Biochemical Evaluation of Antidiabetic properties of Strychnos potatorum Seeds extract studied in High Fat Diet Fed- Low dose Streptozotocin induced experimental type 2 diabetes in Rats

 

Chandiran Sharmila, Krishnamoorthy Renuka, Sorimuthu Pillai Subramanian*

Department of Biochemistry, University of Madras, Guindy Campus, Chennai- 600025

 

ABSTRACT:

 

 

INTRODUCTION:

Diabetes mellitus once considered as a single entity arises due to impairment of glucose homeostasis, now established as a complex metabolic disorder and a major contributor for many of the micro and macrovascular complications. It is mainly characterized by a chronic elevation in both fasting and postprandial blood glucose levels. It arises due to the absolute lack of insulin secretion (T1DM) or insufficient secretion coupled with insulin resistance (T2DM)¹.

 

The chronic hyperglycemia in diabetes is managed through a stepwise addition of therapeutic intervention strategies that comprise of lifestyle modification including appropriate diet and exercise programs and sequential addition of oral antihyperglycemic drugs having a different mechanism of actions and insulin injection. Several drugs are commercially available to control chronic hyperglycemia in diabetes such as an increase in insulin secretion (sulfonylureas), decrease in insulin resistance (Biguanides), control gluconeogenesis (thiazolidines), decrease in the intestinal glucose absorption (α- glucose inhibitors) and insulin are currently prescribed either as monotherapy or combinatorial strategy for the treatment of type 2 diabetes². However, the maintenance of normoglycemia remains a major task in the treatment of patients with diabetes mellitus due to undesirable side effects and drug resistance after prolonged use. Hence, the search for novel therapeutic agents eliciting better antidiabetic activity at low concentration and without having long-term side effects is necessitated.

 

Herbal medicine is the oldest form of medicine known to mankind using the extract of whole plants or various parts of the medicinal plants. The secondary metabolites produced by the plants are known to possess significant pharmacological as well as beneficial effects to humans owing to their structural diversity, biochemical specificity and maximum therapeutic efficacy with minimal side effects for the treatment of both communicable and non communicable diseases³. More than 50% of the marketed medicines are distillations, reproductions or variations of lead molecules that exist in nature. However, only minority natural products have received medical scrutiny for their pharmaceutical properties. The World Health Organization (WHO) has consistently recommended that the traditional medicinal plant treatments warrant systematic evaluation for their chronic toxicity and efficacy.

 

Strychnos potatorum Linn, is one such medicinal plant widely known for its seeds which have been traditionally used for the purification of water. It is a moderate sized tree belongs to the family Loganiaceae and widely distributed in southern and central parts of India, Sri Lanka and Burma. The seeds were traditionally used for the treatment of antidiabetic, and gastrointestinal disorders. However, the medicinal claims lack scientific appraisal for its toxicological, beneficial and pharmacological properties. Recently, we have reported the defluoridization efficacy of S.potatorum seeds⁴. The present study is aimed to evaluate the antidiabetic properties of S.potatorum seeds extract in high fat diet fed- low dose streptozotocin induced experimental type 2 diabetes in rats.

 

MATERIALS AND METHODS:

Chemicals and drugs:

Streptozotocin (STZ) was procured from Sigma Chemicals, St Louis, USA. All other chemicals such as Metformin, Cholesterol and other reagents used in the present study were of analytical grade obtained from SRL chemicals, Bombay, India.

 

Plant Material:

Fresh and matured Strychnos potatorum seeds were procured from an authorized traditional medical shop in Mylapore, Chennai and authenticated by a qualified a taxonomist in the Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai. The seeds were dried under the shadow and coarsely powdered to obtain a 40 mesh range and were stored in an airtight brown container at 5°C until further use. The powdered seeds were delipidated with petroleum ether (60-80°C) for overnight. It was then filtered and soxhalation was performed with 95% ethanol. Ethanol was recovered in a rotary evaporator at 40-50°C under reduced pressure. The gray coloured semi-solid mass obtained was dried under vacuum. The yield was around 18.5% of dry weight.

 

Preliminary phytochemicals screening:

The ethanolic extract of the S.potatorum seeds was subjected to phytochemical screening for the qualitative analysis of various phytoconstituents such as Alkaloids, Sterols, Flavonoids, Glycosides, Saponins, Tannins, Phytosterols, Triterpenoids, Anthraquinones and Phenols⁵.

