Biochemical evaluation of antidiabetic activity of Piper betel leaves extract in alloxan-induced diabetic rats

 

K. Radhika¹, B. Kumaravel², V. Thamizhiniyan¹, S. Subramanian¹*

¹Department of Biochemistry, University of Madras, Guindy Campus, Chennai – 600025,

²Mahatma Gandhi Medical College and Research Institute, Pondicherry – 607402.

*Corresponding Author E-mail: subbus2020@yahoo.co.in

Globally, majority of cultures directly uses plants as medicines as they are rich sources of drugs. Piper betel Linn. (family Piperaceae) commonly known as the betel vine and has a long history of over 2000 yrs. Betel leaf is traditionally known to be useful for the treatment of various diseases. In the absence of systemic studies in the literature, the present study was aimed to evaluate the antidiabetic potential of Piper betel leaves in alloxan-induced experimental diabetes in rats. The altered levels of biochemical parameters such as blood glucose, plasma insulin, haemoglobin, glycosylated haemoglobin, liver and muscle glycogen content, protein, urea, creatinine, uric aicd, AST, ALT and ALP in the diabetic rats were significantly reverted upon oral administration of Piper betel leaves extract for 30 days. The levels of lipid peroxides in the plasma and pancreas of diabetic rats were elevated significantly and were normalized by the administration of Piper betel leaves extract. The activities of pancreatic enzymic antioxidants and the levels of plasma nonenzymatic antioxidants were markedly declined in the diabetic rats. Upon treatment with leaves extract to diabetic rats, these decreased antioxidant levels were significantly improved. Further, the altered levels of lipid profile were significantly improved upon treatment with leaves extract. The results of the study indicates that Piper betel leaves possess antidiabetic activity and the results were comparable with gliclazide, an oral standard hypoglycemic drug. The phyto-constituents present in the leaves may account for the observed pharmacological properties.

 

 

INTRODUCTION:

Diabetes mellitus is one of the most common chronic metabolic disorders that cause nearly 7% of all worldwide deaths annually.¹ Approximately 366 million people are affected with diabetes in the year 2011, and this is expected to rise to 552 million by 2030.² According to the World Health Organization, the prevalence of known diabetes is 5.6% and 2.7% among urban and rural areas, respectively. Around 80% of people with diabetes are in developing countries, of which India and China share the larger contribution. It is estimated that the total number of people with diabetes in 2010 was around 50.8 million in India, and this will increase to 87.0 million by 2030.³ The global increase in the prevalence of diabetes is due to population growth, aging, urbanization and an increase of obesity and physical inactivity. This could have long-lasting adverse effects on a nation’s health and economy, especially for developing countries.

 

Chronic hyperglycemia in diabetes is associated with long-term damage, dysfunction, and eventually the failure of organs, especially eyes, kidneys, nerves, and the cardiovascular system.⁴ Currently several classes of oral antidiabetic drugs such as sulfonylureas, metformin, thiazolidinediones, α-glucosidase inhibitors, incretins, and DPP-4 inhibitors are available for achieving and maintaining long-term glycaemic control. However, all these drugs are often challenging, and have treatment-limiting side effects. Therefore, a significant need for novel anti-diabetic drugs remains.

 

A majority of cultures around the world directly uses plants as medicines. Medicinal plants are considered as resources of valuable drugs. Many of the modern medicines are derived indirectly from medicinal plants for the treatment of various diseases. Piper betel Linn. (family Piperaceae) commonly known as the betel vine is extensively grown in India, Srilanka, Malaysia, Thailand, Taiwan and other Southeast Asian countries and has a long history of over 2000 yrs.⁵ The plant is much popular in India than in any other country, evident from the numerous citations laid down in the ancient literature, particularly the Indian scriptures. Significance of the leaves has been explained in relation to every sphere of human life including social, cultural, religious and even day-to-day life, which is very much relevant even these days. For example, a well-prepared betel quid is still regarded as an excellent mouth freshener and mild vitalizer.

