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Protective potential of Averrhoa bilimbi fruits in ameliorating the hepatic key enzymes in streptozotocin-induced diabetic rats
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: Received 18 October 2016 Received in revised form 10 November 2016 Accepted 18 November 2016
Back ground: Diabetes is a mutifactorial disease which leads to several complications. Currently available drug regimens for management of diabetes have certain drawbacks. Need for safer and effective medicines from natural sources having potent antidiabetic activity. Averrhoa bilimbi Linn. (Oxalidaceae) is a medicinal plant and is reported to possess hypoglycemic activity. Objective: To investigate the antidiabetic potential of Averrhoa bilimbi fruit extract in streptozotocininduced diabetic rats. Methods: Diabetes was induced in male Sprague Dawley rats by single intraperitoneal injection of streptozotocin (STZ) (40 mg/kg body weight). The diabetic rats were treated orally with ethyl acetate fraction of A. bilimbi fruits (ABE) (25 mg/kg body weight) and metformin (100 mg/kg body weight) by 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.
Keywords: Averrhoa bilimbi Linn Diabetes mellitus Carbohydrate metabolism GC–MS analysis Phytomedicine
1. Introduction Diabetes mellitus (DM) is a multifaceted metabolic disorder characterized by chronic hyperglycemia ensuing from defects in insulin secretion, action or both [1]. Persistent hyperglycemia is a stage of the increased blood glucose level of circulating blood system that leads to the development of microvascular and macrovascular complications. Diabetes and its complications create a severe health care crisis worldwide. Globally, the prevalence of diabetes is predicted to grow from 366 million in 2011 to 552 million by 2030 [2].
* Corresponding author at: Department of Biochemistry, University of Kerala, Kariavattom, Thiruvananthapuram 695 581, Kerala, India. 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.
Diabetes results in the derangement of carbohydrate, lipid and protein metabolism [3]. Glucose homeostasis involves the coordinated regulation of pathways that direct glucose production and utilization [4]. In diabetes, the activities of key enzymes of glycolysis and gluconeogenesis are distorted, which leads to chronic hyperglycemia [5]. Effective measures to manage fasting and postprandial glucose levels are crucial in the management of diabetes. Though, numerous oral hypoglycemic drugs are now available, but their extended consumption produces adverse side effects [6]. Hence, there is a need to explore for phytocomponents that regularize hyperglycemia and diabetes-associated complications [7]. Averrhoa bilimbi Linn. (Oxalidaceae), commonly known as cucumber tree or tree sorrel is a widely cultivated plant in India, Indonesia, Sri Lanka, Bangladesh, Myanmar, Malaysia, Central and South America. The whole plant is used for treating coughs, cold,
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itches, rheumatism, whooping cough, hypertension etc. [8,9]. Traditionally A. bilimbi fruits are reported to have antidiabetic activity and no scientific data are available on the antidiabetic properties of the fruits [10,11]. Previous studies showed that the leaf extract of A. bilimbi and its semi-purified fractions possesses hypoglycemic and hypolipidemic properties in Type I diabetic rats when treated both intraperitoneally [12] as well as orally [13,14]. Our previous studies revealed the beneficial effect of aqueous extracts of Averrhoa bilimbi fruits in controlling blood glucose and lipid metabolism and preventing diabetic complications from lipid peroxidation in Type II diabetic rats [15]. In our preliminary study the ethyl acetate fraction of Averrhoa bilimbi fruits was found to be rich in phenolic compounds with superior antioxidant activity [16]. Preliminary invitro studies revealed the potency of ABE fraction. Present study evaluates the antidiabetic potential of ethyl acetate fraction of A. bilimbi fruits by monitoring activities of the key enzymes of carbohydrate metabolism in Type II diabetic rats. 2. Materials and methods 2.1. Chemicals Chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA), Merck Chemical Company (Darmstadt, Germany) and Sisco Research Laboratories (Mumbai, India). 2.2. Extraction of Averrhoa bilimbi fruits Fresh fruits of Averrhoa bilimbi were obtained from Thiruvananthapuram, Kerala, India, during the fruiting season (July– December 2014) and documented by Dr. Valsala Devi, Department of Botany, University of Kerala (Voucher no: KUBH 5865). Care was taken that the fruits, which were whitish-green in color and about 5–7.0 cm in size, were not overripe, spoiled or damaged. Fruits (5 kg) were cut and shade dried at a temperature of 28 C. Shade dried fruits were ground in a blender to give 500 g of fine powder. The aqueous extract was prepared by cold maceration of 500 g powder in 1000 ml of distilled water and lyophilized (Thermo electron corporation, MODUL YOD-230) (yield 26%). 100 g lyophilized A. bilimbi fruit extract was defatted with petroleum ether and the defatted extract was fractionated with ethyl acetate (1:1 v/v) (ABE-yield 5%) was concentrated at room temperature and used for the present study. 2.3. Experimental animals Two months old male Sprague Dawley rats (200–220 g body weight, 35 animals in total) bred in our Department animal house was used for the study. Animals were housed in polypropylene cages and maintained under standard conditions [12-h light/dark cycles, (25 10 C)]. All the animal care was taken as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals and the experimental protocol approved by the Institutional Animal Ethics Committee [IAEC-KU-20/2013-14-BC-SM (22)].
between 200 and 400 mg/dl were considered as diabetic and used for the study. 2.5. Experimental design A dose dependent toxicity study was carried out using ABE at different concentrations (5, 10 and 25 mg/kg body weight). The minimal effective dose was fixed as 25 mg/kg body weight by evaluating the activities of toxicity markers and antioxidant enzymes (results not shown). So for the present study, we have selected ABE at a dose of 25 mg/kg body weight and efficacy of ABE was compared with metformin, a standard oral hypoglycemic drug in streptozotocin induced diabetic rats. The experimental animals were divided into five groups, comprising of seven rats. ABE and metformin were administered intragastrically for 60 days. Group I: Normal control rats Group II: Normal rats treated with ABE (25 mg/kg body weight) Group III: Diabetic control rats Group IV: Diabetic rats treated with ABE (25 mg/kg body weight) Group V: Diabetic rats treated with metformin (100 mg/kg body weight) During the experimental period, body weight, blood glucose and physical examinations were done at regular intervals. The dosage was adjusted every week, according to changes in body weight to maintain similar dose per kg body weight of rat over the entire period of study. After 60 days, the rats were sacrificed by sodium pentothal injection. Blood, liver and pancreas were collected for various experimental analysis. 2.6. Gas chromatography–mass spectrometry (GC–MS) analysis The GC–MS analysis of the extract was performed using Agilent CP 3000 GC/Saturn 2200 MS (Agilent, Palo Alto, CA) equipped with ECD, PFPD and MS detectors. For GC/MS detection, an electron ionization system with ionization energy of 70 eV was used. Helium gas was used as the carrier gas at a constant flow rate of 1 ml/min. The inlet temperature was set at 250 C. The oven temperature was maintained at 100 C for 1.5 min and gradually increased up to 270 C at the rate of 5 C per min 1 ml diluted sample was injected and the scan range was 40–600 m/z. 2.7. Biochemical parameters The fasting blood glucose was monitored by glucometer (One Touch Horizon, Johnson and Johnson). Insulin was estimated by using an ELISA kit (DRG Diagnostics, USA). Hepatic toxicity markers such as alkaline phosphatase (ALP), acid phosphatase (ACP) and gamma glutamyl transferase (GGT) were measured using commercially available kits (Agappe Diagnostics, Ernakulam, India). Glycolytic enzymes viz, hexokinase (EC 2.7.1.1) [18] and pyruvate kinase (2.7.1.40) [19] were assayed. Activities of hepatic gluconeogenic enzymes, glucose-6-phosphatase (EC 3.1.3.9) [20] and fructose-1, 6-bisphosphatase (EC 3.1.3.11) [21] were assayed. 2.8. Histopathological analysis of pancreas
2.4. Induction of diabetes Diabetes was induced in rats by single intraperitoneal injection of freshly prepared streptozotocin (STZ) at a dose of 40 mg/kg body weight in 0.1 M citrate buffer (pH 4.5) [17]. The animals were given 5% glucose in drinking water overnight to overcome the druginduced hypoglycemia. 48 h after STZ injection, blood glucose levels were estimated and rats with blood glucose ranging
The pancreatic samples were set for 48 h in 10% neutral formalin, fixative solution was dehydrated by passing effectively in different mixture of ethyl alcohol-water, cleaned with xylene and embedded in paraffin. Sections of pancreas (5 mm thick) were prepared by using a rotary microtome and then stained with hematoxylin and eosin (H & E) dye, which mounted in a neutral deparaffinated xylene medium for microscopic observations [22].
