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Article
Volume 12, Issue 3, 2023, 79
https://doi.org/10.33263/LIANBS123.079
Assessment of the Aqueous Methanol Extract and Ethyl
Acetate Fraction of Tapinanthus globiferus Leaves and
their Effects on Ulceration and Leukocyte Mobilization in
Wistar Albino Rats
Florence Nworah 1,* , Ifeoma Chukwumma 1, Dionysius Osuji 1, Emmanuel Ezeako 1
1 Pharmacology and Pharm-biology Unit, Department of Biochemistry, University of Nigeria, Nsukka, Enugu State, Nigeria;
* Correspondence: florence.nworah@unn.edu.ng (F.N.); Scopus Author ID 57209409197
Received: 16.11.2021; Accepted: 7.02.2022; Published: 20.04.2022
Abstract: This study aimed to ascertain the anti-ulcerative, leukocyte mobilizing, haematologic, and
antioxidant effect of methanol extract of Tapinanthus globiferus leaves (METGL) and ethyl acetate
fraction of Tapinanthus globiferus leaves (EAFTGL) in rat models. Phytochemical analysis of T.
globiferus leaves recorded substantial levels of phenolics, flavonoids, alkaloids, saponins, steroids,
terpenoids, and tannins. The experimental design was categorized into three phases. Ulcer and
inflammation were inflicted on phases I and II rats using indomethacin and inflammatory stimulus (1
mL of 3 % agar suspension), respectively, treatments with reference drug and different concentrations
(100 and 200 mg/kg body weight) METGL and EAFTGL ensued. In phase III, group 1 (normal control)
received 3 ml/kg bw 3 % tween 80, while groups 2-5 were administered with varied dosages (100 and
200 mg/kg bw) of METGL and EAFTGL, respectively. The results revealed that both METGL and
EAFTGL effectively instituted gastro-protection, exerted a significant (p < 0.05) increase in
hematological indices and antioxidant parameters, and caused a significant (p<0.05) decrease in
leukocyte infiltration. These suggest that T. globiferus leaves may be useful in managing ulcer and
inflammatory conditions, validating its use in traditional medicine.
Keywords: anti-ulcerative; hematologic; antioxidant; leukocyte; phytochemicals.
© 2021 by the authors. This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
1. Introduction
Generally, all living things' health state is largely reflected by their hematological
profile [1]. These include analyzing blood cellular components such as erythrocytes,
thrombocytes, blood proteins, leukocytes, and blood-producing organs. Several hematological
indices such as hematocrit, hemoglobin, and total erythrocyte counts are used to assess the
blood's functional status and oxygen-carrying capacity [2]. Leukocyte mobilization involves
physiological events that occur as part of the several immune responses necessary to avert or
remove initial stimuli and mediators of tissue damage, clear out dead cells resulting from
inflammatory cascades, and initiate tissue repair [3].
Oxidative stress, a situation where the levels of prooxidants exceed the levels of
antioxidant mechanisms needed to keep them in check, has been implicated in the etiology of
many anemic and inflammatory diseases [4–6]. In clinical studies, low levels of antioxidant
enzymes indicate elevated levels of free radicals in the system. Consequently, decreased levels
of these antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, and
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catalase result in the diminution of the reduced glutathione (GHS) levels, H2O2, and build-up
results in oxidative stress and the pathology of several diseases [7].
Peptic ulcers, including gastric and duodenal ulcers, are disruptions in the protective
mucosal lining, caused primarily by an imbalance between offensive effects of hydrochloric
acid, pepsin, and defensive effects of mucus and bicarbonate [8]. The disease, associated with
high morbidity and mortality, is attributed to stress involving using non-steroidal anti-
inflammatory drugs (NSAIDs) and infection by the bacteria Helicobacter pylori [9]. Previous
investigators have correlated increased gastric acid secretion with a higher risk of contracting
ulcers in rat models [10]. Furthermore, research has indicated that pharmacologic agents that
downregulate gastric acid output could confer gastroprotective effects on the mucosa lining
and avert ulcerogenesis, which forms the basis of several anti-ulcerative agents [11].
