BIOCHEMICAL EVALUATION OF ANTIDIABETIC AND ANTIOXIDANT POTENTIALS OF ANNONA SQUAMOSA LEAVES EXTRACTS STUDIED IN STZ INDUCED DIABETIC RATS
HTML Full TextBIOCHEMICAL EVALUATION OF ANTIDIABETIC AND ANTIOXIDANT POTENTIALS OF ANNONA SQUAMOSA LEAVES EXTRACTS STUDIED IN STZ INDUCED DIABETIC RATS
- K. Hayath Basha and S. Subramanian*
Department of Biochemistry, University of Madras, Guindy campus, Chennai, Tamil Nadu, India
ABSTRACT
Diabetes mellitus is characterized by persistent fasting and postprandial blood glucose levels due to inability of the body cells to utilize glucose properly. Though drugs are plenty for the treatment of diabetes, none is found to be ideal due to undesirable side effects and diminution after prolonged use. Hence, search for novel drugs, especially from plant origin continues. Based on folkloric use, the present study was designed to evaluate the antidiabetic and antioxidant potential of Annona squamosa Linn. (Annonaceae) in STZ-induced experimental diabetes in rats. Daily oral administration of Annona squamosa leaves extract (100 mg/kg b.w./day) to diabetic rats for 30 days significantly reduced the levels of blood glucose, glycosylated hemoglobin, urea and creatinine. The observed decrease in the levels of plasma protein, plasma insulin, C-peptide and hemoglobin in the diabetic rats were elevated to near normal by the extract treatment. The altered antioxidant status of diabetic rats were reverted back to near normalcy by the administration of Annona squamosa leaves extract. The efficacy of the Annona squamosa extract was comparable with gliclazide, a known hypoglycemic drug.
Keywords:Annona squamosa,
STZ, Antidiabetic, |
Antioxidant
INTRODUCTION: Diabetes mellitus (DM), a major metabolic disorder in the endocrine system, is characterized by elevated blood glucose levels, alterations in carbohydrate, lipid and protein metabolism. It is becoming the third “killer” of mankind after cancer and cardiovascular diseases, because of its high prevalence, morbidity and mortality 1. The pathogenesis, progress and the possibility of its management by oral administration of hypoglycemic agents have stimulated great interest in recent decades. Numerous therapies designed for the treatment of DM have proven to be fairly effective, but none is ideal due to undesirable side effects and diminution after prolonged use. For a long time, diabetes has been treated orally with several medicinal plants or their extracts based on folk medicine.
Annona squamosa Linn. (Annonaceae) commonly known as custard apple, is well known for its edible fruits. The plant is traditionally used for the treatment of epilepsy, dysentery, cardiac problems, fainting, worm infestation, constipation, hemorrhage, fever, ulcers and also as an abortifacient 2. The aqueous leaves extract of A. squamosa has been reported to ameliorate hyperthyroidism 3, which is often considered as a causative factor for DM. A detailed review of literature afforded no systemic studies carried out on the medicinal properties of A. squamosa levels. Hence, in the present study an attempt has been made to evaluate the antidiabetic and antioxidant potential of A. squamosa leaves extract in STZ-induced experimental diabetes in rats.
MATERIALS AND METHODS:
Plant material: Fresh, mature Annona squamosa leaves were collected during July from a tree in Kolli Hills, Tamil Nadu, India. The plant was identified at the Herbarium of Botany, CAS in Botany, University of Madras. An exemplar specimen was deposited in the department herbarium.
Preparation of plant extract: The Annona squamosa leaves were first washed well with distilled water and dried at room temperature. The dried leaves were powdered in an electrical grinder and stored at 5°C until further use. One hundred grams of powder was extracted with petroleum ether (60-80°C) to remove lipids. It was then filtered, and the filtrate was discarded. The residue was extracted with 95% ethanol by soxhelation. The ethanol was evaporated in a rotary evaporator at 40-50°C under reduced pressure. The yield was 1.9 g/100 g. The extract was subjected to preliminary phytochemical screening for various plant constituents 4.
Experimental animals: Animal experiments were reviewed and approved by the Institutional Animal Ethics Committee of the University of Madras (approval no. 01/022/08). Male Wistar albino rats weighing 160-180 g procured from Tamil Nadu Veterinary and Animal Sciences University, Chennai, India were used. The rats were acclimatized and maintained over husk bedding in polypropylene cages in the central animal house facility of the institution. Throughout the experimental period, the rats were fed with balanced commercial pellet diet (Hindustan Lever Ltd., Bangalore, India) with composition of 5% fat, 21% protein, 55% nitrogen-free extract, and 4% fiber (w/w) with adequate mineral and vitamin levels for the animals. Diet and water were provided ad libitum.
Acute toxicity study: Acute toxicity studies on A. squamosa leaves extract were performed in control rats. Graded doses of the ethanolic extract of A. squamosa leaves extract (100, 250, 500, and 1,000 mg/kg body weight) were administered orally and the animals were observed for 2 weeks following administration 5. Change in body weight gain, food consumption, hematological, macroscopic, and clinical biochemical findings including the activities of pathophysiological enzymes were noted.
