JPHYTO 2016; 5(4): 150-156
© 2016, All rights reserved
Ifeoma Chinwude Ezenyi
School of Pharmacy, University of the
Western Cape, Bellville, South Africa
Ifeoma Chinwude Ezenyi*, Oluchi Nneka Mbamalu, Lucy Balogun, Liberty Omorogbe, Fidelis Solomon
Ameh, Oluwakanyinsola Adeola Salawu
This study examines the effects of a methanol extract of Syzygium guineense leaves in streptozotocin (STZ) -
induced diabetes, evaluates its effect on alpha glucosidase and 2, 2-diphenyl-1-picrylhydrazyl radical. Diabetes
was induced in rats by a single intraperitoneal injection of streptozotocin (60 mg/kg). An oral glucose
tolerance test was performed after diabetes induction and repeated after 14 days of treatment with the extract.
The extract elicited antihyperglycemic action in diabetic rats evidenced by an improved oral glucose tolerance.
A dose of 250 mg/kg of extract significantly (P<0.01, 0.001) enhanced glucose clearance at the end of
treatment period and was comparable with metformin, the group also showed increase in hepatic glycogen
content by 33.9% relative to the diabetic control. Serum biochemical analysis showed that the extract improved
indices of renal and hepatic function by reduction in serum albumin, creatinine, liver enzymes, total and direct
bilirubin. Similarly, the extract reduced serum cholesterol, triglycerides and high density lipoprotein (HDL) in
a non-dose dependent manner; treatment with 250 mg/kg extract caused significant (P<0.05) reduction of
HDL. Groups which received 250 and 500 mg/kg of extract showed reversal of glomerular damage compared
with the diabetic untreated group. The extract also exhibited concentration-dependent antioxidant activity
= 0.2 mg/ml) and statistically significant (P<0.01, 0.001) alpha glucosidase inhibitory effect (IC
ability to inhibit alpha glucosidase, scavenge free radicals and increase intrahepatic glucose uptake and storage.
Alpha glucosidase, Antioxidant, Syzygium guineense.
Diabetes mellitus is a major metabolic disorder and a global threat to health owing to its high prevalence,
morbidity and mortality. Diabetes is currently prevalent in 9% of adults aged 18 years and older and
80% of diabetes-related deaths occur in low and middle-income countries
Health Organization, diabetes will be ranked the seventh leading cause of death by 2030
. Non insulin-
deficient insulin secretion from pancreatic beta cells or failure in insulin action and is characterized by
hyperglycemia, impaired lipid metabolism, defects in redox balance, altered metabolism of major food
. Long term health complications of untreated or poorly managed diabetes include diabetic
free radicals have been recognized to play an important role in the development of diabetic
imbalance, with negative effects on key cellular metabolic processes and organelles; necessitating
adequate control of hyperglycemia to mitigate cellular damage
manage type II diabetes include drug classes such as sulfonylureas, thiazolidinediones, biguanides and
the newer incretin mimetics/enhancers. These groups of drugs stimulate insulin secretion, sensitize
tissues to insulin action or inhibit key carbohydrate metabolizing enzymes among other mechanisms
Although oral antidiabetics are frequently employed to manage type II diabetes, they are occasioned with
safer alternatives especially from natural sources. Herbal remedies have been used in traditional practice
for the treatment of different diseases. For example, Syzygium guineense (Myrtaceae) has been used to
alleviate symptoms of different diseases in some parts of Africa. It is a flowering plant native to the
wooded savannah and tropical forests of Africa and bears edible fruits. Also known as ‘water berry’, the
fruits and other parts of the plant are used locally as charcoal, timber, food, medicine, fodder, bee forage
investigation of extracts of the plant reveal its antibacterial and antihypertensive effects
. In view of
leaf extract in streptozotocin – induced diabetic rats.
MATERIALS AND METHODS
Drugs and chemicals
Methanol, dimethylsulfoxide, 2,2-diphenyl-1-picrylhydazyl (DPPH),
, Merck, France), citric acid (BDH, England),
Germany). Other reagents used were of analytical grade.
