Biomedicine & Pharmacotherapy 86 (2017) 562569

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Modulation of the lipid prole and insulin levels of streptozotocin

induced diabetic rats by ethanol extract of Cnidoscolus aconitifolius
leaves and some fractions: Effect on the oral glucose tolerance of
normoglycemic rats
N.K. Achia,* , O.C. Ohaeria , I.I. Ijeha , C. Eleazub
a
Department of Biochemistry, Michael Okpara University of Agriculture, Umudike, Nigeria
b
Federal University, Ndufu-Alike, Ikwo, Ebonyi State, Nigeria

A R T I C L E I N F O A B S T R A C T

Article history: Background: No study to date has investigated the effect of different polar solvent extracts from
Received 27 October 2016 Cnidoscolus aconitifolius leaves on glycemic control as used in folk medicine. Hence this study which
Received in revised form 15 November 2016 investigated the effect of ethanol extract and fractions of C. aconitifolius leaves on body weights, relative
Accepted 28 November 2016
organ weights, serum levels of glucose, lipid proles and insulin in streptozotocin induced diabetic rats
and on oral glucose tolerance of normoglycemic rats.
Keywords: Methods: The ethanol extract was partitioned using methanol, hexane and chloroform to obtain different
Diabetes
fractions.
Cnidoscolus aconitifolius
Herbal medicine
Results: The ethanol extract, fractions or glibenclamide demonstrated hypoglycemic/therapeutic actions
Glucose tolerance as seen from the reduction of serum glucose but increase in serum insulin and body weights of the
diabetic rats at the end of experimentation following their administration, unlike the diabetic control
that had signicant alteration of these parameters with respect to the normal control. Whereas the
diabetic control had signicant increase in pancreatic weights with no alteration in the heart weights, the
ethanol extract, fractions or glibenclamide had no effect on these organs. The ethanol extract, methanol
fractions or glibenclamide showed better hypoglycemic actions than the n-hexane or chloroform
fractions at the doses used and results obtained were corroborated by histology. Furthermore, the
ethanol extract, n-hexane (at 250 mg/kg) and methanol fractions or glibenclamide improved glucose
tolerance in glucose loaded normal rats. The methanol fraction (500 mg/kg) demonstrated anti-
hypercholesterolemic, anti-hypertriglyceridemic and insulin modulatory properties in a manner akin to
glibenclamide. Acute toxicity study revealed the non toxicity of the plant
Conclusion: The study justies the use of polar solvent extracts of this plant in the management of
diabetes mellitus.
2016 Elsevier Masson SAS. All rights reserved.

1. Introduction
Diabetes mellitus refers to a complex and diverse group of
disorders that affect the metabolism of carbohydrates, lipids and
proteins. It is characterized by high blood glucose levels that result
from an absolute or relative insulin deciency or reduced
sensitivity of the tissues to insulin [1].
The International Diabetes Federation has projected that the
number of people with diabetes in the world will increase from 382
million in 2013 to 592 million by 2035, with 80% of diabetic cases
* Corresponding author.
E-mail address: ngoziachi@outlook.com (N.K. Achi).
occurring in the low and middle income countries if this disease is
not curbed [2].
Alteration of lipid metabolism is one of the factors that have
been implicated in the development of diabetic complications. This
is because in insulin deciency or insensitivity, hormone sensitive
lipase is activated, leading to increased mobilization of free fatty
acids (FFAs) from the peripheral depots with decreased peripheral
utilization. The excess FFAs are catabolized to acetyl CoA. The
acetyl CoA cannot be readily utilized as the availability of
oxaloacetate is reduced and TCA cycle activity is sluggish. The
result is that the excess acetyl CoA is channeled to the pathway of
cholesterol biosynthesis leading to hypercholesterolemia and
hyper-lipidaemia characterized by elevated levels of cholesterol,
triacylglycerol and low density lipoprotein (LDL)-cholesterol [3].

