBB, respectively, for seven days. The animals were
sacrificed on the eighth day after being anesthetized with
chloroform. Blood samples were collected through cardiac
puncture by the principal investigator, after which the pellets
were carefully removed, dried in an oven at 60 °C for 24 h, and
weighed. The blood samples were used for the determination
of hematological parameters and lipid profiles. The change in
granuloma tissue weight was calculated as follows:
The final weight of the pellet - the initial weight of the pellet.
Determination of hematological parameters from the
serum of rats implanted with a cotton pellet
Blood samples used for measurement of hematological parameters were transferred into EDTA (anticoagulant) bottles
and used immediately to measure the full blood count using a
hematology analyzer (Erma PCE 210, Japan).
Determination of lipid profile from the serum of rats implanted with a cotton pellet
The following procedures were used to determine the lipid
profile: Total cholesterol by the Allain, Poon, Chan, Richmond,
& Fu, 1974 method using Quimica Clinical Aplicada (QCA)
commercial kits, triacylglycerols was determined with the
Randox commercial kit using the method of Albers, Warnick &
Chenng (1978), HDL was measured with Quimica Clinical Aplicada (QCA) commercial kits using Albers et al. (1978) methods,
while the polyvinyl sulphate method was used to determine
the LDL.
Estimation of atherogenic/dyslipidemia indices
The following equations were used to calculate the atherogenic/dyslipidemia indices as described by Ogbe et al. (2020).
a. Cardiac risk ratio (CRR) =
Total cholesterol
HDL
b. Atherogenic coefficient (AC) =
c. Classical ratio (CR) =
Total cholesterol-HDL
HDL
LDL
HDL
d. Atherogenic index of plasma (AIP) = log
Triglyceride
HDL
Mechanisms of inflammatory reactions
The following mechanisms of anti-inflammatory activity were
investigated: Acetic acid-induced vascular permeability test
according to Whittle (1964), the extent of membrane stability by Shinde et al. (1999), and anti-platelet aggregatory activity was determined using the Born & Cross (1963) method.
The percentage inhibition of the test substances (fractions/
and standard drug) were calculated relative to the control as
shown in equations below:
a. Inhibition of vascular permeability (%) =
Where: AC = Absorbance of the control while AT = Absorbance
of the fractions/test drug.
b. inhibition of membrane hemolysis (%) =
Where OD1 = absorbance of test sample unheated, OD2 =
absorbance of test sample heated, and OD3 = absorbance of
control sample heated.
c. Inhibition of platelet aggregation (%) =
Where : AT = Absorbance of the fractions / test drug while AC
= Absorbance of the control.
Statistical analysis
The statistical package for social science (SPSS) for windows
version 23 (SPSS Inc., Chicago, IL, USA) was used to analyze the
data obtained using one-way analysis of variance (ANOVA),
and Tukey’s post hoc test. p < 0.05 was taken as the significant
threshold. The results were presented as means ± standard deviation.
RESULTS
Acute toxicity study (LD50)
There were no observed behavioral changes or lethality in
mice administered 10-1,600 mg/kg b. w of each fraction after
24 h, while sedation, weakness, and dullness were observed
in mice given 2,900 and 5,000 mg/kg b. w. of both fractions.
Moreover, death was recorded in mice that received 5,000 mg/
kg body weight of each fraction within 24 h of administration
(Table 1).
Effects of MFBB and CFBB on cotton pellet-induced
granuloma tissue formation
Cotton pellet-induced formation of granuloma tissue was inhibited in groups 3-9 treated with different doses of the fractions and indomethacin. However, groups 4 and 6 treated
with MFBB exhibited a significantly (p < 0.05) higher weight
of granuloma tissue compared with groups 7 and 9 given the
same dose of CFBB (Figure 1).
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Istanbul J Pharm 52 (3): 271-280
Results are presented as mean ± SD (n = 5). Mean values having ‘*’ denotes significant difference at p < 0.05 compared with
negative-control (normal saline).
Table 1. Acute toxicity study (LD50) of the root bark
fraction of B. brieyi.
Dose in mg/
kg body
weight
Number of deaths
recorded with
MFBB
Number of deaths
recorded with
CFBB
10
0/3
0/3
100
0/3
0/3
1000
0/3
0/3
Phase 1
Key:
Group 1: Normal rats not implanted with cotton pellets (baseline).
Group 2: Cotton pellet + treatment with 1 ml/kg body weight
of normal saline (negative control).