Experimental induction of diabetes: Rats were fasted overnight and experimental diabetes was induced by intraperitoneal injection of streptozotocin (STZ) with a single dose of 50 mg/kg body weight. STZ was dissolved in a freshly prepared 0.1 M cold citrate buffer pH- 4.5 6. Control rats were similarly injected with citrate buffer. Because STZ is capable of inducing fatal hypoglycemia as a result of massive pancreatic insulin release, STZ-treated rats were provided with 10% glucose solution after 6 h for the next 24 h to prevent hypoglycemia. Neither death nor any other adverse effect was observed. After 3 days for development and aggravation of diabetes, rats with moderate diabetes (i.e., blood glucose concentration 250 mg/dl) that exhibited glycosuria and hyperglycemia were selected for the experiment.
Dosage fixation study: A suitable optimum dosage schedule was identified by administering the A. squamosa leaves extract at different dosages (50, 100, 150 and 200 mg/kg b.w./day) for 30 days) to STZ induced diabetic rats. At the end of experimental period, all the rats were performed glucose tolerance test as described earlier. After conducting glucose tolerance test, the rats were sacrificed and blood was used to assess the biochemical changes. The dosage of A. squamosa leaves extract, which offered maximum hypoglycemic activity (as elicited by glucose tolerance, levels of blood glucose, glycosylated hemoglobin, blood urea and urine sugar) against streptozotocin-induced diabetic rats, was fixed up for all the subsequent experiments. The optimum dosage for A. squamosa leaves extract was fixed as 100 mg/kg body weight/day for 30 days.
Experimental design: The rats were divided into four groups comprising of six animals in each group as follows:
- Group I : Control rats receiving 0.1 M cold citrate buffer (pH 4.5).
- Group II : Diabetic control rats.
- Group III : Diabetic rats treated with Annona squamosa leaves extract (100mg/kg b.w. /day) in aqueous solution orally for 30 days.
- Group IV : Diabetic rats treated with gliclazide (5mg/kg b.w. /day) in aqueous solution orally for 30 days.
During the experimental period, body weight and blood glucose level of all the rats were determined at regular intervals of time. At the end of the experimental period, the rats were anaesthetized and sacrificed by cervical dislocation. Blood was collected with anticoagulant and used for the preparation of plasma. Blood collected without anticoagulant was used for serum separation.
Biochemical parameters: Whole blood was used for glucose 7 and urea 8 estimation. Plasma was separated and used for insulin and C-peptide assay using radioimmunoassay (RIA) kit for rats (Linco Research, Inc., USA). Levels of hemoglobin and glycosylated hemoglobin were estimated according to methods of Drabkin and Austin 9 and Nayak and Pattabiraman 10 respectively. Plasma was used for proteins assay 11 and serum for determination of creatinine 12. Activities of pathophysiological enzymes such as serum aspartate transaminase (AST), serum alanine transaminase (ALT), and serum alkaline phosphatase (ALP) were assayed by the method of King 13, 14. Levels of vitamin C, vitamin E, and glutathione (GSH) were determined by the methods of Omaye et al., 15 Desai 16 and Sedlak and Lindsay 17 respectively.
Liver tissue was excised, washed in ice-cold saline, and then homogenized in Tris–HCl buffer (pH 7.4) using a Teflon homogenizer. The liver homogenate was then centrifuged at 5,000 9 g to remove cellular debris and supernatant was used for determination of levels of lipid peroxides, hydroperoxides and enzymatic antioxidants. Lipid peroxidation was determined using thiobarbituric acid reactive substances by the method of Ohkawa et al.,18 and hydroperoxides were estimated by the method of Jiang et al.,19. Enzymatic antioxidants such as superoxide dismutase 20, catalase 21 and glutathione peroxidase 22 were assayed.
Histological studies: The liver, kidney and pancreatic tissues were dissected out and washed in ice-cold saline immediately. A portion of the liver, kidney and pancreatic tissues was fixed in 10% buffered neutral formalin solution for histological studies. After fixation, tissues were embedded in paraffin; solid sections were cut at 5μm. Liver and kidney sections were stained with haematoxylin and eosin and pancreas was stained with aldehyde fuschin. The sections were examined under light microscope and photomicrographs were taken 23.
Statistical analysis: All the grouped data were statistically evaluated with SPSS 16.00 software. Hypothesis testing methods included one-way analysis of variance followed by least significant difference (LSD) test. p<0.05 was considered to indicate statistical significance. All results are expressed as mean ± standard deviation (SD) for six rats in each group.
RESULTS:
Phytochemical analysis: Preliminary phytochemical screening proves the presence of biologically active principles like flavonoids, Alkaloids, Triterpenoids and Tannins.
Acute toxicity and dosage fixation: The results of acute toxicity studies of the ethanolic extract of A. squamosa leaves extract (100, 250, 500, and 1,000mg/kg body weight) on the normal rats indicated that the leaves extract was non-toxic up to the maximum dosage of 1000 mg/kg body weight. The optimum dosage for A. squamosa leaves extract was fixed as 100 mg/kg body weight/day for 30 days as the rats showed well improved glucose tolerance and controlled blood glucose, glycosylated hemoglobin and urine sugar (data not shown).