http://dx.doi.org/10.1016/j.biopha.2016.11.133
0753-3322/ 2016 Elsevier Masson SAS. All rights reserved.
 N.K. Achi et al. / Biomedicine & Pharmacotherapy 86 (2017) 562569
The glycation and oxidation of LDL will delay their catabolism and
promote their uptake by macrophages which process is dysregu-
lated. This is the starting point of atherosclerosis leading to
myocardial infarction. Furthermore, alteration in HDL activity
further increases the risk of coronary heart disease in people with
diabetes [4].
The mainstay of the management of diabetes mellitus is
pharmacological (exogenous insulin or hypoglycemic drugs
administration) or non-pharmacological (diet, exercise and
surgery) treatments, all of which measures have their various
shortcomings as per reversing the course of the disease [5]. In
addition, the costs and adverse effects associated with the use of
synthetic drugs in the management of this disease, coupled with
the abundance of plant materials with promising antidiabetic
potentials, have led to the recommendation by the World Health
Organization for the management of diabetes mellitus, using
herbal medicine [6]. Moreover, these medicinal plants play
important roles in the lives of rural people, particularly those in
the remote parts of developing countries that have limited access
to adequate health facilities [3].
Cnidoscolus aconitifolius, known by its English name as tree
spinach, is a perennial plant that is indigenous to Mexico in Central
America. The local names of this plant also vary. For instance, while
it is called efo inyana ipaja by the Yorubas in the Western part of
Nigeria, it is called ogwonuoria by the Ibos in the Eastern part of
Nigeria. In African ethnomedicine, the plant is commonly used in
the management of diabetes mellitus, hypercholesterolemia,
hyperlipidemia, obesity, atherosclerosis and kidney stones [7,8].
In the traditional management of diabetes mellitus and hyper-
cholesterolemia in Nigeria using C. aconitifolius leaves, practi-
tioners often use different polar solvent extracts from the plant to
prepare their traditional medicines. In addition to its therapeutic
value, the leaves of this plant are also eaten as vegetable in soups,
salads in most Nigerian dishes due to the nutritive values [911].
Although C. aconitifolius has been scientically documented as a
hypoglycemic plant, the biochemical basis of its hypoglycemic
action still remains unclear. In a bid to further understand the
biochemical basis of the antidiabetic action of this plant, the
present study therefore sought to investigate the effect of ethanol
extract of C. aconitifolius leaves and its fractions on the body
weights, relative organ weights, serum levels of glucose, lipid
proles and insulin in streptozotocin induced diabetic rats and on
oral glucose tolerance of normoglycemic rats.
2. Materials and methods
2.1. Collection and identication of plant materials
Fresh leaves of Cnidoscolus aconitifolius were collected from the
deciduous forest of Amaekpu in Ohaa Local Government Area of
Abia State, Nigeria. They were identied and authenticated by Mr.
Ibe Ndukwe, a taxonomist in the Hebarium section of the
Department of Forestry and Environmental Management, Michael
Okpara University of Agriculture, Umudike, Abia State, Nigeria. A
voucher specimen with number-MOUAU/107727 was kept at the
herbarium for future reference.
2.2. Preparation of plant extract
The plant extract was prepared as previously described [12].
The leaves were sorted, washed and air dried at room temperature
(25  3  C). Dried leaves were pulverized into ne powder using an
electric blender (Corona-Ref.121, Landers and Qlink Blender, Model
No. OBl15 l 40). The powdered leaves (500 g) were extracted with
2.5 liters of 80% ethanol over night in a big stoppered bottle with
occasional stirring at room temperature. They were then sieved
563
using muslin cloth and ltered using whatman No. 1 lter paper.
The ltrate was concentrated in a rotary evaporator under reduced
pressure at 40  C for 45 min and then lyophilized to get the ethanol
(crude) extract.
2.3. Fractionation of the ethanol extract
The ethanol extract was partitioned successively by column
fractionation using different solvents to obtain the fractions [12].
The sample for the column was prepared by adsorbing 50 g of the
ethanol extract of C. aconitifolius onto 60 g of silica gel
(G 60120 mesh size). The mixture was air dried and carefully
layered on top of the packed silica gel in the column (60 cm length
and 6 cm diameter) for successive partitioning. The extract in the
column was eluted with methanol, n-hexane and chloroform in
increasing order of polarity. Each of the elutes was concentrated
under reduced pressure and labeled as ET, ME, CHL and nHE which
represented the ethanol extract, methanol, chloroform, and n-
hexane fractions respectively.
2.4. Chemicals
Streptozotocin (STZ) was purchased from Sigma-Aldrich
Chemical Company, USA. Glibenclamide was purchased from
Grace and Mercy Pharmaceuticals, Umuahia, Abia State, Nigeria.
Every other chemical used was also of analytical grade.
2.5. Animal experiments
Healthy male albino rats (weighing 120130 g) were purchased
from University of Nigeria, Nsukka (UNN). Ethical approval was
obtained from the College of Veterinary Medicine, Michael Okpara
University of Agriculture, Umudike, Abia State, Nigeria which was
in line with the guidelines for the care and use of laboratory
animals as given by the National Institute of Health [13]. Following
acclimatization of the animals for 14 d to their diets and water, they
were randomized into different groups and maintained under good
laboratory practice (12 h light and dark cycles) with free access to
food (Pzer feeds, Kaduna, Nigeria) and water ad libitum.
2.6. Acute toxicity study
For acute toxicity studies, healthy male mice weighing 20  5 g
were also purchased from UNN. After 2 weeks of acclimatization,
the mice were starved overnight and were orally administered the
extracts of C. aconitifolius in increasing order of: 250, 500, 1000,
2500, 3000, 3500, 4000, 4500, and 5000 mg/kg bwt. The animals
were observed for signs of toxicity for 2 h. After a period of 24 and
48 h, the mice were observed for lethality and LD50 was calculated
using the method of Lorke [14]. The ethanol extract had an LD50 of
4000 mg/kg body weight (bwt), the methanol fraction had an LD50
value of 3500 mg/kg bwt, the n-hexane fraction had an LD50 value
of 2000 mg/kg bwt while that of the chloroform fraction was
obtained as 2500 mg/kg bwt. Based on these values, the doses of
250 and 500 mg/kg were selected for the study.
2.7. Induction of diabetes
Freshly prepared solution of streptozotocin (STZ) (0.42 g
dissolved in 68 ml of freshly prepared cold sodium citrate buffer
0.1 M, pH 4.5) was injected intraperitoneally to the rats at a dosage
of 60 mg/kg bwt at fasting state. To overcome the hypoglycemia
that occurred during the rst 24 h following STZ administration,
the STZ treated rats were orally given 5% glucose solution and
allowed to stay for 24 h after which it was removed [15]. Blood was
collected from the tail and blood glucose was analyzed (at fasting
564 N.K. Achi et al. / Biomedicine & Pharmacotherapy 86 (2017) 562569

