African Journal of Biomedical Research, Vol.

9 (2006); 173 - 187

ISSN 1119 – 5096 © Ibadan Biomedical Communications Group

Full Length Research Article

Morphological Changes and Hypoglycemic Effects
of Annona Muricata Linn. (Annonaceae) Leaf
Aqueous Extract on Pancreatic Β-Cells of
Streptozotocin-Treated Diabetic Rats
Stephen O. Adewole1* and Ezekiel A. Caxton-Martins2
1* 2
Department of Anatomy and Cell Biology,
Full-text available at
http://www.ajbrui.com Faculty of Basic Medical Sciences, College of Health Sciences,
http://www.bioline.br/md Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria
http://www.ajol.com
ABSTRACT
The present study was undertaken to investigate the leaf aqueous extract effects of
Annona muricata Linn. on the morphology of pancreatic β-cells and oxidative stress
induced by streptozotocin (STZ)-diabetic rats. Diabetes mellitus was induced in the
diabetic animal groups B and C by intraperitoneal injections of STZ (75 mg/kg body
weight), while the control group received equal volume of citrate buffer (pH 6.3)
solution intraperitoneally. The rats in group C were given A. muricata leaf aqueous
extract (AME, 100 mg/kg, p.o.) as from day 5 post STZ injections, and stopped on
the 30th day of the study period. The pancreases of the rats were excised and
randomly processed for histological staining and biochemical assays for antioxidant
enzymes [such as glutathione (GSH), superoxide dismutase (SOD), glutathione
peroxidase (GSH-Px), catalase (CAT), malondialdehyde (MDA) and serum nitric
oxide (NO)]. In diabetic state, pancreatic β-cells of STZ-treated group B rats
Received:
histologically demonstrated marked alterations in the micro-anatomy and cellular
June 2006
integrities. The morphology of A. muricata-treated rats’ pancreases showed viable
cellularity with distinct β-cell mass. STZ treatment significantly decreased GSH-Px,
Accepted (Revised):
SOD, GSH, CAT and pancreatic/serum insulin levels (p<0.05). However, STZ
August 2006
treatment increased blood glucose concentrations, MDA, and NO. A. muricata-
Published treated rats showed a significant decrease (p<0.05) in elevated blood glucose, MDA
September, 2006 and NO. Furthermore, A. muricata treatment significantly increased (p<0.05)
antioxidant enzymes’ activities, as well as pancreatic/serum insulin contents. In
conclusion, the findings of the present study indicate that A. muricata treatment has
beneficial effects on pancreatic tissues subjected to STZ-induced oxidative stress by
directly quenching lipid peroxides and indirectly enhancing production of
endogenous antioxidants. Annona muricata protected and preserved pancreatic β-
cell integrity. (Afr. J. Biomed. Res. 9: 173 - 180

Keywords: Annona muricata Leaf Aqueous Extract; morphological Changes; Oxidative

Stress; Antioxidants.

*Correspondence (Present Address): Department of Pharmacology, Faculty of Health

Sciences, University of KwaZulu-Natal, Private Bag X54001, Durban 4000, South Africa.
Tel. : +27 – 31 – 260 – 7767; Fax : +27 – 31 – 260 – 7907
(e-mail): [email protected]

Abstracted by:
African Index Medicus (WHO), CAB Abstracts, Index Copernicus, Global Health Abstracts, Asian Science Index, Index
Veterinarius, Bioline International , African Journals online
 African Journal of Biomedical Research 2006 (Vol. 9) / Adedapo, Arinola, Ige, Adedapo and Salimonu

INTRODUCTION leathery-appearing but tender, inedible bitter skin

from which protrude few or many stubby, or more
Herbal remedies from medicinal plants have been elongated and curved soft, pliable “spines”. The tips
used traditionally in many parts of the world where break off easily when the fruit is fully ripe. The skin
access to formal healthcare is limited. There are is dark-green in the immature fruit, becoming
several reasons why the use of medicinal plants slightly yellowish-green before the mature fruit is
should be studied: herbal remedies may have soft to the touch. Its inner surface is cream-colored
recognizable therapeutic effects (Bailey and Day, and granular and separates easily from the mass of
1989); they may also have toxic side-effect (Keen et snow-white, fibrous, juicy segments – much like
al; 1994). Although, the use of medicinal plants flakes of raw fish – surrounding the central, soft-
provides an indication of beliefs about illness and its pithy core. In aroma, the pulp is somewhat
treatment that may conflict with beliefs of workers pineapple-like, but its musky, subacid to acid flavor
in the formal healthcare system (Morgan and is unique (Schultes and Raffauf, 1990). Most of the
Watkins, 1988). Over the last three decades chronic closely-packed segments are seedless. In each fertile
disorders such as diabetes and hypertension have segment, there is a single oval, smooth, hard, black
emerged as the major causes of adult morbidity and seed, 1.25-2 cm long; and a large fruit may contain
mortality all over the world (Gulliford, 1994). from a few dozen to 200 or more seeds (Morton,
Successful control of health problem stemming from 1980). It is indigenous to most of the warmest
diabetes and hypertension requires active tropical areas in South and North America including
participation of patients in their own care, but as Amazon, A. muricata has become naturalized in
Morgan (1995) has pointed out, health care many countries, and now has a wide distribution
providers’ limited understanding of patients’ throughout tropical and subtropical parts of the
concepts of illness and ideas about treatment are world, including western part of Nigeria.
obstacles to establishing effective patterns of self- All parts of the A. muricata tree are used in
care. natural medicine in the tropics including the bark,
Several studies have described the medicinal leaves, root and fruit-seeds. Generally the fruit and
purposes of Annona muricata and have outlined the fruit juice is taken for worms and parasites, to cool
social history of the plants’ use (Ayensu, 1981). A. fevers, to increase mother’s milk after childbirth
muricata (Linn.) (family, Annonaceae) commonly (lactagogue), and as an astringent for diarrhea and
called “Soursop” is a small, upright evergreen tree dysentery. The crushed seeds are used as a
growing 5 to 6 meters in height. Young branchlets vermifuge and anthelmintic against internal and
are rusty-hairy, the malodorous leaves, normally external parasites and worms. The bark, leaves and
evergreen, are alternate, smooth, glossy, dark green roots are considered sedative, antispasmodic,
on the upper surface, lighter beneath; oblong, elliptic hypoglycemic, hypotensive, smooth muscle relaxant
or narrow-obovate, pointed at both ends, 6 - 20 cm and nervine and a tea is made for various disorders
long and 2 – 6 cm wide. The flowers are borne for those purposes (Holdsworth, 1990). Many
singly, may emerge anywhere on the trunk, branches bioactive compounds and phytochemicals have been
or twigs. They are short stalked, 4 - 5 cm long, found in A. muricata as scientists have been
plump, and triangular-conical; the 3 fleshy, slightly studying its properties since the 1940’s. Its many
spreading, outer petals yellow-green, with 3 close- uses in natural medicine have been validated by this
set inner petals pale-yellow (de Feo, 1992; Vasquez, scientific research (Weniger et al; 1986). The
1990). The fruit is more or less oval or heart-shaped, earliest studies were between 1941 and 1962.
sometimes irregular, lopsided or curved, due to Several studies by different researchers
improper carper development or insect injury. The demonstrated that the leaf, bark, root, stem and seed
size ranges from 10-30 cm long and up to 15 cm in extracts are antibacterial in vitro against numerous
width and the weight may be up to 4.5-6.8 kg. The pathogens (Misas, 1979; Sundarrao, 1993; Heinrich
fruit is compound in and covered with reticulated, 1992) and that the bark has antifungal properties

