Department of Pharmacology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110007, India
Email: [email protected]
Received: 08 Feb 2017 Revised and Accepted: 02 Mar 2017
Objective: To explore the anti-hyperglycemic effect of fruit extract of Terminalia catappa (Indian almond), a potential medicine from plant origin in a diabetic rat model.
Methods: Streptozotocin-induced chronic diabetic rat model was utilized in the study. Three doses of test drug, hydro-alcoholic fruit extract of Terminalia catappa in 20 mg/kg, 30 mg/kg and 40 mg/kg and a standard anti-diabetic drug, glibenclamide (10 mg/kg) was used. The study had a total of nine groups with eight animals in each group. Drugs were given orally every day for 12 w. Blood glucose, body weight and urine volume were measured weekly, glycosylated hemoglobin (HbA1c) was estimated at 12th week in all groups. Data for all parameters were analyzed using one-way ANOVA repeated measures followed by Mann-Whitney test.
Results: Hydro-alcoholic fruit extract of T. catappa significantly decreased blood glucose, urine volume and increased body weight in a dose-dependent manner in diabetic rats. At 12th week, blood glucose level in control, diabetic control, glibenclamide, T. catappa (40 mg/kg) group was 96.25±2.05 mg/dl, 599.75±0.25 mg/dl, 248.25±11.45 mg/dl, 115.00±3.78 mg/dl respectively. Effect of T. catappa in 30 mg/kg and 40 mg/kg dose was significantly more than glibenclamide. At 12th week, HbA1c level in control, diabetic control, glibenclamide, T. catappa (40 mg/kg) was 2.94±0.33 mmol/l, 4.94±0.49 mmol/l, 3.61±0.28 mmol/l, 3.21±0.27 mmol/l. Treatment with T. catappa 30 mg/kg, 40 mg/kg and glibenclamide brought back the level of HbA1c to normal levels. The addition of glibenclamide to T. catappa (40 mg/kg) did not produce any additional effect on blood glucose and HbA1c levels compared to the effect of T. catappa (40 mg/kg) in diabetic rats.
Conclusion: Terminalia catappa fruit extract exhibited a significant anti-hyperglycemic effect in diabetic rats and has a great potential to be used in diabetes.
Keywords: Anti-diabetic activity, Blood sugar, Experimental diabetes, Indian badam, Glycosylated hemoglobin
© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
Diabetes mellitus is a complex metabolic syndrome characterized by increased blood glucose levels, abnormal glucose and lipid metabolism, sub-clinical inflammation and increased oxidative stress. Hyperglycemia, or raised blood sugar, is a common effect of uncontrolled diabetes and is a major contributor to morbidity and mortality.
In recent years, the pharmacologic treatment of diabetes has advanced, but still an adherence to therapy and control of blood sugar remains a challenge. Herbal drugs, as alternatives to modern or allopathic medicines, are becoming popular for their cost effectiveness and comparatively fewer side effects than conventional modern medicine treatment. Furthermore, adherence to therapy amongst the diabetic population with medicines from plant origin may be better than conventional modern allopathic medicines. India has witnessed a tremendous rise in the diabetic population over the last few decades thereby earning itself the distinction of the ‘Diabetic Capital of the World’. With almost 50.9 million people already suffering from diabetes mellitus this fig. is expected to reach up to 80 million by the year 2025 .
Terminalia catappa Linn. (Family–Combretaceae) is usually found in Maharashtra, Karnataka, Tamil Nadu, Andhra Pradesh, and Kerala, West Bengal and other warm regions of India. Terminalia catappais commonly known as Indian almond, Malabar almond and is normally consumed by a lot of people in their daily routine. Many studies have shown that apart from the nutritional benefits, Terminalia catappa plant also possessed a number of medicinal properties such as antimicrobial , anti-inflammatory , analgesic , wound-healing , anti-oxidant , hepatoprotective , anti-cancer  and anti-ageing activities . These activities are widely owed to the virtue of the presence of certain phytochemicals in Terminalia catappa (T. catappa) such as tannins, saponins, phenols and flavonoids, carotenoids. Fruit of T. catappa has been reported to contain glucose, pentosans, corilagin, brevifolin carboxylic acid, β-carotene, cyanidin-3-glucoside, ellagic acid, gallic acid, and tannin [10, 11]. Ellagic acid has shown to have anti-diabetic effect in diabetic rats . The entire green fruit with seed inside has shown to have more phenolic content and other phytochemicals . Therefore, in the present study, we have investigated the anti-diabetic effect of the entire fruit of T. catappa in streptozotocin-induced diabetic rats.
