Okoye and Effiong, 2016

J. basic appl. Res 2(2): 102-108

Hematopoietic and enzyme modulatory effects of aqueous stem bark extract of

Annona Muricata

Okoye J.O.,1* Effiong G.S.2

1*
Histopathology Specialty, Medical Laboratory Science Department, School of Public and Allied Health, Babcock University, Ilishan-
Remo, Ogun State, Nigeria.
2
Medical Laboratory Science Department, Faculty of Health Sciences, Madonna University, Elele Campus, Rivers State, Nigeria.

*corresponding author: okoyej@babcock.edu.ng

Received: 30-3-2016 Abstract: This study evaluated the effects of aqueous stem bark extract of
Revised: 10-4-2016 Annona muricata on the liver of albino rats. A total of 24 male albino rats,
Published: 15-4-2016 randomly divided into 4 groups: 1(control), 2, 3 and 4, were orally administered
0, 25, 50 and 100 mg/kg body weights of the extract, respectively for 14 days.
Keywords:
Annona muricata; Blood samples were analyzed using the Reitman and Frankel,
Enzymes; Cyanmethhaemoglobin methods. Comparing the control and test groups, results
haemoglobin; showed significantly increased Hb (16.40 ± 2.48 to 22.74 ± 3.83), PCV (49.20
Liver; ± 7.40 to 68.20 ± 11.52), organ weight (7.65 ± 1.14 to 8.17 ± 0.93) and relative
PCV,
Phytochemicals weight (5.12 to 5.64). Higher levels of ALP (492.50 ± 169.27 to 568.00 ±
265.67) were observed in group 3 and 4 while lower level was observed in
group 2 (397.16 ± 188.80). Lower levels of ALT (22.50 ± 11.18 to 17.67 ±
3.56) and LDH (145.00 ± 10.41 to 88.83 ± 57.81) were also observed when the
control group was compared with the test groups (p>0.05). Liver microscopy
revealed mild to moderate necrosis in group 3 and 4. The study suggests that
Annona muricata was non-toxic at 25 mg/kg and may possess hematopoietic
and hepatoprotective effects.

1.0 INTRODUCTION extract at 1 mg/ml had in vitro activity against

Annona muricata is a species of the genus Annona herpes simplex 1, while the root had activity
of the custard apple tree family, Annonaceae, against herpes simplex type 2 (Feng, 1962). Many
known mostly for its edible fruit. The fruit is of the acetogenins have demonstrated selective
usually called soursop due to its slightly acidic or toxicity to tumor cells (at very low dosages): lung
sour taste when ripe. A. muricata is native to the carcinoma cell lines; human breast solid tumor
Caribbean and Central America but is now widely lines; prostate adenocarcinoma; pancreatic
cultivated and in some areas, becoming invasive in carcinoma cell lines; colon adenocarcinoma cell
tropical climates throughout the world it is lines; liver cancer cell lines; human lymphoma cell
commonly known as soursop (Morton and Julia, lines; and multi-drug resistant human breast
2007). The soursop also called graviola from the adenocarcinoma (Lannuzel et al., 2003).
Annona muricata tree originated in the lowlands of Acetogenins act by inhibiting NADH oxidase in the
Central America. It was first described by Spanish plasma membranes of cancer cells. This enzyme is
historian Gonzalo Fernández de Oviedo y Valdés in only transiently expressed in ‘normal healthy’ cells.
1526 and was spread around the world by the By inhibiting this enzyme cellular ATP is depleted
Spanish explorers (Lannuzel et al., 2003). The raw which in turn causes inhibition of oxidative
nutritional value of soursop per 100g includes, Vit phosphorylation and cancer cell growth. The
B1 (thiamine), B2 (riboflavin), B3, and C, acetogeninannonacin is able to induce apoptotic
Pathothenic acid, Niacin, Folate, Choline, protein cell death. It enhanced the expression of Bax and
and trace metals such as calcium, Iron, Magnesium, Bad (Feng, 1962).
Phosphorus, Potassium, sodium and zinc (Champy The seeds and bark are considered toxic and
et al., 2005). contain a number of compounds and potentially
The bark, leaves, and roots are considered sedative, poisonous alkaloids such as anonaine, muricine,
antispasmodic, hypotensive anthelmintic, and hydrocyanic acid. This can cause inflammation
antiparasitic, antipyretic, sedative, antispasmodic, problems and neurotoxic effects (Tattersfield and
nervine, hypotensive, anticonvulsant, Padma, 1940; Lannuzel et al., 2003]. Researchers
cardiodepressant and digestive (Antoun, 1993; have suggested that these alkaloids might be linked
Feras, 1999; Lannuzel et al. 2003). The The bark to atypical Parkinson’s disease in countries where
used for Asthenia, asthma, childbirth, cough, heart the seeds are employed as a common herbal
tonic, hypertension, nervine, parasites, sedative etc parasite remedy (Hasrat et al., 1997). The aim of
(Feras, 1999). The stem and bark in an ethanol this study was to determine the dosage at which

