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Tetrorchidium Didymostemon: Phytoconstituents, Antioxidant Potential And

Impact On Hematological Indices And Proinflammatory Cytokines In
Plasmodium Berghei-Infected Mice

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 Tetrorchidium didymostemon…

The Bioscientist Vol. 8(1): 51-67, January 2020 Available online at

http://www.bioscientistjournal.com

OPEN ACCESS

Full Length Research Paper

Tetrorchidium Didymostemon: Phytoconstituents, Antioxidant Potential And Impact On

Hematological Indices And Proinflammatory Cytokines In Plasmodium Berghei-Infected
Mice
1
*Ebohon, O., 1Irabor F., 2Ibukun, O., 3Amiebenomo, R., and 3Ehimwenma, S. O.
1
Biochemistry Program, Department of Biological and Chemical Sciences, Faculty of Natural
and Applied Sciences, Michael and Cecilia Ibru University, PMB 100, Agbarha-Otor, Ughelli
North, Delta State, Nigeria.
2
Department of Biochemistry, Faculty of Basic Medical Sciences, University of Medical
Sciences, P.O. Box 536, Ondo, Ondo State, Nigeria.
3
Malaria Research, Molecular Biology and Toxicology Unit, Department of Biochemistry,
University of Benin, PMB 1154, Benin City, Nigeria.
*Email: ebohonosamudiamen@yahoo.com

ABSTRACT

This study was aimed at evaluating the phytochemical and antioxidant potentials of methanol
extracts of Tetrorchidium didymostemon leaf and stem bark. The study also investigated the
effects of the extracts on hematological and immunomodulatory changes in Plasmodium
berghei-infected mice. Standard methods were used to carry out the phytochemical analysis.
Forty two (42) P. berghei-infected Swiss albino mice were used in this study and the mice were
sacrificed after four days of consecutive administration of T. didymostemon extracts (250 and
500 mg/kg body weight). Hematological indices and proinflammatory cytokines were thereafter
determined. The leaf extract had substantial quantity of phytochemicals and antioxidant potential
than the stem bark extract. White blood cells count (16.80 × 10³ ± 0.23 µL) of the leaf extract
treated mice was significantly (p < 0.05) lower when compared with the negative control (34.70
× 10³ ± 2.71 µL). However, there was no significant (p > 0.05) difference in the red blood cells,
hematocrit and hemoglobin levels of the mice treated with the leaf and stem bark extracts when
compared with the negative control. The leaf extract treated mice at 500 mg/kg body weight had
a significantly (p < 0.05) lower tumor necrosis factor alpha (6.59 ± 0.30 pg/mL) and interferon
gamma (9.41 ± 0.10 pg/mL) levels when compared with the negative control (9.18 ± 1.56 and
10.77 ± 0.61 pg/mL, respectively). This study has demonstrated that methanol extracts of T.

51
Ebohon et al., 2020

didymostemon leaf may ameliorate malaria infection by inducing changes in hematological
profile and proinflammatory cytokines.
Keywords: Tetrorchidium didymostemon, Malaria, Immunomodulatory, Anemia, Phytochemical,
Antioxidant
INTRODUCTION
Despite the reported drop in the number of
deaths resulting from malaria, from
approximately 438,000 in 2015 to 409,000
in 2019 (World Malaria Report, 2015 and
2020), malaria still remains one of the major
causes of death worldwide. This decrease in
death may be as a result of the increased
availability of antimalarial drugs in endemic
regions and the increased usage of
insecticide treated nets and indoor residual
sprays. However, there is a threat to the use
of antimalarial drugs and indoor residual
sprays as Plasmodium falciparum and its
vector has developed resistance to most of
the currently available drugs and residual
sprays, respectively (World Malaria Report,
2020). Presently, there are no K13 propeller
mutation associated with artemisinin
resistance in Africa. However, recent report
from a study in Rwanda identified Pfkelch13
R561H mutation in 7.4% of the studied
population and this mutation was confirmed
to drive artemisinin resistance in vitro
(Uwimana et al., 2020). Similarly, there are
some reported cases of therapeutic failures
after regimen with artemisinin-based
combined therapies in Nigeria (Ebohon et
al., 2019). These treatment failures and a
combination of anemia and cytokine
imbalance already implicated in malaria
infection may lead to drawback in malaria
control programs.
Cytokine imbalance contributes to
the pathological features of malaria such as
Ebohon et al., 2020
Tetrorchidium didymostemon…
cellular activation and activation of
endothelial adhesion molecules (Takahiko et
al., 2011). Macrophages, T cells, natural
killer cells and cytokines contribute either to
reduce malaria infection or cause an increase
in parasitaemia thereby resulting in
complicated malaria (Taylor-Robinson,
1995; Winkler et al., 1998). The periodic
fever associated with this infection have
been linked to spiking levels of
proinflammatory cytokines such as tumour
necrosis factor alpha (TNF-α) (Karunaweera
et al., 1992). Anemia which has been
implicated in malaria infection results from
splenic elimination of infected erythrocytes
and continuous degradation of hemoglobin
in the erythrocytes as this hemoglobin is
required as source of amino acid for parasite
growth.
In Nigeria, the poor populations who
mainly reside in rural areas depend on
herbal medicine for the treatment of malaria
due to the relatively high cost of orthodox
drugs. The use of plant materials for
treatment might be imperative and beneficial
as key important and active ingredients of
antimalarial drugs (artemisinin and quinine
derivatives) are known to have their source
from herbs (Willcox and Bodeker, 2004).
Currently, there is an increasing interest in
the use of medicinal plants for the treatment
and management of ailments and diseases.
Tetrorchidium didymostemon (Baill.) Pax &
K. Hoffm is an evergreen shrub distributed
52