Phase 2
1600
0/3
0/3
2900
0/3
0/3
5000
1/3
1/3
Effects of MFBB and CFBB on hematological parameters
of rats implanted with a cotton pellet
A significant (p < 0.05) decrease in Hb, RBC, WBC, and PCV
with a resultant increase in platelet count was observed in
group 2 (given normal saline) after cotton pellet implantation
compared with group 1. Interestingly, a significant (p < 0.05)
concentration-dependent restoration of Hb, RBC, WBC, and
platelet count occurred in rats administered varying doses of
the fractions of the root bark of B. brieyi and indomethacin. Varied doses of MFBB were efficacious in restoring RBC, WBC, and
platelet counts, compared with CFBB, but the reverse was the
case with Hb and PCV (Table 2).
Group 3: Cotton pellet + treatment with 10 mg/kg body
weight of indomethacin (standard drug).
Groups 4, 5, and 6 rats were treated with 50, 100, and 200 mg/
kg body weight of MFBB, respectively, after cotton pellet implantation.
n=3
Groups 7, 8, and 9 were treated with 50, 100, and 200 mg/kg body
weight of CFBB, respectively, after cotton pellet implantation.
Figure 1. Changes in the weight of granuloma tissue formed after
treatment.
Effects of MFBB and CFBB on lipid profile indices of rats
implanted with a cotton pellet
Implantation of cotton pellets significantly (p < 0.05) altered
the lipid profile indices in group 2 when compared with group
1. Interestingly, groups 3, 4, 5, and 6, treated with indomethacin (50 mg/kg) and MFBB (50, 100, and 200 mg/kg) respectively, had a significant (p < 0.05) decrease in cholesterol, TAG,
and LDL when compared with group 2, except in high-density
lipoprotein, which showed a significant (p < 0.05) increase only
in groups 3 and 6. Moreover, only 200 mg/kg b. w of CFBB was
effective in attenuating all the lipid indices significantly (p <
0.05) compared with group 2. The MFBB was found to be more
potent in decreasing cholesterol and LDL compared with
CFBB, whereas the reverse was the case with TAG (Figure 2).
Table 2. Effects of MFBB and CFBB on concentrations of some serum hematological parameters of rats
implanted with a cotton pellet.
Groups
Hb (g/dl)
RBC (x 106/l)
WBC (x103/l)
PCV (%)
Platelets (x106/l)
1
25.14±2.22
5.44 ±1.37
9560.00±219.09
45.20±5.63
135.00±9.35
2
13.24±1.23
#
#
3.36 ±1.08
#
7860.00±219.09
25.00±3.67
110.00±14.58#
3
22.44±2.09
*
*#
5.24 ±1.75
*#
6760.00±167.33
*
48.00±2.92
116.00±12.94#
4
19.70± 1.08*#
4.34 ±0.58*#
9120.00±228.04*#
34.20±4.60*#
198.00±12.55*#
5
22.38±2.45*
5.00 ±1.12*#
12440.0±260.77*#
36.80±5.54*#
169.00±4.18*#
6
22.94±0.36
6.02 ±2.74
14480.0±109.54
44.20±3.49
125.00±6.12*
7
23.40±1.27*
3.68 ±1.84*#
8280.00±109.54*#
40.20±5.35*
133.00±12.04*
8
23.68±1.61
4.72 ±1.18
10040.0±167.33
*
44.80±3.27
150.00 ±7.90*#
9
24.88±1.18*
5.24 ±1.58*#
9720.00± 178.89*
46.00±0.71*
113.00±12.55*#
*
*
*#
*#
*#
*#
#
*
Results are presented as mean ± SD (n=5). Mean values with ‘#’ denotes significant difference at p < 0.05 compared with baseline while ‘*’ denotes significant difference at p<0.05 compared with negative-control.
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Chukwuma et al. Protective effects of B. brieyi against hemolysis and dyslipidemia
rats). Interestingly, significant dose-dependent restoration of
dyslipidemia was observed in almost all the fractions treated
groups when compared with the untreated control (group 2).
The inhibitory effects of the standard drug were found to be
comparable with groups administered 200 mg/kg b. w of both
fractions. However, the highest inhibitory effects of cardiac
risk ratio (CRR), atherogenic coefficient (AC), and classical ratio
(CR) were recorded in group 6 administered 200 mg/kg b. w of
MFBB (Table 3).
