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Anti-diabetic effect of a novel N-Trisaccharide isolated from Cucumis prophetarum on streptozotocin-nicotinamide induced type 2 diabetic rats.

ABSTRACT

Ethnopharmacological relevance: Cucumis prophetarum (L) is used in traditional Indian medicine for the treatment of inflammation related problems.

Aim of the study: The present investigation was designed to study the effect of N-Trisaccharide (a new compound isolated from the fruit of C. prophetarum (L.)) on hyperglycemia in streptozotocin (STZ)-nicotinamide (NA) induced type 2 diabetic rats.

Materials and methods: Different doses of N-Trisaccharide (25 and 50 mg/kg b.w.) were administered once daily for 28 days to STZ-NA induced diabetic rats. Plasma insulin and glycogen levels were measured. The activities of hexokinase, glucose-6-phosphatase, fructose-1,6- bisphosphatase, glucose-6-phosphate dehydrogenase, glycogen synthase and glycogen phosphorylase were measured. Further, histological studies on pancreas were also carried out.

Results: The active compound at doses of 25 and 50 mg/kg b.w. given orally for 14 days showed 47.7% and 69.3% antihyperglycemic activity, respectively. Treatment at the same doses for 28 days provided complete protection against STZ-NA challenge (65 and 230 mg/kg b.w., respectively), intraperitoneally. N-Trisaccharide significantly (p [less than or equal to] 0.05) increased the plasma insulin and liver glycogen levels in diabetic rats. The altered enzyme activities of carbohydrate metabolism in the liver and kidney of the diabetic rats were significantly (p [less than or equal to] 0.05) improved. Additionally, N- Trisaccharide increased glycogen synthase and decreased glycogen phosphorylase activity in diabetic rats. Histological studies confirmed an increase in insulin level is due to stimulation of injured pancreatic [beta]-cells.

Conclusion: The results of the study suggested that N-Trisaccharide possesses propitious effect on STZ-NA induced type 2 diabetes, indicating its usefulness in diabetes management.

Keywords:

Cucumis prophetarum

Antidiabetic activity

N-Trisaccharide

Streptozotocin-nicotinamide

Type 2 diabetes

Glucose metabolism

Introduction

Diabetes is a leading cause of morbidity and mortality for the world's growing population. The International Diabetes Federation has predicted a worldwide increase from 8.3% to 9.9% by the year 2030, with China and India projected to have the largest number of diabetic cases (IDF 2012). Diabetes is a chronic condition characterized by major derangements in glucose metabolism and disturbances in fat and protein metabolism. Type 2 diabetes is a chronic and progressive illness that particularly targets 80% of the cases. It is one of the primary threats to human health due to increased prevalence and associated disabling complications. At present, the treatment mainly involves a sustained reduction in hyperglycemia using oral hypoglycemic agents besides injectable insulin. However, prominent side-effects of such drugs are the main reason for an increasing number of people seeking alternative therapies that may have less severe or no side-effects, hence the demand has arisen for using a more benign drug (UK Prospective diabetes study group 1995 and Moller 2001).

A number of plants originating from different parts of the world possessing antidiabetic and related beneficial effects have been documented (Kavishankar et al. 2011). Such plants with potential medicinal property are known to stimulate insulin production, secretion, and, enhancement of glucose uptake by adipose or muscle tissues, inhibition of glucose absorption from intestine and glucose production from liver (Hui et al. 2009). Scientific data is lacking on safety and efficacy of these plants and their use in modern medical practice. WHO Expert Committee on diabetes recommends further investigation in this area.

Cucumis prophetarum (L.) (CP) commonly known as Globe cucumber or Wild cucumber is native to Asia and Africa belongs to Cucurbitaceae family, which is mostly a prostate or climbing, monoecious herb. The fruits are ellipsoid, echinate and green with white strips. It is distributed geographically in the Western Ghats of Coorg region, Karnataka, India (Saldanha and Ramesh 1984). Earlier work in our laboratory has shown that, the newly isolated compound N-Trisaccharide, has significant in vitro antidiabetic and antioxidant activities (unpublished data). However, no studies on in vivo antidiabetic activity of this plant were previously reported. Hence, in the present study, efforts were made to evaluate the antidiabetic activity of active compound from aqueous extract of this fruit.

[FIGURE 1 OMITTED]

Materials and methods

Plant material

The fruits of C. prophetarum (L.) were collected during July and August 2011 from Western Ghats of Coorg region (Karnataka, India) and were authenticated by taxonomist. The herbarium with voucher no KU/BL/MK/105 has been deposited in Applied Botany Department, Kuvempu University, Shimoga, India.

