Biomedicine & Pharmacotherapy 85 (2017) 725–732

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Original article

Protective potential of Averrhoa bilimbi fruits in ameliorating the

hepatic key enzymes in streptozotocin-induced diabetic rats
Surya B Kurup, Mini S*
Department of Biochemistry, University of Kerala, Kariavattom, Thiruvananthapuram, Kerala, India

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

Article history: Back ground: Diabetes is a mutifactorial disease which leads to several complications. Currently available
Received 18 October 2016 drug regimens for management of diabetes have certain drawbacks. Need for safer and effective
Received in revised form 10 November 2016 medicines from natural sources having potent antidiabetic activity. Averrhoa bilimbi Linn. (Oxalidaceae) is
Accepted 18 November 2016
a medicinal plant and is reported to possess hypoglycemic activity.
Objective: To investigate the antidiabetic potential of Averrhoa bilimbi fruit extract in streptozotocin-
Keywords: induced diabetic rats.
Averrhoa bilimbi Linn
Methods: Diabetes was induced in male Sprague Dawley rats by single intraperitoneal injection of
Diabetes mellitus
Carbohydrate metabolism
streptozotocin (STZ) (40 mg/kg body weight). The diabetic rats were treated orally with ethyl acetate
GC–MS analysis fraction of A. bilimbi fruits (ABE) (25 mg/kg body weight) and metformin (100 mg/kg body weight) by
Phytomedicine intragastric intubation for 60 days. After 60 days, the rats were sacrificed; blood, liver and pancreas were
collected. Several indices such as blood glucose, plasma insulin, toxicity markers and the activities of
carbohydrate-metabolizing enzymes were assayed. The phytochemicals present in the ABE was
identified by gas chromatography-mass spectrometry analysis.
Results: ABE significantly (p < 0.05) reduced the level of blood glucose and hepatic toxicity markers and
increased plasma insulin in diabetic rats. ABE modulated the activities of carbohydrate-metabolizing
enzymes, significantly increased the activities of hexokinase (59%) and pyruvate kinase (68%) and
reduced the activities of glucose-6-phosphatase (32%) and fructose-1, 6-bisphosphatase (20%). The
histological studies of the pancreas also supported our findings. The results were compared with
metformin, a standard oral hypoglycemic drug. GC–MS analysis of ABE revealed the presence of 11
chemical constituents in the extract.
Conclusions: ABE exerts its antidiabetic effect by promoting glucose metabolism via glycolysis and
inhibiting hepatic endogenous glucose production via gluconeogenesis.
© 2016 Elsevier Masson SAS. All rights reserved.

1. Introduction Diabetes results in the derangement of carbohydrate, lipid and

protein metabolism [3]. Glucose homeostasis involves the
Diabetes mellitus (DM) is a multifaceted metabolic disorder coordinated regulation of pathways that direct glucose production
characterized by chronic hyperglycemia ensuing from defects in and utilization [4]. In diabetes, the activities of key enzymes of
insulin secretion, action or both [1]. Persistent hyperglycemia is a glycolysis and gluconeogenesis are distorted, which leads to
stage of the increased blood glucose level of circulating blood chronic hyperglycemia [5]. Effective measures to manage fasting
system that leads to the development of microvascular and and postprandial glucose levels are crucial in the management of
macrovascular complications. Diabetes and its complications diabetes. Though, numerous oral hypoglycemic drugs are now
create a severe health care crisis worldwide. Globally, the available, but their extended consumption produces adverse side
prevalence of diabetes is predicted to grow from 366 million in effects [6]. Hence, there is a need to explore for phytocomponents
2011 to 552 million by 2030 [2]. that regularize hyperglycemia and diabetes-associated complica-
tions [7].
Averrhoa bilimbi Linn. (Oxalidaceae), commonly known as
cucumber tree or tree sorrel is a widely cultivated plant in India,
* Corresponding author at: Department of Biochemistry, University of Kerala, Indonesia, Sri Lanka, Bangladesh, Myanmar, Malaysia, Central and
Kariavattom, Thiruvananthapuram 695 581, Kerala, India. South America. The whole plant is used for treating coughs, cold,
E-mail address: [email protected] (M. S).

