American Journal of Plant Sciences, 2013, 4, 1834-1838 Published Online September 2013 (
The Effects of Aqueous Extracts of Acalypha wilkesiana
Supplementation and Exercise Training on Hematopoietic
System in Rats
Alfred F. Ehiaghe1,2, Joy I. Ehiaghe2, Igere E. Bright2,3, Iyen I. Roland2
1Department of Haematology, Igbinedion University, Okada, Nigeria; 2Department of Microbiology, Lahor Research Centre, Benin
City, Nigeria; 3Department of Microbiology and Biotechnology, Western Delta University, Oghara, Nigeria.
Received June 14th, 2013; revised July 14th, 2013; accepted August 15th, 2013
Copyright © 2013 Alfred F. Ehiaghe et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The aim of this study is to compare the effects of single and combined oral administration of fresh aqueous extracts of
Acalypha wilkesiana supplementation at 300 mg/ml and exercise training on some immuno indicator parameters in Rats.
The study was carried out in the College of Health Sciences, Department of Hematology, Igbinedion University, Okada
between the Month of June and July, 2012. Following 30 days post-oral administration of extracts on (Group 2 and
Group 3), exercise training on (Group 2 and Group 4) and No treated on Group 1, hematological parameters were de-
termined using the sysmex® Automated Hematological Analyzer, while CD4 and CD8 cells were estimated using Partec
cyflow counter and serum IgG is determined using the ELISA Method. CD4 cells, CD8, and Total WBC count show a
statistically significant increase while Hb concentration and IgG level show a statistically significant decrease in Group
2 and Group 4 (P < 0.05). Total WBC count shows a statistically significant increase against the control Group, while
CD4 cell, CD8 cell count, Hb-concentration and IgG level show a statistically insignificant increase (P > 0.05), Table 2.
Acalypha wilkesiana at a concentration of 300 mg/g/day seems to be immuno-protective in Rats. Single or combine
effects of prolonged exercise and A. wilkesiana produce significant change in some immuno indicators parameters.
However, the molecular mechanism behind their combined effect would require further investigation.
Keywords: Acalypha Wilkesian; Serum IgG; ELISA; CD4 Cells; CD8 Cells; Exercise
1. Introduction
Acalypha wilkesiana is an evergreen Shrub. It grows 3 m
high and 2 m across. The leaves are coppery green with
red splashes of colour, its other names including: A. am-
entaceae and A. tricolor. Its common names are copper
leaf, Joseph’s coat, fire dragon and match-me-if-you-can
[1]. The Hausas of the Northern Nigeria call it “Jiwene”,
while the Yoruba of the Southern Nigeria call it “awor-
oso”. It is propagated by stem cutting at anytime of the
year [1,2].
The phytochemical screening of the leave revealed the
presence of Alkaloids, Carotenoids, Flavonoids, Proteins,
Lipid, Carbohydrate, Reducing sugar, fiber, Saponins
and Tannins, all of which have potential health promot-
ing effects [3,4]. The expressed juice of A. wilkesiana is
used as an antimicrobial agent for the treatment of gas-
trointestinal disorders and fungal skin infection [5]. In
the southern Nigeria, the leaves extracts are used for the
management of hypertension [6]. Its Beta carotene con-
tents act as an antioxidant, which helps to boost the im-
mune system against cancer, cataract and damaging ef-
fects of radiation [3,7].
The immune system has a central role in protecting
against various external disease promoting factors and
perhaps against malignant cells. The immune system re-
gulates itself by means of the helper and suppressor T cells.
Nutrients and other constituents of Medicinal plants play
a contributory role in enhancing immune function [8].