Extract preparation

Fresh fruits were washed, cleaned, and air dried. Whole fruits were homogenized without water in a commercial blender. The fresh juice was filtered using muslin cloth and filtrate was centrifuge at 23,000 x g for 10 min, the supernatant was lyophilized and stored at -20[degrees]C until use.

Isolation of active compound

The active compound was isolated via a novel one-step precipitation process. The compound was precipitated with the addition of methanol: water (4:1) to the crude extract, centrifuge at 8500 x g for 5 min to separate the supernatant layer. The white precipitate was washed twice with methanol and dried using lyophilizer. The purity of the compound was determined by HPLC-DAD experiment showing RT value of 7.26 min (Fig. 1). The structure was elucidated using NMR, 2D NMR, LC-MS/MS and 1R data (Fig. 2).

HPLC and LC-MS/MS analysis

The active compound was analyzed using an Agilent HPLC series 6400 equipped with diode array detector (DAD). The separation was carried out in Zorbax C18 column. The column oven temperature was set at 40[degrees]C with 10 [micro]l of sample injection volume using mobile phase of ultra-pure water (A) (10%) containing 10% formic acid and acetonitrile (B) (90%). The flow rate was 0.5 ml/min with a linear gradient of 3 min run time. A triple quadrupole mass spectrometer equipped with an ESI interface and a Q-array-Octapole mass analyzer was used for LC-MS/MS analysis. Mass spectra were recorded in the range of m/z 0-1000. 13C NMR (400 MHz D2O): 98.4 (C2), 54.8 (C3), 70.1 (C4), 78.3 (C5), 75.2 (C6), 60.7 (C7), 83.8 (C1'), 73.9 (C2'), 72.6 (C3')> 71.5 (C4'), 72.8 (C5'), 74.2 (C6'), 100.2 (C2"), 69.1 (C3"), 71.3 (C4"), 70.7 (C5"), 72.3 (C6"), 61.7 (C7"). 'H NMR (400 MHZ [D.sub.2]O): [delta] 5.3 (H, C2), 4.1 (H, C3), 7.0 (C- N[H.sub.3.sup.+]), 4.2 (H, C4), 3.4 (9H,S-OH,2',3',4',5',6',3",4",5"), 3.02 (H, C5), 4.02 (H, C6), 3.7 (2H, dd, C7a, C7a), 3.5 (2H, dd, C7b, C7b), 2.75 (H, C1'), 3.3 (H, C2'), 3.23 (H, C3'), 3.23 (H, C4'), 3.23 (H, C5'), 3.57 (H, C6'), 5.03 (H, C2"), 3.5 (H, C3"), 3.25 (H, C4"), 3.10 (H, C5"), 3.5 (H, C6"); LC-MS/MS: m/z 504.19 for [M2+ + H]. The molecular formula of the compound is [C.sub.12][H.sub.33][O.sub.15][N.sub.1].

[FIGURE 2 OMITTED]

Experimental animals and induction of diabetes

Albino wistar rats of either sex, weighing 150-180 g were used. Animals were maintained at 22 [+ or -] 2[degrees]C with 12 h light and dark cycle, fed on standard pellet diet supplied by Lipton India Ltd. Animals had free access to diet and water. All animal studies conducted were approved by the Institutional Animal Ethics Committee, University of Mysore (Approval No. UOM/IAEC/4/2012), Mysore, as stated by prescribed guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.

After initial determination of 12 h fasting blood glucose levels (end tail puncture), development of experimental type 2 diabetes was achieved by intraperitoneal administration of Nicotinamide (NA) at 230 mg/kg b.w. dissolved in saline 15 min before Streptozotocin (STZ) at 65 mg/kg dissolved in 0.1 M citrated buffer (pH 4.5) immediately before use. Eight hours after STZ-NA administration, the rats were kept for next 24 h on 15% glucose solution bottles in their cages to prevent hypoglycemia (Masiello et al. 1998).

Determination of antidiabetic activity and change in body mass

Animals having marked hyperglycemia (fasting blood glucose [greater than or equal to] 250 mg/dl), 48 h after STZ-NA treatment were selected for the study. Animals were treated orally for different duration with varying doses of test compounds suspended in water. Fasting blood glucose levels were monitored weekly using dextrostrix with One Touch Select Lifescan Glucometer (glucose oxidase method). Change in body weight of untreated and experimental animals were recorded in fasting state simultaneously.

