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Antidiabetogenic and antioxidant effects of Caralluma attenuata extract on streptozotocin induced diabetes in rats
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 2 5 7 e2 6 2
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Original Article
Antidiabetogenic and antioxidant effects of Caralluma attenuata extract on streptozotocin induced diabetes in rats Pradeep Kumar a,b,*, Alok Sharma d,e, Paresh Varshney c, Chandana Venkateswara Rao f a
Senior Research Scientist, Department of Clinical Pharmacology and Pharmacokinetics, Ranbaxy Laboratories Ltd., GP 5, HSIDC Sector-18, Old Delhi-Gurgaon Road, Gurgaon 122015, India b CMJ University, Modrina Mansion Laitumkhrah, Shillong Meghalaya-793003, India c Research Scientist, Department of Clinical Pharmacology and Pharmacokinetics, Ranbaxy Laboratories Ltd., GP 5, HSIDC Sector-18, Old Delhi-Gurgaon Road, Gurgaon 122015, India d Pharmacognosy and Ethnopharmacology Division, National Botanical Research Institute, Rana pratap Marg, Post Box No: 436 Lucknow 226 001, India e Research Scientist, Dabur Research & Development Centre, Dabur India Ltd., Plot No. 22, Site IV, Sahibabad 201010, India f Senior Scientist, Pharmacognosy and Ethnopharmacology Division, National Botanical Research Institute, Rana pratap Marg, Post Box No: 436 Lucknow 226 001, India
article info
abstract
Article history:
Background/objective: Despite the introduction of hypoglycemic agents from natural and
Received 5 February 2013
synthetic sources, diabetes and its secondary complications continue to be a major prob-
Accepted 14 March 2013
lem in the world population. Many indigenous Indian medicinal plants have been found to
Available online 23 April 2013
successfully manage diabetes. Caralluma attenuata, locally known as ‘Kundaetikommu’, is eaten raw as a cure for diabetes. The potential role of 50% ethanolic extract of C. attenuata
Keywords:
(CAEt) in the treatment of diabetes along with its antidiabetogenic and antioxidant effects
Biochemical study
was studied in streptozotocin induced diabetic rats.
Catalase
Methods: Dried plant materials of C. attenuata were extracted with 50% ethanol and sub-
Lipid peroxidation
jected for phytochemical analysis. Diabetes was induced in experimentally designed rat
Streptozotocin
models using streptozotocin. Diabetic rats were treated with CAEt 100 mg/kg, CAEt 250 mg/
Superoxide dismutase
kg and tolbutamide 10 mg/kg. Fasting blood glucose levels, serum insulin levels, antioxidant activities and lipid profile were measured and statistical analysis was performed on all the data. Results: Oral administration of CAEt (100 and 250 mg/kg) significantly decreased the blood glucose levels and considerably increased the body weight, food intake, liquid intake and glucose tolerance in diabetic rats. The extract significantly decreased thiobarbituric acid reactive substances and subsequently increased the total reduced glutathione levels, superoxide dismutase and catalase activity in the diabetic animals. Treatment by plant extract significantly reduced total cholesterol ( p < 0.05), triglycerides ( p < 0.01), low density lipoproteins ( p < 0.05), free fatty acids ( p < 0.05) and phospholipids ( p < 0.05) levels as well as significantly increased high density lipoproteins levels.
