Beneficial role of Areca catechu nut extract in Alloxan-induced Diabetic Rats

 

L. Kavitha1, B. Kumaravel2, G. Sriram Prasath1 and S. Subramanian1*

1Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600 025, India

2Mahatma Gandhi Medical College and Research Institute, Puducherry-607402.

 

 

ABSTRACT:

Areca catechu, popularly known as “Areca nuts” have been used in traditional herbal medicine for the treatment of various diseases including diabetes. In the absence of systemic scientific data in the literature, the present study was designed to evaluate the hypoglycemic, hypolipidemic and antioxidant properties of Areca catechu nut extract in alloxan-induced experimental diabetes in rats. Phytochemical analysis revealed the presence of alkaloids, flavanoids, carbohydrates, saponins, tannins, phytosterol, terpenoids and phenols. The effect of oral administration of Areca catechu nut extract (250 mg/kg b.w.) on the levels of biochemical parameters was determined in both control as well as experimental groups of rats. The altered levels of biochemical parameters in the diabetic rats were significantly reverted back to near basal values by the administration of Areca catechu nut extract for 30 days. The level of glycogen content was improved upon the extract treatment. The altered activities of serum aminotransferase and alkaline phosphatases were restored to normalcy. The levels of lipid peroxides in the plasma and pancreatic tissues of diabetic rats were elevated significantly and were normalized by the administration of Areca catechu nut extract. The activities of enzymatic antioxidants in pancreatic tissues and the levels of plasma non-enzymatic antioxidants were markedly declined in the diabetic rats. Upon treatment with Areca catechu nut extract to diabetic rats, decreased levels were elevated to near normal values. The altered levels of lipid profile in the diabetic group of rats were reverted back to near normalcy upon the extract treatment. Thus, the results of the study indicate that Areca catechu nut extract possesses antilipidemic, antioxidant effects in addition to antidiabetic activity. The results are comparable with gliclazide, an oral standard hypoglycemic drug. The phytochemicals found to be present present in the Areca catechu nut extract may account for the observed pharmacological properties.

 

KEYWORDS: Areca catechu, antidiabetic, antilipidemic, antioxidant nature.

 

INTRODUCTION:

Diabetes is a chronic disorder resulting in the abnormalities of carbohydrates, proteins, and fat metabolism due to absolute or relative deficiency of insulin secretion with/without varying degree of insulin resistance. According to World Health Organization projections, the prevalence of diabetes is likely to increase by 35% by the year 20251. The world prevalence of diabetes among adults is 6.4%, affecting 285 million adults, in 2010, and will increase to 7.7%, and 439 million adults by 2030. Between 2010 and 2030, there will be a 69% increase in numbers of adults with diabetes in developing countries and a 20% increase in developed countries2.

 


Regardless of the type of diabetes, individuals with diabetes are required to control their blood glucose with medications and/or by adhering to an exercise program and a dietary plan. Most of the currently available oral hypoglycaemic drugs possess adverse side effects. Hence search for novel drugs without side effects still continues. Plant drugs and herbal formulations are frequently considered to be less toxic and free from adverse side effects than synthetic one3. Plants have always been an exemplary source of drugs and many of the currently available drugs have been derived either directly or indirectly from them.

 

Areca Catechu is popularly used in traditional herbal medicines in many parts of the world. The seeds are commonly known in folklore medicine for treatment of various diseases such as dyspepsia, constipation, beriberi and oedema. The seeds contain phenolics and alkaloids such as arecoline, arecaidine and guvacine4. A. catechu constituents exert several beneficial effects on skin, suggesting the possible use in cosmetics industries5.  A. catechu is burned to make charcoal, which is pulverized and added to toothpaste6. The betel leaves and areca nuts are used ceremonially in traditional weddings. Traditionally, a groom might offer the bride's parents betel and areca, the leaf and the nut symbolizing the ideal married couple bound together. In Vietnamese the phrase "matters of betel and areca" is synonymous with marriage.

 

Areca Catechu is found to possess various activities like platelet aggregation activity7, anticancer, anti-inflammatory8, 9,, antidepressant10, antivenom11  and antioxidant activity12. In the absence of systemic studies in the literature, the present study was aimed to evaluate the antidiabetic, antioxidant and antidyslipidemic activity of ethanolic extract of Areca Catechu nut in alloxan induced experimental diabetes in rats

 

MATERIALS AND METHODS:

PLANT MATERIAL

Areca Catechu nuts were collected from a retail shop and authenticated by  a qualified taxonomists and a voucher specimen was deposited at the Department of Biochemistry, University of Madras.