MATERIALS AND METHODS
Hydro-alcoholic extract of the fruit of Terminalia catappa was obtained from standardized, GMP compliant laboratory, Pharmanza Herbal Private Limited, Gujarat with a certificate of analysis.
Glibenclamide and streptozotocin was obtained from Sigma Aldrich, USA. Glycosylated hemoglobin (HbA1c) ELISA kit was obtained from KINESISDx, California, USA. All other chemicals were of analytical grade.
Ethical clearance for the study was obtained from Institutional Animal Ethics Committee, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi (Letter no. IAEC/42/2014). Wistar rats of either sex in the weight range of 180-250 gm were obtained from Central Animal House Facility. Experimental rats were placed in metabolic cages in the animal house of Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India. Animals were housed under carefully controlled standard laboratory conditions of ventilation, humidity and lighting. The animals in the study were maintained at room temperature 23±1 °C with natural light and dark cycle. Animals in the study were fed with standard pellet diet and water available ad libtium. The animals were allowed a period of one week for acclimatization to laboratory conditions before the experiment.
The care of the animals was done in accordance with the guidelines of the committee for the purpose of control and supervision of experimentation on animals (CPCSEA).
Methodology and study design
Diabetes was induced in overnight fasted Wistar rats by single intraperitoneal (i. p.) injection of streptozotocin (STZ) in a dose of 45 mg/kg in 0.1 M citrate buffer and a pH of 4.5 was maintained . Animals with blood glucose levels more than 300 mg/dl were considered diabetic and were included in the present study. All precautions were taken to get the real effect of test drug in diabetic condition, therefore in addition to control, diabetic control, a group treated with standard anti-diabetic drug and per se effect of test drug and standard anti-diabetic group were included and the study period was 12 w. Effect of highest dose of test drug (T. catappa) was also combined with standard anti-diabetic drug (glibenclamide) to see any additive/ potentiating effect. Permission to take eight animals (usual number permitted is six) was obtained from ethical committee. Treatment with all the drugs was started from 2nd day till 12th week and all the drugs were dissolved in water and were given by oral route. Rats in all the groups were fed with normal pellet diet and water ad libtium.
A study conducted by Nagappa et al., have used glibenclamide in 10 mg/kg dose . Hence, the same dose of glibenclamide was used in present study. Earlier study done with methanolic extract of Terminalia catappa (T. catappa) fruit had used 40 mg/kg p. o. for 3 w and had shown significant hypoglycemic activity without any side effects in rats . Therefore, the first dose selected was 40 mg/kg in the present study for T. catappa fruit extract. The next two doses were chosen after the results obtained with 40 mg/kg. Effect of T. catappa on its own (per se) and glibenclamide per se was checked by giving these drugs alone in two groups of experimental animals for 12 w as mentioned below.
Group 1: Control
Group 2: Diabetic control
Group 3: Glibenclamide 10 mg/kg per se
Group 4: Glibenclamide treatment 10 mg/kg in diabetic rats
Group 5: Terminalia catappa 40 mg/kg per se
Group 6: Terminalia catappa 40 mg/kg in diabetic rats
Group 7: Terminalia catappa 30 mg/kg in diabetic rats
Group 8: Terminalia catappa 20 mg/kg in diabetic rats
Group 9: Terminalia catappa 40 mg/kg plus glibenclamide 10 mg/kg in diabetic rats
Blood glucose level in control, diabetic control and treatments groups were measured every 3rd day till 3rd week and thereafter it was measured on a weekly basis. The tail of the rat was pricked with a syringe and using electronic glucometer (Bayer breeze 2) and glucose test strips blood glucose level was measured. Glycosylated Hemoglobin A1c (HbA1c) was estimated at 12th week in all the groups by ELISA kit of KINESISDx following manufacturer’s instructions.