Experimental design

A total of 36 rats were divided into six groups as follows:

Group I: normal untreated rats.

Group II: normal rats treated with 50 mg N-Trisaccharide/kg b.w./day for 28 days.

Group III: diabetic untreated rats.

Group IV: diabetic rats treated with 25 mg N-Trisaccharide/kg b.w./day for 28 days.

Group V: diabetic rats treated with 50 mg N-Trisaccharide/kg b.w./day for 28 days.

Group VI: diabetic rats treated with 25 mg Glibenclamide/kg b.w./day for 28 days.

N-Trisaccharide/Glibenclamide was administered orally to the animals of the respective groups every day morning for 28 days by gastric intubation with a force feeding needle. All the six groups were sacrificed after an overnight fasting by cervical dislocation. Blood was drawn from the heart. Pancreas was collected in 10% formalin solution and immediately processed for histological studies by the paraffin technique. The liver and kidney were removed, washed with chilled saline and small weighed portion of the tissues were immediately processed for determination of glycogen. Ten percent homogenate (w/v) of liver and kidney was prepared in 150mM KCI using homogenizer at 4[degrees]C. The homogenates were centrifuge at 3000 x g for 15 min at 4[degrees]C. The supernatants were frozen at -20[degrees]C until assayed for different enzymes. Blood plasma was recovered by centrifugation at 1000 x g for 10 min at 4[degrees]C.

Biochemical measurements

Plasma insulin level was measured according to the method described by Herbert et al. (1965), using insulin radio immune assay kit obtained from BARC, Mumbai, India. Glycosylated hemoglobin was estimated by the method of Sudhakar Nayak and Pattabiraman (1981). Hepatic glycogen levels were measured by the method of Kemp and Hejnigen (1954). Protein content of enzyme samples was determined colorimetrically (Lowry et al. 1951).

Activities of hexokinase (E.C.2.7.1.1), Glucose-6-phosphatase (E.C.3.1.3.9), Fructose-1,6-bisphosphatase (E.C.3.1.3.11), Glucose-6-phosphate dehydrogenase (E.C.1.1.1.49), Hepatic glycogen synthase and glycogen phosphorylase (E.C.2.4.1.11), were carried out according to the methods of Branstrup et al. (1957), King (1965), Gancedo and Gancedo (1971), Langdon (1966), Leloir and Goldemberg (1962), respectively.

Statistical analyses

The experimental data are expressed as mean [+ or -] SD. Statistical comparisons were performed by one-way analysis of variance (ANOVA) followed by Student's t-test to determine the significant difference between samples with 95% confidence limit.

[FIGURE 3 OMITTED]

Results

Anti-diabetic activity of active compound

Fasting blood glucose of untreated diabetic rats was significantly higher than those of normal control rats (Table 1). Significant decrease in blood glucose levels were observed in diabetic treated rats (325 [+ or -] 46.47 to 99.5 [+ or -] 10.4mg/dl) after treatment with NTrisaccharide at 50mg/kgb.w. The treatment showed significant antihyperglycemic activity (69.3%) by bringing down the blood glucose level to near normal on day 14 and continued in the same level thereafter till 28 days (experimental period) in diabetic rats. At 25 mg/kg b.w., N-Trisaccharide treatment showed 47.7% fall in diabetic treated rats. No hypoglycemic effect was observed in normal treated rats. Treatment of diabetic rats with standard antidiabetic drug glibenclamide at 25 mg/kg b.w. resulted in 62.3% fall in blood glucose level on day 14.

Effect on biochemical parameters

Effect on plasma insulin level and other variables

There was a significant decrease in plasma insulin levels of diabetic untreated group compared to those in normal rats (Fig. 3). The insulin levels were further decreased in untreated diabetic rats after 28 days. In diabetic rats, after treatment with N-Trisaccharide, the insulin level was significantly increased to 16.93 [+ or -] 0.33 IU/ml from an initial value of 8.8 [+ or -] 0.71 IU/ml. An increase from 8.7 [+ or -] 0.75 to 15.01 [+ or -] 0.40 IU/ml was observed in diabetic rats treated with N-Trisaccharide at 25 mg/kg b.w. In the normal treated rats with N-Trisaccharide, there was a slight increase from an initial level of 16 [+ or -] 0.62 to 19.28 [+ or -] 0.56 IU/ml.

