Original Article 287
Authors
S. Rani, S. Sharma, S. Kumar
Affiliation
Division of Pharmacology, Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology, Haryana, India
Key words ▶ antihyperglycemic activity ● ▶ streptozotocin ● ▶ safrole ● ▶ lipase ● ▶ amylase ●
Abstract
▼
In the present investigation anti-diabetic and in-vitro antioxidant potential of safrole were evaluated (100 and 200 mg/kg p.o.) in acute and chronic Streptozotocin-nicotinamide (STZ) induced antihyperglycemic rat model. The oral administration of safrole for 30 days affects the level of blood glucose, glycosylated hemoglobin (HbA1C), total cholesterol (TC), triglycerides (TG), phospholipids, high density lipoprotein (HDL), body weight, insulin level, liver glycogen content, antioxidant parameters, lipase,
Introduction
▼ received 12.09.2013 accepted 22.09.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1357192 Published online: October 16, 2013 Drug Res 2014; 64: 287–295 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence S. Sharma Division of Pharmacology Department of Pharmaceutical Sciences Guru Jambheshwar University of Science and Technology Hisar Haryana-125001 India Tel.: + 91/941/6107 896 Fax: + 91/1662/278 181 sharmask71@rediffmail.com
Diabetes mellitus is a group of metabolic disorder characterized by hyperglycaemia due to defect in insulin secretion or action or both. In this condition the β cells of the pancreas being unable to produce sufficient insulin to prevent hyperglycemia [1]. It is associated with significant changes in the plasma lipid and lipoprotein profile thereby an increased risk of premature atherosclerosis, coronary insufficiency and myocardial infarction exists [2]. Oxidative stress plays a major role in the pathogenesis and development of complications of both types of diabetes mellitus. In diabetes mellitus oxygen free radicals also increases in body which are responsible for oxidative stress. There was 285 million people living with diabetes in 2010, which are increased to 366 million in 2011 and this is expected to reach 552 million by 2030, an increase of 50.7 %. In India, 61.3 million people had diabetes in 2011 and expected that figure is projected to rise to 101.2 million by 2030. There are lots of constituents upon which antidiabetic work is carried out and give the positive response towards the anti-diabetic research. Safrole is a chemical components of camphor oil which also contains a-pinene, camphene,
α-amylase in normal and STZ induced diabetic rats. The oral administration of safrole at dose 100 & 200 mg/kg p.o. significantly improve the diabetic condition in Streptozotocin-induced diabetic rats. In enzymatic assay, the IC50 value of the safrole for α-amylase and lipase was found to be 702.78 and 861.35 μg/ml respectively which was found comparable with the standard drug (ascorbic acid) as 252.12 μg/ml. Further studies can be performed on safrole for mechanistic and toxicological aspects so that it can be investigated as a new substance for the management of various diseases.
b-pinene, sabinene, phellandrene, limonene, 1, 8-cineole, y-terpinene, p-cymene, terpinolene, furfural, camphor, linalool, bornyl acetate, terpinen-4-ol, caryophyllene, borneol, piperitone, geraniol, cinnamaldehyde, methyl cinnamate and eugenol. The brown and yellow camphor oil has very high safrole content (10–80 %). Camphor, a natural product derived from the wood of the tree Cinnamomum camphora, has a long history of use as: analgesic [3], respiratory relief, inhalants [4], nasal decongestant, expectorant [5], effect on liver [6], in tumor [7], contraceptive & sexual performance [8], scabies [9]. The prolonged administration of safrole for a period of 82 weeks in 7–28 days old animals (rats and mice) caused liver tumors in male mice and malignant liver tumors (hepatocellular carcinoma or adenoma or cholangiocarcinoma) in rats of both sexes. Enzymes also play a major role in the treatment of diabetes mellitus as enzymes hydrolyzes starch molecules to give diverse products including dextrin and progressively smaller polymers composed of glucose units within the small intestine which causes hyperglycemia and development of type 2 diabetes mellitus. The inhibition of α-amylase activity is possibility to lower postprandial blood glucose levels [10]. The enzyme
Rani S et al. Antihyperglycemic Potential of Safrole … Drug Res 2014; 64: 287–295
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To Investigate Antihyperglycemic and Antihyperlipidemic Potential of Safrole in Rodents by in-vivo and in-vitro Study
288 Original Article
Experimental Design
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Drugs and chemicals Glibenclamide (Torrent Pharmaceutical, Ahemadabad), streptozotocin, heparin (SRL, India), safrole, orlistat, lipase (Sigma Aldrich, USA), EDTA, soluble starch, iodine, α-amylase, sodium cholate, triolein, lecithin, TES buffer (Hi-media Lab. Pvt Ltd., Mumbai), n-butanol, acetic acid, n-hexane, petroleum ether, ethyl acetate, glucose standard, citric acid, sodium citrate, tris hydrochloride, buffer tablet, sodium lauryl sulphate, thiobarbituric acid, trichloroacetic acid, triton-X, glycogen, ethanol, tween 80, carboxy methyl cellulose, Ellman’s reagent (5,5’- dithiobis(2-nitro-benzoic acid); DTNB), sodium sulphate, methanol, pyridine, anthrone, thiourea, benzoic acid, sodium chloride (SD Fine Chem. Ltd., Mumbai). Safrole was separately dissolved in 1 % of CMC (carboxy methyl cellulose) solution. Volume of oral dosing was 1.0 ml/100 g of animal.
Experimental animals Albino wistar rats (weight 150–200 g) were procured from disease free small animal house, lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar (Haryana). The rats were housed in cages (polycarbonate cage size: 29 × 22 × 14 cm) under laboratory conditions with alternating light and dark cycle of 12 h each. The animals had free access to food and water. The animals were kept fasted 2 h before and 2 h after drug administration. The experimental protocol was approved by institutional animals ethics committee (IAEC) and animal care was done as per the guidelines of committee for the purpose of control and supervision of experiments on animals (CPCSEA), govt. of India (registration no. 0436).
Induction of experimental diabetes using streptozotocin-nicotinamide The animal model of type-2 diabetes mellitus (NIDDM) was induced in overnight fasted animals by a single intraperitoneal injection of STZ (45 mg/kg) followed by nicotinamide injection (110 mg/kg). The nicotinamide injection was given to rats 15 min prior to the STZ injection. Hyperglycemia was confirmed by the elevated blood glucose levels determined at 72 h then on 7th day of the injection. Only rats confirmed with permanent NIDDM were used in the anti-diabetic study [12].
Groups for streptozotocin-nicotinamide induced diabetic rat model Total no. of animals was divided into 9 groups and each group contains 6 animals. Rani S et al. Antihyperglycemic Potential of Safrole … Drug Res 2014; 64: 287–295
Group I: Normal control rat. Group II: Diabetic control: Animals were administered vehicle only. Group III: Diabetic animals were administered with glibenclamide (600 μg/kg, orally). Group VI: Diabetic animals were administered with safrole (100 mg/kg, orally). Group V: Diabetic animals were administered with safrole (200 mg/kg, orally). Group VI: Normal animals were administered with safrole (200 mg/kg, orally).
Glucose tolerance test After 29 days treatments, on 30th day a fasting blood sample was collected from all the groups in heparinized micro-centrifuge tube from retro-orbital plexus. Blood samples were also collected at the time interval of 0, 30, 60, 90, and 120 min after administration of glucose at a concentration of 2 g/kg of body weight.
Biochemical assay The fasted animals were sacrificed by cervical decapitation on 30th day of first treatment. Trunk blood was collected in heparinised micro centrifuged tubes and the plasma obtained by centrifugation at 5 000 rpm for 5 min was used for the determination of plasma glucose, cholesterol, malondialdehyde (MDA), reduced glutathione. While whole blood was used for glycosylated hemoglobin.
Estimation of serum glucose and cholesterol Plasma cholesterol and glucose level were measured by commercial supplied biological kit Erba Glucose Kit (GOD-POD Method) and Erba Cholesterol Kit (CHOD-PAP Method) respectively using Chem. 5 Plus- V2 Auto-analyzer (Erba Mannheim, Germany) in plasma sample prepared as above. Glucose and cholesterol values were calculated as mg/dl blood sample.
Estimation of glycosylated hemoglobin (Hb1ac) Glycosylated hemoglobin was measured using commercial supplied biological kit (Erba Diagnostic) in plasma sample prepared as above using Chem. 5 Plus-V2 Auto-analyzer (Erba Mannheim, Germany). Values are expressed as the percent of total hemoglobin.
Estimation of insulin The serum insulin level was measured by an enzyme-linked immuno-sorbent assay (ELISA) procedure using Mercodia rat insulin ELISA kit. In short, the solid phase 2-site enzyme immunoassay is based on the direct sandwich technique in which 2 monoclonal antibodies are directed against separate antigenic determinants 35 (epitopes) on the insulin molecule. During incubation, insulin in the sample reacts with peroxidase-conjugated anti-insulin antibodies and anti-insulin antibodies bound to the micro titration well. After washing 3 times, unbound enzyme labeled antibody was removed. The bound conjugated insulin was detected by reacting with 3, 3′, 5, 5′-tetramethylbenzidine. The reaction was stopped by adding acid to give a colorimetric end-point, and optical density was measured with a micro plate auto reader (Bio-tek Instrument Inc., USA) at a wavelength of 450 nm. The serum insulin is expressed as μg/l [13].