Effect of A. squamosa leaves extract on basic biochemical parameters: The biochemical parameters (Table 1) such as blood glucose, protein, urea and creatinine in control and experimental groups of rats shows significant increase in the levels of glucose, urea, creatinine and a significant decrease in the levels of plasma total protein in STZ-induced diabetic rats, when compared with control rats. Administration of A. squamosa leaves extract to diabetic rats for 30 days resulted in the restoration of blood glucose, total plasma protein, urea, and creatinine levels towards near normalcy as in gliclazide treated diabetic rats.
TABLE 1: BIOCHEMICAL MARKERS IN CONTROL AND EXPERIMENTAL GROUPS OF RATS
Groups | Control | Diabetic Control | Diabetic + A. squamosa | Diabetic + Gliclazide |
Blood glucose (mg/dl) | 82.63 ± 4.27 | 292.72 ± 12.33 a* | 91.54 ± 2.74 b* | 83.64 ± 4.02 b* |
Total protein (g/dl) | 7.14 ± 0.23 | 4.35 ± 0.35 a* | 6.87 ± 0.35 b* | 6.90 ± 0.34 b* |
Serum creatinine (mg/dl) | 0.71 ± 0.03 | 1.24 ± 0.22 a* | 0.66 ± 0.03 b* | 0.69 ± 0.02 b* |
Blood urea (mg/dl) | 17.44 ± 1.53 | 36.77 ± 2.33 a* | 18.28 ± 1.41 b* | 18.37 ± 0.71 b* |
Results are expressed as mean ± S.D. (n=6). One way ANOVA followed by post hoc test LSD. *p<0.05; the results were compared with a control; b Diabetic control
Effect of A. squamosa leaves extract on Insulin and C-peptide levels: The levels of insulin and C-peptide in control and experimental groups of rats are shown in Fig. 1 and 2 respectively. In diabetic rats, significant decrease was noted in the levels of insulin and C-peptide when compared with the control rats. Administration of A. squamosa leaves extract to diabetic rats increased the levels of insulin and C-peptide as compared with diabetic rats.
FIG. 1: THE LEVELS OF PLASMA INSULIN IN CONTROL AND EXPERIMENTAL GROUPS OF RATS
Results are expressed as mean ± S.D. (n=6). One way ANOVA followed by post hoc test LSD. *p<0.05; the results were compared with a control; b Diabetic control.
FIG. 2: THE LEVELS OF PLASMA C-PEPTIDE IN CONTROL AND EXPERIMENTAL GROUPS OF RATS
Results are expressed as mean ± S.D. (n=6). One way ANOVA followed by post hoc test LSD. *p <0.05; the results were compared with a control; b Diabetic control
Effect of A. squamosa leaves extract on hemoglobin and glycosylated hemoglobin levels: Fig. 3 shows the levels of hemoglobin and glycosylated hemoglobin in control and experimental groups of rats. A significant decrease in the level of hemoglobin and a significant increase in the level of glycosylated hemoglobin were observed in diabetic rats when compared to control rats. Administration of A. squamosa leaves extract to diabetic rats resulted in the restoration of hemoglobin and glycated hemoglobin levels to near normal.
FIG. 3: THE LEVELS OF HAEMOGLOBIN AND GLYCOSYLATED HEMOGLOBIN IN CONTROL AND EXPERIMENTAL GROUPS OF RATS
Units: g/dl for Hemoglobin; % Hemoglobin for Glycosylated hemoglobin. Results are expressed as mean ± S.D. (n=6). One way ANOVA followed by post hoc test LSD. *p <0.05; the results were compared with a control; b Diabetic control
Effect of A. squamosa leaves extract on the activities of pathophysiological enzymes: Table 2 depicts the activities of AST, ALT and ALP in serum of control and experimental groups of rats. There was a significant increase in the activities of AST, ALT and ALP in serum of diabetic rats when compared with control rats. Administration of A. squamosa leaves extract brought down these enzyme activities to near normal.
TABLE 2: THE ACTIVITIES OF PATHOPHYSIOLOGICAL ENZYMES IN CONTROL AND EXPERIMENTAL GROUPS OF RATS
Groups | Control | Diabetic Control | Diabetic + A. squamosa | Diabetic + Gliclazide |
Serum AST | 76.61 ± 2.45 | 133.50 ± 2.54 a* | 81.13 ± 1.80 b* | 78.42 ± 3.38 b* |
Serum ALT | 16.22 ± 0.73 | 35.21 ± 1.74 a* | 15.23 ± 0.55 b* | 14.42 ± 0.91 b* |
Serum ALP | 68.93 ± 1.11 | 107.79 ± 3.02 a* | 69.29 ± 1.93 b* | 71.79 ± 2.50 b* |
The enzyme activities expressed as: AST and ALT - mmoles of pyruvate/min/mg of protein. ALP - mmoles of phenol liberated/min/mg of protein. Results are expressed as mean ± S.D. (n=6). One way ANOVA followed by post hoc test LSD. *p <0.05; the results were compared with a control; b Diabetic control
Effect of A. squamosa leaves extract on the plasma non-enzymatic antioxidants levels: The levels of non-enzymatic antioxidants such as vitamin C, vitamin E and reduced glutathione in plasma of control and experimental groups of rats are as shown in Table 3. A significant decrease in the levels of vitamin C, vitamin E and reduced glutathione were found in diabetic rats when compared with control rats. These alterations were restored back to near normalcy in A. squamosa leaves extract treated diabetic rats which is similar to gliclazide treated diabetic rats.
