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Antidiabetic study of combined extracts of Vernonia amygdalina, Ocimum

gratissimum, and Gongronema latifolium on alloxaninduced diabetic rats

Article · April 2013

DOI: 10.4103/2229-5119.110345

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 Original Article

Antidiabetic study of combined extracts of Vernonia amygdalina,

Ocimum gratissimum, and Gongronema latifolium on alloxan-
induced diabetic rats

Jude E. Okokon, Uwemedimo F. Umoh1, Benjamin A. J. Ekpo1, Emmanuel I. Etim2

ABSTRACT Departments of
Pharmacology and Toxicology,
Introduction: Herbal products are often used as combinations of some herbs for the treatment of diseases
1
Pharmacognosy and
in traditional medicine. The anti-diabetic activities of polyherbal combinations of Vernonia amygdalina (V. Traditional Medicine, and
amygdalina), Gongronema latifolium (G. latifolium), and Ocimum gratissimum (O. gratissimum) used in
2
Pharmaceutical and Medicinal
Chemistry, Faculty of
folkloric management of diabetes. Materials and Methods: The various polyherbal combinations of V.
Pharmacy, University of Uyo,
amygdalina, G. latifolium, and O. gratissimum (100 mg/kg each) were evaluated for anti-diabetic activity in Uyo, Nigeria
alloxan induced diabetic rats. The anti-diabetic activities during acute and prolonged studies were investigated.
Glibenclamide, 10 mg/kg, was used as positive control. Blood glucose level (BGL) was measured at intervals
by using glucometer. Results: Treatment of alloxan diabetic rats with the various herbal combinations caused
significant (P < 0.001) reductions in the BGL of the diabetic rats both in acute and prolonged treatment (2
weeks). The activities of various combined extracts were comparable and more pronounced than that of
glibenclamide and compared to that of glibenclamide in the prolonged study. Conclusion: The anti-diabetic
activities of the various herbal combinations confirm the folkloric use of these polyherbal remedies.

Key words: Antidiabetic, Gongronema latifolium, Ocimum gratissimum, polyherbal, Vernonia amygdalina

INTRODUCTION Ethnobotanical information from some

Access this article online
Website: www.jnatpharm.org
DOI: 10.4103/2229-5119.110345
Quick Response Code:
Address for
correspondence:
Dr. Jude E. Okokon,
Department of
Pharmacology and
Toxicology, Faculty of
Pharmacy, University of
Uyo, Uyo, Nigeria.
E-mail: judeefiom@
yahoo.com
Ethnomedicine is an integral part of the
culture of people of Akwa Ibom State[1] and
about 75% of the people rely on traditional
medicine for health-care delivery.[2] Majority
of the people still patronize herbal remedies
despite the availability of orthodox medicine
in their management of some diseases and
ailments. Besides, polyherbal therapy
(practice) is a common practice in the Ibibio
traditional medicine as combination of roots/
leaves and stem barks of various plants are
often use in the treatment of a single disease.
Moreover, polyherbal therapy is said to be a
current pharmacological principle having the
advantage of producing maximum efficacy
with minimum side effects. [3] According
to Tiwari and Rao,[4] polyherbal therapies
have the synergy, potentiative, agonistic/
antagonistic pharmacological agents within
themselves, that work together in a dynamic
way to produce therapeutic efficacy with
minimum side-effects. Diabetes is one of
the diseases treated by herbalist of Ibibio
tribe using polyherbal combinations.
traditional medicine practioners revealed
that Vernonia amygdalina (V. amygdalina),
Ocimum gratissimum (O. gratissimum),
and Gongronema latifolium (G. latifolium)
constitute bulk of the many polyherbal
combinations use in the area for the
treatment of diabetes. Ajibesin et al.,[1] in
their ethnobotanical survey of Akwa Ibom
State documented the three plants as anti-
diabetic remedies used in the region. Several
workers have reported on the anti-diabetic/
hypoglycemic activities V. amygdalina,[5-7] O.
gratissimum,[6,8-10] and G. latifolium.[6,11-13] In
this study, we investigated the efficacy of the
different combinations of the three plants
on alloxan induced diabetic rats as used in
ethnomedical therapy of diabetes in Akwa
Ibom State to ascertain their potency.
MATERIALS AND METHODS
Plant materials
Fresh leaves of V. amygdalina Del.
(compositae), O. gratissimum Linn.
(Labiaceace), and G. latifolium Benth.