Fig. 4 shows the level of HbA1c (%) and hepatic glycogen levels. The change in body weight of the normal and experimental diabetic rats is shown in Fig. 5. Higher levels of HbA1c and lower levels of hepatic glycogen were observed in diabetic rats as compared to normal controls. Treatment with N-Trisaccharide decreased HbA1c and increased hepatic glycogen levels to near normal. Diabetic rats showed marked reduction in body weight when compared to normal control rats. Treatment with N-Trisaccharide significantly increased the body weight of diabetic rats but not to near normal control rats.

Effect on glucose metabolism and hepatic glycogen enzymes

The effect of oral administration of N-Trisaccharide on carbohydrate metabolic enzymes in liver and kidney of normal and diabetic control rats is illustrated in Tables 2 and 3. Diabetic rats showed decreased activity of hexokinase, and, increased activities of glucose-6-phosphatase, fructose-1,6-bisphosphatase and glucose-6-phosphate dehydrogenase. Treatment with N-Trisaccharide (25 and 50 mg/kgb.w.) brought back these enzyme levels significantly (p [less than or equal to] 0.05) toward normal levels.

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

The effect of N-Trisaccharide on glycogen synthase and glycogen phosphorylase enzymes in the liver of normal and diabetic rats is shown in Table 4. Diabetic rats showed increased activity of glycogen phosphorylase and decreased activity of glycogen synthase. The enzyme levels were significantly (p [less than or equal to] 0.05) reverted to near normal levels after treatment with N-Trisaccharide.

Histologic changes in the pancreas

Pancreatic tissue of normal control (Fig. 6A) and normal treated with N-Trisaccharide at 50 mg/kg b.w. (Fig. 6B) showed the exocrine portion consisting of normal acini (a), endocrine portion with the islets of Langerhans (IL) containing alpha cells ([alpha]) at the periphery of islets, beta cells ((3) in the core and delta cells ([delta]) of a relatively larger size. The normal architecture of the islets in the diabetic rats were shrunken (Fig. 6C) with less number of [beta]-cells. The islets showed vacuolation (v) and irregular hyperchromatic nuclei (hen). In animals treated with 25 and 50 mg/kg b.w. of NTrisaccharide (Fig. 6D and E), the restoration of the normal cellular population and size of islets were noted especially in the central (3-cell region.

Discussion

The use of medicinal plants is increasing due to the extraction and development of many successful drugs and chemotherapeutic agents from plants and their use as traditional rural herbal remedies (Tiwari and Madhusudhana Rao 2000). STZ-NA induced diabetes mellitus is a type 2 diabetic model; this could be due to partial destruction of [beta]-cells of the islets of Langerhans in the pancreas. Type 2 diabetes is the consequence of a number of defects, including impaired insulin secretion by the pancreatic [beta]-cells, resistance of peripheral tissues to the glucose-utilizing effect of insulin, and augmented hepatic glucose production (Shulman 2000).

The efficacy of N-Trisaccharide is comparable to standard antidiabetic drug glibenclamide, and is mediated by improving the glycemic control mechanisms and increasing insulin secretion from remnant pancreatic [beta]-cells. N-Trisaccharide showed better reduction in blood glucose level at 50 mg/kgb.w. against the baseline level of 300-330 mg/dL and did not cause hypoglycaemia or any mortality, proving its safety or no toxic effects. Whereas, glibenclamide causes hypoglycaemia and its treatment could be contraindicated for those with G6PDH deficiency, as it may cause acute haemolysis. The potency of N-Trisaccharide is significant and could be safer to use over-time when compared to glibenclamide and may be attractive alternative to synthetic drugs or reinforcements to currently used treatments.

In the present investigation, treatment for 28 days with N-Trisaccharide showed significant antihyperglycemic activity. Maximum reduction in glucose levels was observed in groups receiving 50 mg/kg b.w. of active compound, as it is evident by the significant increase in the level of insulin in diabetic rats. The possible mechanism for exhibiting antihyperglycemic action in diabetic rats could be due to increased pancreatic secretion of insulin from the existing (8-cells. Similar stimulatory effect on insulin release has been reported by the use of a number of plants possessing antihyperglycemic activity by several workers (Nmila et al. 2000; Sharma et al. 2006; Gupta et al. 2009). STZ-NA induced diabetes is characterized by a severe loss in body weight (Pari and Srinivasan 2010). Due to absolute or relative deficiency of insulin and decreased production of ATP, protein synthesis decreases in all tissues (Murray et al. 2003). This insulin deficiency cause hyperglycemia and when blood glucose level exceeds the renal threshold, glucose excretes in urine. Water accompanies glucose due to osmotic effect and to compensate for this loss of water, thirst center is activated and more water is taken. The loss and ineffective utilization of glucose leads to breakdown of fat and protein. Structural proteins are known to contribute to body weight, the loss or degradation of these structural proteins reflects the reduction in body weight (Ramesh and Pugalendi 2006). The excessive catabolism of protein to provide amino acids for gluconeogenesis during insulin deficiency results in muscle wasting and weight loss in diabetic untreated rats. In this study, diabetic rats showed marked reduction in their body weight. The protective effect of N-Trisaccharide in preventing body weight loss could be as a result of its ability to increase insulin level thereby improving glycemic control.