* Corresponding author. CPP, Ranbaxy Laboratories Ltd., GP 5, HSIDC Sector-18, Old Delhi-Gurgaon Road, Gurgaon, Haryana, India. Tel.: þ91 124 4231001, þ9868715323; fax: þ91 124 4231002. E-mail address: [email protected] (P. Kumar). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.03.021
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j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 2 5 7 e2 6 2
Conclusion: The results showed that 50% ethanolic extract of C. attenuata can be used to overcome diabetic complications by pancreatic b cell regeneration or stimulation of insulin secretion or in other ways. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Diabetes mellitus is a syndrome, initially characterized by a loss of glucose homeostasis resulting from defects in insulin secretion and/or insulin action, which consequently bring about impaired metabolism of glucose and other energyyielding fuels such as lipids and proteins.1 Experimentally induced diabetes in animals has provided considerable insight into the physiological and biochemical derangement of the diabetic state. Significant changes in lipid metabolism and its structure also occur in diabetes.2 Such structural changes are clearly oxidative in nature and associated with development of vascular disease in diabetes.3 In experimental diabetic rats, increased lipid peroxidation has also found to be associated with hyperlipidemia.4 Concurrently, liver and kidney that participate in the uptake, oxidation and metabolic conversion of free fatty acids, synthesis of cholesterol, phospholipids, and triglycerides, are also severely affected during diabetes.5 Many indigenous Indian tropical medicines have been found useful in successfully managing the diabetes. Caralluma attenuata weight (Family: Asclepiadaceae) is a herb growing wild in dry hill slope regions of southern India. Indigenously it is known as ‘Kundaetikommu’, and is eaten raw as a cure for diabetes and the juice of the plant along with black pepper is recommended in the treatment of migraine.6 This plant was found to be a rich source of glycosides and known for its antihyperglycemic activity.7 The hypoglycemic effect of whole plant C. attenuata was investigated in both normal and alloxan induced diabetic rats.8 The knowledge and experimental data base of herbal medicine can provide new functional leads to reduce time, money and toxicity e the three main hurdles in drug development. It is rightly said that ‘laboratories to clinics’ becomes ’clinics to laboratories’ e a true reverse pharmacology approach. The present investigation was undertaken to study the potential effect of the antidiabetogenic activity of CAEt with a view to provide scientific evidence on modern lines and the study is also important for being the first biochemical study on the effects of CAEt in the management of type-I diabetes mellitus.
2.
Materials and methods
2.1.
Test animals
Male Wistar rats (210e250 g) were purchased from the animal house of National Laboratory Animal Centre, Lucknow, India. They were maintained in standard environmental conditions and had free access to feed and tap water ad libitum during quarantine period. The animals were kept fasting overnight
but allowed free access to the water. All studies were performed in accordance with the guidance for care and use of laboratory animals, as adopted and promulgated by the Institutional Animal Care Committee, CPCSEA, India (Reg. No. 222/2000/CPCSEA).
2.2.
Plant materials and preparation of extract
Fresh whole plants of C. attenuata were collected from Ghatkesar, Andhra Pradesh, India. The plant material was identified taxonomically and authenticated by taxonomist in National Botanical Research Institute, Lucknow. The shade dried plant materials were crushed, powdered and exhaustively extracted with 10 volumes of 50% ethanol. The extract was filtered, pooled and concentrated on Rotavapour (Buchi, USA) and dried in lyophilizer (Laboconco, USA) under reduced pressure to obtain 10.6% of residue (CAEt).
2.3.
Preliminary phytochemical screening
Preliminary qualitative phytochemical screening of CAEt gave a positive result for steroids, carbohydrates, triterpenoids, resins, flavanoids, and tannins.
2.4.
Induction of diabetes
Diabetes was induced in rats by injecting a freshly prepared solution of streptozotocin (STZ, 50 mg/kg bw, i.p) in 0.1 M citrate buffer, pH was 4.5. Fasting blood glucose concentration was measured after one week of STZ injection to confirm for induced diabetes. The rats with blood glucose level above 140 mg/dl were considered to be diabetic and were used in the experiment. The animals were kept fasting overnight for dosing as per experimental design.
2.5.
Experiment design
After induction of diabetes, forty rats were divided into five groups equally9 as follows. Group I: (control group): rats of this group received only vehicle solution. Group II: (diabetic group): rats of this group were made diabetic by injecting a freshly prepared solution of streptozotocin (STZ, 50 mg/kg bw, i.p) in 0.1 M citrate buffer/100 g bw/rat. Group III: diabetic rats treated with CAEt 100 mg/kg bw/day orally in 2% gum acacia. Group IV: diabetic rats treated with CAEt 250 mg/kg bw/day orally in 2% gum acacia. Group V: diabetic rats treated with Tolbutamide in 10 mg/kg (sigma USA).
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 2 5 7 e2 6 2
2.6.
Measurement of fasting blood glucose levels
Fasting blood samples were drawn on 1st day after single administration of CAEt and after 7 and 14 days by tail vein puncture under mild ether anesthesia in Eppendroff’s tubes containing 50 ml of anticoagulant (10% trisodium citrate solution) from the normal and STZ-induced diabetic rats. All the animals were sacrificed by decapitation after recording the final body weight. Blood was collected and serum was separated by centrifugation at 5000 rpm for 10 min for insulin assay by enzyme-linked immunosorbent assay (ELISA) technique.