In recent years, because of the myriads of complications associated with synthetic
drugs, which inundates the market, lots of attention has been given to medicinal plants as an
alternative option [12]. This surge in interest in plant-sourced disease therapy is due to their
ubiquity, safety, affordability, and optimal conformity with the several ethical considerations
compared to synthetic drugs. The medicinal properties of these plants are dependent on several
chemically active substances referred to as phytochemicals, which possess definite
physiological activities [13].
Most plant-sourced modern pharmaceutics and food are based on folkloric knowledge
and traditional plant deployment in disease management [14]. Tapinanthus globiferus (Family-
Loranthaceae), commonly known as mistletoe in English, apari in Igbo, afomoonisana in
Yoruba, and Kauchi in Hausa, is a parasitic plant that grows on various trees of the genus,
Kola, Citrus, Pakia, Combretum, Acacia, Aloe and Terminalia as host plants [14]. Tapinanthus
globiferus is an evergreen shrub that forms dark to the yellowish-green drooping bush, which
grows up to about 0.6 - 0.9 m long on the branch of its host tree [15]. Tapinanthus globiferus
is widely employed traditionally in managing many ailments, including inflammations,
malaria, bacterial infections, pains, and ulcers. Recently, several studies have reported that the
extracts exhibit immunomodulatory and anti-inflammatory activities [16,17]. Scant or no work
has been done to elucidate the plant's anti-ulcerative and haematologic effects. This study aims
to investigate the effect of Tapinanthus globiferus leaves extract and fraction on ulceration,
leukocyte migration, antioxidant markers, and hematological indices, lending credence to its
folkloric uses.
2. Materials and Methods
2.1. Materials.
2.1.1. Instruments and equipment.
Water bath, weighing balance, beakers, test tube, adjustable micropipette, measuring
cylinder, syringe, spectrophotometer, cuvette, gloves, magnifying lens, dissecting blade, pH
paper, canula.
2.1.2. Experimental animals.
The use of Albino Wistar rats in this study was carried out according to the National
Institutes of Health guide for the care and use of laboratory animals [18]. Albino Wistar rats
(weighing 45 – 70 g) of either sex were used. The animals were procured from the animal house
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of the Department of Zoology, University of Nigeria Nsukka (UNN), and were kept in a 12-
hour light-dark cycle.
2.2. Methodology
2.2.1. Collection of plant materials.
Fresh leaves of Tapinanthus globiferus, a parasite of the host tree Kola acuminata, were
obtained from Orba in Udenu Local Government Area, Enugu, Nigeria. They were identified
by Mr. A. O. Ozioko, a taxonomist of the Bio-Resources Development and Conservation
Programme, UNN, Enugu.
2.2.2. Preparation of plant extract.
The fresh leaves were airdried under a shade at room temperature (27oC) for 14 days
and then ground into a fine powder using a manual grinder. The powdered leaves of about 1350
g were soaked in 2600 ml of methanol and 650 ml of distilled water in a transparent container
and left to macerate for 48 hours and filtered. After filtration, with Whatman No. 1, a rotary
evaporator was used to concentrate the filtrate until a chocolate-like semi-solid methanol
extract (METGL) was obtained. The weight and percentage yield of the extract was recorded
at 34.69 g and 2.569 % w/w, respectively.
2.2.3. Preparation of plant fractions.
A portion (17 g) of METGL was further subjected to partitioning using n-hexane and
ethyl acetate. Afterward, both fractions were collected and subjected to preliminary studies,
after which the ethyl acetate fraction (EAFTGL) was preferred and used for further studies.
2.2.4. Qualitative and quantitative analysis of Tapinanthus globiferus extract.
The phytochemical screening of the crude pulverized leaves, METGL, and EAFTGL
was carried out following the method described by [19].
2.2.5. Acute toxicity study.
The determination of the oral median lethal dose (LD50) of METGL and EAFTGL was
conducted using [20] with slight modifications. The study deployed thirty-six (36) Swiss albino
mice deprived of food for 18 hours but given access to water. They were split into two (2)
phases made up of six (6) groups of three (3) mice each. Mice in each phase were administered
with varied dosages (0.01, 0.1, 1, 1.6, 2.9, and 5 g/kg) of METGL and EAFTGL, respectively,
via oral intubation. The animals were then examined for behavioral changes and or mortality
for 24-hour. Based on the preliminary toxicity testing results, the doses of the extract for further
studies were decided to be 0.1 and 0.2 g/kg body weight of the rats.