Figure 2. Effects of root barks fractions of B. brieyi on concentrations of
serum lipid profile indices of rats implanted with cotton pellet.
TAG (A), Chol. (B), HDL (C) and LDL (D) stands for triacylglycerol,
cholesterols, high density lipoprotein and low density lipoprotein respectively. Values are presented as mean ± SD (n = 5).
Mean values with ‘#’ denotes significant difference (p < 0.05)
compared with baseline while ‘*’ denotes significant difference
(p < 0.05) compared with negative-control.
Key:
Group 1: Normal rats not implanted with cotton pellets (baseline).
Group 2: Cotton pellet + treatment with 1 ml/kg body weight
of normal saline (negative control).
Group 3: Cotton pellet + treatment with 10 mg/kg body
weight of indomethacin (standard drug).
Groups 4, 5, and 6 rats were treated with 50, 100, and 200 mg/
kg body weight of MFBB, respectively, after cotton pellet implantation.
Groups 7, 8, and 9 were treated with 50, 100, and 200 mg/kg body
weight of CFBB, respectively, after cotton pellet implantation.
Effects of MFBB and CFBB on atherogenic /dyslipidemia
indices in rats
There was an increase in CRR, AC, CR, and AIP in groups implanted with cotton pellets compared with group 1 (normal
Key:
Group 1: Normal rats not implanted with cotton pellets (baseline).
Key:
Group 1: Normal rats not implanted with cotton pellets (baseline).
Group 2: Cotton pellet + treatment with 1 ml/kg body weight
of normal saline (negative control).
Group 3: Cotton pellet + treatment with 10 mg/kg body
weight of indomethacin (standard drug).
Groups 4, 5, and 6 rats were treated with 50, 100, and 200 mg/
kg body weight of MFBB, respectively, after cotton pellet implantation.
Groups 7, 8, and 9 were treated with 50, 100, and 200 mg/kg
body weight of CFBB, respectively, after cotton pellet implantation
Effects of MFBB and CFBB on acetic acid-induced vascular permeability in rats
A significant inhibition of vascular permeability, which was in
a dose-dependent manner, was observed in the rats administered with both fractions. The inhibitory effect (72%) of group
6, given 200 mg/kg of CFBB, was significantly (p < 0.05) higher
compared with the 69.3% inhibition observed in group 4, given the same dose of MFBB. However, the percentage inhibition
(78%) of vascular permeability in the group given the standard
drug, indomethacin, was significantly higher compared with
Table 3. Effects of MFBB and CFBB on atherogenic/dyslipidemia indices in rats implanted with a cotton pellet.
Atherogenic/dyslipidemia indices in rats
Groups
CRR
AC
CR
AIP
1
1.86 (54)*
0.86 (72)*
0.70 (75)*
0.09 (133)*
2
4.10 (-)
3.10 (-)
2.80 (-)
0.27 (-)
3
2.41 (41)*
1.41 (55)*
1.20 (57)*
0.04 (85)*
4
3.22 (21)*
2.22 (28)*
1.94 (31)*
0.17 (37)*
5
3.04 (26)*
2.04 (34)*
1.72 (39)*
0.18 (33)*
6
2.28 (44)*
1.28 (59)*
1.03 (63)*
0.09 (67)*
7
3.39 (17)
2.39 (22)
2.16 (23)*
0.11 (59)*
8
3.15 (23)*
2.15 (30)*
1.88 (33)*
0.15 (44)*
9
2.57 (37)*
1.57 (49)*
1.33 (53)*
0.07(74)*
Values are presented as the mean of 5 rats. Percentage changes (%) in mean values were calculated relative to control and enclosed in parenthesis. Values with * are significantly (p < 0.05) different from control.
CRR, AC, CR, and AIP denote cardiac risk ratio, atherogenic coefficient, classical ratio, and atherogenic index power respectively.
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Istanbul J Pharm 52 (3): 271-280
all the groups administered with different doses of both fractions (Table 4).
time-dependent manner. The inhibition of platelet aggregation by the plant was comparable with that of indomethacin.
Effects of MFBB and CFBB on heat-induced hemolysis of
human red blood cells
The fractions inhibited heat-induced hemolysis of RBC in a reverse concentration-dependent manner. However, MFBB provoked a significantly (p < 0.05) higher membrane stabilization
potential across all concentrations assayed compared with
CFBB and indomethacin (Table 5).