Every week body weight of experimental animals was recorded. The animals in all groups-control, diabetic control and treatment groups were placed on an electronic weighing balance and their weight was taken.
Rats were housed in metabolic cages and their urine volume recorded every day. Average urine volume for every week was calculated.
All data are expressed as mean±standard error of the mean (SEM). A statistician help was taken to apply the suitable statistical test for the study. One way ANOVA with repeated measures was followed by a pairwise comparison between various treatment groups. The pair-wise comparison was done using non-parametric Mann-Whitney test. Statistical analysis was done using SPSS statistics version 20 software.
The phytochemical screening of hydro-alcoholic extract of Terminalia catappa fruit revealed the presence of tannins (10.50%), alkaloids (1.95%), saponins (19.99%) and total polyphenols (12.01%).
Blood glucose level significantly increased in diabetic rats from baseline of 101.88±2.26 mg/dl to 599.75±0.25 mg/dl by 12th week (table 1). Glibenclamide treatment significantly reduced blood glucose level (p<0.01) from 2nd week onwards and the effect further increased significantly (p<0.001) at 12th week, blood glucose level decreased to 248.25±11.45 mg/dl at 12th week. The fruit extract of T. catappa showed dose-dependent effect on reduction of blood sugar in diabetic rats. The effect was observed from 2nd week (p<0.01) and decrease in blood glucose level increased further with time. The blood glucose level was 115.00±3.78 mg/dl at 12th week with 40 mg/kg fruit extract and 152.38±9.97 mg/dl and 262.75±153.74 mg/dl with 30 mg/kg and 20 mg/kg (table 1). Effect of T. catappa fruit extract in the dose of 40 mg/kg on decreasing blood glucose level was statistically greater (p<0.01) compared to 30 mg/kg from 4th week till 12thweek. Effect of 40 mg/kg on blood glucose was statistically significant (p<0.01) than 20 mg/kg at all weeks. Terminalia catappa in 30 mg/kg dose showed significant more decrease (p<0.01) in blood sugar from 6th week onwards as compared to 20 mg/kg in diabetic rats. By 12th week in all the treatment groups though the blood glucose level was statistically lower than diabetic control rats but was still statistically higher than the normal control rats. Effect of glibenclamide on lowering blood glucose level was statistically less than 30 mg/kg and 40 mg/kg of T. catappa fruit. No additive effect of glibenclamide was observed when it was combined with 40 mg/kg fruit of T. catappa (table 1).
Levels of serum HbA1c significantly increased (4.94±0.49 mmol/l) in diabetic rats as compared to normal control (2.94±0.33 mmol/l) at 12th week. Glibenclamide, 30 mg/kg, 40 mg/kg dose of T. catappa fruit extract decreased the level of HbA1c to 3.61±0.28 mmol/l,3.84±0.29 mmol/l, 3.21±0.27 mmol/l and the levels were statistically same as in the control group (fig. 1). However, 40 mg/kg dose of fruit produced more reduction in the level of HbA1c (3.21±0.27 mmol/l) and was statistically lower than diabetic control rats and was statistically equal to control value. The addition of glibenclamide to 40 mg/kg dose of fruit both did not produce any further increase in effect (fig. 1).
