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Indian Journal of Pharmacology, Vol. 43, No. 3, May-June, 2011, pp. 286-290 Research Article Alcoholic leaf extract of Plectranthus amboinicus regulates carbohydrate metabolism in alloxan-induced diabetic rats BC Koti, Aparna Gore, A. H. M. Thippeswamy, A. H. M. Viswanatha Swamy, Rucha Kulkarni Department of Pharmacology, K. L. E. University's College of Pharmacy, Hubli, Karnataka, India Correspondence Address: A. H. M. Thippeswamy, Department of Pharmacology, K. L. E. University's College of Pharmacy, Hubli, Karnataka, India, tips_pg@yahoo.co.in Date of Submission: 25-Jun-2010 Code Number: ph11076 DOI: 10.4103/0253-7613.81520 Abstract Objective: The present investigation was undertaken to explore the possible mechanisms of Plectranthus amboinicus leaf extract in alloxan-induced diabetic rats.
Keywords: Aldolase, alloxan, glucose-6-phosphate dehydrogenase, Plectranthus amboinicus Introduction Diabetes mellitus is chiefly characterized by chronic hyperglycemia, with disturbances of carbohydrate, lipid and protein metabolism resulting from defects in insulin secretion, insulin action or both. Insulin deficiency and/or insulin resistance is associated with the pathogenesis of diabetic dyslipidemia and micro- and macrovascular complications. The commonly encountered acute and late diabetic complications are already responsible for major causes of morbidity, disability and premature deaths in Asian countries. The underlying causes attributed to hyperglycemia ultimately result in oxidative stress, alterations in enzyme activities, protein glycosylation and several structural changes. [1] Alloxan acts as a diabetogenic owing to its ability to destroy the β-cells of the pancreas possibly due to the free radical generation mechanism. Enzyme activities of gluconeogenesis have been shown to increase during the course of diabetes, with a simultaneous increase in the glycogenolytic and lipolytic pathways. Diabetes also has been accompanied with a decrease in the enzyme activities of the glycolytic and pentose phosphate pathways. [2] Plants have always been an exemplary source of drugs, and many of the currently available drugs have been derived directly or indirectly from them. Even the discovery of the widely used hypoglycemic drug metformin came from the traditional approach of using Galega officinalis. There is a need to explore new drugs to modify the course of diabetic complications. In traditional system of medicine, Plectranthus amboinicus is used in the treatment of renal calculi, malarial fever, hepatopathy, chronic asthma, hiccough, helminthiasis and epilepsy. [3],[4] The leaf extract of Plectranthus amboinicus contains chemical constituents like thymol, carvacrol, β-caryophylline, α-pinene, β-pinene, camphor, cymene, camphene, α-terpinene, p-cymene, limonene and α-phellandrene, which are well known essential oils. [5] The plant is credited with antifungal, [6] antileptospiral, [7] rheumatoid arthritis [8] and nephroprotective activity. [9] However, the underlying mechanism by which the glucose level reduces needs to be investigated. The present investigation was undertaken to explore the antidiabetic effect and possible mechanisms of action of the leaf extract of Plectranthus amboinicus in alloxan-induced diabetic rats. Materials and Methods Animals Male Wistar albino rats weighing 150-200 g were used. Animals were kept at room temperature (26 ± 2°C) for 1 week to acclimatize to laboratory conditions before starting the experiment. They were given free access to food and water ad libitum. Plant material Leaves of Plectranthus amboinicus were collected from Hubli, Karnataka, and were authenticated at the Department of Botany of H.S. Kothambri Science Institute, Hubli, and a voucher specimen has been deposited at the herbarium for further reference. Extract Preparation Powdered leaves (80 g) were macerated three times with 750 ml of 95% ethanol. The solvent was filtered and the hydroalcoholic extracts were concentrated in the Buchi rotavapour, which yielded about 8 g of the semisoild extract. This ethanol extract was kept in a refrigerator throughout the experimental duration. Induction of diabetes The model was developed by injecting the rats intraperitoneally with alloxan monohydrate dissolved in sterile saline (0.9% NaCl) at a single dose of 150 mg/kg. To avoid an early fatal hypoglycemic state, a 5% glucose solution was fed for 1 day to all the animals. The animals showing fasting blood glucose levels around 400-450 mg/dl were selected for the study, determined at the 3rd day after alloxan administration. Experimental design A total of 30 rats were divided into five groups of six animals each. The daily oral treatments using fresh suspension of each drug were continued for 15 days, [10] with distilled water serving as the vehicle. Suspensions were