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aqueous stem bark extract of Annona muricata is test, Glycosides by Fehling’s solution test, Protein
detrimental to the body system in relation to some by Millon’s test, Steriods by the chloroform-
haematological and biochemical parameters, and sulphuric acid test and alkaloids by Mayer-
microanatomical architecture of the liver in albino wagner’s test.
rats.
Animals handling
2.0 MATERIALS AND METHODS The experimental animals used for this study were
Study area obtained from the animal house of Madonna
The study was carried out at the animal farm in the University, Elele, Nigeria housed in wire meshed
department of Medical Laboratory Science, Faculty cage under standard conditions (temperature 25 -
of Health Sciences, Madonna University Elele, 29°C, 12 hours light and 12 hours darkness cycles).
Rivers state, Nigeria. The study area is located in
the tropics (with the mean daily temperature of 29 Experimental design
ºC) at the southern part of Nigeria; latitude 5 27-5 This experimental controlled study was carried out
31N and longitude 6 55-7 85E (Gobo, 1988). It is in Madonna University, Elele Campus, River State;
bordered by four neighboring communities namely A total of 24 Male albino rats weighing between
Isiokpo, Umuagwo, Ahoda, and Omoku. 110-202g were used as animal model, housed in
four meshed cages containing 6 rats in each. The
Ethical approval animals were allowed to acclimatize for two weeks
The research received ethical approval from the before the experiment began. They were allowed to
ethics committee of the department of Medical feed on standard rat chow and water freely
Laboratory Science, Faculty of Health Sciences, throughout the period the experiment.
Madonna University, Nigeria (FHS/MLS/10/041). Administration of the aqueous extract of Annona
The experiment was conducted in accordance with muricata stem bark was given orally; Group 1
the Guidelines of the U.S. National Institute of received 2ml of distilled water, Group 2: received
Health (NIH, 1985) on the care and use of 25 mg/kg body weight of Annona muricata extract,
laboratory animals and also in accordance with the Group 3: received 50 mg/kg body weight of
principles of Good Laboratory Procedure (WHO, Annona muricata extract while Group 4: received
1998). 100 mg/kg body weight of Annona muricata. The
experiment lasted for 14 days.
Test sample
Fresh large quantity of stem bark of Annona 2.5 Sample collection and biochemical analyses
muricata tree was gotten from Amucha village in At the end of the experiment (14 days), the animals
Orlu local government of Imo state Nigeria. The were subjected to an overnight fast, weighed using
botanic identification and authentification was a weighing balance (Doran-PC 500), anesthetized
carried out in faculty of pharmacy Madonna by the “Drop method” (JHU, 2009) and blood
University, Elele campus by a taxonomist and a samples taken from their jugular vein. Whole blood
sample keep at the herbarium samples were draw into heparinized capillary tubes
(MU/PHGSY/05/001). and spurn immediately in a microhematocrit
centrifuge (Hawksley hematospin 1400) for 5 mins
Preparation of extract at 1500g as described by Bain et al. (2008) to
Fresh stem bark of Annonaceae muricata was cut estimate pack cell volume (PCV). Haemoglobin
from the tree, dried for one week until there were estimation was carried using the
no evidence of obvious moisture in them, the dried Cyanmethhaemoglobin method as described by
stem bark was grinded into powered form. Six Bain et al. (2008). The remaining blood samples
hundred (600) ml of distilled water was added to were dispensed into plain containers labeled
100 grams of the ground stem bark and allowed to appropriately and allowed to coagulate. The
stand in a refrigerator for 72hrs. The supernatant samples were centrifuged in a Power Spin
was decanted into a beaker, filtered using filter Centrifuge (C858E) for 10 minutes at 1500g within
papers and the water evaporated to dryness in an two hours after collection. The serum obtained was
oven. After drying, the powder form of the extract stored frozen until analyses. Immediately after the
was raped in aluminum foil and preserved in a blood collection the rats were sacrificed and
refrigerator (Francis, 2000; Cheng et al., 2014). dissected. The liver was excised, blotted dry,
weighed on a microbalance sensitive at 0.001mg
Phytochemical analysis (Precisa 125A, Switzerland) and recorded. Liver
The test carried out was based on procedures samples were fixed in 10% formal saline until
outlined by Harbourne (1973) and Evans (2002). histopathological analysis. Biochemical analyses
Estimation of carbohydrates was carried out by the were carried out to determine the serum
Molisch’s test, Saponins by Frothing test, Tannins concentrations and activity of some enzymes such
by Ferric chloride test, Flavanoids by Ammonium as AST and ALT (using the Reitman and Frankel