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lipase that travels all the way down to the intestines promotes breakdown of fats like cholesterol and triglycerides to give di and mono glycerides, glycerol and free fatty acids from ingested food. Researchers and health professionals believe that the inhibition of PL can reduce digestion of fats, hence, their assimilation and absorption. This can mimic a reduced calorie intake in obese patients and help in preventing additional weight gain [11]. The present study was designed to investigate the in-vitro and invivo antihyperglycaemic properties of safrole in normoglycaemic and streptozotocin-induced diabetic rats as this model represent a good resemblance human for type-2 diabetes and show many of the features of the human disease, including hyperphagia, hyperglycaemia, insulin resistance and progressive obesity.
Original Article 289
Malondialdehyde (MDA), an index of free radical generation/ lipid peroxidation, was determined as described by Okhawa et al., (1979). The reaction mixture consisted of 0.2 ml of 8.1 % sodium lauryl sulphate, 1.5 ml of 20 % acetic acid (ph 3.5) and 1.5 ml of 0.8 % aqueous solution of thiobarbituric acid. This reaction mixture was added to 0.2 ml of blood plasma. This mixture was made up to 4.0 ml with distilled water and cooling the contents under running tap water. Then 5.0 ml of n-butanol, pyridine (15:1 v/v) and 1.0 ml of distilled water added to the above mixture. After that centrifuged it at about 3 000 rpm for 10 min. Organic layer was separated out and absorbance was measured at 532 nm using double beam UV-visible spectrophotometer (systronics 2203, Bangalore, India) against a blank. MDA values are calculated using the extinction coefficient of MDA-thiobarbituric acid complex 1.56 × 105 l/mol × cm and expressed as N mol/ mg protein [14].
Estimation of plasma reduced glutathione level Took 2 ml of distilled water and 0.5 ml of trichloroacetic acid in a 10 ml test tube Added this solution into 2.5 ml of blood plasma. The tubes were shaken intermittently for 10–15 min and centrifuged it for 15 min at approximately 3 000 rpm. Supernatant was added with 2 ml of 0.4 m tris-EDTA buffer (ph 8.9) and 0.1 ml DTNB and mixed well. At last absorbance was read within 5 min of the addition of DTNB at 412 nm against a regent blank [15].
Estimation of liver glycogen content Liver glycogen estimation was done by the method as described by [16]. Immediately after excision from the animal, 1 g of the liver was dropped into a previously weighed test tube containing 3 ml of 30 % potassium hydroxide solution. Weight out the liver and tissue was then digested by heating the tube for 20 min in boiling water bath. Then digest tissue was cooled and transferred quantitatively to a 50 ml volumetric flask, Diluted it to the mark with water. Contents of the flask were then thoroughly mixed; a measured portion was then further diluted with water in a second volumetric flask so as to yield a solution of glycogen of 3–30 μg/ml. 5 ml aliquots of the final dilution were then pipette into Evelyn tube and the determination with anthrone was carried out. The amount of glycogen in the aliquot used was then calculated using the following equation: μg of glycogen in aliquot = 100 U/1.11 S U is the optical density of unknown solution. S is the optical density of 100 μg glucose and 1.11 is the factor determined by Morris standard (Morris, 1948) for the conversion of the glucose to the glycogen [17].
soluble starch (500 mg) was dissolved in 25 mL of 0.4 M NaOH and heated for 5 min at 100 °C. After cooling in ice H2O, the solution was adjusted to pH 7 with 2 M HCl, and H2O was added to adjust the volume to 100 ml. Sample solutions were prepared by dissolving each sample in acetate buffer (pH 6.5) to make different concentrations (50–500 μg/ml). The substrate (40 μL) and sample (20 μL) solutions were mixed in a micro-plate well, and the mixtures were pre-incubated at 37 °C for 3 min. Then 20 μL of α-amylase solution (50 μg/ml) was added to each well, and the plate was incubated for 15 min. The reaction was terminated by addition of 80 μL of 0.1 M HCl; then 200 μL of 1 mM iodine solution was added, and the absorbance was measured at 650 nm. Inhibitory activity ( %) was calculated as follows: % inhibition = {1− (absorbance of sample and substrate – absorbance of sample, substrate, and lipase)/(absorbance of substrate – absorbance of substrate and lipase) × 100} IC50 value was obtained by interpolation of graph between concentrations of tested or standard drugs vs. % enzyme inhibition curves. The concentration of an inhibitor required to inhibit 50 % of enzyme activity under the mentioned assay conditions is defined as the IC50 value.
Measurement of pancreatic lipase inhibitory activity Lipase inhibitory activity was evaluated according to the method of Han et al. [20] with slight modifications. Orlistat, a specific pancreatic lipase inhibitor used in the prevention of obesity clinically was used as a positive control for the comparison of tested oils. Substrate solution was prepared by sonication (10 min in an ice bath) of a mixture of glyceryl trioleate (80 mg), lecithin (10 mg), and sodium cholate (5 mg) suspended in 9 mL of 0.1MTES buffer (pH 7.0). Samples were separately dissolved in 0.1 M TES buffer to make 0.2 mg/mL solutions. The substrate (20 μL) and sample solutions (20 μL) in microplate wells were pre-incubated for 3 min; then 10 μL of lipase solution (20 μg/mL) was added to each reaction mixture and incubated for 30 min at 37 °C. The amount of released fatty acid was measured by a LISA Plus micro-plate reader at 550 nm. Inhibitory activity ( %) was calculated as follows: % Inhibition = {1− (absorbance of sample and substrate – absorbance of sample, substrate, and lipase)/(absorbance of substrate – absorbance of substrate and lipase) × 100} IC50 value was obtained by interpolation of graph between concentrations of tested or standard drugs vs. % enzyme inhibition curves. The concentration of an inhibitor required to inhibit 50 % of enzyme activity under the mentioned assay conditions is defined as the IC50 value.
Data analysis Histopathological studies For the histopathological studies, rats were scarified under light ether anesthesia 24 h after the last dosage and the pancreas were removed and washed with normal saline. The pancreas tissue were fixed in 10 % formalin solution, dehydrated with a sequence of ethanol solution and then embedded in paraffin. Sections of pancreas of 5–6 micrometer in thickness were cut, stained with hematoxylin and eosin and then subjected to photo microscopic observation (Digital microphotography Olympus CX41).
Measurement of α-amylase inhibitory activity The α-amylase activity was measured applying the method reported by [18, 19] with little changes. Acarbose was used as the positive control. Substrate solution was prepared as follows:
All the results were expressed as mean ± standard error of mean (SEM). The data of all the groups were analyzed using one-way ANOVA followed by Dunnett’s t-test, the criterion for statistical significance was p < 0.01 as compare to diabetic control.
Results
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Acute hypoglycemic effect of safrole on normoglycaemic rats The effect of the treatment with safrole on the serum glucose in ▶ Fig. 1. The safrole treated normal fasted rats is shown in ● groups at 100 mg/kg and 200 mg/kg p.o. shown a significant decrease in serum glucose level as compared to untreated group. Rani S et al. Antihyperglycemic Potential of Safrole … Drug Res 2014; 64: 287–295
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Estimation of MDA level
290 Original Article Effect of safrole on total cholesterol ▶ Fig. 5 indicated the effect of safrole on The data presented in ●
The safrole treated group shown a marked fall in glucose level in 30–90 min interval. After 90 min the serum glucose level returns to normal value (p < 0.01).
total plasma cholesterol. Total plasma cholesterol was observed higher in diabetic rats compared to normal rats. The administration of the control drugs significantly lowered the total plasma cholesterol concentration as compare to diabetic control. The administration of safrole 100 mg/kg, p.o. and safrole 200 mg/kg, p.o. shows significant (p < 0.01) reduction in total plasma cholesterol level as compare to diabetic control on 10th (acute study) as well as 30th day (chronic study).
Chronic antihyperglycemic effect of safrole in STZ induced diabetic rats Safrole 100 mg/kg p.o. give to STZ diabetic rats for 30th day (chronic study) shown a fall in serum glucose level from 378.94 mg/kg 261.61 mg/kg at 30th day when compared to 1st day value. At safrole 200 mg/kg p.o. to STZ diabetic rats for 30th day shown a fall in serum glucose level from 368.81 mg/kg 155.26 mg/kg at 30th day when compared to 1st day value ▶ Table 1) (● ▶ Fig. 2). (p < 0.01) (●
Effect of safrole on total HDL level ▶ Fig. 6 indicated the effect of safrole on The data presented in ●
The effect of safrole on plasma glucose level after glucose loading at 2 g/kg orally to the normal, diabetic and test animals was ▶ Fig. 3. The blood glucose level rises to a maxexpressed in the ● imum in 30 min after glucose loading. The safrole 100 mg/kg and 200 mg/kg had shown a significant increase in rate of clearance of glucose as compared to untreated group. The safrole treated group shown a marked fall in glucose level in 30, 60, 90, 120 min interval. After 120 min the serum glucose level returns to normal value (p < 0.01).
Effect of safrole on total Triglycerides level ▶ Fig. 7 indicates the effect of safrole on total The data shown in ● plasma triglycerides level. Total plasma triglycerides were observed higher in diabetic rats compared to normal rats. The administration of the control drugs significantly reduced the total plasma cholesterol concentration as compare to diabetic control. The administration of safrole 100 mg/kg, p.o. and 200 mg/kg, p.o. shown significant (p < 0.01) reduction in total plasma triglycerides level as compare to diabetic control on 10th (acute study) as well as 30th day (chronic study).