 

Determination of Total phenolic, flavonoids, carbohydrate and protein contents:

The total phenolic, flavonoid contents of Strychnos potatorum seeds was estimated using gallic acid and quercetin as reference solutions. The total carbohydrate and protein contents in the seeds extract was estimated by the 3, 5-dinitrosalicylic acid method and Lowry et al. (1951) respectively^6,7.

 

Acute toxicity and dosage fixation studies:

Acute toxicity studies were performed as per the OECD guidelines (423) for the determination of acute toxicity of chemicals in normal rats⁸. Graded doses of seeds extract were administered orally. The rats were observed for 4 weeks following the oral administration. The changes in water intake, food consumption, psychomotor activities, changes in body weight gain and changes in the skin, fur, eyes, salivation, diarrhea and lethargy were reliably monitored. Macroscopic observation of vital organs was also performed. Likewise, based on the earlier reports, the dosage fixation studies were carried out by administering graded doses of S. potatorum seeds extract for 30 days to determine the dose-dependent hypoglycemic effect in experimental diabetic rats.

 

 

Experimental animals:

Male Wistar rats weighing about 160–180g, procured from Tamilnadu Veterinary and Animal Sciences University, Chennai, India, were housed in clean, sterile, polypropylene cages ((38×23×15cm) under standard vivarium conditions. The animals were allowed free access to standard rat chow diet (Hindustan Lever Ltd., India) or high fat diet as the case may be and water ad libitum. The composition of the standard rat diet includes 5% fat, 21% protein, 55% nitrogen-free extract and 4% fiber (w/w) with adequate minerals and vitamins for the animals. The animals were acclimatized to the laboratory conditions for 2 weeks before the commencement of experiments. The animal experiments were performed according to the regulations lay down by the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) as well as Institutional Animal Ethics Committee Guidelines.

High-fat diet fed and low-dose STZ-induced Type 2 diabetes:

The High Fat Diet (HFD) was prepared indigenously by using normal pellet diet, raw cholesterol, a mixture of vanaspati ghee and pure coconut oil (2:1). Briefly, the normal rat pellet diet was powdered by grinding and mixed with 2.5% cholesterol and a mixture of vanaspati ghee and coconut oil (5%). The mixture was made into pellet form and orally fed to rats to induce metabolic syndrome⁹. The rats were divided into two dietary regimens by feeding either normal or HFD for the initial period of 2 weeks¹⁰. After 2 weeks of dietary management to develop insulin resistance (metabolic syndrome), the groups of rats fed with HFD were intraperitoneally injected with a freshly prepared low dose of STZ (35mg/kg b.w) dissolved in 0.1M ice-cold citrate buffer, pH 4.5. After 3 days of injection with STZ, the experimental rats were screened for fasting blood glucose level. The experimental rats which show the fasting blood glucose above 250mg/dl were considered as diabetic and chosen for further experimental studies.

 

Experimental Protocol:

The rats were divided into four groups each comprising six rats.

Group 1: Control rats

Group 2: Diabetic rats (HFD fed-low dose STZ; 35 mg/kg b.w.)

Group 3: Diabetic rats treated with S. potatorum seeds extract (500mg/kg b.w.)

Group 4: Diabetic rats treated with metformin (50 mg/kg b.w.).

 

During the experimental period, body weight, blood glucose, food and water consumption and physical examinations were determined at regular intervals. At the end of the treatment period, the rats were fasted overnight, anesthetized and sacrificed by cervical decapitation. The blood was collected with or without anticoagulants for plasma or serum separation, respectively. The liver pancreatic tissues were selectively dissected out and washed in ice-cold saline and used for further experimental studies.