Exercise is any bodily activity that enhances physical
fitness [9]. It improves mental health, helps prevent de-
pression and promote positive self-esteem [10] and re-
duces the level of cortisol which suppresses the immune
system [11]. Physical exercises are generally grouped
into three types, which include, flexibility exercise (such
as stretching, which improves the range of motion on the
muscle and joints), Aerobic exercise (such as cycling,
Copyright © 2013 SciRes. AJPS
The Effects of Aqueous Extracts of Acalypha wilkesiana Supplementation and Exercise Training
on Hematopoietic System in Rats
swimming, walking, skipping rope, running, biking or
playing tennis, which focus on increasing cardiovascular
endurance) and Anaerobic exercise (such as weight train-
ing or sprinting, which increases short-term strength [12-
The aim of this study is to compare the effect(s) of
single and combined oral administration of fresh aqueous
extract of A. wilkesiana supplementation at 300 mg/ml
concentration and exercise training on CD4 cell count,
Immunoglobulin G (IgG) level, Total White Blood cell
count and Hemoglobin concentration in Rats.
2. Materials and Methods
2.1. Study Area
This study was carried out in the college of Health Sci-
ences, Department of Hematology, Igbinedion University,
Okada, located in Ovia-North East Local Government
area of Edo State, coordinates: 6˚300E in Central South-
ern Nigeria between the month of May and July, 2012.
Predominant occupation among the people is farming,
despite the availability of reliable medical service, the
local populaces still rely on the use of herbs as medicines
for both curative and prophylaxis purpose.
2.2. Sample Size
The sample size was calculated using the formula de-
scribed by [15]
N = The minimum number of unit in the study (Minus
T = The treatment component including control group
(Minus one);
B = The Blocking component, representing environ-
mental effect allowed for the design (minus one) B = 0;
E = The degree of freedom (Between 10 and 20).
Using the formula, where E = 20, B = 0, T = 4.
The minimum number of sample size will be 23.
2.3. Study Design
A total of 24 male albino Rats weighing 105 ± 05 g was
purchased from the Animal production and Health De-
partment, Federal University of Technology, Akure,
Ondo State, Nigeria and housed in the experimental Ani-
mal House, College of Health Science, Department of
Hematology, Igbinedion University Okada, Separately in
well ventilated wire-bottom steel cage under hygienic
condition with proper aeration at 25˚C ± 2˚C and a rela-
tive humidity of 45% - 50%. The Rats were randomly
assigned into 4 groups of 6 rats each and fed on standard
Rat diet (10 g/100 g body weight) twice daily and tap
water ad libitum. Prior to commencement of administra-
tion of the extract and exercise training, the Rats were
allowed to stabilize in the Animal House with standard
12 hours light dark cycle, for a period of 14 days and was
treated for 30 days. All studies on the experimental ani-
mals were conducted in accordance with the current Ani-
mal Care Regulations and standards approved by the
Institute for Laboratory Animal Research. The Rats were
marked by ear puncturing system [16].
2.4. Collection and Preparation of Crude
Aqueous Extracts
Samples of fresh leaves of A. wilkesiana were collected
from within the college of Health Science, Igbinedion
University, Okada. After due identification at the Igbin-
edion University Herbarium, the leaves were washed tho-
roughly in tap and sterile distilled water. 30 g of the
washed leaves were soaked for 24 hours and homoge-
nized in a clean electric blender containing 100 ml sterile
distilled water according to the method [17]. These gave
a 3.0 g/10 ml = 300 mg/ml of the homogenate. The ho-
mogenates was shaken for one hour in a rotary flask and
then filter into separate sterile container using a funnel
containing sterile cotton wool and later with Whatman
No. 1 filter paper. The liquid filtrates were transferred
into separate sterile MacCartney bottles and stored in the
refrigerator at 4˚C after daily administration to the ex-
perimental animals.
2.5. Animal Treatment
A total of 24 Rats were randomly assigned into 4 groups
(6 per group) and treated as shown in Table 1.
G2 and G3 were given 1 ml/100 g body weight of the
extract using intragastric tube and adjusted daily with
change in body weight throughout the treatment period
which lasted for 30 days. G2 and G4 were simultane-
ously trained with exercise preconditioning in the form
of mere swimming. The swimming exercise was per-
formed in a 120 cm deep × 80 cm wide cylindrical tank,
with water temperature of 31˚C ± 1˚C. Swimming was
Table 1. Showing the mode of exercise and aqueous extract
administration to the subjects.
