The Effects of Aqueous Extracts of <i>Acalypha wilkesiana</i> Supplementation and Exercise Training on Hematopoietic System in Rats

doi:10.4236/ajps.2013.49225

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American Journal of Plant Sciences, 2013, 4, 1834-1838

http://dx.doi.org/10.4236/ajps.2013.49225 Published Online September 2013 (http://www.scirp.org/journal/ajps)

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.

Email: [email protected]

Received June 14th, 2013; revised July 14th, 2013; accepted August 15th, 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

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,

The Effects of Aqueous Extracts of Acalypha wilkesiana Supplementation and Exercise Training

on Hematopoietic System in Rats

1835

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-

14].

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]

ENBT

where:

N = The minimum number of unit in the study (Minus

one);

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.

GROUPS EXERCISE AQUEOUS EXTRACT

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.

The Effects of Aqueous Extracts of Acalypha wilkesiana Supplementation and Exercise Training

on Hematopoietic System in Rats

1836

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

1837

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.

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