in the West and Central Africa. In Nigeria, it
is called ‘iheni’ in Edo, ‘ofun oke’ in Yoruba
while in neighboring Cameroun it is called
‘efobolo’. It is common at forest edges or
along rivers, swamps and lakesides, from
sea level up to 1700m altitude (Toirambe,
2008). T. didymostemon is a shrub with
white latex, sometimes its latex can be red
or colourless, twigs are slightly zigzag,
leaves alternate on flowering shoots,
opposite on other branches, and it’s
prominently scarred at the nodes. The plant
Figure 1: Stem bark and leaves of T. didymostemon showing its fruiting branches (Toirambe,
2008)
MATERIALS AND METHODS
Experimental design
Completely randomized design was
used in this study. Forty-two (42) male
albino mice of the Swiss strain were
randomly divided using the Rand function in
Microsoft Excel into 6 groups of 7 mice
each. On the first day of the experiment
(termed ‘day 0’), all mice were injected
intraperitoneally with standard inoculums of
P. berghei with 1 × 107 infected
erythrocytes except the basal control group.
The first treatment was done two (2) hours
after injection of mice with the standard
Ebohon et al., 2020
Tetrorchidium didymostemon…
diverse medicinal uses such as its
application to wounds as a hemostatic, and
treatment of fever, malaria, enlarged spleen
etc. has been reported by Burkill (2004) and
Toirambe (2008). Despite the diverse usage
of T. didymostemon, there are shortfalls of
scientific investigations on its extracts.
Hence, this study was designed to evaluate
the phytochemicals and the effects of
methanol extracts of Tetrorchidium
didymostemon leaf and stem bark on
Plasmodium berghei-infected mice.
inoculum (day 0) by means of an oral
cannula and was repeated once daily for four
consecutive days (day 1 – day 4).
Ethical approval
Ethical approval for this study was
granted by the Institutional Ethics Review
Committee, University of Benin (No:
LS19114).
Plant collection and authentication
Leaf and stem bark of Tetrorchidium
didymostemon (Baill.) Pax & K. Hoffm were
collected from the wild in Urhokuosa village
53