OF A. wilkesiana
G1(n = 6) NO NO
G2(n = 6) YES 300 mg/g/day
G3(n = 6) NO 300 mg/g/day
G4(n = 6) YES NO
G1 = Control group (No extract & No Exercise); G2 = 300 mg/g/day ex-
tracts and exercise; G3 = 300 mg/g/day extract; G4 = Exercise only.
Copyright © 2013 SciRes. AJPS
The Effects of Aqueous Extracts of Acalypha wilkesiana Supplementation and Exercise Training
on Hematopoietic System in Rats
Copyright © 2013 SciRes. AJPS
performed for 6 weeks, 5 days per week and one hour per
day as described [18].
2.6. Euthanasia
Overnight prior to euthanasia (Mercy death); the animals
were starved of food. The animals were sacrificed by
cervical dislocation as described [19]. Cardiac blood spe-
cimens were obtained from each Rat by terminal bleed-
ing from the heart. The first half of the blood collected
was transferred into total white blood cell, Hemoglobin
concentration and CD4 cell evaluation. While the second
portion was transferred into an anticoagulant free test
tube and allowed to clot and subsequently centrifuged at
750× g for 15 minutes to obtain serum.
The serum were immediately aliquoted into Eppendorf
tubes placed on ice and immediately stored at 80˚C un-
til serum immunoglobulin G (IgG) is evaluated using
Enzyme Linked Immunosorbent Assay Method.
2.7. Evaluation of Parameters
Hematological parameters (Total white Blood cells and
Hemoglobin Concentration) were determined using the
sysmex® Automated Hematology Analyzer Kx-2IN, sys-
mex corporation, Kobe-Japan. It employs WBC detector
block and WBC/HGB lyse reagent to measure WBC
count and Hemoglobin concentration as described [20].
CD4 and CD8 cell count were estimated using Partec
Cyflow Counter, Germany for the quantification of CD4
T lymphocytes as described [21].
Serum immunoglobin G (IgG) level was assayed by
conventional Sandwich Enzyme Linked Immunosorbent
Assay (ELISA) using 96 well plates coated with antibody
specific for IgG (Pharmacia CAP system IgG FEIA,
Pharmacia, Uppsala, Sweden). The assay system utilizes
two unique antibodies (a mouse monoclonal and a goat
polyclonal) directed against distinct antigenic determi-
nants on the IgG molecule. Into the plastic micro titer
well with anti-IgG (Mouse monoclonal) was added test
sample/control containing IgG to form immune com-
plexes. Anti-IgG (goat polyclonal Enzyme—labeled with
horseradish peroxidase was added to each well and incu-
bated for 45 minutes at room temperature. The IgG mo-
lecule in the sample was sandwiched between the solid
phase and enzyme-labeled antibodies. The wells were
emptied and washed five times to remove unbound-la-
beled antibody an enzyme chromogen was added to the
wells incubated for 15 minutes at room temperature in
the dark, resulting in the development of a blue colour. A
stop solution was added to each well and the intensity of
the developed yellow colour is directly proportional to
the concentration of IgG in the sample. This was read at
450 nm wavelength. Awareness Technology Inc. Palm
City FL 34991, USA.
3. Statistical Analysis
All numerical results were obtained from the four (4)
group (control and treated). Data were presented as mean
± Standard Deviation and Analyzed using one way ana-
lysis of variance (ANOVA) and Tukey-Kramer Multiple
Comparisons Test Using SPSS—18.0 (Statistical pack-
ages for social scientist—version 18.0) statistical pro-
gram. P values < 0.05 were considered significant
4. Results
Our findings revealed that CD4 cells count, CD8 cells
counts, total WBC count shows statistically significant
increase while Hb concentration and IgG level shows a
statistical significant decreased in Group 2 and Group 4
against the control group (G1). In Group 3, the total WBC
count shows a statistically significant increase against
control group (G1) while CD4 cell count, CD8 cell count,
Hb-Concentration and IgG level shows a statistically in-
significant increase against the control group (P > 0.05),
Table 2.
5. Discussion
Our study revealed that administration of 300 mg/ml of A.
wilkesiana to experimental animal shows a statistically
Table 2. Showing the mean ± standard deviation of parameters analyzed in each group.
