.

British   British Journal of Medicine & Medical Research

Journal of   1(1): 24-34, 2011
Medicine & 
Medical  SCIENCEDOMAIN international
Research   www.sciencedomain.org

Effect of Combined Leaf Extracts of Vernonia

amygdalina (Bitter Leaf) and Gongronema latifolium
(Utazi) on the Pancreatic β-Cells of Streptozotocin-
Induced Diabetic Rats

Mfon I. Akpaso1, Item J. Atangwho2, Amabe Akpantah1, Victor A. Fischer1,

Anozeng O. Igiri1 and Patrick E. Ebong2
1
Department of Anatomy, College of Medical Sciences, University of Calabar,
P. M. B. 1115 Calabar, Nigeria.
2
Department of Biochemistry, College of Medical Sciences, University of Calabar,
P. M. B. 1115 Calabar, Nigeria.

Received 12th February 2011

Accepted 25th March 2011
Research Article Available online 28th April 2011

ABSTRACT

The study evaluated the effect of combined extracts of Vernonia amygdalina (VA) and Gongronema
latifolium (GL) on the pancreas of streptozotocin (STZ) induced diabetic Wistar rats. Thirty-two (32)
albino rats were divided equally into 4 groups. Groups A and B which served as normal (NC) and
diabetic (DC) controls respectively, received placebo treatment. The diabetic test groups C and D were
respectively treated with combined extracts of VA and GL (200mg/kg b. w., p. o.) and insulin, (humulin 5
IU/kg, s.c.) for 28 days. Thereafter, the animals were sacrificed and blood and pancreas were collected
for serum glucose and histological evaluation, respectively. Changes in animal weight were also
measured within the period. From the results it was revealed that both the combined extracts and
humulin significantly increased the animals’ body weight (p<0.05) from -10.5% reduction in the DC, to
7.6% and 8.9% respectively. In the same order, serum glucose significantly decreased (p<0.05) by
12.49% and 14.96% after the 28-day treatment compared to DC. The extent of reversal of
hyperglycemia in the extract treated animals compared well with the insulin treated group. The
biochemical results were corroborated with results of histological evaluations: The pancreatic β-cells of
DC animals which were distorted and degenerated with shrunken cell mass as against prominent islet
cells with normal exocrine pancreas of NC animals became rapidly proliferated upon intervention with
the combined extracts, suggesting a possible regeneration of the islet cells. On the otherhand,
_________________________________________________________________________________________________________

* Corresponding author: E-mail: [email protected]; [email protected]

 British Journal of Medicine & Medical Research, 1(1): 24-34, 2011

intervention with humulin did not produce observable differences in the cyto-architecture of the
pancreatic islets compared to the diabetic control, confirming an extra-pancreatic mechanism of insulin.

Keywords: Diabetes mellitus, V. amygdalina, G. latifolium, human insulin, pancreas β-cells regeneration;

1. INTRODUCTION

Diabetes mellitus (DM) is a complex and chronic disease associated with a myriad of debilitating
complications, causes of which are usually multi-factorial. The lesions in the patho-physiology of diabetes
are multiple and therefore would require more than a single drug agent to reverse all or majority of the
aspects of the disease. The effective therapeutic approach should be multimodal and in this light, several
traditional medicinal herbs have been preferred given the plethora of active ingredients present in a single
herb (Tiwari and Rao, 2002, Atangwho et al., 2009).

Medicinal plants have formed the basis of health care throughout the world since the earliest days of
humanity and have remain relevant in both developing and the developed nations of the world for various
chemotherapeutic purposes. The use of plant derived natural compounds as part of herbal preparations
for alternate source of medicament continues to play major roles in chemotherapy especially in third world
countries (Joy et al., 1998). Several studies carried out have shown that traditional medicines could
provide better glycaemic control than currently used conventional drugs (Rates, 2001; Roja and Rao,
2000). Plants by means of secondary metabolism contain a variety of herbal and non-herbal ingredients
that can ameliorate a disease condition by acting on a variety of targets (various modes and
mechanisms) in the host organism. On the basis of the above, polyherbal therapy is considered the
preferred therapeutic approach to management of diabetes mellitus given its multi-factorial pathogenicity
(Tiwari and Rao, 2002; Ebong et al., 2008).