2.3. Experimental design.
The experimental design is shown in Table 1. The study employed the CRD method in
the experimental design. Eight-five (85) Wistar albino rats and thirty-six (36) Swiss albino
mice were used in the study, and the rats were randomly distributed into three phases. Phases
I and II (comprising six (6) groups of five (5) rats each), and phase III (comprising five (5)
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groups of five (5) rats each), were used to evaluate the anti-ulcerative, leukocyte mobilizing,
and haematologic and antioxidant effects of METGL and EAFTGL, respectively. Ulcer and
inflammation were inflicted on phases I and II rats using indomethacin and inflammatory
stimulus (agar suspension), respectively, after treatments with reference drug and different
concentrations (100 and 200 mg/kg body weight) of METGL and EAFTGL have ensued.
In phases I and II, groups 3-6 were administered with different dosages (100 and 200
mg/kg bw) of METGL and EAFTGL, respectively. The normal and standard control groups
(Group 1 and 2) in phase I were administered with 3 ml/kg bw distilled water and 20 mg/kg
bw omeprazole (standard drug), while those in phase II were administered with 3 ml/kg bw 3
% tween 80 and 50 mg/kg bw diclofenac (standard drug), respectively. In phase III, group 1
(normal control) received 3 ml/kg bw 3 % tween 80, while groups 2-5 were administered with
varied dosages (100 and 200 mg/kg bw) of METGL and EAFTGL, respectively.
Table 1. Experimental design.
Groups
Phase I treatments
Phase II treatments
Phase III treatments
1 (NC)
3 ml/kg bw 3% tween 80 (via oral
intubation)
3 ml/kg bw 3% tween 80 (via oral
intubation)
3 ml/kg bw 3% tween 80 (via oral
intubation)
2 (SC)
Indo. + 20 mg/kg bw omeprazole
drug
1 mL of 3 % AS in tween 80 + 50
mg/kg bw diclofenac drug
100 mg/kg bw METGL
3
Indo. + 100 mg/kg bw METGL
1 mL of 3 % AS in tween 80 +100
mg/kg bw METGL
200 mg/kg bw METGL
4
Indo. + 200 mg/kg bw METGL
1 mL of 3 % AS in tween 80+ 200
mg/kg bw METGL
100 mg/kg bw EAFTGL
5
Indo. + 100 mg/kg bw EAFTGL
1 mL of 3 % AS in tween 80+ 100
mg/kg bw EAFTGL
200 mg/kg bw EAFTGL
6
Indo. + 200 mg/kg bw EAFTGL
1 mL of 3 % AS in tween 80 + 200
mg/kg bw EAFTGL
-
Key: NC = normal control, SC = standard control, Indo.= Indomethacin, METGL = methanol extract of
Tapinanthus globiferus leaves, EAFTGL = ethyl acetate fraction of Tapinanthus globiferus leaves, AS=agar
suspension
2.3.1. Induction of ulcer in Phase I.
Animals in phase I was induced with ulcer following the outlined method [21]. Shortly
after starving the animals of food but being allowed access to water for 24-hours, they were
treated with a single dosage (30 mg/kg bw) of indomethacin. About 4-hours after the induction,
various degrees of ulceration were observed and examined.
2.4. Determination of Ulcer Index.
The degree of ulcerations in the rats after induction was estimated following the
procedures outlined by [22]. After 8-hours, animals in all groups were humanely sacrificed and
subjected to ulcer examination. The rats’ abdomens were cut open along the greater curvature,
and their stomachs (gastric mucosa) were rinsed with water, pinned flat on a cork base, and
examined with a convex hand lens (x10). Erosions formed on the glandular portion of the
stomach were counted by scoring the ulcerations on a 0–5 scale grading, based on the gravity
of the lesion. The damaged areas of the mucosal layer were evaluated in the percentage of the
entire surface area of the stomach glandular portion was presented in millimeters square (mm2),
and the mean ulcer index and percentage ulcer inhibition exhibited by the standard drug,
extract, and fractions in each animal was estimated using the formula outlined by [23], as
follows:
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The effect of standard drugs, METGL and EAFTGL, on leukocytes mobilization
stimulated by treatments with an inflammatory stimulus (agar suspension) was evaluated by
adopting the procedures outlined by [24] with slight modifications by [25].
