|Year : 2013 | Volume
| Issue : 1 | Page : 33-38
Evaluation of antidiabetic activity of ethanolic extract of Cedrus deodara (Pinaceae) stem bark in streptozotocin induced diabetes in mice
Pradeep Singh1, RL Khosa2, Garima Mishra1
1 Department of Pharmacognosy, Teerthanker Mahaveer College of Pharmacy, Teerthanker Mahaveer University, Moradabad, Uttar Pradesh, India
2 Department of Pharmacy, Bharat Institute of Technology, Meerut, Uttar Pradesh, India
|Date of Web Publication||30-Dec-2013|
Department of Pharmacognosy, Teerthanker Mahaveer College of Pharmacy, Teerthanker Mahaveer University, Moradabad - 244 001, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Objective: To evaluate the antidiabetic activity of ethanolic extract of bark of Cedrus deodara. Materials and Methods: Ethanolic extract of the Cedrus deodara (EECD) at dose levels of 250 mg/kg and 500 mg/kg body weight was subjected to the streptozotocin induced diabetes mice. The biochemical parameters, serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), cholesterol and triglycerides were also assessed in the experimental animals. Results: The EECD exhibited significant antihyperglycemic activity and also lowers the biochemical parameters like SGPT, SGOT, cholesterol and triglycerides. The ethanolic extract at dose level of 500 mg/kg was found to be more potent than 250 mg/kg in lowering the blood glucose level, almost near to the effect of the standard drug. Conclusion: This study supports the traditional claim and the ethanolic extract of this plant could be added in traditional preparations for the ailment of various diabetes-associated complications.
Keywords: Antidiabetic activity, biochemical parameters, Cedrus deodara, streptozotocin
|How to cite this article:|
Singh P, Khosa R L, Mishra G. Evaluation of antidiabetic activity of ethanolic extract of Cedrus deodara (Pinaceae) stem bark in streptozotocin induced diabetes in mice. Niger J Exp Clin Biosci 2013;1:33-8
|How to cite this URL:|
Singh P, Khosa R L, Mishra G. Evaluation of antidiabetic activity of ethanolic extract of Cedrus deodara (Pinaceae) stem bark in streptozotocin induced diabetes in mice. Niger J Exp Clin Biosci [serial online] 2013 [cited 2019 Jun 17];1:33-8. Available from: http://www.njecbonline.org/text.asp?2013/1/1/33/123961
| Introduction|| |
Diabetes mellitus is a chronic metabolic disorder resulting from insulin deficiency, characterized by hyperglycemia, altered metabolism of carbohydrates, protein, lipid and an increased risk of vascular complication.  According to the international diabetes federation, diabetes affects at least 285 million people worldwide and that number is expected to reach 438 million by the year 2030, with two-thirds of all diabetes cases occurring in low-to middle-income countries. The number of adults with impaired glucose tolerance will rise from 344 million in 2010 to an estimated 472 million by 2030.  Virtually all forms of diabetes mellitus are caused by a decrease in the circulating concentration of insulin and decrease in response of peripheral tissues to insulin. These abnormalities lead to alterations in the metabolism of carbohydrates, lipids, ketones and amino acids.  Although insulin and oral hypoglycemic agents are the major players in the treatment of the diabetes mellitus, they have prominent side effects and fail to significantly alter the course of diabetes. , The World Health Organization has recommended that traditional plant treatment for diabetes warrant further evaluation.  An antidiabetic agent could exert a beneficial effect in the diabetic situation by enhancing insulin secretion and/or by improving/mimicking insulin action.  Nowadays, the use of complementary and alternative medicine and especially the consumption of botanicals have been increasing rapidly worldwide, mostly because of the supposedly less frequent side effects when compared to modern western medicine. 
