Insulinotropic and ß-cell protective action of cuminaldehyde, cuminol and an inhibitor isolated from Cuminum cyminum in streptozotocin-induced diabetic rats.

Cuminum cyminum, a commonly used spice, is known to have anti-diabetic action. The present study aims towards the isolation of bioactive components from C. cyminum and the evaluation of their insulin secretagogue potential with the probable mechanism and ß-cell protective action. The anti-diabetic activity was detected in the petroleum ether (pet ether) fraction of the C. cyminum distillate and studied through in vivo and in vitro experiments. Bioactive components were identified through GC-MS, Fourier transform infrared spectroscopy and NMR analysis. The isolated components were evaluated for their insulin secretagogue action using rat pancreatic islets. Further, the probable mechanism of stimulation of islets was evaluated through in vitro studies using diazoxide, nifedipine and 3-isobutyl-1-methylxanthine. ß-Cell protection was evaluated using the (1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan) (MTT) assay, the alkaline comet assay and nitrite production. The administration of the pet ether fraction for 45 d to streptozotocin-induced diabetic rats revealed an improved lipid profile. Cuminaldehyde and cuminol were identified as potent insulinotrophic components. Cuminaldehyde and cuminol (25 µg/ml) showed 3·34- and 3·85-fold increased insulin secretion, respectively, than the 11·8 mm-glucose control. The insulinotrophic action of both components was glucose-dependent and due to the closure of the ATP-sensitive K (K⁺-ATP) channel and the increase in intracellular Ca²âº concentration. An inhibitor of insulin secretion with potent ß-cell protective action was also isolated from the same pet ether fraction. In conclusion, C. cyminum was able to lower blood glucose without causing hypoglycaemia or ß-cell burn out. Hence, the commonly used spice, C. cyminum, has the potential to be used as a novel insulinotrophic therapy for prolonged treatment of diabetes.

Antidiabetic activity of Kalanchoe pinnata in streptozotocin-induced diabetic rats by glucose independent insulin secretagogue action.

CONTEXT: Kalanchoe pinnata Lam. (Crassulaceae) is used as a traditional medicine worldwide to treat several ailments, including diabetes. However, the mechanism for the antihyperglycemic action is unknown. OBJECTIVE: The present study evaluates the antihyperglycemic and insulin secretagogue potential of Kalanchoe pinnata and assessment of the probable mechanism of action. MATERIALS AND METHODS: Steam distillate of Kalanchoe pinnata leaves was subjected to solvent fractionation and antidiabetic activity was detected in dichloromethane (DCM) fraction. In the in vivo studies, rats were treated with 5 and 10 mg/kg body weight of DCM fraction for 45 days orally. Lipid profile and other biochemical parameters were estimated. The probable mechanism for insulin secretagogue action was evaluated through studies using diazoxide and nifedipine. The bioactive component from DCM fraction was studied using HPTLC, GCMS and IR. RESULTS AND DISCUSSION: Fasting blood glucose values were reduced to 116 mg/dl from 228 mg/dl on treatment with 10 mg/kg body weight of DCM fraction, while glycated hemoglobin improved to 8.4% compared with 12.9% in diabetic controls. The insulin level and lipid profile values were close to normal values. In vitro studies demonstrated a dose-dependent insulin secretagogue action. Insulin secretion was 3.29-fold higher at 10 µg/ml as compared to the positive control. The insulin secretagogue activity was glucose independent and K(+)-ATP channel dependent. The bioactive component of the DCM fraction was identified to be a phenyl alkyl ether derivative. CONCLUSION: The DCM fraction of Kalanchoe pinnata demonstrates excellent insulin secretagogue action and can be useful in treatment of diabetes mellitus.

Antihyperglycemic and antihyperlipidemic effect of Santalum album in streptozotocin induced diabetic rats.

CONTEXT: Santalum album Linn (Santalaceae), commonly known as Sandalwood is used traditionally for its antihyperlipidemic and diuretic activity. OBJECTIVE: This study investigated the antihyperglycemic and antihyperlipidemic effect of long-term oral administration of the Santalum album pet ether fraction in streptozotocin-induced diabetic rats. MATERIALS AND METHODS: Diabetes was induced by a single intraperitoneal injection of streptozotocin at 70 mg/kg body weight. Rats were treated with Santalum album pet ether fraction orally at a dose of 10 µg/kg body weight twice daily for 60 days. Metformin (30 mg/kg body weight) was used as positive control. Lipid profile and glycated hemoglobin were estimated. HPLC profiling of Santalum album pet ether fraction was carried out. RESULTS AND DISCUSSION: Treatment of diabetic rats for 60 days demonstrated reduction in blood glucose level by 140 mg/dl. Metformin treated group showed a decrease in blood glucose by 70 mg/dl, as against an increase in diabetic control group by 125 mg/dl. Total cholesterol (TC), low density lipoprotein (LDL) and triglyceride (TG) levels were decreased by 22, 31 and 44%, respectively, in treated diabetic rats whereas, cardioprotective, high density lipoprotein (HDL) increased by 46%. In case of metformin, the values were 11, 29 and 15% respectively, while HDL increased by 7%. Significant improvement in atherogenic index from 267 to 139% was observed in treated rats. CONCLUSION: Santalum album pet ether fraction has potential antihyperlipidemic activity that can help in overcoming insulin resistance.

Effective inhibition of protein glycation by combinatorial usage of limonene and aminoguanidine through differential and synergistic mechanisms.