Adult Wistar rats of either sex were used for the study. The rats were
housed in steel cages and acclimatized for two weeks to laboratory
conditions before the study. They were maintained on standard rodent
feed and allowed unrestricted access to potable drinking water. All
applicable institutional and international guidelines for the care and
use of animals were adhered to in all procedures
Fresh leaves of S. guineense were collected in October from Suleja,
Niger state, Nigeria and identified by a plant taxonomist. A voucher
specimen was prepared (voucher number: NIPRD/H/6644) and
deposited in the herbarium of the National Institute of Pharmaceutical
Research and Development (NIPRD). The leaves were air-dried
under shade for two weeks then milled mechanically to coarse
A 200 g quantity of S. guineense leaf powder was extracted by
maceration in 80 %v/v methanol (1:8) at room temperature. The
mixture was filtered after 48 h with Whatman filter paper then the
filtrate concentrated under vacuum. The concentrate obtained was
dried on a hot water bath maintained at 50
C. The dry extract (SG)
identification of the presence of free anthraquinone glycosides,
combined anthraquinone glycosides, saponins, terpenes, flavonoids
and alkaloids was carried out in accordance with standard rest
Chromatographic separation was performed using a C
) with a compatible guard
0.01% formic acid/acetonitrile; B, 0.01% formic acid/water, was
filtered through a 0.45 µm filter and degassed prior to use. A 20µL
volume of extract was filtered through a 0.22 mm filter disk and
injected into the column. Flow rate of the mobile phase was
maintained at 0.8 mL/min, and peaks were separated according to the
following linear gradient elution: 0 - 1 min, 82% A/18% B; 1 - 15
min, 82% A /18% B to 75% A/25% B; 15 - 20 min, 75% A /25% B to
65% A /35% B; 20 - 25 min, 65% A/35% B to 40% A/60% B; 25 - 26
min, 40% A/60% B to 82% A/18% B; followed by an equilibration
with 82% A /18% B for 10 min. Wavelength for ultraviolet detection
was 370 nm.
Acute toxicity test in rats was done using a modification of Lorke’s
method, in two phases
each were given orally, 300 and 1000 mg/ kg of body weight of the
methanol extract respectively and monitored for 24 h for physical
signs of toxicity and mortality. The rats were subsequently observed
for two weeks for delayed signs of toxicity and/or mortality. In the
second phase, three groups of three rats each were orally administered
1250, 2500 and 5000 mg/kg respectively and monitored likewise. The
median lethal dose in mice (LD
) was calculated as the geometric
minimum dose that produced 100% mortality.
Fifty rats were fasted overnight on day zero (0) during which they
were granted unrestricted access to potable drinking water. Fresh
streptozotocin solution in ice cold citrate buffer (0.1 M, pH 4.5) was
prepared in aliquots and protected from light. The solution was
immediately injected intraperitoneally to rats on day 1 at a dose of 60
mg/kg of body weight. Thereafter, the rats were granted access to
food and 10%w/v sucrose solution for 48 h. After an overnight fast,
blood glucose was taken at 72 h using an Accu-Chek glucometer
(Roche, Mannheim, Germany) with its corresponding strips. Only rats
with fasting blood glucose concentration (BGC) above 200 mg/dL
were considered diabetic and used for the 2 week study.
The experimental rats were fasted overnight but allowed access to
drinking water. After the fast, the rats were divided into 5 groups
(n=5) and individual and individual pre-treatment BGC values were
recorded. Group 1 was the diabetic control and received distilled
water (1 ml/kg). Other treatment groups comprised diabetic rats
receiving the extract (250, 500 and 1000 mg/kg) and metformin
hydrochloride (100 mg/kg). Thirty minutes after treatment of all the
groups, each rat was administered an oral glucose load (1 g/kg) and
BGCs recorded at 0, 30, 60, 90, 120, 180 and 240 min.