Effect of safrole on glycosylated hemoglobin ▶ Fig. 4 indicated the effect of safrole on The data depicted in ● glycosylated hemoglobin (HbA1c). The HbA1c data for the STZ diabetic group were higher than the normal rats. The chronic administration of the standard drug significantly reduced the HbA1c level in 30 days. The administration of safrole reduces the HbA1c level as compared to the diabetic control. Higher doses produced more reduction HbA1c level indicate dose dependent effect of safrole in diabetic rats (p < 0.01).
Fig. 1 Acute hypoglycaemic effect of safrole on normoglycaemic rats. Values are presented as mean ± S.E.M.; n = 6 in each group. One way ANOVA followed by Dunnett’s test *p < 0.05; **p < 0.01; as compare to control.
Fig. 2 Chronic antihyperglycaemic effect of safrole in STZ induced diabetic rats. Values are presented as mean ± S.E.M.; n = 6 in each group. One way ANOVA followed by Dunnett’s test *p < 0.05; **p < 0.01; as compare to control.
Table 1 Chronic antihyperglycemic effect of safrole in STZ induced diabetic rats. Treatment
Dose
Mean blood glucose concentration (mg/dl) ± S.E.M 1st Day
Normal Diabetic control Glibenclamide Safrole safrole
600 μg/kg 100 mg/kg 200 mg/kg
92.65 ± 1.57 381.14 ± 5.29 304.79 ± 4.58** 378.94 ± 4.25 368.81 ± 3.13
10th Day 97.33 ± 33 369.98 ± 3.94 243.47 ± 5.01** 353.80 ± 2.78 352.50 ± 3.19*
20th Day 96.24 ± 4.03 376.63 ± 3.45 181.10 ± 3.45** 316.29 ± 2.63* 258.67 ± 4.93**
Values are presented as mean ± S.E.M. ; n = 6 in each group. One way ANOVA followed by Dunnett’s test *p < 0.05; **p < 0.01; as compare to control
Rani S et al. Antihyperglycemic Potential of Safrole … Drug Res 2014; 64: 287–295
30th Day 93.11 ± 2.27 366.12 ± 3.15 107.39 ± 3.15** 261.61 ± 2.68* 155.26 ± 3.72**
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total plasma HDL. Total plasma HDL was observed low in diabetic rats compared to normal rats. The administration of the safrole significantly enhances the total plasma HDL concentration as compare to diabetic control. The administration of safrole 100 mg/kg, p.o. and 200 mg/kg, p.o. shown significant (p < 0.01) enhancement in total plasma HDL level as compare to diabetic control on 10th as (acute study) well as 30th day (chronic study). Higher doses increase more HDL level.
Effect of safrole in glucose loaded hyperglycaemic rats
Original Article 291
Fig. 4 Effect of safrole on glycosylated hemoglobin. Values are presented as mean ± S.E.M.; n = 6 in each group. One way ANOVA followed by Dunnett’s test **p < 0.01; as compare to control.
Fig. 5 Effect of safrole on total cholesterol. Values are presented as mean ± S.E.M. ; n = 6 in each group. One way ANOVA followed by Dunnett’s test *p < 0.05; **p < 0.01; as compare to control.
Effect of Safrole on Glutathione Level The plasma glutathione (GSH) level was decreased in STZ rats ▶ Fig. 8. The level of compared to normal animals as shown in ● glutathione was returned to near normal range in STZ diabetic rats treated with glibenclamide and it also shown significant (p < 0.01) enhancement by the safrole 100 mg/kg, p.o. and 200 mg/kg, p.o.
Effect of safrole on antioxidant parameter
Fig. 6 Effect of safrole on total HDL level. Values are presented as mean ± S.E.M. ; n = 6 in each group. One way ANOVA followed by Dunnett’s test *p < 0.05; **p < 0.01; as compare to control.
Fig. 7 Effect of safrole on total triglycerides level. Values are presented as mean ± S.E.M. ; n = 6 in each group. One way ANOVA followed by Dunnett’s test **p < 0.01; as compare to control.
Fig. 8 Effect of safrole on glutathione level. Values are presented as mean ± S.E.M. n = 6 in each group. One way ANOVA followed by Dunnett’s test *p < 0.05; **p < 0.01; as compare to control.
pared to normal rats. Treatment of STZ diabetic rats with safrole resulted in a marked decrease in plasma level of MDA.
Effects of safrole on liver glycogen content The liver glycogen level was found to be low in STZ rats when ▶ Fig. 10. Safrole compared to normal rats as data reflected in ● 100 mg/kg p.o. and 200 mg/kg p.o. increased the liver glycogen content significantly when compared diabetic control rats ▶ Fig. 10. (p < 0.01) ●
The plasma level of malondialdehyde (MDA) of normal and ▶ Fig. 9. Plasma experimental animal in each group is shown in ● MDA level in plasma was increased in STZ diabetic rats comRani S et al. Antihyperglycemic Potential of Safrole … Drug Res 2014; 64: 287–295
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Fig. 3 Effect of safrole in glucose loaded hyperglycaemic rats. Values are presented as mean ± S.E.M.; n = 6 in each group. One way ANOVA followed by Dunnett’s test *p < 0.05; **p < 0.01; as compare to control.
292 Original Article Effects on insulin ▶ Table 2 shows the level of plasma insulin in the control and ● experimental groups of rats. Diabetic rats shown a decrease in plasma insulin compared with normal rats. Oral administration of safrole 100 mg/kg/day and 200 mg/kg/day respectively had shown a significant increase in plasma insulin level in respective ▶ Fig. 11). groups as compared to control rats (●
IC50 value obtained for these enzyme inhibitions are shown ▶ Table 3. in ●
Histopathological study ▶ Fig. 14. Histopathological changes of pancreas are given in ● Haematoxylin & eosin stained tissue sections of control animals’
Effects on body weight An increase in the body weight of normal rats was observed whereas the weight of diabetic control rats decreased from day 1 to day 30. Safrole 100 and 200 mg/kg p.o. when administered to respective groups of diabetic rats shown a significant increase in body weight as compared to the diabetic control group ▶ Table 2). (p < 0.01) (●
In-vitro study Enzymes inhibitory activity of safrole Fig. 11 Effect of safrole on insulin level.
Fig. 9 Effect of safrole on antioxidant parameter. Values are presented as mean ± S.E.M. ; n = 6 in each group. One way ANOVA followed by Dunnett’s test *p < 0.05; **p < 0.01; as compare to control.
Fig. 12 Enzymes inhibitory activity of safrole.
Fig. 10 Effects of safrole on liver glycogen content. Values are presented as mean ± S.E.M. ; n = 6 in each group. One way ANOVA followed by Dunnett’s test *p < 0.05; **p < 0.01; as compare to control.
Fig. 13 Enzymes inhibitory activity of safrole.
Table 2 Effect of safrole on body weight and insulin level. Treatment Normal Diabetic control Glibenclamide Safrole safrole
Dose (mg/kg p.o.)
Initial body weight (g)
Final body weight (g)
Change in weight (g)
Insulin micro U/ml
600 μg/kg 100 mg/kg 200 mg/kg
217.66 221.79 215.77 219.27 225.70
235.73 193.31 239.02 230.15 243.50
18.07 − 28.48 23.25** 10.88** 17.8**
18.23 ± 0.93 8.48 ± 0.82 17.35 ± 0.9 12.16 ± 0.81 14.07 ± 0.6
Values are presented as mean ± S.E.M.; n = 6 in each group. One way ANOVA followed by Dunnett’s test *p < 0.05; **p < 0.01; as compare to control
Rani S et al. Antihyperglycemic Potential of Safrole … Drug Res 2014; 64: 287–295
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The enzyme inhibitory activity of safrole against α-amylase & lipase were determined by using different concentration 200 μg/ ▶ Fig. 12, 13. The ml 400 μg/ml 800 μg/ml and 1 600 μg/ml as in ●
shows normal islets in pancreas histology throughout the study ▶ Fig. 14a). In contrast, STZ administration elicited severe (● injury of pancreas, such as decreasing of the islet cells’ numbers, diminishing of the diameter of pancreatic island, necrosis, degeneration and congestion in the islet of Langerhans cells. The islets were shrunken in diabetic rats when compared with nor▶ Fig. 14b). Treatment given to diabetic rats with the mal rats (● safrole 100 mg/kg shown significant expansion of islets. Further, there was significant reductions in the injuries of pancreas have also been observed but slight vaculation and degeneration was ▶ Fig. 14c, d, glibenclamide (25 mg/kg) shown moderpresent. ● ate expansion of islets and significantly reduced the injuries of ▶ Fig. 14e). These results shown that safrole could be pancreas (● useful in repairing β-cells in pancreatic islet injury. The dosedependent protective effect would be closely related with its improving function.