 

Oral glucose tolerance test (OGTT) and Assessment of HOMA-IR and QUICK-I function:

On the day before sacrifice, an OGTT was performed in both control and experimental groups of rats. The blood samples were withdrawn from the lateral tail vein of rats deprived of food overnight. Successive blood samples were collected at 0, 30, 60, 90, and 120 min following the oral administration of 2g/kg b.w. of glucose solution. Insulin resistance was assessed by QUICKI¹¹) and HOMA-IR scores¹² using the formula,

 

                         Blood glucose (mg/dl) × Insulin (µU/ml)

HOMA-IR    = –––––––––––––––––––––––––––––––––

                                                     405

QUICK-I       =      1 / (log(fasting insulin µU/ml) +

                              log(fasting glucose mg/dl)

 

Assessment of hyperglycemia and assay of clinical marker enzymes:

The levels of fasting blood glucose, hemoglobin, glycosylated hemoglobin, plasma protein, blood urea, uric acid and serum creatinine were determined by established procedures. Plasma insulin and C-peptide were assayed using ELISA kit for rats (Linco Research, Inc., USA). The presence of urine sugar was detected using urine strips. Similarly the activities of pathological marker enzymes such as AST, ALT and ALP in serum were assayed.

 

Assay of key enzymes of carbohydrate metabolism:

A portion of the liver tissue was dissected, washed with ice-cold saline, homogenized in 0.1 M Tris–HCl buffer (pH 7.4) and centrifuged. The supernatant was used for the assay of hexokinase, pyruvate kinase, lactate dehydrogenase, glucose-6-phosphatase, fructose-1,6-bisphosphatase, glucose-6-phosphate dehydrogenase, glycogen synthase and glycogen phosphorylase activity by established procedures. The glycogen content was estimated in wet liver tissue.

 

Statistical analysis:

The results were expressed as mean±SEM of six rats in each group, and the statistical significance was evaluated by one-way Analysis of Variance (ANOVA) using the SPSS (version 16) program followed by least significant difference (LSD). Values were considered statistically significant when p<0.05.

 

RESULTS AND DISCUSSION:

The present study was performed to systematically evaluate the antidiabetic properties of S. potatorum seeds. The yield after delipidation followed by ethanolic extraction of seeds was 18.5% w/w. The phytochemical screening provided the evidence for the presence of biologically active ingredients such as alkaloids, sterols, flavonoids, saponins, tannins, phytosterols, triterpenoids, glycosides and phenolic compounds in the seeds extract. The total phenolic, flavonoids, carbohydrate and protein content in the seeds extract were found to be 16.72± 1.13µg gallic acid equivalents,10.05±0.16µg quercetin equivalents, 4.19±0.28mg/g of the seeds extract and 2.26±0.32mg/g of the seeds respectively. The presence of both primary and secondary metabolites in appreciable amounts in the seeds extract also provides the rationale for its use in traditional medicine¹³. HFD- low dose STZ induced experimental type 2 diabetes in rats resembles most of the clinical features of human type 2 diabetes and hence it was chosen as the animal model to evaluate the anti diabetic properties of the seeds extract¹⁴. The acute and chronic toxicity studies revealed the non toxic nature of the seeds extract up to 2000mg /kg/body weight. Based on the dose fixation studies the optimal dose was fixed as 500 mg/kg/bw/rat/day for 30 days.

 

The body weight gain, levels of food and fluid intake in control and experimental groups of rats are shown in Figs. 1 and 2 respectively. Diabetic rats treated with S. potatorum seeds extract showed an optimal increase in body weight gain when compared to the diabetic rats which have shown a marginal increase in body weight. The observed trivial increase in body weight in the diabetic group of rats indicating the impairment in the major metabolic pathways due to diminished insulin sensitivity coupled with insufficiency¹⁵. The noticeable improvement in body weight gain in diabetic rats treated with S. potatorum seeds extract provide evidence for the synergistic beneficial effect of the individual phytochemicals present  in the S. potatorum seeds extract in maintaining normal homeostasis.

 

Fig. 1 Effect of Strychnos potatorun seeds extract on body weight in experimental groups of rats

Values are given as means ± SEM for six rats in each group. One – way ANOVA followed by post hoc test LSD was done. Values are statistically significant at *p<0.05. The results were ^acompared to control rats and ^bcompared to diabetic rats

 

Fig. 2 Effect of Strychnos potatorun seeds extract on food and fluid consumption in control and experimental groups of rats

Values are given as means ± SEM for six rats in each group. One – way ANOVA followed by post hoc test LSD was done. Values are statistically significant at *p<0.05. The results were ^acompared to control rats and ^bcompared to diabetic rats

 

The levels of food and water consumption were significantly increased in the diabetic group of rats when compared with control groups of rats. However, the increased desire for food and water consumption was normalized after oral treatment with S. potatorum seeds extract which was comparable with metformin. The increased food and water consumption observed in the diabetic group of rats indicate the existence of polyphagic and polydipsic conditions which arises due to persistent hyperglycemia. Polyphagia, polydipsia and polyuria are the classic triads of diabetic symptoms¹⁶. An increased thirst may be due to chronic hyperglycemia which raises the osmolarity of blood and makes it more concentrated. The food and water intake of diabetic groups of rats treated with seeds extract significantly reduced after 30 days of treatment which indicates the antidiabetic properties of seeds extract and the efficacy was comparable with metformin.