Group 1 rats were administered with the vehicle (3 ml/kg bw 3% tween 80), while rats
in groups 2-6 were administered with standard drug (diclofenac) and varied dosages (100 and
200 mg/kg bw) of METGL and EAFTGL, respectively via oral intubation. 3-hours after the
various treatment on the animals were accomplished, animals in groups 2-6 were administered
with 1 mL of 3 % w/v agar suspension in tween 80 solution via intraperitoneal injection. 4-
hours later, the rats were humanely killed; afterward, their peritoneal cavities were washed in
5 ml of 5 % EDTA solution in phosphate-buffered saline. EDTA bottles were used to collect
the recovered peritoneal fluid. The perfusates were used to perform the differential, and total
leukocyte counts with a manual cell counter following Wright’s stain staining procedures.
2.5. Evaluation of the effect of METGL and EAFTGL on hematological Indices and antioxidant
markers in Phase III.
Twenty-five (25) male albino Wistar rats were randomly split into five (5) groups of
five (5) rats each. Group 1 (normal control) was administered with only the vehicle (3 ml/kg
bw 3% tween 80), while groups 2-5 were administered with varied dosages (100 and 200 mg/kg
bw) of METGL and EAFTGL, respectively via oral intubation. The rats were treated daily for
21-days and were closely examined frequently for behavioral changes and other symptoms of
toxicity or mortality during the study period of 24-hours following the last treatment;
evaluation of the effect of METGL and EAFTGL on the hematological indices and antioxidant
markers were performed using blood obtained from direct heart puncture.
2.6. Preparation of serum.
The blood serum was prepared following standard procedures as outlined by [26].
Serum preparation was performed using blood obtained from the treated animals via direct
cardiac puncture using a 21-G needle affixed to a 5 ml syringe after inducing mild chloroform
anesthesia on the animals. Afterward, the blood was left to clot for 30-minutes and then spun
at 2500 rpm for 15-minutes, after which the serum was harvested and used to estimate the
hematological indices and antioxidants parameters.
2.6.1. Haematological indices studies.
2.6.1.1. Determination of hemoglobin concentration.
The determination of the hemoglobin concentration was performed using the
cyanomethaemoglobin method as outlined by [27].
2.6.1.2. Determination of Parked cell volume (PCV).
The microhematocrit method was adopted to determine Parked cell volume (PCV)
following the outlined method [28].
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2.6.1.3. Determination of red blood cell count.
The red blood cell count (RBC) was evaluated visually using a modified Neubauer
counting chamber according to the method of [29], using the formula as follows,
Total RBC count=
2.6.1.4. Determination of Mean corpuscular volume (MCV).
The mean corpuscular volume (MCV) was determined following the method of [30]
with the formula as follows,
MCV =
2.6.1.5. Determination of Mean Corpuscular Haemoglobin (MCH).
The mean corpuscular hemoglobin (MCH) expressed in picograms (pg) (1pg=10-12)
was estimated according to the method of Briggs and Bain (2017) with the formula as follows,
MCH=(Haemoglobin (g/100 ml)×10)/(RBC (million per cu.mm))
2.6.1.6. Determination of Mean Corpuscular Haemoglobin Concentration (MCHC).
The mean corpuscular hemoglobin concentration (MCHC) expressed as a percentage
of packed cells was performed following the outlined procedures of [31], using the formula as
follows,
MCHC =
2.6.1.7. Determination of total white blood cell count.
The white blood cell count (WBC) was estimated visually using a modified Neubauer
counting chamber following the outlined method [29], using the formula as follows,
Total WBC count =
× Depth (mm)
2.6.1.8. Determination of differential white blood cell count.
The differential white blood cell count was performed using Wright’s stain staining
method as outlined by [29].
2.7. Antioxidant markers studies.
2.7.1. Evaluation of lipid peroxidation using thiobarbituric acid reactive substances
(TBARS)
The levels of TBARs in the serum were used to assay for Lipid peroxidation levels in
serum, according to the method outlined by [32]. In this method, 2 M sodium sulfate was used
to prepare the thiobarbituric acid to reduce the interference from sialic acid.
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2.7.2. Evaluation of superoxide dismutase.
