Abstract
The present study was designed to evaluate the antidiabetic and antioxidant potential of Psidium guajava Linn. (Myrtaceae) leaves, an important ingredient in many Ayurvedic preparations, in streptozotocin-induced diabetes in rats. Oral administration of P. guajava leaf extract (300 mg/kg body weight/day) for 30 days to streptozotocin-induced diabetes rats significantly decreased the levels of blood glucose, glycosylated hemoglobin and improved the levels of plasma insulin and hemoglobin. The levels of protein, urea, creatinine, non-enzymatic antioxidants, and the activities of enzymatic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase were markedly altered in liver of STZ-induced diabetic rats. Oral administration of P. guajava for 30 days restored all these biochemical parameters to near control levels. The present study reveals the efficacy of P. guajava leaf extract in the amelioration of diabetes, which may be attributed to its hypoglycemic nature along with its antioxidant potential. The antidiabetic and antioxidant effects of P. guajava leaf were also compared with glyclazide, a standard hypoglycemic drug.
Introduction
Diabetes is a chronic metabolic disorder that continues to pose a major worldwide health problem. It is characterized by absolute or relative deficiencies in insulin secretion and/or insulin action associated with chronic hyperglycemia and disturbances of carbohydrate, lipid, and protein metabolism. Demographic and epidemiological evidence indicates that, unless an effective treatment strategy is developed, there will be a sharp increase in the global prevalence of diabetes (CitationDuckworth, 2001).
It is accepted that oxidative stress results from an imbalance between the generation of free radicals and the availability of antioxidant potential (CitationAbdollahi et al., 2004). Various studies have shown that diabetes is associated with increased formation of free radicals and decrease in antioxidant potential. This leads to oxidative damage of cell components such as proteins, lipids, and nucleic acids. In both type 1 and type 2 diabetes there is increased oxidative stress (CitationNaziroğlu & Butterworth, 2005).
To reach the goal of near normal glycemic control, various kinds of drugs have now been developed and clinically investigated. All the diabetic drugs available now, including sulfonylurea, biguanides, thiazolidinediones drugs modifying the absorption of glucose, insulin (more rapid-acting and more long-acting) have several disadvantages. Hence, the search for novel antidiabetic drugs continues (CitationTakei & Kasatani, 2004).
Plants represent a vast source of potentially useful dietary supplements for improving blood glucose control and preventing long-term complications in type 2 diabetes mellitus. More than 400 plants worldwide have been documented as beneficial in the treatment of diabetes (Nagarajan et al., 1987). The majority of traditional antidiabetic plants await proper scientific and medical evaluation for their ability to improve blood glucose control (CitationSwanston-Flatt et al., 1991). Psidium guajava Linn. (Myrtaceae) is one such plant, and hence in the present study an attempt has been made to evaluate the effects of P. guajava leaf extract on antidiabetic and antioxidant status in rats with diabetes mellitus.
P. guajava has been used as a health tea. The leaves contain copious amounts of phenolic phytochemicals and therefore can be expected to prevent various chronic diseases such as diabetes, cancer, heart diseases (CitationKimura et al., 1985). Many people habitually take a medicinal decoction of P. guajava leaves for the treatment of gastrointestinal disorders (CitationWatt & Branchwizk, 1969), and the safety of P. guajava leaves has been empirically confirmed (CitationHamada & Kitanaka, 1999). Furthermore, P. guajava leaves can prevent arterial sclerosis, thrombosis, and cataract and inhibit senescence of the body and skin (CitationOkuda et al., 1982). The plant is used as astringent, febrifuge, anti-spasmodic and tonic and in the treatment of wounds, ulcers, cholera, diarrhea, vomiting, for swollen gums and ulceration of mouth (CitationLozoya et al., 1990); it has anti-cough, anti-microbial (CitationJaiarj et al., 1999; CitationJaiarj et al., 2000), analgesic, anti-inflammatory (CitationOlajide et al., 1999) and antioxidant properties (CitationQian & Nihorimbere, 2004). It is also reported that the leaf extract contains quercetin which has an effect on the intracellular calcium levels in gastrointestinal smooth muscle (CitationLozoya et al., 1990), in cardiac muscle cell (CitationApisariyakul et al., 1999), and in neuromuscular junctions (CitationRe et al., 1999).
