Therapeutic Potential of Cinnamomum Zeylanicum Extract to Mitigate Hyperglycemia

Background: Nutrition and health have become one of the most intriguing focuses in the world today. Technological advancement, nutritional imbalances and sedentary lifestyle have ascended numerous health issues worldwide. Scientific evidences have provided the chemo-preventive and chemotherapeutic role of dietary phytochemicals to cure these ailments. Cinnamon (Cinnamomumzeylanicum) has been utilized as a potential therapeutic agent in various cultures for centuries. Objective: Trans-cinnamaldehyde (3-phenyl-2-propanal) contributes as a major constituent of cinnamon bark oil approximately about 49.9-62.8% of the total amount and has hypoglycemic, hypocholesterolemic and anticancer potential. Methods: The ethanolic and supercritical fluid 1 National Institute of Food Science and Technology University of Agriculture, Faisalabad 2 National Institute of Food Science and Technology University of Agriculture, Faisalabad 3 National Institute of Food Science and Technology University of Agriculture, Faisalabad 4 Department of Biochemistry University of Agriculture, Faisalabad Date of Submission: 15-03-2017 Date of 1 st Revision Received: 15-05-2017 Date of Acceptance for Publication: 20-06-2017 Conflict of Interest: None Funding Source: None


Introduction
Across the globe, nutritional imbalances in the diet are causing a number of physiological dysfunctions which engrossed the adoption of diet based therapies as an intervention against various infirmities.Amongst principal therapeutic tool of apposite dietary guidelines, the use of functional and nutraceutical food provides an opportunity to alleviate these health problems within the population. 1 Overwhelming evidences from epidemiological and biological studies have illuminated the extensive use of plant based products owing to their rich phytochemistry against numerous ailments.Hence, dietary approbations for the prevention and cure of chronic diseases have emphasized the con-ANNALS VOL 23, ISSUE 2, APR.-JUN.2017 sumption of variety of plant foods. 2 Spices having a virtuous recognition in cultural heritage and food appreciation possess pivotal health linkages and have also been used effectually in ethnic system of medicine.Being common dietary adjunct, spices contribute as a source of numerous bioactive compounds which influence various digestion and metabolic processes.Spices rich in polyphenols, inhibit oxidation processes in food products and exhibit health promoting effects by increasing antioxidative potential of the body on consumption. 3Amongst spices, cinnamon (Cinnamomumzeylanicum) belonging to family Lauraceae has been utilized as a potential therapeutic agent in various cultures for centuries.Cinnamon bark is one of the oldest known spices used against gastrointestinal complaints, chronic bronchitis and inflammation of eyes in ayurvedic medicine for over 6000 years. 4The major constituents of cinnamon bark oil includes 75% cinnamaldehyde, 5% cinnamyl acetate, 3.3% caryophyllene, 2.4% linalool and 2.2% eugenol. 5iabetes mellitus is a chronic metabolic disorder that affects about 4% of the world's population and is expected to upsurge by 5.4% in 2025. 6According to W.H.O.prediction, diabetes will become the 7 th leading cause of mortality worldwide by the end of 2030. 7hey projected the diabetes prevalence with total number of people affected will increase from 171 to 366 million between 2000 and 2030. 8It is also associated with abnormalities in body's metabolism which ultimately leads to numerous other complications like an increased risk of cardiovascular disease, neuropathy, retinopathy and nephropathy in addition to damaging liver, kidney and beta-cells of pancreas.Improvements in glycemic control have proved to be beneficial in reducing the risk of these complications.Consumption of cinnamon has been found effective in improving the glycaemic control and also reduces the onset and progression of diabetes. 9innamaldehyde (3-phenyl-2-propanal) represents the main constituent of the cinnamon bark oil contributing about 49.9 to 62.8% of the total amount. 10It provides protection against metabolic syndromes, cardiovascular complications and diabetes.Functionality of insulin receptors is improved by virtue of enzyme activation (insulin receptor kinase) which is responsible for insulin binding to the cells.Likewise, it is also responsible for hindrance in the enzyme (insulin receptor phosphatase) activity that impede this process, ultimately leading to the maximum phosphorylation of insulin receptor, which is associated with improved insulin sensitivity.Additionally, glucose tolerance is also increased by reducing effect of cinnamaldehyde on activity of hexokinase and glycogen content in the liver and skeletal muscles.Cinnamon polyphenols contribute to the regulation of various proteins like glucose transporter 4 (GLUT4), insulin receptor β and tristetraprolin involved in insulin signal transduction pathway. 11Recent research illustrates that cinnamaldehyde contributes positively towards consumers health by reducing glucose, cholesterol and LDL levels.