174 Protective effects of A. muricata on the morphology of pancreatic β-cells

 African Journal of Biomedical Research 2006 (Vol. 9) / Adedapo, Arinola, Ige, Adedapo and Salimonu

(Lopez Abraham, 1979). Much of the recent oxidase and xanthine oxidase (Guzik et al; 2002;
research on A. muricata has been on a novel set of Etoh et al; 2003). (iii) mitochondria respiratory
phytochemicals (Annonaceous acetogenins) that are chain, during oxidative phosphorylation process,
found in the leaves, seeds and stem which are electrons are transferred from electron carriers
cytotoxic against various cancer cells (Chang, 2001; NADH and FADH2 to oxygen, thus generating ATP
Chang, 2003; Liaw, 2002). in the process (Green et al; 2004).
Streptozotocin (STZ), an antibiotic produced by Annona muricata has a long history of use in
Streptomyces achromogenes, possesses pancreatic β- herbal medicine in the tropical areas in South and
cell cytotoxic effect (Weiss, 1982). Streptozotocin North America including the Amazon and West
has been widely used for inducing diabetes mellitus Africa. All parts of the tree are claimed to be used in
in a variety of animals. STZ causes degeneration and natural medicine, in view of this, the present study
necrosis of pancreatic β-cells (Uchigata et al; 1982; was designed to evaluate the hypoglycemic,
Merzouk et al; 2000). Although the mechanism of morphological changes of pancreatic β-cells and
the β-cell cytotoxic action of STZ is not fully antioxidant effects of A. muricata leaf aqueous
understood, experimental evidence has demonstrated extract in experimental models of diabetes mellitus
that some of its deleterious effects are attributable to in rats.
induction of metabolic processes, which lead on to
an increase in the generation of reactive oxygen MATERIALS AND METHODS
species (ROS) (Chen et al; 1990). Apart from
production of ROS, STZ also inhibits free radical Animal care and monitoring: Healthy, male and
scavenger-enzymes (Kröncke et al; 1995). The female, Wistar rats (Rattus norvegicus) weighing
superoxide radical has been implicated in lipid 250–300 g (averaging 12 weeks old) and normal
peroxidation, DNA damage, and sulfhydryl Mice (Mus domesticus) were used in this study.
oxidation (Tiedge et al; 1997; Matkovics et al; They were housed under standard laboratory
1998). conditions of light, temperature (21±2oC) and
Oxidative stress is the excess formation and/or relative humidity (55±5%). The animals were given
insufficient removal of highly reactive molecules standard rat pellets and tap water ad libitum. The
such as reactive oxygen species (ROS) and reactive rats were randomly divided into three experimental
nitrogen species (RNS) (Turko et al; 2001; Maritim groups: A (control), B (STZ-treated), C (STZ + A.
et al; 2003). ROS include free radicals such as muricata-treated). The control group animals (A)
superoxide (O2-), hydroxyl (OH-), peroxyl RO2), consisted of ten rats, while groups B and C consisted
hydroperoxyl (HRO2-) as well as nonradical species of forty rats. The mice were used for acute toxicity
such as hydrogen peroxide (H2O2), and testing of the crude plant extract, while the rats were
hydrochlorous acid (HOCl) (Evans et al; 2003). used for hypoglycemic and morphological
RNS include free radicals like nitric oxide (NO) and evaluations of the plant’s extract. Maintenance and
nitrogen dioxide (NO2-), as well as nonradicals such treatment of animals were in accordance with the
as peroxynitrite (ONOO-), nitrous oxide (HNO2) and principles of the “Guide for care and use of
alkyl peroxynitrates (RONOO) (Liu et al; 2002). Of laboratory animals in research and teaching”
these reactive molecules, O2-, NO and ONOO- are prepared by the National Institutes of Health (NIH)
the most widely studied species and play important publication 86-23 revised in 1985.
roles in the diabetic cardiovascular complications.
Oxidative stress in diabetes, sources from (i) Plant material : Fresh leaves of Annona muricata
nonenzymatic source resulting from biochemical (Linn.) (family, Annonaceae) (locally known as
oxidation of glucose, glucose reacts with protein in “Soursop in English, and “Abo” in Yoruba language
nonenzymatic manner leading to the development of of Western Nigeria) were collected in Ibadan,
advanced glycation end product (AGE) (Taniyama Nigeria between April and May 2006. The leaves
and Griendling, 2003). (ii) enzymatic sources which were identified, by the Taxonomist/Curator of the
includes nitric oxide synthetase (NOS), NAD(P)H Department of Botany, Obafemi Awolowo