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method), ALP, LDH and Amylase using the kinetic carried out on the data using the Statistical Package
method as described by Sood (2009). Their for Social Sciences (SPSS; version 18). Values
absorbencies were read using a spectrophotometer were reported as Mean ± SD. P-value is significant
(APEI PD-303S). at p<0.01 and p<0.05.

2.7 Automatic tissue processing 3.0 RESULTS

The tissues were processed in an automatic tissue
processor (Jung Histokinette 2000) as follows: Table 1: Phytochemical analysis of Annona muricata
Phytochemical Quantification
Beaker 1 (containing 10% formal saline) for 3 components
hours, Beaker 2 (containing 70% alcohol) for Carbohydrate ++
1hour, Beaker 3 (containing 80% alcohol) for 2 Proteins +
hours, Beaker 4 (containing 90% alcohol) for 2 Glycosides ++
hours, Beaker 5 (containing 95% alcohol) for 2 Tannins +
Saponins +
hours, Beaker 6 (containing 95% alcohol) for 2 Flavonoids ++
hours, Beaker 7 (containing absolute alcohol I) for Steroids +
2 hours, Beaker 8 (containing absolute alcohol II) Alkaloids ++
for 2 hours, Beaker 9 (containing xylene I) for 3
hours, Beaker 10 (containing xylene II) for 3 hours, In table 2 below, the test groups showed
Wax bath I for 3 hours, Wax bath II for 3 hours. insignificant overall increase in the level of Hb and
After to the last processing stage, tissues were PCV when compared with the control group
covered in molten paraffin wax in metallic (p>0.05). However, significant increases were
embedding moulds, allowed to cool and solidify in observed in the level of Hb and PCV when groups
a refrigerator for 15 minutes at 5ºC. The cooled and 2 and 4 were compared with the control group
solidified tissue blocks were trimmed to remove (p<0.05). Insignificant decrease was observed in
excess wax and serially sectioned at 5 μm on a group 2 while insignificant increase were observed
rotary microtome (KEDEE KD- 3668AM). The in groups 3 and 4 when the level of ALP was
sections were floated in 20% alcohol and compared with that of the control group (p>0.05).
transferred to a water bath (HH-420) at 45ºC and Generalized insignificant decreases were observed
picked up at an angle using clean frosted end slides. in the level of ALT when the test groups were
Adequate attachment of tissues to slides was compared with the control group (p>0.05). It also
ensured by placing the tissue and slide on a hot shows significant differences in the level of serum
plate for a duration of 40 minutes. AST within and between the groups (p<0.05).
However, no significant increase was observed
2.7 Haematoxylin and Eosin staining when group 2 was compared with the control group
The tissue sections were dewaxed in 2 changes of nor any significant decrease observed when groups
xylene for 2 minutes each, hydrated through 3 and 4 were compared with group 1 (p>0.05).
decreasing grades of alcohol (that is, 100%, 90%, Overall insignificant decrease was observed in the
80% and 70%) for 2 minutes each, stained in activity of LDH and Amylase when the test groups
Ehrlichs haematoxylin for 30 minutes and rinsed in were compared with the control group (p>0.05).
running tap-water to remove excess stain. The Comparing the weight of the liver, insignificant
sections were differentiated in 1% acid alcohol for increases were observed when the test groups were
a second, blued in running tap water 10 minutes, compared with the control group (p>0.05).
counterstained with 1% eosin for 2 minutes, rinsed
in water, dehydrated in ascending grades of alcohol Table 3 above showed no significant change in
(70%, 80, 95% and absolute), dealcoholized in body weight when the weight of the animals (in
xylene, dried at room temperature and mounted each group) before the commencement of the
using dibuthylphthalate propylene xylene. The experiment was compared with the weight of the
slides were examined under a light microscope and animals after the experiment (p>0.05). The table
photomicrographs were taken using a camera also shows that group two had higher change in
attached to the microscope. body weight when compared with other groups.
Dose dependent increase in the organ: body weight
2.8 Statistical Analysis ratio (relative weight; organ weight divided by
The biochemical data were subjected to some body weight after experiment multiple by 100) was
statistical analysis: analysis of variance (ANOVA), observed in the tests groups when compared with
Partial correlation analysis and Post Hoc test was the control group (p>0.05).

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Figure 1 Figure 2

Figure 3 Figure 4

Figure 1 (Group 1; {control} administered 2 ml of distilled water): photomicrograph of a liver section showing normal tissue microarchitecture. Its shows distinct liver sinusoids (marked
by green arrows), hepatocytes (marked by blue arrow heads), and central vein (marked by black arrow). H&E stained. X400
Figure 2 (group 2; administered 25 mg/kg body weight of Annona muricata): photomicrograph of a liver section showing no obvious pathological change. H&E stained. X400
Figure 3 (group 3; 50 mg/kg body weight of Annona muricata): photomicrograph of a liver section moderate generalized cytoplasmic necrosis. H&E stained. X400
Figure 4 (group 4: administered 100 mg/kg body weight of Annona muricata): photomicrograph of a liver section showing mild generalized cytoplasmic necrosis and stromal erosion
(hepatitis). H&E stained. X400