Polyherbal therapy which is the use of a combination of various agents from different plant sources for
therapeutic purposes is a current pharmacological principle and has the advantage of producing
maximum therapeutic efficacy with minimum side effects (Ebong et al., 2008). This enhanced efficacy is
thought to derive from phytochemicals endowed traditional medicinal plants, since they present exciting
opportunities for the development of new types of therapeutics for the management of diabetes mellitus.
Such phytochemicals include tea polyphenols which suppress Post-Prandial Hyperglycaemia and glucose
transport across the small intestine (Yoshikawa et al., 1999) and saponins which delay glucose transfer
from the stomach to the small intestine (Yuan, 1998; Chatopadhya, 1998). Epicatechin has a restorative
effect on pancreatic β-cells against alloxan damage (Chakravaty, 1982), and plant flavonoids which exert
their antidiabetic activity via antioxidant properties (Bnouham et al., 2006). These reports have
accelerated the global efforts to harness and harvest those medicinal plants that bear substantial amount
of potential phytochemicals showing multiple beneficial effects in combating diabetes and diabetes-
related complications (Tiwari and Rao, 2002).

In our laboratory, the antidiabetic activities of Azadirachta indica (AI), Vernonia amygdalina (VA) and
Gongronema latifolium (GL) have been reported. In a recent report, the chemical components thought to
exert the antidiabetic action were compared (Atangwho et al., 2009a). Although extracts from these plants
have individually demonstrated antidiabetic action, recent evidence from our laboratory show that
antidiabetic efficacy of the extracts is enhanced when given in combination (Ebong et al., 2008; Atangwho
et al., 2009b; Atangwho et al., 2010b). It therefore became absolutely necessary to investigate the
biochemical and histological mechanism(s) of the enhanced action of this combination of extracts.
Accordingly, the present study was set up to investigate the effect of combined extracts of V. amygdalina
and G. latifolium on blood glucose and the histology (morphology) of the pancreas, with a view to
ascertaining whether or not the antidiabetic action is exerted via pancreatic (β-cell) action/modulation.

Vernonia amygdalina (VA) commonly known as bitter leaf is a shrub or small tree of 2 – 5 m belonging to
the family Asteraceae. It has petiolate leaves of about 6 mm diameter and elliptic shape. The leaves are
green with a characteristic odour and a bitter taste (Singha, 1966). In many parts of Nigeria, the plant has

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 British Journal of Medicine & Medical Research, 1(1): 24-34, 2011

been domesticated (Igile et al., 1994)). It is known as ‘Ewuro’ in Yoruba, ‘Etidot’ in Ibibio, ‘Onugbu’ in Igbo
and ‘Chusa-diki’ in Hausa tribes in Nigeria (Egedigwe, 2010). VA grows under a range of ecological
zones in Africa and produces large mass of forage and is drought tolerant; it is found in homes in villages
as fence post and pot-herb (Bonsi et al., 1995). The leaves are used as soup condiment and washed
before eating to get rid of the bitter taste. They are used as vegetable in meals to stimulate the digestive
system, and as a treatment for fever. A wide array of phytochemicals has been shown to be present in
VA. The presence of oxalates, phytates and tannins have been reported (Udensi et al., 2002; Ejoh et al.,
2007; Eleyinmi et al., 2008), as well as flavonoids (Igile et al., 1994; Udensi et al., 2002; Tona et al.,
2004). VA extracts have been shown to exhibit profound ethnomedical and pharmacological properties
viz, anti-diabetic, antimalarial, antihelminthic and antibiotic properties (Farombi, 2003).