Body weight of diabetic rats by 12th week was significantly lower (127.25±2.04g) as compared to control rats (406.00±2.56g). Glibenclamide and T. catappa fruit extract in all the three doses increased body weight in diabetic rats. At 12th week, the body weight of glibenclamide-treated rats was 289.38±7.47g. The body weight in diabetic rats treated with 20 mg/kg, 30 mg/kg and 40 mg/kg fruit extract of T. catappa was 184.38±2.20g, 231±2.49g, 309.63±9.11g respectively. Terminalia catappa in the dose of 20 mg/kg significantly increased body weight in diabetic rats at 8th week (p<0.05); increase in body weight increased with time, i.e., at 10thand at 12th week (p<0.01). However, with 30 mg/kg of T. catappa, an increase in body weight started at 4th week (p<0.01) and this increase remained till 12th week. Similarly, 40 mg/kg dose of T. catappa showed an increase in body weight at 4th week (p<0.01) and the effect on body weight was observed till 12th week in diabetic rats. Administration of T. catappa 40 mg/kg plus glibenclamide in diabetic rats showed a statistically equal effect in increasing body weight when compared with T. catappa 40 mg/kg alone though numerically it was more. Terminalia catappa produced a dose-dependent effect on body weight in diabetic rats.
Effect of 40 mg/kg was statistically more (p<0.01) compared to 30 mg/kg and 20 mg/kg at all weeks. Similarly, the effect of 30 mg/kg of T. catappa was statistically significant than 20 mg/kg throughout the study period on body weight in diabetic rats After 12 w of treatment with all the drugs body weight was still statistically lower than the normal control rats (fig. 2).
Table 1: Effect of different doses of Terminalia catappa fruit extract and glibenclamide on blood glucose (mg/dl) in diabetic rats
|Normal Control||Diabetic Control||Glibenclamide
|T. catappa fruit extract 40 mg/kg||T. catappa fruit extract 30 mg/kg||T. catappa fruit extract 20 mg/kg||T. catappa fruit 40 mg/kg+Glibenclamide|
##p<0.01,###p<0.001 compared with Normal Control; **p<0.01, *** p<0.001 compared with Diabetic Control;+p<0.05,++p<0.01 compared with Glibenclamide treatment; aap<0.01 compared with T. catappa fruit extract 40 mg/kg; bbp<0.01 compared with T. catappa fruit extract 30 mg/kg. Gb-Glibenclamide. Differences were analyzed by One way ANOVA followed by Mann-Whitney test.
Fig. 1: Effect of different doses of Terminalia catappa fruit extract and glibenclamide on HbA1c (mmol/l) in diabetic rats at 12th week study period. (#p<0.05compared with Normal Control; *p<0.05, **p<0.01 compared with Diabetic Control; ap<0.05 compared with T. catappa fruit extract 40 mg/kg. Gb-Glibenclamide. Differences were analyzed by One-way ANOVA followed by Mann-whitney test)
Fig. 2: Effect of different doses of Terminalia catappa fruit extract and glibenclamide on body weight (g) in diabetic rats. (##p<0.01 compared with Normal Control; **p<0.01 compared with Diabetic Control;++p<0.01 compared with glibenclamide treatment; aap<0.01 compared with T. catappa fruit extract 40 mg/kg; bbp<0.01 compared with T. catappa fruit extract 30 mg/kg. Gb-glibenclamide. Differences were analyzed by One-way ANOVA followed by Mann-whitney test)
Induction of diabetes in experimental animals produced a significant increase in urine volume from 2nd week onwards. By 12th week, the urine volume in diabetic control rats was 76.84±1.10 ml as compared to 1.63±0.29 ml in control rats (fig. 3).
Treatment with glibenclamide decreased urine volume significantly from 2nd week onwards, and by 12th week, urine volume decreased to 21.59±0.50 ml. Terminalia catappa fruit extract in diabetic rats produced significant (from 2nd week onwards) dose-dependent reduction with 20 mg/kg, 30 mg/kg and 40 mg/kg in urine volume viz. 21.93±0.21 ml, 10.63±0.42 ml and 6.48±0.61 ml respectively. Effect of 40 mg/kg of T. catappa was significantly more than 20 mg/kg and 30 mg/kg and effect of 30 mg/kg was more than 20 mg/kg of T. catappa fruit extract on the reduction of urine volume.