The activities of superoxide dismutase (SOD) were assayed following the outlined
procedures [33]. Xanthine-xanthine oxidase system was deployed to produce superoxide
radicals, while nitro-blue tetrazolium (NBT) served as an indicator to track the radical flux.
SOD activities were determined by evaluating the extent of inhibition of the reaction unit of
the enzyme exerting 50 % inhibition of NBT reduction. The results were presented in U/mL.
2.7.3. Evaluation of catalase activity.
The levels of catalase activities in serum were evaluated following the modified
procedures [34]. To a test tube containing 2.80 ml of 50 mM potassium phosphate buffer of pH
7.0, 10 L of serum was added. Afterward, 0.1 mL of fresh 30 mM hydrogen peroxide was
added to initiate the reaction, and the hydrogen peroxide decomposition rate was estimated at
240 nm for 5-minutes using a UV spectrophotometer. Catalase activities were estimated with
a molar extinction coefficient of 0.041 mM−1 cm−, using the formula as follows,
Absorbance =concentration ×molar extinction coefficient ×path length
2.7.4. Evaluation of Reduced glutathione (GSH).
The concentrations of reduced glutathione were estimated following the procedures
outlined by [35]. The principle of the essay is centered on the formation of chromophore
products which absorb at 412 nm when 5-5’ dithiobis-2-nitrobenzoic acid (DNTB) reacts with
reduced glutathione (GSH) (a sulfhydryl containing compound). In summary, the procedure is
as follows. Tissue homogenate (50 μL) was diluted by dissolving in 10 mL of 0.1 M phosphate
buffer of pH 8. Afterward, 20 μL of 0.01 M DTNB was dissolved into 3 mL of the diluted
mixture, and the absorbance read at 412 nm against a blank made under akin conditions.
2.7.5. Glutathione peroxidase (GPx).
The glutathione peroxidase (GPx) activities were estimated following the procedures
outlined by [36]. The principle of the assay is stated briefly. Serum (5 μL) was added into a 0.9
mL assay solution at 37oC to initiate the reaction. Afterward, the reaction was observed to
change in the absorbance of NADPH at 340 nm. The assay solution consisted of 0.1 mM
NADPH, 10 μM NaN3, 2 mM GSH, 20 μmol hydrogen peroxide in 20 mM Tris-HCl buffer of
pH 8.0, and 0.4 U/mL GSH-reductase.
2.7.6. Determination of serum antioxidant mineral.
The levels of serum antioxidant minerals were analyzed according to the outline
procedures stated by [37].
2.8. Statistical analysis.
Statistical analysis was made on data using one-way analysis of variance (ANOVA).
Each value represents the mean ± SD (n=5); vertical superscripts with the same alphabets are
non-significant (p > 0.05), while superscripts with different alphabets are significant (p < 0.05).
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3. Results and Discussion
3.1. Results.
3.1.1. Determination of qualitative and quantitative phytochemical screening of the pulverized
leaves of T. globiferus, METGL, and EAFTGL.
Evaluation of the qualitative and quantitative composition of bioactive ingredients
present in the crude extract, METGL, and EAFTGL revealed the presence of essential bioactive
principles such as flavonoids, tannins, phenolics, alkaloids, and terpenoids in both METGL
and EAFTGL, and saponins, reducing sugar, carbohydrates and steroids were only present in
METGL in appreciable levels, as presented in Tables 2 and 3.
Table 2. Shows the results of the qualitative phytochemical screening of the pulverized leaves of T. globiferus,
METGL and EAFTGL.
Phytochemicals
Crude extract
METGL
EAFTGL
Carbohydrates
++
+
-
Reducing sugar
+++
++
-
Flavonoid
++
+
+++
Tannins
+++
+
++
Phenolics
++
+
++
Alkaloids
++
++
+++
Steroids
++
++
-
Terpenoids
++
++
+++
Saponins
++
+++
-
Glycosides
-
-
-
Key: +++ = Highly present++ = Moderately present+ =minimally present- = Absent
Table 3. Shows the results of quantitative phytochemical screening of the pulverized leaves of T. globiferus,
METGL and EAFTGL.