 

PREPARATION OF PLANT EXTRACT

Areca Catechu nuts were dried at room temperature and powdered in an electrical grinder, which was then stored in an airtight container at 5° C until further use. The powdered nuts were delipidated with petroleum ether (60 - 80° C) for overnight. It was then filtered and soxhalation was performed with 95% Ethanol. Ethanol was evaporated in a rotary evaporator at 40 – 50° C under reduced pressure.

 

PHYTOCHEMICAL SCREENING

The ethanolic extract of Areca Catechu nuts were subjected to preliminary phytochemical screening of various plant constituents13.

EXPERIMENTAL ANIMALS

Male albino Wistar rats (150-180 g) were purchased from TANUVAS, MADAVARAM, Chennai. The rats were housed in polypropylene cages lined with husk and kept in Animal house, Department of Biochemistry. It was renewed every 24 hours. The rats were fed with commercial pelleted rats chow (VRK Nutritional Solutions, Maharashtra, India) and had free access to water. The experimental rats were maintained in a controlled environment (12:12 hours light/dark cycle) and temperature (30 ± 2° C). The experiments were designed and conducted in accordance with the ethical norms approved by Ministry of Social Justices and Empowerment, Government of India and Institutional Animal Ethics Committee Guidelines for the investigation of experimental pain in conscious rats. The rats were acclimatized for one week before starting the experiments.

 

INDUCTION OF DIABETES MELLITUS

Diabetes was induced by single intraperitonial injection of alloxan monohydrate dissolved in sterile normal saline at a dose 120 mg/Kg, after overnight fasting to induce hyperglycemia. After 1 hour, the animals were fed on standard pellets and water ad libitum. Rats were supplied with 5% glucose solution for 48 hours after alloxan injection in order to prevent severe hypoglycaemia. After 1 week time for the development and aggravation of diabetes, the rats with moderate diabetes having persistant glycosuria and hyperglycemia (Blood Glucose range of above 250 mg/dL) were considered as diabetic rats and used for the experiment. The treatment was started on the eighth day after alloxan injection and this was considered as first day of treatment.

 

EXPERIMENTAL DESIGN

The rats were divided into 4 groups, each comprising of a minimum of six rats as follows:

Group I       Control rats (Water and food ad libitum).

Group II      Alloxan induced diabetic Rats.

Group III   Diabetic rats treated with Areca Catechu nut extract (250 mg/Kg Body weight/day) in aqueous solution orally for 30 days.

Group IV    Diabetic rats treated with gliclazide (5mg/Kg body weight/day) in aqueous solution orally for 30 days.

 

During the experimental period, body weight and blood glucose levels of all the rats were determined at regular intervals. At the end of the experimental period, the rats were fasted over night, anaesthetized, and sacrificed by cervical dislocation. The blood was collected with or without anticoagulant for plasma or serum separation respectively.

 

Preparation of tissue homogenate

The pancreatic tissues were excised, rinsed in ice- cold saline. The tissues were homogenized in Tris–HCl buffer (100 mM, pH 7.4) at 4°C, in a Potter– Elvehjem homogenizer with a Teflon pestle at 600 rpm for 3 min. The homogenate was then centrifuged at 12,000g for 30 min at 4°C. The supernatant was collected as tissue homogenate, which was used to assay various parameters.

 

Blood glucose level was estimated by the method of glucose oxidase/peroxidase as described by Trinde14 and urea by Natelson et al.15. Plasma was separated and used for insulin assay using ELISA kit for rats. Levels of hemoglobin and glycosylated hemoglobin were estimated according to methods of Drabkin and Austin16 and Nayak and Pattabiraman17, respectively. Plasma was used for protein assay 18 and serum for determination of creatinine19 and uric acid20. Aspartate transaminase(AST), Alanine transaminase(ALT) and Alkaline phosphatase (ALP) were assayed by the method of King et al.21, 22. For the estimation of glycogen, the extraction was carried out by the method of Morales et al. (1973)23. The pancreatic tissue homogenate was centrifuged at 5000g to remove cellular debris and supernatant was used for the determination of lipid peroxides and enzymatic antioxidants. Lipid peroxides were eatimated using thiobarbituric acid reactive substances by the method of Ohkawa et al.24 Levels of vitamin C, vitamin E, ceruloplasmin and glutathione (GSH) were determined by the methods of Omaye et al,25 Desai26, Ravin27, Sedlak and Lindsay28, respectively. Enzymatic antioxidants such as superoxide dismutase29, catalase30, glutathione peroxidase31 in pancreatic supernatant.