in Uhumwonde Local Government Area of
Edo State in April 2019. Authentication of
the plant was done at the Department of
Plant Biology and Biotechnology,
University of Benin, Benin City, Nigeria by
Dr. H.A Akinnibosun and voucher specimen
of the plant (UBHT439) was deposited at the
herbarium of same department. The
authenticated leaf and stem bark were
washed, air dried, ground and stored at room
temperature in an airtight container.
Extraction of plant materials
The plant extracts were prepared by
soaking 300 g of ground leaf and stem bark
of T. didymostemon in 2.5 liters of methanol
(99.8% purity) in a separate air - tight
containers for 72 hours (Ojemekele et al.,
2017). The soaked plant samples in
methanol were stirred thrice daily. The
resulting suspensions were filtered using
Whatman No 1 filter paper into a clean flask
and the filtrates were concentrated using a
rotary evaporator (RE 300, Bibby Scientific,
UK) with reduced pressure at 45˚C and final
concentrates were obtained using silica gel
in a desiccator. Petri dish with the plant
extract was transferred to a desiccator
containing anhydrous silica gel for 24 hours
to completely remove solvent of extraction.
The extracts were thereafter stored in a
sterile container and kept at 4oC till when
needed.
Dosage calculation of leaf and stem bark
extracts
The dosage of plant extracts and
drug used in this study was calculated using
the formula below according to the OECD
guidelines. Using the formula, the calculated
Ebohon et al., 2020
Tetrorchidium didymostemon…
dose was constituted in 1 mL of 0.7%
carboxymethyl cellulose.
Dosage in mg =
Quantification of phytochemicals and in
vitro antioxidant capacity of extracts
Standard protocols described by
Harbone (1998), Sofowora (1993) and
Trease and Evans (1989) were used to
carryout phytochemical screening. Total
phenolics, proanthocyanidins, flavonoids
and tannins contents were determined using
Cicco et al. (2009), Sun et al. (1998),
Miliauskas et al. (2004) and Polshettiwar et
al. (2007) methods respectively. The free
radical scavenging capacity of the extracts
against 1,1–diphenyl–2–picrylhydrazyl
(DPPH) and nitric oxide (NO) radicals were
determined by the methods of Brand-
Williams et al. (1995) and Garrat (1964)
respectively. Ferric Reducing Antioxidant
Potential (FRAP) and phosphomolybdenum
reduction capacity (PRC) were estimated
using Benzie and Strain (1996) and Prieto et
al. (1999) methods respectively.
Procurement of the Experimental animals
Forty-two healthy Swiss albino male
mice weighing between 19 - 23g, obtained
from the Nigerian Institute of Medical
Research (NIMR), Lagos State, Nigeria
were used for this study. The mice were
housed under standard laboratory conditions
of light / dark cycle for 12 h at temperature
27 ± 2ºC in the vivarium of the Department
of Biochemistry, University of Benin. They
had access to commercial mash (Pfizer
Livestock Feeds, Lagos Nigeria) and water
54
 Tetrorchidium didymostemon…

ad libitum. The mice were acclimatized for  Negative control: Was infected with
two weeks before the experiment P. berghei and orally administered
commenced. 0.2 mL of the vehicle (0.7%
carboxymethyl cellulose).
Malaria parasite inoculation into mice
 CqT (10 mg/kg body weight): Was
Chloroquine-sensitive strain
infected with P. berghei and orally
of rodent parasite, Plasmodium berghei
administered 10 mg/kg body weight
NK65 maintained in mice was obtained
of chloroquine phosphate in 0.2 mL
from the Nigerian Institute of Medical
of the vehicle (0.7% carboxymethyl
Research, Lagos, Nigeria. The parasites
cellulose).
were kept alive by continuous intra-
 Leaf (250 mg/kg): Was infected with
peritoneal passage in healthy mice. Blood
P. berghei and orally administered
from a single donor mouse with 25%
250 mg/kg body weight of leaf
parasitaemia was obtained via cardiac
extract in 0.2 mL of vehicle (0.7%
puncture into heparinized tube and was
carboxymethyl cellulose).
diluted with normal saline to have
 Leaf (500 mg/kg): Was infected with
inoculums containing approximately 1 × 107
P. berghei and orally administered
infected erythrocytes. Thereafter, 0.2 mL of
500 mg/kg body weight of leaf
standard inoculums (1x107 Plasmodium
extract in 0.2 mL of vehicle (0.7%
berghei strain NK 65 parasitized
carboxymethyl cellulose).
erythrocytes) were used to infect other mice
where necessary.  Stem bark (250 mg/kg): Was infected
with P. berghei and orally
Treatment of mice administered 250 mg/kg body weight
The Peter’s 4 - day suppressive test of stem bark extract in 0.2 mL of
against chloroquine-sensitive Plasmodium vehicle (0.7% carboxymethyl
berghei NK 65 infection in mice was cellulose).
employed (Peters, 1967). All mice except  Stem bark (500 mg/kg): Was infected
the basal control were injected with P. berghei and orally
intraperitoneally with standard inoculums of administered 500 mg/kg body weight
P. berghei with 1 × 107 infected of stem bark extract in 0.2 mL of
erythrocytes. The first treatment was done vehicle (0.7% carboxymethyl
two (2) hours after inoculation by means of cellulose).
an oral cannula and was repeated once daily
for four consecutive days (day 1 – day 4).
The experimental animals were
Collection of blood samples for analysis
distributed into groups shown below.
The experimental animals were
 Basal control: Was not infected with
subjected to fasting overnight and sacrificed
P. berghei but was administered 0.2
on ‘day’ 5 of the experiment (after 4 days of
mL of the vehicle (0.7%
consecutive administration of extracts and
carboxymethyl cellulose).
drug) (Akanbi, 2015). Blood samples were