Cedrus deodara (Lamb.) G. Don is an ornamental plant, belonging to family Pinaceae. It is an evergreen tree, distributed in the Mediterranean region and the Western Himalayas,  and grows at an altitude of about 1300-3300 m. Leaves are dark green or bluish green, 3-sided, single on elongated shoot, but in dense clusters on arrested branchlets. Cones erect 10-12 cm long and 7-10 cm in diameter. Seeds up to 2 cm long with triangular wings.  C. deodara contains himachald, allohimachalol, himadarol, centdard, isocentdarol, dewarene, dewardiol, dewarenol, taxifolin, cedeodarin, dihydromyricetin, cetrin and cedrinoside. Pharmacologically, it is used as anti-inflammatory and analgesic.  Traditionally, it is used in the treatment of ulcers and skin diseases. The wood powder is used as carminative, diaphoretic, diuretic and in the treatment of fever, urinary disorders  rheumatism, piles, gravels in kidney, snake-bite,  diarrhea and dysentery. 
| Materials and Methods|| |
Stem bark of the C. deodara was collected from Dehradun, India. The taxonomical identification of the plant was done by Dr. D. C. Saini, Scientist 'E', Birbal Sahni Institute of Palaeobotany, Lucknow, India. A voucher specimen with no. BSIP 01 was submitted in Birbal Sahni Institute of Palaeobotany, Lucknow and also in Department of Pharmacognosy, Teerthanker Mahaveer College of Pharmacy, Moradabad.
Preparation of Extracts
Dried and powered plant material (300 g) was successively extracted by soxhlet method with petroleum ether (60-80°) and ethanol (75-80°) for 75 h each. The obtained extracts were evaporated in vacuum rotary evaporator to give residues and their percentage yields were determined.
In order to determine the presence of alkaloid, carbohydrate, flavonoid, proteins, amino acids, phenols, tannins, glycosides and steroids, a preliminary phytochemical study was performed with both the extracts. ,, The standard methods for various phytoconstituents are as follows.
Tests for Alkaloids
2-3 ml test solution may give cream color precipitate with Mayer's reagent to ensure the presence of alkaloids.
Reddish brown colored precipitate may appear when 2-3 ml test solution was added to Dragendorff's reagent to reveal the presence of alkaloids.
2-3 ml test solution may give reddish brown precipitate with Wagner's reagent, which may correspond to the presence of alkaloids.
Yellow colored precipitate may appear when 2-3 ml of test solution was mixed with Hager's reagent.
Tests for Carbohydrates
The test solution was treated with few drops of alcoholic α-naphthol solution. The solution was shaken and concentrated H 2 SO 4 was added slowly through the sides of the test tube. A purple to violet color ring may form at the junction of the two liquids.
Tests for Flavonoids
Shinoda test (magnesium hydrochloride reduction test)
Few fragments of magnesium ribbon and concentrated hydrochloric acid when added drop wise to the test solution, pink to red color may be observed.
Tests for Proteins
3 ml of test solution was mixed with 5 ml of Millon's reagent (Mercuric nitrate in nitric acid containing traces of nitrous acid), a white precipitate may appear which turn red upon gentle heating or precipitate may dissolve giving red color solution.
3 ml test solution was added to a mixture of 4% NaOH and few drops of 1% CuSO 4 . Violet or pink color may appear.
Tests for Amino Acids
3 ml test solution was heated with 3 drops of 5% Ninhydrin solution in boiling water bath for 10 min. Purple or bluish color may indicate the presence of amino acids.
Tests for Tannins and Phenolic Compounds
To 2-3 ml of aqueous or alcoholic extract was added with few drops of 5% Ferric chloride solution: Test solution may show deep blue-black color to confirm the presence of free tannins. A brownish green precipitate may appear if condensed tannins were present.
Lead acetate solution
Test solution may give white precipitate.
Red precipitate may appear.
Dilute NH 4 OH and potassium ferricyanide
Test solution may give red color solution.
Tests for Glycosides
A thick section may show yellow to orange color with sodium picrate.
To the solutions of aqueous or alcoholic extract 1 ml pyridine and 1 ml alkaline sodium nitroprusside solution were added. Pink to red color may appear.
Keller Killiani test (test for deoxy sugars)
2 ml extract of the drug was added to glacial acetic acid containing a trace amount of 0.5% ferric chloride. It was transferred to a small test tube; 0.5 ml of concentrated sulphuric acid was carefully added by the side of the test tube. Reddish brown color if appeared at the junction of the two liquids and upper layer appeared bluish green, may correspond to the presence of glycosides.
Tests for Steroids
A mixture of 2 ml chloroform and 2 ml concentrated sulphuric acid was added to the test solution, shaken well and allowed to stand for some time, chloroform layer may appear red indicating the presence of sterols while acid layer may show greenish yellow colored fluorescence to reveal the presence of triterpenoids.