Discussion
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The aim of the present study was to evaluate the possible protective effects of safrole on serum glucose level and antioxidant defense systems of plasma in streptozotocin induced diabetes rats. The levels of glucose in blood, lipid hydroperoxide, glycogen content, body weight, insulin level GSH level and malonaldehyde were estimated in blood of control and experimental groups of streptozotocin (STZ) induced diabetic rats. In the present study, it was found that there was low level of feed intake as the rats shown poor appetite during the experiment and significant weight loss in diabetic animals (193.31 g) as compared to normal animals (235.73 g). The weight loss may be due to fluid depletion and accelerated breakdown of fats and
Table 3 Enzymes inhibitory activity of safrole. ENZYME ASSAY α-amylase Lipase
IC50 VALUE (μg/ml) Standard
Safrole
252.12 446.76
702.78 861.35
Values are presented as mean as 3 parallel measurements were conducted
adipose muscles [21]. Treatment with 100 mg/kg and 200 mg/ kg p.o. safrole significantly increases food and water intake of diabetic rats. This indicates that 100 mg/kg and 200 mg/kg safrole may improve characteristic symptoms of polyphasia & polydypsia of diabetes mellitus. It has been reported that diabetic STZ rats show significant glucose intolerance when orally administered with 2 g/kg of glucose and also have high insulin level. In the present study also diabetic animals were found to have impaired glucose tolerance with high glucose level after 1 h of glucose load compared to control animals. The results of this study have demonstrated that oral administration of 100 mg/kg and 200 mg/kg p.o. daily over duration of 30 days to STZ-induced type 2 diabetic rats can significantly improve the impaired glucose tolerance compare to diabetic control. These results are consistent with those reported earlier [22] and [23] wherein they have demonstrated the M. charantia may either have insulin-like secretagogue effect [24], it can stimulate peripheral glucose utilization or it may inhibit key gluconeogenic enzymes such as glucose-6-phosphatase and fructose biphosphatase [25]. Abnormalities in lipoproteins are very common in both NIDDM and IDDM. Diabetes leads to alterations in the plasma lipid and lipoprotein profile and increases risk of coronary heart disease [26]. In diabetic control hyper triglyceridemia, increased level of blood cholesterol and low HDL-cholesterol levels were seen when compared with normal control group [27]. The increase in blood cholesterol and triglycerides is due to the action of hormone sensitive lipase, which promotes lipolysis and subsequently increases the level of plasma free fatty acids and triglycerides. These free fatty- acids are catabolized to acetyl CoA which is further channeled to cholesterol synthesis, thus increasing blood cholesterol level. In the present investigation, serum cholesterol and triglyceride levels of diabetic rats were found to be significantly decreased by the treatment with safrole. We have also observed that in STZ induced diabetic rats; the level of HDL-cholesterol was significantly lower. 100 mg/kg and 200 mg/kg p.o. safrole normalized these effects, possibly by controlling the hydrolysis of certain lipoproteins and their selective uptake and metabolism by different tissues. The results of safrole highlight the topic for research and discussion for dose dependent effects of safrole. Hyperglycemia induces the generation of free radicals which can affect antioxidant defenses thus leading to the disruption of cellular Fig. 14 a Pancreas of Control animal shows normal histology. b Pancreas of Diabetic Control animal expressing severe necrosis, degeneration, congestion of islets cell. c Pancreas of Diabetic treated with 25 mg/kg of glibenclamide shows histology with large amount of islets cells as compare to diabetic rats. d Animal treated with 100 mg/kg of safrole shows mild regeneration of islets cell and congestion and vaculation are also present cells. c Pancreas of Diabetic treated with safrole (200 mg/kg) rats indicates repairing of islet of Langerhans but vaculation and degeneration are also present.
Rani S et al. Antihyperglycemic Potential of Safrole … Drug Res 2014; 64: 287–295
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Original Article 293
294 Original Article
Rani S et al. Antihyperglycemic Potential of Safrole … Drug Res 2014; 64: 287–295
Acknowledgement
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The authors are highly grateful to the All India Council for Technical Education, New Delhi (India) for providing research fellowship during research work. The authors have no conflict of interest.
Conflict of Interest
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The authors declare that they have no conflict of interests.
References 1 Rother KI. Diabetes treatment – Bridging the Divide. N Engl J Med 356: 1499–1501 2 Betteridge J. Lipid disorders in diabetes mellitus. In: Pickup JC, Williams G (eds.) Text Book of Diabetes. Blackwell Science Publishers, London: 1997; 1–35 3 Xu H, Blair NT, Clapham DE. Camphor activates and strongly desensitizes the transient receptor potential vanilloid subtype 1 channel in a vanilloid-independent mechanism. J Neur 2005; 25: 8924–8937 4 Jochen GW, Theis MD. Camphorated oil: still endangering the lives of Canadian children. Canadian Medical Association journal 1995; 152: 1821–1824 5 Inoue Y, Takeuchi S. Expectorant-like action of camphor derivatives. Nippon Ika Daigaku Zasshi 1969; 36: 351–354 6 Guilland-Cumming DF, Smith GJ. The effect of camphor on mitochondrial respiration. Experientia 1982; 38: 236–237 7 Goel HC, Roa AR. Radio sensitizing effect of camphor on transplantable mammary adenocarcinoma in mice. Canc Lett 1988; 43: 21–27 8 Akram J, Javed S, Ali AS et al. Effects of camphor oil on sexual behaviour in male rats. Iranian journal of pharmaceutical sciences 2006; 2: 209–214 9 Morsy TA, Rahem MA, El-Sharkawy EM et al. Eucalyptus globulus (camphor oil) against the zoonotic scabies, Sarcoptes scabiei. J Egypt Soc Parasit 2003; 33: 47–53 10 Sudha P, Smita SZ, Shobha YB et al. Potent α-amylase inhibitory activity of Indian Ayurvedic medicinal plants. BMC Compl Alt Med 2011; 11: 5 11 Mukherjee M. Human digestive and metabolic lipases – a brief review. J Mol Catal B: Enzym 2003; 22: 369–376 12 Marudamuthu AS. Leelavinothan P. Effect of pterostilbene on lipids and lipid profiles in Streptozotocin – Nicotinamide induced type 2 diabetes mellitus. J Appl Biomed 2008; 6: 31–37 13 Kumar J, Sharma S, Kumar S. In-vivo anti-diabetic and antioxidant potential of Psoralea corylifolia seeds in STZ induced type-2 diabetic rats. J health sci 2011; 57: 225–235 14 Kumar S, Vasudena N, Sharma S. GC-MS analysis and screening of antidiabetic, antioxidant and hypolipidemic potential of Cinnamomum tamala oil in streptozotocin induced diabetes mellitus in rats. Cardiovas Diabetol 2012; 11: 95 15 Sedlak J, Lindsay RH. Estimation of total protein bound and non protein bound sulfhydryl group in tissue with ellman’s reagent. Anal Biochem 1968; 15: 192–205 16 Scifter S, Dayton S, Molic B et al. The estimation of glycogen with the anthrone reagent. In Archive Biochem 1950; 25: 191 17 Kumar S, Singh R, Vasudeva N et al. Acute and chronic animal models for the evaluation of anti-diabetic agents. Cardiovas Diabetol 2012; 11: 9 18 Xiao Z, Storms R, Tsang A. A quantitative starch-iodine method for measuring alpha-amylase and glucoamylase activities. Anal Biochem 2006; 351: 146–148 19 Yoshikawa M, Shimoda H, Nishida N et al. Salacia reticulata and its polyphenolic constituents with lipase inhibitory and lipolytic activities have mild anti-obesity effects in rats. J Nutr 2002; 132: 1819–1824 20 Han LK, Kimura Y, Okuda H. Reduction in fat storage during chitinchitosan treatment in mice fed a high-fat diet. Int J Obes 1999; 23: 174–179 21 Egesie UG, Adelaiye AB, Ibu JO et al. Safety and hypoglycemic properties of aqueous leave extract of Ocimum gratissium in streptozotocin induced diabetic rabbits Nig J Physiol Sci 2006; 21: 31–35 22 Ahmed I, Cummings E, Adeghate E et al. Beneficial effects and mechanism of action of Momordica charantia fruit juice in the treatment of streptozotocin-induced diabetes mellitus in rats. Mol Cell Biochem 2004; 26: 63–70
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functions, oxidative damage to membranes and increased susceptibility to lipid peroxidation [28]. In this aim, we chose to work on a model of severe experimental diabetes. The ability of those above doses to quench hydroxyl radicals seem to be directly related to the prevention of propagation of the process of lipid peroxidation. It is known that hydroperoxide are primary products of lipid-peroxidation, the lowering effect of lipid hydroperoxide level may be due to an antioxidant activity of safrole. Many plants like mulberry fruit extracts shown their ability effect to scavenge hydroxy radical [29]. In this study the level of hydroperoxide in treated extract groups may be related to scavenging lipid hydroperoxide radical’s activity of one or more of its components, suggesting its potent antilipid peroxidative like previously observed by [30]. In conclusion, the present study shown that safrole possess potent antioxidant activity, which may be directly or indirectly responsible for the hypoglycaemic property. These potent antioxidant properties may contribute towards preventing peroxidative damage. α–amylase is an endo-acting enzymes which catalyzes the hydrolysis of the 1–4-α-glycosidic linkages of starch, amylose, amylopectins, glycogen and various maltodextrins. Natural α-amylase inhibitors from herbal sources offer an attractive therapeutic approach to the treatment of postprandial hyperglycemia by decreasing glucose release from starch and may have potential for use in the treatment of diabetes mellitus and obesity [31]. Many herbal extracts has been reported for their anti-diabetic activities and being used in ayurveda for the treatment of diabetes. Therefore, screening of the enzymes in plant has received more attention. In in-vivo study, the safrole shown anti-diabetic activity, for conformation the in-vitro study is also carried out in this experiment. The medicinal plants or natural products involve retarding the absorption of glucose by inhibiting the carbohydrate hydrolyzing enzymes. In this study, in vitro effect of different concentrations of safrole was evaluated. Amylase inhibition is expressed by the concentration of extract residues that inhibits 50 % of the enzymatic activity (IC50value). In in-vitro studies demonstrate an appreciable α-amylase inhibitory activity of safrole with IC50 values of 702.78 μg/ml. The present study indicated that safrole could be useful in management of postprandial hyperglycemia. Pancreatic Lipase (PL), the principal lipolytic enzyme synthesized and secreted by the pancreas, responsible for the digestion of triacylglycerols into mono- and di-acylglycerols and fatty acids to be absorbed by the body. Researchers and health professionals believe that the inhibition of PL can reduce digestion of fats, hence, their assimilation and absorption. This can mimic a reduced calorie intake in obese patients and help in preventing additional weight gain. PL is responsible for the hydrolysis of 50–70 % of total dietary fats. PL inhibition is one of the most widely studied mechanisms for the determination of the potential efficacy of natural products as anti-obesity agents [32]. Our in vitro studies demonstrate a lipase inhibitory activity of safrole with IC50 values of 861.35 μg/ml. There is a positive correlation between inhibitory activity of safrole and concentrations in enzyme assay medium. The result strongly suggests that safrole decrease the glucose level by inhibiting lipase activity. From present investigation we conclude that safrole has significant inhibitory activity against amylase, lipase which might be helpful in preventing and suppressing the progress of various disorders associated with diabetes mellitus.
