European Journal of Pharmacology 590 (2008) 437–443 Contents lists available at ScienceDirect European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r A novel compound from Casearia esculenta (Roxb.) root and its effect on carbohydrate metabolism in streptozotocin-diabetic rats Govindasamy Chandramohan a, Savarimuthu Ignacimuthu b, Kodukkur Viswanathan Pugalendi a,⁎ a b Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar—608 002, Tamil Nadu, India Division of Ethanopharmacology, Entomology Research Institute, Loyola College, Nungambakkam, Chennai-600 034,Tamil Nadu, India a r t i c l e i n f o Article history: Received 19 July 2007 Received in revised form 19 February 2008 Accepted 20 February 2008 Available online 18 March 2008 Keywords: Casearia esculenta 3-hydroxymethyl xylitol Glucose Insulin Glibenclamide a b s t r a c t Casearia esculenta root (Roxb.) is widely used in traditional system of medicine to treat diabetes in India. An active compound 3-hydroxymethyl xylitol (3-HMX) has been isolated and its optimum dose has been determined in a short duration study and patented. In the present study, the long-term effect of 3-HMX in type 2 diabetic rats has been investigated. An optimum dose of 3-HMX (40 mg/kg body weight) was orally administered for 45 days to streptozotocin-diabetic rats for the assessment of glucose, insulin, hemoglobin (Hb), glycated hemoglobin (HbA1c), hepatic glycogen, and activities of carbohydrate metabolizing enzymes, such as glucokinase, glucose 6phosphatase, fructose 1,6-bisphosphatase and glucose-6-phosphate dehydrogenase and hepatic marker enzymes, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and gammaglutamyl transferase (GGT) in normal and streptozotocin-diabetic rats. 3-HMX at 40 mg dose produced similar effects on all biochemical parameters studied as that of glibenclamide, a standard drug. Histological study of pancreas also confirmed the biochemical findings. These results indicate that 3-hydroxymethyl xylitol, the compound from C. esculenta, possesses antihyperglycemic effect on long-term treatment also. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from the defects in insulin secretion, insulin action, or both. The chronic hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of various organs, especially the eyes, kidneys, nerves, heart, and blood vessels (American Diabetes Association, 2007). Diabetes mellitus is the most common serious metabolic disorder and it is considered to be one of the five leading causes of death in the world (Gipsen and Biessels, 2000). The global prevalence of diabetes mellitus for all age groups was estimated to be 2.8% in 2000 and is projected to rise to 4.4% in 2030 (Wild et al., 2004). The pharmacological agents currently used for treatment of type 2 diabetes include sulfonylureas, biguanide, thiazolidinedione and α-glycosidase inhibitors. These agents, however, have restricted usage due to several undesirable side effects and fail to significantly alter the course of diabetic complications (Rang and Dale, 1991). Renewed attention to alternative medicines and natural therapies has stimulated new wave of research interest in traditional practices, and there is a need to look for more efficacious agents with lesser side effects. Presently, there is a growing interest in herbal remedies due to the side effects associated with the oral hypoglycemic agents for the treatment of diabetes mellitus (Kim et al., 2006). Casearia esculenta Roxb. (Flacourtiaceae) is one such plant in Indian traditional medicine and the plant has been a popular remedy for the treatment of diabetes (Asolkar et al., 1992; Wealth of India, 1992; Yoganarasimhan, 2000). Preliminary research conducted in our laboratory was highly encouraging and revealed a significant blood glucose lowering effect after oral administration of C. esculenta root extract in normal and streptozotocin-diabetic rats and no harmful side effects were observed throughout the study (Prakasam et al., 2002) Further, the active compound, 