Materials and Methods
The dried cinnamon (Cinnamomumzeylanicum) bark was procured from local market and ground to obtain particle sizes in the range of 300-500 µm for further analyses.The reagents (analytical and HPLC grade) and standards were purchased from Merck (Merck KGaA, Darmstadt, Germany) and Sigma-Aldrich (Sigma-Aldrich Tokyo, Japan).For efficacy trial, male Sprague Dawley rats were acquired from National Institute of Health (NIH) Islamabad.housed in the Animal Room of NIFSAT.For biological assay, diagnostic kits were purchased from Sigma-Aldrich, Bioassay (Bioassays Chemical Co.Germany) and Cayman Chemicals (Cayman Europe, Estonia).
The cinnamon extracts were prepared using ethanol and water (50% v/v) for a period of 60 min with constant temperature of 50 o Cfollowing the outlines of Mariod et al. 12] Resultant solvent extract was filtered and recovered through Rotary Evaporator (Eyela, Japan).Cinnamon powder was subjected to supercritical fluid extraction system (SFT-150 Supercritical Fluid Technologies, Inc.) to obtain supercritical fluid extracts using 99.8% pure CO 2 .Accordingly, sample was placed in 100 mL extraction vessel followed by Optimization of CO 2 at 5000 psi while maintaining time and temperature conditions constant. 13he bio evaluation trial was conducted to investigate the therapeutic potential of cinnamaldehyde against hyperglycemia.Purposely, seventymale Sprague Dawley rats were procured from National Institute of Health (NIH) Islamabad and housed in the Animal Room of National Institute of Food Science and Technology, University of Agriculture Faisalabad.Intentionally, rats were acclimatized by feeding on basal diet for a period of one week under environmentally controlled conditions of temperature (23 ± 2ºC) and relative humidity (55 ± 5%) along with 12 hrs light-dark period.At the initiation of study, some rats were sacrificed to get baseline trend of selected biochemical parameters.During efficacy trial of sixty days, two

Study I Normal rats
Study II Hyperglycemic rats types of studies were conducted separately in order to determine the therapeutic effect of conventional solvent extract (CSE) @ 0.5% and supercritical solvent extract (SFE) @ 0.1% on selected parameters such as lipid profile, glucose and insulin level.Study I comprised of rats fed on normal diet, whereas high sucrosediet was administrated to induce hyperglycemia in study II (Table 1).The composition of diets given to rats is presented in Table 3.On the basis of administrated diet, each study was further subdivided into three groups containing 10 rats in every group.During 60 days trials, simultaneous provision of nutraceutical CSE (D 1 ), nutraceutical SFE (D 2 ) diets along with control (D 0 ) were given to respective groups (Table 2).

Studies
Normal rats Hyperglycemic rats It is assured that all the bioefficacy trials were performed in compliance with the institutional guidelines and relevant laws of the National Institute of Food Science and Technology, University of Agriculture Faisalabad, Pakistan.Additionally, allthe experimental modeling embraces dietary and safety plans were reviewed and approved by the institutional committee(s).
Feed intake of individual group of rats was measured daily by subtracting the remaining diet from the total diet during the whole trial.Likewise, water intake of each group was also recorded on daily basis. 14Gain in body weight of experimental rats was measured weekly throughout the study period to monitor any suppressing effect of cinnamaldehyde enriched diets.In each study, the collected sera were evaluated for glucose concentration by GOD-PAP method as described by Sailesh and Padmanabha 15 whereas, insulin level was assessed following the procedure descrybed byAhn et al. 16 Serum lipid profile of rats including total cholesterol, low density lipoprotein (LDL), high density lipoprotein (HDL) and triglycerides were measured according to their respective protocols.Triglycerides and cholesterol level in sera samples were determined by liquid triglyceride (GPO-PAP) and CHOD-PAP method respectively, outlined by Vafa et al. 17 Likewise, HDL of serum samples using cholesterol precipitant method and LDL were analyzed as per guidelines of Alshatwi et al. 18 The resultant data were examined through completely randomized design (CRD) by employing Cohort version 6.1 (Costat-2003).Moreover, level of significance was measured using analysis of variance (ANO-VA) following the principles outlined by Steel et al. 19