DISCUSSION
Effects of root barks fractions of B brieyi on platelet aggregation induced by CaCl2
Both fractions inhibited in vitro platelet aggregation induced
by CaCl2 in a manner comparable with the standard drug, indomethacin. The highest anti-aggregatory activity was recorded at the highest time assayed (150 sec.). The results in Table 6
also reveal that 200 and 400µg/ml of indomethacin reduced
platelet aggregatory response to CaCl2 in a concentration and
Table 5. Effects of MFBB and CFBB on heat-induced
hemolysis of human red blood cells.
Treatments
Control
MFBB
CFBB
Indomethacin
Conc.
(µg/ml)
% inhibition of HRBC
hemolysis
-
0
100
89.08
200
84.62
400
86.32
600
87.12
800
83.92
100
74.76
200
71.86
400
64.36
600
61.65
800
61.96
200
75.36
400
77.46
Results of reported as mean ± SD of triplicate absorbance determination. The inhibition of HRBC hemolysis (%) was calculated
relative to control.
The extent of exudation and proliferation as a result of tissue
degeneration and fibrosis under chronic inflammatory response is measured by the cotton pellet-induced granuloma
model (Kumar et al., 2016; Misra, Varma & Kumar, 2018). Hence,
inhibition of granuloma formation by the fractions suggests
its potency in offering protection against chronic inflammation, which is recognized as the predisposing factor for the
pathogenesis of various forms of cancer (Patil et al., 2019). This
suggests that the fractions inhibited abnormal permeability
of the vascular tissues and mobilization of inflammatory cells
and mediators. It could be possible that the bioactive components present in the plant, which demonstrated antioxidant
and anti-inflammatory properties (Chukwuma et al., 2020b;
Chukwuma et al., 2021), inhibited the release of inflammatory
mediators such as prostaglandin, thereby hindering proliferation of granuloma-forming cells like macrophages, fibroblasts,
and neutrophils (Kumar et al., 2016). Lending credence to this
also is the high phenolic content found in the plant, which has
been reported to inhibit the expression of pro-inflammatory
genes (Chukwuma et al., 2020a).
A prolonged inflammatory response is associated with modification of hematological parameters. Hence, monitoring hematological indices under chronic inflammatory diseases helps to
ascertain the extent of tissue damage. The observed decreases
in Hb, PCV, and RBC counts in groups implanted with cotton
pellets reflect the presence of anemia. Also, the excess release
of ROS in inflammatory reactions degrades hemoglobin and
lipid components of cells. Interestingly, the significant restoration of Hb, RBC, WBC, and PCV in groups treated with the
fractions when compared with group 2 might be due to antioxidative compounds found in the fractions which inhibited
the release of ROS (Odo et al., 2017; Chukwuma et al., 2020a;
Chukwuma et al., 2020b) and hence, preserved the integrity of
the cell membrane from hemolysis of RBC. This concurs with
the report of Haddouchi, Chaouche, Saker, Ghellai, & Boudjemai (2021) who also studied the antioxidant potential of polyphenolic compounds. Moreover, the increase in WBC count
suggests the potential of the plant in maintaining the integrity of the rats’ immune system while the observed increase in
Table 4. Percentage inhibition of vascular permeability by MFBB and CFBB.
Groups
Treatments
Dosage (mg/kg)
Absorbance (610 nm)
Inhibition (%)
1
Control
-
0.274 ± 0.006
0
2
Indomethacin
50
0.059 ± 0.005*
78.47
3
MFBB
100
0.128 ± 0.006*
53.28
200
0.084 ± 0.003*
69.34
100
*
0.144 ± 0.003
47.45
200
0.076 ± 0.004*
72.26
4
5
6
CFBB
Results are presented as mean ± SD n = 5. The absorbance of the treatment groups was used to calculate % inhibition relative to control. Absorbance with ‘*’ is significantly different (p < 0.05) compared with the control.
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Chukwuma et al. Protective effects of B. brieyi against hemolysis and dyslipidemia
Table 6. Effects of MFBB and CFBB on platelet aggregation induced by CaCl2.
Conc.