2.7.
Glucose tolerance test
After overnight fasting, on the day the animals were sacrificed, a zero-min blood sample was taken from tip of tail vein of all the rats: control (Group I), diabetic (Group II), CAEt (Group III), CAEt (Group IV) and tolbutamide (Group V). The rats of all groups were given glucose (2 g/kg) 30 min after dosing and blood samples were collected at 30th and 90th min for the measurement of glucose levels by single touch glucometer after the administration of glucose.
2.8.
Serum insulin level
Serum insulin was measured10 using ELISA kit from Boehringer Mannheim Diagnostic, Mannheim, Germany. The intraassay variation was 4.9%. As the samples were run at a time there was no inter-assay variation. The insulin level in serum was expressed in mIU/ml.
2.9.
Antioxidant activities
Lipid peroxidation in liver and kidney were estimated colorimetrically by thiobarbituric acid reactive substances (TBRAS)11 and hydroperoxides.12 Glutathione (GSH) was estimated using Beutler method,13 glutathione reductase (GSH-R) was estimated using the method of Horn.14 Superoxide dismutase (SOD) was measured by using Kakkar’s15 method. Catalase (CAT) activity was measured by using the rate of decomposition of H2O2 by method of Aebi.16 All these estimations were made in both liver and kidney.
2.10.
Estimation of lipid profile
Total cholesterol (TC), high density lipoproteins (HDL) cholesterol, Triglyceride (TG) levels in serum were measured spectrophometrically by Allian Buccolo method.17 Lowdensity lipoprotein (LDL) cholesterol was calculated by Friedewald’s method.18 Free fatty acid content was estimated by Hron’s method19 and phospholipids were estimated by Folch’s method.20
2.11.
Statistical analysis
All the data was presented as mean S.E.M and analyzed by paired-t-test using SPSS software package (SPSS, Cary, NC, USA).
3.
259
Results
The present investigation highlights the antidiabetogenic and antioxidant efficacy of C. attenuata extract. The antidiabetic potency has been evaluated by the measurement of parameters like body weight, water and fluid intake, fasting blood glucose level, intravenous glucose tolerance along with serum insulin level. It was concluded that there was a significant decrease ( p < 0.01) in body weight, food and liquid intake of diabetic group as compared to the control group. After administration of CAEt there was a significant recovery of these parameters toward the control level. Treatment of CAEt to streptozotocin diabetic animals resulted in a complete recovery of fasting blood glucose level and the animals were able to tolerate the exogenous glucose load compared with normal controls (Table 1). There was a significant increase in blood glucose level ( p < 0.05) in diabetic rats when compared with normal controls. CAEt also showed significant reduction ( p < 0.01) in serum glucose level in STZ diabetic rats (Table 1). The antioxidant efficacy was, contrary, based on the measurement of free radical scavenging enzymes viz. TBARS, GSH, GSH-R, SOD and CAT. Table 2 shows the levels of TBARS, GSH and GSH-R in liver and kidney of control and experimental animals ( p < 0.001). A significant elevation in tissues TBARS and significant reduction in GSH, and GSH-R was observed in the diabetic control rats as compared to the normal control rats. Oral administration of CAEt (100 and 250 mg/kg bw) for three weeks shows significant reduction in TBARS and increase in GSH-R in both liver and kidney ( p < 0.001). With respect to GSH there was a significant increase in the glutathione in the liver and kidney. Table 2 also cite the activities of the enzymatic antioxidants SOD and CAT in liver and kidney ( p < 0.001). Activities of these enzymes decreased significantly in the diabetic control rats as compared to the normal control ( p < 0.001). Oral administration of CAEt (100 mg and 250 mg/kg) for 3 weeks significantly reversed these enzymes to near normal values. The various parameters of blood lipid profile of severely diabetic rats were tested before and after treatment. The effect of CAEt 100 and 250 mg/kg on TC, TG and LDL levels are shown in Table 2. A significant increase in TG ( p < 0.01), TC ( p < 0.05) and LDL ( p < 0.05) levels was observed in diabetic controls as compared to normal controls. Treatment by CAEt significantly reduced TC ( p < 0.05), TG ( p < 0.01), LDL (p < 0.05), free fatty acids ( p < 0.05) and phospholipids ( p < 0.05) levels as well as significantly increased HDL levels.