Phytochemicals
Crude extract (μg/g)
METGL (μg/g)
EAFTGL (μg/g)
Carbohydrates
295.94 ± 1.33c
720.61 ± 45.67a
ND
Reducing sugar
477.68 ± 61.22d
1000.70 ± 62.79a
ND
Flavonoid
1637.50 ± 0.00e
792.60 ± 79.07a
3599.17 ± 141.34c
Tannins
85.79 ± 6.92b
203.03 ± 2.12a
167.54 ± 11.34a
Phenolics
3764.44 ± 76.50g
7254.59 ± 5335.72c
4759.14 ± 414.83d
Alkaloids
2211.11 ± 38.49f
4255.01 ± 335.06b
9277.78 ± 133.68e
Steroids
2.19±0.21a
0.42±0.00a
ND
Terpenoids
472.88 ± 23.35d
693.05 ± 6.14a
705.40 ± 33.47b
Saponins
315.15 ± 2.14b
2061.30 ± 171. 82b
ND
Values are mean ± standard error. Means with different superscript letters within a row are significantly different
(p < 0.05).
3.1.2. Acute toxicity test.
The result of the acute toxicity test is presented in Table 4. The result shows that both
METGL and EAFTGL did not exhibit any lethal effect, behavioral changes, or mortality in
mice up to 5000 mg/kg.
Table 4. Shows the result of acute toxicity test of METGL and EAFTGL on albino mice.
Groups
Number of mice
used
Dose (mg/kg)
Mortality (%) of
METGL
Mortality (%) of
EAFTGL
1
3
10
0
0
2
3
100
0
0
3
3
1000
0
0
4
3
1600
0
0
5
3
2900
0
0
6
3
5000
0
0
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3.1.3. Effect of METGL and EAFTGL on indomethacin-induced ulceration in albino rats.
The result of the effect of METGL and EAFTGL on indomethacin-induced ulceration
in rats is presented in Table 5. The result shows that indomethacin effectively induced
ulceration in rats; however, groups treated with reference drug, and varied dosages (100 and
200 mg/kg bw) of METGL and EAFTGL, respectively, exhibited various percentage inhibition
of ulceration. Further, rats treated with high doses (200 mg/kg bw) of METGL and EAFTGL
respectively showed significantly (p < 0.05) greater % ulcer inhibition (62.94 and 58.82 %)
than the standard control group, and this was not significantly (p > 0.05) different compared to
the % ulcer inhibition (73.52 %) exerted by the reference drug (omeprazole).
Table 5. Effect of METGL and EAFTGL on indomethacin-induced ulceration in albino rats.
Groups
Mean Ulcer Index (µL)
% Ulcer Inhibition
Positive control
1.70 ± 0.84b
---
Standard control
0.45 ± 0.33a
73.52
100 mg/kg bw METGL
1.13 ± 0.62ab
33.82
200 mg/kg bw METGL
0.63 ± 0.40a
62.94
100 mg/kg bw EAFTGL
1.08 ± 0.45ab
36.76
200 mg/kg bw EAFTGL
0.70 ± 0.41a
58.82
Values are mean ± standard error. Means with different superscript letters within a column are significantly
different (p < 0.05).
3.1.4. Effect of METGL and EAFTGL on hematological indices and leukocyte migration in
rats.
The effect of METGL and EAFTGL on hematological indices in rats is presented in
Table 6 and Figure 1.
Figure 1. Effect of the aqueous methanol extract and ethyl acetate fractions of T. globiferus on the
hematological profile of Wistar albino rats.
The result showed that METGL and EAFTGL produced various effects on the serum's
hematological parameters and leukocytes population. The WBC, MCV, MCH, MCHC,
eosinophils, lymphocyte, and neutrophil levels were more effectively reduced by week 4 when
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compared to the control, while hemoglobin, RBC, and PCV were non-significantly (p > 0.05)
reduced across both treatments.
Table 6. shows the effects of aqueous MeOH extract and E.A fraction of T. globiferus on leukocyte
mobilization in Wistar albino rats.