state) 72 h later and then on day 7 post STZ injection, using a blood
glucose meter (ACCU-CHEK Active, Germany). Rats with fasting
blood glucose levels 200 mg/dl after 7 d of STZ induction were
considered to be diabetic and were used for the study.
2.8. Experimental procedure
Both normal and rats with stable diabetes were assigned into
eleven groups of six rats each. The experimental design and
treatments administered are shown in Table 1.
The body weights of the rats were recorded on a daily basis. At
the end of 28 d, the rats were fasted overnight and euthanized on
the 29th day by cervical dislocation. Blood was collected by cardiac
puncture into plain tubes and allowed to clot. Sera were harvested
from the clotted blood samples following centrifugation at
3000  g for 20 min for the analysis of glucose, lipid prole and
insulin.
The organs (pancreas and heart) were excised and weighed.
Thereafter, a portion of the pancreas from the control groups and
one from any of two extract groups was xed in 10% formalin for
histology.
The percentage change in the body weights of the rats was
calculated as:
{Final body weight Initial body weight}/Final body weight}
 100
Furthermore, the relative organ weights of the rats were
calculated as follows:
Relative heart weight (g/100 g) = {Total heart weight/Final body
weight}  100
Relative pancreas weight (g/100 g) = {Total pancreatic weight/
Final body weight}  100 [16,17].
Glucose was assayed for in the sera of the rats using Randox
Assay Kits. Insulin was assayed for in the sera using the Enzyme
Linked Immunosorbent Assay technique [18] and results were
expressed in mIU/ml. Serum total cholesterol, triacylglycerol and
HDL-cholesterol concentrations were analyzed using the Randox
assay diagnostic kits following the methods described by as Tietz
[19] and NCEP [20]. LDL-cholesterol and VLDL-cholesterol levels of
the rats were calculated using the method of Friedewald et al. [21]
2.9. Histological assay
The histological assay of the pancreas was carried out using the
method of Dunn [22].
2.10. Oral glucose tolerance test in normal rats
Sixty rats were divided into ten groups of 6 rats each. The blood
glucose of the rats was determined after overnight fast. The
animals in group 1 were administered normal saline. Groups 2 and
3 were administered ethanol extract (250 and 500 mg/kg), Groups
4 and 5 were administered methanol fraction (250 and 500 mg/kg),
Groups 6 and 7 were administered n-hexane fraction (250 and
500 mg/kg), Groups 8 and 9 were administered chloroform fraction
(250 and 500 mg/kg) while Group 10 was given the standard drug,
glibenclamide (2.5 mg/kg) by means of oral gavage. Glucose
(2.5 g/kg) was fed 30 min after administration of the extract or
fractions. Blood was collected from the tail-tip of the rats at 0, 30,
60, 90 and 120 min respectively after extract administration and
their glucose levels were immediately estimated using a
glucometer (ACCU-CHEK Active, Germany).
2.11. Statistical analysis
Data were analyzed statistically using the Statistical Package for
Social Sciences (SPSS) version 20.00. One way analysis of variance
was used for comparison of means. Differences between means
were considered to be signicant when P < 0.05.
3. Results
The weights and percentage yields of the ethanol extract and
fractions are shown in Fig. 1. The weight of the ethanol extract was
98.1 g with a percentage yield of 19.62. The chloroform fraction
weighed 14.01 g with a percentage yield of 28.02; the methanol
fraction weighed 19.2 g with a percentage yield of 38.4 while the n-
hexane fraction weighed 13.15 g with a percentage yield of 26.3.
Result of acute toxicity study revealed that the ethanol extract
of C. aconitifolius leaves had an LD50 of 4000 mg/kg bwt, the
methanol fraction had an LD50 of 3500 mg/kg bwt, the n-hexane
fraction had an LD50 of 2000 mg/kg bwt while the LD50 of the
chloroform fraction was obtained as 2500 mg/kg bwt.
The serum glucose levels of the rats investigated in this study
are shown in Table 2. The serum glucose of the diabetic control at
the end of experimentation was elevated (P < 0.05) compared with
the normal control. Administration of the ethanol extracts,
fractions or glibenclamide attenuated (P < 0.05) the elevated
serum glucose levels of the diabetic rats compared with the
diabetic control.
The effect of administration of different extracts of C.
aconitifolius on the oral glucose tolerance of normoglycemic rats
is shown in Table 3. The blood glucose of the normal control peaked
to 121.68 mg/dl at 1 h post oral glucose loading but declined to
83.5 mg/dl after another 1 h.
The blood glucose of the rats administered the ethanol extracts
(250 and 500 mg/kg) peaked to 100.33 and 103.22 mg/dl respec-
tively at 1 h post oral glucose loading but declined to 100.28 and
87.11 mg/dl after further 1 h.