55
Ebohon et al., 2020

collected from the animals via aortic
puncture under chloroform vapour into plain
sterile bottles to obtain serum and EDTA
tubes to obtain plasma for hematological
analysis. Blood samples in the plain tubes
were allowed to stand for 40 minutes to clot
before it was centrifuged (DLAB, DM0412)
for 15 min at 2000 rpm to obtain the serum.
After blood sample collection, the
anaesthetized mice were euthanized via
cervical dislocation and death was
confirmed. The carcasses of the mice were
disposed of appropriately using a burial pit.
Hematological analysis and serum
cytokine assay
Hematological parameters such as
hematocrit (HCT), hemoglobin (Hb), red
blood cells (RBC), white blood cells
(WBC), platelet (PLT) counts etc. were
analyzed using an automated hematological
RESULTS
Table 1: Phytochemical composition of the methanol extracts of leaf and stem bark of
Tetrorchidium didymostemon
Phytochemicals
Flavonoids
Phenolics
Cardiac glycosides
Terpenoids
Tannins
Saponins
Sterols
Quinones
Anthraquinones
Coumarins
Phlobatannins
Alkaloids
KEY: +++ = Present in very high amount; ++ = Present in high amount; + = Present in low amount; – = Not detected
Ebohon et al., 2020
Tetrorchidium didymostemon…
analyzer (Beckman Coulter JT series
Hematological Analyser). Tumor necrosis
factor alpha (TNF-α) and interferon gamma
(IFN-γ) were estimated with Elabscience®
ELISA Kit using the sandwich-ELISA
method. The micro ELISA plate provided in
the kits were pre-coated with an antibodies
specific to mice TNF-α and IFN-γ.
Statistical analysis
Statistical analysis was performed
using the statistical package for social
sciences (SPSS) for windows, version 16.0
(SPSS Inc., Chicago, IL, USA). The results
obtained were expressed as mean ± SEM.
One way analysis of variance (ANOVA) test
was used to determine significance
differences between the groups and post hoc
multiple comparison test was done using
Tukey's HSD (honest significant difference).
Statistical significance was declared when P
value was less than 0.05.
Leaf Stem bark
++ +
+ +
+++ ++
+ +
++ +
+ +
+ +
+ ++
- -
++ +
+ -
+ +
56
 Tetrorchidium didymostemon…

Table 2: Total phenolic, flavonoid, tannin and proanthocyanidin contents of Tetrorchidium

didymostemon leaf and stem bark extracts

Assays Leaf Stem bark

Total phenolics (mg Gallic Acid Equivalent / g extract) 42.08 ± 0.59a 46.19 ± 0.48b
Total flavonoids (mg Quercertin Equivalent / g extract) 35.91 ± 0.53a 3.01 ± 0.26b
Total tannins (mg Tannic Acid Equivalent/ g extract) 33.21 ± 0.23a 19.70 ± 0.36b
Total proanthocyanidins (mg Ascorbic Acid Equivalent / g 29.90 ± 1.02a 2.32 ± 0.10b
extract)
Values with different alphabet as superscript on same row are significantly different between means (p < 0.05), while those with same alphabet
are not significantly different.

Figure 2: DPPH radical scavenging capacity of methanol extracts of the leaf and stem bark of
Tetrorchidium didymostemon. Values are expressed as mean ± SEM, n = 3/group.

57
Ebohon et al., 2020
 Tetrorchidium didymostemon…

Figure 3: Nitric oxide (NO) radical scavenging potential of methanol extracts of the leaf and
stem bark of Tetrorchidium didymostemon. Values are expressed as mean ± SEM, n = 3/group.