Groups Paramete rs G1 G2 G3 G4
CD4 cells (Cells/μl) 600 ± 0.02 650 ± 0.03S 601 ± 0.02NS 650 ± 0.04S
CD8 cells (Cells/μl) 300 ± 0.01 500 ± 0.04S 301 ± 0.01NS 450 ± 0.03S
Total WBC (Cells/μl) 5000 ± 1.4 8000 ± 0.9S 5300 ± 0.6S 7500 ± 0.7S
Hb.Conc. (g/dl) 8.0 ± 0.3 7.5 ± 0.01S 8.2 ± 0.02NS 7.0 ± 0.02S
IgG (mg/dl) 1400 ± 0.2 800 ± 0.04S 1405 ± 0.8NS 650 ± 0.4S
All values are expressed as Mean ± Standard deviation of the 6 animals in each group. Keys: WBC = White Blood cell; Hb. Conc. = Hemoglobin concentration;
IgG = Immunoglobulin G; G1 = Control group (No extract & No Exercise); G2 = 300 mg/g/d extracts and exercise; G3 = 300 mg/g/d extract; G4 = Exercise
only; S = P < 0.05; NS = P > 0.05.
The Effects of Aqueous Extracts of Acalypha wilkesiana Supplementation and Exercise Training
on Hematopoietic System in Rats
insignificant alteration in CD4, CD8, Total WBC, Hb
and IgG level, Table 2. This may be due to the fact that
at a concentration of 300 mg/ml, the extract seems to
have some immuno protective effects or non toxic effects
on the indicator parameters. This is in line with these
findings. Its Beta Carotene contents acts as an antioxi-
dant, which help to boost the immune system against
cancer, cataract and damaging effects of radiation [3,7].
The expressed juices of the extract are used as an antim-
icrobial agent for the treatment of infection [5].
As seen in Table 2, the increase in TWBC after the
bout of exercise with or without the extracts may be due
to an adaptive mechanisms by the immune system to
remove the damaged tissue caused by the prolong exer-
cise in the experimental animal. This had been reported
by these authors. The immune system response to the da-
mage done by exercise peak 2 to 7 days after exercise,
which is the period during which most of the adaptation
that lead to greater fitness occurs, this effect may be to
some extent protective against diseases which are associ-
ated with oxidative stress and provides partial explana-
tion for the lower incidence of major diseases and better
health for those who undertake regular exercise [22,23].
The Leucocytosis is due to Neutrophilia and the recruit-
ment of B and T cells to the peripheral blood after acute
moderate exercise [24].
In Table 2, the fall in the CD4/CD8 ratio may be due
to the transient change in the lymphocyte subset, which
favours the proliferation of the CD8, which are involved
in the cytotoxic mediated cellular immune response. This
is in accordance with these findings. The fall in CD4/
CD8 ratio is mainly due to an increase in the number of
CD8 T cells, the change in the lymphocyte subsets is
transient, basal level usually being reached within one
and half hour after exercise [25].
Table 2 shows a decrease in the level of immuno-
globulin G (IgG) after the exercise with or without the
aqueous extract of A. wilkesiana may be due to the im-
muno suppressive effects of the increase production of
cortisol occasioned by the stress response during exer-
cising to exhaustion. This has been reported by these Au-
thors. Glucocorticoids (cortisol) are potent modulators of
the immune system with immunosuppressive effects [26].
Several stressors (exercise) have been associated with a
shift in cytokine production toward the anti-inflamma-
tory pattern with Glucocorticoids as the proposed me-
diators of this shift [26].
At diagnosis, the mean hemoglobin concentration shows
a statistically significant reduction in G2 and G4 against
the control group (G1), Table 2. This might be due to the
oxidative stress induced by the prolong exercise on the
matured red blood cells of the experimental animals. This
is in accordance with these findings. A decreased in the
Hemoglobin concentration is majorly due to Oxidative
damage to the red blood cells arising from an imbalance
between reactive oxygen species production and antioxi-
dant level. Factors such as decreased in red cell survival
and reduced erythropoietin response by the bone marrow
erythroid cell can induce anemia [27,28].