Gongronema latifolium is a tropical rainforest plant belonging to the Ascepiadaceae family

(Chattopadhyay, 1998; 1999; Chattopadhayay et al., 1992). It is a shrub, with milky or less often, clear
latex. The leaves are simple, opposite or occasionally whorled, very rarely alternate, usually without
obvious stipules, and the margins are nearly always entire (Bingtao et al., 1977) G. latifolium is a
perennial edible plant with soft and pliable stem. It is widely used in the West African sub region for a
number of medicinal and nutritional purposes. In the South-Eastern States of Nigeria, the plant is known
locally as “Utazi” and used primarily as a staple vegetable/ spice (Ugochukwu et al., 2003; Morebise et
al., 2002; Ugochukwu and Babady, 2002). G. latifolium has a very bitter taste and its phytochemical
composition indicates that it contains saponins (Morebise et al., 2002). In some African cultures it is used
as a spice to support the pancreas (Okafor and Ham, 1999), and in the United States it is also a
constituent of a DM Tea blend marketed for the maintenance of healthy blood glucose levels.

Consequently, the aim of this study is to investigate the efficacy of the extracts of Vernonia amygdalina
and Gongronema latifolium in combination on the blood sugar and pancreas of streptozotocin induced
diabetic Wistar rats.

2. MATERIALS AND METHODS

2.1 COLLECTION AND PREPARATION OF PLANT EXTRACTS

Four hundred grammes (400g) each of V. amygdalina and G. latifolium leaves harvested from the
Endocrine Research Farm of the University of Calabar were separately homogenized in 80% ethanol
(V/V) using an electric homogenizer (QL4-18L40). The homogenates were allowed in a refrigerator for 48
hours at 4˚C after which, they were filtered using a cheese material and afterwards WhatMan No. 1 filter
paper. The filtrates were separately concentrated at 37- 40˚C under reduced pressure, using a rotary
evaporator to one-tenth of their original volumes. These were then allowed in a water bath for evaporation
to dryness yielding 49.1g (12.28%) and 26.7g (6.68%) for V. amygdalina and G. latifolium, respectively.

2.2 EXPERIMENTAL ANIMALS

Thirty-two (32) male albino rats weighing 140-180g were used for the work. The animals were obtained
from the animal house of the Department of Pharmocology, University of Calabar and allowed to
acclimatize in the animal house of the Department of Human Anatomy, Faculty of Basic Medical
Sciences, University of Calabar for about 3 weeks prior to experimentation. They were kept in properly
ventilated cages, where bedding was replaced every two days, at a room temperature of about 27oC and
12 hour light/dark cycle. The animals were fed with growers’ marsh and water obtained from tap ad
libitum.

2.3 INDUCTION OF EXPERIMENTAL DIABETES

Diabetes was induced by intra-peritoneal injection of streptozotocin (STZ) (Sigma St. Louis, M.O. USA)
(Batch No. U925) at a dose of 65mg/kg b.w., reconstituted in normal saline. Prior diabetes induction, the
animals were fasted for 12 hours. Confirmation of diabetes was done seven days after STZ treatment
(Fasting Blood Sugar), using One Touch glucometer (Lifescan, Inc 1995 Milpas, California, USA). Blood

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 British Journal of Medicine & Medical Research, 1(1): 24-34, 2011

sample for the FBS determination was obtained from tail puncture of the rats, and animals with FBS ≥
200mg/dl were considered diabetic and included in the study as diabetic animals.

2.4 EXPERIMENTAL DESIGN

Animal grouping and treatment schedule is as shown in Table 1. The dosages of plant extracts and
insulin used were according to the methods of Ebong et al., (2006) and Sonia and Scrinivasan (1999)
respectively. Plant extracts were administered twice daily via orogastric intubation for 28 days (at 6.00 am
and 6.00pm) and insulin subcutaneously once a day post prandial. At the end of the 28 days, food was
withdrawn from the rats and they were fasted overnight, but with free access to water. They were then
euthanized under chloroform vapour and sacrificed. The pancreas was surgically removed, immediately
blotted using filter paper to remove traces of blood, weighed with an analytical balance then fixed in 10%
formal saline preparatory to histological processing. Whole blood was also collected via cardiac puncture
using sterile syringes into plain tubes and allowed for about two hours to clot. This was centrifuged at
3000rpm for 10mins and serum separated from the clotted blood and used for glucose assay.