TABLE 3: THE LEVELS OF VITAMIN C, VITAMIN E AND REDUCED GLUTATHIONE IN PLASMA OF CONTROL AND EXPERIMENTAL GROUPS OF RATS
Groups | Control | Diabetic Control | Diabetic + A. squamosa | Diabetic + Gliclazide |
Vitamin C (mg/dl) | 1.73 ± 0.13 | 0.60 ± 0.40 a* | 1.46 ± 0.10 b* | 1.63 ± 0.50 b* |
Vitamin E (mg/dl) | 1.20 ± 0.02 | 0.52 ± 0.06 a* | 0.92 ± 0.05 b* | 0.98 ± 0.03 b* |
Reduced Glutathione (mg/dl) | 28.73 ± 1.41 | 16.29 ± 1.46 a* | 24.94 ± 0.91 b* | 26.95 ± 0.82 b* |
Results are expressed as mean ± S.D. (n=6). One way ANOVA followed by post hoc test LSD. *p <0.05; the results were compared with a control; b Diabetic control
Effect of A. squamosa leaves extract on TBARS and hydroperoxides levels in liver tissues: The levels of TBARS and hydroperoxides in liver tissues of control and experimental groups of rats are presented in Fig. 4 and 5 respectively. Diabetic rats showed marked increase in TBARS and hydroperoxides when compared with control rats. Administration of both A. squamosa leaves extract as well as gliclazide to diabetic rats tends to bring the concentration of TBARS and hydroperoxides significantly to near normal levels.
Effect of A. squamosa leaves extract on the liver enzymatic antioxidants: The activities of SOD, CAT and GPx in liver tissues of control and experimental groups of rats are represented in Table 4. A significant decrease in the activities of these antioxidant enzymes was found in liver tissues of diabetic rats when compared with control rats. The altered activities of antioxidant enzymes were brought back to near normalcy by A. squamosa leaves extract as well as gliclazide treated diabetic rats.
TABLE 4: ACTIVITIES OF ANTIOXIDANT ENZYMES IN LIVER TISSUES OF CONTROL AND EXPERIMENTAL GROUPS OF RATS
Groups | Control | Diabetic Control | Diabetic + A. squamosa | Diabetic + Gliclazide |
SOD | 13.48±0.43 | 6.97 ± 0.18a* | 11.86 ± 0.46b* | 13.01 ± 0.66b* |
CAT | 76.52±2.85 | 43.37 ± 2.4a* | 71.16 ± 0.9b* | 75.50 ± 3.15b* |
GPx | 11.64±0.83 | 5.53 ± 0.67a* | 10.21 ± 0.43b* | 10.92 ± 0.48b* |
Activities were expressed as: 50% of inhibition of epinephrine auto oxidation/mg protein/ min for superoxide dismutase (SOD); μmoles of hydrogen peroxide decomposed per min per mg of protein for Catalase (CAT); μmoles of glutathione oxidized per min per mg of protein for Glutathione peroxidase (GPx). Results are expressed as mean ± S.D. (n=6). One way ANOVA followed by post hoc test LSD. *p <0.05; the results were compared with a control; b Diabetic control
FIG. 4: THE LEVELS OF LIPID PEROXIDE IN LIVER TISSUES OF CONTROL AND EXPERIMENTAL GROUPS OF RATS
Units: nmoles of TBARS/100 g of wet tissue. Results are expressed as mean ± S.D. (n=6). One way ANOVA followed by post hoc test LSD. *p<0.05; the results were compared with a control; b Diabetic control
FIG. 5: THE LEVELS OF HYDROPEROXIDE IN LIVER TISSUES OF CONTROL AND EXPERIMENTAL GROUPS OF RATS
Units: nmoles/100 g of wet tissue. Results are expressed as mean ± S.D. (n=6). One way ANOVA followed by post hoc test LSD. *p <0.05; the results were compared with a control; b Diabetic control
PLATE 1 (A-D): HISTOPATHOLOGICAL OBSERVATIONS IN THE LIVER TISSUE OF CONTROL AND EXPERIMENTAL GROUPS OF RATS (HE, 100X)
PLATE 2 (A-D): HISTOPATHOLOGICAL OBSERVATIONS IN THE KIDNEY TISSUE OF CONTROL AND EXPERIMENTAL GROUPS OF RATS (HE, 100X)
PLATE 3 (A-D): HISTOPATHOLOGICAL OBSERVATIONS IN THE PANCREATIC TISSUE OF CONTROL AND EXPERIMENTAL GROUPS OF RATS (ALDEHYDE FUSCHIN, 320X)
DISCUSSION: The herbal extracts contain different phytochemicals with wide range of biological activity. For example, phytochemicals such as saponins, terpenoids, flavonoids and tannins found to inhibit cancer cell proliferation, regulate inflammatory, immune response and protect against lipid peroxidation. Most of the phytochemicals have the ability to inhibit lipid peroxidation and also possess hypoglycemic and hypolipidemic properties 24.