Four experimental groups were used for the study and treated once
daily for 14 days as follows: Group 1 served as the diabetic control
and received the aqueous vehicle alone (1ml/kg). Groups 2 and 3 were
treated with 250 and 500 mg/kg of extract prepared in aqueous vehicle
while group 4 was treated with metformin hydrochloride (100 mg/kg).
Oral glucose tolerance test was repeated again for all the groups at the
end of the study period, as described previously.
At the end of the treatment period, the rats were euthanized by
chloroform inhalation. Blood samples collected from each rat by
cardiac puncture were dispensed into plain tubes, allowed to clot and
centrifuged at 3500 rpm for 10 min. The serum was stored at -4°C and
used for evaluation of biochemical parameters including electrolytes,
creatinine, urea, alanine transaminase (ALT), aspartate transaminase
(AST), alkaline phosphatase (ALP), lipids, total and conjugated
bilirubin, using commercial kits (Randox Laboratories, Antrim, UK).
Effect of extract administration on kidney, liver weight and liver
The kidneys and liver of each rat was excised carefully and their
weights were calculated relative to body weight of the rat on the same
day. Approximately 1 g of tissue was cut from each liver for
estimation of glycogen content expressed as gram per gram (g/g) of
Kidneys and pancreas obtained from the rats were fixed in 10%
formal saline for at least 48 h. These were then processed routinely
and the tissues were embedded in paraffin wax. Histological sections
Sample slides bearing codes were examined by a pathologist blinded
to the study design and treatment groups to identify histological
A chromogenic method described previously was used
. Briefly, 20
with 80 μL of 100 mM phosphate buffer solution (pH 6.8) and 40 μL
of enzyme solution (0.76 unit/mL). After addition of 40 μL of p-
nitrophenyl-α-D-glucopyranoside (5 mM), the mixture was further
incubated for 15 min. The reaction was stopped by addition of 20 μL
of 200 mM sodium carbonate and p-nitrophenol generated was
measured at 405 nm using a microplate reader (GF-M3000, England).
Test incubations were carried out in triplicate. Blank and control
incubations were prepared by replacing enzyme and extract solution
with 80 μL buffer solution and 20 μL DMSO in blank and control
incubations respectively. Enzyme inhibition (%) was calculated using
Where A = absorbance of the control without test samples, and B =
concentration of extract necessary to inhibit enzyme activity by 50%
) was calculated by linear regression where the abscissa (x)
average inhibition (%) of enzyme activity.
The test was performed as described previously with slight
prepared in methanol and kept away from light. A 150 µL volume of
this solution was added to 50 µl of various concentrations (0.1565 - 5
mg/mL) of extract prepared in methanol. After 30 min of incubation
in the dark, absorbance was taken at 492 nm. Free radical scavenging
activity was calculated using:
100 − [
As − Ab
= Net absorbance of sample, A
= Absorbance of
the initial DPPH absorbance by 50% (EC
) was calculated by linear
the ordinate (y) represents the average percentage (%) scavenging
Results were expressed as mean ± SEM. Statistical analysis was
carried out using one-way analysis of variance (ANOVA) using
Graph Pad Prism 5.0 software. The data obtained was further
subjected to dunnet’s post hoc test; differences between treated groups
and the untreated control were accepted as significant at P<0.05.
The extract did not cause any obvious toxicity at all the doses used in
both phases of the acute toxicity test. No mortality was recorded
within 2 weeks following administration of extract doses up to 5000
mg/kg. Orally administered extract produced dose-dependent decrease
in blood sugar in diabetic rats in an oral glucose tolerance test after
diabetes injection. All the diabetic groups showed impaired tolerance
to oral glucose seen as elevated blood sugar concentration for over
240 min following glucose administration (Table 1). The extract
produced dose-dependent decrease in blood glucose concentration
with time and a maximum dose of 1000 mg/kg of extract produced
31.35% reduction at 240 min relative to the control. This effect was
observed to be higher than the effect produced by metformin. In a
repeated glucose tolerance test after 14-day treatment, the extract (250
hyperglycemic peak in diabetic rats, as seen in the rapid drop in
compared to the diabetic control (Table 2). A similar effect was also
produced by metformin (100 mg/kg). The onset of action was half an
hour after extract administration and lasted for over 2 h in a time
dependent manner, as blood glucose concentration decreased to
NDNT: Non diabetic non treated group; *P<0.05; **P<0.01(Data analysis and post hoc test)
Serum biochemical analysis revealed that the extract did not
dependent reduction of serum creatinine compared to the diabetic
control (Table 3). Also, the extract caused a reduction in serum levels
of liver marker enzymes, total and direct bilirubin and albumin; most
of these reductions were observed to be dose-dependent (Table 4).