Original Article 295
28 Giugliano D, Ceriello A, Paolisso G. Oxidative stress and diabetic vascular level and the P.E. Fraction showed a moderate increase in GSHcomplications. Diab Car 1996; 19: 257–267 29 Song-Hwan B, Hyung-Joo S. Antioxidant activities of five different mulberry cultivars in Korea. LWT 2007; 40: 955–962 30 Pavana P, Sethupathy S, Santa K et al. Effects of Tephrosia purpurea aqueous seed extract on blood glucose and antioxidant enzyme activities in streptozotocin induced diabetic rats. Afr J Trad CAM 2009; 6: 78–86 31 Gallaher D, Schneeman BO. Nutritional and metabolic response to plant inhibitors of digestive enzymes. Adv Exp Med Biol 1986; 199: 167–184 32 Yasser B, Mohammad M, Mohammad H et al. Screening of Some Medicinal Plants for their Pancreatic Lipase Inhibitory Potential. Jord J Pharm Sci 2011; 4: 81
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23 Sarkar S, Pranava M, Manita R. Demonstration of the hypoglycemic action of Momordica charantia in a validated animal model of diabetes. Pharmacological Research 2007; 33: 1–4 24 Karunanayake EH, Jeevathayaparan S, Tennekoon KH. Effect of Momordica charantia fruit juice on streptozotocin-induced diabetes in rats. J Ethnopharmacol 1990; 30: 199–204 25 Bailey CJ, Day C, Turner SL et al. Cerasee, a traditional treatment for diabetes. Studies in normal and streptozotocin diabetic mice. Diabetes Res 1985; 2: 81–84 26 Betteridge J. Lipid disorders in diabetes mellitus. In: Pickup JC, Williams G (eds.). Text Book of Diabetes. Blackwell Science Publishers, London: 1997; 1–35 27 Taskinen M. Quantitative and qualitative lipoprotein abnormalities in diabetes mellitus. Diabetes 1992; 41: 12–17
Rani S et al. Antihyperglycemic Potential of Safrole … Drug Res 2014; 64: 287–295
Authors
S. Rani, S. Sharma, S. Kumar
Affiliation
Division of Pharmacology, Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology, Haryana, India
Key words ▶ antihyperglycemic activity ● ▶ streptozotocin ● ▶ safrole ● ▶ lipase ● ▶ amylase ●
Abstract
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In the present investigation anti-diabetic and in-vitro antioxidant potential of safrole were evaluated (100 and 200 mg/kg p.o.) in acute and chronic Streptozotocin-nicotinamide (STZ) induced antihyperglycemic rat model. The oral administration of safrole for 30 days affects the level of blood glucose, glycosylated hemoglobin (HbA1C), total cholesterol (TC), triglycerides (TG), phospholipids, high density lipoprotein (HDL), body weight, insulin level, liver glycogen content, antioxidant parameters, lipase,
Introduction
▼ received 12.09.2013 accepted 22.09.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1357192 Published online: October 16, 2013 Drug Res 2014; 64: 287–295 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence S. Sharma Division of Pharmacology Department of Pharmaceutical Sciences Guru Jambheshwar University of Science and Technology Hisar Haryana-125001 India Tel.: + 91/941/6107 896 Fax: + 91/1662/278 181 sharmask71@rediffmail.com
Diabetes mellitus is a group of metabolic disorder characterized by hyperglycaemia due to defect in insulin secretion or action or both. In this condition the β cells of the pancreas being unable to produce sufficient insulin to prevent hyperglycemia [1]. It is associated with significant changes in the plasma lipid and lipoprotein profile thereby an increased risk of premature atherosclerosis, coronary insufficiency and myocardial infarction exists [2]. Oxidative stress plays a major role in the pathogenesis and development of complications of both types of diabetes mellitus. In diabetes mellitus oxygen free radicals also increases in body which are responsible for oxidative stress. There was 285 million people living with diabetes in 2010, which are increased to 366 million in 2011 and this is expected to reach 552 million by 2030, an increase of 50.7 %. In India, 61.3 million people had diabetes in 2011 and expected that figure is projected to rise to 101.2 million by 2030. There are lots of constituents upon which antidiabetic work is carried out and give the positive response towards the anti-diabetic research. Safrole is a chemical components of camphor oil which also contains a-pinene, camphene,
α-amylase in normal and STZ induced diabetic rats. The oral administration of safrole at dose 100 & 200 mg/kg p.o. significantly improve the diabetic condition in Streptozotocin-induced diabetic rats. In enzymatic assay, the IC50 value of the safrole for α-amylase and lipase was found to be 702.78 and 861.35 μg/ml respectively which was found comparable with the standard drug (ascorbic acid) as 252.12 μg/ml. Further studies can be performed on safrole for mechanistic and toxicological aspects so that it can be investigated as a new substance for the management of various diseases.
b-pinene, sabinene, phellandrene, limonene, 1, 8-cineole, y-terpinene, p-cymene, terpinolene, furfural, camphor, linalool, bornyl acetate, terpinen-4-ol, caryophyllene, borneol, piperitone, geraniol, cinnamaldehyde, methyl cinnamate and eugenol. The brown and yellow camphor oil has very high safrole content (10–80 %). Camphor, a natural product derived from the wood of the tree Cinnamomum camphora, has a long history of use as: analgesic [3], respiratory relief, inhalants [4], nasal decongestant, expectorant [5], effect on liver [6], in tumor [7], contraceptive & sexual performance [8], scabies [9]. The prolonged administration of safrole for a period of 82 weeks in 7–28 days old animals (rats and mice) caused liver tumors in male mice and malignant liver tumors (hepatocellular carcinoma or adenoma or cholangiocarcinoma) in rats of both sexes. Enzymes also play a major role in the treatment of diabetes mellitus as enzymes hydrolyzes starch molecules to give diverse products including dextrin and progressively smaller polymers composed of glucose units within the small intestine which causes hyperglycemia and development of type 2 diabetes mellitus. The inhibition of α-amylase activity is possibility to lower postprandial blood glucose levels [10]. The enzyme
Rani S et al. Antihyperglycemic Potential of Safrole … Drug Res 2014; 64: 287–295
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To Investigate Antihyperglycemic and Antihyperlipidemic Potential of Safrole in Rodents by in-vivo and in-vitro Study
288 Original Article
Experimental Design
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Drugs and chemicals Glibenclamide (Torrent Pharmaceutical, Ahemadabad), streptozotocin, heparin (SRL, India), safrole, orlistat, lipase (Sigma Aldrich, USA), EDTA, soluble starch, iodine, α-amylase, sodium cholate, triolein, lecithin, TES buffer (Hi-media Lab. Pvt Ltd., Mumbai), n-butanol, acetic acid, n-hexane, petroleum ether, ethyl acetate, glucose standard, citric acid, sodium citrate, tris hydrochloride, buffer tablet, sodium lauryl sulphate, thiobarbituric acid, trichloroacetic acid, triton-X, glycogen, ethanol, tween 80, carboxy methyl cellulose, Ellman’s reagent (5,5’- dithiobis(2-nitro-benzoic acid); DTNB), sodium sulphate, methanol, pyridine, anthrone, thiourea, benzoic acid, sodium chloride (SD Fine Chem. Ltd., Mumbai). Safrole was separately dissolved in 1 % of CMC (carboxy methyl cellulose) solution. Volume of oral dosing was 1.0 ml/100 g of animal.
Experimental animals Albino wistar rats (weight 150–200 g) were procured from disease free small animal house, lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar (Haryana). The rats were housed in cages (polycarbonate cage size: 29 × 22 × 14 cm) under laboratory conditions with alternating light and dark cycle of 12 h each. The animals had free access to food and water. The animals were kept fasted 2 h before and 2 h after drug administration. The experimental protocol was approved by institutional animals ethics committee (IAEC) and animal care was done as per the guidelines of committee for the purpose of control and supervision of experiments on animals (CPCSEA), govt. of India (registration no. 0436).
Induction of experimental diabetes using streptozotocin-nicotinamide The animal model of type-2 diabetes mellitus (NIDDM) was induced in overnight fasted animals by a single intraperitoneal injection of STZ (45 mg/kg) followed by nicotinamide injection (110 mg/kg). The nicotinamide injection was given to rats 15 min prior to the STZ injection. Hyperglycemia was confirmed by the elevated blood glucose levels determined at 72 h then on 7th day of the injection. Only rats confirmed with permanent NIDDM were used in the anti-diabetic study [12].