Table 2: mean comparison of the effect of aqueous stem bark extract of Annona muricata on haematological, biochemical and organ
weight based on group treatments.
*Mean
Groups Hb PCV ALP ALT AST LDH Amylase Liver wt
Group 1 16.40 ± 2.48 49.20 ± 7.40 492.50 ± 169.27 22.50 ± 11.18 72.00 ± 13.88 145.00 ± 10.41 220.33 ± 27.95 7.65 ± 1.14
Group 2 22.74 ± 3.83 68.20 ± 11.52 397.16 ± 188.80 18.50 ± 5.32 85.33 ± 5.81 132.00 ± 19.42 192.50 ± 37.56 8.17 ± 0.93
0.008* 0.008* 0.417 0.369 0.061 0.539 0.188 0.358
Group 3 19.72 ± 3.34 59.20 ± 10.03 501.00 ± 153.90 17.67 ± 3.56 66.83 ± 12.95 140.83 ± 36.91 179.00 ± 30.65 7.97 ± 0.99
0.137 0.135 0.942 0.280 0.452 0.843 0.057 0.585
Group 4 20.75 ± 3.56 62.17 ± 10.68 568.00 ± 265.67 19.17 ± 7.81 58.00 ± 12.19 88.83 ± 57.81 180.50 ± 43.29 7.72 ± 0.63
0.047* 0.048* 0.519 0.452 0.051 0.014* 0.067 0.904
G2 vs G3 0.173 0.176 0.377 0.850 0.012* 0.676 0.516 0.736
G2 vs G4 0.342 0.336 0.153 0.880 0.001* 0.051 0.563 0.422
G3 vs G4 0.619 0.633 0.567 0.734 0.204 0.021* 0.942 0.665
F-value 3.131 3.124 0.748 0.474 5.772 3.078 1.757 0.373
P-value 0.053 0.053 0.536 0.704 0.005* 0.051 0.188 0.773

difference is significant at P<0.05. Analysis of variance and Post Hoc test, N=24, n=6
Keys: Hb= Haemoglobin, PCV= Packed Cell Volume, ALP= Alkaline Phosphatase, AST= Aspartate transaminase, ALT= Alanine transaminase, Lactate Dehydrogenase

Table 3: T-test analysis comparing animal weight before and after the experiment
Groups Wt. Before Wt. After Mean diff. in weight Organ/ Body wt P-value T-value
1 139.00±19.62 149.33±31.46 10.33 5.12 0.421 0.877
2 132.33±18.65 150.33±27.22 18 5.43 0.207 1.447
3 131.60±26.16 144.40±29.27 12.8 5.52 0.464 0.809
4 133.66±26.39 136.83±25.74 3.17 5.64 0.871 0.170
*Mean difference is significant at P<0.05. Student t-test, N=24, n=6
Key: WT= weight, diff= difference