4.
Discussion
Following hypothesis has been proposed for the mode of action of the C. attenuata extract. Streptozotocin induced diabetes mellitus is associated with the generation of reactive oxygen species (ROS) causing oxidative damage. STZ diabetic animals may exhibit most of the diabetic complications mediated through oxidative stress.21 Lipid peroxidation is a free radical induced process leading to oxidative deterioration of polyunsaturated fatty acids. Under Physiologic condition, low concentrations of lipid peroxides are found in tissues.22 It
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Table 1 e Effect of CAEt on fasting blood glucose level, serum glucose level and glucose tolerance in diabetic rats. Group
*Fasting blood glucose level (mg/dl) 0 day
I II III IV V
81.2 78.6 80.1 81.4 79.0
4.20 5.20 4.20 4.50 4.10
7th day 79.6 338.9 266.1 259.3 170.4
**Serum glucose level (mg/dl)
14th day
4.10 4.60a 4.70y 4.50z 4.10z
76.9 422.3 130.3 134.2 97.6
0 day
4.30 4.90a 4.10y 4.70y 4.40
83.30 241.48 243.31 242.67 235.53
13.21 24.63 22.92 36.16 32.41
7th day 82.33 231.56 190.46 181.35 103.82
11.59 12.26c 13.27x 16.31x 12.67y
14th day 83.36 228.72 158.58 146.37 93.37
12.72 13.09c 19.53y 14.25y 13.67y
***Blood glucose tolerance (mg/100 ml) Basic value 79.25 81.28 84.53 80.23 64.48
I II III IV V
30 min
0.10 0.91 0.70 0.81 1.13
80.23 144.59 131.37 137.56 82.87
90 min
1.53 1.76c 2.92x 1.11x 1.66z
80.91 114.71 106.73 104.93 56.94
1.15 1.60c 1.89y 1.01y 2.40z
*Values are mean S.E.M. for 6 animals in each group. xp < 0.05, yp < 0.01 and zp < 0.001 when compared with diabetic controls ap < 0.05. **The values represent the means S. E. M for six rats per group. xp < 0.05 and yp < 0.01 compared to diabetic control group. cp < 0.001 as compared to normal. ***The values represent the means S. E. M for six rats per group. xp < 0.05, yp < 0.01 and zp < 0.001 compared to diabetic control group. cp < 0.001 as compared to normal.
has been proposed that antioxidants that maintain the concentration of reduced glutathione may restore the cellular defense mechanisms, block lipid peroxidation and thus protect the tissue damage against oxidative damage.23 Our results showed that in diabetic control animals the level of TBARS was high due to increased lipid peroxidation. CAEt reduced the TBARS levels in both liver and kidney, which may be due to the free radical scavenging action of the active ingredients present in CAEt. CAEt inhibited the lipid peroxidation process effectively. The decrease in GSH level in liver during diabetes is probably due to its increased utilization by the hepatic cells which could be the result of decreased synthesis or increased
degradation of GSH by oxidative stress in diabetes.23 We have also observed the decrease in GSH in liver and kidney. The treatment with C. attenuata significantly altered the GSH and GSH-R to be comparable with the control group. SOD and CAT are two major scavenging enzymes that remove the toxic free radical in vivo. SOD scavenges the superoxide ions produced as cellular by-products. SOD is a major defense for aerobic cells in combating the toxic effects of superoxide radicals.24 CAT reduces hydrogen peroxide produced by disputation reaction and preventing generation of hydroxyl radicals thereby protecting the cellular constituents from oxidative damage in peroxisomes. Reduced activities of SOD and CAT in liver and kidney have been observed
Table 2 e Effect of CAEt on various biochemical parameters and antioxidant enzyme activity in diabetic rats. Group I II III IV V
*TC 60.4 122.5 82.0 84.2 82.1
3.4 2.3a 2.0x 1.7x 1.2x
*HDL 29.2 27.5 33.4 30.2 29.9
*LDL
1.6 0.5 1.1y 0.5y 0.4y
14.3 64.2 21.0 23.4 24.1
2.4 1.9a 2.1x 1.9x 0.8x
*TG 93.0 155.4 76.3 81.2 101.9
*Phospholipids 2.8 1.0a 0.8y 1.5y 0.9y