6. Conclusion
A. wilkesiana at a concentration of 300 mg/g/day seems
to be immuno-protective in Rats. But the single or com-
bine effects of prolonged exercise and A. wilkesiana pro-
duce significant change in some immuno indicators pa-
rameters. The immune system is merely responding to
the damage done by the exercise bout, during which most
of the adaptation leads to greater fitness, if balance diet
and proper resting are observed after exercise. But the
molecular mechanism behind their combined effect would
require further investigation.
[1] S. Christman, “Alalypha Wilkesiana LC,
Florida,” 2004.
Http/www.floridata.Com/ref/Asalpha wil.cfm
[2] E. F. Ciliman, Acalypha Wilkesiana Environmental Hor-
ticulture Department, Florida Cooperative Extension Ser-
vice, Institute of Food and Agricultural Sciences, Univer-
sity of Florida, Fact Sheet FPS, 1999.
[3] S. K. Basil, J. E. Thomas and S. N. Acharya, “Prospects
for Growth in Global Nutraceutical and Functional Food
Markets: A Canadian Perspective,” Austral Journal of
Basil Applied Science, Vol. 1, No. 4, 2007, pp. 637-649.
[4] AOAC (Association of Official Analytical Chemists),
“Official Method of Analysis of the AOAC,” 18th Edition,
Association of Official Analytical Chemists, Washington
DC, 2006.
[5] A. O. Ogundaini, “From Greens into Medicine: Taking a
Lead from Nature,” In: Inaugural Lecture Series 176,
OAU Press Limited, Ile-Ife, 2005, pp. 12-15.
http:/ pharmacy.aogund.pdf
[6] J. C. Ikewuchi, A. Anyadiegwu, E. V. Ugono and S. O.
Okungbowa, “Effect of Acalypha Wilkesiana on Plasma
Sodium and Potassium Concentration of Normal Rab-
bits,” Pakistan Jorunal of Nutrition, Vol. 7, No. 1, 2008,
pp. 130-132. doi:10.3923/pjn.2008.130.132
[7] B. Best, “Phytochemical as Nutraceutical,” 2006.
http// nutreut/phytochemical.html
[8] O. M. Kandil, T. H. Abdellah and A. Elkhadi, “Garlic and
the Immune System in Humans: Its Effects on Natural
Killer Cells,” Federal Procedures, Vol. 46, 1987, p. 441.
[9] M. J. Stampfer, F. B. Hu, J. E. Manson, E. B. Rimm and
W. C. Willet, “Primary prevention of Coronary Heart Dis-
ease in Women through Diet and Lifestyle,” New Eng-
land Jorunal of Medicine, Vol. 343, No. 1, 2000, pp. 16-
Copyright © 2013 SciRes. AJPS
The Effects of Aqueous Extracts of Acalypha wilkesiana Supplementation and Exercise Training
on Hematopoietic System in Rats
22. doi:10.1056/NEJM200007063430103
[10] F. B. Hu, J. Manson, M. Stampfer and C. Graham, “Diet,
Lifestyle and Risk of Type 2 Diabetes Mellitus in
Women,” New England Journal of Medicine, Vol. 345,
No. 11, 2001, pp. 790-797. doi:10.1056/NEJMoa010492
[11] A. Cornil, G. Decoster and J. Copinschi, “Effects of
Muscular Exercise on the Plasma Level of Cortisol in
Man,” European Journal of Endocrinology, Vol. 10, 1965,
pp. 60-63.
[12] O. O’Connor, M. Crowe and W. Spinks, “Effects of
Static Stretching on Leg Capacity during Cycling,” Turin,
Vol. 46, No. 1, 2005, pp. 52-56.
[13] J. Wilmore and H. Knittgen, “Aerobic Exercise and En-
durance Improving Fitness for Health Benefits,” The
Physician and Sports Medicine, Vol. 31, No. 5, 2003, p.
45. doi:10.3810/psm.2003.05.367
[14] N. De Vos, N. Singh, D. Ross and T. Stavrines, “Optimal
Load for Increasing Muscle Power during Explosive Re-
sistance Training on Older Adults,” The Journals of Ger-
ontology, Vol. 60A, No. 5, 2005, pp. 638-647.