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Table 4: Correlation analysis between the haematological phytochemicals components (table 1). The
and biochemical parameters
phytochemical components of the stem bark extract
Parameters r-value p-value Type of correlation
Hb vs PCV 1.000 0.000 Significant positive may have led to the lower serum enzyme levels by
ALP vs LDH -0.467 0.038 Significant negative reducing the overall cellular damage associated
ALP vs Amylase 0.448 0.047 Significant positive with some metabolic processes in the liver and
LDH vs Amylase -0.672 0.001 Significant negative pancreas.
*Mean difference is significant at P<0.05 and P<0.01. Partial
correlation analysis, N=24, n=6
The result of the study revealed increase in body
and organ weight (tables 2 and 3) at 25 mg/kg body
weight of the aqueous stem bark extract Annona
4.0 DISCUSSION
muricata. It could be argued that the concomitant
Annona muricata have been reported to possess
increase is non-pathological. This is supported by
many therapeutic properties ranging from antiulcer
the fact that there were higher values of the
to anticancer potency. Its use in the field of
haematological parameters, lower serum levels of
alternative medicine is with some controversies.
ALP and ALT, and lower LDH activity when
Some studies have assessed its potency at 10 mg/kg
compared with the control (table 3). The higher
on tumours with some level of success (Sundarrao
level of Hb and PCV observed in the test groups
et al., 1993), while some studies reported some
may be attributed to the presence of the blood
cytotoxicity following its administration of the seed
forming nutritional value of Annona muricata
and fruit juice. However, little or no work has been
previously reported by Champy et al. (2005). This
done to ascertain the effect of the stem bark extract
is supported by the strong positive correlation
on the entire body system. This study was carried
observed between Hb and PCV (table 4). The
out to determine the effects of aqueous stem bark
observed higher insignificant level of AST could be
extract of Annona muricata in relation to some
physiological. Furthermore, the absence of tissue
hematological and biochemical parameters, body
damage in figure 2 suggests that the plant extract
and organ weight and liver architecture in albino
could be beneficial to the body at 25 mg/kg body
rats in a bid to finding its safe doses.
weight. However, the decrease in body weight and
increase in organ: body weight ration in group 4
The result of this study shows that aqueous stem
when compared with other groups (table 3)
bark extract of Annona muricata contain more
suggests that aqueous stem back extract of Annona
carbohydrate, alkaloids, flavonoids and glycosides
muricata could be toxic at 100 mg/kg body weight.
than the leaf extract as earlier reported by
This observation is in line with the findings of
Vijayameena et al.(2013). The flavonoids observed
Agbai and Nwanegwo (2013) who reported similar
in table 1 have been shown to have antioxidant
decrease in body weight following the
activity, free radical scavenging capacity, coronary
administration of methanolic leaf extract of Annona
heart disease prevention, hepatoprotective, anti-
muricata.