3-hydroxymethyl xylitol (3-HMX) was isolated on the basis of bioassay-guided fractionation technique, its efficacy and optimum dose was determined in a 15 day short duration study. Administration of 3-HMX at 20, 40 and 80 mg/kg body weight gave significant reduction of plasma glucose in streptozotocin-diabetic rats. Since 3-HMX at 40 mg dose gave a maximum improvement on body weight, and decreased plasma glucose level, it was fixed as the optimum dose (Chandramohan et al., 2007). In the present study, we have investigated the long-term efficacy of 3-HMX on glucose, insulin, hemoglobin, glycated hemoglobin, hepatic glycogen content, activities of carbohydrate metabolizing enzymes, hepatic enzymes and histological changes of pancreas in normal and streptozotocin-diabetic rats. The structure of 3-HMX is depicted below (Fig. 1). 2. Materials and methods 2.1. General ⁎ Corresponding author. Tel.: +91 4144 238343; fax: +91 4144 239141. E-mail address: drkvp@sify.com (K.V. Pugalendi). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.02.082 Thin layer chromatography was used to access the reactions and purity of products. Melting point was determined on a Boetius 438 G. Chandramohan et al. / European Journal of Pharmacology 590 (2008) 437–443 Fig.1. Structure of the 3-hydroxymethyl xylitol. Microheating table and mettler-FP5 melting apparatus and is uncorrected. 1H and 13C NMR spectra were recorded with a NMR studies were performed in AL-300 MHz, JEOL spectrometer. (1H), 75 MHZ (13C) and chemical shift were given in ppm. IR spectrum was recorded in Shimadzu by KBr pellet method and mass spectra on a Shimadzu with temperature of EI method. The entire spectrum was taken in the Nicholas Piramal India Limited, Ennore, Chennai, Tamil Nadu, India. 2.2. Plant material The root of C. esculenta was collected from Kolly Hills, Namakkal District, Tamil Nadu, India. The plant was botanically identified and authenticated in the Department of Botany, Annamalai University, Annamalainagar, Tamil Nadu, India and a voucher specimen (No. AU 2145) was deposited at the herbarium of Botany. The root of the plant was air dried at 25–28 °C and the dried root was ground into fine powder with auto-mix blender. rats, over a period of 2 h i.e. fasting and postprandial glucose level. Fraction 1 exhibited a significant reduction in plasma glucose while fraction 2 showed no activity. Then fraction 1 was treated with hot water (65 ± 5 °C) and filtered (Fraction 1a). The remaining residue was labeled as fraction 1b. Fraction 1a was freeze-dried and lyophilized to obtain a white amorphous compound (1.6 g), with a sweet taste and a melting point of 128 °C, which exhibited significant reduction of plasma glucose. Fraction 1b did not show any significant reduction on plasma glucose. Fraction 1a was spotted on a precoated silica gel 60 F254, 0.25 mm thick TLC plate (Merck) and run in acetonitrile and water (8.5:1.5) system. A single spot was obtained confirming the purity of the compound. The structure of the active principle was determined on the basis of FT-IR, 1H NMR, 13C NMR and MS. 2.4. Animals Male albino rats of Wistar strain with the body weight ranging from 180 to 200 g were procured from Central Animal House, Department of Experimental Medicine, Rajah Muthiah Medical College and Hospital, Annamalai University, and they were maintained in an air conditioned room (25 ± 1 °C) with a 12 h light: 12 h dark cycle. Feed and water were provided ad libitum. Studies were carried out in accordance with Indian National Law on Animal Care and Use. Institutional Animal Ethics Committee of Rajah Muthiah Medical College and Hospital (Reg No.160/1999/CPCSEA), Annamalai University, Annamalainagar, provided ethical clearance. 2.5. Chemicals Streptozotocin was purchased from Sigma-Aldrich, St. Louis, USA. All other chemicals were of analytical grade and obtained from E. Merck or Himedia, Mumbai, India. 