 

Oral Glucose Tolerance Test (OGTT)

At the end of the experimental period, a fasting blood sample was collected from all the groups of rats to perform oral glucose tolerance test. Rats were administered orally with 2 g/kg glucose solution. Blood samples were collected at 30, 60, 90 and 120 minutes after glucose administration and the levels of blood glucose was determined.

 

Lipid profile

Plasma was used for the estimation of lipid profile. Cholesterol content was estimated by the method of Parekh and Jung32. Triglyceride was estimated by the method of Rice33. HDL Cholesterol fraction was separated by the precipitation techniques of Burstein and Scholnick34 and the cholesterol content was determined.

 

RESULTS:

Table 1 shows the qualitative analysis of phytochemicals present in the ethanolic extract of Areca Catechu nut. Phytochemical evaluation revealed the presence of alkaloids, flavanoids, carbohydrates, saponins, tannins, phytosterol, terpenoids and phenols.

 

Table 1 Phytochemical screening of A. catechu nut extract

PHYTOCONSTITUENTS

INFERENCE

Alkaloids

+

Flavonoids

+

Carbohydrates

+

Glycosides

-

Saponins

+

Tannins

 

+

Phytosterol

+

Triterpenoids

-

Anthraquinones

+

Phenols

+

 

Table 2, shows the changes of body weight in control and experimental group of rats. Diabetic rats exhibited reduction in body weight. However, diabetic rats treated with the nut extract as well as gliclazide showed an improvement in body weight.

 

Table 2: Effect of A. catechu nut extract on changes in body weight of experimental groups of rats after 30 days treatment.

Groups

Body weight (g)

Initial

Final

Control

166.18 ± 4.04

209.72 ± 5.21

Diabetic

169.36 ± 2.58

147.19 ± 7.35*

Diabetic + A. catechu extract

164.27 ± 3.05

182.56 ± 4.41@

Diabetic + gliclazide

165.54 ± 4.12

191.16 ± 3.36@

Values are given as mean ± SD for groups of six rats in each. Values are statistically significant at p < 0.05. Statistical significance was compared within the groups as follows:  *compared with control, @ compared with diabetic rats.

 

Table 3 shows the changes in the levels of blood glucose, after oral administration of glucose (2g/ kg) in control and experimental rats. The data of OGTT revealed that the blood glucose value in control rats reach peak at 60 minutes after the oral glucose load and gradually return backs to normal levels after 120 minutes. In diabetic control rats, the peak increases in blood glucose concentration was observed after 60 minutes and stayed high over the next 60 minutes. Treatment with Areca Catechu showed definite lower peak blood glucose values, 60 minutes after glucose load also elicit lower values almost at the end of 120 minutes.

 

 


 

Table 3.  Effect of A. catechu nut extract on the blood glucose level (mg/dl) in the experimental groups of rats receiving an oral glucose load.

Groups

Fasting

30 min

60 min

90 min

120 min

Control

93.25 ± 5.16

134.21 ± 6.44

168.77 ± 9.07

139.06 ± 11.14

99.85 ± 9.25

Diabetic

266.69 ± 17.56*

303.33 ± 23.16*

376.48 ± 30.51*

342.37 ± 25.14*

324.46 ± 21.96*

Diabetic + A. catechu extract

150.61 ± 13.76@

190.48 ± 19.24@

243.41 ± 21.93@

198.67 ± 19.55@

160.01 ± 14.99@

Diabetic + gliclazide

141.55 ± 9.25@

172.52 ± 17.04@

224.95 ± 18.18@

176.38 ± 13.89@

155.07 ± 10.51@

Values are given as mean ± SD for groups of six rats in each. Values are statistically significant at p < 0.05. Statistical significance was compared within the groups as follows:  *compared with control, @ compared with diabetic rats.

Table 4 Effect of A. catechu nut extract on the levels of blood glucose, plasma insulin, hemoglobin, glycosylated hemoglobin, and urine sugar in the experimental groups of rats.

Groups

Glucose (mg/dl)

Insulin (µU/ml)

Hemoglobin (g/dl)

Glycosylated hemoglobin (%)

Urine sugar

Control

99.69 ± 10.05

15.88 ± 2.54

14.96 ± 2.48

6.01 ± 1.42

Nil

Diabetic

283.08 ± 22.56*

5.03 ± 2.02*

10.08 ± 1.67*

13.11 ± 1.92*

+++

Diabetic + A. catechu extract

146.42 ± 11.24@

11.51 ± 1.95@

12.47 ± 2.49@

8.12 ± 1.89@

Nil

Diabetic + gliclazide

131.20 ± 14.21@

12.19 ± 1.63@

13.18 ± 2.09@

7.06 ± 2.03@

Nil

Values are given as mean ± SD for groups of six rats in each. Values are statistically significant at p < 0.05. Statistical significance was compared within the groups as follows:  *compared with control, @ compared with diabetic rats.