HbA1c levels are monitored as a reliable index of glycemic control in diabetes. Glycosylation of hemoglobin increases in diabetes mellitus and the amount of increase is directly proportional to fasting blood glucose levels (Babu et al. 2007). In this study, diabetic rats showed high levels of HbA1c. Administration of N-Trisaccharide to diabetic rats prevented the increase in glycosylated hemoglobin significantly and this could be due to decrease in glucose levels. Very recently Anand (Ananda Prabu et al. 2012) reported that oral administration of aqueous extract of Biophytum sensitivum reduced HbA1c levels in STZ-NA induced diabetic rats.

Glycogen level in tissues such as liver and skeletal muscle corresponds to the insulin activity as it causes glycogen deposition by stimulating glycogen synthase and inhibiting glycogen phosphorylase. By insulin treatment, recovery of glycogen levels in tissues (liver and muscle) occurs as a result of increased influx of glucose in the liver (Vats et al. 2004). Treatment with N-Trisaccharide for 28 days significantly increased the hepatic glycogen levels in STZ-NA induced diabetic rats, indicating the presence of insulin, which could be due to secretagogue activity of N-Trisaccharide.

In general, hyperglycemia results from increased hepatic glucose production plus decreased hepatic glycogen synthesis and glycolysis, which are the major symptoms in type-2 diabetes and this seems to be the consequence of the low hexokinase and high glucose-6-phosphatase activities in diabetic state (Guignot and Mithieux 1999). One of the key enzymes in the catabolism of glucose is hexokinase; an insulin dependent enzyme which plays an important role by phosphorylating glucose to glucose-6-phosphate in the cell system (Laakso et al. 1995). In the present study, due to insulin deficiency, the activities of both hepatic and kidney hexokinase were decreased in diabetic rats as compared to normal rats. Similar results were reported by others (Bopanna et al. 1997; Rayidi 2011). Administration of N-Trisaccharide to diabetic rats enhanced the hexokinase activity in liver and kidney, which could be due to increased insulin secretion and thereby greater uptake of glucose from blood by liver cells and increased glycolysis for energy production leading to decreased blood glucose levels.

[FIGURE 6 OMITTED]

Under normal conditions, insulin suppresses glucose-6phosphatase and fructose-1,6-bisphosphatase, the important regulatory enzymes of gluconeogenic enzymes. Glucose-6phosphatase, the key enzyme in the homeostatic regulation of blood glucose level, catalyzes the terminal step in gluconeogenesis and glycogenolysis (Berg et al. 2001). Fructose-1,6-bisphosphatase, catalyzes one of the irreversible steps in gluconeogenesis and serves as a site for the regulation of the process (Tillmann et al. 2002). The activity of these gluconeogenic enzymes increases in the liver of diabetic rats (Baquer et al. 1998), which could be due to insulin deficiency. Diabetic rats treated with N-Trisaccharide and glibenclamide exhibited lowered activity levels of these two enzymes, which may be due to higher secretion of insulin.

Glucose-6-phosphate dehydrogenase (G6PDH) is the enzyme, which maintains intracellular glucose-6-phosphate at optimum levels by diverting it into pentose phosphate pathway, thus maintaining normal blood sugar level. Insulin is reported to increase the activity of G6PDH in a dose dependent manner (Weber and Convery 1966). The activity of G6PDH significantly gets decreased in diabetic rats as reported by Shibib et al. (1993). Treatment of diabetic rats with N-Trisaccharide restored the activity of G6PDH to near normal, which could be due to increased level of insulin.

Liver plays an important role in buffering postprandial blood sugar level and is involved in glycogen storage. In diabetic condition, the normal capacity of liver to synthesize glycogen gets impaired (Sirag 2009). In STZ-NA induced diabetic rats, the activation of glycogen synthase from synthase phosphatase is defective (Kirana and Srinivasan 2008). In the present study, the activity of glycogen phosphorylase increased and that of glycogen synthase decreased in diabetic rats. In diabetic rats treated with N-Trisaccharide, the liver glycogen was brought back to near normal level and this could be due to increased secretion of insulin that enhances glycogenesis.