2.3. Isolation and identification of the active compound (Chandramohan et al., 2007) 2.6. Experimental induction of diabetes Using percolation method, 2 kg of C. esculenta root powder was extracted with 6 L of benzene [1:3 w/v). The residue left after extraction with benzene was further extracted with alcohol (1:3 w/v) for 72 h. The filtrate was concentrated using a rotary evaporator at room temperature (32 ± 2 °C) and centrifuged. The solid matter obtained was washed with diethyl ether, dried at room temperature (5.7 g) and labeled as fraction 1. The remaining alcohol portion was labeled as fraction 2. Fractions 1 and 2 were tested for plasma glucose lowering activity in normal and streptozotocin-diabetic The animals were rendered diabetes by a single intraperitoneal injection of streptozotocin (40 mg/kg body weight) in freshly prepared citrate buffer (0.1 M, pH 4.5) after an overnight fast. Streptozotocin injected animals were given 20% glucose solution for 24 h to prevent initial drug-induced hypoglycemic mortality. Streptozotocin injected animals exhibited massive glycosuria (determined by Benedict's qualitative test) and diabetes in streptozotocin rats was confirmed by measuring the fasting plasma glucose concentration, 96 h after injection with streptozotocin. The animals with plasma glucose Fig. 2. IR spectrum of 3-hydroxymethyl xylitol. G. Chandramohan et al. / European Journal of Pharmacology 590 (2008) 437–443 439 above 240 mg/dl were considered to be diabetic and used for the experiment. The supernatants were separated and used for various biochemical estimations. 2.7. Experimental design 2.9. Biochemical analysis The animals were randomly divided into five groups of six animals each. Feeding was started by 9 a.m. and 3-HMX or glibenclamide (dissolved in water) were administered post-orally using intragastric tube at 10.00 a.m. The duration of treatment was 45 days. Plasma glucose was estimated by the method of Trinder using a reagent kit (Trinder, 1969). Hemoglobin (Hb) and glycated hemoglobin (HbA1c) were estimated by the method of Drabkin and Austin (1932) and Sudhakar and Pattabiraman (1981), respectively. The plasma insulin in the rat was measured by the method of Burgi et al. (1988). Glucokinase, glucose 6-phosphatase, fructose 1,6-bisphosphatase and glucose-6-phosphate dehydrogenase were assayed in the tissues by the methods of Brandstrup et al. (1957), Koide and Oda (1959), Gancedo and Gancedo (1971) and Bergmeyer (1984), respectively. Glycogen content was determined as described by Morales et al. (1975). The activities of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) were estimated (by using commercially available kits), by the method of Reitman and Frankel (1957). The activities of serum alkaline phosphatase (ALP) and γ-glutamyl transferase (γ-GT) were estimated by the methods of Kind and King (1954) and Rosalki and Rau (1972), respectively. Histological studies of pancreas were done by the method of Pearse (1981). Group I Normal control (water) Group II Normal control + 3-HMX (40 mg/kg body weight) in water Group III Diabetic control Group IV Diabetic rats + 3-HMX (40 mg/kg body weight) in water Group V Diabetic rats + glibenclamide (600 μg/kg body weight) in water 2.8. Sample collection After 45 days of treatment, the animals were fasted for 12 h, anaesthetized between 8:00 a.m. to 9:00 a.m. each morning using ketamine (24 mg/kg body weight, intramuscular injection), and sacrificed by decapitation. Blood was collected in a dry test tube and allowed to coagulate at ambient temperature for 30 min. Serum was separated by centrifugation at 2000 rpm for 10 min for the estimation of serum ALT, AST, ALP and GGT. Blood was collected in tubes with a mixture of potassium oxalate and sodium fluoride (1:3) for the estimation of plasma insulin, glucose, and ethylenediamine tetra acetic acid (EDTA) for the estimation of hemoglobin, glycated hemoglobin. Liver and kidney were immediately dissected out, washed in ice-cold saline to remove the blood. Tissues were sliced into pieces and homogenized in an appropriate buffer (pH 7.0) in cold condition to give 20% homogenate (w/v). The homogenates were centrifuged at 1000 rpm for 10 min at 0 °C in cold centrifuge. 