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Fig. 1. GC–MS analysis of ethyl acetate fraction of Averrhoa bilimbi fruits.
3.2. Changes in body weight and ratio of liver/pancreas to 100 g body weight
2.9. Statistical analysis The values were expressed as mean SEM. The statistical analysis was carried out by one-way analysis of variance using SPSS (version 17) statistical analysis programme. Duncan’s post hoc multiple comparison tests were used to determine significant differences among groups. p < 0.05 was considered to be significant. 3. Results 3.1. GC–MS analysis The mass spectrum of ABE is shown in Fig. 1. Eleven compounds were identified in the ABE extract by the GC–MS analysis. To the best of our knowledge, the identified compounds were not reported previously in this plant. Compounds present in ABE were identified by comparison of their mass spectra with a built in National Institute of Standards and Technology (NIST). Identified compounds include Phenelzine, Phenyl acetic acid, 1,3-Propanediol, 2,2-dimethyl-, Benzeneacetamide, 2,4-Di-tert-butylphenol, Tetrapentacontane, 1,54-dibromo-, 2-Cyclohexen-1-one 4-hydroxy-3,5,5-trim, L-Ascorbyl-6-dipalmitate, g-Stearolactone, 1–Hentetracontanol and Octadecanoic acid, 12-hydroxy-, methyl ester. The active principles with their retention time (RT), molecular formula, molecular weight (MW) and peak area% were presented in Table 1.
Changes in body weight and liver/pancreas ratio to 100 g body weight in control and experimental rats were depicted in Tables 2 and 3. There was no significant difference among the groups during initial body weight estimation. The diabetic rats showed significant (p < 0.05) decrease in the body weight and liver/pancreas ratio when compared with normal control. Treatment with ABE and metformin to diabetic rats, significantly (p < 0.05) increased the body weight and liver/pancreas ratio. Changes in body weight and liver/pancreas ratio were comparable in diabetic rats treated with ABE and metformin. The treatment with ABE to normal rats resulted in no significant change in the body weight and liver/pancreas ratio as compared to normal control rats. 3.3. Blood glucose and insulin Fig. 2. shows the levels of blood glucose in control and experimental rats. Blood glucose was significantly (p < 0.05) increased in diabetic control rats (307.50 3.36 mg/dl) as compared with normal control. The administration of ABE and metformin for a period of 60 days significantly (p < 0.05) decreased blood glucose (148.63 5.53 mg/dl and 155.80 5.80 mg/dl) in diabetic rats. Blood glucose levels were comparable in diabetic rats treated with ABE and metformin. However, the administration of
Table 1 Chemical composition of ABE Extract. RT
Name
Molecular Formula
Molecular Weight
Peak Area %
2.064
Phenelzine [Hydrazine, (2-phenylethyl)-] 1,3-Propanediol, 2,2-dimethylPhenyl acetic acid Benzeneacetamide 2,4-Di-tert-butylphenol Tetrapentacontane, 1,54-dibromo2-Cyclohexen-1-one, 4-hydroxy-3,5,5-trim L-Ascorbyl-6-dipalmitate [l-(+)-Ascorbic acid-6-dihexadecanoate] g-Stearolactone [2(3H)-Furanone, dihydro-5-tetradecyl-] 1 Hentetracontanol Methyl ricinoleate [Octadecanoic acid, 4-hydroxy-, methyl ester]
C8H12N2
136.19
2.64
C5H12O2 C8H8O2 C8H9NO C14H22O C54H110 C13H18O3 C38H68O8
104.15 136.15 135.16 206.32 759.45 222.28 652.94
1.10 2.46 2.60 2.00 0.07 2.90 9.42
C18H34O2
282.46
0.90
C4H84O C19H36O3
593.10 312.48
0.91 0.20
5.757 8.198 10.901 13.478 19.368 21.727 25.875 28.445 32.066 35.360 RT: Retention Time.
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Table 2 Change in Body Weight (g). Groups
I II III IV V
Body Weight (g) Initial (0th day)
Final (60th day)
220.37 8.21 221.40 8.24 220.37 8.21 223.45 8.32 219.35 8.17
292.13 10.88 297.25 11.07 153.75 5.73a 236.25 8.76b 230.13 8.60b
Change in 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, STZinduced 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.