 

Table 5   Effect of A. catechu nut extract on the levels of liver and muscle glycogen content in the experimental groups of rats.

Groups

Glycogen (mg glucose/g tissue)

Liver

Skeletal muscle

Control

41.42 ± 3.62

7.93 ± 0.82

Diabetic

19.51 ± 2.08*

3.46 ± 0.47*

Diabetic + A. catechu extract

31.71 ± 3.46@

5.57 ± 0.62@

Diabetic + gliclazide

34.98 ± 2.75@

5.90 ± 0.86@

Values are given as mean ± SD for groups of six rats in each. Values are statistically significant at p < 0.05. Statistical significance was compared within the groups as follows:  *compared with control, @ compared with diabetic rats.

 

Table 6 Effect of A. catechu nut extract on the levels of protein, urea, creatinine and uric acid in plasma of  experimental groups of rats.

Groups

Protein (g/dl)

Urea (mg/dl)

Creatinine (mg/dl)

Uric acid (mg/dl)

Control

8.02 ± 1.09

24.81 ± 2.15

1.17 ± 0.13

2.34 ± 0.90

Diabetic

5.65 ± 0.72*

46.53 ± 4.08*

2.96 ± 0.31*

5.36 ± 1.18*

Diabetic + A. catechu extract

6.65 ± 0.88@

31.99 ± 3.12@

1.36 ± 0.18@

3.24 ± 0.86@

Diabetic + gliclazide

7.21 ± 0.84@

29.92 ± 2.98@

1.21 ± 0.12@

2.96 ± 1.10@

Values are given as mean ± SD for groups of six rats in each. Values are statistically significant at p < 0.05. Statistical significance was compared within the groups as follows:  *compared with control, @ compared with diabetic rats.

 


Table 4 depicts the effect of oral administration of A. catechu nut extract on the levels of blood glucose, plasma insulin, hemoglobin, glycosylated hemoglobin, and urine sugar in the  control as well as experimental groups of rats. The elevated levels of blood glucose, glycosylated hemoglobin in the diabetic group of rats were reverted to near normal level by the administration of A. catechu nut. Conversely, the decreased levels of plasma insulin, hemoglobin in diabetic group of rats were elevated by the administration of A. catechu nut extract to diabetic rats for 30 days. Urine sugar which was present in the diabetic group of rats was absent in A. catechu nut extract as well as gliclazide treated diabetic group of rats.

 

Table 5 depicts the level of liver and muscle glycogen content in control and experimental group of rats. The significant decrease in liver and muscle glycogen content were observed in diabetic rats when compared with normal control rats and the level was brought back nearer to normal by oral administration of Areca Catechu nut extract as well as gliclazide.

 

The effect of oral administration of Areca Catechu nut extract on the levels of total protein, urea, uric acid and creatinine are presented in Table 6. The altered levels of these parameters were reverted back to near normalcy upon the treatment with the nut extract.

 

Table 7 depicts the level of serum enzymes such as AST, ALT and ALP in normal control and experimental group of rats. The increased activities of these marker enzymes were reverted back to near normalcy upon the treatment with the nut extract.

 


 

Table 7 Effect of A. catechu nut extract on the activity of AST, ALT and ALP in the serum of experimental groups of rats.

Groups

AST

ALT

ALP

Control

65.42 ± 6.34

18.16 ± 2.39

81.91 ± 10.16

Diabetic

111.99 ± 14.98*

46.28 ± 4.71*

153.26 ± 18.64*

Diabetic + A. catechu extract

93.16 ± 10.04@

24.93 ± 3.62@

100.05 ± 11.78@

Diabetic + gliclazide

80.52 ± 8.31@

20.98 ± 2.02@

98.64 ± 10.11@

 

Table 8   Effect of A. catechu nut extract on the level of TBARS in plasma, pancreas and liver of experimental groups of rats.

Groups

TBARS

Plasma

Pancreas

Liver

Control

4.17 ± 0.69

40.24 ± 4.75

1.70 ± 0.36

Diabetic

8.12 ± 1.61*

78.26 ± 9.44*

4.37 ± 0.70*

Diabetic + A. catechu  extract

5.29 ± 1.16@

58.29 ± 6.81@

2.49 ± 0.31@

Diabetic + gliclazide

5.04 ± 1.02@

55.99 ± 7.43@

2.65 ± 0.39@

Units: mM/100 g in tissues; nM/ml in plasma. Values are given as mean ± SD for groups of six rats in each. Values are statistically significant at p < 0.05. Statistical significance was compared within the groups as follows:  *compared with control, @ compared with diabetic rats.