Table 1
Study groups and treatments.

Group Oral administration

Group 1 (Normal control) Normal saline (1 ml/kg)
Group 2 (Diabetic control) Normal saline (1 ml/kg)
Group 3 (Study group 1) Ethanol extract (250 mg/kg)
Group 4 (Study group 2) Ethanol extract (500 mg/kg)
Group 5 (Study group3) Methanol fraction (250 mg/kg)
Group 6 (Study group 4) Methanol fraction (500 mg/kg)
Group 7 (Study group 5) n-hexane fraction (250 mg/kg)
Group 8 (Study group 6) n-hexane fraction (500 mg/kg)
Group 9 (Study group 7) Chloroform fraction (250 mg/kg)
Group 10 (Study group 8) Chloroform fraction (500 mg/kg)
Group 11 (Positive control) Glibenclamide (2.5 mg/kg) Fig. 1. Weights and percentage yields of extracts.
 N.K. Achi et al. / Biomedicine & Pharmacotherapy 86 (2017) 562569 565

Table 2
Serum glucose levels of rats (mg/dl).

Glucose

Groups/Treatments 1st Day 7th Day 14th Day 21st Day 28th Day
Normal Control (1 ml/kg) 97.19  1.29 98.60  2.33 121.68  1.39 91.17  6.33 100.35  0.66
Diabetic Control (1 ml/kg) 261.76  3.34* 274.77  1.98* 288.11  2.10* 289.79  1.10* 286.32  3.00*
Ethanol (250 mg/kg) 255.51  1.33 252.51  1.99a 226.96  2.7a 225.11  1.11a 195.71  2.55a
Ethanol (500 mg/kg) 259.11  0.32 250.14  1.90a 209.94  3.10a 180.44  1.93a 163.19  1.69a
Methanol (250 mg/kg) 251.18  2.57a 249.88  1.21a 221.33  1.09 a 201.22  3.12a 158.70  3.00 a
Methanol (500 mg/kg) 256.84  2.33 230.44  2.92a 210.18  2.43a 179.00  1.23a 105.29  2.76a
n-Hexane (250 mg/kg) 246.31  2.19a 246.21  2.74a 246.33  3.51a 240.12  2.22a 239.22  3.00a
n-Hexane (500 mg/kg) 251.18  1.21a 250.31  2.17a 243.21  2.11a 253.00  2.65a 240.00  2.37a
Chloroform (250 mg/kg) 254.99  2.33 250.96  1.50a 251.59  1.13a 250.21  2.12a 251.00  2.32a
Chloroform (500 mg/kg) 253.02  1.35a 247.17  2.91a 243.10  1.39a 250.50  1.87a 251.70  3.12a
Glibenclamide (2.5 mg/kg) 256.00  1.92 237.16  2.91a 227.72  3.45a 157.33  1.82a 137.33  1.15a

Values are mean  SD. *P < 0.05 in comparison with normal control. aP < 0.05 in comparison with diabetic control.

Table 3
Effect of C. aconitifolius extracts on oral glucose tolerance in glucose-loaded normal rats.

Blood glucose (mg/dl)

Groups/Fractions 0 min 30 min 60 min 90 min 120 min

Normal control 99.39  2.93 98.60  0.33 121.68  1.39 91.17  1.33 83.50  0.66
Ethanol (250 mg/kg) 98.11  2.32 92.59  1.33* 123.11  2.20 100.33  1.21* 100.28  2.53*
Ethanol (500 mg/kg) 93.23  1.11 90.11  1.70* 117.54  2.00 103.22  1.53* 87.11  1.11
Methanol (250 mg/kg) 96.88  2.57 89.81  0.71* 119.33  0.59 86.11  2.62 79.23  1.25
Methanol (500 mg/kg) 94.30  2.20 80.72  2.32* 116.10  2.53 81.00  1.25* 65.25  2.33*
n-Hexane (250 mg/kg) 96.52  2.21 94.31  1.44 120.33  3.51 119.12  2.22* 100.11  2.00*
n-Hexane (500 mg/kg) 99.10  1.92 96.44  2.00 104.11  2.10* 120.00  2.35* 114.00  2.37*
Chloroform (250 mg/kg) 94.99  2.93 97.08  1.69 120.59  1.33 125.32  2.12* 121.00  3.32*
Chloroform (500 mg/kg) 93.02  1.69 92.67  2.01* 128.10  1.19 120.35  1.67* 118.12  1.32*
Glibenclamide (2.5 mg/kg) 96.00  1.11 82.11  2.11* 116.92  3.45 85.53  1.62 68.92  2.25*

Values are mean  SD. *P < 0.05 in comparison with normal control.