ABE to normal rats resulted in no significant (p < 0.05) change in the levels of blood glucose. The levels of plasma insulin in control and experimental rats were depicted in Fig. 3. The diabetic rats showed significant (p < 0.05) decrease in the level of plasma insulin when compared with normal control. Treatment with ABE and metformin to diabetic rats, significantly (p < 0.05) increased plasma insulin level (3.45-fold and 2.3-fold) when compared to diabetic control. ABE treatment showed a significant increase in insulin level than metformin treatment in diabetic rats. The treatment with ABE to normal rats resulted in no significant change in the level of plasma insulin as compared to normal control rats. 3.4. Hepatic toxicity markers Liver toxicity markers were assayed to assess hepatic injury. The activities of alkaline phosphatase (ALP), acid phosphatase (ACP) and gamma glutamyl transferase (GGT) were significantly altered in the diabetic control group, indicating damage to hepatocytes. The hepatic toxicity markers in normal rats treated with ABE did not show any statistical difference in comparison with normal control rats. Treatment with ABE and metformin significantly (p < 0.05) lowered the activities of these enzymes. Superior effect was seen in diabetic rats treated with ABE than metformin (Table 4). 3.5. Carbohydrate metabolizing enzymes Tables 5 and 6 summarizes the activities of hexokinase (HK), pyruvate kinase (PK), glucose-6-phosphatase (G6Pase) and fructose-1, 6-bisphosphatase (F 1, 6 BPase) in the control and experimental rats. STZ-induced diabetic rats showed a significant (p < 0.05) decrease in the activities of HK and PK along with the increase in the activities of G6Pase and F 1, 6 BPase in comparison
with the normal control. Treatment with ABE resulted in marked elevation in the activities of HK-59% and PK-68% and decreased in the activities of G6Pase-32% and F 1, 6 BPase-20% in hepatic tissue as compared with diabetic control. The modulatory effect of these enzymes by ABE was found to be comparable to the reference drug, metformin. Administration of ABE to normal rats did not show any significant effect in the activities of HK, PK, G6Pase and F 1, 6 BPase as compared with normal control rats. 3.6. Histopathological analysis of pancreas The histopathological analysis of pancreas in normal control and normal rats treated with ABE showed normal acini and islets with normal round or elongated structural intactness with their nucleus (Fig. 4A and B). While the STZ-induced diabetic group (Fig. 4C) exhibited loss of structural integrity of islets, reduced number of b-cells and moderate levels of inflammation, whereas diabetic rats treated with ABE and metformin restored the cellular morphology to almost normal levels (Fig. 4D and E). 4. Discussion Diabetes mellitus is characterized by a progressive dysfunction of pancreatic b-cells with a decline in insulin secretion leading to persistent hyperglycemia. STZ-induced hyperglycemia in experimental animals has been widely used as a valuable experimental model to study the effect of different hypoglycemic agents [23]. The STZ at lower doses (40 mg/kg body weight) destruct pancreatic b-cells moderately in rats, leading to deficient insulin secretion causing type 2 diabetic model [24,25]. Averrhoa bilimbi is a medicinal plant used by the folk healers as an antidiabetic agent. This study was conducted to evaluate the antidiabetic effect of ethyl acetate fraction of Averrhoa bilimbi fruits (ABE) in normal and STZ-induced diabetic rats by evaluating glucose profile, activity of hepatic enzymes, viz, hexokinase, pyruvate kinase, glucose-6-phosphatase and fructose-1, 6bisphosphatase as well as the capability of ABE to stimulate insulin secretion. Dehydration and loss of body weight has been reported to be associated with diabetes [26,27]. Loss of body weight in diabetes is due to dehydration, loss of carbohydrates and the excessive break down of tissue proteins and fat [28–30]. In agreement with the above reports, the body weight and tissue weight (liver and pancreas) were significantly decreased in STZ-induced diabetic rats. Treatment with ABE and metformin for 60 days significantly increased the body weight and tissue weight which indicates the better control of the hyperglycemic state in diabetic rats. The experimentally induced diabetic rats showed rigorous hyperglycemia consistent with a decrease in the endogenous insulin secretion and release. In the present study, fasting blood glucose was elevated significantly in diabetic control rats and this 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 II III IV V
3.07 0.11 3.18 0.12 1.03 0.04a 1.95 0.07b 1.85 0.07b
0.66 0.02 0.68 0.03 0.31 0.01a 0.49 0.02b 0.47 0.02b
4.74 0.18 4.75 0.18 3.38 0.13a 4.06 0.15b 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.
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Table 5 Hepatic glycolytic enzymes.
Fig. 2. Blood glucose. Values are expressed as mean SEM of seven rats in each group; significance accepted at P < 0.05. aStatistically significant as compared to normal group. bStatistically significant as compared to 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 weight/day; V, STZ-induced diabetic rats treated with metformin at a dose of 100 mg/kg body weight/day.
Groups
Hexokinase (mg glucose phosphorylated/ min/mg protein)
Pyruvate kinase (units/mg protein)
I II III IV V
16.58 0.62 17.22 0.67 7.21 0.27a 11.51 0.42b 11.25 0.41b
35.80 1.27 36.22 1.34 12.61 0.47a 21.19 0.78b 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 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, STZinduced 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.
Table 6 Hepatic gluconeogenic enzymes.
Fig. 3. Plasma Insulin. Values are expressed as mean SEM of seven rats in each group; significance accepted at P < 0.05. aStatistically significant as compared to normal group. bStatistically significant as compared to diabetic group. cStatistically 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 weight/day; V, STZ-induced diabetic rats treated with metformin at a dose of 100 mg/kg body weight/day.
Table 4 Hepatic toxicity markers. Groups
ALP (U/L)
ACP (U/L)
GGT (U/L)
I II III IV V
9.23 0.34 8.97 0.34 15.53 0.58a 11.69 0.44b 13.02 0.32b,c
121.27 4.23 117.88 4.39 164.00 6.11a 135.30 5.04b 148.63 5.53b,c
39.28 1.53 38.03 1.48 83.35 3.28a 59.80 2.37b 71.45 2.83b,c
Hepatic toxicity markers.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, STZinduced 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. c Statistically significant as compared to ABE treated diabetic group.
metformin showed a significant decrease in the level of blood glucose, which may be due to increased release of insulin from the existing and/or regenerated pancreatic b-cells [31,32]. Histopathology of the pancreas also confirms these findings.
Groups
Glucose-6-phosphatase (mmol of Pi liberated/ h/mg protein)
Fructose 1,6-bis-phosphatase (mmol of Pi liberated/ h/mg protein)
I II III IV V
25.29 0.94 24.63 0.92 65.26 2.43a 44.21 1.65b 41.51 1.55b
77.56 2.89 74.83 2.79 137.94 5.14a 109.50 4.08b 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, STZinduced 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.