 

Table 9 Effect of A. catechu nut extract on the activity of SOD, Catalase and GPx, and the level of GSH in pancreas of experimental groups of rats.

Groups

SOD

Catalase

GPx

GSH

Control

5.05 ± 1.69

15.23 ± 2.10

6.37 ± 1.01

26.09 ± 2.65

Diabetic

1.42 ± 0.40*

5.85 ± 1.42*

3.09 ± 0.31*

12.44 ± 1.61*

Diabetic + A. catechu  extract

3.82 ± 0.90@

12.16 ± 1.47@

4.73 ± 0.64@

20.69 ± 2.27@

Diabetic + gliclazide

3.96 ± 0.87@

13.10 ± 1.08@

5.39 ± 0.91@

22.09 ± 2.74@

 

Table 10   Effect of A. catechu nut extract on the activity of SOD, Catalase and GPx, and the level of GSH in liver of experimental groups of rats.

Groups

SOD

Catalase

GPx

GSH

Control

16.04 ± 2.47

70.81 ± 8.31

11.16 ± 1.09

38.19 ± 5.64

Diabetic

5.05 ± 0.71*

30.19 ± 3.54*

3.74 ± 0.69*

21.89 ± 2.82*

Diabetic + A. catechu  extract

10.95 ± 1.22@

61.02 ± 6.09@

8.16 ± 0.89@

31.56 ± 4.08@

Diabetic + gliclazide

12.13 ± 1.50@

65.45 ± 5.95@

9.01 ± 1.04@

32.60 ± 4.59@

 


The enzyme activities are expressed as: AST and ALT µmoles of pyruvate liberated /h/mg of protein; ALP µmoles of phenol liberated/min/mg of protein. Values are given as mean ± SD for groups of six rats in each. Values are statistically significant at p < 0.05. Statistical significance was compared within the groups as follows:  *compared with control, @ compared with diabetic rats.

 

The level of TBARS in plasma, pancreas and hepatic tissues of control and experimental group of rats are presented in table 8. Diabetic rats showed marked increase in TBARS when compared with control rats. Upon treatment of Areca Catechu nut extracts as well as gliclazide to the diabetic rats showed significant decrease in the levels of TBARS when compared with diabetic rats.

 

Table 9 and 10 shows the activity of antioxidant enzymes such as SOD, Catalase, glutathione peroxidase and reduced glutathione in pancreatic and liver tissues respectively in normal control and experimental group of rats. A significant decrease in the activity of antioxidant enzymes was observed in alloxan induced diabetic rats. Upon treatment with ethanolic extract of Areca Catechu nut as well as gliclazide to alloxan induced diabetic rats restored the level of antioxidant enzymes to normal.

 

Activity is expressed as: 50% of inhibition of epinephrine autooxidation/min/mg of protein for SOD; µmoles of hydrogen peroxide decomposed/min/mg of protein for catalase; µmoles of glutathione oxidized/min/mg of protein for GPx; mg/100 g tissue for GSH. Values are given as mean ± SD for groups of six rats in each. Values are statistically significant at p < 0.05. Statistical significance was compared within the groups as follows:  *compared with control, @ compared with diabetic rats.

 

Activity is expressed as: 50% of inhibition of epinephrine autooxidation/min/mg of protein for SOD; µmoles of hydrogen peroxide decomposed/min/mg of protein for catalase; µmoles of glutathione oxidized/min/mg of protein for GPx; mg/100 g tissue for GSH. Values are given as mean ± SD for groups of six rats in each. Values are statistically significant at p < 0.05. Statistical significance was compared within the groups as follows:  *compared with control, @ compared with diabetic rats.

 

The levels of non enzymatic antioxidant such as Vitamin E, Vitamin C, Ceruloplasmin and reduced glutathione in plasma of control and experimental group of rats are shown in Table 11. The diminished levels of non-enzymatic antioxidants in the diabetic group of rats were significantly improved to near normal values by the oral administration of Areca Catechu nut extract as well as gliclazide, after 30 days of treatment.


 

Table 11   Effect of A. catechu nut extract on the levels of vitamin C, vitamin E, ceruloplasmin and GSH in plasma of  experimental groups of rats.