Effect of 30 mg/kg and 40 mg/kg dose of hydroalcoholic fruit extract was statistically greater than glibenclamide. Administration of T. catappa 40 mg/kg plus glibenclamide in diabetic rats showed a significant decrease (p<0.01) in urine volume from 2nd week onwards till 12th week when compared with T. catappa 40 mg/kg alone (fig. 3).
Fig. 3: Effect of different doses of Terminalia catappa fruit extract and glibenclamide on urine volume (ml) in diabetic rats. (##p<0.01compared with Normal Control; **p<0.01 compared with Diabetic Control; ++p<0.01 compared with Glibenclamide treatment; aap<0.01 compared with T. catappa fruit extract 40 mg/kg; bbp<0.01 compared with T. catappa fruit extract 30 mg/kg. Gb-Glibenclamide. Differences were analyzed by One-way ANOVA followed by Mann-whitney test)
The prevalence of diabetes mellitus is increasing at an alarming rate and the development of medicines from the natural origin or functional food are need of the hour as they are associated with less side effects and the compliance to medication could be more. Adherence to therapy is important as diabetes is a chronic disease and requires long-term therapy for control of blood sugar. Therefore, we have used a chronic diabetic rat model of 12 w. Earlier study conducted with Terminalia catappa was for three weeks’ duration . The present study showed the anti-hyperglycemic effect of hydro-alcoholic fruit extract of Terminalia catappa in streptozotocin-induced diabetic rats. Streptozotocin leads to the destruction of pancreatic β-cells by causing alkylation of DNA leading to insufficient production of insulin thereby increasing blood glucose levels [16, 17]. In our study, blood glucose level in the diabetic control group remained significantly high as compared to control group throughout the study period of 12 w.
Glibenclamide, a standard anti-diabetic drug reduced blood glucose level in diabetic rats. Similar results with glibenclamide are reported in previous studies indicating the reduced blood glucose level in diabetic rats which was suggested to be due to a stimulatory effect on the initial release of insulin and inhibition of ATP-sensitive K+channels [18, 19].
Hydro-alcoholic fruit extract of T. catappa in three doses (20 mg/kg, 30 mg/kg and 40 mg/kg) showed a significant dose-dependent reduction in blood glucose level compared to diabetic control rats. Terminalia catappa (T. catappa) contains alkaloids, flavonoids, tannins, saponosides, sterols, carbohydrates, proteins, carotenoids (β-carotene), fat, vitamins, triterpenes, thiocyanates, cardiac glycosides, cyanogenic glycosides and para-hydroxybenzoic acid [10, 11, 20]. Earlier, Nagappa et al. (2011) have shown the anti-hyperglycemic effect of methanolic extract of T. catappa fruit and have postulated the role of β-carotene in causing regeneration of pancreatic beta cells in Wistar rats . Polyphenols have shown antihyperglycemic effect by protecting β-cell function and promoting insulin action and secretion [21, 22, 23]. Phytoconstituents like flavonoids, tannins and triterpenoids have shown inhibitory effect on α-glucosidase and α-amylase activity thereby inhibiting intestinal Na+dependent glucose transporters (SGLT1 and SGLT2) leading to decreased digestion and intestinal absorption of dietary carbohydrates [24, 25, 26]. A study conducted by Salehi et al. (2013) reported the anti-diabetic effect of ten plants containing phenols in different extracts by inhibition of α-glucosidase and α-amylase activity in diabetic rats . Thus, in the present study, the anti-hyperglycemic activity of hydro-alcoholic fruit extract of T. catappa may be attributed due to the presence of high concentration of tannins and other polyphenols that are known to regenerate pancreatic β cells, protect β cell function and promote insulin action, secretion and decrease in absorption thereby decreasing blood glucose levels.