Diabetes is frequently associated with the elevated activities of liver toxicity marker enzymes like ALP, ACP and GGT in serum, 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 increased in STZ-induced diabetic group. But ABE and metformin administration reduced the activities of ALP, ACP and GGT in diabetic condition and the effect was superior for ABE. In addition, the activities of liver toxicity markers were not altered in the normal rats treated with ABE group, in comparison with the normal control group. It indicates that ABE treatment was safe and has no significant toxicity. Several reports have shown that hexokinase is the most sensitive marker of the glycolytic pathway in diabetic rats [34,35]. Hexokinase phosphorylates glucose to glucose-6-phosphate and helps in the maintenance of glucose homeostasis [36]. Hexokinase is an insulin sensitive enzyme and is almost totally prevented or inactivated in the diabetic rat liver in the absence of insulin [37,38]. Treatment with ABE and metformin significantly increased the activity of hexokinase which indicates the effective utilization of glucose and direct stimulation of glycolysis in tissues with increased glucose elimination from the blood circulation. Pyruvate kinase is a regulatory enzyme ofglycolysis, catalyzes the conversion of phosphoenol pyruvate to pyruvate with the release of ATP [39]. PK deficiency results in the accumulation of the glycolytic intermediates chiefly 2, 3 bisphosphoglycerate
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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, STZinduced 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 through the inhibition of hexokinase [40]. Consistent with the above reports PK activity was significantly decreased in diabetic rats. Treatment with ABE and metformin showed a remarkable increase in plasma insulin that induces a decrease in ATP, a known allosteric inhibitor of pyruvate kinase, raises the pyruvate kinase activity in diabetic rats. Increased hepatic gluconeogenesis is a key step in the pathogenesis of type 2 diabetes, which is synchronized primarily by gluconeogenic enzymes [41]. Glucose-6-phosphatase (G6Pase), a key enzyme in the homeostatic regulation of blood glucose catalyzes the conversion of glucose-6-phosphate to free glucose as the final step in gluconeogenesis and glycogenolysis [42]. Fructose1, 6-bisphosphatase (F1, 6 BPase) catalyzes one of the irreversible steps in gluconeogenesis and serves as a spot for the regulation of gluconeogenesis [43]. In agreement with the above reports, the activities of G6Pase and F 1, 6 BPase were significantly increased in STZ-induced diabetic group. Upon oral administration of ABE and metformin the activities of hepatic gluconeogenic enzymes were significantly decreased, which reveal the reduced endogenous glucose production. ABE may play a crucial role in maintaining the fasting blood glucose level compared to metformin treatment. Plasma insulin levels were found to increase significantly in diabetic rats treated with ABE, which may be a result of the significant reduction in the level of gluconeogenic enzymes.The regulation of gluconeogenic flux by the extract might be one of the possible mechanisms for its antihyperglycemic nature. Previous studies have shown that the pancreas was damaged in STZ-induced diabetic rats [44]. Consistent with the above report histopathology of diabetic pancreas revealed the loss of structural
integrity of islets, reduced number of pancreatic b-cells and moderate levels of inflammation, but ABE and metformin treated diabetic pancreas showed near the normal structure of pancreatic islet cells and also preserved pancreatic b-cell integrity. The present findings suggest that ABE may regenerate b-cells and has a protective effect on b-cells from glucose toxicity. The antihyperglycemic activity of ABE may be mediated through insulin release from the remnant b-cells and increasing insulin sensitivity [45]. The compounds in the ABE were identified by GC–MS analysis. These include Phenelzine, Phenyl acetic acid, 1,3-Propanediol, 2,2dimethyl-, Benzeneacetamide, 2,4-Di-tert-butylphenol, Tetrapentacontane, 1,54-dibromo-, 2-Cyclohexen-1-one, 4-hydroxy-3,5,5trim, L-Ascorbyl-6-dipalmitate, g-Stearolactone, 1-Hentetracontanol and Octadecanoic acid, 12-hydroxy-, methyl ester. Ascorbyl palmitate, as the most abundant component of the ABE, has been reported to protect the human RBC from oxidative damage [46]. Ascorbyl palmitate has been reported to have anticancer activity by inhibiting hepatocellular development and erythrocyte polyamine levels in ODS rats [47]. Phenyl aceticacid is identified to have antihyperglycemic activity by regulating gluconeogenesis and insulin secretion [48]. 4-di-tert-butylphenol has been reported to have an antidiabetic effect in STZ-induced diabetic rats by regulating glucose, insulin and lipid peroxidation [49]. Phenelzine, a monoamino oxidase inhibitor is recognized to have a hypoglycemic effect by regulating fasting blood glucose in diabetes mellitus [50–52]. Benzeneacetamide, a hydroxyl steroid dehydrogenase inhibitor has been reported to have hypoglycemic effect in diabetic rats by inhibiting the mRNA levels and activities of two key enzymes in hepatic glucose production, phosphoenol pyruvate
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carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) [53,54]. The hypoglycemic effect of ABE may be attributed by the cumulative effect of these compounds. 5. Conclusion Our study clearly indicates that ABE has potential antihyperglycemic activity in STZ-induced diabetic rats. The protective effect of ABE against severe hyperglycemia may be due to the improvement in the peripheral glucose utilization, modulating glycolysis and gluconeogenesis. The beneficial antidiabetic effect of ABE was comparable with known standard hypoglycemic drug metformin. The chemical compounds identified by GC–MS analysis may be responsible for the anti-hyperglycemic activity. Thus, the dietary supplementation of Averrhoa bilimbi fruits may be helpful for the management of diabetes and prevention of diabetic complications. Conflict of interest statement The authors declare that there are no conflicts of interest. Author contributions Surya. B. Kurup designed and conducted the experiment, analyzed the results and prepared the manuscript; S. Mini was responsible for the very concept and design of the experiment and the interpretation of the data. Acknowledgements This work was financially supported by Promotion of University Research and Scientific Excellence (PURSE) programme, Department of Science and Technology (DST), Government of India. References [1] S. Akkati, K.G. Sam, G. Tungha, Emergence of promising the rapies in Diabetes mellitus, J. Clin. Pharmacol. 51 (2011) 796–804. [2] International Diabetes Federation, One adult in ten will have diabetes by 2030 Brussels: International Diabetes Federation; http://www.idf.org/mediaevents/press- releases/2011/diabetes-atlas-5th-edition, 2014. [3] Leelavinothan Pari, Subramani Srinivasan, Antihyperglycemic effect of diosmin on hepatic key enzymes of carbohydrate metabolism in streptozotocin-nicotinamide-induced diabetic rats, Biomed. Pharmacother. 64 (2010) 477–481. [4] R. Sundaram, R. Naresha, P. Shanthi, P. Sachdanandam, Modulatory effect of green tea extract on hepatic key enzymes of glucose metabolism in streptozotocin and high fat diet induced diabetic rats, Phytomedicine 20 (2013) 577–584. [5] S. Sivakumar, S.P. Subramanian, D-pinitol attenuates the impaired activities of hepatic key enzymes in carbohydrate metabolism of streptozotocin-induced diabetic rats, Gen. Physiol. Biophys. 28 (2009) 233–241. [6] K.B. Park, M. Lee, Y.H. Kim, T. Han, W. Yi, D.H. Lee, et al., Discovery of a novel phenylethyl benzamide glucokinase activator for the treatment of type 2 diabetes mellitus, Bioorg. Med. Chem. Lett. 23 (2013) 537–542. [7] R.B. Kasetti, S.A. Nabi, S. Swapna, C. Apparao, Cinnamicacid as one of the antidiabetic active principle(s) from the seeds of Syzygium alternifolium, Food Chem. Toxicol. 50 (2012) 1425–1431. [8] S.H. Goh, C.H. Chuah, J.S.L. Mok, E. Soepadmo, Malaysian Medicinal Plants for the Treatment of Cardiovascular Diseases, vol. 63, Pelanduk, Malaysia, 1995. [9] M.M. Mackeen, A.M. Ali, S.H. El Sharkawy, M.Y. Manap, K.M. Salleh, N.H. Lajis, et al., Antimicrobial and cytotoxic properties of some Malaysian traditional vegetables (ulam), Int. J. Pharmacogn. 35 (3) (1997) 174–178. [10] H.C.U. Ong, M. Nordiana, Malay ethno-medico botany in machang kelantan, Malaysia, Fitoterapia 70 (1999) 502–513. [11] A.N. Alsarhan, N. Sultana, M.R.A. Kadir, T. Aburjai, Ethno pharmacological survey of medicinal plants in Malaysia, the KangkarPulai region, Int. J. Pharmacol. 8 (8) (2012) 679–686. [12] B.K. Tan, P. Fu, P.W. Chow, A. Hsu, Effects of A. bilimbi on blood sugar and food intake in streptozotocin-induced diabetic rats, Phytomedicine 3 (1996) 271. [13] P. Pushparaj, C.H. Tan, B.K. Tan, Effects of Averrhoa bilimbi leaf extract on blood glucose and lipids in streptozotocin-diabetic rats, J. Ethnopharmacol. 72 (2000) 69–76.