Groups

Vitamin C

Vitamin E

Ceruloplasmin

GSH

Control

1.49 ± 0.15

0.69 ± 0.09

12.29 ± 1.62

30.74 ± 3.99

Diabetic

0.50 ± 0.09*

0.32 ± 0.04*

5.06 ± 0.89*

14.99 ± 2.45*

Diabetic + A. catechu  extract

0.98 ± 0.12@

0.55 ± 0.07@

9.95 ± 1.46@

22.86 ± 2.87@

Diabetic + gliclazide

1.02 ± 0.14@

0.59 ± 0.05@

10.41 ± 1.88@

25.15 ± 3.06@

Units: mg/dl. Values are given as mean ± SD for groups of six rats in each. Values are statistically significant at p < 0.05. Statistical significance was compared within the groups as follows:  *compared with control, @ compared with diabetic rats.

 

Table 12 Effect of A. catechu nut extract on the levels of total cholesterol, triglycerides, LDL-cholesterol and HDL-cholesterol in the plasma of experimental groups of rats.

Groups

Total cholesterol

Triglycerides

LDL

HDL

Control

92.51 ± 11.84

62.29 ± 8.46

50.05 ± 6.12

28.95 ± 2.08

Diabetic

169.12 ± 18.65*

152.07 ± 14.07*

124.45 ± 9.61*

14.64 ± 1.62*

Diabetic + A. catechu extract

103.87 ± 14.18@

90.15 ± 10.05@

72.69 ± 7.49@

22.03 ± 1.78@

Diabetic + gliclazide

97.18 ± 11.92@

84.46 ± 8.09@

60.17 ± 6.59@

23.91 ± 2.11@

Units: mg/dl. Values are given as mean ± SD for groups of six rats in each. Values are statistically significant at p < 0.05. Statistical significance was compared within the groups as follows:  *compared with control, @ compared with diabetic rats.


Table 12 depicts the level of total cholesterol, triglycerides and lipoproteins (LDL and HDL) levels of normal control and experimental group of rats. The elevated levels of lipid and lipoproteins (LDL) and reduced level of HDL cholesterol was observed in diabetic rats than normal control and the level was restored back nearer to the normal value was achieved by administration of Areca Catechu nut extract as well as gliclazide.

 

DISCUSSION:

The therapeutic values of plants lie in phytochemical constituents that exert distinct physiological activities in the human body. Highly coloured vegetables and fruits found to be highest in phytoconstituents, but tea, chocolate, nuts, flax nuts, and olive oil are excellent sources as well. Phytochemical analysis of A. catechu nut extract revealed the presence of alkaloids, flavanoids, carbohydrates, saponins, tannins, phytosterol, terpenoids and phenols. These phytochemicals were speculated to account for the observed pharmacological effects of the extract.

 

Alloxan acts as a cytotoxin for beta-cells of the islet of langerhans, causes diabetes by inducing β cell necrosis35. The reactive oxygen species mediates the cytotoxic action with the increase in cytosolic calcium concentration, leading to rapid beta-cells destruction36. Diabetic rats exhibit gradual weight loss as compared with the normal group. This process is due to muscle wasting and depletion of protein in tissues. A decrease in body weight was observed in diabetic group indicating the increased proteolysis. Diabetic rats treated with Areca Catechu nut extract and gliclazide for 30 days showed a significant improvement in body weight as compared to diabetic animals, which shows the beneficial effects of the extract in controlling the muscular wasting. The results of the present study are in agreement with those of previous reports37.

 

Diabetes mellitus is characterized by decreased glucose tolerance due to low secretion of insulin or its action. Oral glucose tolerance test (OGTT) is a test of immense value in favour of using fasting plasma glucose concentration to facilitate the diagnosis of diabetes mellitus. OGTT revealed that the blood glucose levels in control rats reach peak at 60 minutes after the oral glucose load and gradually return backs to normal levels after 120 minutes. In diabetic control rats, the peak increases in blood glucose concentration was observed after 60 minutes and remained high over the next 60 minutes. However, oral treatment with Areca Catechu showed definite lower peak blood glucose values, 60 minutes after glucose load also gives lower values almost at the end of 120 minutes indicating the improved glucose tolerance

 

Blood glucose estimation provides valuable information about the severity and therapeutic control of diabetes mellitus. In diabetes, the blood glucose levels are drastically elevated which results from reduced glucose utilization by various tissues. Alloxan induction causes specific damage in β cells and thus exerts a pronounced increase in blood glucose concentration. It is well established that gliclazide is used as an antihyperglycemic drug, which increases the insulin secretion from pancreas and it is often used as a standard drug in alloxan induced diabetic models to compare the antidiabetic property of various plant extracts. Oral administration of Areca Catechu nut extract to alloxan induced diabetic rats resulted in reduction in blood glucose level indicating the hypoglycemic nature of the nut extract.