Lipid profile results show that the 250 mg/kg extract decreased
cholesterol, triglycerides and significantly (P<0.05) reduced serum
HDL (Table 5). These changes were however observed to be non
Table 2: Effect of extract on oral glucose tolerance test after 14-day treatment
*P<0.05, **P<0.01, ***P<0.001(Data analysis and post hoc test)
Table 3: Effect of extract on serum electrolytes and creatinine
*P<0.05, **P<0.01 (Data analysis and post hoc test)
Table 5: Effect of extract on serum lipid profile
Table 6: Antioxidant activity of S. guineense leaf extract
= 0.2 mg/ml.
Table 7: Alpha glucosidase inhibitory activity of S. guineense ethanol
mean absorbance due to
2.615 ± 0.04**
2.367 ± 0.01***
2.168 ± 0.04***
1.793 ± 0.04***
0.649 ± 0.04***
= 6.15 mg/ml. **P<0.01; **P<0.001 (Data analysis and post hoc test)
Table 8: Effect of extract on absolute kidney, liver weight and liver glycogen
Relative kidney weight
Relative liver weight (g)
(gram/gram of tissue)
Value in parenthesis (%) represents percentage increase in liver glycogen content relative to the diabetic control group. **P<0.01(Data analysis and post hoc test)
significantly affect electrolyte and urea levels but elicited dose-
An antioxidant effect was observed to be produced by the extract in a
concentration-dependent manner (Table 6). A maximum radical
scavenging effect was produced at a concentration of 1.25 mg/mL of
extract and a concentration of 0.2 mg/ml was estimated to be the
efficient concentration required to elicit 50 % radical scavenging
). Similarly, the extract elicited significant (P < 0.01,
tested concentrations and the concentration required to inhibit enzyme
activity by 50 % (IC
) was estimated to be 6.15 mg/ml (Table 7).
groups revealed that the extract did not significantly alter the absolute
and relative weights of these organs. Further studies however showed
that liver glycogen content was elevated in diabetic rats treated with
250 mg/kg extract, compared to the diabetic untreated control (Table
VIII). Histopathological analysis of kidneys of the diabetic untreated
control revealed complete loss of nuclei within the collecting duct
which appeared dense with epithelial destruction and glomerular
atrophy. Diabetic groups which received metformin, 250 and 500
mg/kg of extract showed normal glomeruli although the nuclei within
collecting duct appeared slightly enlarged or diffuse (Figure 1).
Pancreatic tissue of the diabetic untreated group appeared to have
fewer acinar cells with widened interstitial spaces compared to the
normoglycemic and extract-treated groups (Figure 2).
Alkaloids, flavonoids, saponins and terpenoids were detected in the
extract, whereas glycosides and anthraquinone derivatives where not
detected. High performance liquid chromatoghy fingerprint of the
extract revealed rutin and quercitrin as some of the polyphenolic
constituents (Figure 3). Three unknown, prominent constituents with
retention times of 3.538, 4.661 and 13.218 min respectively were also
present in the extract.