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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: Received 18 October 2016 Received in revised form 10 November 2016 Accepted 18 November 2016
Back ground: Diabetes is a mutifactorial disease which leads to several complications. Currently available drug regimens for management of diabetes have certain drawbacks. Need for safer and effective medicines from natural sources having potent antidiabetic activity. Averrhoa bilimbi Linn. (Oxalidaceae) is a medicinal plant and is reported to possess hypoglycemic activity. Objective: To investigate the antidiabetic potential of Averrhoa bilimbi fruit extract in streptozotocininduced diabetic rats. Methods: Diabetes was induced in male Sprague Dawley rats by single intraperitoneal injection of streptozotocin (STZ) (40 mg/kg body weight). The diabetic rats were treated orally with ethyl acetate fraction of A. bilimbi fruits (ABE) (25 mg/kg body weight) and metformin (100 mg/kg body weight) by 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.
Keywords: Averrhoa bilimbi Linn Diabetes mellitus Carbohydrate metabolism GC–MS analysis Phytomedicine
1. Introduction Diabetes mellitus (DM) is a multifaceted metabolic disorder characterized by chronic hyperglycemia ensuing from defects in insulin secretion, action or both [1]. Persistent hyperglycemia is a stage of the increased blood glucose level of circulating blood system that leads to the development of microvascular and macrovascular complications. Diabetes and its complications create a severe health care crisis worldwide. Globally, the prevalence of diabetes is predicted to grow from 366 million in 2011 to 552 million by 2030 [2].
* Corresponding author at: Department of Biochemistry, University of Kerala, Kariavattom, Thiruvananthapuram 695 581, Kerala, India. 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.
Diabetes results in the derangement of carbohydrate, lipid and protein metabolism [3]. Glucose homeostasis involves the coordinated regulation of pathways that direct glucose production and utilization [4]. In diabetes, the activities of key enzymes of glycolysis and gluconeogenesis are distorted, which leads to chronic hyperglycemia [5]. Effective measures to manage fasting and postprandial glucose levels are crucial in the management of diabetes. Though, numerous oral hypoglycemic drugs are now available, but their extended consumption produces adverse side effects [6]. Hence, there is a need to explore for phytocomponents that regularize hyperglycemia and diabetes-associated complications [7]. Averrhoa bilimbi Linn. (Oxalidaceae), commonly known as cucumber tree or tree sorrel is a widely cultivated plant in India, Indonesia, Sri Lanka, Bangladesh, Myanmar, Malaysia, Central and South America. The whole plant is used for treating coughs, cold,
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itches, rheumatism, whooping cough, hypertension etc. [8,9]. Traditionally A. bilimbi fruits are reported to have antidiabetic activity and no scientific data are available on the antidiabetic properties of the fruits [10,11]. Previous studies showed that the leaf extract of A. bilimbi and its semi-purified fractions possesses hypoglycemic and hypolipidemic properties in Type I diabetic rats when treated both intraperitoneally [12] as well as orally [13,14]. Our previous studies revealed the beneficial effect of aqueous extracts of Averrhoa bilimbi fruits in controlling blood glucose and lipid metabolism and preventing diabetic complications from lipid peroxidation in Type II diabetic rats [15]. In our preliminary study the ethyl acetate fraction of Averrhoa bilimbi fruits was found to be rich in phenolic compounds with superior antioxidant activity [16]. Preliminary invitro studies revealed the potency of ABE fraction. Present study evaluates the antidiabetic potential of ethyl acetate fraction of A. bilimbi fruits by monitoring activities of the key enzymes of carbohydrate metabolism in Type II diabetic rats. 2. Materials and methods 2.1. Chemicals Chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA), Merck Chemical Company (Darmstadt, Germany) and Sisco Research Laboratories (Mumbai, India). 2.2. Extraction of Averrhoa bilimbi fruits Fresh fruits of Averrhoa bilimbi were obtained from Thiruvananthapuram, Kerala, India, during the fruiting season (July– December 2014) and documented by Dr. Valsala Devi, Department of Botany, University of Kerala (Voucher no: KUBH 5865). Care was taken that the fruits, which were whitish-green in color and about 5–7.0 cm in size, were not overripe, spoiled or damaged. Fruits (5 kg) were cut and shade dried at a temperature of 28 C. Shade dried fruits were ground in a blender to give 500 g of fine powder. The aqueous extract was prepared by cold maceration of 500 g powder in 1000 ml of distilled water and lyophilized (Thermo electron corporation, MODUL YOD-230) (yield 26%). 100 g lyophilized A. bilimbi fruit extract was defatted with petroleum ether and the defatted extract was fractionated with ethyl acetate (1:1 v/v) (ABE-yield 5%) was concentrated at room temperature and used for the present study. 2.3. Experimental animals Two months old male Sprague Dawley rats (200–220 g body weight, 35 animals in total) bred in our Department animal house was used for the study. Animals were housed in polypropylene cages and maintained under standard conditions [12-h light/dark cycles, (25 10 C)]. All the animal care was taken as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals and the experimental protocol approved by the Institutional Animal Ethics Committee [IAEC-KU-20/2013-14-BC-SM (22)].
between 200 and 400 mg/dl were considered as diabetic and used for the study. 2.5. Experimental design A dose dependent toxicity study was carried out using ABE at different concentrations (5, 10 and 25 mg/kg body weight). The minimal effective dose was fixed as 25 mg/kg body weight by evaluating the activities of toxicity markers and antioxidant enzymes (results not shown). So for the present study, we have selected ABE at a dose of 25 mg/kg body weight and efficacy of ABE was compared with metformin, a standard oral hypoglycemic drug in streptozotocin induced diabetic rats. The experimental animals were divided into five groups, comprising of seven rats. ABE and metformin were administered intragastrically for 60 days. Group I: Normal control rats Group II: Normal rats treated with ABE (25 mg/kg body weight) Group III: Diabetic control rats Group IV: Diabetic rats treated with ABE (25 mg/kg body weight) Group V: Diabetic rats treated with metformin (100 mg/kg body weight) During the experimental period, body weight, blood glucose and physical examinations were done at regular intervals. The dosage was adjusted every week, according to changes in body weight to maintain similar dose per kg body weight of rat over the entire period of study. After 60 days, the rats were sacrificed by sodium pentothal injection. Blood, liver and pancreas were collected for various experimental analysis. 2.6. Gas chromatography–mass spectrometry (GC–MS) analysis The GC–MS analysis of the extract was performed using Agilent CP 3000 GC/Saturn 2200 MS (Agilent, Palo Alto, CA) equipped with ECD, PFPD and MS detectors. For GC/MS detection, an electron ionization system with ionization energy of 70 eV was used. Helium gas was used as the carrier gas at a constant flow rate of 1 ml/min. The inlet temperature was set at 250 C. The oven temperature was maintained at 100 C for 1.5 min and gradually increased up to 270 C at the rate of 5 C per min 1 ml diluted sample was injected and the scan range was 40–600 m/z. 2.7. Biochemical parameters The fasting blood glucose was monitored by glucometer (One Touch Horizon, Johnson and Johnson). Insulin was estimated by using an ELISA kit (DRG Diagnostics, USA). Hepatic toxicity markers such as alkaline phosphatase (ALP), acid phosphatase (ACP) and gamma glutamyl transferase (GGT) were measured using commercially available kits (Agappe Diagnostics, Ernakulam, India). Glycolytic enzymes viz, hexokinase (EC 2.7.1.1) [18] and pyruvate kinase (2.7.1.40) [19] were assayed. Activities of hepatic gluconeogenic enzymes, glucose-6-phosphatase (EC 3.1.3.9) [20] and fructose-1, 6-bisphosphatase (EC 3.1.3.11) [21] were assayed. 2.8. Histopathological analysis of pancreas
2.4. Induction of diabetes Diabetes was induced in rats by single intraperitoneal injection of freshly prepared streptozotocin (STZ) at a dose of 40 mg/kg body weight in 0.1 M citrate buffer (pH 4.5) [17]. The animals were given 5% glucose in drinking water overnight to overcome the druginduced hypoglycemia. 48 h after STZ injection, blood glucose levels were estimated and rats with blood glucose ranging
The pancreatic samples were set for 48 h in 10% neutral formalin, fixative solution was dehydrated by passing effectively in different mixture of ethyl alcohol-water, cleaned with xylene and embedded in paraffin. Sections of pancreas (5 mm thick) were prepared by using a rotary microtome and then stained with hematoxylin and eosin (H & E) dye, which mounted in a neutral deparaffinated xylene medium for microscopic observations [22].