Groups for streptozotocin-nicotinamide induced diabetic rat model Total no. of animals was divided into 9 groups and each group contains 6 animals. Rani S et al. Antihyperglycemic Potential of Safrole … Drug Res 2014; 64: 287–295
Group I: Normal control rat. Group II: Diabetic control: Animals were administered vehicle only. Group III: Diabetic animals were administered with glibenclamide (600 μg/kg, orally). Group VI: Diabetic animals were administered with safrole (100 mg/kg, orally). Group V: Diabetic animals were administered with safrole (200 mg/kg, orally). Group VI: Normal animals were administered with safrole (200 mg/kg, orally).
Glucose tolerance test After 29 days treatments, on 30th day a fasting blood sample was collected from all the groups in heparinized micro-centrifuge tube from retro-orbital plexus. Blood samples were also collected at the time interval of 0, 30, 60, 90, and 120 min after administration of glucose at a concentration of 2 g/kg of body weight.
Biochemical assay The fasted animals were sacrificed by cervical decapitation on 30th day of first treatment. Trunk blood was collected in heparinised micro centrifuged tubes and the plasma obtained by centrifugation at 5 000 rpm for 5 min was used for the determination of plasma glucose, cholesterol, malondialdehyde (MDA), reduced glutathione. While whole blood was used for glycosylated hemoglobin.
Estimation of serum glucose and cholesterol Plasma cholesterol and glucose level were measured by commercial supplied biological kit Erba Glucose Kit (GOD-POD Method) and Erba Cholesterol Kit (CHOD-PAP Method) respectively using Chem. 5 Plus- V2 Auto-analyzer (Erba Mannheim, Germany) in plasma sample prepared as above. Glucose and cholesterol values were calculated as mg/dl blood sample.
Estimation of glycosylated hemoglobin (Hb1ac) Glycosylated hemoglobin was measured using commercial supplied biological kit (Erba Diagnostic) in plasma sample prepared as above using Chem. 5 Plus-V2 Auto-analyzer (Erba Mannheim, Germany). Values are expressed as the percent of total hemoglobin.
Estimation of insulin The serum insulin level was measured by an enzyme-linked immuno-sorbent assay (ELISA) procedure using Mercodia rat insulin ELISA kit. In short, the solid phase 2-site enzyme immunoassay is based on the direct sandwich technique in which 2 monoclonal antibodies are directed against separate antigenic determinants 35 (epitopes) on the insulin molecule. During incubation, insulin in the sample reacts with peroxidase-conjugated anti-insulin antibodies and anti-insulin antibodies bound to the micro titration well. After washing 3 times, unbound enzyme labeled antibody was removed. The bound conjugated insulin was detected by reacting with 3, 3′, 5, 5′-tetramethylbenzidine. The reaction was stopped by adding acid to give a colorimetric end-point, and optical density was measured with a micro plate auto reader (Bio-tek Instrument Inc., USA) at a wavelength of 450 nm. The serum insulin is expressed as μg/l [13].
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lipase that travels all the way down to the intestines promotes breakdown of fats like cholesterol and triglycerides to give di and mono glycerides, glycerol and free fatty acids from ingested food. Researchers and health professionals believe that the inhibition of PL can reduce digestion of fats, hence, their assimilation and absorption. This can mimic a reduced calorie intake in obese patients and help in preventing additional weight gain [11]. The present study was designed to investigate the in-vitro and invivo antihyperglycaemic properties of safrole in normoglycaemic and streptozotocin-induced diabetic rats as this model represent a good resemblance human for type-2 diabetes and show many of the features of the human disease, including hyperphagia, hyperglycaemia, insulin resistance and progressive obesity.
Original Article 289
Malondialdehyde (MDA), an index of free radical generation/ lipid peroxidation, was determined as described by Okhawa et al., (1979). The reaction mixture consisted of 0.2 ml of 8.1 % sodium lauryl sulphate, 1.5 ml of 20 % acetic acid (ph 3.5) and 1.5 ml of 0.8 % aqueous solution of thiobarbituric acid. This reaction mixture was added to 0.2 ml of blood plasma. This mixture was made up to 4.0 ml with distilled water and cooling the contents under running tap water. Then 5.0 ml of n-butanol, pyridine (15:1 v/v) and 1.0 ml of distilled water added to the above mixture. After that centrifuged it at about 3 000 rpm for 10 min. Organic layer was separated out and absorbance was measured at 532 nm using double beam UV-visible spectrophotometer (systronics 2203, Bangalore, India) against a blank. MDA values are calculated using the extinction coefficient of MDA-thiobarbituric acid complex 1.56 × 105 l/mol × cm and expressed as N mol/ mg protein [14].
Estimation of plasma reduced glutathione level Took 2 ml of distilled water and 0.5 ml of trichloroacetic acid in a 10 ml test tube Added this solution into 2.5 ml of blood plasma. The tubes were shaken intermittently for 10–15 min and centrifuged it for 15 min at approximately 3 000 rpm. Supernatant was added with 2 ml of 0.4 m tris-EDTA buffer (ph 8.9) and 0.1 ml DTNB and mixed well. At last absorbance was read within 5 min of the addition of DTNB at 412 nm against a regent blank [15].
Estimation of liver glycogen content Liver glycogen estimation was done by the method as described by [16]. Immediately after excision from the animal, 1 g of the liver was dropped into a previously weighed test tube containing 3 ml of 30 % potassium hydroxide solution. Weight out the liver and tissue was then digested by heating the tube for 20 min in boiling water bath. Then digest tissue was cooled and transferred quantitatively to a 50 ml volumetric flask, Diluted it to the mark with water. Contents of the flask were then thoroughly mixed; a measured portion was then further diluted with water in a second volumetric flask so as to yield a solution of glycogen of 3–30 μg/ml. 5 ml aliquots of the final dilution were then pipette into Evelyn tube and the determination with anthrone was carried out. The amount of glycogen in the aliquot used was then calculated using the following equation: μg of glycogen in aliquot = 100 U/1.11 S U is the optical density of unknown solution. S is the optical density of 100 μg glucose and 1.11 is the factor determined by Morris standard (Morris, 1948) for the conversion of the glucose to the glycogen [17].
soluble starch (500 mg) was dissolved in 25 mL of 0.4 M NaOH and heated for 5 min at 100 °C. After cooling in ice H2O, the solution was adjusted to pH 7 with 2 M HCl, and H2O was added to adjust the volume to 100 ml. Sample solutions were prepared by dissolving each sample in acetate buffer (pH 6.5) to make different concentrations (50–500 μg/ml). The substrate (40 μL) and sample (20 μL) solutions were mixed in a micro-plate well, and the mixtures were pre-incubated at 37 °C for 3 min. Then 20 μL of α-amylase solution (50 μg/ml) was added to each well, and the plate was incubated for 15 min. The reaction was terminated by addition of 80 μL of 0.1 M HCl; then 200 μL of 1 mM iodine solution was added, and the absorbance was measured at 650 nm. Inhibitory activity ( %) was calculated as follows: % inhibition = {1− (absorbance of sample and substrate – absorbance of sample, substrate, and lipase)/(absorbance of substrate – absorbance of substrate and lipase) × 100} IC50 value was obtained by interpolation of graph between concentrations of tested or standard drugs vs. % enzyme inhibition curves. The concentration of an inhibitor required to inhibit 50 % of enzyme activity under the mentioned assay conditions is defined as the IC50 value.
Measurement of pancreatic lipase inhibitory activity Lipase inhibitory activity was evaluated according to the method of Han et al. [20] with slight modifications. Orlistat, a specific pancreatic lipase inhibitor used in the prevention of obesity clinically was used as a positive control for the comparison of tested oils. Substrate solution was prepared by sonication (10 min in an ice bath) of a mixture of glyceryl trioleate (80 mg), lecithin (10 mg), and sodium cholate (5 mg) suspended in 9 mL of 0.1MTES buffer (pH 7.0). Samples were separately dissolved in 0.1 M TES buffer to make 0.2 mg/mL solutions. The substrate (20 μL) and sample solutions (20 μL) in microplate wells were pre-incubated for 3 min; then 10 μL of lipase solution (20 μg/mL) was added to each reaction mixture and incubated for 30 min at 37 °C. The amount of released fatty acid was measured by a LISA Plus micro-plate reader at 550 nm. Inhibitory activity ( %) was calculated as follows: % Inhibition = {1− (absorbance of sample and substrate – absorbance of sample, substrate, and lipase)/(absorbance of substrate – absorbance of substrate and lipase) × 100} IC50 value was obtained by interpolation of graph between concentrations of tested or standard drugs vs. % enzyme inhibition curves. The concentration of an inhibitor required to inhibit 50 % of enzyme activity under the mentioned assay conditions is defined as the IC50 value.
Data analysis Histopathological studies For the histopathological studies, rats were scarified under light ether anesthesia 24 h after the last dosage and the pancreas were removed and washed with normal saline. The pancreas tissue were fixed in 10 % formalin solution, dehydrated with a sequence of ethanol solution and then embedded in paraffin. Sections of pancreas of 5–6 micrometer in thickness were cut, stained with hematoxylin and eosin and then subjected to photo microscopic observation (Digital microphotography Olympus CX41).