http://dx.doi.org/10.1016/j.biopha.2016.11.088
0753-3322/© 2016 Elsevier Masson SAS. All rights reserved.
726 S.B. Kurup, M. S / Biomedicine & Pharmacotherapy 85 (2017) 725–732

itches, rheumatism, whooping cough, hypertension etc. [8,9]. between 200 and 400 mg/dl were considered as diabetic and
Traditionally A. bilimbi fruits are reported to have antidiabetic used for the study.
activity and no scientific data are available on the antidiabetic
properties of the fruits [10,11]. 2.5. Experimental design
Previous studies showed that the leaf extract of A. bilimbi and its
semi-purified fractions possesses hypoglycemic and hypolipi- A dose dependent toxicity study was carried out using ABE at
demic properties in Type I diabetic rats when treated both different concentrations (5, 10 and 25 mg/kg body weight). The
intraperitoneally [12] as well as orally [13,14]. Our previous studies minimal effective dose was fixed as 25 mg/kg body weight by
revealed the beneficial effect of aqueous extracts of Averrhoa evaluating the activities of toxicity markers and antioxidant
bilimbi fruits in controlling blood glucose and lipid metabolism and enzymes (results not shown). So for the present study, we have
preventing diabetic complications from lipid peroxidation in Type selected ABE at a dose of 25 mg/kg body weight and efficacy of ABE
II diabetic rats [15]. In our preliminary study the ethyl acetate was compared with metformin, a standard oral hypoglycemic drug
fraction of Averrhoa bilimbi fruits was found to be rich in phenolic in streptozotocin induced diabetic rats.
compounds with superior antioxidant activity [16]. Preliminary The experimental animals were divided into five groups,
invitro studies revealed the potency of ABE fraction. comprising of seven rats. ABE and metformin were administered
Present study evaluates the antidiabetic potential of ethyl intragastrically for 60 days.
acetate fraction of A. bilimbi fruits by monitoring activities of the Group I: Normal control rats
key enzymes of carbohydrate metabolism in Type II diabetic rats. Group II: Normal rats treated with ABE (25 mg/kg body weight)
Group III: Diabetic control rats
2. Materials and methods Group IV: Diabetic rats treated with ABE (25 mg/kg body
weight)
2.1. Chemicals Group V: Diabetic rats treated with metformin (100 mg/kg body
weight)
Chemicals were purchased from Sigma Aldrich (St. Louis, MO, During the experimental period, body weight, blood glucose
USA), Merck Chemical Company (Darmstadt, Germany) and Sisco and physical examinations were done at regular intervals. The
Research Laboratories (Mumbai, India). dosage was adjusted every week, according to changes in body
weight to maintain similar dose per kg body weight of rat over the
2.2. Extraction of Averrhoa bilimbi fruits entire period of study. After 60 days, the rats were sacrificed by
sodium pentothal injection. Blood, liver and pancreas were
Fresh fruits of Averrhoa bilimbi were obtained from Thiruva- collected for various experimental analysis.
nanthapuram, Kerala, India, during the fruiting season (July–
December 2014) and documented by Dr. Valsala Devi, Department 2.6. Gas chromatography–mass spectrometry (GC–MS) analysis
of Botany, University of Kerala (Voucher no: KUBH 5865). Care was
taken that the fruits, which were whitish-green in color and about The GC–MS analysis of the extract was performed using Agilent
5–7.0 cm in size, were not overripe, spoiled or damaged. Fruits CP 3000 GC/Saturn 2200 MS (Agilent, Palo Alto, CA) equipped with
(5 kg) were cut and shade dried at a temperature of 28  C. Shade ECD, PFPD and MS detectors. For GC/MS detection, an electron
dried fruits were ground in a blender to give 500 g of fine powder. ionization system with ionization energy of 70 eV was used.
The aqueous extract was prepared by cold maceration of 500 g Helium gas was used as the carrier gas at a constant flow rate of
powder in 1000 ml of distilled water and lyophilized (Thermo 1 ml/min. The inlet temperature was set at 250  C. The oven
electron corporation, MODUL YOD-230) (yield 26%). temperature was maintained at 100  C for 1.5 min and gradually
100 g lyophilized A. bilimbi fruit extract was defatted with increased up to 270  C at the rate of 5  C per min 1 ml diluted
petroleum ether and the defatted extract was fractionated with sample was injected and the scan range was 40–600 m/z.
ethyl acetate (1:1 v/v) (ABE-yield 5%) was concentrated at room
temperature and used for the present study. 2.7. Biochemical parameters