2.10. Tissue sampling for histological study For histological study, the pancreas was immediately dissected out and washed with cold physiological saline, followed by formalin (10% formaldehyde). Pancreas was excised immediately and fixed in 10% formalin. 2.11. Statistical analysis Values were given as means ± SD for six rats in each group. Data were analyzed by one-way analysis of variance followed by Duncan's Fig. 3. Possible mechanism of 3-HMX in streptozotocin-diabetic rats. 440 G. Chandramohan et al. / European Journal of Pharmacology 590 (2008) 437–443 Table 1 Effect of 3-HMX on body weight and plasma glucose level in normal and streptozotocin-diabetic rats Name of the group Body weight (g) Normal control Normal + 3-HMX (40 mg/kg body weight) Diabetic control Diabetic + 3-HMX (40 mg/kg body weight) Diabetic + glibenclamide (600 µg/kg body weight) Plasma glucose (mg/dl) 0 day 45th day 183.32 ± 3.69 184.31 ± 3.18 182.61 ± 4.71 182.45 ± 3.63 184.34 ± 5.79 196.41 ± 3.54a 193.60 ± 3.48a,b 154.59 ± 5.24c 191.70 ± 3.18b 195.14 ± 3.76a,b Change (%) 7.14 (+) 5.04 (+) 15.34 (−) 5.09 (+) 5.85 (+) 0 day 45th day 77.40 ± 2.28 78.54 ± 1.89 246.54 ± 3.44 248.49 ± 2.78 253.67 ± 4.10 83.15 ± 6.33a 69.83 ± 6.45b 292.45 ± 4.85c 122.21 ± 6.05d 117.39 ± 5.94d Change (%) 6.91 (+) 11.08 (−) 18.62 (+) 50.81 (−) 53.72 (−) Values are given as means ± S.D. from six rats in each group. Values in parenthesis indicate the percentage glycemic changes. Values not sharing a common superscript vertically differ significantly at P b 0.05 (DMRT). Multiple Range Test (DMRT) using SPSS version 10 (SPSS, Chicago, IL). The limit of statistical significance was set at P b 0.05. 3. Results Using bioassay-guided fractionation technique a single active compound was isolated. Structural determination of the active compound was done using different spectral techniques and it was confirmed as 3-hydroxymethyl xylitol. The compound was identified based on the following evidences: MS: m/z = 182 [M]+, [M + 1] 183, 133, 115, 103, 85. Fig. 2 shows the IR (neat) max/cm: 3369, 2942 and 1455 indicating the presence of corresponding functional groups (–OH) (–CHCH2) and (–CH). 1H NMR (400 MHz, D2O) δ/ppm: 3.5 (d, –CH2–CHOH, J = 6.78 Hz), 3.8 (t, 2H, CHOH, J = 6.32 Hz). 13C NMR (400 MHz D2O) δ/ppm: 63 (CH2OH), 70 (CHOH), 70.8 (CHOH) (Fig. 3). Table 1 shows the effect of administration 3-HMX for 45 days on body weight and plasma glucose in normal and streptozotocindiabetic rats. Body weight significantly decreased and plasma glucose significantly increased in diabetic rats. Both, 3-HMX or glibenclamide, significantly improved the body weight and brought down the plasma glucose towards normal level. Normal rats treated with 3-HMX also decreased significantly the plasma glucose level but not up to hypoglycemic level. Table 2 shows the levels of plasma insulin, Hb, and HbA1c in normal and diabetic rats. Plasma insulin and Hb significantly decreased, and HbA1c increased significantly in diabetic rats, and treatment with 3HMX or glibenclamide reversed these values to near normalcy. Table 3 shows the activities of carbohydrate metabolizing enzymes and the hepatic glycogen content in the liver of normal and diabetic rats. Glucokinase and glucose 6-phosphate dehydrogenase activities, and glycogen content decreased significantly in the liver of diabetic rats. Oral administration of 3-HMX or glibenclamide reversed these parameters to near normalcy. Table 4 shows the activities of gluconeogenic enzymes in the liver and kidney of normal and diabetic rats. Glucose 6-phosphatase and fructose 1, 6-bisphosphatase activities increased significantly in the Table 2 Effect of 3-HMX on plasma insulin, blood hemoglobin and glycated hemoglobin in normal and streptozotocin-diabetic rats Name of the group Insulin (µU/ml) Hemoglobin (g/dl) Glycated hemoglobin (mg/g of Hb) Normal control Normal + 3-HMX (40 mg/kg body weight) Diabetic control Diabetic + 3-HMX (40 mg/kg body weight) Diabetic + glibenclamide (600 µg/kg body weight) 17.31 ± 0.83a 18.23 ± 0.81b 13.67 ± 0.76a 14.31 ± 0.71a 0.45 ± 0.03a 0.41 ± 0.03a 5.88 ± 0.44c 16.17 ± 0.48d 6.26 ± 0.47b 11.51 ± 0.88c 1.16 ± 0.08b 0.56 ± 0.04c 16.49 ± 0.64d 12.72 ± 0.97d 0.52 ± 0.03c Values are given as means ± S.D. from six rats in each group. Values not sharing a common superscript vertically differ significantly at P b 0.05 (DMRT). liver and kidney of diabetic rats, and these activities decreased significantly on treatment with 3-HMX or glibenclamide. Table 5 shows the activities of serum liver enzymes AST, ALT, ALP and γ-GT in the normal and diabetic rats. The activities of AST, ALT, ALP and γ-GT increased significantly in diabetic rats. Oral administration of 3-HMX or glibenclamide reversed significantly these parameters to towards normalcy. Histological examination of pancreas showed the normal histology in normal rat and normal rat treated with 3-HMX (Figs. 4 and 5). Diabetic pancreas showed shrinkage of islets and growth of adipose tissue (Fig. 6). Treatment with 3-HMX or glibenclamide reduced these changes in the pancreas (Figs. 7 and 8). 4. Discussion Streptozotocin-induced diabetes is characterized by a severe loss in body weight (Al-Shamaorry et al., 1994), which might be the result of protein wasting due to unavailability of carbohydrate as an energy source (Chen and Ianuzzo, 1982). Oral administration of 3-HMX improved the body weight in diabetic rats, which might be via glyceamic control. Mattila et al. (1998), who studied on a related compound, xylitol, reported an increase of body weight in streptozotocin-diabetic rats after receiving 20% dietary supplementation of xylitol. In the present study, streptozotocin-diabetic rats showed significantly decreased plasma glucose level on treatment with 3-HMX, which was similar to glibenclamide. 3-HMX might bring about glucose lowering action through stimulation of surviving β-cells of islets of Langerhans to release more insulin. This was clearly evidenced by the increased levels of plasma insulin in diabetic rats treated with 3-HMX and also reduced shrinkage of islet and decreased growth of adipose tissue in pancreas. Prakasam et al. (2002) reported that the C. esculenta root extract possessed antihyperglycemic activity and Table 3 Effect of 3-HMX on carbohydrate metabolic enzyme activities and glycogen content in the liver of normal and streptozotocin-diabetic rats Name of the group Glucokinase (Ua/h/mg protein) Glucose 6-phosphate Glycogen (mg/100 g tissue) dehydrogenase (Ub/mg protein) Normal control Normal + 3-HMX (40 mg/kg body weight) Diabetic control Diabetic + 3-HMX (40 mg/kg body weight) Diabetic + glibenclamide (600 µg/kg body weight) 0.293 ± 0.021a 0.321 ± 0.023b 4.41 ± 0.33a 4.49 ± 0.26a 57.26 ± 4.28a 59.67 ± 4.35a 0.091 ± 0.006c 0.248 ± 0.018d 2.68 ± 0.20b 3.21 ± 0.24c 15.20 ± 0.94b 49.63 ± 3.11c 0.268 ± 0.020d 3.36 ± 0.25c 53.13 ± 4.17a,c Values are given as means ± S.D. from six rats in each group. Values not sharing a common superscript vertically differ significantly at P b 0.05 (DMRT). a µmol of glucose phosphorylated per hour. b nmol of NADPH formed per minute. G. Chandramohan et al. / European Journal of Pharmacology 590 (2008) 437–443 441 Table 4 Effect of 3-HMX on gluconeogenic enzyme activities in the liver and kidney of normal and streptozotocin-diabetic rats Name of the group Normal control Normal + 3-HMX (40 mg/kg body weight) Diabetic control Diabetic + 3-HMX (40 mg/kg body weight) Diabetic + glibenclamide (600 µg/kg body weight) Glucose 6-phosphatase (Ua/min/mg protein) Fructose 1,6-bisphosphatase (Ub/h/mg protein) Liver Liver Kidney 0.181 ± 0.013a 0.192 ± 0.015a 0.155 ± 0.011b 0.172 ± 0.012a Kidney 0.426 ± 0.032a 0.755 ± 0.057a 0.407 ± 0.031a 0.739 ± 0.056a 0.486 ± 0.037c 0.297 ± 0.022b 0.786 ± 0.060b 1.187 ± 0.090b 0.241 ± 0.020d 0.239 ± 0.018c 0.598 ± 0.045c 0.938 ± 0.071c 0.216 ± 0.016d 0.221 ± 0.017c 0.498 ± 0.038d 0.844 ± 0.064d Values are given as means ± S.D. from six rats in each