Beta-cell population of the islets of Langerhans in pancreas reflects the production and secretion of insulin. In the present study, the destruction of [beta]-cells was observed in STZ-NA induced diabetic rats. Control rats showed normal cellular population and size of [beta]-cells. Restoration of [beta]-cells in the islets of Langerhans was observed in the diabetic rats treated with N-Trisaccharide. This correlates the increased level of insulin in diabetic treated group compared to diabetic untreated group. The possible mechanism of action by which the novel N-Trisaccharide brings about its antidiabetic action may be by stimulation of insulin secretion in injured [beta]-cells.

Conclusion

Administration of N-Trisaccharide significantly increased plasma insulin level in diabetic rats, which could be due to stimulatory effect on the remnant [beta]-cells thereby increasing insulin secretion. This may be as a consequence of significant reduction in the level of gluconeogenic enzymes or due to the increased utilization of glucose by the activities of hexokinase, glucose-6phosphate dehydrogenase and glycogen synthase that can cause the decrease in the concentration of glucose in blood.

The present investigation confirms the enhanced antidiabetic effect of N-Trisaccharide on STZ-NA induced type 2 diabetic rats.

http://dx.doi.org/10.1016/j.phymed.2013.12.002

ARTICLE INFO

Article history:

Received 29 January 2013

Received in revised form 29 October 2013

Accepted 20 December 2013

Conflict of interest

The authors have declared no conflict of interest.

Acknowledgements

The investigators are grateful for the financial assistance provided by University Grants Commission, New Delhi, India, to carry out this research work. GBK is grateful to Prof. Antony Williams, Royal Society of Chemistry, USA, for elucidating the structure of the compound. Also, the authors are thankful to HSc, Bangalore for NMR and IR facilities and Department of Studies in Zoology, University of Mysore, Mysore, for providing animals and infrastructure facilities.

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G.B. Kavishankar (a,b), N. Lakshmidevi (a), *

(a) DOS in Microbiology, University of Mysore, Manasagangotri, Mysore 570 006, Karnataka, India

(b) DOS in Biochemistry, University of Mysore, Manasagangotri, Mysore 570 006, Karnataka, India

* Corresponding author. Tel.: +91 90081 77435.

E-mail address: nlakshmidevi7@gmail.com (N. Lakshmidevi).
Table 1
Effect of N-Trisaccharide on the fasting blood glucose levels in
normal and experimental diabetic rats.

Croups                                        Blood glucose level
                                         (mg/dl) at weekly interval

                                         Day 0

Normal                                    87 [+ or -] 11.7 (a)
Normal + N-Trisaccharide (50 mg/kg)       84 [+ or -] 11.46 (b)
Diabetic                                 303 [+ or -] 38.0
Diabetic + N-Trisaccharide (25 mg/kg)    306 [+ or -] 30.68 (c)
Diabetic + N-Trisaccharide (50 mg/kg)    325 [+ or -] 46.47 (c)
Diabetic + Glibenclamide (25 mg/kg)      313 [+ or -] 28.0 (c)

Croups                                       Blood glucose level
                                         (mg/dl) at weekly interval

                                         Day 7

Normal                                      90 [+ or -] 16.46 (a)
Normal + N-Trisaccharide (50 mg/kg)        103 [+ or -] 9.8 (b)
Diabetic                                 301.3 [+ or -] 24.0
Diabetic + N-Trisaccharide (25 mg/kg)      179 [+ or -] 22.0 (b,c)
Diabetic + N-Trisaccharide (50 mg/kg)      131 [+ or -] 36.5 (b,c)
Diabetic + Glibenclamide (25 mg/kg)        131 [+ or -] 18.0 (b,c)

Croups                                       Blood glucose level
                                         (mg/dl) at weekly interval

                                         Day 14

Normal                                   94.5 [+ or -] 10.42 (a)
Normal + N-Trisaccharide (50 mg/kg)       101 [+ or -] 7.63 (b)
Diabetic                                  320 [+ or -] 37.65
Diabetic + N-Trisaccharide (25 mg/kg)     160 [+ or -] 15.0 (b,c)
Diabetic + N-Trisaccharide (50 mg/kg)    99.5 [+ or -] 10.4 (b)
Diabetic + Glibenclamide (25 mg/kg)       118 [+ or -] 13.92 (b,c)

Croups                                       Blood glucose level
                                         (mg/dl) at weekly interval