 

The betel leaves are very nutritive and contain substantial amount of vitamins and minerals. Approximately six leaves with a little bit of slaked lime is said to be comparable to about 300 ml of cow milk particularly for the vitamin and mineral nutrition. Betel leaf is traditionally known to be useful for the treatment of various diseases like Halitosis, boils and abscesses, conjunctivitis, constipation, headache, hysteria, itches, mastitis, mastoiditis, leucorrhoea, otorrhoea, ringworm, swelling of gum, rheumatism, abrasion, cuts and injuries etc,.⁵ Evidences suggest that Piper betel possess various pharmacological properties include anticancer, antimutagenic, anti-amoebic, anti-giardial, anti-inflammatory, mosquito larvicidal, antimicrobial, immunomodulatory, antiulcerogenic, radioprotective, antileishmanial, and antifungal activity.^6-12 In the absence of systemic literature, the present study was aimed to evaluate the antidiabetic activity of Piper betel leaves in alloxan-induced diabetic rats.

 

MATERIALS AND METHODS:

Plant material

Piper betel leaves were collected from Kumbakonam, Thanjavur District, Tamil Nadu, India. The plants were identified and authenticated and a voucher specimen was deposited at the Department of Biochemistry, University of Madras, Chennai.

 

Preparation of plant extract

The Piper betel leaves 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 root was 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 Piper betel leaves were subjected to preliminary phytochemical screening of various plant constituents.¹³

 

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 experimental diabetes in rats

Rats were induced diabetes by single intraperitonial injection of alloxan monohydrate dissolved in sterile normal saline at a dose 120 mg/Kg, after 18 hours fasting to induce hyperglycemia.¹⁴ After 1 hour alloxan administration, 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 grouped into 4 groups, comprising of 6 rats in each group as follows:

Group 1: Control Rats (Water and food ad libitum).

Group 2: Alloxan induced diabetic Rats.

Group 3: Diabetic Rats treated with Piper betel leaves extract (250 mg/Kg Body weight/day) in aqueous solution orally for 30 days.

Group 4: Diabetic Rats treated with gliclazide (5mg/Kg body weight/day) inaqueous solution orally for 30 days.

 

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

 

Plasma insulin was assayed by using ELISA kit for rats. Blood glucose level was estimated by the method of glucose oxidase/ peroxidase as described by Trinder¹⁵; plasma protein by Lowry’s et al.¹⁶; urea by Natelson et al.¹⁷; hemoglobin and glycosylated hemoglobin by the methods of Drabkin and Austin,¹⁸ and Nayak and Pattabiraman,¹⁹ respectively. The levels of creatinine²⁰ and uric acid,²¹ and the activities of AST²², ALT²² and ALP²³ in serum were also assayed.

 

 Oral Glucose Tolerance Test (OGTT)

At the end of the experimental period, fasting blood glucose was monitored after overnight fasting of rats. Then rats were orally administered with glucose solution (2 g/kg bw) and the levels of blood glucose in all the group of rats at 30, 60, 90 and 120 minutes after glucose administration was measured using Glucometer.

Assay of oxidative stress markers and antioxidants in pancreas and plasma

The pancreatic tissues were excised, rinsed in ice-cold saline and 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 and used for the determination of lipid peroxides and enzymatic antioxidants. Lipid peroxides were determined by the method of Ohkawa et al.²⁴ Enzymatic antioxidants such as superoxide dismutase,²⁵ catalase,²⁶ and glutathione peroxidase²⁷ in pancreatic supernatant were assayed. Further, the levels of lipid peroxides, and the non-enzymatic antioxidants such as vitamin C, vitamin E, ceruloplasmin and glutathione (GSH) in plasma were determined by the methods of Omaye et al.²⁸; Desai²⁹; Ravin³⁰; Sedlak and Lindsay,³¹ respectively.