The blood glucose of the rats administered the methanol
fractions (250 and 500 mg/kg) peaked to 119.33 and 116.10 mg/dl
respectively at 1 h post oral glucose loading but declined to 79.23
and 65.25 mg/dl respectively after another 1 h.
The blood glucose of the rats administered the n-hexane
fraction (250 and 500 mg/kg) peaked to 120.33 and 104.11 mg/dl at
1 h post oral glucose loading. While the rats administered the
fraction at 250 mg/kg recorded a decline in blood glucose
(100.11 mg/dl) after further 1 h of oral glucose loading, the rats
administered the fraction at 500 mg/kg had elevated blood glucose
(114 mg/dl) after further 1 h of oral glucose loading.
The blood glucose of the rats administered the chloroform
fraction (250 and 500 mg/kg) achieved a maximum peak (120.59
and 128.1 mg/dl respectively) at 1 h post oral glucose loading.
While the rats administered the fraction at 250 mg/kg had elevated
blood glucose (121 mg/dl) after further 1 h, the rats administered
the fraction at 500 mg/kg had a decline in blood glucose
(118.12 mg/dl) after further 1 h. On the other hand, the rats
administered glibenclamide recorded their maximum peak in
blood glucose (116.92 mg/dl) 1 h after oral glucose loading but had
a decline in blood glucose (68.92 mg/dl) after further 1 h.
The lipid prole in the sera of the rats investigated in this study
is shown in Table 4.
The total cholesterol levels of the diabetic control were
signicantly elevated (P < 0.05) compared with the normal control.
Administration of the ethanol extracts (250 and 500 mg/kg), most
of the fractions at either doses (except chloroform at 250 and
500 mg/kg) or glibenclamide (2.5 mg/kg) resulted in signicant
reduction (P < 0.05) of total cholesterol levels of the diabetic rats
compared with the diabetic control. However, chloroform at both

Table 4
Lipid prole in the sera of rats (mg/dl).

Lipid prole

Groups/Treatments TC TG HDL-C LDL-C VLDL

Normal Control 116.46  1.11 71.00  0.87 29.09  1.39 73.17  2.33a 14.20  0.45
Diabetic Control 185.33  2.34* 157.15  1.98* 15.11  2.10* 138.79  1.10* 31.43  1.11*
Ethanol (250 mg/kg) 178.21  1.12 b 174.70  1.99b 17.76  2.70 125.51  1.81b 34.94  2.55
Ethanol (500 mg/kg) 166.41  0.68 b 125.65  1.90b 20.91  3.10 120.37  0.93b 25.13  1.19b
Methanol (250 mg/kg) 150.67  2.57 b 136.05  1.42b 24.40  1.19 b 99.06  2.00 b 27.21  1.13b
Methanol (500 mg/kg) 135.41  0.16bc 97.85  2.12bc 29.50  1.33 bc 86.34  1.02 bc 19.57  0.76bc
n-Hexane (250 mg/kg) 177.39  2.00b 143.55  3.51 b 16.12  2.22 132.56  0.97 b 28.71  0.99b
n-Hexane (500 mg/kg) 178.11  2.01 b 153.75  2.17 16.92  1.11 130.44  1.15 b 30.75  1.66
Chloroform (250 mg/kg) 191.22  1.00 b 118.55  1.50b 17.23  1.23 150.28  1.12 b 23.71  2.02b
Chloroform (500 mg/kg) 195.33  0.77 b 134.00  2.91b 16.93  1.19 151.60  0.87 b 26.80  1.12b
Glibenclamide (2.5 mg/kg) 132.30  2.17b 150.67  2.57 b 26.13  1.91b 87.79  1.36b 18.38  1.12b

Values are mean  SD. *P < 0.05 in comparison with normal control; bP < 0.05 in comparison with diabetic control; cP > 0.05 in comparison with glibenclamide.
566 N.K. Achi et al. / Biomedicine & Pharmacotherapy 86 (2017) 562569

Table 5
Body weights of rats (g).

Body weights

Groups/Treatments 1st Day 7th Day 14th Day 21st Day 28th Day
Normal Control (1 ml/kg) 130.00  1.42 143.11  1.57 154.33  3.32 175.41  1.34 210.00  1.47
Diabetic Control (1 ml/kg) 128.12  2.72 101.55  2.87* 92.79  3.27* 70.34  1.98* 68.47  2.90*
Diabetic + ethanol (250 mg/kg) 129.41  1.55 137.73  1.33b 140.61  1.54a 152.71  1.87a 169.61  1.55a
Diabetic + ethanol (500 mg/kg) 120.95  1.22 132.17  1.67b 151.19  2.50a 163.66  1.93a 181.84  2.30a
Diabetic + methanol (250 mg/kg) 130.02  1.54 135.43  1.33b 149.99  0.99a 165.33  1.96a 185.44  3.11a
Diabetic + methanol (500 mg/kg) 126.53  2.11 134.64  1.54b 154.76  1.98a 183.65  2.67a 199.04  2.45a
Diabetic + n-hexane (250 mg/kg) 129.11  2.33 125.32  3.00b 129.55  1.88a 126.73  2.44a 127.55  1.72a
Diabetic + n-hexane (500 mg/kg) 128.76  2.00 120.69  2.66b 124.44  0.96a 128.65  1.76a 128.99  1.59a
Diabetic + chloroform (250 mg/kg) 130.88  3.00 126.53  2.54b 126.11  1.21a 127.32  1.39a 129.74  1.17a
Diabetic + chloroform (500 mg/kg) 127.71  1.06 120.11  2.3b 127.33  2.67a 129.11  1.81a 126.88  2.41a
Diabetic + Glibenclamide (2.5 mg/kg) 130.23  1.33 137.55  1.52b 138.23  2.00a 167.44  1.47a 189.56  1.39a
a,b
Values are mean  SD. *P < 0.05 in comparison with normal control; P < 0.05 in comparison with diabetic control.

