Introduction
Diabetes mellitus is one of the major global health and economic problem, characterized by high levels of blood glucose resulting from defects in insulin production, insulin action, or both. Diabetes has affected 6% of the world’s population [
1,
2]. Type II diabetes accounts for 90–95% of all diabetic cases [
2]. Long-term complications viz; cardiomyopathy, angiopathy, nephropathy etc. are a major cause of morbidity in patients with diabetes mellitus. Hyperlipidemia and oxidative stress frequently co-exist with diabetes mellitus [
3]. The increased blood glucose levels in diabetes produce superoxide anions, which generate hydroxyl radicals via Haber Weiss reaction, resulting in peroxidation of membrane lipids and protein glycation causing oxidative damage of cell membranes. These radicals further damage other important biomolecules including carbohydrates, proteins and deoxyribonucleic acid (DNA) [
4]. Antioxidants play an important role to protect the human body against damage caused by reactive oxygen species. Hence compounds with both hypoglycemic and antioxidant properties would be useful antidiabetic agent.
Some studies have suggested that essential oils may be useful in the treatment of insulin resistance and type II diabetes mellitus, and various oils have been used as therapeutic agents for years without any significant adverse health effects.
Cinnamomum tamala (Buch.-Ham.) Nees & Eberm (Tejpat) (Lauraceae), a volatile oil containing tree is commercially known as Indian cassia. It is used in traditional medicines as an astringent, stimulant, diuretic, carminative and in cardiac disorders [
5]. The leaves of
Cinnamomum tamala have been reported to possess antidiabetic, antioxidant [
6], antidiarrhoeal [
7], antihyperlipidemic [
8], antioxygenic [
9], anti-inflammatory [
10], acaricidal [
11], hepatoprotective [
12], gastroprotective [
13], antibacterial and immunomodulatory activities [
14]. The essential oil from
Cinnamomum species can be extracted easily by hydro distillation [
15]. The oil has been widely used as a flavoring agent and additives for centuries in the food industries. As far as we know, the effect of oil on the blood profiles in diabetic models has not been studied. In light of these findings, we carried out this study for the evaluation of antidiabetic, hypolipidemic and antioxidant potential of the CTO.
Materials and methods
Drugs and chemicals
The drugs and chemicals used in the study were glibenclamide (Torrent Pharmaceutical, Ahmadabad), streptozotocin, heparin (SRL, India), EDTA (Hi-media Lab. Pvt Ltd., Mumbai, India), 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, India). All the chemicals used in the study were of analytical grade.
Preparation of oil
The dried leaves of Cinnamomum tamala procured from local market of Hisar which were identified and authenticated by Dr. H. B. Singh, Head, Raw Materials Herbarium and Museum, National Institute of Science Communication and Information Resources (Ref. NISCAIR/RHMD/Consult/-2011-12/1858/158), Delhi (India). The leaves were cut in to small pieces and oil was extracted with the help of Clevenger apparatus. The percentage yield of the oil was found to be 0.45%.
Gas chromatography–mass spectrometry (GC-MS) analysis
The GC-MS analysis of the essential oil was performed using Agilent 7890A GC system equipped with MS detector 5975C inert XL EI/CI MSD having automatic sampler CTC analysis CombiPAL robotic arm. For GC/MS detection, an electron ionization system with ionization energy of 70 eV was used. Helium gas was used as the carrier gas at a constant flow rate of 1 ml/min. The inlet temperature was set at 270°C. The specification of the capillary column used was Agilent 19091S-433: 1548, 52849 HP-5MS 5% Phenyl Methyl Silox 30 m × 250 μm x 0.25 μm HP-5MS. The oven temperature was programmed from 80°C to 300°C. The diluted samples (1/100, v/v, in Hexane) of 2 μL were injected.
Identification of constituents
The relative percentage amount of each component was calculated by comparing its average peak area to the total areas. The oils components were identified by matching their recorded mass spectra with the data bank mass spectra (Search library Database/W9N08.L) and by comparing their retention indices relative to a series of n-hydrocarbons (C7–C23) with literature values [
16].
Experimental animals
Healthy male albino wistar rats (150–250 g, 60–90 days old) were procured from Disease Free Small Animal House, Chaudhary Charan Singh Haryana Agriculture University, Hisar (Haryana). The rats were housed in (Polycarbonate cage size: 29 × 22 × 14 cm) under laboratory standard conditions (25 ± 3°C:35–60% humidity) with alternating light and dark cycle of 12 h each and were feed fed with a standard rat pellet diet (Hindustan Lever Ltd, Mumbai, India) and water ad libitum. The experimental protocol was approved by Institutional Animals Ethics Committee (IAEC) and animal care was taken as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Govt. of India (Registration No. 0436).
Acute toxicity studies
Healthy adult albino wistar rats of both sex, starved overnight were divided in to eight groups (n = 6) and were orally fed with the oil of
Cinnamomum tamala in the increasing dose of 10, 50, 100, 200, 500, 1000, 1500 and 2000 mg/kg body weight. The rats were observed continuously for 2 h for behavioral changes and after 24 and 72 h for any lethality [
17].