 

Determination of Liver and muscle glycogen content

The level of glycogen content in liver and muscle was estimated as described by Morales et al. (1973).³² Briefly, Glycogen was precipitated from the alkali extract of the tissues by adding 1:3 volume of 95% ethanol and a drop of 1 M ammonium acetate and was kept in a boiling water bath for 5 min. After cooling, the samples were shaken and placed in a freezer overnight. The precipitated glycogen was then collected by centrifugation at 3,000g for 40 min. The precipitate was dissolved in water, then precipitated with alcohol and centrifuged again. The final precipitate was dissolved in water and heated for 5 min in a boiling water bath. Then the samples were cooled in an ice-bath, anthrone reagent was added and heated for 20 min in a boiling water bath. Again the samples were cooled to room temperature and the green colour developed was read at 640 nm in a Shimadzu spectrophotometer. The levels of glycogen were expressed as mg of glucose/g of wet tissue.

 

Assay of plasma lipid profile

Cholesterol content was estimated by the method of Parekh and Jung.³³ Triglyceride was estimated by the method of Rice.³⁴ HDL Cholesterol fraction was separated by the precipitation techniques of Burstein and Scholnick and the cholesterol content was determined.³⁵

 

RESULTS:

The presence of phytochemical constituents such as alkaloids, flavanoids, saponins, tannins, phytosterol, Anthraquinones and phenols in the ethanolic extract of Piper betel leaves depicted in Table1.

 

Table 1   Phytochemical screening of Piper betel leaves extract

Table 2    Effect of P. betel leaves extract on changes in body weight of experimental groups of rats after 30 days treatment.

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.

 

Graph 1 represents the levels of blood glucose at fasting and up to 2 h, after oral administration of glucose (2g/ kg) in control and experimental groups of rats. The results revealed that blood glucose values in control rats reach peak at 60 minutes after the oral glucose load and gradually return backs to basal levels at the end of 120 minutes. In diabetic rats, the blood glucose concentration was significantly greater than the control values at 30 min and peak 60 minutes, and remains higher even over the next 60 minutes indicates impaired glucose tolerance. Treatment with leaves extract to diabetic rats showed improved glucose tolerance.

 

Graph 1. Effect of Piper betel leaves extract on the blood glucose level in the experimental groups of rats receiving an oral glucose load.

 

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 depicts the effect of Piper betel leaves extract on the levels of blood glucose, plasma insulin, hemoglobin, glycosylated hemoglobin, and urine sugar in the experimental groups of rats. The elevated levels of blood glucose, glycosylated hemoglobin in the diabetic group of rats were significantly reverted by the administration of Piper betel leaves extract. Conversely, the decreased levels of plasma insulin, hemoglobin in diabetic group of rats were elevated by the administration of extract compared to normal rats. Urine sugar which is present in the diabetic group of rats was absent in extract as well as gliclazide treated diabetic group of rats.

 

Table 3.  Effect of Piper betel leaves extract on the levels of blood glucose, plasma insulin, hemoglobin, glycosylated hemoglobin, and urine sugar in the experimental groups of rats.

 

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 Piper betel leaves extract on the levels of protein, urea, creatinine and uric acid in plasma of experimental groups of rats.

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 Piper betel leaves extract on the activity of AST, ALT and ALP in the serum of experimental groups of rats.

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 effect of oral administration of Piper betel leaves extract on the levels of total protein, urea, uric acid and creatinine are presented in Table 4. The altered levels of these parameters were reverted back to near normalcy upon the treatment with the fruit extract.   

 

Table 5 depicts the level of activities of serum enzymes such as AST, ALT and ALP in normal control and experimental group of rats. The increased levels of these enzymes were reverted back to near normalcy upon the treatment with the fruit extract.

 

 

The level of TBARS in plasma and pancreas of control and experimental group of rats are presented in Table 6. Diabetic rats showed marked increase in TBARS when compared with control rats. Upon treatment of Piper betel leaves extract as well as gliclazide to the diabetic rats showed significant decrease in the levels of TBARS when compared with diabetic rats.

 

Table 6. Effect of Piper betel leaves extract on the level of TBARS in plasma and pancreas of experimental groups of rats.

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 7 shows the levels of activities of antioxidant enzymes such as SOD, Catalase, and glutathione peroxidase in pancreatic tissues of normal control and experimental group of rats. A significant decrease in the level of antioxidant enzymes was observed in alloxan induced diabetic rats. Upon treatment with ethanolic extract of Piper betel leaves extract as well as gliclazide to alloxan induced diabetic rats restored the level of antioxidant enzymes to normal.