2 ml extract was mixed with chloroform and 1-2 ml acetic anhydride was added to it, the solution was boiled and cooled, concentrated sulphuric acid was added from the side of the test tube which may show first red then blue and finally green color.
Acute Toxicity Study
Acute oral toxicity study was performed as per Organization for Economic Co-operation and Development-423 guidelines (acute toxic class method). Albino mice (n = 6) of either sex selected by random sampling technique were kept fasting for overnight providing only water, after that the extract was administered orally at the dose level of 5 mg/kg body weight and observed for 14 days. If mortality was observed in 2-3 animals, then the dose administered was assigned as toxic dose. If mortality was observed in one animal, then the same dose was repeated again to confirm the toxic dose. If mortality was not observed, the procedure was repeated for further higher doses such as 50, 100, 200, 400, 800 and 1600 mg/kg body weight. 
Animals and Treatment
Healthy Swiss albino mice of either sex (25 ± 5 g), with no prior drug treatment, were used in the studies. The animals were housed in a group of four in separate cages under controlled conditions of temperature (22 ± 2°C). All animals were given standard diet (Golden feed, New Delhi) and water regularly. Animals were further divided in four groups with six animals in each group. The animal study was performed in Teerthanker Mahaveer College of Pharmacy, TMU, Moradabad, India, after approval from the Institutional Animal Ethics Committee of Teerthanker Mahaveer College of Pharmacy, TMU, Moradabad (Reg No. 1205/c/08/CPCSEA).
Oral Glucose Tolerance Test
Mice divided into four groups (n = 6) were administered with distilled water 10 mL/kg, glibenclamide (standard drug) 10 mg/kg and ethanolic extract of Cedrus deodara (EECD) at a dose of 250 mg/kg and 500 mg/kg p.o., respectively.  All the animals were given glucose (2 g/kg) 60 min after dosing. Blood samples (upto 0.2 ml each) were collected from the tail vein just prior to (0 h) and at 30, 60, 90 and 120 min after glucose loading and glucose level were estimated. 
Streptozotocin (STZ) Induced Diabetes
Diabetes was induced in mice by intraperitoneal injection of STZ at a dose of 60 mg/kg body weight,  dissolved in 0.1M cold citrate buffer (pH = 4.5). Diabetes was confirmed by the determination of fasting blood glucose concentration.
Mice were divided into five groups of six mice each. Groups 1 and 2 served as the vehicle and standard (treated with 10 mg/kg/day glibenclamide). Group 3 served as diabetic untreated control (STZ). Groups 4 and 5 were treated with extracts at the dose level of 250 mg/kg and 500 mg/kg p.o., respectively, for 21 days.
Finally, on day 21, blood was collected to perform various hematological and biochemical parameters. The freshly prepared solutions were orally administered for a period of 21 days. Body weights and blood glucose analysis (with the help of Gluco check) was done weekly on overnight fasted animals. At the end of the experimental period, the animals were fasted overnight and blood (0.2 ml) was collected for various biochemical estimations. The animals were sacrificed by cervical decapitation. Liver was dissected, immediately rinsed with ice cold saline and immediately frozen and stored at 80°C for further biochemical analysis. 
After termination of the experiment, mice from all the groups were anesthetized and dissected out. Pancreas was taken in formaldehyde solution and histological preparations were made. 5 μm thick sections were cut and stained with hematoxylene and eosin. 
The values were expressed as mean ± standard error of the mean. The results were analyzed for statistical significance using one-way analysis of variance followed by Bonferroni t-test. Levels of aP < 0.05, bP < 0.01 and cP < 0.001 were considered significant.
| Results|| |
Preliminary phytochemical tests of EECD showed the presence of carbohydrates, alkaloids, glycosides, flavonoides and phenolic compounds.
Acute Toxicity Study
The result of the acute oral administration of EECD in various doses of 50, 100, 200, 400, 800 and 1600 mg/kg indicated no mortality up to 7 days after treatment.
