Effect of Cassia auriculata flowers on blood sugar levels, serum and tissue lipids in streptozotocin diabetic rats

Tags: diabetic rats, Singapore Med, lipid metabolism, body weight, glycosylated haemoglobin, flowers, pentose phosphate pathway, World Health Organisation, metabolic derangements, beneficial effect, diabetes, hormone sensitive lipase, Streptozotocin, blood glucose, Annamalai University, lipid, diabetic state, Diabetes mellitus, Cassia Auriculata, Blood Sugar Levels, serum lipids, Uma Maheswari J. Hypoglycemic, Clin Chem Acta, Clin Chem, Pari L. Hypoglycemic, Galdieri M. Multiple, animal tissues, J Biol Chem, Bruni C. Determination, J Ethnopharmacol, Bogle GJ., glucose metabolism, colorimetric method, carbohydrate metabolism, free fatty acids, Clin Med, NADPH, fat metabolism, cellular activity
Content: Singapore Med J 2002 Vol 43(12) : 617-621
ORIGINAL ARTICLE
Effect of Cassia Auriculata Flowers on Blood Sugar Levels, Serum and Tissue Lipids in Streptozotocin Diabetic Rats L Pari, M Latha
ABSTRACT Aim of the study: The main aim was to demonstrate the effects of Cassia auriculata flowers on blood glucose and lipid levels in experimental diabetic rats. Methodology: Aqueous extract of Cassia auriculata flowers was administered orally and different doses of the extract on blood glucose, haemoglobin, glycosylated haemoglobin, serum and tissue lipids, hexokinase and glucose6-phosphatase in streptozotocin-induced diabetic rats were studied. Glibenclamide was used as standard reference drug. Results: Cassia auriculata flower extract (CFEt), at doses of 0.15, 0.30 and 0.45 g/kg body weight for 30 days, suppressed the elevated blood glucose and lipid levels in diabetic rats. Cassia auriculata at 0.45 g/kg was found to be comparable to glibenclamide. Conclusion: Our findings indicate that the Cassia auriculata flowers possess antihyperlipidaemic effect in addition to antidiabetic activity. Keywords: Blood glucose, Cassia auriculata, Carbohydrate enzymes, Insulin, Lipids Singapore Med J 2002 Vol 43(12):617-621 INTRODUCTION Diabetes mellitus is characterised by hyperglycaemia together with biochemical alterations of glucose and lipid metabolism(1). Liver is an insulin dependent tissue, which plays a pivotal role in glucose and lipid homeostasis and is severely affected during diabetes(2). Liver participates in the uptake, oxidation and metabolic conversion of free fatty acids, synthesis of cholesterol, phospholipids and triglycerides. During diabetes a profound alteration in the concentration and composition of lipid occurs(3). Decreased glycolysis, impeded glycogenesis and increased gluconeogenesis are some of the changes of glucose metabolism in the diabetic liver(4).
Many traditional plant treatments for diabetes mellitus are used throughout the world(5). Few of the traditional plant treatments for diabetes have received scientific scrutiny, and the World Health Organisation has recommended that this area warrants attention(6). This paper describes the study of Cassia auriculata L. (Cesalpinaceae, COMMON NAME: Tanner's Cassia) a common plant in Asia, has been widely used in traditional medicine as a cure for rheumatism, conjunctivitis and diabetes(7). In addition, Cassia auriculata has been widely used in Ayurvedic medicine as `Avarai Panchaga Choornam' and the main constituent of Kalpa herbal tea, has come under extensive study in the light of its antidiabetic effects. We have recently reported the antiperoxidative effect of Cassia auriculata flowers in streptozotocin diabetic rats(8). This study was thus initiated with the aim of evaluating the effects of an aqueous extract of Cassia auriculata flowers on the blood glucose level, serum and tissue lipids in streptozotocin diabetic rats. MATERIALS AND METHODS Animals All the experiments were carried out with male Wistar rats aged seven to eight weeks (180-200 g), obtained from the Central Animal House, Rajah Muthiah Medical College, Annamalai University, India. The animals were housed in polypropylene cages and provided with water and standard pellet diet (Karnataka Agro Food Corporation Limited, Bangalore, India) ad libitum. The animals used in the present study were approved by the ethical committee, Annamalai University. Chemicals Streptozotocin was obtained from Himedia Laboratory Limited, Mumbai, India. All Other reagents used were of analytical grade. Plant Material Cassia auriculata flowers were collected freshly from Neyveli, Cuddalore District, Tamil Nadu, India. The
Department of Biochemistry Faculty of Science Annamalai University Annamalai Nagar Tamil Nadu-608 002 India L Pari, MSc, MPhil, PhD Reader M Latha, MSc, MPhil, PhD Scholar Correspondence to: Dr L Pari Tel: +914144 38343 Fax: +914144 22265 Email: [email protected] sancharnet.in
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Table I. Blood glucose, plasma insulin, total haemoglobin, glycosylated haemoglobin, changes in body weight and urine sugar of normal and experimental animals.