In the present study, we have found that most of the biologically active phytochemicals were present in the ethanolic leaves extract of A. squamosa. The antidiabetic and antioxidant properties of A. squamosa leaves extract may be due to the presence of such phytochemicals. Streptozotocin- induction cause specific damage to islet b-cells and thus exerts a pronounced increase in the concentrations of blood glucose. It is well established that gliclazide produce hypoglycemia and is often used as a standard drug in STZ-induced moderate diabetic models to compare the antidiabetic properties of a variety of compounds 25. Administration of A. squamosa leaves extract to STZ-induced diabetic rats resulted in a significant reduction in blood glucose level.
Insulin deficiency is manifested in a number of biochemical and physiological alterations. Insulin estimations and more specifically assessment of C-peptide are generally accepted as an index of β-cell function. In the present study, we have observed a significant decrease in the levels of insulin and C-peptide in streptozotocin-induced diabetic rats. C-peptide deficiency is a contributing pathogenic factor in type 1 diabetic complications. C-peptide promotes insulin action at low hormone concentration and inhibits it at high hormone levels suggesting a modulatory effect by C-peptide on insulin signaling. C-peptide has insulinomimetic effects on its own by activating insulin receptor and increases glycogen synthesis and amino acid uptake. Oral administration of A. squamosa leaves extract increased the levels of insulin and C-peptide and decreased the levels of blood glucose. This decreased level of blood glucose might be due to the increased formation of glycogen and amino acid uptake by C-peptide activity 26. The increased level of insulin as well as C-peptide in A. squamosa leaves extract treated diabetic rats might be due to the activation of remnant β-cells of the pancreas.
During diabetes the excess glucose present in blood reacts non-enzymatically with hemoglobin to form glycosylated hemoglobin (HbA1C). As a result, the total hemoglobin level is decreased in diabetic rats 27. The rate of glycosylation is proportional to the concentration of blood glucose 28. Hence, estimation of glycosylated hemoglobin is a well-accepted biochemical parameter useful for the diagnosis and management of the disease. The increased glycated hemoglobin is associated with loss of b-cell function and has been implicated in the complications of diabetes mellitus 29. Glycated hemoglobin (HbA1C) was found to increase in patients with diabetes mellitus and the amount of increase being directly proportional to the fasting blood glucose level 30. Oral administrations of A. squamosa leaves extract as well as gliclazide tend to decrease the level of glycosylated hemoglobin by improving the blood glucose homeostasis.
During diabetes, there is increased protein catabolism with flow of amino acids into liver, which feeds gluconeogenesis 31. STZ-induced diabetic rats manifest a negative nitrogen balance related to enhanced proteolysis particularly in the muscles coupled with lowered protein synthesis. The accelerated proteolysis of uncontrolled diabetes occurs as a result of deranged glucagon-mediated regulation of cyclic AMP formation in insulin deficiency 32. This readily accounts for the observed decrease in the total protein content in diabetes mellitus. Administration of A. squamosa leaves extract to diabetic rats significantly improves the total plasma protein level to near normal.
Elevated protein catabolism with inflow of amino acids to the liver in diabetes facilitates urea synthesis thereby resulting in hyperuremia 31. Thus the hyperuremia in blood reflects either increased synthesis of urea or its decreased excretion. Administration of A. squamosa leaves extract to diabetic rats decreased the level of blood urea to near normal and was comparable with gliclazide treated rats.
Creatinine is a break-down product of creatine phosphate in muscle, and is usually produced at a fairly constant rate by the body. Creatinine is chiefly filtered out of the blood by the kidneys. If the filtering ability of the kidney is deficient, blood creatinine levels rise. Therefore, creatinine levels in blood and urine may be used to assess the renal function. Oxidative stress in diabetes causes renal dysfunction 33. The observed increase in creatinine level in diabetic rats is mainly due to renal dysfunction and is altered to near normal by oral administration of A. squamosa leaves extract for 30 days.
The clinical and diagnostic values associated with changes in serum enzyme activities such as AST, ALT and ALP have long been recognized. The increased activity of these enzymes during diabetic condition is probably due to the alterations in the normal function of the cell.[34] Increase in serum ALT activity is almost always due to hepatocellular damage followed by cardiac damage and is usually accompanied by an increase in AST activity 35. The normalization of the activities of AST and ALT by the administration of A. squamosa leaves extract as well as gliclazide to STZ induced diabetic rats indicates amelioration of cellular dysfunction and tissue damage caused by hyperglycemia. Alkaline phosphatase is a non-specific hepatic marker enzyme. ALP and ACP activities were markedly increased in insulin deficient animals leading to tissue necrosis.[36] The altered activities were reverted back to near normal by A. squamosa leaves extract as well as glyclazide treatment. The reversal of AST, ALT and ALP activities in A. squamosa leaves extract treated diabetic rats towards near normalcy indicates the tissue protective and non-toxic nature of the A. squamosa leaves extract.