doses signicantly increased (P < 0.05) the total cholesterol levels

Table 6
of the diabetic rats when compared with the diabetic control.
Relative organ weights of rats (g/100 g) (wet weight).
The triacylglycerol levels of the diabetic control were signi-
cantly elevated (P < 0.05) in comparison with the normal control. Groups/Treatments Pancreas Heart
Administration of the ethanol extracts, most of the fractions Normal Control (1 ml/kg) 0.382  0.014 0.304  0.012
(except n-hexane at 500 mg/kg) or glibenclamide resulted in Diabetic Control (1 ml/kg) 0.698  0.061* 0.302  0.059
Diabetic + ethanol (250 mg/kg) 0.588  0.046 0.303  0.029
signicant (P < 0.05) reduction of the triacylglycerol levels of the
Diabetic + ethanol (500 mg/kg) 0.578  0.316 0.306  0.034
diabetic rats compared with the diabetic control. Diabetic + methanol (250 mg/kg) 0.458  0.056 0.301  0.022
The HDL-cholesterol levels of the diabetic control were reduced Diabetic + methanol (500 mg/kg) 0.388  0.026 0.303  0.037
(P < 0.05) compared with the normal control. While administra- Diabetic + n-hexane (250 mg/kg) 0.674  0.03 0.305  0.019
tion of the ethanol extract (at 250 and 500 mg/kg), the n-hexane Diabetic + n-hexane (500 mg/kg) 0666  0.016 0.307  0.024
Diabetic + chloroform (250 mg/kg) 0.689  0.036 0.306  0.044
and chloroform fractions (at 250 and 500 mg/kg) did not
Diabetic + chloroform (500 mg/kg) 0.682  0.016 0.305  0.054
signicantly affect (P > 0.05) the HDL-cholesterol levels of the Diabetic + glibenclamide (2.5 mg/kg) 0.488  0.058 0.306  0.036
diabetic rats, administration of the methanol fractions (250 and
Values are mean  SD. *P < 0.05 in comparison with normal control.
500 mg/kg) or glibenclamide signicantly increased (P < 0.05) the
HDL-cholesterol levels of the diabetic rats compared with the
diabetic control. The relative heart weights of the diabetic control were not
The LDL-cholesterol levels of the diabetic control were signicantly different (P > 0.05) from that of the normal control. In
signicantly increased (P < 0.05) when compared with the normal addition, the relative heart weights of the diabetic rats adminis-
control. Administration of the ethanol extracts, the fractions or tered the ethanol extracts, the fractions or glibenclamide were also
glibenclamide at the doses used in the study resulted in signicant not signicantly different (P > 0.05) from that of the diabetic
(P < 0.05) reduction of LDL-cholesterol levels of the diabetic rats control.
compared with the diabetic control The insulin levels in the sera of the rats investigated in this
The VLDL-cholesterol levels of the diabetic control were study are shown in Fig. 2. The insulin levels of the diabetic control
signicantly increased (P < 0.05) when compared with the normal were signicantly reduced (P < 0.05) when compared with the
control. Administration of the ethanol extract (at 500 mg/kg), most normal control.
of the fractions at either (except n-hexane at 500 mg/kg and Whereas administration of the ethanol extracts (250 and
chloroform at 250 and 500 mg/kg) or glibenclamide resulted in 500 mg/kg), methanol fractions (250 and 500 mg/kg) or gliben-
signicant reduction (P < 0.05) of VLDL-cholesterol levels of the clamide signicantly increased (P < 0.05) the insulin levels of the
diabetic rats in comparison with the diabetic control. However, the diabetic rats, administration of the n-hexane (250 and 500 mg/kg)
chloroform fraction at both doses signicantly increased (P < 0.05) and chloroform (250 and 500 mg/kg) fractions did not signicantly
the VLDL-cholesterol levels of the diabetic rats compared with the
diabetic control.
The body weights of the rats investigated in this study are
shown in Table 5. The body weights of the rats in the respective
groups were not signicantly different from each other (P > 0.05)
by the 1st day of experimentation. However, by the last day of
experimentation, the body weights of the diabetic control were
signicantly decreased (P < 0.05) compared with the normal
control whereas the body weights of the diabetic rats administered
the ethanol extracts, the fractions or glibenclamide were
signicantly increased (P < 0.05) compared with the diabetic
control.
The relative organ weights of the rats investigated in this study
are shown in Table 6. The relative pancreatic weights of the
diabetic control were signicantly increased (P < 0.05) compared
with the normal control.
Fig. 2. Insulin levels in the sera of rats. adMeans with different superscripts are
Administration of the ethanol extract, the fractions or
signicantly different (P < 0.05) across the groups. N Rats-Normal rats; D RATS-
glibenclamide did not signicantly (P > 0.05) affect the pancreatic Diabetic rats; ET-Ethanol; MET-Methanol; nHE-n-Hexane; CHL-Chloroform; GLIB-
weights of the rats compared with the diabetic control. Glibenclamide.
 N.K. Achi et al. / Biomedicine & Pharmacotherapy 86 (2017) 562569
affect (P > 0.05) the insulin levels of the diabetic rats when
compared with the diabetic control.
The histology of the pancreas of the rats investigated in this
study is shown in Fig. 3.