                                         Day 21

Normal                                      92 [+ or -] 11.62 (a)
Normal + N-Trisaccharide (50 mg/kg)         97 [+ or -] 8.0 (b)
Diabetic                                   330 [+ or -] 41.0
Diabetic + N-Trisaccharide (25 mg/kg)      132 [+ or -] 13.4 (b,c)
Diabetic + N-Trisaccharide (50 mg/kg)      105 [+ or -] 6.0 (b,c)
Diabetic + Glibenclamide (25 mg/kg)      113.6 [+ or -] 8.56 (b,c)

Croups                                       Blood glucose level
                                         (mg/dl) at weekly interval

                                         Day 28

Normal                                     92 [+ or -] 12.37 (a)
Normal + N-Trisaccharide (50 mg/kg)        86 [+ or -] 6.0 (b)
Diabetic                                  354 [+ or -] 14.0
Diabetic + N-Trisaccharide (25 mg/kg)     123 [+ or -] 10.5 (b,c)
Diabetic + N-Trisaccharide (50 mg/kg)      96 [+ or -] 7.0 (b)
Diabetic + Glibenclamide (25 mg/kg)      99.8 [+ or -] 4.51 (b,c)

Values are mean [+ or -] SD (n = 6).

Values not sharing a common superscript letter differ significantly
at p < 0.05.

Table 2
Effect of N-Trisaccharide on enzyme activities of carbohydrate
metabolism in liver of different group of experimental rats.

Croups                                   Hexokinase (1) *

Normal                                    0.16 [+ or -] 0.017 (a)
Normal + N-Trisaccharide (50 mg/kg)       0.18 [+ or -] 0.015 (b,c)
Diabetic                                  0.08 [+ or -] 0.011
Diabetic + N-Trisaccharide (25 mg/kg)     0.13 [+ or -] 0.013 (b,c)
Diabetic + N-Trisaccharide (50 mg/kg)     0.14 [+ or -] 0.010 (b,c)
Diabetic + Glibenclamide (25 mg/kg)      0.139 [+ or -] 0.012 (b,c)

Croups                                   Glucose-6-phosphatase (2)

Normal                                   0.052 [+ or -] 0.010 (a)
Normal + N-Trisaccharide (50 mg/kg)      0.056 [+ or -] 0.011 (b)
Diabetic                                 0.148 [+ or -] 0.011
Diabetic + N-Trisaccharide (25 mg/kg)    0.085 [+ or -] 0.012 (b,c)
Diabetic + N-Trisaccharide (50 mg/kg)    0.075 [+ or -] 0.011 (b,c)
Diabetic + Glibenclamide (25 mg/kg)      0.082 [+ or -] 0.010 (b,c)

Croups                                   Fructose-1,6-
                                         bisphosphatase (3)

Normal                                   0.044 [+ or -] 0.011 (a)
Normal + N-Trisaccharide (50 mg/kg)      0.046 [+ or -] 0.010 (b)
Diabetic                                 0.129 [+ or -] 0.010
Diabetic + N-Trisaccharide (25 mg/kg)    0.053 [+ or -] 0.010 (b)
Diabetic + N-Trisaccharide (50 mg/kg)    0.059 [+ or -] 0.010 (b,c)
Diabetic + Glibenclamide (25 mg/kg)      0.081 [+ or -] 0.012 (b,c)

Croups                                   Glucose-6-phosphate
                                         dehydrogenase (4)

Normal                                   0.324 [+ or -] 0.054 (a)
Normal + N-Trisaccharide (50 mg/kg)       0.44 [+ or -] 0.04 (b,c)
Diabetic                                   0.2 [+ or -] 0.025
Diabetic + N-Trisaccharide (25 mg/kg)     0.27 [+ or -] 0.04 (b,c)
Diabetic + N-Trisaccharide (50 mg/kg)     0.31 [+ or -] 0.03 (b)
Diabetic + Glibenclamide (25 mg/kg)       0.29 [+ or -] 0.014 (b)

Values expressed as mean [+ or -] SD (n = 6).

Values not sharing a common superscript letter differ significantly
at p < 0.05.

* (1) [micro]moles of glucose phosphorylated/h/mg protein, (2)
[micro]moles of Pi liberated/h/mg protein, (3) [micro]moles of Pi
liberated/h/mg protein, (4) U/mg protein.

Table 3
Effect of N-Trisaccharide on enzyme activities of carbohydrate
metabolism in kidney of different group of experimental rats.