Table 3: Ferric Reducing Antioxidant Potential (FRAP), Phosphomolybdate Reduction Capacity

(PRC) and IC50 values of leaf and stem bark extracts of Tetrorchidium didymostemon

Assays Leaf Stem bark Ascorbic acid

DPPH IC50 (µg/mL) 41.29a 87.03b 4.12c
NO IC50 (mg/mL) 0.89a 7.10b 0.43c
FRAP (µM Fe (II)/ g extract) 391 ± 11a 454.50 ± 2.5b 1178 ± 10c
PRC (mg Ascorbic Acid Equivalent/ g 179.94 ± 2.47a 80.27 ± 2.32b -
extract)
Values with different alphabet as superscript on same row are significantly different between means (p < 0.05), while those with same
alphabets are not significantly different. DPPH = 1,1–Diphenyl–2–picrylhydrazyl and NO = Nitric oxide

58
Ebohon et al., 2020
 Tetrorchidium didymostemon…

Table 4: Effect of methanol extracts of the leaf and stem bark of T. didymostemon on
hematological indices of P. berghei-infected mice.

Parameters Basal Negative CqT (10 Leaf Leaf Stem bark Stem bark
Control Control mg/kg) (250 (500 (250 (500
mg/kg) mg/kg) mg/kg) mg/kg)
WBC (x10³µL) 5.90 ± 34.7 ± 10 ± 13.5 ± 16.80 ± 26.50 ± 31.10 ±
0.50a 2.71e 0.58ab 0.29bc 0.23c 1.73d 1.73e
LYM (x10³µL) 4.83 ± 24.60 ± 8.07 ± 12.13 ± 12.90 ± 13.17 ± 19.10 ±
0.09a 2.89d 0.15a 0.52b 0.79b 0.38b 0.67c
MON 0.20 ± 2.27 ± 1.27 ± 2.10 ± 1.83 ± 3.90 ± 4.07 ±
(x10³µL) 0.06a 0.09d 0.03b 0.06d 0.07c 0.12e 0.12e
GRAN 0.30 ± 2.87 ± 1.23 ± 2.23 ± 1.47 ± 5.00 ± 4.23 ±
(x10³µL) 0.06a 0.09d 0.12b 0.09c 0.09b 0.29f 0.12e
LYM (%) 84.40 ± 81.57 ± 76.00 ± 81.80 ± 79.70 ± 63.30 ± 67.50 ±
2.08c 1.87 bc
0.75 b
0.99bc 4.38bc 2.23a 2.41a
MON (%) 12.23 ± 15.30 ± 13.13 ± 15.07 ± 13.30 ± 14.40 ± 14.60 ±
0.58a 0.64 d
0.35 ab
0.32cd 0.29abc 0.46bcd 0.91bcd
GRAN (%) 8.30 ± 12.53 ± 9.73 ± 13.77 ± 11.70 ± 15.87 ± 14.17 ±
0.44a 0.76 c
0.33 a
0.20d 0.20b 0.87d 0.58
RBC (x10⁶µL) 9.68 ± 5.34 ± 9.92 ± 5.41 ± 6.68 ± 5.49 ± 5.40 ±
0.40b 0.02 a
0.58 b
0.11a 0.90a 0.20a 0.06a
HGB (g/dL) 15.20 ± 9.10 ± 13.60 ± 9.35 ± 9.20 ± 7.57 ± 7.40 ±
0.10d 0.06b 0.06c 0.26b 1.10b 0.33a 0.06a
HCT (%) 50.87 ± 27.47 ± 40.50 ± 28.47 ± 31.13 ± 25.20 ± 25.90 ±
2.07c 0.42a 1.19b 0.53a 4.67a 0.70a 1.73a
MCV (fl) 52.57 ± 49.07 ± 43.57 ± 51.13 ± 47.07 ± 45.67 ± 47.33 ±
1.66c 1.73bcd 0.78a 1.52cd 1.33abc 0.71ab 0.52abc
MCH (pg) 14.80 ± 13.67 ± 14.73 ± 16.97 ± 13.93 ± 13.83 ± 13.40 ±
0.55b 0.23ab 0.61b 0.47c 0.23ab 0.20ab 0.17a
MCHC (g/dL) 29.90 ± 27.43 ± 29.70 ± 31.80 ± 30.10 ± 29.83 ± 29.10 ±
1.06ab 0.49a 1.03ab 1.31b 1.03ab 0.87ab 0.42ab
PDW (%) 16.90 ± 16.30 ± 16.47 ± 16.37 ± 16.13 ± 16.80 ± 15.97 ±
0.21a 0.32 a
0.78 a
0.24a 0.43a 0.29a 0.44a
PLT (µL) 1098.50 ± 323.00 ± 1048.67 ± 833.50 ± 864.50 ± 520.50 ± 347.00 ±
51.10c 11.55a 1.33c 29.73bc 172.92c 18.76ab 23.09a
PCT (%) 0.67 ± 0.29 ± 0.61 ± 0.58 ± 0.74 ± 0.41 ± 0.31 ±
0.03cd 0.01 a
0.03 c
0.02c 0.02d 0.01b 0.02ab
MPV (fl) 6.03 ± 6.87 ± 6.10 ± 7.03 ± 6.97 ± 6.50 ± 7.20 ±
0.03a 0.12 b
0.06 a
0.12b 0.12b 0.21ab 0.26b
PDW (fl) 7.20 ± 8.90 ± 7.17 ± 8.80 ± 8.93 ± 7.93 ± 8.90 ±
0.25a 0.12 b
0.29 a
0.15b 0.15b 0.12ab 0.40b
Values are mean ± SEM, n = 6 mice/ group. Values with different alphabet on the same row as superscript are significantly different between
means (p < 0.05) while those with same alphabet are not significantly different. Basal control = Not infected with P. berghei, Negative control =
P. berghei infected but not treated, CqT = Chloroquine treated.