Induction of diabetes
Type II diabetes mellitus (NIDDM) was induced in overnight fasted animals by a single intraperitoneal injection of 50 mg/kg STZ in 0.1 M citrate buffer (pH-4.5) in a volume of 1 ml/kg body weight. Diabetes was developed and stabilized over a period of 7 days. Diabetes was confirmed by the elevated blood glucose levels determined at 72 h and on 7th day after injection. Only rats confirmed with permanent NIDDM were used in the antidiabetic study. Blood was collected by intraocular route [
18].
Experimental design
After the induction and confirmation of diabetes, Rats were divided into the following groups comprising six rats in each group.
For acute antihyperglycemic model
In the acute antihyperglycemic models the study was carried out for 4 hours to check whether the plant have some effect or not.
-
Group 1 Normal rats were administered 2% Dimethyl sulfoxide (DMSO).
-
Group 2 Diabetic control rats were administered 2% Dimethyl sulfoxide (DMSO).
-
Group 3 Diabetic animals were administered glibenclamide (0.6 mg/kg p.o).
-
Group 4 Diabetic animal were administered orally 100 mg/kg of CTO.
-
Group 5 Diabetic animal were administered orally 200 mg/kg of CTO.
-
Group 6 Diabetic animal were administered orally 20 mg/kg of Cinnamaldehyde.
For chronic antihyperglycemic model
In the chronic antihyperglycemic models the study was carried out for 28 days to study the various parameters of the diabetes and hyperlipidemia to confirm the antidiabetic, antioxidant and hypolipidemic activity of
Cinnamomum tamala oil and its main constituent cinnamaldehyde in streptozotocin induced diabetes in rats.
-
Group 7 Normal rats were administered 2% Dimethyl sulfoxide (DMSO).
-
Group 8 Diabetic control rats were administered 2% Dimethyl sulfoxide (DMSO).
-
Group 9 Diabetic animals were administered glibenclamide (0.6 mg/kg p.o).
-
Group 10 Diabetic animal were administered orally 100 mg/kg of CTO.
-
Group 11 Diabetic animal were administered orally 200 mg/kg of CTO.
-
Group 12 Diabetic animal were administered orally 20 mg/kg of Cinnamaldehyde.
Sample collection
Blood sample
The 24 h fasted animals were sacrificed by cervical decapitation on 29th day of treatment. Trunk blood was collected in heparinized tubes and the plasma was obtained by centrifugation at 5000 rpm for 5 min. for the determination of biochemical parameters; glucose, insulin, cholesterol, glycosylated hemoglobin, malondialdehyde (MDA), reduced glutathione (GSH) etc.
Collection of organs
The rats were anaesthetized by using the overdose of anesthesia, and tissue sample were taken for assessment of biochemical parameters.
Estimation of plasma 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-analyser (Erba Mannhein 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-analyser (Erba Mannhein Germany). Values are expressed as the percent of total hemoglobin.
Estimation of liver glycogen content
Liver glycogen estimation was done by the method as described by Seifter
et al. (1950) [
19]. 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. The weight of the liver sample was determined. The tissue was then digested by heating the tube for 20 min in boiling water bath, and following this the digest was cooled, transferred quantitatively to a 50 ml volumetric flask, and diluted to the mark with water. The contents of the flask were then thoroughly mixed and 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. Five 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:
(1)
U is the optical density of unknown solution. S is the optical density of the 100 μg glucose and 1.11 is the factor determined by Morris in 1948 for the conversion of the glucose to the glycogen.
Serum insulin assay by ELISA kit
Serum insulin level was measured by an enzyme-linked immunosorbent assay (ELISA) procedure using Mercodia rat insulin ELISA kit. Briefly, the solid phase two-site enzyme immunoassay is based on the direct sandwich technique in which two 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 three 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.
Discussion
The aim of the study was to evaluate the antidiabetic, antihyperlipidemic and antioxidant potential of the CTO and its active constituent cinnamaldehyde in STZ induced diabetes in rats. Diabetes mellitus causes a disturbance in the uptake of glucose as well as glucose metabolism. A dose of STZ as low as 50 mg/kg produces an incomplete destruction of pancreatic beta cells even though the rats become permanently diabetic [
28]. After treatment with a low dose of STZ many beta cells survive and regeneration is also possible [
29]. Hyperglycemia generates abnormally high levels of free radicals by autoxidation of glucose and protein glycation, and oxidative stress has been reported to be a positive factor of cardiovascular complications in STZ-induced diabetes mellitus [
30]. Hyperglycemia is associated with the generation of reactive oxygen species (ROS) causing oxidative damage particularly to heart, kidney, eyes, nerves, liver, small and large vessels and gastrointestinal system [
31]. The increased levels of plasma glucose in diabetic rats were lowered by CTO and cinnamaldehyde administration. The antihyperglycemic action of cinnamaldehyde results from the potentiation of insulin from existing beta cells of the islets of Langerhans [
32].