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Fig. 1. GC–MS analysis of ethyl acetate fraction of Averrhoa bilimbi fruits.
3.2. Changes in body weight and ratio of liver/pancreas to 100 g body weight
2.9. Statistical analysis The values were expressed as mean SEM. The statistical analysis was carried out by one-way analysis of variance using SPSS (version 17) statistical analysis programme. Duncan’s post hoc multiple comparison tests were used to determine significant differences among groups. p < 0.05 was considered to be significant. 3. Results 3.1. GC–MS analysis The mass spectrum of ABE is shown in Fig. 1. Eleven compounds were identified in the ABE extract by the GC–MS analysis. To the best of our knowledge, the identified compounds were not reported previously in this plant. Compounds present in ABE were identified by comparison of their mass spectra with a built in National Institute of Standards and Technology (NIST). Identified compounds include Phenelzine, Phenyl acetic acid, 1,3-Propanediol, 2,2-dimethyl-, Benzeneacetamide, 2,4-Di-tert-butylphenol, Tetrapentacontane, 1,54-dibromo-, 2-Cyclohexen-1-one 4-hydroxy-3,5,5-trim, L-Ascorbyl-6-dipalmitate, g-Stearolactone, 1–Hentetracontanol and Octadecanoic acid, 12-hydroxy-, methyl ester. The active principles with their retention time (RT), molecular formula, molecular weight (MW) and peak area% were presented in Table 1.
Changes in body weight and liver/pancreas ratio to 100 g body weight in control and experimental rats were depicted in Tables 2 and 3. There was no significant difference among the groups during initial body weight estimation. The diabetic rats showed significant (p < 0.05) decrease in the body weight and liver/pancreas ratio when compared with normal control. Treatment with ABE and metformin to diabetic rats, significantly (p < 0.05) increased the body weight and liver/pancreas ratio. Changes in body weight and liver/pancreas ratio were comparable in diabetic rats treated with ABE and metformin. The treatment with ABE to normal rats resulted in no significant change in the body weight and liver/pancreas ratio as compared to normal control rats. 3.3. Blood glucose and insulin Fig. 2. shows the levels of blood glucose in control and experimental rats. Blood glucose was significantly (p < 0.05) increased in diabetic control rats (307.50 3.36 mg/dl) as compared with normal control. The administration of ABE and metformin for a period of 60 days significantly (p < 0.05) decreased blood glucose (148.63 5.53 mg/dl and 155.80 5.80 mg/dl) in diabetic rats. Blood glucose levels were comparable in diabetic rats treated with ABE and metformin. However, the administration of
Table 1 Chemical composition of ABE Extract. RT
Name
Molecular Formula
Molecular Weight
Peak Area %
2.064
Phenelzine [Hydrazine, (2-phenylethyl)-] 1,3-Propanediol, 2,2-dimethylPhenyl acetic acid Benzeneacetamide 2,4-Di-tert-butylphenol Tetrapentacontane, 1,54-dibromo2-Cyclohexen-1-one, 4-hydroxy-3,5,5-trim L-Ascorbyl-6-dipalmitate [l-(+)-Ascorbic acid-6-dihexadecanoate] g-Stearolactone [2(3H)-Furanone, dihydro-5-tetradecyl-] 1 Hentetracontanol Methyl ricinoleate [Octadecanoic acid, 4-hydroxy-, methyl ester]
C8H12N2
136.19
2.64
C5H12O2 C8H8O2 C8H9NO C14H22O C54H110 C13H18O3 C38H68O8
104.15 136.15 135.16 206.32 759.45 222.28 652.94
1.10 2.46 2.60 2.00 0.07 2.90 9.42
C18H34O2
282.46
0.90
C4H84O C19H36O3
593.10 312.48
0.91 0.20
5.757 8.198 10.901 13.478 19.368 21.727 25.875 28.445 32.066 35.360 RT: Retention Time.
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Table 2 Change in Body Weight (g). Groups
I II III IV V
Body Weight (g) Initial (0th day)
Final (60th day)
220.37 8.21 221.40 8.24 220.37 8.21 223.45 8.32 219.35 8.17
292.13 10.88 297.25 11.07 153.75 5.73a 236.25 8.76b 230.13 8.60b
Change in 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, STZinduced 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.