 

The blood glucose levels in certain specific intervals after the oral administration of glucose (2g/kg b.w.) in control and the experimental group of rats are presented in Fig. 3. In control group of rats, the blood glucose level reached the maximum peak at 60 min after an oral glucose load and then gradually relapsed back to near normal levels at 120 min indicating the maintenance of normoglycemia, whereas in the case of STZ-induced diabetic group of rats, the peak increase in blood glucose concentration was observed after 60 min and remained high over the next 60 min. Oral administration of seeds extracts as well as metformin to HFD-STZ induced diabetic rats showed a significant decrease in blood glucose concentration at 60 and 120 min when compared with the diabetic group of rats. The impaired glucose tolerance observed in the diabetic group of rats was altered to near physiological range by the treatment with seeds extract indicating its blood glucose lowering effect which in turn may be due to the insulin stimulatory or insulin mimetic properties of seeds extract.

 

In the diabetic group of rats, the levels of fasting blood glucose and HbA1c were significantly increased with a concomitant decrease in the levels of plasma insulin and hemoglobin (Table 1) Treatment with seeds extract significantly decreased the altered levels of fasting blood glucose and HbA1c in experimental diabetic groups of rats. Similarly, the levels of plasma insulin and hemoglobin were improved to the physiological range upon oral administration with seeds extract for 30 days. The urine sugar which was present in the diabetic group of rats was absent in experimental groups of rats treated with seeds extract and as well as metformin.

 

Fig. 3 Effect of oral administration of Strychnos potatorun seeds extract on OGTT in control and experimental groups of rats

Values are given as means ± SEM for six rats in each group. One – way ANOVA followed by post hoc test LSD was done. Values are statistically significant at *p<0.05. The results were ^acompared to control rats and ^bcompared to diabetic rats

 

The observed rise in insulin level along with decreased levels of glycosylated hemoglobulin in seeds extract treated diabetic rats, evidencing that the seeds extract treatment improved the pancreatic β-cell mass and function. The absence of sugar in the urine samples of S. potatorum seeds extract treated rats evidenced its renoprotective effect.

 

The increased levels of urea, uric acid and creatinine and reduced levels of plasma protein in diabetic rats were restored to physiological levels in seeds extract as well as metformin treated diabetic rats (Table 2). Experimentally induced diabetes in rat model indicates several alterations of amino acid metabolism, which may be attributed to increased muscle proteolysis, reduced protein synthesis, an energy-dependent process in the liver, and stimulated hepatic gluconeogenesis utilizing gluconeogenic amino acids. This readily accounts for the observed decrease in the total protein content in diabetic rats. Oral administration of seeds extract to diabetic rats significantly controls proteolysis caused by insulin deficiency and improves total protein levels to near normalcy indicating the beneficial and pharmacological properties of seeds extract. Further, the supraphysiological concentration of glucose in the diabetic state causes severe derangement in protein metabolism which results in the development of negative nitrogen balance. This, in turn, increases the levels of both urea and creatinine which acts as a diagnostic marker for assessing renal impairment and drug-induced toxicity¹⁷. The observed alteration in the levels of blood urea and serum creatinine in the group of diabetic rats reverted to near normalcy by treatment with seeds extract, indicating renal protective nature of the extract during glucose toxicity.

Table 1: Levels of blood glucose, hemoglobin, glycosylated hemoglobin (HbA1c), plasma insulin and urine sugar in control and experimental groups of rats.

Units: mg/dl for blood glucose, g/dl for hemoglobin, % hemoglobin for HbA1c, µU/ml for plasma insulin, pmol/ml for plasma C-peptide, +++ indicates more than 2% sugar.

 

Results are expressed as mean ± S.E.M [n=6]. One-way ANOVA followed by post hoc test LSD. Values are statistically significant at ^@ P<0.05; ^#P<0.01; *P<0.001. The results were compared with ^aControl rats, ^bDiabetic rats, ^cDiabetic rats treated with metformin.

