Substitution of Combined Extracts of Gongronema latifolium and Nauclea
latifolia with Insulin Requirement in the Management of Type I Diabetes
Grace Sylvester Effiong,
Herbert O.C. Mbagwu,
Imoh E. Udoh,
Nsikan M. Udo,
Item J. Atangwho,
Patrick E. Ebong,
Ekpo N. Asuquo
Lucy A. Wilson
Polyherbal therapy, sometimes called polyherbalism-a combination of herbs or
phytochemicals from more than one source, has today become accepted as an effective
therapeutic approach in sourcing medicament for degenerative ailments. The present
study, investigated the effect of combined extracts from Gongronema latifolium
(GL) and Nauclea latifolia (NL) on some hormonal indices of normal and
diabetic rats. Type 1 diabetes was induced with streptozotocin (50 mg kg-1
b.w.) and the hormonal parameters were measured using standard methods. Measured
c-peptide levels showed significant increase in diabetic rats treated with the
combined extracts and only GL relative to non-diabetic control. Whereas there
were significant increase (p<0.05) in serum triiodothyronine (T3)
and tetraiodothyronine (T4) concentrations of diabetic control when
compared to normal control, administration of both combined extracts and insulin
caused significant reduction (p<0.05) in T3 and T4
relative to the diabetic control, although, this observation differed in the
non diabetic test animals. Measured serum insulin level was significantly reduced
(p<0.05) in untreated diabetic rats relative to normal control, administration
of only GL and its combination with NL extracts also reduced the level in a
manner similar to insulin administration. On the contrary, normal rats that
received similar treatments showed significant increase (p<0.05) in serum
insulin level. Therefore, combined extracts of Gongronema latifolium
and Nauclea latifolia may mimic insulin in its antihyperglycemic action
at least in rats.
to cite this article:
Grace Sylvester Effiong, Herbert O.C. Mbagwu, Imoh E. Udoh, Nsikan M. Udo, Item J. Atangwho, Patrick E. Ebong, Ekpo N. Asuquo and Lucy A. Wilson, 2013. Substitution of Combined Extracts of Gongronema latifolium and Nauclea
latifolia with Insulin Requirement in the Management of Type I Diabetes. Research Journal of Medicinal Plants, 7: 107-112.
Received: February 18, 2013;
Accepted: March 12, 2013;
Published: July 02, 2013
The use of folk medicine in Africa and most Asian countries is prevalent, and
the searches for herbal cures are the common practices especially among the
low income groups and peasants. According to Okoli et
al. (2007), in the past four decades, Africans had been using the traditional
medicine for their health care before the introduction of orthodox medicine.
Focho et al. (2009) reported on the usage of
traditional medicine by 80% of developing countries for their health care. Gongronema
latifolium (GL) (Asclepiadaceae) and Nauclea latifolia (NL) (Rubiaceae)
are two plants used traditionally in the management of diabetes in the African
sub-region and Asia. Gongronema latifolium (Asclepiadaceae) is a perennial
edible plant with soft and pliable stem. In the West African sub-region, GL
is widely used for both medicinal and nutritional purposes (Nwanjo
et al., 2006). Apart from hypoglycemic activity, GL also possesses
hepatoprotective and hypolipidaemic effects (Ugochukwu and
Babady, 2003; Ugochukwu et al., 2003; Nwanjo,
2005 and Nwanjo and Alumanah, 2005). On the other
hand, Nauclea latifolia is from the family Rubiaceae. Traditional medicine
practitioners profusely use NL, commonly known as Pin cushion tree. Gidado
et al. (2005) reported the effect of N. latifolia on blood
glucose levels in diabetic rats while the hypoglycemic activity is reported
by Gidado et al. (2008).
Although, there are many investigations reported on GL and NL concerning antidiabetic
action, few literatures are available on their mechanism(s) of action in hypoglycemia
and antihyperglycemia. Moreover, the combined extracts of GL and NL had not
undergone biochemical studies. Atangwho et al. (2010)
reported on a recent pharmacological concept that has the advantage of producing
maximum therapeutic efficacy with minimum side effects.
Consequently, this present study investigated in part the mode by which extracts
of Gongronema latifolium and Nauclea latifolia singly and in combination
mediate or exert their anti-diabetic effects by evaluating some serum hormonal
indices of diabetic rats given single and combined extracts from these plants.