Protective effects of A. muricata on the morphology of pancreatic β-cells 175

 African Journal of Biomedical Research 2006 (Vol. 9) / Adedapo, Arinola, Ige, Adedapo and Salimonu

University, Ile-Ife, Nigeria, as those of A. muricata diabetic ‘test’ rats. 1, 2, 3, 4 and 8 h following oral
Linn. (family, Annonaceae). Voucher specimen of administrations of the test compound to the animals,
the plant has been deposited in the Herbarium of the blood glucose concentrations (Gt) were again
University’s Botany Department. determined and recorded. In each case and for each
dose, the rats were restrained in a cage, and blood
Preparation of Annona muricata leaf aqueous samples (0.02 ml) were collected from the tail vein
extract: A. muricata fresh leaves were air-dried at of each rat for blood glucose analysis. Percentage
room temperature. One kilogram of the air-dried glycemic variation was calculated as a function of
leaves of the plant was milled into fine powder in a time (t) by applying the formula:
Waring blender, department of Pharmacognosy, % glycemic change = Go – Gt x 100/Go
Obafemi Awolowo University, Ile-Ife, Nigeria. The [where Go and Gt represent glycemic values before
powdered leaf was macerated in distilled water and (i.e., 0-time or 0-h glycemic values), and glycemic
extracted twice, on each occasion with 2.5 1itre of values at 1, 2, 3, 4 and 8 hours after, oral
distilled water at room temperature for 48 h. The administrations of ‘test’ compound respectively].
combined aqueous extract solubles were
concentrated to dryness under reduced pressure at Induction of experimental diabetes: Diabetes
60oC±1oC in a rotary evaporator. The resulting mellitus was induced (in groups B and C ‘test’ rats)
aqueous extract was freeze-dried, finally giving by single intraperitoneal injection of STZ (75
26.23 g (i.e., 2.62% yields) of a light green, powdery mg/kg), freshly dissolved in 0.1mol/l citrate buffer
crude aqueous leaf extract of A. muricata (AME). (Rossini et al; 1978). Control rats were injected with
Aliquot portions of the crude plant extract residue only citrate buffer solution (pH 6.3)
were weighed and dissolved in distilled water for intraperitoneally. The ‘test’ animals in groups B and
use on each day of our experiments. C became diabetic within 48 hours after STZ
administration. Diabetic state was confirmed by
Acute toxicity testing: The median lethal dose measuring basal blood glucose concentration 48
(LD50) of A. muricata leaf aqueous extract (AME) hours after STZ injection. Diabetes was allowed to
was determined in the rat using a modified method develop and stabilize in these STZ-treated rats over
of Lorke, (1983). Mice fasted for 16 h were a period of 3-5 days. All animals in groups A, B and
randomly divided into groups of eight mice each. C were kept and maintained under laboratory
Graded doses of AME (50, 100, 200, 400, 800, 1600 conditions of temperature, humidity, 12-h day – 12-
and 3200 mg/kg) were separately administered h night cycle; and were allowed free access to food
intraperitoneally (i.p.) to the mice in each of the test (standard pellet diet) and water ad libitum. Before
groups. Each of the mice in the control group was the commencement of our experiments, both the
treated only with distilled water (3 ml/kg, i.p.). The control normal (normoglycemic) and STZ-treated,
mice in both the test and control groups were then diabetic (hyperglycemic) test rats were fasted for 16-
allowed free access to food and water, and observed h, but still allowed free access to water throughout.
over a period of 48 h for signs of acute toxicity. The At the end of the 16-h fasting period – taken as 0
number of deaths (caused by the extract) within this time (i.e., 0 h) – blood glucose levels (initial
period of time was noted and recorded. Log dose- glycemia, G0) of the fasted normal and STZ-treated,
response plots were constructed for the plant’s diabetic rats were determined and recorded. Fasted
extract, from which the LD50 of the leaf aqueous STZ-treated rats with blood glucose concentration
extract was determined. ≥18 mmol/L were considered to be diabetic, and
used in this study. The test compound [i.e., Annona
Evaluations of hypoglycemic activity of A. muricata leaf aqueous extract (AME, 100 mg/kg
muricata leaf aqueous extract (AME): The test p.o.)] was administered orally (by gastric intubation)
compound (i.e., A. muricata leaf aqueous extract to the group C fasted diabetic rats. The
(AME, 50-400 mg/kg) were administered orally to administration of plant’s aqueous extract was
the groups of fasted normal (control) and fasted commenced as from the 5th day post STZ injections.