ABE to normal rats resulted in no significant (p < 0.05) change in the levels of blood glucose. The levels of plasma insulin in control and experimental rats were depicted in Fig. 3. The diabetic rats showed significant (p < 0.05) decrease in the level of plasma insulin when compared with normal control. Treatment with ABE and metformin to diabetic rats, significantly (p < 0.05) increased plasma insulin level (3.45-fold and 2.3-fold) when compared to diabetic control. ABE treatment showed a significant increase in insulin level than metformin treatment in diabetic rats. The treatment with ABE to normal rats resulted in no significant change in the level of plasma insulin as compared to normal control rats. 3.4. Hepatic toxicity markers Liver toxicity markers were assayed to assess hepatic injury. The activities of alkaline phosphatase (ALP), acid phosphatase (ACP) and gamma glutamyl transferase (GGT) were significantly altered in the diabetic control group, indicating damage to hepatocytes. The hepatic toxicity markers in normal rats treated with ABE did not show any statistical difference in comparison with normal control rats. Treatment with ABE and metformin significantly (p < 0.05) lowered the activities of these enzymes. Superior effect was seen in diabetic rats treated with ABE than metformin (Table 4). 3.5. Carbohydrate metabolizing enzymes Tables 5 and 6 summarizes the activities of hexokinase (HK), pyruvate kinase (PK), glucose-6-phosphatase (G6Pase) and fructose-1, 6-bisphosphatase (F 1, 6 BPase) in the control and experimental rats. STZ-induced diabetic rats showed a significant (p < 0.05) decrease in the activities of HK and PK along with the increase in the activities of G6Pase and F 1, 6 BPase in comparison
with the normal control. Treatment with ABE resulted in marked elevation in the activities of HK-59% and PK-68% and decreased in the activities of G6Pase-32% and F 1, 6 BPase-20% in hepatic tissue as compared with diabetic control. The modulatory effect of these enzymes by ABE was found to be comparable to the reference drug, metformin. Administration of ABE to normal rats did not show any significant effect in the activities of HK, PK, G6Pase and F 1, 6 BPase as compared with normal control rats. 3.6. Histopathological analysis of pancreas The histopathological analysis of pancreas in normal control and normal rats treated with ABE showed normal acini and islets with normal round or elongated structural intactness with their nucleus (Fig. 4A and B). While the STZ-induced diabetic group (Fig. 4C) exhibited loss of structural integrity of islets, reduced number of b-cells and moderate levels of inflammation, whereas diabetic rats treated with ABE and metformin restored the cellular morphology to almost normal levels (Fig. 4D and E). 4. Discussion Diabetes mellitus is characterized by a progressive dysfunction of pancreatic b-cells with a decline in insulin secretion leading to persistent hyperglycemia. STZ-induced hyperglycemia in experimental animals has been widely used as a valuable experimental model to study the effect of different hypoglycemic agents [23]. The STZ at lower doses (40 mg/kg body weight) destruct pancreatic b-cells moderately in rats, leading to deficient insulin secretion causing type 2 diabetic model [24,25]. Averrhoa bilimbi is a medicinal plant used by the folk healers as an antidiabetic agent. This study was conducted to evaluate the antidiabetic effect of ethyl acetate fraction of Averrhoa bilimbi fruits (ABE) in normal and STZ-induced diabetic rats by evaluating glucose profile, activity of hepatic enzymes, viz, hexokinase, pyruvate kinase, glucose-6-phosphatase and fructose-1, 6bisphosphatase as well as the capability of ABE to stimulate insulin secretion. Dehydration and loss of body weight has been reported to be associated with diabetes [26,27]. Loss of body weight in diabetes is due to dehydration, loss of carbohydrates and the excessive break down of tissue proteins and fat [28–30]. In agreement with the above reports, the body weight and tissue weight (liver and pancreas) were significantly decreased in STZ-induced diabetic rats. Treatment with ABE and metformin for 60 days significantly increased the body weight and tissue weight which indicates the better control of the hyperglycemic state in diabetic rats. The experimentally induced diabetic rats showed rigorous hyperglycemia consistent with a decrease in the endogenous insulin secretion and release. In the present study, fasting blood glucose was elevated significantly in diabetic control rats and this 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 II III IV V
3.07 0.11 3.18 0.12 1.03 0.04a 1.95 0.07b 1.85 0.07b
0.66 0.02 0.68 0.03 0.31 0.01a 0.49 0.02b 0.47 0.02b
4.74 0.18 4.75 0.18 3.38 0.13a 4.06 0.15b 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.
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Table 5 Hepatic glycolytic enzymes.
Fig. 2. Blood glucose. Values are expressed as mean SEM of seven rats in each group; significance accepted at P < 0.05. aStatistically significant as compared to normal group. bStatistically significant as compared to 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 weight/day; V, STZ-induced diabetic rats treated with metformin at a dose of 100 mg/kg body weight/day.
Groups
Hexokinase (mg glucose phosphorylated/ min/mg protein)
Pyruvate kinase (units/mg protein)
I II III IV V
16.58 0.62 17.22 0.67 7.21 0.27a 11.51 0.42b 11.25 0.41b
35.80 1.27 36.22 1.34 12.61 0.47a 21.19 0.78b 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 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, STZinduced 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.
Table 6 Hepatic gluconeogenic enzymes.
Fig. 3. Plasma Insulin. Values are expressed as mean SEM of seven rats in each group; significance accepted at P < 0.05. aStatistically significant as compared to normal group. bStatistically significant as compared to diabetic group. cStatistically 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 weight/day; V, STZ-induced diabetic rats treated with metformin at a dose of 100 mg/kg body weight/day.
Table 4 Hepatic toxicity markers. Groups
ALP (U/L)
ACP (U/L)
GGT (U/L)
I II III IV V
9.23 0.34 8.97 0.34 15.53 0.58a 11.69 0.44b 13.02 0.32b,c
121.27 4.23 117.88 4.39 164.00 6.11a 135.30 5.04b 148.63 5.53b,c
39.28 1.53 38.03 1.48 83.35 3.28a 59.80 2.37b 71.45 2.83b,c
Hepatic toxicity markers.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, STZinduced 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. c Statistically significant as compared to ABE treated diabetic group.
metformin showed a significant decrease in the level of blood glucose, which may be due to increased release of insulin from the existing and/or regenerated pancreatic b-cells [31,32]. Histopathology of the pancreas also confirms these findings.
Groups
Glucose-6-phosphatase (mmol of Pi liberated/ h/mg protein)
Fructose 1,6-bis-phosphatase (mmol of Pi liberated/ h/mg protein)
I II III IV V
25.29 0.94 24.63 0.92 65.26 2.43a 44.21 1.65b 41.51 1.55b
77.56 2.89 74.83 2.79 137.94 5.14a 109.50 4.08b 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, STZinduced 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.