28 Journal of Natural Pharmaceuticals, Volume 4, Issue 1, January-June, 2013

 Okokon, et al.: Antidiabetic activities of polyherbal combinations on diabetic rats
(Asclapiadaceace) were collected in August, 2008 from
the Medicinal plants farm of Faculty of Pharmacy,
University of Uyo, Uyo, Nigeria. The plant was identified
and authenticated by Dr. Margaret Bassey, a taxonomist
in the Department of Botany, University of Uyo, Uyo,
Nigeria.
Extraction
The fresh leaves (2 kg) of each plant were air-dried
separately on laboratory table for 2 weeks and reduced to
powder. The powder (100 g) of each plant was macerated
in 95% ethanol (300 mL) for 72 h (cold extraction). The
liquid filtrates obtained were concentrated in vacuo at
40°C. The extracts were stored in a refrigerator at 4°C
until used for experiment reported in this study.
Animals
Albino Wistar rats (155-165 g) of either sex were obtained
from the University of Uyo animal house. They were
maintained on standard animal pellets (Guinea Feed) and
water ad libitum. Permission and approval for animal
studies were obtained from the College of Health Sciences
Animal Ethics Committee, University of Uyo.
Induction of diabetes
The animals were fasted overnight and diabetes was
induced by a single intraperitoneal injection of a freshly
prepared solution of alloxan (150 mg/kg body weight)
in ice cold 0.9% sodium chloride (NaCl) saline solution.
The animals were allowed to drink 5% glucose solution
overnight to overcome the drug-induced hypoglycemia.
Control rats were injected with normal saline alone.
After 72 h for the development of diabetes, rats with
moderate diabetes having glycosuria and hyperglycemia
(blood glucose level (BGL) range above 200 mg/dl) were
considered as diabetic and used for the drug treatment.
The leaf extracts of the three plants in aqueous solution
were administered to rats orally through a gavage at
a concentration of 100 mg/kg body weight/rats/day for
14 days.
Experimental design
The animals were divided into six groups of six rats each
and treated as follows:
• Group I: Diabetic rats administered orally with
combined leaf extracts of G. latifolium, V. amygdalina,
and O. gratissimum (GVOE) at a dose of 100 mg/kg/
day each extract in aqueous solution for 14 days.
• Group II: Diabetic rats given leaf extracts of O.
gratissimum and G. latifolium (OGE) at a dose
of 100 mg/kg/day in aqueous solution orally for
14 days.
• Group III: Diabetic rats administered orally with leaf
extracts of G. latifolium and V. amygdalina (GVE) at a
dose of 100 mg/kg/day each extract in aqueous solution
for 14 days.
Journal of Natural Pharmaceuticals, Volume 4, Issue 1, January-June, 2013
• Group IV: Diabetic rats administered orally with leaf
extract of O. gratissimum and V. amygdalina (OVE)
at a dose of 100 mg/kg/day each extract in aqueous
solution for 14 days.
• Group V: Diabetic rats given glibenclamide (10 mg/kg/
day) for 14 days in aqueous solution orally for 14 days.
• Group VI: Diabetic control rats.
The fasting BGLs of all the rats were recorded at regular
intervals during the experimental period. For acute
study, the BGL was monitored after 1, 3, 5, and 7 h of
administration of a single dose of the extract and at
the end of 1, 3, 5, 7, 10, 12, and 14 days for prolonged
treatments. The BGL was monitored in the blood of
the diabetic rats by tail tipping method. The blood was
dropped on the dextrostix reagent pad and was inserted
into microprocessor digital blood glucometer and the
readings were noted.[14]
Statistical analysis
Data are reported as mean ± standard error of the mean
(SEM) and were analyzed statistically using one-way
analysis of variance followed by Tukey–Kramer multiple
comparison test and values of P < 0.001 and 0.05 were
considered significant.[15]
RESULTS
During acute study of the effects of the combined
extracts on BGL of the diabetic rats, all the groups
treated with combined leaf extracts exhibited
significant (P < 0.001) reductions in BGL compared
to control and the effects were more pronounced
than those of the group treated with glibenclamide
(10 mg/kg). Though, all the combined extracts-treated
groups had comparable activities, the group treated
with the combined leaf OGE had the most significant
reduction after 7 h (80.97%,7 h), followed by those
treated with GVE (79.21%, 7 h), GVOE (76.93%,
7 h), and OVE (74.90%,7 h) [Table 1].
During prolonged study, the combined extracts-treated
groups and glibenclamide treated group produced
sustained significant (P < 0.001) reduction in BGL of
the diabetic compared to control [Table 2]. The effect of
the combined extracts and that of glibenclamide were
comparable on day 15. A more pronounced effect was
observed in the group treated with a combined extracts
of the three plants, i.e., GVOE.
DISCUSSION
The anti-diabetic effects of different combinations of
leaf extracts of G. latifolium, V. amygdalina and O.
gratissimum use in polyherbal therapy of diabetes
29
 Okokon, et al.: Antidiabetic activities of polyherbal combinations on diabetic rats