MATERIALS AND METHODS
Collection and preparation of plants
extracts: Fresh but matured Gongronema latifolium leaves were collected
from a cultivated land at Ibiaku Itam, Nigeria while Nauclea latifolia
was collected in Calabar, Nigeria in March, 2012. They were authenticated by
Dr. E.G. Amanke in the Department of Botany, University of Calabar, Nigeria
and voucher specimen deposited in the Department of Botany herbarium, University
of Calabar. The leaves were rinsed severally with clean tap water to remove
particles and debris and thereafter allowed to completely drain. The ethanol
extract was prepared using the wet method of extraction; one kilogramme of the
fresh leaves of the plants were separately cut and chopped into pieces with
a knife on a chopping board, blended in 1.5 L of ethanol (96%) with an electric
blender and transferred into amber colored bottle and kept in cool (4°C)
dark compartment for 72 h. These were then filtered using a cheese cloth and
thereafter with Whatman No. 1 filter paper to obtain a homogenous filtrate.
These filtrate were then concentrated in vacuo using a rotary evaporator
at 37-40°C to about one tenth the original volume. The concentrates were
allowed open in a water bath (40°C) to dryness yielding 30.00 and 78.95
g of greenish brown and brown oily substances of G. latifolium and N.
latifolia, respectively. They were dried completely in a desiccator containing
a self-indicating silica gel and then refrigerated at 2-8°C until used.
Animals and experimental design: Sixty male Albino Wistar rats of 150-200
g obtained from the animal house of the Department of Pharmacology and Toxicology,
Faculty of Pharmacy of the University of Uyo, Nigeria were used. They were kept
in clean cages (wooden bottom and wire mesh top), maintained under standard
laboratory conditions (Temperature 25±5°C, Relative humidity 50-60%
and a 12/12 h light/dark cycle) and were allowed free access to standard diet
(Vital Feed from Grand Cereals and Oil Mills Limited, Jos, Plateau State of
Nigeria) and water ad libitum. Animals were acclimatized for 14 days
in the animal house of the Department of Biochemistry, University of Calabar,
Nigeria, before each of the experiments. The 60 rats were divided into 5 parallel
groups consisting of a diabetic and non-diabetic pair of 6 animals each (Table
|| Treatment schedule for diabetic and non diabetic rats
|GL: Gongronemal latifolium, D: Diabetic, NL: Naucela
latifolia, ND: Non-diabetic
Experimental induction of diabetes: Diabetes was induced in an overnight
fasted animals by a single intraperitoneal injection of freshly prepared solution
of streptozotocin (Sigma, USA) 50 mg kg-1 b.wt. in 0.1 M cold sodium
citrate buffer pH 4.5 (Ghoraishian, 2006; Rao
and Naidu, 2010). The animals were considered as being diabetic if the blood
glucose value were >200 mg dL-1 on the third day after streptozotocin
injection and were used in the experiment. This was estimated using One Touch
Glucometer (Lifescan, inc 1995).
Experimental protocol: Diabetic and non-diabetic animals were grouped
as shown in Table 1 and accordingly, treated with extracts
and/or insulin. The dosages of the plant extracts were as determined from preliminary
work in the laboratory whereas insulin dose, NPH (5 U kg-1 b.wt.)
was as previously used by Atangwho (2008) and also chosen
to simulate human regimen. The plant extracts were administered via gastric
intubation, twice per day (6:00 a.m.: 6:00 p.m.) and insulin once per day post
prandial (6:00 p.m.). The animals were treated for 21 days.
Collection of samples for analysis: At the end of the 21 days, food
was withdrawn from the rats and they were fasted overnight but had free access
to water. They were then euthanized under chloroform vapour and sacrificed.
Whole blood was collected via cardiac puncture using sterile syringes and needles,
emptied into plain tubes and allowed to clot for about two hours. The clotted
blood was thereafter centrifuged at 3,000 rpm for 10 min to recover serum from
clotted cells. Serum was separated with sterile syringes and needles and stored
frozen until used for hormonal analysis.
Estimation of serum hormonal indices: Elisa kits for hormonal assays
including c-peptide, tri- and tetra-iodothyronine were purchased from Monobind
Inc. Lake Forest, CA 92630. U.S.A. and insulin kit from DRG Instruments, GmbH,
Germany, Division of DRG International, Inc Frouenbergergstr.18, D-35039 Marburg.
Serum was used for the estimation of various hormonal parameters: c-peptide
based on Eastham (1985), (T3) based on Gharib
et al. (1971), (T4) based on DIALAB Method and insulin
based on Starr et al. (1978).
Statistical analysis: The results were analyzed for statistical significance
by one way ANOVA using the SPSS statistical program and Post Hoc Test
(LSD) between groups using MS excel program. All data were expressed as Mean±SEM.
p-values<0.05 were considered significant.