Non-enzymatic antioxidants such as vitamin C (ascorbic acid) vitamin E (α-tocopherol), reduced glutathione (GSH) may work synergistically in cellular antioxidant defense 37. Vitamin C is one of the four dietary antioxidants, the other three being vitamin E, vitamin A and selenium. Also, vitamin C regenerates vitamin E from its oxidized form 38. Vitamin C has been recognized as an outstanding plasma antioxidant and its depletion leads to formation of hydroperoxides even when the other antioxidants are still present 39. The levels of plasma vitamin C were found to be lowered in diabetic rats.
Thus, the elevation in glucose concentration may depress natural antioxidant like vitamin C or due to decrease in GSH levels, since GSH is required for recycling of vitamin C. Vitamin E is a lipophilic antioxidant and inhibits lipid peroxidation, scavenging lipid peroxyl radicals to yield lipid hydroperoxides and the a-tocopheroxyl radial 40. Vitamin E is also responsible for protecting poly unsaturated fatty acid (PUFA) against lipid peroxidation and its deficiency in diabetes may be due to their exhaustion during detoxification of free radicals produced by membrane lipid peroxidation 41. Diabetic rats administered with A. squamosa leaves extract which contains antioxidant phytochemicals significantly reduces the generated free radicals thereby maintaining the normal levels of vitamin C and vitamin E.
GSH is an essential antioxidant for recycling of vitamin E and C 42. During diabetes the decreased levels of vitamin C diminish the recycling of vitamin E 43. GSH has a multifaceted role in antioxidant defense. It is a direct scavenger of free radicals as well as a co-substrate for glutathione peroxidase activity and as a cofactor for many enzymes and also act as a conjugates in endo and xenobiotic reactions 44. The present study confirms with the finding that decreased level of glutathione in diabetes may represent increased utilization due to oxidative stress.
Several studies support the hypothesis that in diabetes, chronic hyperglycemia increases the polyol pathway as well as advanced glycation end products (AGEs) formation and free radical generation rates, leading to increased GSH oxidation. A relative depletion of NADPH due to aldose reductase activation and secondary to reduced production through the pentose cycle impairs GSH generation and leads to depletion of this free radical scavenger 45.
Glutathione reductase (GR) plays a major role in regenerating endogenous GSH from GSSG, thus maintaining the balance between the redox couple. Decrease in the activity of G6PD in diabetes results in reduced availability of NADPH and hence decreased levels of GSH. Reversal of hyperglycemia and scavenging of free radicals by the A. squamosa leaves extract may itself decrease the oxidative stress and thus may enhance glutathione content.
The endogenous antioxidant enzymes such as SOD, CAT and GPx are responsible for the detoxification of deleterious oxygen radicals. SOD has been postulated as one of the most important enzymes in the enzymatic antioxidant defense system which catalyses the dismutation of superoxide radicals to produce H2O2 and molecular oxygen hence diminishing the toxic effects caused by these radicals 46. Catalase (CAT) is a hemeprotein which catalyses the reduction of hydrogen peroxides and protects the tissues from highly reactive hydroxyl radicals. The superoxide anion has been known to inactivate CAT, which is involved in the detoxification of hydrogen peroxide. GPx plays a primary role in minimizing oxidative damage. It has been proposed that GPx is responsible for the detoxification of H2O2 in low concentration whereas catalase comes into play when GPx pathway is reaching saturation with the substrate. GPx catalyze the reduction of hydrogen peroxide and hydroperoxides to non-toxic metabolites.
The reduction in SOD and catalase activities in diabetic condition may be due to direct glycation of enzyme protein 47. The increase in superoxide radical in diabetes may inhibit the activity of catalase and glutathione peroxidase 48, 49. GPx function is concert with GSH in decomposing hydrogen peroxide generated from free radical mediated reactions. Due to decrease in the concentration of GSH, the activity of GPx is also decreased in diabetes. The diminished activity of GPx in diabetes elevates hydrogen peroxide and lipid peroxides leading to the accumulation of these oxidants and thus the subsequent oxidation of lipids. It has been concluded that the restoration of antioxidant defense system by the administration of A. squamosa leaves extract may be attributed mainly due to the presence of antioxidant phytochemicals.
The tremendous increase in TBARS and hydroperoxides in liver tissues suggests an increase in oxygen radicals that could be due to either increased production or decreased detoxification which could be due to persistent hyperglycemia in diabetes 50. Maintenance of persistent normoglycemia by the administration of A. squamosa leaves extract may attenuate lipid peroxidation in tissues and thus prevent tissue damage.