2.3. Experimental animals The fasting blood glucose was monitored by glucometer (One
Touch Horizon, Johnson and Johnson). Insulin was estimated by
Two months old male Sprague Dawley rats (200–220 g body using an ELISA kit (DRG Diagnostics, USA). Hepatic toxicity markers
weight, 35 animals in total) bred in our Department animal house such as alkaline phosphatase (ALP), acid phosphatase (ACP) and
was used for the study. Animals were housed in polypropylene gamma glutamyl transferase (GGT) were measured using com-
cages and maintained under standard conditions [12-h light/dark mercially available kits (Agappe Diagnostics, Ernakulam, India).
cycles, (25  10  C)]. All the animal care was taken as per the Glycolytic enzymes viz, hexokinase (EC 2.7.1.1) [18] and pyruvate
guidelines of the Committee for the Purpose of Control and kinase (2.7.1.40) [19] were assayed. Activities of hepatic gluconeo-
Supervision of Experiments on Animals and the experimental genic enzymes, glucose-6-phosphatase (EC 3.1.3.9) [20] and
protocol approved by the Institutional Animal Ethics Committee fructose-1, 6-bisphosphatase (EC 3.1.3.11) [21] were assayed.
[IAEC-KU-20/2013-14-BC-SM (22)].
2.8. Histopathological analysis of pancreas
2.4. Induction of diabetes
The pancreatic samples were set for 48 h in 10% neutral
Diabetes was induced in rats by single intraperitoneal injection formalin, fixative solution was dehydrated by passing effectively in
of freshly prepared streptozotocin (STZ) at a dose of 40 mg/kg body different mixture of ethyl alcohol-water, cleaned with xylene and
weight in 0.1 M citrate buffer (pH 4.5) [17]. The animals were given embedded in paraffin. Sections of pancreas (5 mm thick) were
5% glucose in drinking water overnight to overcome the drug- prepared by using a rotary microtome and then stained with
induced hypoglycemia. 48 h after STZ injection, blood glucose hematoxylin and eosin (H & E) dye, which mounted in a neutral
levels were estimated and rats with blood glucose ranging deparaffinated xylene medium for microscopic observations [22].
 S.B. Kurup, M. S / Biomedicine & Pharmacotherapy 85 (2017) 725–732 727

Fig. 1. GC–MS analysis of ethyl acetate fraction of Averrhoa bilimbi fruits.

2.9. Statistical analysis 3.2. Changes in body weight and ratio of liver/pancreas to 100 g body
weight
The values were expressed as mean  SEM. The statistical
analysis was carried out by one-way analysis of variance using SPSS Changes in body weight and liver/pancreas ratio to 100 g
(version 17) statistical analysis programme. Duncan’s post hoc body weight in control and experimental rats were depicted in
multiple comparison tests were used to determine significant Tables 2 and 3. There was no significant difference among the
differences among groups. p < 0.05 was considered to be groups during initial body weight estimation. The diabetic rats
significant. showed significant (p < 0.05) decrease in the body weight and
liver/pancreas ratio when compared with normal control.
3. Results Treatment with ABE and metformin to diabetic rats, signifi-
cantly (p < 0.05) increased the body weight and liver/pancreas
3.1. GC–MS analysis ratio. Changes in body weight and liver/pancreas ratio were
comparable in diabetic rats treated with ABE and metformin.
The mass spectrum of ABE is shown in Fig. 1. Eleven compounds The treatment with ABE to normal rats resulted in no significant
were identified in the ABE extract by the GC–MS analysis. To the change in the body weight and liver/pancreas ratio as compared
best of our knowledge, the identified compounds were not to normal control rats.
reported previously in this plant. Compounds present in ABE
were identified by comparison of their mass spectra with a built in 3.3. Blood glucose and insulin
National Institute of Standards and Technology (NIST). Identified
compounds include Phenelzine, Phenyl acetic acid, 1,3-Propane- Fig. 2. shows the levels of blood glucose in control and
diol, 2,2-dimethyl-, Benzeneacetamide, 2,4-Di-tert-butylphenol, experimental rats. Blood glucose was significantly (p < 0.05)
Tetrapentacontane, 1,54-dibromo-, 2-Cyclohexen-1-one 4-hy- increased in diabetic control rats (307.50  3.36 mg/dl) as com-
droxy-3,5,5-trim, L-Ascorbyl-6-dipalmitate, g-Stearolactone, pared with normal control. The administration of ABE and
1–Hentetracontanol and Octadecanoic acid, 12-hydroxy-, methyl metformin for a period of 60 days significantly (p < 0.05) decreased
ester. The active principles with their retention time (RT), blood glucose (148.63  5.53 mg/dl and 155.80  5.80 mg/dl) in
molecular formula, molecular weight (MW) and peak area% were diabetic rats. Blood glucose levels were comparable in diabetic rats
presented in Table 1. treated with ABE and metformin. However, the administration of