Measurement of α-amylase inhibitory activity The α-amylase activity was measured applying the method reported by [18, 19] with little changes. Acarbose was used as the positive control. Substrate solution was prepared as follows:
All the results were expressed as mean ± standard error of mean (SEM). The data of all the groups were analyzed using one-way ANOVA followed by Dunnett’s t-test, the criterion for statistical significance was p < 0.01 as compare to diabetic control.
Results
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Acute hypoglycemic effect of safrole on normoglycaemic rats The effect of the treatment with safrole on the serum glucose in ▶ Fig. 1. The safrole treated normal fasted rats is shown in ● groups at 100 mg/kg and 200 mg/kg p.o. shown a significant decrease in serum glucose level as compared to untreated group. Rani S et al. Antihyperglycemic Potential of Safrole … Drug Res 2014; 64: 287–295
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Estimation of MDA level
290 Original Article Effect of safrole on total cholesterol ▶ Fig. 5 indicated the effect of safrole on The data presented in ●
The safrole treated group shown a marked fall in glucose level in 30–90 min interval. After 90 min the serum glucose level returns to normal value (p < 0.01).
total plasma cholesterol. Total plasma cholesterol was observed higher in diabetic rats compared to normal rats. The administration of the control drugs significantly lowered the total plasma cholesterol concentration as compare to diabetic control. The administration of safrole 100 mg/kg, p.o. and safrole 200 mg/kg, p.o. shows significant (p < 0.01) reduction in total plasma cholesterol level as compare to diabetic control on 10th (acute study) as well as 30th day (chronic study).
Chronic antihyperglycemic effect of safrole in STZ induced diabetic rats Safrole 100 mg/kg p.o. give to STZ diabetic rats for 30th day (chronic study) shown a fall in serum glucose level from 378.94 mg/kg 261.61 mg/kg at 30th day when compared to 1st day value. At safrole 200 mg/kg p.o. to STZ diabetic rats for 30th day shown a fall in serum glucose level from 368.81 mg/kg 155.26 mg/kg at 30th day when compared to 1st day value ▶ Table 1) (● ▶ Fig. 2). (p < 0.01) (●
Effect of safrole on total HDL level ▶ Fig. 6 indicated the effect of safrole on The data presented in ●
The effect of safrole on plasma glucose level after glucose loading at 2 g/kg orally to the normal, diabetic and test animals was ▶ Fig. 3. The blood glucose level rises to a maxexpressed in the ● imum in 30 min after glucose loading. The safrole 100 mg/kg and 200 mg/kg had shown a significant increase in rate of clearance of glucose as compared to untreated group. The safrole treated group shown a marked fall in glucose level in 30, 60, 90, 120 min interval. After 120 min the serum glucose level returns to normal value (p < 0.01).
Effect of safrole on total Triglycerides level ▶ Fig. 7 indicates the effect of safrole on total The data shown in ● plasma triglycerides level. Total plasma triglycerides were observed higher in diabetic rats compared to normal rats. The administration of the control drugs significantly reduced the total plasma cholesterol concentration as compare to diabetic control. The administration of safrole 100 mg/kg, p.o. and 200 mg/kg, p.o. shown significant (p < 0.01) reduction in total plasma triglycerides level as compare to diabetic control on 10th (acute study) as well as 30th day (chronic study).
Effect of safrole on glycosylated hemoglobin ▶ Fig. 4 indicated the effect of safrole on The data depicted in ● glycosylated hemoglobin (HbA1c). The HbA1c data for the STZ diabetic group were higher than the normal rats. The chronic administration of the standard drug significantly reduced the HbA1c level in 30 days. The administration of safrole reduces the HbA1c level as compared to the diabetic control. Higher doses produced more reduction HbA1c level indicate dose dependent effect of safrole in diabetic rats (p < 0.01).
Fig. 1 Acute hypoglycaemic effect of safrole on normoglycaemic rats. Values are presented as mean ± S.E.M.; n = 6 in each group. One way ANOVA followed by Dunnett’s test *p < 0.05; **p < 0.01; as compare to control.
Fig. 2 Chronic antihyperglycaemic effect of safrole in STZ induced diabetic rats. Values are presented as mean ± S.E.M.; n = 6 in each group. One way ANOVA followed by Dunnett’s test *p < 0.05; **p < 0.01; as compare to control.
Table 1 Chronic antihyperglycemic effect of safrole in STZ induced diabetic rats. Treatment
Dose
Mean blood glucose concentration (mg/dl) ± S.E.M 1st Day
Normal Diabetic control Glibenclamide Safrole safrole
600 μg/kg 100 mg/kg 200 mg/kg
92.65 ± 1.57 381.14 ± 5.29 304.79 ± 4.58** 378.94 ± 4.25 368.81 ± 3.13
10th Day 97.33 ± 33 369.98 ± 3.94 243.47 ± 5.01** 353.80 ± 2.78 352.50 ± 3.19*
20th Day 96.24 ± 4.03 376.63 ± 3.45 181.10 ± 3.45** 316.29 ± 2.63* 258.67 ± 4.93**
Values are presented as mean ± S.E.M. ; n = 6 in each group. One way ANOVA followed by Dunnett’s test *p < 0.05; **p < 0.01; as compare to control
Rani S et al. Antihyperglycemic Potential of Safrole … Drug Res 2014; 64: 287–295
30th Day 93.11 ± 2.27 366.12 ± 3.15 107.39 ± 3.15** 261.61 ± 2.68* 155.26 ± 3.72**
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total plasma HDL. Total plasma HDL was observed low in diabetic rats compared to normal rats. The administration of the safrole significantly enhances the total plasma HDL concentration as compare to diabetic control. The administration of safrole 100 mg/kg, p.o. and 200 mg/kg, p.o. shown significant (p < 0.01) enhancement in total plasma HDL level as compare to diabetic control on 10th as (acute study) well as 30th day (chronic study). Higher doses increase more HDL level.
Effect of safrole in glucose loaded hyperglycaemic rats
Original Article 291
Fig. 4 Effect of safrole on glycosylated hemoglobin. Values are presented as mean ± S.E.M.; n = 6 in each group. One way ANOVA followed by Dunnett’s test **p < 0.01; as compare to control.
Fig. 5 Effect of safrole on total cholesterol. Values are presented as mean ± S.E.M. ; n = 6 in each group. One way ANOVA followed by Dunnett’s test *p < 0.05; **p < 0.01; as compare to control.
Effect of Safrole on Glutathione Level The plasma glutathione (GSH) level was decreased in STZ rats ▶ Fig. 8. The level of compared to normal animals as shown in ● glutathione was returned to near normal range in STZ diabetic rats treated with glibenclamide and it also shown significant (p < 0.01) enhancement by the safrole 100 mg/kg, p.o. and 200 mg/kg, p.o.
Effect of safrole on antioxidant parameter
Fig. 6 Effect of safrole on total HDL level. Values are presented as mean ± S.E.M. ; n = 6 in each group. One way ANOVA followed by Dunnett’s test *p < 0.05; **p < 0.01; as compare to control.
Fig. 7 Effect of safrole on total triglycerides level. Values are presented as mean ± S.E.M. ; n = 6 in each group. One way ANOVA followed by Dunnett’s test **p < 0.01; as compare to control.
Fig. 8 Effect of safrole on glutathione level. Values are presented as mean ± S.E.M. n = 6 in each group. One way ANOVA followed by Dunnett’s test *p < 0.05; **p < 0.01; as compare to control.
pared to normal rats. Treatment of STZ diabetic rats with safrole resulted in a marked decrease in plasma level of MDA.
Effects of safrole on liver glycogen content The liver glycogen level was found to be low in STZ rats when ▶ Fig. 10. Safrole compared to normal rats as data reflected in ● 100 mg/kg p.o. and 200 mg/kg p.o. increased the liver glycogen content significantly when compared diabetic control rats ▶ Fig. 10. (p < 0.01) ●
The plasma level of malondialdehyde (MDA) of normal and ▶ Fig. 9. Plasma experimental animal in each group is shown in ● MDA level in plasma was increased in STZ diabetic rats comRani S et al. Antihyperglycemic Potential of Safrole … Drug Res 2014; 64: 287–295
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Fig. 3 Effect of safrole in glucose loaded hyperglycaemic rats. Values are presented as mean ± S.E.M.; n = 6 in each group. One way ANOVA followed by Dunnett’s test *p < 0.05; **p < 0.01; as compare to control.
292 Original Article Effects on insulin ▶ Table 2 shows the level of plasma insulin in the control and ● experimental groups of rats. Diabetic rats shown a decrease in plasma insulin compared with normal rats. Oral administration of safrole 100 mg/kg/day and 200 mg/kg/day respectively had shown a significant increase in plasma insulin level in respective ▶ Fig. 11). groups as compared to control rats (●
IC50 value obtained for these enzyme inhibitions are shown ▶ Table 3. in ●
Histopathological study ▶ Fig. 14. Histopathological changes of pancreas are given in ● Haematoxylin & eosin stained tissue sections of control animals’
Effects on body weight An increase in the body weight of normal rats was observed whereas the weight of diabetic control rats decreased from day 1 to day 30. Safrole 100 and 200 mg/kg p.o. when administered to respective groups of diabetic rats shown a significant increase in body weight as compared to the diabetic control group ▶ Table 2). (p < 0.01) (●
In-vitro study Enzymes inhibitory activity of safrole Fig. 11 Effect of safrole on insulin level.
Fig. 9 Effect of safrole on antioxidant parameter. Values are presented as mean ± S.E.M. ; n = 6 in each group. One way ANOVA followed by Dunnett’s test *p < 0.05; **p < 0.01; as compare to control.