78.3 151 86.1 89.4 98.2
6.3 11.2a 5.3y 7.6 1.6
*Free fatty acids 79.1 154.1 85.2 84.2 84.3
6.2 10.3a 5.6y 5.4 7.4
Effect of CAEt on antioxidant enzyme activity in diabetic rats **TBARS (Nmoles/ mg protein) I II III IV V
6.64 7.45 7.10 6.92 6.79
0.05 0.09c 0.08y 0.08z 0.06z
**Glutathione (Nmoles/ mg protein) 39.68 23.82 32.25 34.50 38.78
1.82 2.10b 1.24y 2.16z 1.55z
**Gluthione oxidase (unit/mg protein) 208.82 214.20 217.51 217.86 216.50
0.62 0.32b 0.78y 0.69y 0.72z
**CAT (unit/mg protein) 29.39 14.57 21.24 25.35 27.23
2.48 1.09c 1.74x 2.22y 2.12y
**SOD (unit/mg protein) 3.20 19.16 32.28 37.28 1.10
0.48 0.20a 9.12x 0.10x 8.8x
*Values are mean S.E.M. for 6 animals in each group. ap < 0.05, bp < 0.01 when compared with diabetic controls. xp < 0.001 as compared to normal group. **The values represent the means S. E. M for six rats per group. xp < 0.05, yp < 0.01 and zp < 0.001 compared to diabetic control group. a p < 0.05bp < 0.01cp < 0.001 as compared to normal.
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 2 5 7 e2 6 2
during diabetes and this may result in a number of deleterious effects due to the accumulation of superoxide radicals and hydrogen peroxide.25 C. attenuata and tolbutamide treated rats showed decreased lipid peroxidation that is associated with increased activity of SOD and CAT. Insulin also plays an important role in the metabolism of lipids. Insulin is a potent inhibitor of lipolysis. Since it inhibits the activity of the hormone sensitive lipases in adipose tissue and suppresses the release of free fatty acids,26 during diabetes, enhanced activity of this enzyme increases lipolysis and releases more free fatty acids in to the circulation. Increased fatty acids concentration also increases the boxidation of fatty acids, producing more acetyl CoA and cholesterol during diabetes. In normal condition, insulin increases the receptor-mediated removal of LDL-cholesterol while the decreased activity of insulin during diabetes causes hypercholesterolemia. Hypercholesterolemia and hypertriglyceridemia have been reported to occur in diabetic rats.27 The increased concentration of cholesterol could result in a relative molecular ordering of the residual phospholipids resulting in a decrease in membrane fluidity. The increased concentration of free fatty acids in liver and kidney may be due to lipid breakdown and this may cause increased generation of NADPH, which results in the activation of NADPH dependent microsomal lipid peroxidation. Liver and kidney phospholipids were increased in diabetic control rats. Phospholipids are present in cell membrane and make up vast majority of the surface lipoprotein forming a lipid bilayer that acts as an interface with both polar plasma environment and non-polar lipoprotein of lipoprotein core.28 Phospholipids are vital part of biomembrane rich in polyunsaturated fatty acids, which are susceptible substrate for free radicals such as O2and OH radicals. Increased phospholipids levels in tissues were reported in streptozotocin diabetic rats.29 Administration of C. attenuata decreased the levels of tissue free fatty acids and phospholipids. Accumulation of triglycerides is one of the risk factors in coronary heart disease. The significant increase in the level of triglycerides in liver and kidney of diabetic control rats may be due to the lack of insulin as under normal condition insulin activates the enzyme lipoprotein lipase and hydrolysis triglycerides.30 CAEt reduces triglycerides in tissues of streptozotocin-induced diabetic rats and hence may prevent the progression of coronary heart disease. It is interesting to note that CAEt brought down the elevated level of TC, LDL and VLDL cholesterol and TG in diabetic animals to nearly normal level.
5.