Table 1
Chemical composition of ABE Extract.

RT Name Molecular Formula Molecular Peak Area

Weight %
2.064 Phenelzine C8H12N2 136.19 2.64
[Hydrazine, (2-phenylethyl)-]
5.757 1,3-Propanediol, 2,2-dimethyl- C5H12O2 104.15 1.10
8.198 Phenyl acetic acid C8H8O2 136.15 2.46
10.901 Benzeneacetamide C8H9NO 135.16 2.60
13.478 2,4-Di-tert-butylphenol C14H22O 206.32 2.00
19.368 Tetrapentacontane, 1,54-dibromo- C54H110 759.45 0.07
21.727 2-Cyclohexen-1-one, 4-hydroxy-3,5,5-trim C13H18O3 222.28 2.90
25.875 L-Ascorbyl-6-dipalmitate C38H68O8 652.94 9.42
[l-(+)-Ascorbic acid-6-dihexadecanoate]
28.445 g-Stearolactone C18H34O2 282.46 0.90
[2(3H)-Furanone, dihydro-5-tetradecyl-]
32.066 1 Hentetracontanol C4H84O 593.10 0.91
35.360 Methyl ricinoleate C19H36O3 312.48 0.20
[Octadecanoic acid, 4-hydroxy-, methyl ester]

RT: Retention Time.

728 S.B. Kurup, M. S / Biomedicine & Pharmacotherapy 85 (2017) 725–732

Table 2 with the normal control. Treatment with ABE resulted in marked
Change in Body Weight (g).
elevation in the activities of HK-59% and PK-68% and decreased in
Groups Body Weight (g) the activities of G6Pase-32% and F 1, 6 BPase-20% in hepatic tissue
Initial (0th day) Final (60th day)
as compared with diabetic control. The modulatory effect of these
enzymes by ABE was found to be comparable to the reference drug,
I 220.37  8.21 292.13  10.88
II 221.40  8.24 297.25  11.07
metformin. Administration of ABE to normal rats did not show any
III 220.37  8.21 153.75  5.73a significant effect in the activities of HK, PK, G6Pase and F 1, 6 BPase
IV 223.45  8.32 236.25  8.76b as compared with normal control rats.
V 219.35  8.17 230.13  8.60b