Groups
Body weight (g)
Initial
Final
Fasting Blood Glucose (mg/dl)
Plasma insulin (µU/ml)
Haemoglobin (g/dl)
Glycosylated haemoglobin (mg/gHb)
Urine sugarA
Normal Diabetic control Diabetic + Cassia auriculata (0.15g/kg)
196 ± 10.40 201 ± 15.70 193 ± 17.70
208 ± 9.80 151 ± 13.66··· 198 ± 15.33***
97.50 ± 8.04a 232.00 ± 15.40b 216.66 ± 20.80b
16.03 ± 1.04a 4.35 ± 0.95b 4.90 ± 0.41b
12.85 ± 0.72a 5.60 ± 0.45b 6.91 ± 0.61c
0.22 ± 0.01a 0.81 ± 0.07b 0.68 ± 0.03c
Nil +++ ++
Diabetic + Cassia auriculata (0.30g/kg) Diabetic + Cassia auriculata (0.45g/kg) Diabetic + Glibenclamide (600 µg/kg)
198 ± 18.30 202 ± 19.68 195 ± 11.80
208 ± 10.32*** 214 ± 12.72*** 206 ± 13.43***
158.60 ± 14.20c 113.3 ± 10.30ad 124.6 ± 10.32d
7.05 ± 0.64c 14.16 ± 0.67d 12.70 ± 0.65e
9.54 ± 0.93d 11.5 ± 0.91e 10.36 ±1.01d
0.48 ± 0.04d
+
0.37 ± 0.04e
NIL
0.47 ± 0.04d TRACE
Values are given as mean ± S.D for six rats in each group. Values not sharing a common superscript letter differ significantly at p<0.05 (DMRT). Duncan procedure, Range for the level 2.89, 3.03, 3.13, 3.20, 3.25. Diabetic control was compared with normal, ··· p<0.001. experimental groups were compared with diabetic control, *** p<0.001. A - Indicates 0.25% sugar and (+ + +) indicates more than 1% sugar.
Table II. Changes in levels of cholesterol, free fatty acids, triglycerides and phospholipids in serum of normal and experimental animals.
Groups
Cholesterol (mg/100 ml)
Free fatty acids (mg/100 ml)
Triglycerides (mg/100 ml)
Phospholipids (mg/100 ml)
Normal Diabetic control Diabetic + Cassia auriculata (0.45 g/kg) Diabetic + Glibenclamide (600 µg/kg)
74.00 ± 1.49a 98.66 ± 4.03b 83.46 ± 2.18c 90.26 ± 1.37d
69.43 ± 4.06a 83.86 ± 6.67b 75.06 ± 1.55c 78.51 ± 0.87d
44.53 ± 3.36a 62.83 ± 1.50b 53.93 ± 2.70c 58.46 ± 1.70d
80.25 ± 1.57a 98.75 ± 4.28b 85.50 ± 2.86c 90.00 ± 2.12d
Values are given as mean ± S.D for six rats in each group. Values not sharing a common superscript letter differ significantly at p<0.05 (DMRT). Duncan procedure, Range for the level 2.95, 3.09, 3.20.