Table 1. Experimental design

Group No. of Treatments Dosage

rats
A: Normal control 8 Normal saline 0.2 ml

B: Diabetic control 8 Normal saline 0.2 ml

C: Diabetic extract treated 8 V. amygdalina and G. 200 mg/kg b.w.
group latifolium leaf extracts (100mg/kg b.w. each)
D: Diabetic Standard 8 Humulin 5 IU/kg b.w.
control, insulin treated

2.5 GLUCOSE ESTIMATION

Randox-assay kit (GOD-PAP) method based on Barham and Trinder (1972) was used. The principle
involves the enzymatic oxidation of glucose in sample by the enzyme Glucose oxidase which generates
hydrogen peroxide and gluconic acid. The concentration of H2O2 released is proportional to initial amount
of glucose in the sample and it reacts under catalysis of peroxidase, with phenol and 4-amino phenazone
to form a red violet quinoneimine dye whose colour intensity reflects the concentration of glucose in the
sample.

2.6 HISTOPATHOLOGICAL STUDIES

The fixed pancreatic tissues were sectioned (5-micron thickness) and sections firstly stained with basic
dyes, of Heamatoxylin and Eosin (H&E) according to Conn (Conn, 1946) procedure and later pancreatic
sections were specifically stained for beta cells by the aldehyde fuschin procedure (Gomori aldehyde
method) and photomicrographs (x 400) developed (see result section).

2.7 STATISTICAL ANALYSIS

Glucose and weight measurements are presented as mean ± SE. One way Analysis of Variance
(ANOVA) and the LSD post hoc test were used to analysis the data (p<0.05).

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 British Journal of Medicine & Medical Research, 1(1): 24-34, 2011

3. RESULTS

3.1 MORPHOLOGICAL AND BIOCHEMICAL OBSERVATION

From tables 2 and 3 it can be said that, there was a significant variation (p<0.05) in the weight and serum
glucose concentration of the animals among the treatment groups. Body weight of diabetic control
animals was decreased significantly by -10.5% (p<0.05) when compared to the initial body weight,
whereas the normal control and the treated groups showed a significant increases (p<0.05) in the final
body weight. The extent of increase in body weight of the extract treated animals (7.6%) was similar
(p>0.05) when compared to the Normal (6.8%) and Standard (8.9%) controls. As a converse to the effect
of diabetes on weight, there was a significant increase, 53.23% (p<0.05) in the serum glucose level of the
untreated diabetic animals relative to the normal control. Decrease in serum glucose of animals treated
with plant extracts (12.49%) was significant (p<0.05) and it was observably similar to that of the standard
control animals, humulin (14.96%). Combined extracts of VA and GL mimic insulin in these two respects.

Table 2. Variation in body weight of the animals in the various experimental/ treatment groups

Group Initial (g) Final (g) % Increase

A 148.93±2.92 170.58±3.51b 6.8
a,c
B 147.58±3.05 119.73±2.71 -10.5
C 146.98±3.07 171.49±2.28b 7.6d
D 147.23±2.23 176.20±6.02b 8.9d

Values are expressed as Mean ± SEM, n = 8

Table 3. Variation in serum glucose of the animals in the various experimental/ treatment groups

Group Initial serum Final serum glucose % Decrease

glucose
A 69.83±5.31 65.62± 2.47 3.11

B 74.65±4.02 244.62±8.64c,b -53.23

C 75.83±2.32 59.00±2.09d 12.49

D 73.33±3.61 54.25±1.33d 14.96

Values are expressed as Mean ± SEM, n=8, a= significant decrease at p<0.05 vs initial value
b= significant increase at p<0.05 vs initial value, c= p<0.05 vs NC, d= p<0.05 vs DC.

3.2 HISTOLOGICAL OBSERVATIONS

Effect of the combined extracts on the cyto-architecture of the pancreas in this work was studied using
H/E staining technique and the Gomori Aldehyde Fuschin (GAF) staining method for pancreatic islet cells.
Stained sections were examined using the light microscope.

3.2.1 HAEMATOXYLIN EOSIN (H & E) STAIN

Normal Control (PLATE 1a): The pancreatic ducts are properly outlined; the islet cells are prominent and
well circumscribed. Also, there are clearly defined secretory acini, centoracinar cells and excretory ducts.