prepared using 0.3% w/v sodium carboxy methylcellulose in distilled water and used for the experimental purpose. The volume of the suspension or vehicle was 5 ml/kg. All the groups received daily treatment orally between 08.00 and 09.00 h. Group I: Normal control (vehicle treated with 5 ml/kg of distilled water) Group II: Diabetic control (vehicle treated with 5 ml/kg of distilled water) Group III: Diabetic rats + ethanol extract of Plectranthus amboinicus (PAEE) 200 mg/kg Group IV: Diabetic rats + PAEE 400 mg/kg Group V: Diabetic rats + glibenclamide 600 μg/kg [11] At the end of the experimental period, the animals were deprived of food overnight and sacrificed under mild anesthesia. Blood and liver tissues were collected immediately for biochemical analyses. The liver tissue was dissected, washed in ice cold saline and blotted on filter paper. The liver was homogenized (10% w/v) with 0.1 M Tris-HCl buffer, pH 7.4 using a homogenizer. The tissue homogenate was then centrifuged at a speed of 3000 rpm for 15 min at room temperature and the supernatant was kept at 2°C for further biochemical analysis. Biochemical Assays Blood was withdrawn retro-orbitally from the inner canthus of the eye with the help of a capillary tube under mild ether inhalation anesthesia at between 08.00 and 09.00 h. Blood samples were collected in Eppendroff′s tubes and allowed to clot for 10 min. Serum was separated by centrifuging the samples at 3000 rpm for 10 min and stored in a refrigerator until the analysis. Glucose estimation along with their body weight was recorded in all the groups prior to treatment and 1 h after the respective treatment on the first, fourth, seventh, 10th and 15 th days of the experiment. Blood glucose was determined by Trinder′s glucose oxidase method. Determination of glucose-6-phosphatase Glucose-6-phosphatase was assayed by the method described by Cori and Cori. [12] The reaction mixture contained 3 ml of potassium citrate (0.1 M, pH 6.8), 5 ml glucose-6-phosphate (0.01 M, pH 6.8) and 2 ml homogenate. Blank was prepared using 5 ml of distilled water instead of glucose-6-phosphate. Both the tubes were incubated at 30 o C. After 1 h, 1 ml of 10% trichloro acetic acid was mixed and filtered. Inorganic phosphate was determined in 1 ml of the filtrate. Determination of fructose-1,6-diphosphatase Fructose-1,6-diphosphatase was determined by the method of Gancedo and Gancedo. [13] The reaction mixture at a final volume of 2 ml contained 1.2 ml of Tris-HCl buffer (0.1 M, pH 7.0), 0.1 ml of fructose-1,6-diphosphatase (0.05 M), 0.25 ml of MgCl 2 (0.1 M), 0.1 ml of KCl (0.1 M), 0.25 ml of EDTA (0.001 M) and 0.1 ml of homogenate. Incubate the reaction mixture for 5 min at 37 o C. One milliliter of 10% TCA was added and centrifuged. Inorganic phosphate was determined in 1 ml of the filtrate. Determination of phosphoglucose isomerase enzyme The activity of the Phosphoglucose isomerase (PGI) enzyme was measured according to the method described by Horrocks et al. [14] One milliliter of freshly prepared buffered substrate (glucose-6-phosphate in 10 M borate buffer, pH 7.8) was pipetted into two large test tubes named "test" and "blank". 0.1 ml of the homogenate was added in both the tubes with or without incubation exactly for 30 min at 37 o C in a water bath. At the same time, in the test tubes named "standard" and "standard blank," 1 ml of the standard solution (fructose in 0.25% benzoic acid) and 1 ml of distilled water were added. Nine milliliters of chromogenic agent (HCl, resorcinol-thiourea reagent and distilled water, 7:1:1) was added to four tubes and placed in a 75°C water bath for 15 min. The absorbance was measured at 410 nm. The calculation of the activity is given by (Test - blank) / (Standard-blank) x 100 nmoles of fructose formed/min Determination of aldolase enzyme Aldolase enzyme was assayed by the method described by Sibley et al. [15] The reaction mixture contained 1 ml of Tris buffer (0.1 M, pH 8.6), 0.25 ml fructose-1,6-diphosphatase solution (0.05 M, pH 8.6) and 0.25 ml hydrazine solution (0.56 M, pH 8.6) in a test tube at 38 o C. To this, sufficient water was added to make the volume to 2.5 ml. 0.5 ml of the homogenate was added to this mixture and the reaction was stopped after 30 min by the addition of 2 ml of 10% trichloroacetic acid. The blank was prepared in a similar manner, except that the fructose-1,6-diphosphatase was added after trichloroacetic acid. The tubes were centrifuged. One milliliter of NaOH (0.75 N) was added in 1 ml of the supernatant. After 10 min, dinitrophenyl hydrazine was added and the tubes were placed in a 38 o C water bath for 10 min. Seven milliliters of NaOH was added to give a total volume of 10 ml. The absorbance was read at 540 nm after 10 min of addition of alkali. The percentage transmission was plotted against mm 3 Fructose-1,6-diphosphate (FDP) split/h. The