59
Ebohon et al., 2020
 Tetrorchidium didymostemon…

Table 5: Effect of methanol extracts of T. didymostemon on TNF-α and IFN-γ levels in P.

berghei-infected mice

Groups TNF-α (pg/mL) IFN-γ (pg/mL)

Basal control 6.40 ± 1.31c 8.13 ± 0.89de
Negative control 9.18 ± 1.56a 10.77 ± 0.61a
Chloroquine (10 mg/kg) 6.58 ± 1.35bc 7.38 ± 0.93e
Leaf (250 mg/kg) 6.65 ± 0.15bc 8.83 ± 0.10de
Leaf (500 mg/kg) 6.59 ± 0.30bc 9.41 ± 0.10cd
Stem bark (250 mg/kg) 8.32 ± 0.11ab 10.73 ± 0.48c
Stem bark (500 mg/kg) 7.92 ± 0.43abc 11.73 ± 0.41b
Values are mean ± SEM, n = 6 mice/ group. Values with different alphabet on the same column as superscript are significantly different between
means (p < 0.05) while those with same alphabet are not significantly different. Basal control = Not infected with P. berghei, Negative control =
P. berghei infected but not treated, CqT = Chloroquine treated.

DISCUSSION rural dwellers especially in developing

Phytochemicals and antioxidant capacity countries depend heavily on medicinal
of Tetrorchidium didymostemon plants for the treatment and management of
Phytochemical screening of several ailments. These medicinal plants
methanol extracts of Tetrorchidium contain numerous bioactive constituents
didymostemon leaf and stem bark revealed which are used for therapeutic purposes and
wide range of phytochemicals (Table 1). T. also as chemical entities for synthetic drugs.
didymostemon leaf extract had a
significantly (p < 0.05) higher amount of In this study, T. didymostemon leaf
flavonoids, tannins and proanthocyanidins extract was a better scavenger of DPPH and
content when compared with the stem bark nitric oxide radicals when compared with
extract (Table 2). the stem bark extract (Figure 2 and 3). The
leaf extract also had a lower half-maximal
Proanthocyanidins are condensed inhibitory concentration (IC50) values which
tannins (Prakash and Gupta, 2009) and the corresponds to increased radical scavenging
higher amount of tannins in the leaf extract potential (Table 3). Similarly, the leaf
compared with the stem bark extract was extract had a better phosphomolybdate
consistent with that of proanthocyanidins. reduction capacity (Table 3). The higher
Bioactive compounds such as flavonoids, bioactive compounds such as flavonoids,
tannins and phenolic acids have been tannins and proanthocyanidins present in the
reported to possess anti-inflammatory, leaf extract may be responsible for its
antiatherosclerotic and anticarcinogenic excellent radical scavenging properties
activity (Khan et al., 2012). Hence, the compared with the stem bark extract. When
presence of these phenolic compounds in in excess, free radicals such as reactive
this plant may be responsible for its oxygen species, reactive nitrogen species,
effective usage in ethnomedicinal practices. nitric oxide etc. contribute to the
pathological features of diseases as they
The earliest medicine known to man result in oxidative stress. Antioxidants help
are from medicinal plants and till date, most
60
Ebohon et al., 2020