plant was identified and authenticated at the Herbarium of Botany Directorate in Annamalai University. A voucher specimen (No.231) was deposited in the Botany Department of Annamalai University. Preparation of plant extract Five hundred g of Cassia auriculata flowers were extracted with 1,500 ml of water by the method of continuous hot extraction at 60єC for six hours and evaporated. The residual extract was dissolved in water and used in the study(9). Induction of experimental diabetes A freshly prepared solution of streptozotocin (45 mg/kg i.p) in 0.1 M citrate buffer, pH 4.5 was injected intraperitoneally in a volume of 1 ml/kg. After 48 hours of streptozotocin administration, rats with moderate diabetes having glycosuria and hyperglycaemia (i.e. with a blood glucose of 200300 mg/dl) were taken for the experiment(10). Experimental procedure In the experiment, a total of 36 rats (30 diabetic
surviving rats, six normal rats) were used. The rats were divided into six groups of six rats each. Group 1: Normal untreated rats. Group 2: Diabetic control rats given 1 ml of aqueous solution daily using an intragastric tube for 30 days. Group 3: Diabetic rats given CFEt (0.15 g/kg body weight) in 1ml of aqueous solution daily using an intragastric tube for 30 days. Group 4: Diabetic rats given CFEt (0.30 g/kg body weight) in 1 ml of aqueous solution daily using an intragastric tube for 30 days. Group 5: Diabetic rats given CFEt (0.45 g/kg body weight) in 1 ml of aqueous solution daily using an intragastric tube for 30 days. Group 6: Diabetic rats given glibenclamide (600 µg/ kg body weight)(11) in 1 ml of aqueous solution daily using an intragastric tube for 30 days. At the end of 30 days, the animals were deprived of food overnight and sacrificed by decapitation. Blood was collected in two different tubes (i.e.,) one with anticoagulant- potassium oxalate and sodium fluoride for plasma and another without
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Table III. Changes in levels of cholesterol, free fatty acids, triglycerides and phospholipids in liver of normal and experimental animals.
Groups
Cholesterol (mg/100 g wet tissue)
Free fatty acids (mg/100 g wet tissue)
Triglycerides (mg/100 g wet tissue)
Phospholipids (g/100 g wet tissue)
Normal
329.04 ± 2.88a
607.70 ± 30.68a
347.88 ± 13.04a
1.66 ± 0.11a
Diabetic control
512.70 ± 5.88b
915.22 ± 50.27b
621.35 ± 8.40b
2.54 ± 0.08b
Diabetic + Cassia auriculata (0.45 g/kg) Diabetic + Glibenclamide (600 µg/kg)
420.14 ± 4.40c 441.98 ± 5.36d
774.09 ± 46.86c 806.67 ± 25.30c
442.98 ± 13.05c 530.19 ± 11.70d
2.02 ± 0.05c 2.29 ± 0.10d
Values are given as mean ± S.D for six rats in each group. Values not sharing a common superscript letter differ significantly at p<0.05 (DMRT). Duncan procedure, Range for the level 2.95, 3.09, 3.20.
Table IV. Changes in activities of hexokinase and glucose-6-phosphatase in liver of normal and experimental animals.
Groups
Hexokinase (unitsA/g protein)
Glucose- 6-phosphatase (unitsB/mg protein)
Normal Diabetic control Diabetic + Cassia auriculata (0.45 g/kg) Diabetic + Glibenclamide (600 µg/kg)
146.66 ± 6.09a 107.48 ± 5.74b 128.70 ± 9.44c 123.20 ± 5.40c
0.168 ± 0.013a 0.242 ± 0.023b 0.186 ± 0.011ac 0.200 ± 0.008c
Values are given as mean ± S.D for six rats in each group. Values not sharing a common superscript letter differ significantly at p<0.05 (DMRT). Duncan procedure, Range for the level 2.95, 3.09, 3.20. A - µ moles of glucose phosphosylated/min. B - µ moles of Pi liberated/min.