value obtained was represented in μmols as 22.4 mm 3 of FDP = 1 μmol of FDP = 2 μmols of triose phosphate (glyceraldehyde) formed. Determination of glucose-6-phosphate dehydrogenase The activity of glucose-6-phosphate dehydrogenase was determined by the method of Ellis et al. [16] The incubation mixture contained 1 ml of Tris-HCl buffer (0.05 M, pH 7.5), 0.1 ml MgCl 2 (0.1 M), 0.1 ml of nicotinamide adenine dinucleotide phosphate (0.1 M), 0.5 ml of phenazine methosulfate (0.1 M), 0.4 ml of dye solution and 0.1 ml of homogenate. The mixture was allowed to stand for 10 min at 37 o C. The reaction was initiated by the addition of 0.5 ml of glucose-6-phosphate. Absorbance was read at 640 nm using water as a blank for 5 min at 1 min interval. The enzyme activity was expressed in units by multiplying the change in absorbance/min by the factor 6/17.6, the molar extinction co-efficient of the reduced co-enzyme. Determination of inorganic phosphate Inorganic phosphate was measured according to the method of Horwitt. [17] One milliliter of the filtrate was mixed with 8.75 ml of distilled water and 2.5 ml of solution E (15% trichloroacetic acid). After 5 min, the mixture was centrifuged and filtered. 1.25 ml of the supernatant was added to 12.5 ml of solution A (10% trichloroacetic acid) and 1.25 ml of solution B (1% ammonium molybdate). After 7 min, 1.25 ml of solution D (stannous chloride in HCl) was mixed. Readings were taken at 660 nm after 15 min. The standards representing different concentrations of phosphorus (2 mg, 4 mg, 6 mg, 8 mg and 10 mg) in 100 ml were prepared using KH 2 PO 4 solution and processed in the same manner as the serum sample. The standard curve was plotted using concentrations of phosphorus (mg/100 ml) versus absorbance. Enzyme activities were obtained in the respective units by using a conversion factor. Statistical Analysis The results were expressed as mean ± S.E.M. The results obtained from the present study were analyzed using one-way ANOVA followed by Dunnett′s multiple comparison tests. Data were computed for statistical analysis using the Graph Pad Prism Software. Differences between the data were considered significant at P < 0.05. Results The antihyperglycemic effect of graded doses of PAEE on fasting blood glucose is shown in [Table - 1]. Diabetic control rats showed a significant elevation (P < 0.001) in fasting blood glucose on successive days of the experiment as compared with their basal values, which was maintained over a period of 2 weeks. Daily oral treatment with PAEE showed a significant reduction (P < 0.001) in blood glucose on successive days of the experiment as compared with their basal values. The most pronounced antihyperglycemic effect was obtained with a dose of 400 mg/kg. The activities of glucose-6-phosphatase and fructose-1,6-diphosphatase enzymes were significantly increased (P < 0.001) in diabetic control rats as compared with control animals. Oral administration with PAEE reversed the values to normal (P < 0.01), as shown in [Table - 2]. Enzymes like PGI and glucose-6-phosphate dehydrogenase showed a significant decrease (P < 0.001), whereas the aldolase enzyme exhibited a significant increase (P < 0.001) in their levels in diabetic control rats compared with normal control rats. The activities of PGI and glucose-6-phosphate dehydrogenase resulted in a significant increase (P < 0.001), with a significant decrease (P < 0.001) in the activities of aldolase in the PAEE-treated rats, as shown in [Table - 3]. Discussion Alloxan, a β-cytotoxin, has demonstrated severe physiological and biochemical derangements of the diabetic state. The alloxan rats exhibited severe glucose intolerance and metabolic stress as well as hyperglycemia due to a progressive oxidative insult interrelated with a decrease in endogenous insulin secretion and release. [18] Several studies show that treatment with antioxidants might be an effective strategy for reducing diabetic complications due to disproportionate generation of free radicals. Studies focusing on the mechanisms of oxidative stress in diabetes have been carried out in order to develop a causal antioxidant therapy. In this study, diabetic rats exhibited a severe hyperglycemia. Treatment with PAEE has successively reduced the serum glucose level measured at the first, fourth, seventh and 10 th days and at the end of the study both in normal and in diabetic rats. A decrease in the serum glucose level in normal rats was found to be an indication of hypoglycemic action of the PAEE treatment. The 400 mg/kg dose shows a consistent decrease in serum glucose. High levels of oxidative cytotoxicity have been linked to glucose oxidation, lipid abnormalities and non-enzymatic glycation of proteins, which contribute to the development of diabetic complications. [19] Increased glucooxidation results due to increased aldose reductase