group. Values not sharing a common superscript vertically differ significantly at P b 0.05 (DMRT). a µmol of Pi liberated per hour. b µmol of Pi liberated per minute. insulin secretory effects in streptozotocin-diabetic rats. The present study shows that the compound, 3-hydroxymethyl xylitol, present in C. esculenta is responsible for antihyperglycemic activity. In this context, reports are available on a related compound, xylitol, which is present in many vegetables and fruits (Makinen and Derling, 1980). Khalid and Rahman (1984) have reported that xylitol possesses insulin secretory effect in isolated rat islets of Langerhans. Xylitol is a nontoxic substance, natural sweetener without the bad side effects and artificial sugar substitute that is suitable for diabetic patients (Sherill Sellman, 2003). Further, Mattila et al. (1998), reported that 20% dietary supplementation of xylitol, increased insulin level and decreased glucose to 28% in streptozotocin-diabetic rats after 90 days. But our compound decreased glucose up to 50.81% in streptozotocin-diabetic rats in 45 days at 40 mg/kg body weight. The plasma glucose lowering activity was comparable with glibenclamide (53.72% of reduction), a standard hypoglycemic drug. Insulin generally has an anabolic effect on protein metabolism in that it stimulates protein synthesis and retards protein degradation (Murray et al., 2000), which may be responsible for the increased level of Hb in 3-HMX. In uncontrolled or poorly controlled diabetes, there is an increased glycosylation of a number of proteins, including Hb (Alberti and Press, 1982). HbA1c was 3.4–5.8% of total Hb in normal human red blood cells (Paulsen, 1973) and it was found to increase in diabetic patients up to 16% (Koeing et al., 1976). The level of HbA1c is monitored as a reliable index of glycemic control in diabetes (Gabbay, 1976) and useful in the management of diabetes mellitus. The HbA1c level reflects the average blood glucose concentration over the preceding 6–8 weeks (Murray et al., 2000). In our study also, Hb decreased Fig. 4. Normal rats showing islets with acini. and HbA1c increased in diabetic rats and, treatment with 3-HMX or glibenclamide brought back Hb and HbA1c values to near normal levels, as a result of improved glycemic control. Glycogen is the primary intracellular storable form of glucose and its level in various tissues is a direct reflection of insulin activity as insulin promotes intracellular glycogen deposition by stimulating glycogen synthase and inhibiting glycogen phosphorylase (Golden et al., 1979). The liver glycogen content is markedly decreased in diabetic animals (Bollen et al., 1998), which are in proportion to insulin deficiency (Stalmans et al., 1997). Diabetic rats treated with 3-HMX brought back liver glycogen to near normal level, which could be due to increased secretion of insulin. The liver is an important organ that plays a pivotal role in glycolysis and gluconeogenesis. A partial or total deficiency of insulin causes derangement in carbohydrate metabolism that decreases activity of several key enzymes including glucokinase, phosphofructokinase and pyruvate kinase (Hikino et al., 1989), resulting in impaired peripheral glucose utilization and augmented hepatic glucose production. In our study, glucokinase activity was decreased in the liver of diabetic rats, which may be due to a deficiency of insulin and, treatment with 3HMX or glibenclamide elevated the activity of glucokinase. 