Change in Body weight. Values are expressed as mean  SEM of seven rats in each 3.6. Histopathological analysis of pancreas
group; significance accepted at P < 0.05. I, normal control rats; II, normal rats
treated with ethyl acetate fraction of Averrhoa bilimbi Linn fruits (ABE); III, STZ-
The histopathological analysis of pancreas in normal control
induced diabetic rats; IV, STZ-induced diabetic rats treated with ethyl acetate
fraction of Averrhoa bilimbi Linn fruits (ABE) at a dose of 25 mg/kg body weight/day; and normal rats treated with ABE showed normal acini and islets
V, STZ-induced diabetic rats treated with metformin at a dose of 100 mg/kg body with normal round or elongated structural intactness with their
weight/day. nucleus (Fig. 4A and B). While the STZ-induced diabetic group
a
Statistically significant as compared to normal group. (Fig. 4C) exhibited loss of structural integrity of islets, reduced
b
Statistically significant as compared to diabetic group.
number of b-cells and moderate levels of inflammation, whereas
diabetic rats treated with ABE and metformin restored the cellular
ABE to normal rats resulted in no significant (p < 0.05) change in
morphology to almost normal levels (Fig. 4D and E).
the levels of blood glucose.
The levels of plasma insulin in control and experimental rats
4. Discussion
were depicted in Fig. 3. The diabetic rats showed significant
(p < 0.05) decrease in the level of plasma insulin when compared
Diabetes mellitus is characterized by a progressive dysfunction
with normal control. Treatment with ABE and metformin to
of pancreatic b-cells with a decline in insulin secretion leading to
diabetic rats, significantly (p < 0.05) increased plasma insulin level
persistent hyperglycemia. STZ-induced hyperglycemia in experi-
(3.45-fold and 2.3-fold) when compared to diabetic control. ABE
mental animals has been widely used as a valuable experimental
treatment showed a significant increase in insulin level than
model to study the effect of different hypoglycemic agents [23].
metformin treatment in diabetic rats. The treatment with ABE to
The STZ at lower doses (40 mg/kg body weight) destruct pancreatic
normal rats resulted in no significant change in the level of plasma
b-cells moderately in rats, leading to deficient insulin secretion
insulin as compared to normal control rats.
causing type 2 diabetic model [24,25].
Averrhoa bilimbi is a medicinal plant used by the folk healers as
3.4. Hepatic toxicity markers
an antidiabetic agent. This study was conducted to evaluate the
antidiabetic effect of ethyl acetate fraction of Averrhoa bilimbi fruits
Liver toxicity markers were assayed to assess hepatic injury. The
(ABE) in normal and STZ-induced diabetic rats by evaluating
activities of alkaline phosphatase (ALP), acid phosphatase (ACP)
glucose profile, activity of hepatic enzymes, viz, hexokinase,
and gamma glutamyl transferase (GGT) were significantly altered
pyruvate kinase, glucose-6-phosphatase and fructose-1, 6-
in the diabetic control group, indicating damage to hepatocytes.
bisphosphatase as well as the capability of ABE to stimulate
The hepatic toxicity markers in normal rats treated with ABE did
insulin secretion.
not show any statistical difference in comparison with normal
Dehydration and loss of body weight has been reported to be
control rats. Treatment with ABE and metformin significantly
associated with diabetes [26,27]. Loss of body weight in diabetes is
(p < 0.05) lowered the activities of these enzymes. Superior effect
due to dehydration, loss of carbohydrates and the excessive break
was seen in diabetic rats treated with ABE than metformin
down of tissue proteins and fat [28–30]. In agreement with the
(Table 4).
above reports, the body weight and tissue weight (liver and
pancreas) were significantly decreased in STZ-induced diabetic
3.5. Carbohydrate metabolizing enzymes
rats. Treatment with ABE and metformin for 60 days significantly
increased the body weight and tissue weight which indicates the
Tables 5 and 6 summarizes the activities of hexokinase (HK),
better control of the hyperglycemic state in diabetic rats.
pyruvate kinase (PK), glucose-6-phosphatase (G6Pase) and fruc-
The experimentally induced diabetic rats showed rigorous
tose-1, 6-bisphosphatase (F 1, 6 BPase) in the control and
hyperglycemia consistent with a decrease in the endogenous
experimental rats. STZ-induced diabetic rats showed a significant
insulin secretion and release. In the present study, fasting blood
(p < 0.05) decrease in the activities of HK and PK along with the
glucose was elevated significantly in diabetic control rats and this
increase in the activities of G6Pase and F 1, 6 BPase in comparison
may be due to pancreatic b cell damage. Rats treated with ABE and

Table 3
Ratio of liver/pancreas to 100 g body weight.