176 Protective effects of A. muricata on the morphology of pancreatic β-cells

 African Journal of Biomedical Research 2006 (Vol. 9) / Adedapo, Arinola, Ige, Adedapo and Salimonu

bicinchoninic acid (BCA) protein assay reagent

Histological procedures: Pancreatic tissues were (Pierce Chemical Company, Rockford, IL).
excised from sacrificed animals, weighed, and fixed
in aqueous Bouin’s solution for 48 h and were Catalase Activity (CAT): The activity of catalase
sequentially embedded in paraffin wax blocks (CAT) was measured using its perioxidatic function
according to the standard procedure, sectioned at 5 μ according to the method of Johansson and Borg
thickness. They were further deparaffined with (1988). 50 µL potassium phosphate buffer (250 mM,
xylol, and histologic observations were performed pH 7.0) was incubated with 50 µL methanol and 10
after staining for functional pancreatic tissues by µL hydrogen peroxide (0.27%). The reaction was
Aldehyde fuchsin trichrome method described by initiated by addition of 100 µL of enzyme sample
Stevens (1982). The slides were examined using with continuous shaking at room temperature
light microscope. (20oC). After 20 minutes, reaction was terminated by
addition of 50 µL of 7.8 M potassium hydroxide.
Biochemical assays 100 µL of purpald (4-Amino-3-hydrazino-5-
mercapto-1,2,4-triazole, 34.2 mM in 480 mM HCl)
Blood Glucose and serum insulin estimations: was immediately added, and the mixture was again
Blood samples were obtained by repeated needle incubated for 10 minutes at 20oC with continuous
puncture of the same tail tip vein. Samples were shaking. Potassium peroxidate (50 µL 65.2 mM)
obtained 1 day before STZ-treatment, and on various was added to obtain a coloured compound. The
days after induction of diabetes mellitus. Diabetes absorbance was read at 550 nm in a
was allowed to develop and stabilize in these STZ- spectrophotometer. Results are expressed as
treated rats over a period 5 days. Blood glucose micromoles of formaldehyde produced/mg protein.
concentrations were determined by means of Bayer
Elite® Glucometer, and compatible blood glucose Reduced GSH and oxidized glutathione GSSG
test strips (Henry, 1984). activities: Pancreatic GSH and GSSG contents were
The mean fasting blood glucose levels for normal, measured as described by Hissin and Hilf (1973).
nondiabetic rats were found to vary between For measuring GSH contents, 4 ml of pancreatic
4.01±0.04 and 4.20±0.13 mmol/L. Fasted STZ– homogenate was precipitated by adding 1 ml of 25%
treated rats with blood glucose concentrations ≥18 metaphosphoric acid and centrifuged at 10,000 x g
mmol/L were considered to be diabetic, and used in (Ultracentrifuge, Hitachi, Japan) for 30 min.
this study. Serum insulin concentrations were Supernatant was diluted 20 times with same buffer
determined by an enzyme-linked immunosorbent and 100 μl of orthopthaldehyde (OPT) was added. In
assay (ELISA) using a commercial kit (Crystal addition, for GSSG assay, 0.5 ml supernatant was
Chem, Chicago, III). incubated at room temperature with 200 μl of 0.04
mol/l N-ethylmaleimide solution for 30 min and to
Preparation of pancreas homogenates: The rats this mixture 4.3 ml of 0.1 mol/l NaOH was added. A
were sacrificed by cervical dislocation and pancreata 100 μl sample of this mixture was taken for the
were excised, rinsed in ice-cold physiological saline, measurement of GSSH using the exact procedure
and homogenized with Potter Elvehjem described above for GSH assay except that 0.1 mol/l
homogenizer. 10% homogenates were prepared in NaOH was used as the diluent instead of phosphate
6.7 mM phosphate buffer, pH 7.4 and centrifuged at buffer. Samples were incubated at room temperature
10,000 rpm for 10 min at 4oC, and the supernatant for 15 min and fluorescence was measured using
was used for antioxidant enzyme assays. For the spectrofluorometer (Tecan Spectra Fluor Plus
determination of lipid peroxidation, pancreatic Germany) at 350 nm (Ex)/420 nm (Em).
tissues were homogenized in 1.15% KCl solution to
obtain a 10% (w/v) homogenate. Protein content of Superoxide Dismutase Activity (SOD): Pancreatic
pancreas homogenates was determined by using SOD activity was assayed by the method of Kakkar
et al. (1984). Reaction mixture contained 1.2 ml of

Protective effects of A. muricata on the morphology of pancreatic β-cells 177

 African Journal of Biomedical Research 2006 (Vol. 9) / Adedapo, Arinola, Ige, Adedapo and Salimonu