Results
Efficacy studies were performed in vivoon male Sprague Dawley rats in order to determine the functional and nutraceuticalimportance of cinnamon extract against hyperglycemia.For this purpose, rodent experimental modeling was used instead of human subjects because of appropriate supervision, easy handling, feasibility ofcontrolled environmental conditions and Mean values for (study I) explicated feed intake 13.92 ± 0.46, 14.45 ± 0.63 and 14.87 ± 0.52 g/rat/day at initiation that raised to 18.65 ± 0.71, 19.12 ± 0.67 and 19.43 ± 0.56 g/rat/day for D 0 , D 1 and D 2 , respectively at the completion of study.The Figure 1 illustrated maximum feed intake at initiation in D 0 (16.78 ± 0.62 g/rat/day) followed by D 1 (16.21 ± 0.65 g/rat/day) and D 2 (15.49± 0.58 g/rat/day) whereas similar trend was observed at the termination of study II i.e.D 0 (24.59 ± 0.47 g/rat/day) trailed by D 1 (22.34 ± 0.45 g/rat/day) and D 2 (21.16 ± 0.44 g/rat/day).Means relating to drink intake revealed a gradual rise as a function of treatments and time.In this context, minimum intake was noticed in D 0 followed by D 1 and D 2 i.e. 18.51 ± 0.37 & 23.37 ± 0.64, 18.69 ± 0.32 & 24.18 ± 0.56 and 18.74 ± 0.41 & 24.63 ± 0.59 mL/rat/day, respectively at 1 st and 8 th week of the study I. Conversely, in study II (hyperglycemic rats), D 0 expounded the highest drink intake 22.49 ± 1.03 mL/rat/day followed by D 1 (22.37 ± 0.95 mL/rat/day) and D 2 (22.31 ± 0.98 mL/g/ rat) at initiation of the trial.Whereas, increase in drink intake was recorded due to the effect of time as 31.62 ± 0.78, 30.13 ± 0.89 and 29.20 ± 0.83 mL/rat/day in D 0 , D 1 and D 2 , respectively at termination (Figure 2).3).However, at the end of trial resultant body weights were 207 ± 6.63, 225.89 ± 7.14 and 238.12 ± 8.49 g/rat for D 0 , D 1 and D 2 , respectively.
Means for glucose in study I (normal rats) showed values 90.98 ± 5.07, 89.14 ± 4.72 and 87.75 ± 4.13 mg/dL for D 0 , D 1 and D 2 groups, respectively.Mean glucose concentration for D 0 group in study II was 138.45 ± 6.69 mg/dL that declined substantially to 124.69 ± 5.45 mg/dL in D 1 .Whereas the lowest glucose level was noticed for D 2 as 122.32 ± 5.18 mg/dL in hyperglycemic rats (Table 4).It is obvious from results that diets containing nutraceutical SFE (D 2 ) performed better against glucose related deformities than diets containing nuteraceutical CSE (D 1 ) and control diet (D 0 ).The Figure 4 depicted highest percent decline in glucose for study II i.e. 9.94 and 11.65% for D 1 and D 2 , respectively.However, study I showed a non-significant decline in glucose level in groups; D 1 (2.02%) and D 2 (3.55%) as compared to control.
Means related to study I (