Groups Liver weight/100 g body weight Pancreas weight/100 g body weight Ratio of liver/pancreas/100 g body weight
I 3.07  0.11 0.66  0.02 4.74  0.18
II 3.18  0.12 0.68  0.03 4.75  0.18
III 1.03  0.04a 0.31  0.01a 3.38  0.13a
IV 1.95  0.07b 0.49  0.02b 4.06  0.15b
V 1.85  0.07b 0.47  0.02b 4.01  0.15b

Ratio of liver/pancreas to 100 g body weight. Values are expressed as mean  SEM of seven rats in each group; significance accepted at P < 0.05. I, normal control rats; II,
normal rats treated with ethyl acetate fraction of Averrhoa bilimbi Linn fruits (ABE); III, STZ-induced diabetic rats; IV, STZ-induced diabetic rats treated with ethyl acetate
fraction of Averrhoa bilimbi Linn fruits (ABE) at a dose of 25 mg/kg body weight/day; V, STZ-induced diabetic rats treated with metformin at a dose of 100 mg/kg body weight/
day.
a
Statistically significant as compared to normal group.
b
Statistically significant as compared to diabetic group.
 S.B. Kurup, M. S / Biomedicine & Pharmacotherapy 85 (2017) 725–732 729

Table 5
Hepatic glycolytic enzymes.

Groups Hexokinase Pyruvate

(mg glucose kinase
phosphorylated/ (units/mg protein)
min/mg protein)
I 16.58  0.62 35.80  1.27
II 17.22  0.67 36.22  1.34
III 7.21  0.27a 12.61  0.47a
IV 11.51  0.42b 21.19  0.78b
V 11.25  0.41b 19.61  0.73b

Glycolytic enzymes.Values are expressed as mean  SEM of seven rats in each

group; significance accepted at P < 0.05.I, normal control rats; II, normal rats treated
with ethyl acetate fraction of Averrhoa bilimbi Linn fruits (ABE); III, STZ-induced
Fig. 2. Blood glucose. Values are expressed as mean  SEM of seven rats in each diabetic rats; IV, STZ-induced diabetic rats treated with ethyl acetate fraction of
group; significance accepted at P < 0.05. aStatistically significant as compared to Averrhoa bilimbi Linn fruits (ABE) at a dose of 25 mg/kg body weight/day; V, STZ-
normal group. bStatistically significant as compared to diabetic group. I, normal induced diabetic rats treated with metformin at a dose of 100 mg/kg body weight/
control rats; II, normal rats treated with ethyl acetate fraction of Averrhoa bilimbi day.
a
Linn fruits (ABE); III, STZ-induced diabetic rats; IV, STZ-induced diabetic rats treated Statistically significant as compared to normal group.
b
with ethyl acetate fraction of Averrhoa bilimbi Linn fruits (ABE) at a dose of 25 mg/kg Statistically significant as compared to diabetic group.
body weight/day; V, STZ-induced diabetic rats treated with metformin at a dose of
100 mg/kg body weight/day.
Table 6
Hepatic gluconeogenic enzymes.

Groups Glucose-6-phosphatase Fructose 1,6-bis-phosphatase

(mmol of Pi liberated/ (mmol of Pi liberated/
h/mg protein) h/mg protein)
I 25.29  0.94 77.56  2.89
II 24.63  0.92 74.83  2.79
III 65.26  2.43a 137.94  5.14a
IV 44.21  1.65b 109.50  4.08b
V 41.51  1.55b 107.02  3.93b