(µg/ml)
% inhibition of platelet aggregation at different time intervals
0s
30s
60s
90s
120s
150s
50.37 ± 4.01
50.00± 3.20
50.13 ±2.71
50.52 ± 3.21
50.53± 2.34
50.53 ± 2.17
MFBB
100
200
48.97 ± 3.75
48.39 ± 2.89
48.52 ± 4.50
48.64 ± 2.90
48.63± 2.10
48.77± 4.00
400
42.49 ± 5.31
42.34 ± 3.42
42.47± 2.01
42.90 ± 1.78
42.68± 1.90
42.64± 1.21
600
17.08 ± 2.65
15.04 ± 1.87
15.49± 3.42
15.63 ± 2.92
17.55± 2.17
15.00± 3.10
800
28.67 ± 1.09
38.02 ± 3.40
27.38 ± 3.11
27.59 ± 3.12
27.41± 3.20
27.51 ± 4.12
100
5.69 ± 0.21
7.25 ± 1.23
7.23 ± 0.98
7.35 ± 2.96
6.93± 0.18
6.03± 0.97
200
24.05 ± 2.47
21.95 ± 2.00
21.72 ± 3.17
22.22 ± 1.32
21.99± 3.19
21.43± 4.12
CFBB
400
34.11 ± 3.33
33.33 ± 1.34
33.45 ± 2.00
33.92 ± 3.45
34.27± 2.14
34.15± 2.93
600
48.71 ± 4.52
49.07 ± 2.05
49.20 ± 1.15
49.33 ± 5.10
49.46± 3.71
49.46± 1.87
800
56.46 ± 2.90
57.05 ± 3.11
54.63 ± 5.12
57.34 ± 0.78
57.37± 2.09
57.40 ± 3.12
INDO
400
34.96 ± 4.53
30.93 ± 2.18
27.65 ± 1.26
27.02 ± 1.23
26.56± 3.11
25.49 ± 1.23
600
47.76± 3.90
41.46 ± 4.57
36.96 ± 2.13
36.15 ± 2.30
35.17± 1.90
35.51 ± 2.17
Indo. Stands for indomethacin. Percentage inhibition of platelet aggregation was calculated relative to control.
platelets suggests its wound healing properties since platelets
are known to be involved in the healing of damaged tissues
(Anyasor, Okanlawon & Ogunbiyi, 2019).
A chronic inflammatory response activates acute phase
proteins that alter lipid metabolism, resulting in a decrease
in HDL, impairment of reverse cholesterol transport,
changes in apolipoproteins, and changes in cholesterol efflux regulatory proteins (Essawy, Abo-elmatty, Ghazy, Badr,
& Sterner, 2014; Esteve, Ricart, & Fernández-Real, 2005).
Normalization of these lipid anomalies in this study was
demonstrated by the decreases in total cholesterol, TAG,
and LDL and an increase in HDL after treatment. This potency could be attributed to the antioxidant compounds
identified in the plant in the preliminary studies by Odo
et al. (2017) which previous studies have reported to be
antioxidant molecules (Chakraborty et al., 2021). Notably,
squalene found in the fraction is a cardioprotective agent,
an enhancer of WBC, and increases fecal excretion which
reduces the concentration of cholesterol (Odo et al. 2017).
Also, the most abundant compound found in the fraction, 9-octadecanoic acid (oleic acid), helps in preventing
atherosclerosis due to its efficacy in lowering LDL (Nkwocha, Odo & Umeakuana, 2019). In the same vein, a previous study by Chukwuma et al. (2021) demonstrated the
phospholipase A2 inhibitory effects of the plant, which
prevents the breakdown of the lipid membrane. So, this
suggests that MFBB and CFBB could be very helpful in reducing the onset of cardiovascular disease since studies
have shown that a significant lowering of LDL-cholesterol
and a rise in HDL-C are reliable biochemical biomarkers for
the prevention of atherosclerosis and ischemic conditions
(Ikumawoyi, Awodele, Rotimi, & Fashina, 2016).
Emerging evidence has shown that chronic inflammatory diseases orchestrate the atherosclerotic vasculopathy involved in
the pathophysiology of cardiovascular diseases (CVD) (Acay et
al., 2014). The use of lipid profiles alone to determine the prevalence and severity of CVD has been questioned. Hence, the
use of atherogenic/dyslipidemia indices, mainly atherogenic
index of plasma (AIP), which estimate the balance between
atherogenic and other non-atherogenic factors, has proven to
be a better predictor of CVD than lipid profile (Acay et al., 2014;
Ogbe et al., 2020). The observed decrease in CRR, AC, CR, and
AIP in this study suggests ameliorating effects of the fractions
in averting inflammatory-induced dyslipidemia. The decrease
in atherogenic/dyslipidemia indices in this study could be attributed to a decrease in LDL and an increase in HDL. Lipids
accumulate in macrophages during inflammation to form lipid
foam cells. These cells form fatty streaks when they accumulate in the walls of the arteries, causing atherosclerotic plague
(Esteve, Ricart, & Fernández-Real, 2005). Agents that subvert infiltration of inflammatory cells into the adipose tissues help to
prevent excessive production of cytokines and adipose lipids
which potentiate these lipid metabolism disorders (Esteve et
al., 2004).