To study the effect of CTO a preliminary investigation was carried out using acute antihyperglycemic model which revealed the significant reduction in glucose level. Therefore further chronic antihyperglycemic model for a period of 28 days to study the effect on various other parameters viz. insulin level, liver glycogen content, glycosylated hemoglobin, total plasma cholesterol, triglyceride and antioxidant parameters were estimated for all treated groups and compared against diabetic control group. The plasma glucose lowering activity was compared with glibenclamide, a standard hypoglycemic drug. Glibenclamide has been used for many years to treat diabetes, to stimulate insulin secretion from pancreatic beta cells [
33]. From the results of the present study, it appears that still insulin producing cells are functioning and the stimulation of insulin release could be responsible for most of the metabolic effects. It may be suggested that the mechanism of action of CTO is similar to glibenclamide. The glucose lowering activity of CTO may be related to both pancreatic (enhancement of insulin secretion) and extra pancreatic (peripheral utilization of glucose) mechanism. The hyperglycemic activity was almost similar to cinnamaldehyde thereby the major constituent responsible for this activity of CTO may be cinnamaldehyde.
An increase in the level of glycosylated hemoglobin (HbA1c) in the diabetic control group of rats is due to the presence of large amount of blood glucose which reacts with hemoglobin to form glycosylated hemoglobin [
34]. Oxidative stress increases due to the activation of transcription factors, advanced glycated end products (AGEs), and protein kinase C. If diabetes is persistent for long time, the glycosylated hemoglobin is found to increase [
35]. The level of HbA
1C was decreased after the administration of CTO and its main constituent cinnamaldehyde as compared to diabetic control group. The effect of cinnamon in clinical study is also reported in which the mean HbA1c was significantly decreased (P < 0.005) in the cinnamon group (8.22% to 7.86%) compared with placebo group (8.55% to 8.68%). Thus the Cinnamon supplementation could be considered as an additional dietary supplement option to regulate blood glucose level along with conventional medications to treat type 2 diabetes mellitus [
36]. In diabetes mellitus, the loss of body weight is caused by increase in muscle wasting and catabolism of fat and proteins [
37]. Due to insulin deficiency protein content is decreased in muscular tissue by proteolysis [
38]. A decrease in body weight was registered in case of diabetic control group rats while in tested groups the weight loss was reversed. Fatty acid mobilisation from adipose tissue is sensitive to insulin. Insulin’s most potent action is the suppression of adipose tissue lipolysis [
39]. A rise in plasma insulin concentration of only 5 IU/ml inhibits lipolysis by 50%, whereas a reduction in basal insulin levels result in a marked acceleration of lipolysis [
40]. We demonstrated that CTO increased plasma insulin concentrations in diabetic rats. Insulin levels higher than those of the control group may result in inhibition of lipolysis and decreased plasma triglyceride and cholesterol levels. Some studies suggest that the antihyperglycemic action of traditional antidiabetic plant extracts may be due in part to decreased glucose absorption in vivo [
41]. This mechanistic explanation may also apply to the actions of CTO in lowering the triglyceride and cholesterol level.
The conversion of glucose to glycogen in the liver cells is dependent on the extracellular glucose concentration and on the availability of insulin which stimulates glycogen synthesis over a wide range of glucose concentration [
35]. Diabetes reduces activity of glycogen synthase thereby affecting the glycogen storage and synthesis in rat liver and skeletal muscle [
27]. Oral administration of CTO 200 mg/kg body weight significantly increased hepatic glycogen levels in diabetic rats possibly because of the reactivation of the glycogen synthase system as a result of increased insulin secretion. In the clinical study the use of species of cinnamon (
Cinnamomum zeylanicum) showed a beneficial effect on glycemic control (both HbA1c and Fating plasma glucose) and the short term (<4 months) effects of the use of cinnamon on glycaemic control looks promising [
42]. The effect of
Cinnamomum zeylanicum is also reported on gastric emptying, arterial stiffness, postprandial lipemia, glycemia, and appetite responses to high-fat breakfast [
43]. Further the work can be explored for mechanism of action.
Conclusion
In conclusion, the present study showed that oral administration of Cinnamomum tamala oil and its main constituent has potential antidiabetic, antihyperlipidemic and antioxidant effect in STZ induced diabetes in rats in our model systems. The potent antioxidant activity may be responsible for the antihyperglycemic and antihyperlipidemic effects. This investigation reveals the potential of CTO for use as a natural oral agent with antidiabetic, antihyperlipidemic and antioxidant effects.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SK designed and planned the study; carried out experimental work, biochemical analysis, statistical analysis, interpretation and discussion of results related to their part of the work. SS and NV designed and planned the study; drafted and revised the manuscript. NV checked and corrected the English language. All authors read and approved the final manuscript.