 

Table 2: Effect of  S.potatorum seeds extract on the levels of plasma protein, blood urea, serum uric acid and serum creatinine in control and experimental groups of rats.

Units: g/dl for plasma protein, mg/dl for blood urea, serum uric acid and serum creatinine.

Results are expressed as mean ± S.E.M [n=6]. One-way ANOVA followed by post hoc test LSD. Values are statistically significant at ^@ P<0.05; ^#P<0.01; *P<0.001. The results were compared with ^aControl rats, ^bDiabetic rats, ^cDiabetic rats treated with Metformin

 

Fig. 4 Effect of Strychnos potatorun Seeds extract treatment on HOMA-IR and QUICK-I

Values are given as means ± SEM for six rats in each group. One – way ANOVA followed by post hoc test LSD was done. Values are statistically significant at *p<0.05. The results were ^acompared to control rats and ^bcompared to diabetic rats

 

In the present study, the increased levels of serum uric acid observed in diabetic rats were restored to near normalcy by the administration of seeds extract indicating the free radical scavenging activity of seeds extract. An increase in creatinine level usually occurs simultaneously with an increase in blood urea nitrogen. Serum creatinine concentration is often used as a variable not only to assess impairment of kidney function but also as a clinical endpoint to detect treatment related toxic effects of compounds on the kidney in experimental animals. In the present study, the oral treatment with seeds extract significantly reduced the serum creatinine level. Therefore, it may be concluded that the early renal changes occurred in the diabetic rats were significantly improved by the oral administration of seeds extract.

 

The extent of insulin insufficiency and resistance was evidenced by fasting insulin levels and HOMA-IR as well as QUICK I (Fig.4). Diabetic rats exhibit severe insulin resistance and marked hyperglycemia. A significantly improved HOMA-IR and QUICK I in S. potatorum seeds extract treated diabetic rats evidenced the efficacy of S. potatorum seeds extract in restoring insulin sensitivity.

 

Figure 5 represents the activities of AST, ALT and ALP in the serum of control and experimental groups of rats. The increased activities of these physiological enzymes were restored to near normal levels in S. potatorum seeds extract as well as metformin treated diabetic rats. The observed increase in activity of these enzymes in the serum of diabetic rats may be due to the leakage of these enzymes from the cytosol into the bloodstream as a consequence of tissue damage. The reversal of AST, ALT and ALP activities in S. potatorum seeds extract treated diabetic rats towards near normalcy indicates the tissue protective nature of the seeds extract.

 

Fig. 5 Effect of Strychnos potatorun Seeds extract treatment on the activities of AST, ALT and ALP in the serum of experimental groups of rats

Enzyme activities are expressed as Units: AST and ALT - µmoles of pyruvate liberated/h/mg protein ALP - µMOLES of phenol liberated/min/mg protein. Results are expressed as mean ± SEM [n= 6]. One-way ANOVA followed by post hoc test LSD was done. Values are statistically significant at *p<0.05. The results were ^acompared to control rats and ^bcompared to diabetic rats

 

The activities of regulatory enzymes of carbohydrate metabolism such as hexokinase, Pyruvate Kinase (PK) and Lactate dehydrogenase (LDH) in liver tissue of control and experimental groups of rats are presented in Tables 3. The data obtained evidence that the activities of both hexokinase and PK were significantly decreased in the hepatic tissue of diabetic rats. On the other hand, the activity of LDH in diabetic rats was significantly elevated when compared to control rats. Oral treatment with seeds extract modulates the activity of the regulatory enzymes in hepatic tissues of diabetic rats and the efficacy was comparable with metformin.

 

Table 3: Effect of S.potatorum seeds extract on the activities of Hexokinase, Lactate dehydrogenase and Pyruvate kinase in liver tissues of control and experimental groups of rats.

Units are expressed as: µmoles of glucose-6-phosphate formed/h/mg of protein for hexokinase, mU/mg of protein for pyruvate kinase.  Values are given as mean ± S.E.M [n=6] for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. Statistical significance was compared within the groups as follows: ^acontrol rats; ^bdiabetic control rats; ^cdiabetic rats treated with Metformin. Values are statistically significant at ^@P<0.05; ^#P<0.01; ^*P<0.001.