anticoagulant for serum separation. Plasma and serum were separated by centrifugation. Liver was immediately dissected out, washed in ice cold saline, patted dry and weighed. Analytical Procedure Fasting blood glucose was estimated by O-toluidine method (Sasaki et al)(12). Plasma insulin level was assayed by Enzyme Linked Immunosorbent Assay (ELISA) kit, using human insulin as standard. Haemoglobin was estimated by the method of Drabkin and Austin(13) and glycosylated haemoglobin by the method of Sudhakar Nayak and Pattabiraman(14). Lipids were extracted from serum and tissues by the method of Folch et al(15). Total cholesterol and triglycerides were estimated by the method of Zlatkis et al(16) and Foster and Dunn(17) respectively. Free fatty acids and phospholipids were analysed by the method of Falholt et al(18) and Zilversmit et al(19). Hexokinase and glucose-6-phosphatase were assayed by the method of Brandstrup et al(20) and Koida and Oda(21). statistical analysis All values were expressed as the mean obtained from a number of experiment (n). Data from all the tables of normal animals, diabetic control animals, reference drug treated and CFEt treated animals were compared
by ANOVA followed by Duncan's Multiple Range Test (DMRT)(22). RESULTS Blood glucose and Plasma insulin Table I shows the levels of blood glucose, plasma insulin, total haemoglobin, glycosylated haemoglobin, changes in body weight and urine sugar of normal and experimental rats. There was a significant elevation in blood glucose and glycosylated haemoglobin levels, while the plasma insulin and total haemoglobin levels decreased significantly in streptozotocin diabetic rats when compared with normal rats. Administration of CFEt and glibenclamide tends to bring the parameters significantly towards the normal. The effect of CFEt at a dose of 0.45 g/kg body weight was more highly significant than 0.15 and 0.30 g/kg body weight and therefore the dose was used for further biochemical studies. In diabetic rats, the urine sugar was (+++) but in the case of CFEt treated rats at a dose of 0.15 and 0.30 g/kg body weight showed decreased urine sugar (++) and (+) respectively. CFEt at a dose of 0.45 g/kg body weight, showed urine sugar as seen in normal rats. These effects were compared with glibenclamide. Serum and tissue lipids The effect of CFEt on serum and tissue lipids of
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normal and experimental rats is summarised in Table II and III respectively. A marked increase in the frequency of cholesterol, free fatty acids, triglycerides and phospholipids were observed in diabetic control rats. Treatment with CFEt significantly reduced the lipid levels. Hepatic hexokinase and glucose-6-phosphatase The activities of carbohydrate enzymes are represented in Table IV. Activity of hexokinase in liver decreased markedly while the glucose-6-phosphatase activity increased significantly in diabetic control rats. Treatment with CFEt in diabetic rats increased the hexokinase activity and decreased the glucose6-phosphatase activity. DISCUSSION Streptozotocin is well known for its selective pancreatic islet -cell cytotoxicity and has been extensively used to induce diabetes mellitus in animals. It interferes with cellular metabolic oxidative mechanisms . (23) Intraperitoneal administration of streptozotocin (45 mg/kg) effectively induced diabetes in normal rats as reflected by glycosuria, hyperglycaemia, polyphagia, polydipsia and body weight loss when compared with normal rats(24). In our present study we have observed that an aqueous extract of Cassia auriculata flower can reverse these effects. The possible mechanism by which CFEt brings about its antihyperglycemic action may be by potentiation of pancreatic secretion of insulin from -cell of islets or due to enhanced transport of blood glucose to peripheral tissue. This was clearly evidenced by the increased level of insulin in diabetic rats treated with CFEt. In this context a number of other plants have also been reported to have antihyperglycemic and insulin-release stimulatory effect(25,26). We have observed a decrease in total haemoglobin during diabetes and this may be due to the formation of glycosylated haemoglobin. Increase in the level of haemoglobin in animals given CFEt may be due to decreased level of blood glucose and glycosylated haemoglobin. CFEt administration to streptozotocin dosed animals reversed the weight loss. The ability of CFEt to recover body weight loss seems to be due to its antihyperglycemic effect. Excess of fatty acids in serum produced by the streptozotocin-induced diabetes promotes conversion of excess fatty acids into phospholipids and cholesterol in liver. These two substances along with excess triglycerides formed at the same time in liver may be discharged into blood in the form of