sodium pyrophosphate buffer (0.052 mM, pH 7.0), standard. Results are expressed as nmoles
0.1 ml of phenazine methosulphate (PMS) (186 TBARS/mg tissue.
μM), 0.3 ml of nitro blue tetrazolium (NBT) (300
μM). 0.2 ml of the supernatant obtained after Nitric Oxide (NO)
centrifugation (1500 x g, 10 min followed by 10,000 Serum nitrite/nitrate levels were determined by
x g, 15 min) of 5% pancreatic homogenate was converting the nitrate to nitrite, using enzyme nitrate
added to reaction mixture. Enzyme reaction was reductase followed by addition of Griess reagent to
initiated by adding 0.2 ml of NADH (780 μM) and colorimetrically quantify the nitrite concentration
stopped precisely after 1 min by adding 1 ml of (Green et al; 1982). The serum was diluted 1:5 in
glacial acetic acid. Amount of chromogen formed PSB before a 25-µl aliquot was added to a mixture
was measured by recording color intensity at 560 of 25 µl nitrate reductase (1.5 U/ml) and 25 µl of
nm. Results are expressed as units/mg protein. NADPH (0.134 mg/ml), both prepared in 40 mM
Tris, pH 7.6. The samples were thereafter incubated
Glutathione Peroxidase Activity (GSH-Px): at room temperature for 3 hours. Following this
Glutathione peroxidase (GSH-Px) activity was period, 100 µl of Griess reagent (1:1 mixture of 1%
measured by NADPH oxidation, using a coupled sulphanilamide in 5% phosphoric acid and 0.1%
reaction system consisting of glutathione, naphyly-ethylenediamine) was added and incubated
glutathione reductase, and cumene hydroperoxide for a further 10 min at room temperature; the
(Tappel, 1978). 100 µL of enzyme sample was absorbency of the samples was measured at 540 nm
incubated for five minutes with 1.55 ml stock with a 650 nm reference. The concentration of
solution (prepared in 50 mM Tris buffer, pH 7.6 nitrite/nitrate was determined from a standard curve
with 0.1 mM EDTA) containing 0.25 mM GSH, of sodium nitrate and calculated as µM
0.12 mM NADPH and 1 unit glutathione reductase. nitrite/nitrate.
The reaction was initiated by adding 50 µL of
cumene hydroperoxide (1 mg/ml), and the rate of Immunohistochemistry
disappearance of NADPH with time was determined The animals were sacrificed by cervical dislocation,
by monitoring absorbance at 340 nm. One unit of some at the end of the 16 hours fasting period, and
enzyme activity is defined as the amount of enzyme others on various days following STZ
that transforms 1 µmol of NADPH to NADP per administration. Pancreatic tissues were excised and
minute. Results are expressed as units/mg protein. weighed after the fat and lymphnodes had been
removed. The splenic parts of the pancreas of each
Lipid Peroxidation contents (LPO): The product rat were fixed in aqueous Bouin’s solution, and
of the reaction between malondialdehyde (MDA) embedded in paraffin. Each pancreatic block was
and thiobarbituric acid reactive substances (TBARS) serially sectioned (5 μ) throughout its length to
were measured by a modified method of Ohkawa et avoid any bias due to changes in islet distribution or
al., (1979). For each sample to be assayed, four cell composition, and thereafter mounted on slides.
tubes were set up containing 100, 150, 200 and 250 For each pancreas, 10 sections were randomly
µL of tissue homogenate, 100 µL of 8.1% SDS, 750 chosen at a fixed interval through the block (every
µL of 20% acetic acid, and 750 µL of 0.8% aqueous 30th section), a procedure that has been shown to
solution of TBA. The volume was made up to 4 ml ensure that selected sections are representative of the
with distilled water, mixed thoroughly and heated at whole pancreas (Mossavat et al; 1997). Sections
95oC for 60 minutes. After cooling, 4 ml of n- were immunostained for insulin, using a peroxidase
butanol was added to each tube, the contents mixed indirect labeling technique. The sections were
thoroughly, and then centrifuged at 3000 rpm for 10 incubated for 1 h with guinea-pig anti-insulin serum
minutes. The absorption of the clear upper (n- (final dilution 1:1,000, Ref. 64-104-1; Aurora, OH).
butanol) layer was read at 532 nm. 1, 1, 3, 3 Thereafter, sections were incubated for 45 min with
tetraethoxy propane (97%) was used as the external peroxidase-conjugated rabbit anti-guinea pig IgG
(final dilution 1:20; Dako, Carpinteria, CA). The

178 Protective effects of A. muricata on the morphology of pancreatic β-cells

 African Journal of Biomedical Research 2006 (Vol. 9) / Adedapo, Arinola, Ige, Adedapo and Salimonu

activity of the antibody-peroxidase complex was

revealed with 3,3’-diaminobenzidine- RESULTS
tetrahydrochloride, using a peroxidase substrate kit
(DAB; Biosys-Vector, Compiegne, France). A Characteristics of diabetic state: Forty-eight hours
standard concentration of hematoxylin was added as after STZ administration, all animals that had been
a counterstain. treated with STZ displayed glucosurea,
hyperglycemia, hypoinsulinemia and a moderate
Pancreatic insulin contents: The splenic regions of the loss of body weight. Blood glucose concentrations
pancreatic tissues from euthanized rats were weighed and and serum insulin levels of the STZ-treated
homogenized, on various experimental days, in acid- experimental animals are shown in Table 1. The
ethanol solution (75% ethanol, 23.5% distilled water,
baseline weight of the rats at the beginning of the
1.5% concentrated HCl). After overnight incubation at
4oC, the suspensions were centrifuged, and the study was similar in all groups. At the end of the
supernatants were collected and assayed for insulin study period (2 months), diabetic animals in group B
contents, using a competitive ELISA kit (Kekow et al; presented with weight loss. The initial and final
1988). Plates were coated with rabbit anti-guinea-pig Ig body weights were not significantly (p<0.05)
secondary Ab (Organon Teknoka, Durhan, NC), followed different in control and AME-treated groups.
by incubation with a guinea-pig anti-human insulin Ab
(Cortex Biochem, San Leandro, CA). Following two Blood glucose and serum insulin concentrations:
washing steps, various extract dilutions or insulin Mean blood glucose concentrations and serum
standards (Linco Research, St. Louis, MO) were mixed insulin levels of the STZ-treated experimental
with constant concentration of HRP-conjugated rat insulin
animals are shown in Table 1. In our control set of
(Organon Teknika) for 4 h at room temperature, or at 4oC
overnight, before competitive capturing was allowed for 3 experiments, pretreatment of the rats with citrate
h. After washing five times, Sigma FAST OPD tablets buffer alone did not significantly modify (p > 0.05)
(Sigma, St. Louis, MO) were used as substrate. Results the serum insulin and blood glucose concentrations.
were analyzed using ceres 900 C ELISA-reader. As shown in Table 2. Fig. 1, there was a gradual rise
in the blood glucose concentrations of the STZ-
Statistical Analysis treated rats as from day 1 following injection of
The data obtained were expressed as means (±SEM), STZ, and the values were significantly higher (p <
and analyzed using repeated measures of variance. 0.05) than those of control animals (Table 1).
The differences between the means were analyzed
statistically with one-way analysis of variance
(ANOVA; 95% confidence interval). Values of
p<0.05 were taken to imply statistical significance.