Histology of the islet cells of the normal control (3a) showed
normal islet cells with no structural changes. Overall histology
indicated normal islet cells.
Histology of the islet cells of the diabetic control (3b) showed
evidence of degeneration and necrotic changes in the islet cells
with shrunken cells.
Histology of the islet cells of the diabetic rats treated with the
methanol fraction (500 mg/kg bwt) (3c) showed evidence of
regeneration of the islet cells.
Histology of the islet cells of the diabetic rats treated with the
ethanol extract (500 mg/kg bwt) (3d) showed evidence of
regeneration of the islet cells with gradual repair of the pancreas.
Histology of the islet cells of the diabetic rats treated with the
chloroform fraction (500 mg/kg bwt) (3e) showed evidence of with
brosis and strands in the islet cells.
Fig. 3. Histoarchitecture of the pancreas of rats.
567
Histology of the islet cells of the diabetic rats treated with the n-
hexane fraction (250 mg/kg bwt) (3f) revealed continuous
degradation and necrotic changes with shrunken islet cells.
Histology of the islet cells of the diabetic rats treated with
glibenclamide (250 mg/kg bwt) (3 g) showed evidence of regener-
ation of normal islet cells.
4. Discussion
Several species of plants have been recognized as having
benecial properties in the management of diabetes. The higher
yield in the methanol fraction compared with the chloroform and
n-hexane fractions may be associated with higher polarity of
methanol that may have enabled its greater extractability of the
hydrophilic constituents of the crude extract than chloroform or
n-hexane.
According to Kalu et al. [23], a substance with an LD50 of
1000 mg/kg body weight and above when administered orally to
rats and mice is regarded as being safe. Therefore, the high LD50
568 N.K. Achi et al. / Biomedicine & Pharmacotherapy 86 (2017) 562569
obtained in this study for all the extracts of Cnidoscolus aconitifolius
leaves, suggest the non toxicity of this plant. Current nding
justies the wide spread use of this plant as a vegetable food and in
different ethno-therapeutic interventions [10].
The STZ rat model of diabetes is one of the most commonly used
models of human diseases because apart from being reproducible,
it also mimics many of the complications of human diabetes [24].
The diabetogenic effect of STZ in rats as seen in the present study
agrees with earlier reports of Ohaeri [15].
The ethanol extracts, the fractions or glibenclamide demon-
strated hypoglycemic actions as was evident from the reduction of
serum glucose levels of the diabetic rats at the end of experimen-
tation following their administration.
However, the ethanol extracts, the methanol fractions or
glibenclamide showed better hypoglycemic actions than the
n-hexane or chloroform fractions at the doses they were used in
the study. This may be associated with better extractability of the
bioactive compounds with hypoglycemic action in the ethanol
extracts and methanol fractions than in the chloroform and hexane
fractions. Furthermore, since the traditional use of this plant in the
management of diabetes by Nigerian traditional practioners
involves extraction with polar solvents, current nding justify
the use of polar solvent extracts from this plant in the management
of diabetes in Nigerian ethnomedicine.
To further explore the biochemical basis of the use of this plant
in the management of diabetes mellitus, the effect of the ethanol
extracts or fractions on oral glucose tolerance was also studied in
glucose-loaded normal rats.
Oral glucose tolerance test measures an individuals ability to
utilize ingested glucose over a period of time. It is a standard
protocol that is used to diagnose diabetes [25].
For normal tolerance to blood glucose, the blood glucose at 2 h
post oral glucose loading is about 110 mg/dl while for impaired
tolerance, blood glucose rises above 110 mg/dl due to lack or
insensitivity of insulin which results in reduced glucose uptake and
its utilization by the peripheral tissues. Since impaired oral glucose
tolerance is indicative of predisposition of an organism to diabetes,
medicinal plants that possess antidiabetic actions will improve
glucose tolerance, thereby arresting the progression of impaired
glucose tolerance to diabetes [26].
Therefore, the ndings of this study which indicated that the
blood glucose levels of the rats administered the ethanol extracts,
methanol fraction and n-hexane fraction (at 250 mg/kg) or
glibenclamide declined to values  100 mg/dl, suggest the ability
of these extracts to improve impaired glucose tolerance. This
nding therefore explains the higher hypoglycemic action
demonstrated by these extracts, fractions or glibenclamide in this
study compared with other fractions. Furthermore, the results of
this study which showed that the blood glucose levels of the rats
administered the n-hexane (500 mg/kg) and chloroform fractions
(250 and 500 mg/kg), were elevated after 2 h of oral glucose
loading with values obtained > 100 mg/dl suggest that these
extracts do not have the ability to improve impaired glucose