lipoproteins(27). The abnormal high concentration of serum lipids in the diabetic subject is due, mainly to increase in the mobilisation of free fatty acids from the peripheral fat depots, since insulin inhibits the hormone sensitive lipase. Hypercholesterolemia and hypertriglyceridemia have been reported to occur in streptozotocin diabetic rats(28,29) and significant increase observed in our experiment was in accordance to these studies. The marked hyperlipidaemia that characterise the diabetic state may therefore, be regarded as a consequence of the uninhibited actions of lipolytic hormones on the fat depots(30). The antihyperlipidaemic effect of CFEt may be due to the down regulation of NADPH and NADH, a cofactor in the fat metabolism. Higher activity of glucose-6-phosphatase provides H+ which binds with NADP+ in the form of NADPH and is helpful in the synthesis of fats from carbohydrates. When glycolysis slows down because of cellular activity, the pentose phosphate pathway still remain active in liver to breakdown glucose that continuously provides NADPH which converts acetyl radicals into long fatty acid chains. CFEt may be capable of oxidising NADPH. Enhanced hexokinase activity in CFEt treated rats suggests greater uptake of glucose from blood by the liver cells. Activities of enzymes suggest that enhanced lipid metabolism during diabetes is shifted towards carbohydrate metabolism and it enhances the utilisation of glucose at the peripheral sites. One of the possible actions of CFEt may be due to its inhibition of endogenous synthesis of lipids. Metabolic aberration in streptozotocin diabetic rats suggest a high turnover of triglycerides and phospholipids. CFEt may antagonise the metabolic aberration and thereby restore the normal metabolism by tilting the balance from high lipids to high carbohydrate turnover. Alteration of fatty acid composition by increased lipid levels contribute to lowering the resistance of tissues and higher rate of Oxidative stress. Decreased activity of glucose6-phosphatase through pentose phosphate shunt results in high reduced glutathione to oxidised glutathione ratio (GSH/GSSG)(27), which is coupled with conversion of NADPH to NADP. CFEt may produce high NADP+ which results in down regulation of lipogenesis and lower risk of the tissues for oxidative stress and high resistance for diabetes. It can be concluded from the data that CFEt significantly reduces the levels of serum and tissue lipids, which are actively raised in streptozotocin diabetes rats. CFEt has beneficial effect on plasma insulin and hexokinase activity. Moreover
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its antihyperlipidaemic effect could represent a protective mechanism against the development of atherosclerosis. REFERENCES 1. Arky RA. Clinical correlates of metabolic derangements of diabetes mellitus in: Kozak GP. (Ed.), Complications of Diabetes mellitus, Saunders WB. Philadelphia, 1982; 16-20. 2. Seifter S, England S. Energy metabolism, In: Arias I, Popper H, Schacter D, et al(Eds.). The Liver: Biology and Pathobiology, Rauen Press, New York, 1982; 219-49. 3. Sochor M, Baquer NZ, McLean P. Glucose over and under utilisation in diabetes: COMPARATIVE STUDIES on the change in activities of enzymes of glucose metabolism in rat kidney and liver. Mol Physiol 1985; 51-68. 4. Baquer NZ. Glucose over utilisation and under utilization in diabetes and effects of antidiabetic compounds. Ann Real Acad Farm 1998; 64:147-80. 5. Swanston Flatt SK, Day C, Bailey CJ, Flatt RR. Traditional plant remedies for diabetes. Studies in the normal and streptozotocin diabetic mice. Diabetologia 1990; 33:462-4. 6. WHO Expert Committee on diabetes mellitus second report. Technical Report Series 646. World Health Organisation. Geneva 1980; 61. 7. Joshi SG. Cesalpinaceae -- Cassia auriculata.Text book of medicinal plants. 2000; 119. 8. Pari L, Latha M. Antidiabetic effect of Cassia auriculata flowers: Effect on Lipid Peroxidation in streptozotocin diabetes rats. Pharmaceutical Biology 2002 (In press). 9. Jain SR. Hypoglycemic principle in the Musa sapientum and its isolation. Planta Medica 1968; 1:43-7. 10. Siddique O, Sun Y, Lin JC, Chum YW. Facilitated transdermal transport of insulin. J Pharm Sci 1987; 76:341-5. 11. Pari L, Uma Maheswari J. Antihyperglycemic activity of Musa Sapentium flower: Effect on lipid peroxidation in alloxan diabetic rats. Phytother Res 2000; 14:1-3. 12. Sasaki T, Matzy S, Sonal A. Effect of acetic acid concentration on the colour reaction in the O-toluidine boric acid method for blood glucose estimation. Rinsho Kagaku 1972; 1:346-53. 13. Drabkin DL, Austin JM. Spectrophotometric constants for common haemoglobin derivatives in human, dog and rabbit blood. J Biol Chem 1932; 98:719-33. 14. Sudhakar Nayak S. Pattabiraman TN. A new colorimetric method for the estimation of glycosylated haemoglobin. Clin Chem Acta 1981; 109:267-74.