Table 1.
Various parameters recorded in control, STZ-treated and STZ + AME-treated diabetic rats just before and after treatment.
Values presented represent the means (± SEM) of 10 observations.
Parameters Control STZ-treated STZ + AME-treated
Body weight (g) Initial 242±12 239±14a 241±17a
Final 249±11 197±17b 244±13a
Pancreatic weight (g) Initial 0.31±1.2 0.29±1.5 0.29±1.4
Final 0.31±1.9 0.22±1.4 0.30±1.5
Blood glucose (mmol/l) Initial 4.1±0.1 19.2±0.5c 4.1±0.2a
c
Final 4.0±0.2 21.5±0.7 4.7±0.1a
d
Serum insulin (μU/ml) Initial 13.9±1.3 9.3±1.3 10.6±0.3a
d
Final 13.7±1.1 5.3±1.5 12.2±0.1a
Values are expressed as means (±SEM) of 10 rats. Insignificant difference (p>0.05) between all groups. b,c,d Significant
a

difference (p<0.05) between treatment and control groups.

Protective effects of A. muricata on the morphology of pancreatic β-cells 179

 African Journal of Biomedical Research 2006 (Vol. 9) / Adedapo, Arinola, Ige, Adedapo and Salimonu

Table 2.
Changes in blood glucose concentrations and serum/pancreatic insulin contents in control, STZ-treated and
STZ + AME-treated diabetic rats.

Control Treated
Days 0 10 20 30 40 50 60

Blood glucose concentration (mmol/L)

STZ-treated 4.0±0.2 19.1±0.7b 19.5±0.1b 20.9±0.3a 21.6±0.3b 21.7±0.5b 21.2±0.3b
STZ + AME-treated 4.1 ±0.1 9.8±0.3b 5.2±0.2 a 5.4±0.5a 5.1±0.2a 4.7±0.3a 4.7 ±0.1 a

Serum insulin concentration (μU/ml)

STZ-treated 13.9±1.3 9.3±1.5 7.3±1.4c 6.7±1.3c 5.9±1.3c 5.7 ±1.5c 5.3±1.2c
STZ + AME-treated 13.7±1.1 10.6±1.2a 10.8±1.9a 11.9±1.3a 11.9±1.5a 12.1±1.5a 12.2±1.4a

Pancreatic insulin content (μU/mg)

STZ-treated 16.3±1.1 10.9±1.7 8.3±1.5d 7.1±1.9d 7.3±1.6d 6.4±1.3d 5.2±1.7d
a a a a a
STZ + AME-treated 18.2±1.5 12.4±1.3 14.7±1.9 13.1±1.6 15.2±1.2 15.2±1.4 16.2±1.9a
Values are expressed as means (±SEM) of 10 rats. a Insignificant difference (p>0.05) between all groups. b,c,d Significant
difference (p<0.05) between treatment and control groups.

Furthermore, high levels of blood glucose

Table 3. concentrations of the STZ-treated rats were
Pancreatic tissue CAT (μmol/mg protein), GSH (U/g persistently observed throughout the study period
protein) SOD (U/mg protein), GSH-Px (U/mg protein), (21.6±0.3 mmol/l). The blood glucose
MDA (nmol/mg protein), Insulin (μU/mg protein) and concentrations in the AME-treated group, (5.6
serum NO (μmol/l) of all groups, A (control), B (STZ-
mmol/l) significantly reduced in value (p < 0.05).
treated) and C (STZ- +AME-treated) rats.

Parameters Control STZ- STZ + Biochemical findings:

treated AME-
treated Table 3 shows the effects of A. muricata on
Pancreatic 0.42±0.3 0.21±0.4b 0.43±0.1a biochemical variables suggestive of oxidative stress
CAT in STZ-treated animals. There was clear evidence
Pancreatic 6.42±1.3 3.53±0.2b 5.6±0.9a that STZ-induced pancreatic injury was associated
GSH with free radical injury and oxidative stress.
Pancreatic 26.9±1.8 15.7±2.3b 25.4±2.3a Oxidative stress was characterized by increased lipid
SOD peroxidation and/or altered non-enzymatic and
Pancreatic 0.49±0.4 0.28±0.3b 0.51±0.2a enzymatic antioxidant systems (Zima et al; 2001).
GSH-Px
Effects of STZ and STZ + AME treatments on
Pancreatic 88±13 138±17b 92±11a
pancreatic tissue’s CAT, GSH, SOD, GSH-Px,
MDA
Pancreatic 15.2±0.4 7.5±0.3b 14.8±0.7a
MDA, insulin and serum NO are presented in Table
Insulin 3. The pancreatic antioxidant activity of CAT, GSH,
Serum 4.12±0.58 8.38±1.12b 4.57±0.14a SOD, GSH-Px and pancreatic insulin significantly
NO decreased (p<0.05), while pancreatic MDA and
Values are expressed as means (±SEM) of 10 rats for all serum NO significantly increased in the STZ-treated,
groups. a Insignificant difference (p>0.05) between all diabetic group of rats. The control group rats
groups. b Significant difference (p<0.05) in the same row maintained optimal value activity of the antioxidants
between treatment and control groups. studied. Annona muricata treatment significantly
(p<0.05) decreased the elevated NO and MDA, but

180 Protective effects of A. muricata on the morphology of pancreatic β-cells

 African Journal of Biomedical Research 2006 (Vol. 9) / Adedapo, Arinola, Ige, Adedapo and Salimonu