 

 Insulin is the key hormone responsible for metabolizing sugar and preventing the accumulation of glucose in blood stream. In diabetic rats, there is decreased insulin level when compared to control rats. The decreased insulin level depends upon the degree of β cells destruction. Administration of Areca Catechu nut extract as well as gliclazide to alloxan induced diabetic rats shows improved insulin level. This indicates that the Areca Catechu extract has antidiabetic activity by potentiating the stimulation of insulin release from the bound form or from the remnant pancreatic β cell.

 

Glycosylated haemoglobin (HbA1c) is a standard biochemical marker in assessment of diabetes. Glycated haemoglobin is a unique form of haemoglobin used primarily to identify the average plasma glucose concentration over prolonged periods of time. It is formed in non-enzymatic pathway by hemoglobin’s normal exposure to persistent high plasma levels of glucose38. The studies show that HbA1c is an important marker in assessing a patient’s risk of microvascular complications and hypoglycaemia. Hence, measurement of both HbA1c and blood glucose levels are now used in the routine management of diabetic individuals39 .Diabetic rats showed higher levels of glycated hemoglobin indicating their poor glycemic control. There was a significant elevation in the levels of glycosylated haemoglobin, while the level of total haemoglobin decreased during diabetes compared to normal control rats. Oral administration of Areca Catechu extract brought back to near normal indicating the improved glycemic control. Urine sugar which was present in the diabetic groups of rats was found to be absent in the rats treated with extract indicating the improved glucose homeostasis.

 

Liver plays a unique role in controlling carbohydrate metabolism by maintaining glucose concentrations in a normal range over both short and long periods of times. Liver produces glucose by breaking down glycogen (glycogenolysis) and by de novo synthesis of glucose (gluconeogenesis) from non-carbohydrate precursors such as lactate, amino acids and glycerol. In the present study the experimental diabetic rats treated with Areca Catechu nut extract as well as gliclazide treated groups restored the level of hepatic glycogen by means of decreasing the activity of glycogen phosphorylase and increasing the activity of glycogen synthase. It indicates that the defective glycogen storage of the diabetic state was corrected by Areca Catechu which might be due to the improved glucose homeostasis

 

A variety of proteins are subjected to non-enzymatic glycation and contributes to the long-term complications of  diabetes40 . The observed decrease in total protein content in diabetic rats may be ascribed to (i) decreased amino acid uptake; (ii) greatly decreased concentration of variety of essential amino acids; (iii) increased conversion rate of glycogenic amino acids to carbon dioxide and water; and (iv) reduction in protein synthesis secondary to a decreased amount and availability of mRNA41 .

 

Urea is the main end product of protein catabolism in the body. Accumulation of urea nitrogen in experimental diabetes may due to the enhanced breakdown of both liver and plasma proteins42. Diabetes causes disturbance in renal function and blood urea level was significantly increased in diabetic rats. Oral administration of Areca Catechu nut extracts reduced the level of urea in alloxan induced diabetic rats indicating the prophylactic role of the extract in protein metabolism. Our results are in concordance with the recent reports43.

 

Uric acid is one of the main antioxidant present in the body and diabetes causes reduced levels of uric acid44. The extract reflects the antioxidant potential as it reduced the oxidative stress and increase in uric acid level. Diabetic rats exhibited higher uric acid levels indicating the increased oxidative stress. However, oral administration of Areca Catechu nut extract normalized the levels of uric acid indicating the free radical scavenging action of the extract.

 

Creatinine is a byproduct of the breakdown of creatine and phosphocreatine, which are considered as an energy storage compounds in muscle. The serum creatinine concentration may vary based on a number of factors including diet composition, muscle mass and gender. Serum creatinine values also depend on the ability of the kidney to excrete creatinine. An elevation in creatinine usually occurs simultaneously with an increase in blood urea nitrogen. In the present study, the oral treatment with extract significantly reduced the serum creatinine level. Therefore, it may be concluded that the early renal changes occurred in the diabetic rats were significantly improved by the oral administration of the nut extract.