Diabetes is frequently associated with the elevated activities of liver toxicity marker enzymes like ALP, ACP and GGT in serum, 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 increased in STZ-induced diabetic group. But ABE and metformin administration reduced the activities of ALP, ACP and GGT in diabetic condition and the effect was superior for ABE. In addition, the activities of liver toxicity markers were not altered in the normal rats treated with ABE group, in comparison with the normal control group. It indicates that ABE treatment was safe and has no significant toxicity. Several reports have shown that hexokinase is the most sensitive marker of the glycolytic pathway in diabetic rats [34,35]. Hexokinase phosphorylates glucose to glucose-6-phosphate and helps in the maintenance of glucose homeostasis [36]. Hexokinase is an insulin sensitive enzyme and is almost totally prevented or inactivated in the diabetic rat liver in the absence of insulin [37,38]. Treatment with ABE and metformin significantly increased the activity of hexokinase which indicates the effective utilization of glucose and direct stimulation of glycolysis in tissues with increased glucose elimination from the blood circulation. Pyruvate kinase is a regulatory enzyme ofglycolysis, catalyzes the conversion of phosphoenol pyruvate to pyruvate with the release of ATP [39]. PK deficiency results in the accumulation of the glycolytic intermediates chiefly 2, 3 bisphosphoglycerate
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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, STZinduced 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 through the inhibition of hexokinase [40]. Consistent with the above reports PK activity was significantly decreased in diabetic rats. Treatment with ABE and metformin showed a remarkable increase in plasma insulin that induces a decrease in ATP, a known allosteric inhibitor of pyruvate kinase, raises the pyruvate kinase activity in diabetic rats. Increased hepatic gluconeogenesis is a key step in the pathogenesis of type 2 diabetes, which is synchronized primarily by gluconeogenic enzymes [41]. Glucose-6-phosphatase (G6Pase), a key enzyme in the homeostatic regulation of blood glucose catalyzes the conversion of glucose-6-phosphate to free glucose as the final step in gluconeogenesis and glycogenolysis [42]. Fructose1, 6-bisphosphatase (F1, 6 BPase) catalyzes one of the irreversible steps in gluconeogenesis and serves as a spot for the regulation of gluconeogenesis [43]. In agreement with the above reports, the activities of G6Pase and F 1, 6 BPase were significantly increased in STZ-induced diabetic group. Upon oral administration of ABE and metformin the activities of hepatic gluconeogenic enzymes were significantly decreased, which reveal the reduced endogenous glucose production. ABE may play a crucial role in maintaining the fasting blood glucose level compared to metformin treatment. Plasma insulin levels were found to increase significantly in diabetic rats treated with ABE, which may be a result of the significant reduction in the level of gluconeogenic enzymes.The regulation of gluconeogenic flux by the extract might be one of the possible mechanisms for its antihyperglycemic nature. Previous studies have shown that the pancreas was damaged in STZ-induced diabetic rats [44]. Consistent with the above report histopathology of diabetic pancreas revealed the loss of structural
integrity of islets, reduced number of pancreatic b-cells and moderate levels of inflammation, but ABE and metformin treated diabetic pancreas showed near the normal structure of pancreatic islet cells and also preserved pancreatic b-cell integrity. The present findings suggest that ABE may regenerate b-cells and has a protective effect on b-cells from glucose toxicity. The antihyperglycemic activity of ABE may be mediated through insulin release from the remnant b-cells and increasing insulin sensitivity [45]. The compounds in the ABE were identified by GC–MS analysis. These include Phenelzine, Phenyl acetic acid, 1,3-Propanediol, 2,2dimethyl-, Benzeneacetamide, 2,4-Di-tert-butylphenol, Tetrapentacontane, 1,54-dibromo-, 2-Cyclohexen-1-one, 4-hydroxy-3,5,5trim, L-Ascorbyl-6-dipalmitate, g-Stearolactone, 1-Hentetracontanol and Octadecanoic acid, 12-hydroxy-, methyl ester. Ascorbyl palmitate, as the most abundant component of the ABE, has been reported to protect the human RBC from oxidative damage [46]. Ascorbyl palmitate has been reported to have anticancer activity by inhibiting hepatocellular development and erythrocyte polyamine levels in ODS rats [47]. Phenyl aceticacid is identified to have antihyperglycemic activity by regulating gluconeogenesis and insulin secretion [48]. 4-di-tert-butylphenol has been reported to have an antidiabetic effect in STZ-induced diabetic rats by regulating glucose, insulin and lipid peroxidation [49]. Phenelzine, a monoamino oxidase inhibitor is recognized to have a hypoglycemic effect by regulating fasting blood glucose in diabetes mellitus [50–52]. Benzeneacetamide, a hydroxyl steroid dehydrogenase inhibitor has been reported to have hypoglycemic effect in diabetic rats by inhibiting the mRNA levels and activities of two key enzymes in hepatic glucose production, phosphoenol pyruvate
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carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) [53,54]. The hypoglycemic effect of ABE may be attributed by the cumulative effect of these compounds. 5. Conclusion Our study clearly indicates that ABE has potential antihyperglycemic activity in STZ-induced diabetic rats. The protective effect of ABE against severe hyperglycemia may be due to the improvement in the peripheral glucose utilization, modulating glycolysis and gluconeogenesis. The beneficial antidiabetic effect of ABE was comparable with known standard hypoglycemic drug metformin. The chemical compounds identified by GC–MS analysis may be responsible for the anti-hyperglycemic activity. Thus, the dietary supplementation of Averrhoa bilimbi fruits may be helpful for the management of diabetes and prevention of diabetic complications. Conflict of interest statement The authors declare that there are no conflicts of interest. Author contributions Surya. B. Kurup designed and conducted the experiment, analyzed the results and prepared the manuscript; S. Mini was responsible for the very concept and design of the experiment and the interpretation of the data. Acknowledgements This work was financially supported by Promotion of University Research and Scientific Excellence (PURSE) programme, Department of Science and Technology (DST), Government of India. References [1] S. Akkati, K.G. Sam, G. Tungha, Emergence of promising the rapies in Diabetes mellitus, J. Clin. Pharmacol. 51 (2011) 796–804. [2] International Diabetes Federation, One adult in ten will have diabetes by 2030 Brussels: International Diabetes Federation; http://www.idf.org/mediaevents/press- releases/2011/diabetes-atlas-5th-edition, 2014. [3] Leelavinothan Pari, Subramani Srinivasan, Antihyperglycemic effect of diosmin on hepatic key enzymes of carbohydrate metabolism in streptozotocin-nicotinamide-induced diabetic rats, Biomed. Pharmacother. 64 (2010) 477–481. [4] R. Sundaram, R. Naresha, P. Shanthi, P. Sachdanandam, Modulatory effect of green tea extract on hepatic key enzymes of glucose metabolism in streptozotocin and high fat diet induced diabetic rats, Phytomedicine 20 (2013) 577–584. [5] S. Sivakumar, S.P. Subramanian, D-pinitol attenuates the impaired activities of hepatic key enzymes in carbohydrate metabolism of streptozotocin-induced diabetic rats, Gen. Physiol. Biophys. 28 (2009) 233–241. [6] K.B. Park, M. Lee, Y.H. Kim, T. Han, W. Yi, D.H. Lee, et al., Discovery of a novel phenylethyl benzamide glucokinase activator for the treatment of type 2 diabetes mellitus, Bioorg. Med. Chem. Lett. 23 (2013) 537–542. [7] R.B. Kasetti, S.A. Nabi, S. Swapna, C. Apparao, Cinnamicacid as one of the antidiabetic active principle(s) from the seeds of Syzygium alternifolium, Food Chem. Toxicol. 50 (2012) 1425–1431. [8] S.H. Goh, C.H. Chuah, J.S.L. Mok, E. Soepadmo, Malaysian Medicinal Plants for the Treatment of Cardiovascular Diseases, vol. 63, Pelanduk, Malaysia, 1995. [9] M.M. Mackeen, A.M. Ali, S.H. El Sharkawy, M.Y. Manap, K.M. Salleh, N.H. Lajis, et al., Antimicrobial and cytotoxic properties of some Malaysian traditional vegetables (ulam), Int. J. Pharmacogn. 35 (3) (1997) 174–178. [10] H.C.U. Ong, M. Nordiana, Malay ethno-medico botany in machang kelantan, Malaysia, Fitoterapia 70 (1999) 502–513. [11] A.N. Alsarhan, N. Sultana, M.R.A. Kadir, T. Aburjai, Ethno pharmacological survey of medicinal plants in Malaysia, the KangkarPulai region, Int. J. Pharmacol. 8 (8) (2012) 679–686. [12] B.K. Tan, P. Fu, P.W. Chow, A. Hsu, Effects of A. bilimbi on blood sugar and food intake in streptozotocin-induced diabetic rats, Phytomedicine 3 (1996) 271. [13] P. Pushparaj, C.H. Tan, B.K. Tan, Effects of Averrhoa bilimbi leaf extract on blood glucose and lipids in streptozotocin-diabetic rats, J. Ethnopharmacol. 72 (2000) 69–76.
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