to protect cells against the damaging effects
of these free radicals such as hydroxyl
radicals, singlet oxygen, peroxyl radicals,
peroxynite and super oxide which results in
cellular damage (Mattson and Cheng, 2006).
Effect of T. didymostemon extracts on
hematological indices of P. berghei-
infected mice
The result of this study showed no
significant differences (p > 0.05) in the RBC
and HCT level of P. berghei-infected mice
treated with extracts when compared with
the untreated infected mice (negative
control) (Table 4). However, the leaf extract
treated mice had a slightly higher RBC
count than the negative control (Table 4).
Complete blood count provides information
on hematological status in disease condition
Also, the stem bark extract may have
probably suppressed the process of
erythropoiesis. The low hemoglobin
concentration observed in the negative
control and the stem bark extracts treated P.
berghei-infected mice (Table 4) may be
attributed to their high parasite load as the
growing parasites have been reported to feed
on the hemoglobin. In red blood cell, the
parasite catabolizes hemoglobin in its acidic
food vacuole in other to meet its
requirement for growth and further invade
other cells (Sigala and Goldberg, 2014).
The high HCT in the chloroquine
treated group may be due to its
antiplasmodial activity and decrease in RBC
hemolysis (Table 4). Previous study have
identified higher HCT, RBC and HGB
levels in chloroquine-treated P. berghei-
infected mice and it compares favourably
with basal control (Kamkumo et al., 2020).
Ebohon et al., 2020
Tetrorchidium didymostemon…
(Lathia and Joshi, 2014), and this is an
important index of the physiological and
pathological status of man. Hematological
parameters such as red blood cell (RBC),
packed cell volume (PCV) or hematocrit
(HCT), hemoglobin (HGB), white blood cell
(WBC) and platelet (PLT) counts are usually
altered during malaria infection. The
parasiticidal activity of the leaf extract (500
mg/kg) and chloroquine may be responsible
for the increase in RBC count. Whereas, the
low RBC count of the negative control and
the stem bark extract treated groups may be
a reflection of the severity of the infection
which may have led to increase in splenic
clearance, red blood cell destruction, or
immune mediated destruction of P. berghei-
infected erythrocytes.
Saponins are one of the constituents present
in the leaf and stem bark extracts of T.
didymostemon and the presence of this
phytochemical may be responsible for the
low HCT, RBC and HGB levels of the
extracts treated P. berghei-infected mice.
Saponins are known to have strong
hemolytic effect on the RBC (Yang et al.,
2005). They cause hemolysis by increasing
the permeability of the plasma membrane
(Dewick, 2002). Our findings is not in
agreement with the study of Adetutu et al.
(2016) who observed an increase in RBC,
HGB and HCT following treatment of P.
berghei-infected mice with methanol
extracts of Launaea taraxacifolia and
Amaranthus viridis leaves. The extracts
were unable to prevent the drastic reduction
in RBC, HGB and HCT associated with
malaria infection. This implies that the
extract may not play useful role in
61