also significantly increased (p<0.05) the reduced improvement in the islet of Langerhans with distinct
antioxidant enzyme activities. Also, AME proved cellularity changes, majority of the cells showed
significantly better in restoring the altered activity of viable islet of Langerhans, with increase in
antioxidant enzymes like CAT, GSH, SOD, GSG-Px granulation (Plate 1a).
and MDA, NO and insulin towards their normal
values in the pancreas. Immunohistochemical findings
Immunohistochemistry staining of the pancreatic
Histopathological findings tissues before STZ injection showed the presence of
Histologically, in diabetic rats with no treatment, the a strong islet insulin immunoreactivity at a level of
most consistent findings in the sections of pancreatic 0.59 islet/mm2 of total pancreatic tissue. This was
tissues were breakdown of micro-anatomical limited to cytoplasmic staining of individual β-cells
features such as extensive β-cell degranulation, (Fig. 4A). The majority of islets from AME-treated
decreased cellular density, and an indistinct boarder pancreases stained positive for insulin suggesting
between the endocrine and exocrine regions. Also that the architecture of AME-treated rats was normal
there was a diffused degenerative and necrotic (Fig. 4C). In contrast, islet cells from diabetic rats
changes, and shrunken in the islet of Langerhans were architecturally distorted, containing
(Plate 1b). The nucleus of necrotic cells showed significantly fewer insulin-positive cells (Fig. 4B).
either pyknosis of marginal hyperchromasia, and the Quantitative image analysis was used to assess the
cytoplasm were filled by hydropic changes (Plate proportion of insulin positive cells per islet in
1b). The α-cells, exocrine pancreatic acinar control, STZ-treated and STZ + AME-treated
epithelium, ductal and connective tissues appeared pancreatic sections. The percentage of islets stained
normal (Plate 1a and 1b). Identifiable pancreatic for insulin were 76.35 ± 19%, 42.26 ± 3.2% and
islets of diabetic rats as from day 10 were of low 68.56 ± 24% (p < 0.05) respectively (Fig. 4A, 4B
cellularity, although there was no evidence of and 4C). Together, these findings further support
inflammatory cell infiltration (Plate 1b). In diabetic that AME protected islets from destruction.
rats treated with A muricata, there was a remarkable

A B C

Plate 1
Aldehyde fuchsin trichrome staining of the pancreatic tissues. Arrows show β-cells with purple colour, while arrowheads
show α-cells in orange colour. (C) Control group: showing normal cells in the islet of Langerhans and showing distinct
granules filling the entire islet of Langerhans that are strongly stained purple, α-cells are seen mostly at the periphery. (B)
Diabetic untreated group: shrunken islet of Langerhans displaying degenerative and necrotic changes, nuclear shrinkage
and pyknosis were evident with cytoplasmic vesiculation in the centre of the islet of Langerhans, decreased cellular
density. (A) STZ+AME-treated: still retained substantial amount of β-cells with moderate staining granules, and distinct
endocrine and exocrine boarder, α-cell still highly populated within the islet of Langerhans X 160.

Protective effects of A. muricata on the morphology of pancreatic β-cells 181

 African Journal of Biomedical Research 2006 (Vol. 9) / Adedapo, Arinola, Ige, Adedapo and Salimonu

A B C

Plate 2
Immunohistochemistry staining of the pancreatic tissues, represented by dark granules. (A) Control group: showing
normal β-cells in the islet of Langerhans that are strong staining with the anti-insulin antibody. Immunoperoxidase,
haematoxylin counterstain. (B) Diabetic untreated group: showing shrunking islet with weak immunoreactive β-cells in
the islet of Langerhans. Immunoperoxidase, haematoxylin counterstain. (C) STZ-AME treated: still retained substantial
amount of β-cells with remarkable anti-insulin antibody staining granules. Immunoperoxidase heamatoxylin
counterstain X 160.

Of interest is the stability of pancreatic insulin Generally, the fruit and fruit juice are taken for
content of the AME-treated rats (15.6±2.1 μU/mg), worms and parasites, to cool fevers, to increase
in contrast with the STZ-treated rats, pancreatic mother’s milk after childbirth, and as an astringent
insulin contents were ~65-fold higher (Table 2). for diarrhea and dysentery. The crushed seeds are
This stability in pancreatic insulin values was also used against internal and external parasites, head lice
observed to reflect in the immunohistochemistry and worms. The bark, leaves, and roots are
staining (Fig. 4C). Immunohistochemistry staining considered sedatives, antispasmodic, hypoglycemic,
intensity was an evidence of protective effects/or hypotensive, diuretics, neuralgia (Hasrat et al;
regenerative processes leading to increase in β-cell 1997). Phytochemically, many active compounds
mass, regaining its normal immunostaining for and chemicals have been found in A. muricata, as
insulin and functional status up to the day of scientists have been studying its properties since the
normoglycemia. 1940s. The tree is rich in ellagic acid, tannins,
lactones, and isoquinoline alkaloids. The leaf, stem,
DISCUSSION bark and seeds also contain varying amounts of a
novel group of chemicals believed to be biologically
The aim of the present study was to demonstrate the active, called Annonaceous acetogenins (Tormo et
efficacy of Annona muricata in the reduction of al; 2003; Kojima, 2004), and this include:
blood glucose concentration as well as to determine annocatalin, annohexocin, annomonicin,
the recovery in altered biochemical variables, annomontacin, annomuricin, annomutacin,
indicative of oxidative stress and organ damage in annonacin to mention a few. Purdue researchers
STZ-treated rats. Ethnobotanically, all parts of A. reported that 14 different acetogenins tested thus far
muricata tree have been used medicinally in the demonstrate potent ATP-blocking properties and
tropics, including the bark, leaves, roots, fruit, and were more potent against multi-drug resistant
fruit seeds (Mors, 2000; Haddock, 1994). Different (MDR) cancer cells (Oberlies et al; 1995). A tumor
properties and uses are attributed to the different cell needs energy to grow and reproduce and by
parts of the A. muricata by the Traditional herbal inhibiting energy to the cell, it can no longer run its
medicine practitioners (Padma et al; 2001). pump and expel attacking agents (Yuan et al; 2003).