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Diabetic control (PLATE 2a): The secretory acini and centroacinar cells are present, the lobules are
distorted and the Islet cells are necrotic and appeared degenerated.
Vernonia amygdalina and Gongronema latifolium treated (PLATE 3a): The pancreatic islet cells are
clearly defined and prominent, the excretory duct is present, loblues are well defined and the acinar cells
are present.
Humulin (PLATE 4a): Presence of vacuolations, with necrotic acinar cells and Islet cells which appeared
degenerated.

1a. H & E 1b. GAF

PLATE 1. Photomicrographs (x 400) of pancreas of Normal Control rats given placebo treatment
H&E= Haematoxylin and eosin, GAF=Gomori Aldehyde Fuschin stain

2a. H&E 2b. GAF

PLATE 2. Photomicrographs (x 400) of pancreas of Diabetic rats given placebo treatment
H&E= Haematoxylin and eosin, GAF=Gomori Aldehyde Fuschin stain

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 British Journal of
o Medicine & Me
edical Research,, 1(1): 24-34, 201
11

3.2.2 GO
OMORI ALDEHYDE FUS
SCHIN STAIN

Normal coontrol (PLATE E 1b): Islet ce

ells are clearlyy defined, prominent and well
w circumscrribed.
Diabetic control
c (PLAT TE 2b): Islet ce
ells appear with
w distortion of the pancre eatic cyto-arch
hitecture indiccating
cytotoxic action
a of streptozotocin.

3a. H&E 3b.. GAF

PLATE 3:
3 Photomicrrograph (x 40 00) of pancre
eas of Diabettic rats treate
ed with combbined extrac
cts of
atifolium 200
V. amygdallina and G. la 0mg/kg b.w. (100mg/kg
( b.w. of each extract).
e
H&E=
H Haemato
oxylin and eosin
n, GAF=Gomo
ori Aldehyde Fu
uschin stain

4a.. H&E 4b. GAF

PLATE 4.. Photomicro

ograph (x400
0) of pancrea
as of Diabetic
c rats treated
d with
Humulin (insulin) 5IU/k
kg b.w.
H&E=
H Haemato
oxylin and eosin
n, GAF=Gomo
ori Aldehyde Fu
uschin stain

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 British Journal of Medicine & Medical Research, 1(1): 24-34, 2011

Vernonia amygdalina and Gongronema latifolium treated (PLATE 3b): The islet cells are present,
indicating regeneration of hitherto destroyed β-cells.
Humulin (PLATE 4b): The Islet cells are distorted, appearing degenerated with presence of vacuolations,
and hence insulin may not have any effect on the pancreas.

4. DISCUSSION

The results from this study revealed significant loss of weight of untreated diabetic rats compared to non-
diabetic animals. This may be due to the loss in muscle and adipose tissue resulting from excessive
breakdown of tissue protein and fatty acids (Granner, 1996). Glycosuria is known to cause a significant
loss of calories for every gram of glucose excreted and presumably this loss results in severe weight loss
in spite of increased appetite, especially when it is coupled with loss of muscle and adipose tissue due to
excessive breakdown of protein. Weight loss is one of the symptoms of diabetes mellitus occurring
especially when glycaemic control is poor. Studies have equally reported significant weight reduction in
untreated diabetic rats (Ahmed et al., 2005).

However, the animals treated with humulin tended to gain more weight than the extract treated group.
This agrees with the report of Makimattila et al., (1999) that improved glycaemic control by insulin
promotes weight gain by decreasing both metabolic rate and glycosuria. Such severe weight loss was
prevented in the extract treated group probably due to interaction of several bioactives. However the
extracts treated animals showed appreciable increase in weight compared to the diabetic control group.
This appreciation in weight indicates that the treatment allowed the tissues to access the glucose both to
supply energy and spared some to build tissue materials required for growth. Studies by Neminibio-audia
(2003) showed that treatment with extracts of root VA resulted in appreciation in weight of the animals
after 14 days.

The histology of the pancreas in the non-diabetic group showed normal pancreatic tissues with endocrine
and exocrine parts. The endocrine portion showed normal islet cells. However, in the diabetic control
animals, there was a marked distortion of the pancreatic cyto-architecture with necrotic and degenerated
islet cells. This is in line with the report by Bolkent et al. (2000) and Noor et al. (2008) that streptozocin
damages the pancreatic tissues especially the islet cells, resulting in a prototype of Type 1 diabetes. STZ
induced pancreatic lesions that resulted in full blown diabetes (Szkudelski, 2001).