The pathological changes observed in liver [Plate 1(a-d)], kidney [Plate 2(a-d)] and pancreas [Plate 3(a-d)] of STZ diabetic rats may be due to the hyperglycemia and its mediated oxidative stress. A. squamosa leaves extract resulted in glucose homeostasis and attenuation of oxidative stress by optimization of antioxidant enzymes, which could have protected tissue damages. The histological evidence authenticated the extent of tissue injury by streptozotocin and the protection offered to hepatic, renal and pancreatic b cells by leaves extract and also revealed the non-toxic nature of A. squamosa leaves extract.
From the results of the present study it may be concluded that the antidiabetic and antioxidant nature of A. squamosa leaves might be due to the presence of biologically active phytochemicals present in the extract. Further, the present study demonstrates the scientific rationale for the use of A. squamosa leaves in the traditional medicine for the treatment of diabetes.
References
- Li WL, Zheng HC, Bukuru J, De Kimpe N: Natural medicines used in the traditional Chinese medical system for therapy of diabetes mellitus. J Ethnopharmacol 2004; 92:1-21.
- Nadkarni AK: Indian Materia Medica. Popular Prakashan, Mumbai, India, Vol.1, 2000: pp. 116.
- Sunanda P, Anand K: Possible amelioration of hyperthyroidism by the leaf extract of Annona squamosa. Curr Sci 2003; 84:1402-1404.
- Harborne JB: Phytochemical methods: a guide to modern techniques of plant analysis. Chapman and Hall, New York, 3rd edition, 1998.
- Mancebo A, Scull I, Gonzales Y, Arteaga ME, Gonzales BO, Fuentes D, Hernandez O, Correa M: Ensayo de toxicidad a dosis repetidas (28 dias) por via oral del extracto acuoso de Morinda citrifolia en rata Sprague Dawley. Rev Toxicol 2002; 19:73-77.
- Rakieten N, Rakieten ML, Nadkarni MV: Studies on the diabetogenic action of streptozotocin (NSC-37917). Cancer Chemother Rep 1963; 29:91-98.
- Sasaki T, Matsy S, Sonae A: Effect of acetic acid concentration on the colour reaction in the O-toluidine boric acid method for blood glucose estimation. Rinsh Kagaku 1972; 1:346-353.
- Natelson S, Scott ML, Beffa C: A rapid method for the estimation of urea in biologic fluids. Am J Clin Pathol1951; 21:275-281.
- Drabkin DL, Austin JM: Spectrophotometeric constants for common hemoglobin derivatives in human, dog and rabbit blood. J Biol Chem 1932; 98:719-733.
- Nayak SS, Pattabiraman TN: A new colorimetric method for the estimation of glycosylated hemoglobin. Clin Chim Acta1981; 109:267-274.
- Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265-275.
- Brod J, Sirota JH: The Renal Clearance of Endogenous "Creatinine" In Man, J Clin Invest 1948; 27:645-654.
- King J: The transferase alanine and aspartate transaminase. In: Practical Clinical Enzymology. D.Van Nostrand Company, London, 1965a: 363-395.
- King J: The hydrolases-acid and alkaline phosphatases. In: Practical Clinical Enzymology. D.Van Nostrand Company, London, 1965c: 199-208.
- Omaye ST, Turnbull JD, Sauberlich HE: Selected methods for the determination of ascorbic acid in animal cells, tissues, and fluids. Methods Enzymol 1979; 62:3-11.
- Desai ID: Vitamin E analysis methods for animal tissues. Methods Enzymol1984; 105:138-147.
- Sedlak J, Lindsay RH: Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal Biochem 1968; 25:192-205.
- Ohkawa H, Ohishi N, Yagi K: Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979; 95:351-358.
- Jiang ZY, Hunt JV, Wolff SP: Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein. Anal Biochem 1992; 202:384-389.
- Misra HP, Fridovich I: The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 1972; 247:3170-3175.
- Takahara S, Hamilton HB, Neel JV, Kobara TY, Ogura Y, Nishimura ET: Hypocatalasemia: a new genetic carrier state. J Clin Invest 1960; 39:610-619.
- Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG: Selenium: biochemical role as a component of glutathione peroxidase. Science 1973; 179:588-590.
- Gordon K, Bradbury P: Microtomy and paraffin sections. Bancroft JD, Stevens A, editors. Theory and Practice of Histological Techniques. Churchill Livingston, New York, 3rd edition, 1990: 61-80.
- Johnson MB, Heineke EW, Rhinehart BL, Shietz MJ, Bambart RL, Robinson KM: Antioxidant with marked lipid and glucose lowering activity in diabetic rats and mice. Diabetes; 42:1179-1186.
- Andrade Cetto A, Wiedenfeld H, Revilla MC, Sergio IA: Hypoglycemic effect of Equisetum myriochaetum aerial parts on streptozotocin diabetic rats. J Ethnopharmacol 2000; 72:129-133.
- Grunberger G, Qiang X, Li Z, Mathews ST, Sbrissa D, Shisheva A, Sima AA: Molecular basis for the insulinomimetic effects of C-peptide. Diabetologia 2001; 44:1247-1257.