The release of immune cells and mediators in the presence of
a stimulus dilates the blood vessels to enhance the mobilization of vascular components to the inflamed region (Chen et
al., 2018; Altan et al., 2020). This study investigated the inhibitory effects of MFBB and CFBB on a vasodilator, acetic acid.
Acetic acid stimulates mast cells, which enhances the release
of inflammatory agents responsible for dilating blood vessels
such as prostaglandins, histamine, serotonin, bradykinin, and
leukotrienes (Kumar et al., 2016; Patil et al., 2019). The observed
inhibition of vascular permeability by MFBB and CFBB suggests
277
Istanbul J Pharm 52 (3): 271-280
that they could suppress the exudative phase of inflammation,
which would avert tissue damage. This potency could also be
due to its inhibitory effect on phospholipase A2 and prostaglandin synthase, which hinders the release of inflammatory
mediators including A2, PGD2, PGE2, and PG12, involved in
vaso-dilation (Chukwuma et al., 2021). Perhaps the bioactive
compounds found in the fractions stabilize cell membranes, as
shown in their high inhibition of heat-induced membrane stabilization study. Conversely, the plants ability to inhibit membrane hemolysis may be due to their high antioxidant activity,
as demonstrated in the plants’ in vitro and in vivo antioxidant
studies (Chukwuma et al., 2020a, Chukwuma et al., 2020b).
Plants with antioxidant activity have been reported to be key
anti-inflammatory drug targets since they prevent the leakage
of fluid into the peritoneum, thereby suppressing inflammation. This will also avert the biochemical cascade involved in
chronic inflammation, such as granuloma tissue formation.
Furthermore, an increase in thromboxane and platelet-activating factor production, which causes aggregation of platelets,
is a marker of inflammation and a pharmacological target in
the management of inflammatory diseases (Sokeng, Rokeya,
Hannan, Ali, & Kamtchouing, 2013; Gros, Ollivie & Ho- Tin-Noe,
2014). Platelet aggregation helps in cellular hemostasis. However, excessive aggregation of platelets in inflammatory reactions leads to thrombotic diseases (Chukwunelo et al., 2019).
Interestingly, both MFBB and CFBB had anti-platelet aggregatory capacity. Platelet aggregation can be inhibited through
the inactivation of intracellular signaling pathways or by blocking membrane receptors (Mykola, Ganna & Gennadiy, 2015).
This suggests that the fractions inhibited activation of COX-1,
thereby limiting the synthesis of thromboxane and plateletactivating factors from arachidonic acid. Hence, the fractions
could help in circumventing factors that predispose one to
chronic inflammation-induced diseases such as cardiovascular
diseases.
Author Contributions: Conception/Design of Study- I.F.C., L.U.S.E.,
V.N.O.; Data Acquisition-I.F.C., V.O.A., F.I.N.; Data Analysis/Interpretation- I.F.C., V.O.A., F.N.N., L.U.S.E.; Drafting Manuscript- I.F.C., V.O.A.,
F.N.N., F.I.N.; Critical Revision of Manuscript- L.U.S.E.; Final Approval
and Accountability I.F.C., V.O.A., F.N.N., L.U.S.E.
Conflict of Interest: The authors have no conflict of interest to declare.
Financial Disclosure: The authors declared no financial support.
Acknowledgement: The authors are highly grateful to Mr. Chukwuma Odo, who provided funds used for the research work.
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CONCLUSIONS
The results of this study show that the root bark fractions of B.
brieyi inhibited the exudation and proliferation of granulomaforming cells, thereby limiting the formation of granuloma tissues. It also demonstrated the potential to inhibit hemolysis
and hyperlipidemic aberrations of blood cells and membrane
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the discovery of new anti-inflammatory agents.
Peer-review: Externally peer-reviewed.
Informed Consent: Written consent was obtained from the participants.
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