Oral Glucose Tolerance in Normal Mice
EECD bark when administered 60 min prior to glucose loading produce a significant reduction in the rise in blood glucose level at 90 min after glucose administration which is shown in [Table 1]. Dose dependent glucose reduction was observed in animals treated with standard glibenclamide (10 mg/kg) and ethanolic extracts (250 mg/kg and 500 mg/kg) of C. deodara bark at 0 min, 30 min, 60 min, 90 min and 120 min compared to normal control group [Table 1].
|Table 1: Effect of ethanolic extract of Cedrus deodara bark on oral glucose tolerance in normal mice|
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Effect of Eecd Bark on Fasting Blood Glucose Level and Body Weight in STZ Induced Diabetic Mice
The effect of repeated oral administration of EECD bark on blood glucose levels in STZ induced diabetic mice and body weight is given in [Table 2] and [Table 3].
|Table 2: Effect of ethanolic extract of Cedrus deodara bark on streptozotocin induced diabetes mice|
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|Table 3: Effect of ethanolic extract of Cedrus deodara bark on body weight of STZ induced diabetic mice|
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EECD bark administered in 250 mg/kg and 500 mg/kg doses levels to STZ treated diabetic mice showed a significant reduction in blood glucose levels which are related to the duration of treatment.
Highest reduction was observed on day 21. EECD bark at the dose of 500 mg/kg exhibited significant glucose lowering effect in diabetic mice (P < 0.001) when compared to standard.
STZ produced significant loss of body weight as compared to normal animals during the study. Diabetic control continued to lose weight till the end of the study while EECD bark treated groups showed improvement in body weight compared to diabetic control.
Effect of EECD Bark on Serum Glutamic Pyruvic Transaminase (SGPT), Serum Glutamic Oxaloacetic Transaminase (SGOT), Total Cholesterol and Triglyceride
EECD bark treated groups showed a reduction in biochemical parameters like SGPT, SGOT, cholesterol and triglyceride compared to the diabetic control as shown in [Table 4].
|Table 4: Effect of ethanolic extract of Cedrus deodara bark on biochemical parameters|
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Results showed that the extract enhanced the regeneration of Islets of Langerhans More Details in the pancreas and restoration of normal cellular size of the islet with hyperplasia [Figure 1].
|Figure 1: Effect of ethanolic extract of Cedrus deodara at 250 mg/kg and reference compound on pancreas. (a) Normal architecture of the pancreas, round islet appearing as a lightly stained cluster of cells (arrow head) compared to the surrounding exocrine pancreatic tissue (asterisk), and blood capillaries (arrow). (b) Degenerative and necrotic changes due to streptozotocin demonstrating a relatively small and atrophic islet (arrow head). (c) Regeneration of islets of Langerhans showing (d and e). Restoration of normal cellular size of the islets of Langerhans with hyperplasia|
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| Discussion|| |
The fundamental mechanism underlying hyperglycemia involves over-production (excessive hepatic glycogenolysis and gluconeogenesis) and decreased utilization of glucose by the tissues.  Persistent hyperglycemia, the common characteristic of diabetes can cause most diabetic complications. In all patients, treatment should aim to lower blood glucose to near normal levels. In our investigation, oral glucose tolerance test studies revealed that the ethanolic extract of bark of C. deodara has the capacity to lower blood glucose levels. The diabetic syndrome in mice administered STZ is characterized by stable moderate hyperglycemia, glucose intolerance, loss of body weight and altered but significant glucose stimulated insulin secretion. In antidiabetic study, the data indicates that the EECD treatment significantly reduced the blood glucose levels and caused a gain in body weights of the diabetic mice towards the normal level in the 21 days of the study period in a dose dependant manner. The ethanolic extract also decreases the level of biochemical parameters like SGPT, SGOT, cholesterol and triglycerides in similar dose dependant manner. Our histopathological study showed that treatment with EECD caused enhanced islet regeneration in the pancreas and restoration of normal cellular size of the islet with hyperplasia. It is thus apparent that the hypoglycemic effect may be probably brought about by pancreatic mechanism. The analysis of histological parameters from treated animals further confirmed that the given extract is effective and could act in the management of diabetes.
| Conclusion|| |
It was concluded from the study that there are ample evidence to support that the EECD bark exhibited potent antidiabetic activity in STZ induced diabetic mice. However, further studies are needed to identify the chemical constituent responsible for antidiabetic activity and to elucidate exact mechanism of action for antidiabetic activity.
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[Table 1], [Table 2], [Table 3], [Table 4]
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