 

Table 7. Effect of Piper betel leaves extract on the activity of SOD, Catalase and GPx, in pancreas of experimental groups of 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. 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 8. 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 Piper betel leaves extract as well as gliclazide, after 30 days of treatment.

Table 8. Effect of Piper betel leaves extract on the levels of vitamin C, vitamin E, ceruloplasmin and GSH in plasma of experimental groups of rats.

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 9 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 Piper betel leaves extract as well as gliclazide.

 

Table 9. Effect of Piper betel leaves extract on the levels of liver and muscle glycogen content in the experimental groups of rats.

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 10 depicts the levels of total cholesterol, triglycerides and lipoproteins (LDL and HDL) cholesterol levels of normal control and experimental group of rats. The elevated levels of total cholesterol, triglycerides and LDL-cholesterol and reduced level of HDL-cholesterol was observed in diabetic rats was restored back nearer to the normal after oral administration of Ficus Piper betel leaves extract as well as gliclazide.

 

DISCUSSION:

The symptoms observed in diabetic rats include polydipsia, polyphagia, glycosuria and loss in body weight could be due to dehydration and catabolism of proteins.³⁶ Increased catabolic reactions leading to muscle wasting might also be the cause for the observed reduced weight gain.³⁷ Oral administration of Piper betel leaves extract for 30 days to diabetic rats significantly improved the body weight. This indicates the beneficial role of Piper betel leaves in diabetes.

Chronic impairment in glucose homeostasis contributes to the progression of vascular complications during diabetes.³⁸ Effective glycemic control plays vital role in early intervention and prevention of diabetic complications.³⁹ In the present study, oral administration of leaves extract to diabetic rats significantly improved the glucose tolerance. Further, the altered levels of FBG, Hb, HbA1c and glycogen content in liver and muscle tissues were significantly improved in diabetic rats upon treatment with leaves extract indicates the antihyperglycemic nature of the Piper betel leaves.

 

Insulin, a hormone secreted by pancreatic beta cells improves peripheral tissues glucose uptake, utilization and storage thereby maintains blood glucose level within a narrow range. It has been demonstrated that insulin deficiency and or resistance to the action of insulin in diabetes leads to disruption in carbohydrate, lipid and protein metabolism. Alloxan and streptozotocin-induced diabetes is characterized by apoptosis of pancreatic beta cells leading to decreased systemic levels of insulin, which ultimately results in hyperglycemia.⁴⁰ In the present study, increase in plasma insulin levels of diabetic rats treated with leaves extract might be due to improved glucose stimulated insulin secretion from remnant beta cells or regeneration of pancreatic beta cells. 

 

Hyperglycemia mediated oxidative stress is a major contributory factor for the progression of diabetic complications.⁴¹ The most commonly recognized effect of oxidative stress is the oxidation and damage of macromolecules such as proteins, lipids, nucleic acids, thereby contributing to cellular injury, energetic deficit, and the acceleration of cell death. In diabetes, significant increases in the levels of lipoperoxidation products and decrease of both enzymatic and nonenzymatic antioxidants have been reported, and the presence of oxidative stress has been judged by these indices. Under normal physiological conditions the levels of ROS is balanced by the systemic antioxidant defense.

 

Table 10. Effect of Piper betel leaves extract on the levels of total cholesterol, triglycerides, LDL-cholesterol and HDL-cholesterol in the plasma of experimental groups of rats.

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.

 

The most efficient cellular enzymatic antioxidants are superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase, while non-enzymatic antioxidants include vitamin C, vitamin E, and glutathione. These antioxidant defenses are extremely essential since they represent the direct removal of free radicals, thereby protection of tissues from oxidative damage. Oral administration of leaves extract significantly reduced the levels of lipid peroxides in plasma and pancreatic tissues and improved the levels of activities of SOD, Catalase, GPx, and vitamin C, vitamin E, GSH and ceruloplasmin indicating the antioxidant potential of leaves. 