Effect of treatment with GL, NL, GL and NL combined and insulin on serum hormonal
indices including C-peptide, triiodothyronine (T3), tetraiodothyronine
(T4) and insulin of non-diabetic and diabetic experimental animals
are shown on Table 2. GL and NL combined treated rats (2.10±0.04)
showed a significant increase (p<0.05) in C-peptide concentration compared
to NC (1.13±0.10).
|| Effect of treatment on some selected hormonal indices in
non diabetic and diabetic rats
|*p<0.05 vs. NC, a: p<0.05 vs. DC, b: p<0.05 vs. Insulin,
c: p<0.05 vs. GL+NL, Mean ± SE, n: 6, D: Diabetic rats, ND: Non-diabetic
rats, DC: Diabetic control, NC: Normal control
However, treatment with extracts of only GL, NL or insulin increased non significantly
the C-peptide levels in diabetic rats compared to NC. The extracts treated non-diabetic
rats showed a significant increases (p<0.05) in C-peptide concentration (GLND
= 3.20±0.06; NLND = 2.57±0.42; (GL+NL)ND
= 2.40±0.38: Where ND = Non diabetic rats) in comparison with NC while
insulin treated rats indicated a non significant difference (p>0.05) compared
to NC. Serum triiodothyronine (T3) decreased significantly in treated
diabetic (GLD = 1.57±0.12; NLD = 1.33±0.02;
(GL+NL)D = 1.25±0.02 Where, D = Diabetic rats) and non-diabetic
(GLND = 1.33±0.48; NLND = 1.42±0.61; (GL+NL)ND
= 1.27±0.61) rats in this study with the exception of insulin treated
non-diabetic rats which showed a non significant decrease compared to NC (3.38±0.32).
The diabetic rats serum tetraiodothyronine (T4) recorded a significant
increase (p<0.05) in DC (8.07±0.26), while a non significant difference
(p>0.05) was observed in treatment with NL singly and when combined with
GL but GL alone (1.67±0.02) and insulin (1.87±0.04) treated rats
indicated a significant decrease (p<0.05) in comparison with NC (2.68±0.27).
In the non-diabetic rats, T4 showed a significant increase in the
individual treated extracts (GLND = 3.30±0.06; NLND
= 3.30±0.10) while the combined extract group and insulin treated rats
recorded a non significant increase compared to NC. There were significant decreases
in serum insulin level of diabetic rats compared to NC (37.72±1.51) in
both test (GLD = 16.63±2.95; NLD = 27.10±6.37;
(GL+NL)D = 21.65±0.16) and control (DC = 13.33±0.02)
groups but NL showed a non significant decrease, also there were non significant
decreases in serum insulin of treated non-diabetic rats compared to NC but NL
individual treatment (29.55±1.51) indicated a significant decrease (p<0.05)
when compared to NC (37.72±1.51).
A type 1 model of diabetes such as was induced by STZ in this experiment is
usually characterised by decreased circulatory insulin whose concentration parallels
that of c-peptide. This was in consonance with this work; it is well established
that c-peptide has a longer half-life of about 3-4 times that of insulin (Starr
et al., 1978; Champe et al., 2005)
and so can be retained in blood in higher concentrations compared to insulin.
Infact, according to Karam and Forsham (1994), 100 pmol
L-1 of c-peptide can still be found in humans plasma after an overnight
fast despite the degrading of insulin by insulinase.
No significant changes in serum C-peptide levels were observed in both test
and control animals, however, treatment with the combined extracts of GL and
NL increased significantly (p<0.05) the C-peptide levels both in diabetic
and non-diabetic rats compared to their respective controls. The NL treatment
reduced the diabetic increased C-peptide levels similar to NC, suggesting that
NL may therefore contain some C-peptide mimetic phytochemicals, T3
and T4 concentration in serum of untreated diabetic rats were increased
significantly. Often, T3 and T4 which are diabetogenic
hormones increased in diabetes thereby sustaining and exerbating hyperglycemia
(Brandenburg, 2008; Mayes, 2000)
and usually, this is antagonistic to insulin. This probably may explain the
significantly decreased T3 and T4 levels in insulin treated
diabetic rats indicated in this study. This observation was similar to the report
of Atangwho et al. (2010).
Treatment with extracts of GL in diabetic rats caused significant decrease
in serum insulin similar to insulin treatment in diabetic rats as opposed to
non significant increase in only NL treated diabetic and non-diabetic rats.
These two extracts when combined reduced insulin levels compared to NC. GL may
therefore contain some insulin mimetic phytochemicals, whose action was complemented
when combined with extracts of NL and moreso further strengthens our proposition
that GL and its combination with NL may have potent insulin mimetic action,
since in these groups, T3 concentration was significantly reduced
just like insulin. Also, this was in consonance with the work of Atangwho
et al. (2010). It also confirms the principle that polyherbal therapy
is said to have the advantage of producing maximum therapeutic efficacy.
Treatment with combined extracts of GL and NL may therefore have the potentials
to be substituted for insulin requirement in the management of type I diabetes.
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