Conclusion
On the basis of above results, it could be concluded that CAEt has a potent anti-diabetogenic effect in diabetic rats. It may be stated that this composite extract contains the active antihyperglycemic agent (s) that can be used to overcome diabetic complications by pancreatic b cell regeneration or stimulation of insulin secretion or in other ways. These findings could lead identification of novel molecule from C. attenuata, which serves as a good adjuvant in the present armamentarium of diabetic complications.
261
Conflicts of interest All authors have none to declare.
Acknowledgments The authors are thankful to the director of NBRI for providing necessary facilities and resources to carry out the research work.
references
1. Scheen AJ. Drug treatment of non- insulin dependent diabetes mellitus in the 1990s. Achievements and future developments. Drugs. 1997;54(3):355e368. 2. Sochar M, Baquer NZ, Mclean P. Glucose under utilization in diabetes. comparative studies on the changes in the activities of enzymes of glucose metabolism in rat kidney and liver. Mol Physiol. 1985;7:51e68. 3. Baynes JW, Thorpe SR. Role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes. 1999;48(1):1e4. 4. Morel DW, Chisolm GM. Antioxidant treatment of diabetic rats inhibits lipoprotein oxidation and cytotoxicity. J Lipid Res. 1989;30(12):1827e1834. 5. Seifter S, Englard S. Energy metabolism. In: Arias IM, Boyer JL, Fausto N, Jakoby WB, Schachter DA, Shafritz DA, eds. The Liver: Biology and Pathobiology. 3rd ed. New York: Raven Press; 1994:323e364. 6. Srinivasacharyulu Y. Yogarathnakaram. 2nd ed. Nellore, India: Swatantra Press; 1931. 7. Venkatesh S, Reddy GD, Reddy BM, Ramesh M, Rao AV. Antihyperglycemic activity of Caralluma attenuata. Fitoterapia. 2003;74(3):274e279. 8. Jayakar B, Rajkapoor B, Suresh B. Effect of Caralluma attenuata in normal and alloxan induced diabetic rats. J Herb Pharmacother. 2004;4(1):35e40. 9. Sharma A, Kaithwas G, Vijaykumar M, Unnikrishnan MK, Rao CV. Antihyperglycemic and antioxidant potential of polysaccharide fraction from Portulaca oleracea seeds against streptozotocin-induced diabetes in rats. J Food Biochem. 2012;36(3):378e382. 10. Bu¨rgi W, Briner M, Franken N, Kessler AC. One-step sandwich enzyme immunoassay for insulin using monoclonal antibodies. Clin Biochem. 1988;21(5):311e314. 11. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95(2):351e358. 12. Jamall IS, Smith JC. Effects of cadmium on glutathione peroxidase, superoxide dismutase, and lipid peroxidation in the rat heart: a possible mechanism of cadmium cardiotoxicity. Toxicol Appl Pharmacol. 1985;80(1):33e42. 13. Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Clin Med. 1963;61:882e888. 14. Horn HD. Glutathione reductase. In: Bergmyer HU, ed. Methods in Enzymatic Analysis. New York: Academic Press; 1963:875e879. 15. Kakkar P, Das B, Viswanathan PN. A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys. 1984;21(2):130e132. 16. Catalase Aebi H. In: Bergmeyer HU, ed. Methods of Enzymatic Analysis. 3rd ed. Weinheim: Verlag Chemie; 1983:273e286.
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17. Allain CC, Poon LS, Chan CS, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem. 1974;20(4):470e475. 18. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972;18(6):499e502. 19. Hron WT, Menahan LA. A sensitive method for the determination of free fatty acids in plasma. J Lipid Res. 1981;22(2):377e381. 20. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957;226(1):497e509. 21. Ozturia Y, Altan VM, Yildizoglu A. Effect of experimental diabetes and insulin on smooth muscle functions. Pharma Rev. 1996;48:69. 22. Loew D, Kaszkin M. Approaching the problem of bioequivalence of herbal medicinal products. Phytother Res. 2002;16(8):705e711. 23. Rauscher FM, Sanders RA, Watkins 3rd JB. Effects of new antioxidant compounds PNU-104067F and PNU-74389G on antioxidant defense in normal and diabetic rats. J Biochem Mol Toxicol. 2000;14(4):189e194.