The present study evaluated the antidiabetic and antioxidant potential of P. guajava leaf extract against streptozotocin-induced experimental diabetes in rats. The efficacy was compared with a standard drug, glyclazide.
Materials and methods
Chemicals
Streptozotocin was procured from Sigma (St. Louis, MO). All other chemicals used in this study were of analytical grade.
Plants
Mature, healthy and fresh leaves of P. guajava were collected during the month of March 2007 from a plant in Tenkasi, Tamil Nadu, India. The plant was identified and authenticated by V. Kaviyarasan, Centre for Advanced Studies in Botany, University of Madras, and a voucher specimen was deposited at the Herbarium of Botany.
Preparation of extract
The P. guajava leaves were dried at room temperature, powdered, and stored at 5°C until needed. A 100 g of the powder was defatted with 500 mL of petroleum ether (60–80°C) overnight, and it was then extracted with 500 mL of 95% ethanol by Soxhalation. Ethanol was evaporated in a rotary evaporator at 40–50°C under reduced pressure. The yield of the plant extract was 3.8% w/w.
Animals and diet
The animal experiments were reviewed and approved by the Institutional Animals Ethics Committee (IAEC) of University of Madras (Approval No. 01/030/04). Male Wistar albino rats, weighing 160-180 g, were procured from Tamil Nadu Veterinary and Animal Sciences University, Chennai, India. The rats were housed in a well-ventilated animal house under a constant 12 h light-dark cycle at 21–23°C. Throughout the experimental period, the rats were fed a balanced commercial pellet diet (Hindustan Lever, Bangalore, India) with a 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.
Experimental design
The rats were fasted overnight and experimental diabetes was induced by intraperitoneal injection of streptozotocin, a single dose of 50 mg/kg body weight. Streptozotocin was dissolved in a freshly prepared 0.1 M cold citrate buffer, pH 4.5 (CitationRakieten et al., 1963). The control rats were similarly injected with citrate buffer. Because streptozotocin is capable of inducing fatal hypoglycemia as a result of massive pancreatic insulin release, the streptozotocin 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 three days for the 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 (CitationCanepa et al., 1990). The animals were placed into four groups, comprising of six animals in each group as follows. Group I: control rats tested with citrate buffer alone; group II: streptozotocin-induced diabetic rats; group III: diabetic rats treated with P. guajava leaf extract (300 mg/kg body weight) in aqueous solution for 30 days; group IV: diabetic rats given a single dose of glyclazide (5 mg/kg body weight) in aqueous solution for 30 days (CitationPulido et al., 1997).
The change in body weight gain in all the groups of rats were recorded at regular intervals.
After 30 days of treatment, the rats were fasted overnight and sacrificed by cervical dislocation. Blood was collected with and without anticoagulant. The whole blood was used for the estimation of glucose (CitationSasaki et al., 1972) and urea (CitationNatelson et al., 1951). The plasma was separated and used for the assay of insulin using the RIA kit (for rats) supplied by Linco Research, Inc., St. Charles, MO, USA. The levels of hemoglobin and glycosylated hemoglobin were estimated according to methods of CitationDrabkin and Austin (1932) and CitationNayak and Pattabiraman (1981). Plasma was separated and used for the assays of protein (CitationLowry et al., 1951) and creatinine (CitationBrod & Sirota, 1948). Liver tissue was excised, rinsed in ice-cold saline and then homogenized in Tris-HCl buffer (pH 7.4) using a Teflon homogenizer. The liver homogenate was then centrifuged in a cooling centrifuge at 5,000 g to remove the debris, and the supernatant was used for the analysis of non-enzymatic antioxidants according to the method of CitationOmaye et al. (1979) (vitamin C), CitationDesai (1984) (vitamin E) and CitationSedlak and Lindsay (1968) for glutathione (GSH). The enzymatic antioxidants such as superoxide dismutase (SOD) (CitationMisra & Fridovich, 1972), catalase (CAT) (CitationTakahara et al., 1960), glutathione peroxidase (GPx) and glutathione S-transferase (GST) were assayed as described previously (CitationRotruck et al., 1973; CitationHabig et al., 1974).