It is well evident that glycosylated hemoglobin (HbA1c) level is increased in diabetic condition [28, 29]. In the present study, diabetic control rats had significantly higher HbA1c level compared to control rats. Glibenclamide treatment in diabetic rats reduced HbA1c levels and is similar to results obtained in previous studies [30, 31]. In the present study, treatment of diabetic rats with fruit extract of T. catappa reduced HbA1c levels significantly compared to diabetic control rats. Sampath and Mani, (2015) have reported reduced HbA1c level in diabetic rats treated with tannin enriched extract . Polyphenols have also shown to reduce digestion and intestinal absorption of dietary carbohydrates and promote insulin secretion . Our test drug, hydroalcoholic fruit extract of T. catappa have a high content of tannins and other polyphenols which may be responsible for decreasing the higher HbA1c level in diabetic rats.
In our study, body weight was significantly decreased in diabetic control rats compared to control rats. Diabetes produced a significant reduction in body weight in rats due to loss of tissue proteins and increased muscle wasting in diabetic condition [34, 35]. Treatment with glibenclamide in diabetic rats has been shown to reduce blood glucose and prevention of muscle wasting which probably is responsible for the increase in body weight . Treatment of diabetic rats with hydroalcoholic fruit extract of T. catappa in different doses significantly increased body weight compared to diabetic control rats. Our test drug hydro-alcoholic fruit extract of T. catappa contain tannins and other polyphenols which decrease blood glucose level and are postulated to be responsible for increasing body weight in diabetic rats. Sampath and Mani, (2015) have reported increased body weight in diabetic rats treated with tannin enriched extract of Terminalia chebula and Punica granatum .
Excessive urination is a symptom of diabetes and is due to the inability of the renal tubules to absorb glucose filtered in the glomeruli leading to osmotic diuresis . Similarly, in our study urine volume was significantly increased in diabetic rats compared to control rats. Glibenclamide treated diabetic rats showed a significant reduction in urine volume compared to diabetic control rats and the results of the present study are in agreement with the studies done earlier [37, 38]. Dubey et al. (2013) have shown that treatment of diabetic rats with oleanolic acid decreased blood glucose level and increased glomerular filtration rate .
Diniz et al. (2012) have shown that saponins of Ampelozizyphus amazonicus reduce renal atrial natriuretic peptides (ANP) and/or have a stimulatory effect on renal ATP ase activity and reduce urine volume in rats . Hydro-alcoholic fruit extract of T. catappa in 20 mg/kg, 30 mg/kg and 40 mg/kg dose showed a significant dose-dependent reduction in urine volume compared to diabetic control rats and could be due to the presence of polyphenols like tannins, saponins and flavonoids which reduce blood glucose levels.
The hydro-alcoholic fruit extract of Terminalia catappa (Indian almond) showed a reduction in blood glucose, HbA1c, urine volume and an increase in body weight in diabetic rats. Anti-hyperglycemic effect of fruit extract of T. catappa can be attributed to the presence of tannins, saponins and polyphenols which could be responsible for its anti-diabetic effect in rats. Recently the use of medicines from plant origin and functional foods has been considered as a new approach in the prevention and management of diabetes and its complications. Our test drug, Indian almond fruit (Terminalia catappa) has a great potential as an anti-diabetic drug. Further work is warranted to explore the mechanism of action of this potential anti-diabetic drug which will give additional evidence to use Terminalia catappa for the treatment of diabetes and diabetes-induced complications.
The authors would like to acknowledge the financial support(Project number Z.28015/13/2013-HPC (EMR-AYUSH-A) for the project (part of the work is reported here) received from Central Council for Research in Ayurveda and Siddha (CCRAS), Ministry of AYUSH, Government of India.
Prof. Anita Kotwani conceived the idea and designed the protocol of the study. Prof. Kotwani is the Principal Investigator of the project. Mr. Tapan Behl is Senior Research Officer on the project, conducted the experiments and drafted the manuscript. Prof. Kotwani critically revised the manuscript. Both the authors have approved the final version of the manuscript.
CONFLICT OF INTERESTS
The authors declare no conflict of interest
How to cite this article