Table 1: Effect of polyherbal combinations on blood glucose levels of alloxan diabetic rats after a single dose
Drug Dose (mg/kg) Blood glucose level (mg/dl) (mean±SEM)
Ini al 1h 3h 5h 7h
Control 240.3 ± 1.05 245.2 ± 1.39 262.9 ± 0.75 264.7 ± 0.95 266.2 ± 0.95
Extracts
GVOE 100/extract 247.0 ± 2.83 216.6 ± 2.13* 150.0 ± 3.32* 91.0 ± 2.00* 57.0 ± 2.24*
OGE 100/extract 260.0 ± 2.86 167.5 ± 1.30* 164.0 ± 3.65* 67.0 ± 1.09* 49.5 ± 1.59*
GVE 100/extract 252.5 ± 2.50 105.0 ± 2.20 100.5 ± 2.23 78.5 ± 2.65* 52.5 ± 1.45*
OVE 100/extract 247.0 ± 0.24 149.0 ± 3.28* 112.0 ± 1.36* 69.3 ± 2.25* 62.0 ± 1.11*
Glibenclamide 10 243.8 ± 1.42 169.4 ± 2.38* 162.4 ± 2.11* 148.2 ± 0.51* 130.3 ± 0.75*
*P<0.001 when compared to control. n=6 per group. GVOE: Gongronema la folium, Vernonia amygdalina and Ocimum gra ssimum combined extracts, OGE: Ocimum
gra ssimum and Gongronema la folium combined extracts, GVE: Gongronema la folium and Vernonia amygdalina combined extracts, OVE: Ocimum gra ssimum and Vernonia
amygdalina combined extracts, SEM: Standard error of the mean

Table 2: Effect of polyherbal combinations on blood glucose levels of alloxan diabetic rats during prolonged treatment
Treatment Dose (mg/kg) Blood glucose level (mg/dl) (mean±SEM)
Ini al 1st day 3rd day 5th day 7th day 10th day 12th day 15th day
Control 240.0 ± 1.05 245.2 ± 1.39 260.1 ± 1.51 263.2 ± 0.98 265.2 ± 1.92 265.3 ± 1.26 268.7 ± 1.34 270.2 ± 0.82
Extracts
GVOE 100/extract 247.0 ± 2.83 162.0 ± 1.27* 86.0 ± 2.00* 78.3 ± 1.19* 72.6 ± 1.26* 61.8 ± 1.47* 51.5 ± 1.53* 51.0 ± 1.83*
OGE 100/extract 260.0 ± 2.86 175.5 ± 1.75* 85.0 ± 1.73* 76.8 ± 1.90* 71.0 ± 1.15* 66.5 ± 1.50* 63.4 ± 1.53* 56.3 ± 2.41*
GVE 100/extract 252.5 ± 2.50 186.5 ± 2.91* 92.3 ± 1.08* 86.5 ± 1.54* 81.2 ± 1.59* 74.4 ± 1.15* 63.8 ± 1.03* 53.2 ± 2.14*
OVE 100/extract 247.0 ± 0.24 154.0 ± 1.12* 91.4 ± 1.44* 89.7 ± 1.64* 77.5 ± 1.12* 68.8 ± 2.42* 62.5 ± 2.91* 57.9 ± 1.36*
Glibenclamide 10 243.8 ± 1.42 13.1 ± 2.28* 78.2 ± 1.26* 73.8 ± 1.43* 67.6 ± 1.12* 65.4 ± 1.33* 60.3 ± 3.11* 55.4 ± 1.25*
*P<0.001 when compared to control, n=6 per group. GVOE: Gongronema la folium, Vernonia amygdalina and Ocimum gra ssimum combined extracts, OGE: Ocimum
gra ssimum and Gongronema la folium combined extracts, GVE: Gongronema la folium and Vernonia amygdalina combined extracts, OVE: Ocimum gra ssimum and Vernonia
amygdalina combined extracts, SEM: Standard error of the mean