[15] J. Kirkwood and H. Robert, “The UFAW Handbook on
the Care and Management of Laboratory and Other Re-
search Animal,” Wiley, Blackwell, 2010, p. 29.
[16] Institute for Laboratory Animal Research Council, “Guide
for the Care and Use of Laboratory Animals,” 8th Edition,
Institute for Laboratory Animal Research Council, Divi-
sion on Earth and Life Studies, American Academy of
Sciences, National Research Council of the National Aca-
demics. The National Academies Press, Washington DC,
1996, pp. 11-31.
[17] A. Sofowora, “Medicinal Plants and Traditional Medicine
in Africa,” John Wiley, Chichester, 1982, p. 179.
[18] M. Hashemi, M. Bayat, A. R. Azizi-Saraji and M. En-
terzari, “The Effects of Swimming Exercise on Experi-
mental Diabetic Myopathy in Rats,” World Journal of
Zoology, Vol. 4, No. 3, 2009, pp. 216-222.
[19] J. Ochei and A. Kolhatkar, “Methods of Euthanasia,” In: J.
Ochei and A. Kolhatkar, Eds., Medical Laboratory Sci-
ence, Theo ry and Practice, McGraw-Hill Publishing Com-
pany Limited, New Delhi, 2006, p. 1221.
[20] O. I. Samuel, N. Thomas, O. U. Ernest, N. N. Imelda, N.
S. Elvis and E. Ifeyinwa, “Comparison of Hematological
Parameters Determined by the Sysmex KX-2IN Auto-
mated Hematology Analyzer and the Manual Count,”
BMC Clinical Pathology, Vol. 10, 2006, pp. 3-5.
[21] Partec Cyflow Counter (PCC), “Typical Steps of Particle
Analysis Using Partec Cyflow Counter,” Instrument Ope-
rating Manual, Partec GmbH OHO-Hann-str 32, Munster,
2010, pp. 5-8.
[22] P. Tiidus, “Radical Species in Inflammation and Over-
training,” Canadian Journal of Pharmacology, Vol. 76,
No. 6, 1998, pp. 533-538. doi:10.1139/y98-047
[23] C. Leeuwenburgh and J. Heinecke, “Oxidative Stress and
Antioxidants in Exercises,” Curriculum of Medical Che-
mistry, Vol. 8, No. 7, 2007, pp. 829-838.
[24] D. C. Nieman, S. L. Nehlsen-Cannarella and K. M. Do-
nohue, “The Effect of Acute Moderate Exercise on Leu-
kocyte and lymphocyte Subpopulation,” Medical Science
Sport Exercise, Vol. 22, 1991, pp. 578-585.
[25] B. K. Pedersen, N. Tvede and F. R. Hansin, “Modulation
of Natural Killer Cell Activitity in Peripheral Blood by
Physical Exercise,” Scandinavian Journal of Immunology,
Vol. 72, 1998, pp. 673-678.
[26] E. Elinav, G. Pinsker, R. Safadi, O. Pappo, M. Bromberg
and E. Anis, “Association between Consumption of Her-
balife-Nutritional Supplements and Acute Hepatotoxi-
city,” Journal of Hepatology, Vol. 47, No. 4, 2007, pp.
514-520. doi:10.1016/j.jhep.2007.06.016
[27] F. A. Ehiaghe, I. J. Ehiaghe, S. T. Aladenika, S. M. O.
Etikerentse, A. I. Ikusemoro, B. H. Oladeinde, E. O.
Osakue, S. S. Enitan and J. K. Fadairo, “The Characteris-
tics of Pulmonary Tuberculosis amongst Patients Attend-
ing Chest Clinic, Including Age, Sex, Occupation and
Hemoglobin Concentration in Benin City, Nigeria,” Open
Journal of Clinical Diagnostics, Vol. 3, 2013, pp. 14-18.
[28] O. Ebrahim, P. I. Folb and J. P. Robson, “Blunted Ery-
thropoietin Response to Anemia in Tuberculosis,” Euro-
pean Journal of Hematology, Vol. 55, No. 4, 1995, pp.
251-254. doi:10.1111/j.1600-0609.1995.tb00267.x
Copyright © 2013 SciRes. AJPS