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Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Original Article
Antidiabetogenic and antioxidant effects of Caralluma attenuata extract on streptozotocin induced diabetes in rats Pradeep Kumar a,b,*, Alok Sharma d,e, Paresh Varshney c, Chandana Venkateswara Rao f a
Senior Research Scientist, Department of Clinical Pharmacology and Pharmacokinetics, Ranbaxy Laboratories Ltd., GP 5, HSIDC Sector-18, Old Delhi-Gurgaon Road, Gurgaon 122015, India b CMJ University, Modrina Mansion Laitumkhrah, Shillong Meghalaya-793003, India c Research Scientist, Department of Clinical Pharmacology and Pharmacokinetics, Ranbaxy Laboratories Ltd., GP 5, HSIDC Sector-18, Old Delhi-Gurgaon Road, Gurgaon 122015, India d Pharmacognosy and Ethnopharmacology Division, National Botanical Research Institute, Rana pratap Marg, Post Box No: 436 Lucknow 226 001, India e Research Scientist, Dabur Research & Development Centre, Dabur India Ltd., Plot No. 22, Site IV, Sahibabad 201010, India f Senior Scientist, Pharmacognosy and Ethnopharmacology Division, National Botanical Research Institute, Rana pratap Marg, Post Box No: 436 Lucknow 226 001, India
article info
abstract
Article history:
Background/objective: Despite the introduction of hypoglycemic agents from natural and
Received 5 February 2013
synthetic sources, diabetes and its secondary complications continue to be a major prob-
Accepted 14 March 2013
lem in the world population. Many indigenous Indian medicinal plants have been found to
Available online 23 April 2013
successfully manage diabetes. Caralluma attenuata, locally known as ‘Kundaetikommu’, is eaten raw as a cure for diabetes. The potential role of 50% ethanolic extract of C. attenuata
Keywords:
(CAEt) in the treatment of diabetes along with its antidiabetogenic and antioxidant effects
Biochemical study
was studied in streptozotocin induced diabetic rats.
Catalase
Methods: Dried plant materials of C. attenuata were extracted with 50% ethanol and sub-
Lipid peroxidation
jected for phytochemical analysis. Diabetes was induced in experimentally designed rat
Streptozotocin
models using streptozotocin. Diabetic rats were treated with CAEt 100 mg/kg, CAEt 250 mg/
Superoxide dismutase
kg and tolbutamide 10 mg/kg. Fasting blood glucose levels, serum insulin levels, antioxidant activities and lipid profile were measured and statistical analysis was performed on all the data. Results: Oral administration of CAEt (100 and 250 mg/kg) significantly decreased the blood glucose levels and considerably increased the body weight, food intake, liquid intake and glucose tolerance in diabetic rats. The extract significantly decreased thiobarbituric acid reactive substances and subsequently increased the total reduced glutathione levels, superoxide dismutase and catalase activity in the diabetic animals. Treatment by plant extract significantly reduced total cholesterol ( p < 0.05), triglycerides ( p < 0.01), low density lipoproteins ( p < 0.05), free fatty acids ( p < 0.05) and phospholipids ( p < 0.05) levels as well as significantly increased high density lipoproteins levels.
* Corresponding author. CPP, Ranbaxy Laboratories Ltd., GP 5, HSIDC Sector-18, Old Delhi-Gurgaon Road, Gurgaon, Haryana, India. Tel.: þ91 124 4231001, þ9868715323; fax: þ91 124 4231002. E-mail address: [email protected] (P. Kumar). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.03.021
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Conclusion: The results showed that 50% ethanolic extract of C. attenuata can be used to overcome diabetic complications by pancreatic b cell regeneration or stimulation of insulin secretion or in other ways. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Diabetes mellitus is a syndrome, initially characterized by a loss of glucose homeostasis resulting from defects in insulin secretion and/or insulin action, which consequently bring about impaired metabolism of glucose and other energyyielding fuels such as lipids and proteins.1 Experimentally induced diabetes in animals has provided considerable insight into the physiological and biochemical derangement of the diabetic state. Significant changes in lipid metabolism and its structure also occur in diabetes.2 Such structural changes are clearly oxidative in nature and associated with development of vascular disease in diabetes.3 In experimental diabetic rats, increased lipid peroxidation has also found to be associated with hyperlipidemia.4 Concurrently, liver and kidney that participate in the uptake, oxidation and metabolic conversion of free fatty acids, synthesis of cholesterol, phospholipids, and triglycerides, are also severely affected during diabetes.5 Many indigenous Indian tropical medicines have been found useful in successfully managing the diabetes. Caralluma attenuata weight (Family: Asclepiadaceae) is a herb growing wild in dry hill slope regions of southern India. Indigenously it is known as ‘Kundaetikommu’, and is eaten raw as a cure for diabetes and the juice of the plant along with black pepper is recommended in the treatment of migraine.6 This plant was found to be a rich source of glycosides and known for its antihyperglycemic activity.7 The hypoglycemic effect of whole plant C. attenuata was investigated in both normal and alloxan induced diabetic rats.8 The knowledge and experimental data base of herbal medicine can provide new functional leads to reduce time, money and toxicity e the three main hurdles in drug development. It is rightly said that ‘laboratories to clinics’ becomes ’clinics to laboratories’ e a true reverse pharmacology approach. The present investigation was undertaken to study the potential effect of the antidiabetogenic activity of CAEt with a view to provide scientific evidence on modern lines and the study is also important for being the first biochemical study on the effects of CAEt in the management of type-I diabetes mellitus.