In the groups treated with the extracts, there was proliferation of the islet cells; the cyto-architecture of the
pancreas was almost normal, with proliferation of the islet cells and other cells of the pancreas. This
agrees with our earlier report on administration of extracts of VA alone (Atangwho et al., 2010a). However
the recovery in this previous report was partial. The present observation in which the extract is co-
administered with GL appears to be complete and holistic, suggesting a synergistic interaction of the
abundant phytochemicals and antioxidants from the two different sources. Previous studies by Ebong et
al. (2006) reported this possible synergistic action using the extracts of Azadirachta indica and Vernonia
amygdalina. These observations on the effect of the extracts on the pancreatic lesions could also explain
the antihyperglycaemic/ hypoglycaemic effects of these extracts as the regenerated islet cells could
cause an increase in insulin production and secretion.

Vernonia amygdalina extract contains active ingredients such as vernoniosides, glucosides, (VA)
flavonoids and antioxidants (Jisaka et al., 1993) which may be responsible for their potentials in reversing
pancreatic damage caused by STZ in experimental animals. Several studies have demonstrated the
antioxidant properties and components of VA and GL. VA is reported to be rich in antioxidants
composition (Igile et al., 1995) whereas studies by Nwanjo et al. (2006) have demonstrated the
antioxidant properties of Gongronema latifolium.

Diabetes mellitus is known to be associated with the generation of Reactive Oxygen Species (ROS) and
this affects the antioxidant reaction catalysed by ROS scavenging enzymes, resulting in tissue damage.
Studies by Kesavulu et al. (2000) demonstrated the defects in ROS scavenging enzymes in DM. A plant

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extract that has the ability to ameliorate or reverse pancreatic lesions that results in hypoglycaemia would
by one mechanism or the other address the ROS generation or process. This means that such plants
must possess antioxidant properties which would reverse the cytotoxic cycle of STZ in the pancreas or
mob up the ROS in circulation.

The pancreatic islet cells of the insulin treated animals appeared distorted and degenerated with
vacuolations in the exocrine portions. There was indeed no observable difference in the pancreatic islet
cyto-architecture compared to the diabetic control group. This is in accordance with earlier reports by
Pirola et al., (2004) that insulin does not act directly on the pancreas to reduce blood sugar levels but on
peripheral tissues, facilitating the uptake of glucose into the cells, thereby reducing blood glucose levels.
The hyperglycaemia observed in the Diabetic Control Group suggests that the insulin producing cells of
the pancreatic islets were destroyed. This agrees with reports by Rodrigues et al., (1999) that STZ
selectively destroys insulin producing cells of the pancreas and it induces a prototype of Type 1 diabetes.

Extracts from the leaves of V. amygdalina and G. latifolium when combined produced reduction in
glucose level similar in extent to insulin treatment insulin (Humulin). Plant extracts are known to contain
phytochemicals including tannins, saponins, polyphenols and alkaloids and dietary fibre which are said to
contribute to their blood sugar lowering effect (Tiwari and Rao, 2002). Micronutrients such as venadium
which have insulin-like activities are present in some plant extracts as well and this can equally account
for their hypoglycaemic potency. Yeh et al. (2003) stated that majority of plant extracts exert their blood
sugar lowering effects via insulin-like micronutrients present in the extracts. Besides the presence of
phytochemicals and micronutrients in plant extracts which exert direct effect on blood sugar, the
proliferation/regeneration of the pancreatic islet cells which was evident in this study could also account
for the reduction in blood glucose of treated animals. Treatment with insulin (Humulin) significantly
reduced the blood glucose to levels below normal control. This is in accordance with reports by Rother,
(2007) that treatment with insulin is usually a common cause of hypoglycaemic attacks in diabetic
subjects.

5. CONCLUSION

Combined extracts of VA and GL may exert their antidiabetic action via pancreatic islet regeneration.
However, viability of the regenerated cells with respect to production and secretion of insulin is necessary
to validate this observation.

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