 

Aminotransferases, such as alanine aminotransferase and aspartate aminotransferase measure the concentration of intracellular hepatic enzymes that have leaked into the circulation and serve as a marker of hepatocyte injury. Alkaline phosphatises act as markers of biliary function and cholestasis. It is hypothesized that elevation in ALT, AST and ALP are considered as predictors of diabetes. The elevation of these pathophysiological enzymes may also reflect the damage of the hepatic cells. Kim et al. concluded that the elevation in AST and ALT levels may be due to the destructive changes in the hepatic cells as a result of toxaemia45. On the other hand, other investigators have postulated that diabetes could induce defects in sarcolemmal enzymatic activities46 which leads finally to such effects.  Diabetic rats exhibited higher levels of these serum enzymes indicating the liver damage. Oral administration of Areca Catechu extract to alloxan induced diabetic rats resulted in gradual normalization of the activities of AST, ALT and ALP suggesting the non toxic nature as well as organ protective role of the extract.

 

Free radicals are generated as by-products of normal cellular metabolism; however, several conditions are known to disturb the balance between ROS production and cellular defense mechanisms. This imbalance can result in cell dysfunction and destruction resulting in tissue injury. The intensified free radical production during experimental diabetes resulted in the elevated levels of lipid peroxides and hydroperoxides by oxidative degradation of polyunsaturated fatty acids. Diabetic rats showed marked increase in TBARS when compared with normal rats. Treatment of Areca Catechu nut extracts as well as gliclazide to the diabetic rats showed significant decrease in the levels of TBARS when compared with diabetic rats  indicating the free radical scavenging potential of the extract

 

The increase in the level of ROS in diabetes could be due to their increased production and/ or decreased destruction by nonenzymic and enzymic catalase (CAT), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD)] antioxidants 47. The level of these antioxidant enzymes critically influences the susceptibility of various tissues to oxidative stress and is associated with the development of complications in diabetes. Also this is particularly relevant and harmful for the β cells, which is among those tissues that have the lowest levels of intrinsic antioxidant defenses48. Enzymatic antioxidant activities such as superoxide dismutase, catalase and glutathione peroxidise decrease in liver, kidney and heart tissues of patients with diabetes mellitus49. Oral administration of ethanolic extract of Areca Catechu nut restored the activities of enzymatic antioxidants which reflects the antioxidant potential of the extract.

 

Vitamin C and vitamin E, often referred to as “antioxidant vitamins” that have been suggested to limit oxidative damage in humans and lower the risk of certain chronic diseases such as diabetes mellitus. Vitamin C is a key antioxidant that particularly protects the lipids from peroxidative damage by aqueous solution. Ceruloplasmin is a free radical scavenger as well as a late acute phase reactant protein. It is involved in iron metabolism50. Further, it has also been suggested that the levels of trace elements, especially copper, might be increased in the diabetic state. In the present study, it was found that the levels of non enzymatic antioxidants such as Vitamin E, Vitamin C, ceruloplasmin and reduced glutathione in plasma of alloxan induced diabetic rats was significantly decreased. Oral administration of Areca Catechu nut extracts as well as gliclazide to alloxan induced diabetic rats resulted in a marked increased in the levels of these non enzymatic antioxidants suggesting the free radicals scavenging potential of Areca Catechu nut extract which in turn may be responsible for its anti hyperglycemic property.

 

Diabetes mellitus is associated with higher incidence of dyslipidemia51. DM is associated with a cluster of interrelated plasma lipid and lipoprotein (LP) abnormalities that are all recognized as predictors for coronary heart disease, including reduced plasma levels of high density lipoprotein cholesterol particles and elevated plasma levels of TG 52. The total cholesterol, triglycerides and lipoprotein levels were brought back to normal in Areca Catechu extract given rats when compared to diabetic rats. In the present study, the altered lipid profile was normalized upon treatment with Areca Catechu nut extract exemplifying the hypolipidemic role of the extract.

 

CONCLUSION:

The present study also warrants further studies to isolate and characterising potent molecules for diabetes and its lipids associated complications. In conclusion, the secondary metabolites present in the extract may synergistically act to improve the complications observed in the experimental diabetes. The results of the present study support the use of Areca Catechu nut extract for the treatment of diabetes mellitus. It indicates that the extract possess antidiabetic activity which is evident from Oral glucose tolerance test and other clinically significant biochemical parameters. The antioxidant property of the extract is well established from the improved levels of enzymatic and non enzymatic antioxidants. The hypolipidemic property of the extract is evident from the normalized levels of lipid profile. The data also provides a scientific rationale for the use of Areca nuts in the traditional medicine for the treatment of various ailments.

 

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Received on 02.01.2013

Modified on 15.01.2013

Accepted on 20.01.2013

© A&V Publication all right reserved

Research Journal of Pharmacognosy and Phytochemistry. 5(2): March-April 2013, 100-108