 

The levels of activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatise (ALP) in serum generally predict the severity of liver damage. Because these enzymes are found in liver cells and can leak into the blood if the hepatocytes are damaged. The serum increase in aminotransferases levels in diabetes is due to the cellular damage caused by glucotoxicity. Leibovitch et al. (1991) observed increased levels of serum ALP in pathological conditions involving the kidneys and liver.⁴² The observed significantly decreased activities of AST, ALT and ALP in diabetic rats treated with extract compared with the diabetic control group indicating the tissue protective nature of Piper betel leaves.

 

During diabetes, there is an increased protein catabolism with inflow of amino acids to liver, which feed gluconeogenesis and accelerate ureagenesis, resulting in hypoproteinemia and hypoalbuminemia.⁴³ Consequently, the levels of protein and urea were found to be altered in diabetic rats. In diabetes, renal vein thrombosis and fibrin degradation causes significant decrease in glomerular filtration rate (GFR), and increase in blood uric acid and creatinine. The change in serum creatinine concentration strongly suggested impairment of kidney function in diabetes. An observed decrease in the levels of total protein and increased levels of urea, uric acid and creatinine in alloxan-induced diabetic rats were significantly altered upon treatment with Piper betel leaves extract and this may be due to the protection of renal tissues from glucotoxicity.

 

Diabetes mellitus is also associated with hyperlipidemia with profound alteration in the concentration and composition of lipids.⁴⁴ Changes in the concentrations of the lipids with diabetes mellitus contribute to the development of vascular complications.⁴⁵ The elevated level of serum lipids in diabetes mellitus is mainly due to an increase in the mobilization of free fatty acids from the peripheral fat depots, since insulin inhibits the hormone sensitive lipase. The marked hyperlipidaemia that characterizes the diabetic state may therefore be regarded because of the uninhibited actions of lipolytic hormones on the fat depots.⁴⁶ Excess of fatty acids in plasma promotes the liver conversion of fatty acids to TG, phospholipids and cholesterol. These substances are discharged into the blood results in dyslipidemia.⁴⁷ The elevated levels of cholesterol, triglycerides and LDL- cholesterol and reduced level of HDL-cholesterol observed in diabetic rats were restored significantly after oral administration of Piper betel leaves extract exemplify the antidysplipidemic property of the leaves.

 

In conclusion, the observed normalization in the glucose tolerance, antioxidant status, lipid profile in diabetic rats evidences the antidiabetic property of Piper betel leaves. The phyto-constituents present in the leaves might account for the pharmacological action. The results of the present study shed evidence for the use of betel leaves in the traditional medicine.  Further, studies are in progress to isolate the bioactive principles responsible for the observed antidiabetic activity.

 

REFERENCES:

1.        International Diabetes Federation, IDF Diabetes Atlas, International Diabetes Federation, Brussels, Belgium, 5th edition, 2011.

2.        Whiting DR, Guariguata L, Weil C and Shaw J. IDF diabetes atlas: global estimates of the prevalence of diabetes for 2011 and 2030. Diabetes research and clinical practice. 94(3); 2011: 311-321.

3.         Shaw JE, Sicree RA and Zimmet PZ. Global estimates of the prevalence of diabetes for 2010 and 2030. Diabetes research and clinical practice. 87(1); 2010: 4-14.

4.        American Diabetes Association, Diagnosis and classification of diabetes mellitus. Journal of Diabetes Care. 33(supplement 1); 2010: S62–S69.

5.        Guha P. Betel Leaf: The Neglected Green Gold of India.  Journal of Human Ecology. 19(2); 2006: 87-93.

6.        Rai MP, Thilakchand KR, Palatty PL, Rao P, Rao S, Bhat HP and Baliga MS. Piper betel Linn (betel vine), the maligned Southeast Asian medicinal plant possesses cancer preventive effects: time to reconsider the wronged opinion. Asian Pacific journal of cancer prevention. 12(9); 2011: 2149-2156.

7.        Kanjwani DG, Marathe TP, Chiplunkar SV and Sathaye SS. Evaluation of immunomodulatory activity of methanolic extract of Piper betel. Scandinavian journal of immunology. 67(6); 2008: 589-593.