mg/kg extract, D: Diabetic + 500 mg/kg extract, E: Diabetic+ metformin groups
250 mg/kg extract, D: Diabetic + 500 mg/kg extract, E: diabetic+ metformin groups
and H. quercetin
The absence of mortality at 5000 mg/kg shows that the extract has a
of diabetes by injection of streptozotocin generates free radicals and
causes breaks in DNA of pancreatic beta cells and these results in
their selective destruction, producing a type II diabetes mellitus
hyperglycemia and is characterized by increased glycosylation of
haemoglobin, lipid peroxidation and reduced glutathione activity
The blood glucose lowering effect of S. guineense leaf extract
observed in the oral glucose test before sub acute treatment may be
attributed to improved tissue uptake and storage of glucose, similar to
the mechanism of action of metformin
continuous treatment with the extract may also stimulate residual beta
cells to secrete insulin and improve tissue insulinotropic responses
such as glycogen synthesis and storage, facilitating systemic glucose
clearance. This is likely as some Syzygium species have also been
reported to improve insulin sensitivity and promote insulin-mediated
glucose uptake and storage in liver and adipose tissue
antioxidant activity exhibited by the extract may also mitigate disease
progression, as reactive oxygen species are involved in the
development of diabetic complications. Extracts of S. guineense
leaves have been shown to possess strong antioxidant effects and this
supports the antioxidant effect of the extract seen in our study
Tissue damage in diabetes is mediated by free radicals which act on
ultimately leading to extensive membrane damage and dysfunction
. Also, there is an increased, uninhibited mobilization of free fatty
in serum lipids. Hence, the lipid lowering effects and antioxidant
activity of the extract potentially contributes to its antidiabetic effects
observed in this study. The anti-dyslipidemic effect of the extract may
be a secondary one, following the simulation of insulin release and
action which increase in lipoprotein lipase activity and lowers plasma
The elevation of serum biomarker enzymes such as ALT, AST and
ALP in untreated diabetes is an indication of impaired liver function
due to hepatic damage induced by hyperglycemia
. The ability of
its ability to alleviate the oxidative degenerative effects of
streptozotocin on hepatocytes. This finding is supported by a previous
report on the antioxidant effects of S. guineense on oxidative stress in
reducing effect of the extract may also be attributed to its ability to
ameliorate the progression of renal dysfunction in diabetes. Diabetic
nephropathy is a leading cause of end stage renal failure and a
relatively common complication of diabetes mellitus that ocurs when
there is progessive oxidative renal injury and fibrosis, which manifests
in early stage of disease as albuminuria and inscrease in serum
. The extract likely scavenges free radicals generated in
in the restorative effect in kidneys of extract-treated groups where
glomerular damage was reversed, similar to the metformin-treated
to significantly prevent increase in blood glucose concentration
following a meal. Notably, drugs which inhibit carbohydrate
metabolizing enzymes like alpha glucosidase are commonly used in
combination with regulated diet to control post prandial
. They prevent the breakdown of carbohydrates such
intestinal brush borders and retard the release of large quantities of
glucose from the intestine into bloodstream and its absorption
following a meal
. In recent years, some plants have been known to
attention for their potential for development as antihyperglycemic
elucidates it as a promising agent in this regard. This effect may be
related to the phytochemicals contained within the some species of
Syzygium as species like S. cumini and S.aromaticum reportedly show
alpha glucosidase inhibitory activity
. Secondary metabolites
shown to have antihyperglycemic effect in other plant extracts. They
may exert their effects by acting singly or synergistically to improve
glucose homeostasis and oxidative metabolism in diabetes
of a glucose transporter protein
. Of the polyphenols; rutin,
chromatogram of the extract, rutin and quercetin have been reviewed
as promising oral antidiabetic agents
scavenge free radicals and may contribute to the antioxidant effects
produced by the extract in this study.
This study shows that S. guineense methanol leaf extract shows
potential for development as an antidiabetic agent. Antioxidant,
enzyme inhibitory activities and tissue glucose uptake are likely
mechanisms through which its antidiabetic effects are mediated.
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HOW TO CITE THIS ARTICLE
Ezenyi IC, Mbamalu ON, Balogun L, Omorogbe L, Ameh FS, Salawu OA.
Antidiabetic potentials of Syzygium guineense methanol leaf extract. J