182 Protective effects of A. muricata on the morphology of pancreatic β-cells

 African Journal of Biomedical Research 2006 (Vol. 9) / Adedapo, Arinola, Ige, Adedapo and Salimonu

The increasing prevalence of diabetes is reaching However, subsequent discovery that insulin
epidemic proportion worldwide. Diabetes is a major administration was not necessary for the reduction of
threat to global public health that is rapidly glycosuria and indeed for the control of the disease
escalating (WHO, 2000). It is estimated that more raised ‘queries’ about the role of insulin. The later
than 170 million people are suffering from diabetes demonstration that diabetes had developed in
globally and this number is expected to double by persons whose serum insulin levels were not only
2030, and the greatest increase in prevalence is, adequate, but excessive, further confounded the
however, expected to occur in Asia and Africa, ideas and thinking of both clinicians and researchers
follow the trend of urbanization and lifestyle in the field of diabetes. Birth of the concept of
changes (WHO, 1999). Diabetes and its multiple insulin resistance then took place and provided an
complications are extremely burdensome on the answer to some of the questions. Nevertheless
health and economics of countries worldwide. In insulin production by the beta cells continued to
developing countries adherence to therapies is as attract attention and β-cell failure in type 2 diabetes
low as 20%, resulting in poor health outcomes at a is now being demonstrated (Knowler et al; 2002).
very high cost to society, governments and families What must be of further interest now, however, is
(Caro et al; 1987). If not successfully managed, concept of incorporation of scientifically proved
diabetes along with other chronic diseases will herbal medicine.
become the most expensive problem faced by the The three main classes of biological macromolecules
health care systems. Because of its associated that are susceptible to free radical attack under
morbidity and mortality, it is exerting a major diabetic condition are lipids, nucleic acids and
pressure on the healthcare system, and with a better proteins; the attacks affects the entire tissues in the
understanding of the pathophysiology, a new care body and suffer oxidative damage (Halliwell and
perspective and reform in the training of the health Gutteridge, 1989). Primary effect of lipid
workforce is essential. The new care perspective peroxidation is decreased membrane fluidity, which
advocates for the use of the Innovative Care for alters membrane properties and can significantly
Chronic Conditions framework which integrates disrupt membrane-bound proteins (Tappel, 1975).
prevention, control and treatment of the disease The oxidation of proteins includes oxidation of
across multiple levels of health care system sulfhydryl groups, reduction of disulfides, oxidative
particularly in low resources settings. In Africa, adduction of amino acid residues close to metal-
many plants have been reported to possess binding sites via metal-catalyzed oxidation,
antidiabetic or hypoglycemic activities (Ojewole, reactions with aldehydes, protein-protein cross-
2001; Iwu, 1993; Marles and Farnsworth, 1995). linking, and peptide fragmentation (Standman and
Indeed, hundreds of plants are now being used Oliver, 1991; Starke-Reed and Oliver, 1989).
traditionally for the management of both insulin- Oxidative damage to nucleic acids includes adducts
dependent (IDDM) and noninsulin-dependent of base and sugar groups, single- and double-strand
(NIDDM) diabetes mellitus. To date, however, only breaks in the backbone, and cross-links to other
a few of these plants have been subjected to molecules, this causes damage to all four bases and
scientific scrutiny either in laboratory animals or in thymine-tyrosine cross-link (Dizdaroglu, 1992;
human subjects (Ross, 1999). Halliwell and Dizdaroglu, 1992). In the present
Although diabetes has been recognized since study we used aqueous extracts, obtained from A.
antiquity, and treatments of various efficacy have muricata leaves and studied its effect on pancreatic
been known since the middle ages, the elucidation of β-cells. Our observations demonstrated that STZ-
the pathogenesis of diabetes occurred mainly in the induced oxidative stress and β-cell damage as
20th century (Nathan et al; 2005). The discovery of revealed by histological and immunohistochemical
the action and importance of insulin in the staining results. Depletion of endogenous
metabolism of glucose, not surprisingly, led to the antioxidants and generation of free radicals were
belief that lack of insulin was the critical factor in also noted which are in consonance with the findings
the development of the disease (Patlak, 2002). of Buttke and Sandstorm (1994). A. muricata caused

Protective effects of A. muricata on the morphology of pancreatic β-cells 183

 African Journal of Biomedical Research 2006 (Vol. 9) / Adedapo, Arinola, Ige, Adedapo and Salimonu

significant decrease in elevated blood glucose and an pancreatic tissues. Nevertheless, A. muricata
increase in serum insulin concentrations as well as exhibited antioxidant activity, and is able to
pancreatic insulin contents in STZ-treated diabetic diminish and/or prevent, pancreatic oxidative
rats. Histologically, A. muricata partly protected damage produced by STZ. An increased
pancreatic β-cell integrity. It saved the tissue from consumption of antioxidants in the diet of
the usual shrunken islets, denegation, degranulation, individuals is strongly recommended, so that when
hydropic and necrotic changes peculiar to STZ- an individual is subjected to greater oxidative stress,
induced pancreatic injuries. Immunohistochemistry he/she would have better antioxidant defense
staining was evident of significantly better characteristics, thus counteracting the effects of any
restoration of altered micro-anatomy of islet of pro-oxidant. However, further studies are needed
Langerhans of STZ-treated diabetic rats. We also before antioxidants can be used safely as food
analyzed the intracellular level and activities of additives and supplements.
GSH, SOD, CAT, GSH-Px, MDA, NO and insulin
contents and we found that STZ treatment caused Acknowledgements
significant increases in blood glucose concentration, The authors are grateful to Professor J.A.O. Ojewole for
MDA and NO generation, and decreased GSH, his expertise and useful advice in some of the procedures.
SOD, CAT, GSH-Px, activities as well as insulin We also thank Messrs Adeogun Oludele, Doherty O.
Wiston for their technical assistance in plants processing.
levels when compared with control rats. Decreases
Ms Kogie for her excellent technical assistance in tissue
in GSH, SOD, CAT and GSH-Px, and simultaneous processing.
increases in MDA and NO activities, reflect
susceptibility of pancreas to STZ’s significant
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Protective effects of A. muricata on the morphology of pancreatic β-cells 187