Groups
WBC
NEU
LYM
MON
EOS
1
68.50±11.48c
62.50±11.0a
36.00±10.58a
1.50±1.00a
ND
2
57.50±6.40ab
60.50±7.19a
37.50±5.97a
1.50±1.00a
0.50±1.00a
3
53.50±6.19ab
56.00±12.44a
43.00±12.49a
1.00±1.16a
ND
4
50.50±5.97ab
66.50±4.73a
32.25±3.30a
1.00±1.16a
0.25±0.50a
5
42.50±4.12a
57.00±9.31a
42.00±8.49a
1.00±1.16a
ND
6
54.00±10.19ab
65.50±7.72a
33.50±7.00a
1.00±1.16a
ND
Key: WBC = White blood cells, NEU = Neutrophils, LYM = Lymphocytes, EOS = Eosinophils, MON =
Monocytes, Values are expressed as mean ± standard deviation (n=5). Vertical superscripts with the same
alphabets are non-significant, while superscripts with different alphabets are significant.
3.1.5. Effect of METGL and EAFTGL on antioxidant markers.
Figure 2 shows the effect of METGL and EAFTGL on antioxidant markers. The result
revealed that treatments with METGL and EAFTGL exhibited a stabilizing effect on
antioxidant enzyme activities of superoxide dismutase, catalase, GSH, and GPx, which was not
significant (p > 0.05) different across the group throughout the duration of the study. Also, by
week 4, the levels of MDA were significantly (p < 0.05) reduced in all the groups treated with
varied dosages of METGL and EAFTGL. In contrast, a non-significant (p > 0.05) difference
in the serum mineral (selenium, zinc, and copper) levels was observed in all the treatment
groups when compared to the normal control group.
Figure 2. Effect of METGL and EAFTGL on the mean serum antioxidants of Wistar albino rats.
3.2. Discussion.
In traditional medicine, Tapinanthus globiferus manages several disease conditions,
including stomachache, ulcer, chronic fever, hypertension, tinnitus, asthma, inflammations,
dyspepsia, relief pain, epilepsy, cough, and cancer [14]. Analysis of the bioactive
phytochemicals present in the leave crude extract, methanol extract, and fraction revealed the
presence of flavonoids, tannins, phenolics, alkaloids, and terpenoids in both METGL and
EAFTGL. In contrast, saponins, reducing sugar, carbohydrates and steroids were only present
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in METGL (Table 2). The detection of alkaloids, tannins, saponins, flavonoids, and steroids in
the plant material agrees with the submissions of [15] and [17], which reported akin results
using the plant. As mentioned earlier, these active components in varying amounts in the plant
may be involved with the plant’s high activity for managing various ailments. According to
[38], alkaloids have many pharmacological properties, including antiasthma, cholinomimetic,
vasodilatory, anti-arrhythmic, and analgesic properties. [38] further proposed that the several
pharmacological activities exhibited by medicinal plants could be attributed to the presence of
flavonoids, which inhibit platelet aggregation. This event has been implicated in the etiology
of most inflammatory cascades and diseases. Saponins and flavonoids have been reported to
exhibit anticonvulsant effects [39]. Generally, the presence of many hydroxyls groups in the
chemical structures of tannins, phenolics, and flavonoids greatly favors antioxidant capacity
[40]. The result of phytochemical screening of METGL and EAFTGL in this study will
rightfully guide future researchers on the choice of the plant extract to be deployed for further
study or isolation of the bioactive ingredients.
As presented in Table 5, the result of the study reveals that NSAIDs (indomethacin)
were effective in inducing ulceration in rats. NSAIDs, which are one the most readily available
analgesics, have been effective in their administration but have been reported to cause erosion
on the gastrointestinal (GIT) mucosa, extending into deeper parts of the GIT wall [41]. NSAIDs
confer their therapeutic effects via mediating the inhibition of the biosynthesis of
prostanoids and their derivatives which are pivotal in exerting cytoprotective effects in gastric
mucosa and maintaining the mucosa integrity [41]. These lead to an increased susceptibility to
mucosal injury. In this study, NSAID-induced peptic ulcer was employed to study the ulcer-
protective effect of METGL and EAFTGL. The administration of METGL and EAFTGL in
varying doses (100 and 200 mg/kg bw) respectively led to an increase in % ulcer inhibition of
NSAID-induced ulceration in the stomach of Wistar rats.