8.        Nair R and Chanda S. Antimicrobial Activity of Terminalia catappa, Manilkara zapota and Piper betel Leaf Extract. Indian journal of pharmaceutical sciences. 70(3); 2008: 390-393.

9.        Chang MC, Uang BJ, Tsai CY, Wu HL, Lin BR, Lee CS, Chen YJ, Chang CH, Tsai YL, Kao CJ and Jeng JH. Hydroxychavicol, a novel betel leaf component, inhibits platelet aggregation by suppression of cyclooxygenase, thromboxane production and calcium mobilization. British journal of pharmacology. 152(1); 2007: 73-82.

10.     Bhattacharya S, Banerjee D, Bauri AK, Chattopadhyay S, Bandyopadhyay SK. Healing property of the Piper betel phenol, allylpyrocatechol against indomethacin-induced stomach ulceration and mechanism of action. World journal of gastroenterology. 13(27); 2007: 3705-3713.

11.     Kumarasinghe SP, Karunaweera ND, Ihalamulla RL, Arambewela LS and Dissanayake RD. Larvicidal effects of mineral turpentine, low aromatic white spirits, aqueous extracts of Cassia alata, and aqueous extracts, ethanolic extracts and essential oil of betel leaf (Piper betle) on Chrysomya megacephala. International journal of dermatology. 41(12); 2002: 877-880.

12.     Chou CC and Yu RC. Effect of Piper betle L. and its extracts on the growth and aflatoxin production by Aspergillus parasiticus. Proceedings of the National Science Council, Republic of China. Part B. 8(1); 1984: 30-35.

13.     Harborne JB. Phytochemical methods. A guide to modern techniques of plant analysis. Chapman and Hall, New York, 1998; 3rd ed.

14.     Yamamoto H, Uchigata Y and Okamoto H. Streptozotocin and alloxan induce DNA strand breaks and poly(ADP-ribose) synthetase in pancreatic islets. Nature. 294(5838); 1981: 284-286.

15.     Trinder P. Determination of glucose in blood using glucose oxidase with an alternate oxygen acceptor. Annuals of Clinical Biochemistry. 6; 1969: 24-27.

16.     Lowry OH, Rosebrough NJ, Farr AL and Randall RJ, Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry. 193 (1); 1951: 265-275.

17.     Natelson S, Scott Ml and Beffa C. A rapid method for the estimation of urea in biologic fluids. American Journal of Clinical Pathology. 21 (3); 1951: 275-281.

18.     Drabkin DL and Austin JH. Spectrophotometric constants for common hemoglobin derivatives in human, dog and rabbit blood. The Journal of Biological Chemistry. 98; 1932: 719-733.

19.     Nayak SS and Pattabiraman TN. A new colorimetric method for the estimation of glycosylated haemoglobin. Clinica Chimica Acta. 109 (3); 1981: 267-274.

20.     Brod J and Sirota JH, The renal clearance of endogenous creatinine in man. The Journal of Clinical Investigation. 27 (5); 1948: 645-654.

21.     Caraway WT. Determination of uric acid in serum by a carbonate method. American Journal of Clinical Pathology. 25 (7); 1955: 840-845.

22.     King J. The transaminases: alanine and aspartate transaminases. In: Practical clinical enzymology. Van Nostrand Reinhold, London, (1965a); 199-208.

23.     King J. The hydrolases - acid and alakaline phosphatises. In: Practical clinical enzymology. Van Nostrand Reinhold, London, (1965b) 199-208.

24.     Ohkawa H, Ohishi N and Vagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry. 95; 1979: 351-358.

25.     Misra HP and Fridrovich T. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. The Journal of Biological Chemistry. 247; 1972: 3170-3175.

26.     Takahara S, Hamilton HB, Neel JV, Kobara TY et al, Hypocatalasemia: a new genetic carrier state. Journal of Clinical Investigation. 39; 1960: 610-619.

27.     Rotruck JT, Pope AL, Ganther HE, Swanson AB et al, Selenium: biochemical role as a component of glutathione peroxidise. Science. 179; 1973: 588-590.