 

Hexokinase is the key enzyme involved in the phosphorylation of glucose into glucose-6-phosphate. Diminished activity of hexokinase in diabetes could cause decreased glycolysis and decreased utilization of glucose for energy production. Oral administration of seeds extract to diabetic rats resulted in a significant increase in the activity of hexokinase. The restored levels of plasma insulin and glucose in seeds extract treated HFD-STZ-induced diabetic rats may be attributed to the increased hepatic hexokinase activity. Pyruvate Kinase is a ubiquitously expressed, rate-controlling terminal glycolytic enzyme that catalyzes the conversion of phosphoenolpyruvate to pyruvate with the generation of ATP. The altered activity of PK during diabetic conditions could be expected to diminish the metabolism of glucose and ATP production. The decreased activity of PK readily accounts for the decreased rate of glycolysis and an increase in the gluconeogenic pathway which are considered as essential for the maintenance for normoglycemia. Lactate dehydrogenase is a terminal glycolytic enzyme that plays an indispensable role in the interconversion of pyruvate to lactate to yield energy under anaerobic conditions¹⁸. The decreased activity of LDH in tissues could be important to ensure that a high proportion of both pyruvate and NADH, supplied by glycolysis is subsequently oxidized in mitochondria. Indeed, elevated LDH levels observed in the experimental diabetic animals are associated with impaired glucose-stimulated insulin secretion.Thus, increased activity of LDH interferes with normal glucose metabolism. However, treatment with seeds extract to diabetic rats reverted with the LDH activity to near normalcy most probably by regulating the proportion of pyruvate and NADH thereby promoting the mitochondrial oxidation of (pyruvate) glucose.Tables 5 depicts the activities of glucose-6-phosphatase (G6Pase), fructose-1, 6-bisphosphatase (F-1, 6 BPase), and glucose-6-phosphate dehydrogenase (G6PDH) in the liver tissues of control and experimental groups of rats respectively. Type 2 diabetes induced rats showed significantly increased activities of G6Pase and F-1,6 BPase whereas the activity of G6PDH was significantly decreased. The altered activities were reverted to near physiological levels by oral administration of seeds extract and the efficacy was comparable with metformin. 

 

Glucose-6-phosphatase is an important enzyme in regulating the gluconeogenic and glycogenolytic pathways¹⁹ and its activity is increased significantly in the liver tissues. STZ has been shown to increase the expression of G6Pase mRNA, which contributes to the increased activity of this enzyme in diabetes mellitus. The activation of G6Pase is due to the state of insulin deficiency, since under normal condition insulin function as a suppressor of gluconeogenic enzymes. Diabetic rats administered with seeds extract positively modulated the activity of G6Pase and this might probably be due to an increase in the levels of insulin. Fructose-l,6-bisphosphatase is a key regulatory enzyme in the gluconeogenic pathway. It catalyzes the hydrolysis of D-fructose-1,6-bisphosphate to D-fructose-6-phosphate and inorganic phosphate in the presence of a divalent metal ion, such as magnesium, manganese, or zinc. This metabolic reaction is necessary to achieve a reversal of glycolysis. The activity of the enzyme is regulated by two inhibitors namely, AMP and fructose-2,6-bisphosphate and is increased in the state of hyperglycemia in the of liver tissues of diabetic rats²⁰.

 

Table 4: Activities of glucose-6-phosphatase, fructose-1, 6-bisphosphatase and glucose-6-phosphate dehydrogenase in liver tissues of control and experimental groups of rats.

Units are expressed as: µmoles of Pi liberated/h/mg of protein for glucose-6-phosphatase and fructose-1,6-bisphosphatase and µmoles of NADPH/min/mg of protein for glucose-6-phosphate dehydrogenase.

Values are given as mean ± S.E.M for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. Statistical significance was compared within the groups as follows: ^acontrol rats; ^bdiabetic control rats; ^cdiabetic rats treated with metformin.

Values are statistically significant at ^@P<0.05; ^#P<0.01; ^*P<0.001.

 

Table 5: Level of glycogen content and activities of glycogen synthase and glycogen phosphorylase in liver tissues of control and experimental groups of rats.

Units are expressed as: mg of glucose/g wet tissue for glycogen, µmoles of UDP formed/h/mg protein for glycogen synthase and µmoles Pi liberated/h/mg protein for glycogen phosphorylase.