inflammatory and anticancer activities, while some
flavonoids exhibit potential antiviral activities
The insignificant increase in the body and organ
(Kumar et al., 2013). They are also known to
weights at 50 mg/kg body weight of aqueous stem
inhibit specific enzymes, stimulate some hormones
bark extract Annona muricata, could be said to be
and neurotransmitters (Haysteen, 2002). Saponins
of some benefit but the moderate distortion of
are a group of naturally occurring plant glycosides
tissue integrity observed in figure 3 suggests
which possess significant anticancer properties by
otherwise. However, it could still be argued that at
cell cycle arrest and apoptosis (Man et al., 2010).
this dosage, the plant extract was without serious
Tannins possess anticancinogenic and
damaging effect since higher values of the
antimutagenic potentials which is pertinent in
haematological parameters and lower serum levels
protecting cellular oxidative damage including lipid
of AST, ALT and LDH activity were observed
peroxidation. They are also known to modulate
when compared with the control (table 2). The
immune responses and produce liver necrosis
higher serum level of ALP further undermines the
(Chung et al., 1998). The phytochemical analysis
potency and health benefit of the plant extract at 50
also confirms that the stem bark contains alkaloids
and 100 mg/kg body weight. The reason for the
(table 1) as reported by Tattersfield and Padma
latter increase is still unclear, yet in consonance
(1940) and Lannuzel et al. (2003). These alkaloids
with the reports of Agbai and Nwanegwo (2013).
have are said to possess antitumour, diuretic, anti-
The inverse (negative correlation) relationship
inflammatory, hyponoanalgesic and antidepressant
between ALP and LDH (table 4) suggests that the
properties (Aberoumond et al., 2012) and at the
higher ALP may be from extrahepatic architectural
same time they posses some cytotoxic potency. The
changes (Kim and Wyckoff, 2001). In addition, the
lower serum levels and activity of some of the
positive correlation observed between ALP and
enzymes evaluated in this study may be linked to
Amylase, and the negative correlation between
the inhibitory effect by the quantified

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LDH and amylase (table 4) following Practical Haematology:10th edition. Churchill

administration of the extract is suggestive of Living Stone. Philadephia.
pancreatic effect. Champy, P., Melot, A., Guérineau, E.V., Gleye, C.,
Fall, D. et al. (2005). Quantification of
The result of the study also showed higher values acetogenins in Annona muricata linked to
of the haematological parameters and lower levels atypical parkinsonism in guadeloupe.
of other biochemical parameters when group 4 was Movement Disorders, 20 (12), 1629–33. PMID:
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insignificant, when compared with the rest of the Alternative staining using extracts of hibiscus
groups (table 3). The observable change in liver (Hibiscus rosa-sinensis L.) and red beet (Beta
architecture (figure 4) and higher liver weight vulgaris L.) in diagnosing ova of intestinal
(table 2), and higher organ: body weight ratio nematodes (Trichuris trichiura and Ascaris
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