Statistical analysis
All the grouped data were statistically evaluated with SPSS version 10.00 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 the results were expressed as mean ± SD for six rats in each group.
Results
shows the change in body weight gain in control and experimental groups of rats. The body weight was decreased in diabetic rats and administration of P. guajava leaves extract improved the body weight in streptozotocin-induced diabetes.
Table 1. Body weight changes in control and experimental groups of rats.
depicts the levels of blood glucose, plasma insulin, hemoglobin, and glycosylated hemoglobin of control and experimental groups of rats. The diabetic rats showed a significant increase in the levels of glucose, glycosylated hemoglobin and a significant decrease in the levels of insulin and hemoglobin. Administration of P. guajava as well as glyclazide to diabetic rats restored the changes in the levels of glucose, insulin, glycosylated hemoglobin and hemoglobin to near normal levels.
Table 2. Levels of blood glucose, hemoglobin and glycosylated hemoglobin in control and experimental groups of rats.
shows the total protein, urea and creatinine in control and experimental groups of rats. The increased levels of blood urea, plasma creatinine and significant decreased level of total protein observed in diabetic rats were reverted back to near normal levels in P. guajava as well as glyclazide treated rats.
Table 3. Levels of total protein, blood urea and plasma creatinine in control and experimental groups of rats.
The levels of non-enzymatic antioxidants such as vitamin C, vitamin E and GSH in liver of control and experimental groups of rats are shown in . A significant decrease in the levels of vitamin C, vitamin E, and reduced glutathione were observed in liver of STZ-induced diabetic rats. After administration of P. guajava to diabetic rats, all the values were restored to near normal levels when compared to the control group of rats.
Table 4. Levels of vitamin C, vitamin E and reduced glutathione in liver of control and experimental groups of rats.
shows the activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione S-transferase in liver of control and experimental groups of rats. During diabetes there was a significant reduction in the activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione S-transferase in liver. Treatment with P. guajava and glyclazide restored the activities of these enzymes to near normal in streptozotocin-induced diabetic rats.
Table 5. Activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione S-transferase in liver of control and experimental groups of rats.
Discussion
Diabetes mellitus is a disorder that results in a reduction of endogenous antioxidants and an increase in oxidative stress in the human body. Antioxidants have been shown to reduce the risk of diabetes onset (CitationMontonen et al., 2004), improve glucose disposal (CitationYlonen et al., 2003), and improve some of the associated complications (CitationDe Young et al., 2004). Oxidative stress has been implicated in the pathogenesis and progression of many degenerative disorders, including naturally occurring and chemically induced diabetes mellitus (CitationKakkar et al., 1995). In addition to increased production of free radicals, antioxidant defense systems are disturbed in diabetes mellitus (CitationCunningham et al., 1991).
Diabetes mellitus causes a drastic change in body weight (CitationAl-Shamanoy et al., 1994) and it may be due to excessive breakdown of the tissue proteins and lipids due to insulin insufficiency. The improvement in body weight in P. guajava leaf extract-treated diabetic rats may be due to the improvement in metabolic activity of the system to maintain glucose homeostasis.
Blood glucose is an index for the diagnosis of diabetes mellitus. During diabetes, the blood glucose levels are drastically increased which results from reduced glucose utilization by various tissues, which is a typical condition of insulinopenic diabetes (CitationSoling & Kleineke, 1976). In the present study, oral administration of P. guajava leaf extract significantly decreased the levels of blood glucose and increased the levels of insulin in streptozotocin-induced diabetic rats. Anti-diabetic effect of medicinal plant extract is generally dependent upon the degree of β-cell destruction (CitationGrover & Vats, 2001). P. guajava leaves may bring about its anti-diabetic effect through insulin secretion from the remnant β-cells or from regenerated β-cells. This was clearly evidenced by the increased level of insulin in diabetic rats treated with P. guajava extract.