It was also recorded that rats administered with a high dosage (200 mg/kg bw) of
METGL and EAFTGL, respectively exhibited significantly (p < 0.05) greater % ulcer
inhibition (62.94 and 58.82 %) akin to the standard control group, as it was not significantly (p
> 0.05) different compared to the % ulcer inhibition (73.52 %) exerted by the reference drug
(omeprazole). This suggests that the extracts and fraction of Tapinanthus globiferus leaves
possess anti-ulcerative potentials and lend credence to the folkloric use of the plant leaves in
managing gastrointestinal ailments. [42] demonstrated that pharmacological agents that
possess bioactive ingredients with antioxidant potentials such as flavonoids and polyphenolic
compounds may protect against gastric damage. Some of these substances are highly contained
in plant materials. Thus, the anti-ulcerogenic activities of MEFTGL and EAFTGL may be due
to the high number of unsaturated aromatic rings and compounds and phenolic groups present
in the plant material, which may result in the effective inhibition of inflammatory enzymes
implicated in the pathology of peptic ulcer disease. This corroborates with the assertion of [43]
that antioxidant activities of plants have been attributed to the presence of beneficial
phytoconstituents and such as flavonoids and other bioactive ingredients that work
synergistically to protect the plant against the myriads of deleterious biotic and environmental
factors.
The present study showed that METGL and EAFTGL produced various effects on the
serum's hematological parameters and leukocytes population. The WBC, MCV, MCH, MCHC,
eosinophils, lymphocyte, and neutrophil levels were more effectively reduced by week 4 when
compared to the control, while hemoglobin, RBC, and PCV were non-significantly (p > 0.05)
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reduced across both treatments. This suggests that METGL and EAFTGL exhibited a good and
healthy effect on rats. [44] reported that leukocytes form an important element in the defense
system of the animal body against viral, bacterial, and parasitic infections, while an increased
red blood cell count is because of freedom from diseases and opined that increased RBC is
correlated with a disease-free state in animals. In this study, the WBC and neutrophil levels
were more effectively reduced by 100 mg/kg bw administration of EAFTGL, while 200 mg/kg
bw of METGL reduced lymphocytes more effectively. The WBC components, such as
leukocytes, lymphocytes, monocytes, and neutrophils of any animal, are a function of the
immunity and the animal’s resistance to some vulnerable diseases [2]. Oxygen supply in the
blood is reflected by hemoglobin concentration. This, therefore, implies that the concentration
of hemoglobin in the blood would serve efficiently in diagnosing disease conditions. The non-
significantly reduced hemoglobin levels after administration of treatments in this study could
indicate that no challenge to cellular respiration and metabolic processes was encountered. Red
blood cells and hemoglobin function in the efficient transport of oxygen into and carbon
dioxide out of the body and, in concert with #PCV (the percentage of RBC in blood), indicate
oxygen-carbon dioxide balance status [45,46]. The non-significant influence of METGL and
EAFTGL of the leaves on the rats’ hemoglobin, RBC, and PCV concentrations have been
interpreted as an absence of adverse effect on rats’ hematological properties [47–49]. [2] also
reported that hematological indices (MCV, MCH, and MCHC) are said to indicate secondary
responses of an organism to irritants. A low level of MCV, MCH, and MCHC signifies a
normal condition of the blood of rats. In this study, the values of MCV, MCH, and MCHC
decreased slightly with increasing concentrations of METGL and EAFTGL.
As presented in Figure 2, the result also revealed that METGL and EAFTGL exhibited
stabilizing effect on antioxidant enzymes activities of superoxide dismutase, catalase, GSH,
and GPx, and this was not significantly (p > 0.05) different across the group throughout the
duration of the study. Also, by week 4, the levels of MDA were significantly (p < 0.05) reduced
in all the groups treated with reference drugs and varied dosages of METGL and EAFTGL,
while a non-significant (p > 0.05) difference in the serum mineral (selenium, zinc, and copper)
levels was observed in all the treatment groups when compared to the standard and normal
control group. This conforms to the findings of [39], which also reported an appreciable
antioxidant effect on catalase and superoxide dismutase assay. In the same vein, [50] reported
that Tapinanthus globiferus leaves extract may represent a good antioxidant candidate source
under its elevated iron chelating capacity.
4. Conclusions
The present study evinces that oral administration of T. globiferus leaves exhibits anti-
ulcer and antioxidant activities and anti-inflammatory properties, which are likely adduced to
their high phenolic and flavonoid contents.
Funding
This research received no external funding.
Acknowledgments
This research has no acknowledgment.
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Conflicts of Interest
The authors declare no conflict of interest.
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