Hepatic Gluconeogenic enzymes.Values are expressed as mean  SEM of seven rats

in each group; significance accepted at P < 0.05. I, normal control rats; II, normal
rats treated with ethyl acetate fraction of Averrhoa bilimbi Linn fruits (ABE); III, STZ-
induced diabetic rats; IV, STZ-induced diabetic rats treated with ethyl acetate
fraction of Averrhoa bilimbi Linn fruits (ABE) at a dose of 25 mg/kg body weight/
day; V, STZ-induced diabetic rats treated with metformin at a dose of 100 mg/kg
Fig. 3. Plasma Insulin. Values are expressed as mean  SEM of seven rats in each body weight/day.
a
group; significance accepted at P < 0.05. aStatistically significant as compared to Statistically significant as compared to normal group.
b
normal group. bStatistically significant as compared to diabetic group. cStatistically Statistically significant as compared to diabetic group.
significant as compared to ABE treated diabetic group. I, normal control rats; II,
normal rats treated with ethyl acetate fraction of Averrhoa bilimbi Linn fruits (ABE);
III, STZ-induced diabetic rats; IV, STZ-induced diabetic rats treated with ethyl
acetate fraction of Averrhoa bilimbi Linn fruits (ABE) at a dose of 25 mg/kg body Diabetes is frequently associated with the elevated activities of
weight/day; V, STZ-induced diabetic rats treated with metformin at a dose of
liver toxicity marker enzymes like ALP, ACP and GGT in serum,
100 mg/kg body weight/day.
which might be mostly due to the out flow of these enzymes from
the liver cytosol into the bloodstream [33]. Consistent with the
above reports, the activities of ALP, ACP and GGT were significantly
Table 4
Hepatic toxicity markers. increased in STZ-induced diabetic group. But ABE and metformin
administration reduced the activities of ALP, ACP and GGT in
Groups ALP (U/L) ACP (U/L) GGT (U/L)
diabetic condition and the effect was superior for ABE. In addition,
I 9.23  0.34 121.27  4.23 39.28  1.53 the activities of liver toxicity markers were not altered in the
II 8.97  0.34 117.88  4.39 38.03  1.48
normal rats treated with ABE group, in comparison with the
III 15.53  0.58a 164.00  6.11a 83.35  3.28a
IV 11.69  0.44b 135.30  5.04b 59.80  2.37b normal control group. It indicates that ABE treatment was safe and
V 13.02  0.32b,c 148.63  5.53b,c 71.45  2.83b,c has no significant toxicity.
Hepatic toxicity markers.Values are expressed as mean  SEM of seven rats in each
Several reports have shown that hexokinase is the most
group; significance accepted at P < 0.05. I, normal control rats; II, normal rats sensitive marker of the glycolytic pathway in diabetic rats
treated with ethyl acetate fraction of Averrhoa bilimbi Linn fruits (ABE); III, STZ- [34,35]. Hexokinase phosphorylates glucose to glucose-6-phos-
induced diabetic rats; IV, STZ-induced diabetic rats treated with ethyl acetate phate and helps in the maintenance of glucose homeostasis [36].
fraction of Averrhoa bilimbi Linn fruits (ABE) at a dose of 25 mg/kg body weight/day;
Hexokinase is an insulin sensitive enzyme and is almost totally
V, STZ-induced diabetic rats treated with metformin at a dose of 100 mg/kg body
weight/day. prevented or inactivated in the diabetic rat liver in the absence of
a
Statistically significant as compared to normal group. insulin [37,38]. Treatment with ABE and metformin significantly
b
Statistically significant as compared to diabetic group. increased the activity of hexokinase which indicates the effective
c
Statistically significant as compared to ABE treated diabetic group. utilization of glucose and direct stimulation of glycolysis in tissues
with increased glucose elimination from the blood circulation.
metformin showed a significant decrease in the level of blood Pyruvate kinase is a regulatory enzyme ofglycolysis, catalyzes
glucose, which may be due to increased release of insulin from the the conversion of phosphoenol pyruvate to pyruvate with the
existing and/or regenerated pancreatic b-cells [31,32]. Histopa- release of ATP [39]. PK deficiency results in the accumulation of the
thology of the pancreas also confirms these findings. glycolytic intermediates chiefly 2, 3 bisphosphoglycerate
730 S.B. Kurup, M. S / Biomedicine & Pharmacotherapy 85 (2017) 725–732

Fig. 4. Photomicrographs of hematoxylin-eosin staining of pancreatic tissues of control and experimental groups of rats. Diabetes was induced by single intraperitoneal
injection of STZ (40 mg/kg body weight). ABE (25 mg/kg body weight/day) was orally administered daily for 60 days. At the end of experimental period, rats were sacrificed.
Pancreatic tissues were sectioned for the histological studies. A, normal control rats; B, normal rats treated with ethyl acetate fraction of Averrhoa bilimbi Linn fruits (ABE); C,
STZ-induced diabetic rats; D, STZ-induced diabetic rats treated with ethyl acetate fraction of Averrhoa bilimbi Linn fruits (ABE) at a dose of 25 mg/kg body weight/day; E, STZ-
induced diabetic rats treated with metformin at a dose of 100 mg/kg body weight/day.