were evaluated in this study. The results showed
that the various combinations: G. latifolium and O.
gratissimum; G. latifolium and V. amygdalina; G.
latifolium, V. amygdalina, and O. gratissimum; and
O. gratissimum and V. amygdalina, had significant
antihyperglycemic activity which was more than that
of glibenclamide (in acute study) and comparable in
effect to glibenclamide (during prolonged study). Within
the treatment period (14 days) the various combined
extracts’ effects demonstrated synergism as the extracts
appeared to complement each other thereby reducing
the BGL of the diabetic rats to normal. This was clearly
demonstrated in the group administered combination
of the three extracts during prolonged study. The
results of this study collaborates the advantages
of polyherbal articulated by Tiwari and Rao. [4]
Furthermore, the results of this study support earlier
reports of anti-diabetic potentials of these plants.[5-10]
Different mechanism of actions of anti-diabetic plants
have been proposed such as potentiation of insulin
effect either by increasing the pancreatic secretion
of insulin from the cells of islets of Langerhans or its
release from bound insulin,[16] inhibition of hepatic
glucose production,[17] inhibition of intestinal glucose
absorption, [18] or correction of insulin resistance.[19]
These three extracts may have exerted their anti-
diabetic effects by utilizing one or more of the above
mechanisms. A combination of these mechanisms could
have resulted in the significant anti-diabetic activity
observed in this study, which is likely to be sustained
and better than that of a single extract. Moreover,
sulfonyl ureas, including glibenclamide, produce
hypoglycemia in normal as well as diabetic animals
by stimulating the pancreatic -cells to release more
insulin.[20] These extracts’ combinations maybe working
through a similar mechanism like glibenclamide as the
results gotten from the combinations and glibenclamide
were comparable. Secondary metabolites have been
reported to be involved in anti-diabetic activity of
many plants.[21] Reports have shown that these plants
have various phytochemical constituents in common
such as alkaloids, steroids, glycosides, saponins,
tannins, terpenes, and fl avonoids.[8,22,23] In addition
to anti-diabetic properties of these plants, each of the
plants has been reported of different activities geared
towards alleviation of complications usually associated
with diabetes. For example, V. amygdalina has been
reported to be hepatoprotective[24] and antioxidant,[25]
G. latifolium, antioxidant, hepatoprotective and
hypolipidaemic, [11,26] O. gratissimum, antioxidant [27]
and others. All these activities complements the anti-
diabetic activities of these plants and are advantageous
to antagonize and resolve any side effects any of
the extracts could have posed. The effectiveness of
polyherbal combination has been reported widely. V.
amygdalina and Azadirachta indica combination has
been shown to be very effective.[3]

30 Journal of Natural Pharmaceuticals, Volume 4, Issue 1, January-June, 2013

 Okokon, et al.: Antidiabetic activities of polyherbal combinations on diabetic rats
CONCLUSION
The finding of this study confirms the efficacy of the
various polyherbal combinations used traditionally by
herbalists of Ibibio tribe of Akwa Ibom State, Nigeria
for the management of diabetes which are of advantage.
Further studies into polyherbal combinations used in the
management of diabetes are encouraged to support the
advantage of polytherapy.
ACKNOWLEDGMENT
The authors are grateful to Mr. Nsikan Malachy of Pharmacology
and Toxicology Department, University of Uyo, Uyo, Nigeria for
his technical assistance.
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Cite this article as: Okokon JE, Umoh UF, Ekpo BA, Etim EI. Antidiabetic
study of combined extracts of Vernonia amygdalina, Ocimum gratissimum,
and Gongronema latifolium on alloxan-induced diabetic rats. J Nat Pharm
2013;4:28-31.
Source of Support: Nil. Conflict of Interest: No.
31