ameliorating anaemia induced by malaria
infection.
Also, it suggests that the plant
extract may be hemotoxic thereby resulting
in decreased RBC, HGB and HCT levels.
Previous study on the sub-acute toxicity of
T. didymostemon leaf have shown that at
higher doses, the extract resulted in decrease
in RBC, HGB and HCT levels (Ebohon et
al., 2020).
White blood cell (WBC) is part of
the immune system which helps the body to
fight disease / infection and an increase in
WBC has been linked to severe malaria. In
this study, the WBC count of mice in the
negative control was significantly (p < 0.05)
higher in comparison to the treated groups
(Table 4). The authors suggest that the high
WBC count of the infected mice (negative
control) may be a reflection of the severity
of the malaria infection since WBCs are
mobilized to fight infections.
Similar study conducted by
Kamkumo et al. (2020) reported higher
WBC in mice infected without treatment
when compared with treated mice. Aside
parasite load, high WBC count as seen in the
stem bark extract treated mice may suggest
toxicity and hence lead to immunological
reaction. The decrease in platelet (PLT)
count in the negative control and stem bark
treated mice (Table 4) may be as a result of
thrombocytopenia which is a common
complication in malaria infection (Lacerda
et al., 2011). Oxidative stress can take part
in the genesis of thrombocytopenia in
malaria infection by increasing brittleness,
causing dysfunction in the receptor and loss
of membrane elasticity, resulting in
Ebohon et al., 2020
Tetrorchidium didymostemon…
functional impairment of thrombocytes
(Percário et al., 2012).
Immunomodulatory potential of T.
didymostemon extracts in P. berghei-
infected mice
The high level of proinflammatory
cytokines (IFN-γ and TNF-α) observed in
the negative control and the stem extracts
treated mice when compared with the leaf
extracts and the chloroquine treated mice in
this study (Table 5) may be linked to rising
and high level of parasitaemia, an indication
that the proinflammatory cytokines were
mobilized to fight the infection. Parasite
density has been observed to correlate with
cytokines levels (Belnoue et al., 2008).
The spike in cytokine levels may
also be caused by the rupture of infected
erythrocytes which is responsible for some
of the clinical manifestations of malaria
(Tuteja, 2007). The higher levels of IFN-γ
and TNF-α in the negative control and stem
bark treated group (Table 5) may be initially
beneficial by reducing parasite load and later
harmful by decreasing humoral response
(Julius et al., 2013). Whereas, the reduced
cytokines level noticed in the basal control,
chloroquine and the leaf extract (500 mg/kg)
treated groups may be as a result of either
the absence of infection (basal control) or
reduced parasite load (extracts / chloroquine
treated groups).
T. didymostemon leaf extract may
not have stimulated TNF-α synthesis during
P. berghei infection as previous study by
Ebohon et al. (2020) observed that the leaf
extract did not result in significant changes
in the expression of hepatic and renal TNF-α
genes. Thus, T. didymostemon
62

antiplasmodial potential may be linked to its
plasmocidal properties rather than its
immunomodulatory capacity.
Studies have shown that cytokines
along with natural killer cells, T cells and
macrophages contribute either to
ameliorating malaria infection or cause an
increase in parasitaemia thereby leading to
complicated malaria (Taylor-Robinson,
1995; Winkler et al., 1998). Early secretion
of proinflammatory cytokines that promotes
parasites death by macrophages and
thereafter, prevents immune mediated
damage, contributes to the successful
resolution of malaria infection
(Desruisseaux et al., 2008). Inflammatory
cytokines can contribute to the severity and
immunopathology of malaria (Boström et
al., 2012) through cellular activation and
activation of endothelial adhesion molecules
(Takahiko et al., 2011). Cytokines like
tumor necrosis factor alpha (TNF-α), up-
regulates intercellular adhesion molecule 1
(ICAM-1) expression on the cerebral
vascular endothelium thereby increasing
cytoadhesion of the parasitized infected
erythrocytes (Idro, et al., 2010).
Proinflammatory cytokines such as
TNF-α, IFN- γ and IL-12 have also been
reported to confer protection against malaria
infection (Torre et al., 2002). These
cytokines are involved in the inhibition of
parasite growth and stimulation of
phagocytosis so as to enhance parasite
clearance (Boström et al., 2012).
Ebohon et al., 2020
Tetrorchidium didymostemon…
CONCLUSION
The findings from this study have
shown that the leaf extract of T.
didymostemon has more phytochemicals and
higher potency in scavenging free radicals
when compared with the stem bark extract.
The study also supports the ethnomedicinal
uses of T. didymostemon in the management
and treatment of malaria infection. Although
the extracts showed limited potency in
reducing P. berghei-induced anemia, the
leaf extract was however able to reduce
TNF-α level in infected mice. The authors
suggest identification, isolation, and
characterization of the bioactive
compound(s) responsible for this activity.
FUNDING
This research was privately funded without
external support.
ABBREVIATIONS
HCT = Hematocrit, HGB = Hemoglobin,
WBC = White Blood Cells, RBC = Red
Blood Cells, LYM = Lymphocytes, MON =
Monocytes, GRAN = Granulocytes, MCH =
Mean Corpuscular Hemoglobin, RDW =
Red Cell Distribution Width, PCT =
Plateletcrit, PDW = Platelet Distribution
Width, MCV = Mean Corpuscular Volume,
MCHC = Mean Corpuscular Hemoglobin
Concentration, PLT = Platelets, MPV =
Mean Platelet Volume
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