(2, 3-BPG) which results in the destruction of glycolytic flux integrity of islets, reduced number of pancreatic b-cells and
through the inhibition of hexokinase [40]. Consistent with the moderate levels of inflammation, but ABE and metformin treated
above reports PK activity was significantly decreased in diabetic diabetic pancreas showed near the normal structure of pancreatic
rats. Treatment with ABE and metformin showed a remarkable islet cells and also preserved pancreatic b-cell integrity. The
increase in plasma insulin that induces a decrease in ATP, a known present findings suggest that ABE may regenerate b-cells and has a
allosteric inhibitor of pyruvate kinase, raises the pyruvate kinase protective effect on b-cells from glucose toxicity. The anti-
activity in diabetic rats. hyperglycemic activity of ABE may be mediated through insulin
Increased hepatic gluconeogenesis is a key step in the release from the remnant b-cells and increasing insulin sensitivity
pathogenesis of type 2 diabetes, which is synchronized primarily [45].
by gluconeogenic enzymes [41]. Glucose-6-phosphatase (G6Pase), The compounds in the ABE were identified by GC–MS analysis.
a key enzyme in the homeostatic regulation of blood glucose These include Phenelzine, Phenyl acetic acid, 1,3-Propanediol, 2,2-
catalyzes the conversion of glucose-6-phosphate to free glucose as dimethyl-, Benzeneacetamide, 2,4-Di-tert-butylphenol, Tetrapen-
the final step in gluconeogenesis and glycogenolysis [42]. Fructose- tacontane, 1,54-dibromo-, 2-Cyclohexen-1-one, 4-hydroxy-3,5,5-
1, 6-bisphosphatase (F1, 6 BPase) catalyzes one of the irreversible trim, L-Ascorbyl-6-dipalmitate, g-Stearolactone, 1-Hentetraconta-
steps in gluconeogenesis and serves as a spot for the regulation of nol and Octadecanoic acid, 12-hydroxy-, methyl ester. Ascorbyl
gluconeogenesis [43]. In agreement with the above reports, the palmitate, as the most abundant component of the ABE, has been
activities of G6Pase and F 1, 6 BPase were significantly increased in reported to protect the human RBC from oxidative damage [46].
STZ-induced diabetic group. Upon oral administration of ABE and Ascorbyl palmitate has been reported to have anticancer activity by
metformin the activities of hepatic gluconeogenic enzymes were inhibiting hepatocellular development and erythrocyte polyamine
significantly decreased, which reveal the reduced endogenous levels in ODS rats [47]. Phenyl aceticacid is identified to have anti-
glucose production. ABE may play a crucial role in maintaining the hyperglycemic activity by regulating gluconeogenesis and insulin
fasting blood glucose level compared to metformin treatment. secretion [48]. 4-di-tert-butylphenol has been reported to have an
Plasma insulin levels were found to increase significantly in antidiabetic effect in STZ-induced diabetic rats by regulating
diabetic rats treated with ABE, which may be a result of the glucose, insulin and lipid peroxidation [49]. Phenelzine, a
significant reduction in the level of gluconeogenic enzymes.The monoamino oxidase inhibitor is recognized to have a hypoglyce-
regulation of gluconeogenic flux by the extract might be one of the mic effect by regulating fasting blood glucose in diabetes mellitus
possible mechanisms for its antihyperglycemic nature. [50–52]. Benzeneacetamide, a hydroxyl steroid dehydrogenase
Previous studies have shown that the pancreas was damaged in inhibitor has been reported to have hypoglycemic effect in diabetic
STZ-induced diabetic rats [44]. Consistent with the above report rats by inhibiting the mRNA levels and activities of two key
histopathology of diabetic pancreas revealed the loss of structural enzymes in hepatic glucose production, phosphoenol pyruvate
 S.B. Kurup, M. S / Biomedicine & Pharmacotherapy 85 (2017) 725–732 731

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