Protein glycation is a major mechanism for establishing secondary complication in diabetes mellitus. Effective inhibition of this process can prevent progression of the disorder into secondary complications. Aminoguanidine (AMG) and limonene (LM) are known protein glycation inhibitors. The aim of the present study was to demonstrate their differential mechanisms of action and to study whether combinatorial therapy can act synergistically and lower dosage, and thereby lower toxicity in treatment of secondary complications in diabetes. Glycation in the presence of 2M urea was inhibited by 23% with AMG and by 66% with LM. AMG is more effective than LM in reducing protein carbonyl formation. SPR studies revealed binding of LM reduces affinity of BSA for glucose. LM demonstrated an increase by 2°C in thermal transition in DSC studies as against reduction by 0.4°C by AMG proving that LM can effectively stabilize the protein structure. Combinatorial treatment of AMG and LM prevented α-helix to ß-sheet transitions in BSA at 100µM and inhibited AGE related fluorescence and pentosidine formation by 80 and 90% respectively. The combination can reduce dosage of AMG by almost twenty times, paving the way for effective protein glycation inhibition without toxicity.

Strong inhibition of the polyol pathway diverts glucose flux to protein glycation leading to rapid establishment of secondary complications in diabetes mellitus.

BACKGROUND: Polyol pathway and protein glycation are implicated in establishing secondary complications in diabetes. Their relative contribution to the process needs to be evaluated. It is essential to understand why some aldose reductase inhibitors (ARIs) trials are successful while some have failed and to study their effect on protein glycation. METHODS: Aldose reductase (AR) was assayed using xylose as substrate; protein glycation was evaluated using total and specific fluorescence, fructoseamine and protein bound carbonyl content (PCO) measurements. Long term studies were carried out on streptozotocin induced diabetic rats for evaluation of urine parameters, tissue fluorescence. Anti-cataract action was studied by lens culture studies. RESULTS: Epalrestat, a commercial ARI was also found to possess potent glycation inhibitory action. Long term experiments revealed strong protein glycation with higher concentration of citronellol (ARI) demonstrating shift in glucose flux. Treatment with epalrestat and limonene revealed improved urine parameters and tissue fluorescence. Lens culture studies revealed cataract formation at higher inhibition of AR while no lens opacity was observed at lower citronellol concentration and with limonene and epalrestat. CONCLUSION: Strong inhibition of AR shifts the glucose flux to protein glycation causing damage. ARIs possessing protein glycation inhibition are more useful in amelioration of secondary complications.

A novel mechanism for antiglycative action of limonene through stabilization of protein conformation.

Inhibition of protein glycation is known to ameliorate secondary complications in diabetes. In the present study antiglycative properties of limonene, a natural product, were evaluated using BSA as a model protein. AMG (aminoguanidine) was used as a positive control. Measurement of total AGEs (Advanced Glycation End-products) and specific AGEs revealed that limonene could inhibit protein glycation to the extent of 56.3% and 75.1% respectively at 50 µM concentration as against 54.4% and 82.2% by AMG at 1 mM. Congo red binding and CD (Circular Dichroism) analysis revealed inhibition of α-helix to ß-sheet transition wherein 18.5% ß-sheet structures were observed in glycated BSA (bovine serum albumin) as against 4.9% with limonene. Glycation of protein in the presence of urea was enhanced by 18%, while in the presence of limonene it was reduced by 23% revealing the stabilizing effect of limonene. Electrophoretic mobility was similar to the normal control and a zeta potential value of -12.1 mV as against -15.1 mV in diabetic control was observed. Inhibition of glycation in limonene treated samples was confirmed through LC-MS analysis wherein AGEs such as pentosidine, CML (N(ε)-(carboxymethyl)lysine), CEL (N(ε)-(carboxyethyl)lysine), MOLD (methylglyoxal-lysine dimer) and imidazolone observed in glycated samples were absent in limonene treated samples. PatchDock studies revealed that limonene could bind to the major glycation sites IB, IIA and IIB sub domains and AMG to the IIIA sub domain. Thus limonene is a potent protein glycation inhibitor that prevents protein glycation through a novel mechanism of stabilization of protein structure through hydrophobic interactions.

Aegle marmelos Correa leaf extract prevents secondary complications in streptozotocin-induced diabetic rats and demonstration of limonene as a potent antiglycating agent.

OBJECTIVES: To study the antiglycating, antidiabetic and antioxidant properties of Aegle marmelos Correa leaf extract and identify the bioactive constituent. METHODS: The effect of the chloroform extract of Aegle marmelos Correa was studied in streptozotocin-induced diabetic rats through evaluation of biochemical parameters. Antiglycation activity was assessed in vitro through measurement of total and specific advanced glycation end products, protein carbonyl formation and collagen solubility tests. Antioxidant potential was evaluated using the ferric-reducing antioxidant power assay and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assays. Identification of the bioactive component was attempted through silica gel column chromatography and GC-MS analysis. RESULTS: In-vivo studies for 60 days revealed that the extract prevented kidney damage and other secondary complications. The chloroform extract at 16 µg could inhibit protein glycation by 44.33% and pentosidine formation by 59.31%, and could effectively inhibit protein carbonyl formation. It could scavenge DPPH radicals up to 85.26% (IC50: 26 µg). Bio-guided fractionation revealed limonene as the bioactive component, which could account for the antiglycating activity shown by the chloroform extract. CONCLUSION: The chloroform extract of Aegle marmelos demonstrated antidiabetic antiglycating and antioxidant activity, effectively preventing kidney damage and establishment of cataracts. Limonene is reported for the first time as possessing potent antiglycating activity and is non-toxic at the concentration used.