3-HMX administration increased insulin level which, in turn, activated glucokinase, thereby increasing the utilization of glucose leading to decreased blood sugar level. A decrease in the activity of glucose 6-phosphate dehydrogenase may also slow down the pentose phosphate pathway in diabetic conditions (Abdel-Rahim et al., 1992). Diabetic rats treated with 3-HMX showed significantly increased liver glucose 6-phosphate dehydrogenase activity, via increased secretion of insulin, which might increase the influx of glucose into the pentoses monophosphate shunt and this resulted in an increased production of the reducing agent, NADPH, with concominant decrease in oxidative stress (Ugochukwu and Babady, 2002). Table 5 Effect of 3-HMX on serum ALT, AST, ALP and γ-GT activities in the normal and streptozotocin-diabetic rats Name of the group ALT (IUa/l) AST (IUa/l) ALP (IUb/l) γ-GT (IUc/l) Normal control 25.13 ± 1.91a 75.70 ± 5.76a 81.14 ± 6.17a 15.03 ± 1.14a Normal + 3-HMX 22.26 ± 1.69a 72.16 ± 5.49a 78.37 ± 5.96a 14.89 ± 1.13a (40 mg/kg body weight) Diabetic control 62.22 ± 4.76b 121.48 ± 9.29b 141.14 ± 10.98b 26.64 ± 2.02b Diabetic + 3-HMX 31.43 ± 2.39c 89.13 ± 6.78c 98.64 ± 7.51c 19.12 ± 1.45c (40 mg/kg body weight) Diabetic + glibenclamide 28.28 ± 2.15c,a 84.14 ± 6.40c 89.74 ± 6.83d 16.14 ± 1.22a (600 µg/kg body weight) Values are given as means ± S.D. from six rats in each group. Values not sharing a common superscript vertically differ significantly at P b 0.05 (DMRT). a μmol of pyruvate liberated per hour. b μmol of phenol liberated per minute. c μmol of p-nitroanilide liberated per minute. Fig. 5. Normal rats with 3-HMX showing expansion of islets and acini that are preserved. 442 G. Chandramohan et al. / European Journal of Pharmacology 590 (2008) 437–443 Fig. 6. Diabetic rats showing growth of adipose tissue and shrinkage of islets. Fig. 8. Diabetic rats with glibenclamide showing reduction in adipose tissue. Pancreatic islets within normal limit. Glucose 6-phosphatase (G6Pase) is a crucial enzyme for the final step of gluconeogenesis or glycogenolysis in which it catalyzes the hydrolysis of glucose 6-phosphate (G6P) to glucose and phosphate. Glucose is transported out of the liver to increase blood glucose concentration. Normally insulin inhibits the hepatic glucose production by suppressing G6Pase and fructose 1, 6-bisphosphatase activity (Chen et al., 2000; Wiernsperger and Bailey, 1999). In diabetic rats, administration of 3-HMX decreased the activities G6Pase and fructose 1, 6-bisphosphatase thereby decreasing gluconeogenesis. Ohaeri (2001) found that liver is necrotized in streptozotocininduced diabetic rats. Therefore, increase in the activities of AST, ALT, ALP and γ-GT in plasma may be mainly due to the leakage of these enzymes from the liver cytosol into the blood stream (Navarro et al., 1993), which gives an indication on the hepatotoxic effect of streptozotocin. ALP is a membrane bound enzyme and its alteration is likely to affect the membrane permeability and produce derangement in the transport of metabolites (Ahmed et al., 1999). Serum GGT has been widely used as an index of liver dysfunction. Administration of 3-HMX lowered the serum AST, ALT, ALP and γ-GT activities in diabetic rats. 3-HMX treated with normal rats did not show any significant change in the activity when compared with normal control rats. In this context Truhaut et al. (1977) reported that, from 1.25 g/kg to 10 g/kg administration of xylitol to normal rats did not observe any hepatotoxicity after 2 weeks. Histological study of pancreas showed the growth of adipose tissue and shrinkage of islets in diabetic rats. Treatment with 3-HMX or glibenclamide reduced shrinkage of islets and decreased the growth of adipose tissue in pancreas. having long-term antidiabetic effect and its activity is similar to glibenclamide. This compound showed no toxic effect on measurement of serum hepatic enzymes. 5. Conclusion In conclusion, our results showed that 3-HMX markedly reduced hyperglycemia in streptozotocin-diabetic rats due to increased insulin secretion and inhibition of gluconeogenesis. This investigation reveals that the active compound 3-hydroxymethyl xylitol from C. esculenta is Fig. 7. Diabetic rats with 3-HMX showing reduction in adipose tissue. Pancreatic islets within normal limit. Acknowledgements The financial assistance from Indian Council of Medical Research, New Delhi in the form of Senior Research Fellow to the author G. Chandramohan is gratefully acknowledged. 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