- Sheela CG, Augusti KT: Antidiabetic effects of S-allyl cysteine sulphoxide isolated from garlic Allium sativum Linn. Indian J Exp Biol 1992; 30:523-526.
- Monnier VM, Cerami A: Non-enzymatic glycosylation and browning of proteins in diabetes. Clin Endocrinol Metab 1982; 11:431-452.
- Yates AP, Laing I: Age-related increase in haemoglobin A1c and fasting plasma glucose is accompanied by a decrease in beta cell function without change in insulin sensitivity: evidence from a cross-sectional study of hospital personnel. Diabet Med 2002; 19:254-258.
- Koenig RJ, Peterson CM, Jones RL, Saudek C, Lehrman M, Cerami A: Correlation of glucose regulation and hemoglobin AIc in diabetes mellitus. N Engl J Med 1976; 295:417-420.
- Rannels DE, Marker DE, Morgan HE : Biochemical actions of hormones. In: G. Litwack (ed), Academic Press, New York, Vol. 1, 1997: 135-195.
- Dighe RR, Rojas FJ, Birnbaumer L, Garber AJ: Glucagon-stimulable adenylyl cyclase in rat liver. The impact of streptozotocin-induced diabetes mellitus. J Clin Invest 1984; 73:1013-1023.
- Ha H, Lee HB: Oxidative stress in diabetic nephropathy: basic and clinical information. Curr Diab Rep 2001; 3:282-287.
- Kumar JS, Menon VP: Peroxidative changes in experimental diabetes mellitus. Indian J Med Res 1992; 96:176-181.
- Rao GM, Morghom LO, Kabur MN, Ben Mohmud BM, Ashibani K: Serum glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels in diabetes mellitus. Indian J Me Sci 1989; 43:118-121.
- Hough S, Avioli LV, Teitelbaum SL, and Fallon MD: Alkaline phosphatase activity in chronic streptozotocin-induced insulin deficiency in the rat: effect of insulin replacement. Metabolism 1981; 30:1190-1194.
- Meister A: Glutathione-ascorbic acid antioxidant system in animals. J Biol Chem 1994; 269:9397-9400.
- Buettner GR: The pecking order of free radicals and antioxidants: lipid peroxidation, alpha-tocopherol, and ascorbate. Arch Biochem Biophys 1993; 300:535-543.
- Frei B, England L, and Ames BN: Ascorbate is an outstanding antioxidant in human blood plasma. Proc Natl. Acad. Sci. USA 1989; 86:6377-6381.
- Stahl W, Sies H: Antioxidant defense: vitamins E and C and carotenoids. Diabetes 1997;46:S14-18.
- Sharma A, Kharb S, Chugh SN, Kakkar R, Singh GP: Effect of glycemic control and vitamin E supplementation on total glutathione content in non-insulin-dependent diabetes mellitus. Ann Nutr Metab 2000; 44:11-13.
- Constantinescu A, Han D, Packer L: Vitamin E recycling in human erythrocyte membranes. J Biol Chem 1993; 268:10906-10913.
- Lambelet P, LOliger J: The fate of antioxidant radicals during lipid autooxidation. I. The tocopheroxyl radicals. Chem Phys Lipids 1984; 35:185-198.
- Gregus Z, Fekete T, Halászi E, Klaassen CD: Lipoic acid impairs glycine conjugation of benzoic acid and renal excretion of benzoylglycine. Drug Metab Dispos 1996; 24:682-688.
- Domínguez C, Ruiz E, Gussinye M, Carrascosa A: Oxidative stress at onset and in early stages of type 1 diabetes in children and adolescents. Diabetes Care 1998; 21:1736-1742.
- Baynes JW: Reactive oxygen in the aetiology and complications of diabetes. Drug, diet and disease, Mechanistic approach to diabetes. In: Ioannides C, Flatt PR editors. Ellis Horwood Limited, Hertfordshire, Vol. 2, 1995: 2003-2231.
- Yan H, Harding JJ: Inactivation and loss of antigenicity of esterase by sugars and a steroid. Biochim Biophys Acta 1999; 1454:183-190.
- Kono Y, Fridovich I: Superoxide radical inhibits catalase. J Biol Chem 1982; 257:5751-5754.
- Blun J, Fridovich I: Inactivation of glutathione peroxidase by superoxide radicals. Arch Biochem Biophys 1985; 240:500-508.
- Griesmacher A, Kindhauser M, Andert SE, Schreiner W, Toma C, Knoebl P, et al: Enhanced serum levels of thiobarbituric-acid-reactive substances in diabetes mellitus. Am J Med 1995; 98:469-475.
Article Information
20
643-655
642
1275
English
Ijpsr
S. K. Hayath Basha and S. Subramanian*
Department of Biochemistry, University of Madras, Guindy campus, Chennai, Tamil Nadu, India
16 November, 2010
27 January, 2011
12 February, 2011
http://dx.doi.org/10.13040/IJPSR.0975-8232.2(3).643-55
01 March, 2011