Values are given as mean ± S.E.M for groups of six rats in each. One-way ANOVA followed by post hoc test LSD. Statistical significance was compared within the groups as follows: ^acontrol rats; ^bdiabetic control rats; ^cdiabetic rats treated with metformin.

Values are statistically significant at ^@P<0.05; ^#P<0.01; ^*P<0.001.

 

Oral administration of seeds extract as well as metformin may primarily modulate and regulate the activities of this gluconeogenic enzyme, either through the regulation by cAMP or inhibition of glycolysis and gluconeogenesis.

 

Glucose-6-phosphate dehydrogenase (G6PDH), the first and rate limiting enzyme, catalyzes the oxidation of glucose-6-phosphate to 6-phospho gluconate and at the same time reducing NADP⁺ to generate NADPH in the pentose phosphate pathway (PPP) that supplies reducing energy to the cells. The PPP has multiple important functions in cells including, i) production of pentose phosphates for nucleotide biosynthesis and 3-7 carbon sugars or sugar phosphates for many other uses and ii) production of reducing equivalence in the form of NADPH for a huge range of biosynthetic functions and for antioxidant defense. Several studies have shown that G6PDH activity is decreased in the liver and other tissues in diabetes. The impaired activity of G6PDH likely play a critical role in the pathogenesis of diabetes and treatment with seeds extract regulate G6PDH activity through its insulin mimetic activities and/or regulatory action on intracellular redox status.

 

Table 7 represents the levels of liver glycogen and the activities of glycogen synthase and glycogen phosphorylase in control and experimental groups of rats. Diabetic rats showed a considerable decrease in the liver glycogen content and glycogen synthase activity and a concomitant increase in glycogen phosphorylase activity. Oral treatment with seeds extract to the diabetic group of rats restored the level of liver glycogen and the activities of glycogen synthase as well as glycogen phosphorylase to physiological range. Glycogen is the primary intracellular storage form of glucose in the system and its level in various tissues are a direct reflection of insulin action. Insulin promotes intracellular glycogen deposition by stimulating the activity of glycogen synthase and inhibiting glycogen phosphorylase activity. STZ is known to cause the selective destruction of insulin producing β‐cells of the pancreas; it is rational that the glycogen level in the tissues decreases as the process of glycogenesis depends on insulin action for the influx of glucose²¹. In general, increased hepatic glucose production, decreased hepatic glycogen synthesis and glycolysis are the major contributory factors of type 2 diabetes that result in chronic hyperglycemia²². In the present study, the oral administration of S. potatorum seeds extract to diabetic rats improved the glycogen content and normalized the altered activities of glycogen metabolizing enzymes in the liver tissue, which is due to improved insulin secretion and decreased insulin resistance which in turn facilitated the glucose utilization and storage.

 

Thus, the data obtained clearly evidenced the modulatory effect of Strchnos potatorum seeds extract in ameliorating the dysregulation of carbohydrate metabolism through the modulation of the activities of carbohydrate and glycogen metabolizing enzymes Additionally, the decreased activities of pathophysiological enzymes such as AST, ALT and ALP in the diabetic rats treated with the seeds extract evidenced the non-toxic as well as tissue protective nature of seeds.

 

CONCLUSION:

Oral treatment with S. potatorum seeds extract improves glucose homeostasis and exerts the insulin-sensitizing effect in HFD–fed STZ-induced diabetic rats which is evident from the results of fasting blood glucose level, OGTT, plasma insulin, HOMA-IR and QUICK I values. The results of the present study also established the fact that S. potatorum seeds extract increased the glycogen content by modulating the activities of glycogen metabolizing enzymes suggesting the effective utilization of glucose which in turn may be due to improved insulin sensitivity. Oral treatment with S. potatorum seeds extract to experimental diabetic rats significantly improved the glycemic status by regulating the activities of key enzymes involved in the carbohydrate metabolism. Further studies are in progress to understand the molecular mechanisms involved in the action of S. potatorum seeds extract in improving insulin sensitivity as well as its antidiabetic properties.

 

ACKNOWLEDGMENT:

The financial support provided by the University Grants Commission (UGC), New Delhi, India in the form of Research Fellowship is gratefully acknowledged.

 

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

 

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Accepted on 22.11.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(6):2615-2623.