Fig. 12 Enzymes inhibitory activity of safrole.
Fig. 10 Effects of safrole on liver glycogen content. Values are presented as mean ± S.E.M. ; n = 6 in each group. One way ANOVA followed by Dunnett’s test *p < 0.05; **p < 0.01; as compare to control.
Fig. 13 Enzymes inhibitory activity of safrole.
Table 2 Effect of safrole on body weight and insulin level. Treatment Normal Diabetic control Glibenclamide Safrole safrole
Dose (mg/kg p.o.)
Initial body weight (g)
Final body weight (g)
Change in weight (g)
Insulin micro U/ml
600 μg/kg 100 mg/kg 200 mg/kg
217.66 221.79 215.77 219.27 225.70
235.73 193.31 239.02 230.15 243.50
18.07 − 28.48 23.25** 10.88** 17.8**
18.23 ± 0.93 8.48 ± 0.82 17.35 ± 0.9 12.16 ± 0.81 14.07 ± 0.6
Values are presented as mean ± S.E.M.; n = 6 in each group. One way ANOVA followed by Dunnett’s test *p < 0.05; **p < 0.01; as compare to control
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The enzyme inhibitory activity of safrole against α-amylase & lipase were determined by using different concentration 200 μg/ ▶ Fig. 12, 13. The ml 400 μg/ml 800 μg/ml and 1 600 μg/ml as in ●
shows normal islets in pancreas histology throughout the study ▶ Fig. 14a). In contrast, STZ administration elicited severe (● injury of pancreas, such as decreasing of the islet cells’ numbers, diminishing of the diameter of pancreatic island, necrosis, degeneration and congestion in the islet of Langerhans cells. The islets were shrunken in diabetic rats when compared with nor▶ Fig. 14b). Treatment given to diabetic rats with the mal rats (● safrole 100 mg/kg shown significant expansion of islets. Further, there was significant reductions in the injuries of pancreas have also been observed but slight vaculation and degeneration was ▶ Fig. 14c, d, glibenclamide (25 mg/kg) shown moderpresent. ● ate expansion of islets and significantly reduced the injuries of ▶ Fig. 14e). These results shown that safrole could be pancreas (● useful in repairing β-cells in pancreatic islet injury. The dosedependent protective effect would be closely related with its improving function.
Discussion
▼
The aim of the present study was to evaluate the possible protective effects of safrole on serum glucose level and antioxidant defense systems of plasma in streptozotocin induced diabetes rats. The levels of glucose in blood, lipid hydroperoxide, glycogen content, body weight, insulin level GSH level and malonaldehyde were estimated in blood of control and experimental groups of streptozotocin (STZ) induced diabetic rats. In the present study, it was found that there was low level of feed intake as the rats shown poor appetite during the experiment and significant weight loss in diabetic animals (193.31 g) as compared to normal animals (235.73 g). The weight loss may be due to fluid depletion and accelerated breakdown of fats and
Table 3 Enzymes inhibitory activity of safrole. ENZYME ASSAY α-amylase Lipase
IC50 VALUE (μg/ml) Standard
Safrole
252.12 446.76
702.78 861.35
Values are presented as mean as 3 parallel measurements were conducted
adipose muscles [21]. Treatment with 100 mg/kg and 200 mg/ kg p.o. safrole significantly increases food and water intake of diabetic rats. This indicates that 100 mg/kg and 200 mg/kg safrole may improve characteristic symptoms of polyphasia & polydypsia of diabetes mellitus. It has been reported that diabetic STZ rats show significant glucose intolerance when orally administered with 2 g/kg of glucose and also have high insulin level. In the present study also diabetic animals were found to have impaired glucose tolerance with high glucose level after 1 h of glucose load compared to control animals. The results of this study have demonstrated that oral administration of 100 mg/kg and 200 mg/kg p.o. daily over duration of 30 days to STZ-induced type 2 diabetic rats can significantly improve the impaired glucose tolerance compare to diabetic control. These results are consistent with those reported earlier [22] and [23] wherein they have demonstrated the M. charantia may either have insulin-like secretagogue effect [24], it can stimulate peripheral glucose utilization or it may inhibit key gluconeogenic enzymes such as glucose-6-phosphatase and fructose biphosphatase [25]. Abnormalities in lipoproteins are very common in both NIDDM and IDDM. Diabetes leads to alterations in the plasma lipid and lipoprotein profile and increases risk of coronary heart disease [26]. In diabetic control hyper triglyceridemia, increased level of blood cholesterol and low HDL-cholesterol levels were seen when compared with normal control group [27]. The increase in blood cholesterol and triglycerides is due to the action of hormone sensitive lipase, which promotes lipolysis and subsequently increases the level of plasma free fatty acids and triglycerides. These free fatty- acids are catabolized to acetyl CoA which is further channeled to cholesterol synthesis, thus increasing blood cholesterol level. In the present investigation, serum cholesterol and triglyceride levels of diabetic rats were found to be significantly decreased by the treatment with safrole. We have also observed that in STZ induced diabetic rats; the level of HDL-cholesterol was significantly lower. 100 mg/kg and 200 mg/kg p.o. safrole normalized these effects, possibly by controlling the hydrolysis of certain lipoproteins and their selective uptake and metabolism by different tissues. The results of safrole highlight the topic for research and discussion for dose dependent effects of safrole. Hyperglycemia induces the generation of free radicals which can affect antioxidant defenses thus leading to the disruption of cellular Fig. 14 a Pancreas of Control animal shows normal histology. b Pancreas of Diabetic Control animal expressing severe necrosis, degeneration, congestion of islets cell. c Pancreas of Diabetic treated with 25 mg/kg of glibenclamide shows histology with large amount of islets cells as compare to diabetic rats. d Animal treated with 100 mg/kg of safrole shows mild regeneration of islets cell and congestion and vaculation are also present cells. c Pancreas of Diabetic treated with safrole (200 mg/kg) rats indicates repairing of islet of Langerhans but vaculation and degeneration are also present.
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Original Article 293
294 Original Article
Rani S et al. Antihyperglycemic Potential of Safrole … Drug Res 2014; 64: 287–295
Acknowledgement
▼
The authors are highly grateful to the All India Council for Technical Education, New Delhi (India) for providing research fellowship during research work. The authors have no conflict of interest.
Conflict of Interest
▼
The authors declare that they have no conflict of interests.
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functions, oxidative damage to membranes and increased susceptibility to lipid peroxidation [28]. In this aim, we chose to work on a model of severe experimental diabetes. The ability of those above doses to quench hydroxyl radicals seem to be directly related to the prevention of propagation of the process of lipid peroxidation. It is known that hydroperoxide are primary products of lipid-peroxidation, the lowering effect of lipid hydroperoxide level may be due to an antioxidant activity of safrole. Many plants like mulberry fruit extracts shown their ability effect to scavenge hydroxy radical [29]. In this study the level of hydroperoxide in treated extract groups may be related to scavenging lipid hydroperoxide radical’s activity of one or more of its components, suggesting its potent antilipid peroxidative like previously observed by [30]. In conclusion, the present study shown that safrole possess potent antioxidant activity, which may be directly or indirectly responsible for the hypoglycaemic property. These potent antioxidant properties may contribute towards preventing peroxidative damage. α–amylase is an endo-acting enzymes which catalyzes the hydrolysis of the 1–4-α-glycosidic linkages of starch, amylose, amylopectins, glycogen and various maltodextrins. Natural α-amylase inhibitors from herbal sources offer an attractive therapeutic approach to the treatment of postprandial hyperglycemia by decreasing glucose release from starch and may have potential for use in the treatment of diabetes mellitus and obesity [31]. Many herbal extracts has been reported for their anti-diabetic activities and being used in ayurveda for the treatment of diabetes. Therefore, screening of the enzymes in plant has received more attention. In in-vivo study, the safrole shown anti-diabetic activity, for conformation the in-vitro study is also carried out in this experiment. The medicinal plants or natural products involve retarding the absorption of glucose by inhibiting the carbohydrate hydrolyzing enzymes. In this study, in vitro effect of different concentrations of safrole was evaluated. Amylase inhibition is expressed by the concentration of extract residues that inhibits 50 % of the enzymatic activity (IC50value). In in-vitro studies demonstrate an appreciable α-amylase inhibitory activity of safrole with IC50 values of 702.78 μg/ml. The present study indicated that safrole could be useful in management of postprandial hyperglycemia. Pancreatic Lipase (PL), the principal lipolytic enzyme synthesized and secreted by the pancreas, responsible for the digestion of triacylglycerols into mono- and di-acylglycerols and fatty acids to be absorbed by the body. Researchers and health professionals believe that the inhibition of PL can reduce digestion of fats, hence, their assimilation and absorption. This can mimic a reduced calorie intake in obese patients and help in preventing additional weight gain. PL is responsible for the hydrolysis of 50–70 % of total dietary fats. PL inhibition is one of the most widely studied mechanisms for the determination of the potential efficacy of natural products as anti-obesity agents [32]. Our in vitro studies demonstrate a lipase inhibitory activity of safrole with IC50 values of 861.35 μg/ml. There is a positive correlation between inhibitory activity of safrole and concentrations in enzyme assay medium. The result strongly suggests that safrole decrease the glucose level by inhibiting lipase activity. From present investigation we conclude that safrole has significant inhibitory activity against amylase, lipase which might be helpful in preventing and suppressing the progress of various disorders associated with diabetes mellitus.
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