Glycosylated hemoglobin acts as an important marker for diabetes mellitus and indicates the extent of glycation of proteins. The increased blood glucose in diabetic rats reacts with hemoglobin to form glycosylated hemoglobin (CitationKoenig et al., 1976). This leads to a decrease in the level of total hemoglobin. The amount of increase in glycosylated hemoglobin is directly proportional to excess glucose present in the blood (CitationAl-Yassin & Ibrahim, 1981). Administration of P. guajava leaf extract to diabetic rats decreased the levels of glycosylated hemoglobin by virtue of its hypoglycemic activity. This normalization of glycosylated hemoglobin indicates decreased glycation of proteins and confirms the anti-diabetic potential of P. guajava.
The increased blood glucose in diabetes leads to several derangements in the protein metabolism in diabetic rats. This results in the development of a negative nitrogen balance. Impairment of nitrogen balance increases the level of urea and creatinine (CitationAsayama et al., 1994). Their levels act as a marker for renal dysfunction (CitationAlmdal & Vilstrup, 1988) as well as for assessing the toxicity for the drug (CitationBraunlich et al., 1997). Treatment of diabetic rats with the P. guajava leaf extract brought back their levels to near normal level. These results indicate the efficacy of the extract to prevent kidney damage.
Reduced glutathione is a potent free-radical scavenger. CitationDas (1997) has reported that reduced glutathione protects the cells against chemically induced diabetes. Many workers have found that liver reduced glutathione concentration in diabetic rats was markedly decreased (CitationRavi et al., 2004), which contributes to the pathogenesis and complications associated with chronic diabetes. Treatment of diabetic rats with the P. guajava leaves extract resulted in the elevation of the reduced glutathione levels suggesting that the P. guajava decreased oxidative damage.
Oxidative stress in diabetes coexists with a reduction in the antioxidant defense system and subsequently leads to the long term complications of diabetes (CitationBaynes, 1991). Antioxidants, such as vitamin C and E, have been shown to reduce the oxidative stress in experimental diabetes (CitationGarg & Bansal, 2000). In our study, decreased vitamin C and vitamin E levels were found in the liver of diabetic rats. This may be due to increased level of free-radical formation as well as decrease in the antioxidant defensive in diabetes. These vitamins also directly scavenge reactive oxygen species and up-regulate the activities of antioxidant enzymes (CitationFang et al., 2002). After oral administration of P. guajava leaf extract to diabetic rats, the altered levels were brought back to near normal. Further, the observed antioxidant activity might be expected to the presence of phytochemicals reported in the leaves of P. guajava (CitationQian & Nihorimbere, 2004).
The enzymatic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase are crucial components of the antioxidant defense system in the body. They are cellular antioxidant enzymes, which are involved in the reduction of reactive oxygen species and peroxides produced in the living organism as well as in the detoxification of certain compounds of exogenous origin, thus playing a primary role in the maintenance of a balanced redox status (CitationEvan & Littlewood, 1998). The decreased activities of enzymatic antioxidants in streptozotocin-induced diabetic rats have been reported (CitationRavi et al., 2004). Similar results have been obtained in the present study. Oral administration of P. guajava leaf extract to streptozotocin-induced diabetic rats resulted in increased activity of superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase enzymes. This may be attributed to the free-radical scavenging and antidiabetic activities of the leaf extract of P. guajava.
Conclusion
From the results obtained, it could be concluded that P. guajava leaf extract improves the antidiabetic and antioxidant status in the streptozotocin-induced diabetic rats. The presence of tannins, triterpenes and three types of flavonoids such as quercetin, avicularin, guaijaverin in P. guajava leaves maybe partially responsible for its antidiabetogenic properties. Therefore, supplementing a balanced diet with P. guajava leaf extract may provide health-promoting effects. In conclusion, treatment with P. guajava exhibits a beneficial effect on blood glucose homeostasis as well as a protective effect against streptozotocin-induced diabetes and thus provides a rationale for the use of this plant leaves in Ayurvedic medical treatment.
Acknowledgements
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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