28.     Omaye ST, Turnbull JD and Sauberlich HE. Selected methods for the determination of ascorbic acid in animal cells, tissues, and fluids. Methods in Enzymology. 62; 1979: 3- 11.

29.     Desai JD In: Parker (ed), Methods in enzymology, vol. 105, Academic Press, New York, 1984, pp.138.

30.     Ravin HA.  An improved colorimetric enzymatic assay of ceruloplasmin, The Journal of Laboratory and Clinical Medicine. 58; 1961: 161-168.

31.     Sedlak J and Lindsay RH, Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Analytical Biochemistry. 25; 1968: 192–205.

32.     Morales MA, Jabbagy AJ and Terenizi HR. Mutations affecting accumulation of Neurospora glycogen. News letter. 20; 1973: 24-25.

33.     Parekh AC and Jung DH. Cholesterol determination with ferric acetate-uranium acetate and sulphuric acid ferrous sulphate reagents. Analytical Chemistry. 42; 1970: 1423- 1427.

34.     Rice EW. In: Roedrick P and McDonal RP, editors, Standard methods in clinical chemistry. Academic Press, New York, 1970, pp. 215.

35.     Burstein M, Scholnick HR and Morfin R.  Rapid method for the isolation of lipoproteins from human serum by precipitation with polyanions, The Journal of Lipid Research. 11; 1970: 583-595.

36.     Hakim ZS, Patel BK and Goyal RK. Effects of chronic ramipril treatment in streptozotocin-induced diabetic rats. Indian journal of physiology and pharmacology. 41(4); 1997: 353-360.

37.     Rajkumar L, Srinivasan N, Balasubramanian K and Govindarajulu P. Increased degradation of dermal collagen in diabetic rats. Indian journal of experimental biology. 29(11); 1991: 1081-1083.

38.     Ceriello A. Impaired glucose tolerance and cardiovascular disease: the possible role of post-prandial hyperglycemia. American heart journal. 147(5); 2004: 803-807.

39.     Ratner RE. Glycemic control in the prevention of diabetic complications. Clinical cornerstone. 4(2); 2001: 24-37.

40.     Lenzen S. The mechanisms of alloxan- and streptozotocin-induced diabetes. Diabetologia. 51(2); 2008: 216-226.

41.     Farhangkhoee H, Khan ZA, Barbin Y and Chakrabarti S. Glucose-induced up-regulation of CD36 mediates oxidative stress and microvascular endothelial cell dysfunction. Diabetologia. 48(7); 2005: 1401-1410.

42.     Leibovitch I, Ben-Chaim J, Ramon J and Goldwasser B. Increased serum alkaline phosphatase activity: a possible indicator of renal damage. Journal of clinical laboratory analysis. 5(6); 1991: 406-409.

43.     Exton JH, Corbin JG and Harper SC. Control of gluconeogenesis in liver. V. Effects of fasting, diabetes, and glucagon on lactate and endogenous metabolism in the perfused rat liver. The Journal of biological chemistry. 247(16); 1972: 4996-5003.

44.     Odetola AA, Akinloye O, Egunjobi C, Adekunle WA and Ayoola AO. Possible antidiabetic and antihyperlipidaemic effect of fermented Parkia biglobosa (JACQ) extract in alloxan-induced diabetic rats. Clinical and experimental pharmacology & physiology. 33(9); 2006: 808-812.

45.     Howard BV, Savage PJ, Bennion LJ and Bennett PH. Lipoprotein composition in diabetes mellitus. Atherosclerosis. 30(2); 1978: 153-162.

46.     al-Shamaony L, al-Khazraji SM and Twaij HA. Hypoglycaemic effect of Artemisia herba alba. II. Effect of a valuable extract on some blood parameters in diabetic animals. Journal of ethnopharmacology. 43(3); 1994: 167-171.

47.     Goldberg IJ. Clinical review 124: Diabetic dyslipidemia: causes and consequences. The Journal of clinical endocrinology and metabolism. 86(3); 2001: 965-971.

 

 

Received on 02.01.2013         Modified on 14.01.2013

Accepted on 17.01.2013         © AJRC All right reserved