15. Folch J, Less M, Solane SGH. A simple method for isolation and purification of total lipids from animal tissues. J Biol Chem 1957; 26:497-509. 16. Zlatkis A, Zak B and Bogle GJ. A method for the determination of serum cholesterol. J Clin Med 1953; 41:486-92. 17. Foster LB, Dunn RT. Stable reagents for determination of serum triglycerides by colorimetric hantzsch condensation method. Clin Chem 1973; 19:338-40. 18. Falholt K, Falholt W, Lund B. An easy colorimetric method for routine determination of free fatty acids in plasma. Chem Acta 1973; 46:105-11. 19. Zilversmit DB, Davis AK. Micro determination of phospholipids by TCA precipitation. J Lab Clin Med 1950; 35:155-61. 20. Brandstrup N, Kirk JE, Bruni C. Determination of hexokinase in tissues. J Gerontol 1957; 12:166-71. 21. Koida H, Oda T. Pathological occurrence of glucose-6-phosphatase in liver disease. Clin Chem Acta 1959; 4:554-61. 22. Bennet P, Franklin NH. Statistical analysis in chemistry and chemical industry. New York: John Wiley and Sons, USA. 208-27. 23. Papaccio G, Pisanti FA, Latronico MV, Ammendola E, Galdieri M. Multiple low dose and single high dose treatments with streptozotocin do not generate nitric oxide. J Cell Biochem 2000; 77(1):82-91. 24. Calabresi P, Chabner BA. Antineoplastic agents. In Goodman A, Rall JW (Eds.). The pharmacological basis of therapeutics. 8th Edition Pergmann Press, New York. 1209-63. 25. Prince PSM, Menon VP, Pari L. Hypoglycemic activity of Syzigium cumini seeds: Effect on lipid peroxidation in alloxan diabetic rats. J Ethnopharmacol 1998; 61:1-7. 26. Pari L, Uma Maheswari J. Hypoglycemic effect of Musa sapreitum L. in alloxum induced diabetic rats. J Ethnopharmacal 1999; 68:321-5. 27. Bopanna KN, Kannan J, Sushma G, Balaraman R, Rathod SP. Antidiabetic and antihyperlipaemic effects of neem seed kernel powder on alloxan diabetic rabbits. Indian J Pharmacol 1997; 29:162-7. 28. Sharma SR, Dwivedi SK, Swarup D. Hypoglycemic and hypolipidaemic effects of Cinnamomum tomala nees leaves. Ind J Exp Biol 1996; 34:372-4. 29. Pushparaj P, Tan CH, Tan BKH. Effects of Averrhoa bilimli leaf extract on blood glucose and lipids in streptozotocin diabetic rats. J Ethnopharmacol 2000; 72:69-76. 30. Goodman LS, Gilman A. The pharmacological basis of therapeutics, 7th Edition. Mac Millan, New York, 1985; 1490-510.

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