Groups                                   Hexokinase (1) *

Normal                                   0.138 [+ or -] 0.014 (a)
Normal + N-Trisaccharide (50 mg/kg)      0.169 [+ or -] 0.02 (b,c)
Diabetic                                 0.049 [+ or -] 0.012
Diabetic + N-Trisaccharide (25 mg/kg)    0.119 [+ or -] 0.014 (b,c)
Diabetic + N-Trisaccharide (50 mg/kg)     0.13 [+ or -] 0.012 (b)
Diabetic + Glibenclamide (25 mg/kg)       0.13 [+ or -] 0.012 (b)

Groups                                   Glucose-6-phosphatase (2)

Normal                                   0.048 [+ or -] 0.014 (a)
Normal + N-Trisaccharide (50 mg/kg)      0.043 [+ or -] 0.011 (b)
Diabetic                                 0.118 [+ or -] 0.02
Diabetic + N-Trisaccharide (25 mg/kg)    0.046 [+ or -] 0.011 (b)
Diabetic + N-Trisaccharide (50 mg/kg)    0.052 [+ or -] 0.010 (b)
Diabetic + Glibenclamide (25 mg/kg)      0.043 [+ or -] 0.009 (b)

Groups                                   Fructose-1,6-bisphosphatase (3)

Normal                                   0.042 [+ or -] 0.012 (a)
Normal + N-Trisaccharide (50 mg/kg)      0.036 [+ or -] 0.012 (b)
Diabetic                                 0.126 [+ or -] 0.012
Diabetic + N-Trisaccharide (25 mg/kg)    0.054 [+ or -] 0.009 (b)
Diabetic + N-Trisaccharide (50 mg/kg)    0.049 [+ or -] 0.011 (b)
Diabetic + Glibenclamide (25 mg/kg)      0.048 [+ or -] 0.011 (b)

Groups                                   Glucose-6-phosphate
                                         dehydrogenase (4)

Normal                                     0.3 [+ or -] 0.09 (a)
Normal + N-Trisaccharide (50 mg/kg)      0.428 [+ or -] 0.053 (b,c)
Diabetic                                  0.14 [+ or -] 0.011
Diabetic + N-Trisaccharide (25 mg/kg)    0.259 [+ or -] 0.037 (b)
Diabetic + N-Trisaccharide (50 mg/kg)    0.314 [+ or -] 0.029 (b)
Diabetic + Glibenclamide (25 mg/kg)      0.282 [+ or -] 0.02 (b)

Values expressed as mean [+ or -] SD (n = 6)

Values not sharing a common superscript letter differ significantly at
p < 0.05.

(1) [micro]moles of glucose phosphorylated/h/mg protein, (2)
[micro]moles of Pi liberated/h/mg protein, (3) [micro]moles of Pi
liberated/h/mg protein, (4) U/mg protein.

Table 4
Effect of N-Trisaccharide on enzyme activities of Glycogen synthase
and Glycogen phosphorylase in liver of different group of experimental
rats.

Groups                                    Glycogen synthase (1) *

Normal                                    786.66 [+ or -] 29.09 (a)
Normal + N-Trisaccharide (50 mg/kg)       758.16 [+ or -] 28.67 (b,c)
Diabetic                                  478.33 [+ or -] 24.34
Diabetic + N-Trisaccharide (25 mg/kg)     634.16 [+ or -] 20.74 (b,c)
Diabetic + N-Trisaccharide (50 mg/kg)     661.83 [+ or -] 29.57 (b,c)
Diabetic + Glibenclamide (25 mg/kg)       636.66 [+ or -] 25.36 (b,c)

Groups                                    Glycogen phosphorylase (2)

Normal                                       598 [+ or -] 27.36 (a)
Normal + N-Trisaccharide (50 mg/kg)          551 [+ or -] 31.01 (b,c)
Diabetic                                   737.5 [+ or -] 22.16
Diabetic + N-Trisaccharide (25 mg/kg)        631 [+ or -] 23.6 (b,c)
Diabetic + N-Trisaccharide (50 mg/kg)     532.66 [+ or -] 22.99 (b,c)
Diabetic + Glibenclamide (25 mg/kg)          606 [+ or -] 13.97 (b)

Values expressed as mean [+ or -] SD (n = 6).

Values not sharing a common superscript letter differ significantly at
p < 0.05.

* (1) [micro]moles UDP formed/h/mg protein, (2) [micro]moles of Pi
liberated/h/mg protein.
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Author:Kavishankar, G.B.; Lakshmidevi, N.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Apr 15, 2014
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