tolerance. This nding may also explain the lower antidiabetic
actions demonstrated by these fractions in this study.
Hypercholesterolemia and hypertriglyceridemia are recognized
complications of diabetes mellitus arising from increased mobili-
zation of free fatty acids from the peripheral deposits in insulin
deciency and which is characterized by elevated levels of
cholesterol, triacylglycerol, LDL-cholesterol and VLDL-cholesterol
but decreased levels of HDL-cholesterol and this explains the
elevated levels of cholesterol, triacylglycerol, LDL-cholesterol and
VLDL-cholesterol but decreased levels of HDL-cholesterol in the
diabetic control [3].
The reduction of total cholesterol and VLDL-cholesterol by the
extracts, fractions or glibenclamide; the reduction of
triacylglycerol by the extracts, some of the fractions or glibencla-
mide; the increase in HDL-cholesterol levels by the methanol
fractions or glibenclamide; and the reduction of VLDL-cholesterol
by the ethanol extract (at 500 mg/kg), most of the fractions (except
n-hexane at 500 mg/kg) or glibenclamide suggest the possible
anti-hyperlipidemic actions of some of these extracts or fractions
or glibenclamide. However, the methanol fractions (at 250 and
500 mg/kg) consistently showed anti-hypercholesterolemic and
anti- hypertriglyceridemic properties when compared with the
disease control. Furthermore, the anti-hypercholesterolemic and
anti- hypertriglyceridemic action demonstrated by the methanol
fraction (500 mg/kg) was akin to that of glibenclamide.
Reduction in body weight in the diabetic rats as observed in the
study could be attributed to increased muscle wasting due to
degradation of structural proteins and unavailability of carbohy-
drates for utilization as an energy source [27]. However, the
increased body weights of the diabetic rats following administra-
tion of the extracts, fractions or glibenclamide, suggest increased
synthesis of tissue proteins as a result of glycemic control [28].
Hyperglycemia has been hypothesized to stimulate increase in
pancreatic b- cell mass in animal models of diabetes which may be
the explanation for the increased pancreatic weights of the
diabetic control. However, the non signicant effect on the
pancreatic weights of the diabetic rats following treatment with
the extracts, fractions or glibenclamide may be attributed to the
duration of the treatment.
The non-signicant changes in the relative heart weights of the
rats in the three groups can be attributed to the non-susceptibility
of the heart to STZ assault as the heart expresses GLUT 4
transporter [3].
STZ model of diabetes could result in partial or total destruction
of the b-cells of the pancreas with resultant decline in serum levels
of insulin as a result of decreased synthesis [29] which explains the
decreased serum levels of insulin in the diabetic control. However,
the increased serum levels of insulin as observed following
treatment with the ethanol extracts, methanol fractions or
glibenclamide (unlike the n-hexane or chloroform fractions that
had no effect on insulin) suggest activation of some b-cells in the
pancreas [30]. Furthermore, treatment of the diabetic rats with the
methanol fraction (at 500 mg/kg) increased the insulin levels of the
rats to near control levels and in a manner, akin to glibenclamide.
Results obtained with the ethanol extracts, the methanol
fractions or glibenclamide, corroborate our ndings on their effects
on the oral glucose tolerance of glucose loaded normal rats.
However, while the result obtained for the n-hexane fraction at
250 mg/kg which did not corroborate its effect on the oral glucose
tolerance of glucose loaded normal rats cannot be explained
presently, it is noteworthy.
The ndings of the histopathological examination revealed that
treatment of the diabetic rats with the ethanol extract (500 mg/kg),
the methanol fraction (500 mg/kg) or glibenclamide (at 2.5 mg/ kg)
led to reversal of the histoarchitecture of the pancreatic tissue of
the rats when compared with the diabetic control. To the best of
our knowledge, this is the rst report on the improvement of
glucose tolerance with different polar solvent extracts of this plant
and stimulation of insulin secretion in diabetic subjects with the
extracts. These ndings lead us to suggest a possible rationale for
the hypoglycemic action of this plant to be repair of the islet cells of
the pancreas leading to increased release of insulin as evidenced in
this study.
5. Conclusions
The ethanol extracts and the methanol fractions showed better
hypoglycemic actions than the n-hexane or chloroform fractions at
the doses they were used in this study which justify the use of
 N.K. Achi et al. / Biomedicine & Pharmacotherapy 86 (2017) 562569
polar solvent extracts of this plant by traditional practitioners in
the management of diabetes mellitus. Finally, the current study
suggests the safety in consumption of this plant.
Conict of interest
The authors declare that there are no conicts of interest.
Acknowledgement
This research received no external fund from any organization.
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