pathway activity leading to accumulation of sorbitol and fructose, NADP redox imbalances as well as alterations in signal transduction. [20] Carbohydrates, particularly glucose, are an important source of fuel for living organisms. The three major metabolic abnormalities that contribute to hyperglycemia in diabetes mellitus are defective glucose-induced insulin secretion, increased hepatic glucose output and inability of insulin to stimulate glucose uptake in the peripheral target tissues. Pancreatic β-cells appropriately alter their rates of insulin secretion in response to fluctuations in the levels of these calorigenic molecules, with glucose playing the dominant role in the regulation of insulin secretion. Ultimately, glycolysis and gluconeogenesis are two reciprocally regulated pathways to prevent wasteful glucose operation at the same time. Gluconeogenesis converts the carbohydrate as well as non-carbohydrate precursors to glucose or glycogen and clear metabolic products of the other organs. Glucose-6-phosphatase and fructose-1,6-disphosphatase are the important regulatory enzymes in the gluconeogenic pathway, which get altered during the course of uncontrolled diabetes. Glucose-6-phosphatase catalyzes the conversion of glucose-6-phosphate to glucose and, simultaneously, provides hydrogen, which binds with NADP + in the form of NADPH and enhances the synthesis of fats from carbohydrates, i.e. lipogenesis. [21] The generation of fructose-6-phosphate from fructose-1,6-diphosphate is catalyzed by fructose-1,6-diphosphatase, which determines whether or not a tissue is capable of resynthesizing glycogen from pyruvate triosephosphates. [22] The activities of these two enzymes are found to be increased in the diabetic control rats compared with the normal rats. Treatment with PAEE significantly decreases the activity of these two enzymes in the liver of diabetic rats. Conversely, glycolytic enzymes such as PGI and aldolase are prime enzymes in glucose utilization. The conversion of G6P to F6P is reversibly catalyzed by the enzyme PGI. Aldolase catalyzes reversible aldol condensation in glycolysis. As compared with the normal control values, the mean levels of hepatic PGI were seen significantly decreased, whereas the activity of hepatic aldolase was significantly increased in the diabetic controls. [23] Administration of PAEE to alloxan-induced rats resulted in an increased activity of serum PGI. A significant decrease in the activity of aldolase was found in diabetic rats treated with PAEE. Glucose-6-phosphate dehydrogenase catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconate, with a simultaneous oxidation of NADP + to NADPH. This maintains adequate levels of glutathione in its reduced form and helps to overcome oxidative stress. [24] The significant increase in the activity of glucose-6-phosphate dehydrogenase in PAEE 200 mg/kg- and 400 mg/kg-treated hyperglycemic rats suggests that the hydrogen shuttle systems and the redox state of the cell becomes more oxidized, which results in the increased formation of NADPH for increased utilization in lipogenesis and in turn activates the enzyme, as NADPH is a strong inhibitor of glucose-6-phosphate dehydrogenase. [25] A sequential metabolic correlation between decrease in the activity of enzymes of gluconeogenesis, increase in the activity of glycolytic enzymes, increased hydrogen shuttle reactions and normoglycemia stimulated by PAEE suggests the possible biochemical mechanism through which glucose homeostasis is regulated. The present study further reveals that this may be through an increase in insulin production very similar to the metformin-like action. In this study, in aloxanized rats, PAEE produced a significant and dose-dependent antidiabetic activity effect as compared with Trigonella foenum gracum, Eugenia jambolana and Ocinum sanctum beginning from the first day and progressing till the end of the 15 th day. [26] In conclusion, treatment with PAEE (200 mg/kg and 400 mg/kg) altered the metabolism in the liver of diabetic animals. Hepatic levels of G6Pase, FDPase, PGI and aldolase enzymes arrived to acceptable values after completion of treatment. Decreased gluconeogenesis and improved utilization of glucose by peripheral tissues suggests a metformin-like action of plant treatment. [27] Plectranthus amboinicus seems to have a promising value for the development of potent phytomedicines for diabetes, which may be due to the presence of flavonoids. Acknowledgment The authors thank the Principal, K.L.E. University′s College of Pharmacy, Hubli, India, for providing the necessary facilities to carry out the work. References
Copyright 2011 - Indian Journal of Pharmacology The following images related to this document are available:Photo images[ph11076t2.jpg] [ph11076t3.jpg] [ph11076t1.jpg] |
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