Discussion
In a study, Shatwan et al. 20 assessed the effect of cinnamon extract on feed intake of male Wistar rats.They were of the view that feed intake was significantly higher (35.99 ± 3.17 and 30.23 ± 3.58 g) in diabetic and cinnamon treated rats as compared to control (25.93 ± 1.33 g) group.This increment in feed intake was due to the increase of neuropeptides YmRNA and reduced leptin receptors activity with inadequate insulin supply ultimately reducing the weight gain in diabetic rats.The previous findings of Anand et al. 21further strengthened the present results.They deduced the significant increase of about 4.37 times (175 ± 16 mL/day) in fluid intake of untreated diabetic rats when compared with control (40 ± 3.2 mL/day).They also illuminated the inverse relation between the oral administration of cinnamon @ 20 mg/kg body weight and fluid intake, which resulted in decreased (74 ± 8 mL/ day) fluid intake in diabetic rats.The results of present research regarding body weight in cinnamaldehyde (CND) administered male wistar rats are in agreement with the findings of Anand et al. 22 They reported significant decrease 37.9% in body weight of STZ induced diabetic rats after 60 days induction.They also noticed the restoration in body weight 209 ± 10 g after oral intake of cinnamaldehyde (20 mg/kg) to diabetic rats near to control 224 ± 16 g.
The results relating to glucose reduction with cinnamon polyphenols are in harmony with the earlier work of Cheng et al. 23 In a 12 weeks trial, they obse-ANNALS VOL 23, ISSUE 2, APR.-JUN.2017 rved significant reduction in fasting blood glucose (FBG) level as 0.3, 14.6 and 18.9%, respectively in a dose dependent manner.In hyperglycemic condition, insulin resistance causes dysregulation of gluconeogenesis in hepatic tissues which results in elevated glucose level.Cinnamaldehyde helps to reduce blood glucose level by increasing insulin release and inhibiting the gene expression of phosphoenolpyruvatecarboxykinase & glucose-6-phosphatase.Later, Sailesh and Padmanabha, 24 examined the hypoglycemic effects of cinnamon bark extract in alloxan induced diabetic rats and inferred that cinnamon polyphenols reduced blood glucose level in a 21 days trial.The role of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) to scavenge oxygen free radicals and pancreatic tissue protection form oxidative damage is well known.In diabetic mice, cinnamon polyphenols enhances SOD and GSH-Px activities and decreases malondialdehyde (MDA) contents in pancreatic tissue thus provide beneficial effects against hyperglycemia. 25n a research study, Ismail 26 evaluated the variations in insulin hormone level with provision of different levels i.e. 100 and 200 mg/kg body weight of cinnamon extracts.He expounded progressive increase in the serum insulin level in a dose dependent manner with minimum insulin level 0.89 ± 0.13 ng/mL in untreated diabetic rats which substantially increased to 2.43 ± 0.12 ng/mL in rats treated with cinnamon extract @ 200 mg/kg which further confirmed our results.
The present trend for serum cholesterol reduct-ion is strengthened by the efficacy study of IM et al. 27 Ethanolic extract of cinnamon polyphenols elucidated a marked reduction in hypertriglyceridemia and hypercholesterolemia with 42.79% dec-rease in total cholesterol level in group treated with 70% polyphenol contents as compared to diabetic control.According to another study, cinnamon pro-vides protection against hypercholesterolemia by reducing the serum cholesterol level from 91.50 ± 9.26 to 76.75 ± 5.46 mg/dL in high cholesterol fed and cinnamontreated white male albino rats. 28innamon ability to reduce serum LDL cholesterol level is supported by the work of Askari et al. 29 They concluded that cinnamon intervention results in substantial diminution 55.8 ± 40.8 mg/dL in LDL cholesterol as compared to placebo control 90.3 ± 17.7 mg/dL in patients suffering from non-alcoholic fatty liver disease.The outcomes of Hasanein et al. 30 strengthened the results of recent study as they observed an elevated level of HDL as 34.06 ± 1.08 mg/dL in rats fed with high fat diet treated with aqueous cinnamon extracts.In a study researchers explicated the boosting effect of cinnamon polyphenols on antioxidant ability of diabetic mice against hyperlipidemia.They also recorded substantial reduction in serum triglyceride level of CPS (cinnamon polyphenols) treated mice at the end of study. 31

Conclusion
Prevention of various metabolic syndromes by using different dietary patterns has become a major health concern worldwide especially in developing countries like Pakistan.In this milieu, cinnamon (Cinnamomumzeylanicum) provides a wide range of nutraceuticals especially cinnamaldehyde.The current study was an effort to assess the therapeutic worth of cinnamaldehyde to cope with hyperglycemia.In present study,reduction in glucose level was more in rats fed with nutraceutical SFE diet (D 2 ) as compared to nutraceutical CSE diet (D 1 ) followed by control diet (D 0 ).In the nutshell, cinnamaldehyde enrichment/ supplementation should be encouraged at mass level and consumer awareness regarding diet based therapies should be promoted.

Fig. 4 :
Fig. 4: Percent Decrease in Glucose as Compared to Control.

Fig. 5 :
Fig. 5: Percent Increase in Insulin as Compared to Control.

Table 1 :
Different Studies Conducted during Efficacy Trials.

Table 3 :
Composition of Experimental Diet.

Table 4 :
Effect of Diets on Glucose (mg/dL) of Rats.

Table 5 :
Effect of Diets on Insulin (µU/mL) of Rats.

Table 5
to the effect of diets.Means for HDL values in study I were 33.46 ± 1.93, 34.15 ± 2.08 and 34.37 ± 2.15 mg/dL in D 0 , D 1 and D 2 groups, respectively.Nonetheless in study II, lowest HDL level was recorded in D 0 (42.68 ± 1.51 mg/dL) that significantly uplifted to 43.99 ± 1.27 and 44.20 ± 1.69 mg/dL in D 1 and D 2 groups.