2.
Materials and methods
2.1.
Test animals
Male Wistar rats (210e250 g) were purchased from the animal house of National Laboratory Animal Centre, Lucknow, India. They were maintained in standard environmental conditions and had free access to feed and tap water ad libitum during quarantine period. The animals were kept fasting overnight
but allowed free access to the water. All studies were performed in accordance with the guidance for care and use of laboratory animals, as adopted and promulgated by the Institutional Animal Care Committee, CPCSEA, India (Reg. No. 222/2000/CPCSEA).
2.2.
Plant materials and preparation of extract
Fresh whole plants of C. attenuata were collected from Ghatkesar, Andhra Pradesh, India. The plant material was identified taxonomically and authenticated by taxonomist in National Botanical Research Institute, Lucknow. The shade dried plant materials were crushed, powdered and exhaustively extracted with 10 volumes of 50% ethanol. The extract was filtered, pooled and concentrated on Rotavapour (Buchi, USA) and dried in lyophilizer (Laboconco, USA) under reduced pressure to obtain 10.6% of residue (CAEt).
2.3.
Preliminary phytochemical screening
Preliminary qualitative phytochemical screening of CAEt gave a positive result for steroids, carbohydrates, triterpenoids, resins, flavanoids, and tannins.
2.4.
Induction of diabetes
Diabetes was induced in rats by injecting a freshly prepared solution of streptozotocin (STZ, 50 mg/kg bw, i.p) in 0.1 M citrate buffer, pH was 4.5. Fasting blood glucose concentration was measured after one week of STZ injection to confirm for induced diabetes. The rats with blood glucose level above 140 mg/dl were considered to be diabetic and were used in the experiment. The animals were kept fasting overnight for dosing as per experimental design.
2.5.
Experiment design
After induction of diabetes, forty rats were divided into five groups equally9 as follows. Group I: (control group): rats of this group received only vehicle solution. Group II: (diabetic group): rats of this group were made diabetic by injecting a freshly prepared solution of streptozotocin (STZ, 50 mg/kg bw, i.p) in 0.1 M citrate buffer/100 g bw/rat. Group III: diabetic rats treated with CAEt 100 mg/kg bw/day orally in 2% gum acacia. Group IV: diabetic rats treated with CAEt 250 mg/kg bw/day orally in 2% gum acacia. Group V: diabetic rats treated with Tolbutamide in 10 mg/kg (sigma USA).
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 2 5 7 e2 6 2
2.6.
Measurement of fasting blood glucose levels
Fasting blood samples were drawn on 1st day after single administration of CAEt and after 7 and 14 days by tail vein puncture under mild ether anesthesia in Eppendroff’s tubes containing 50 ml of anticoagulant (10% trisodium citrate solution) from the normal and STZ-induced diabetic rats. All the animals were sacrificed by decapitation after recording the final body weight. Blood was collected and serum was separated by centrifugation at 5000 rpm for 10 min for insulin assay by enzyme-linked immunosorbent assay (ELISA) technique.
2.7.
Glucose tolerance test
After overnight fasting, on the day the animals were sacrificed, a zero-min blood sample was taken from tip of tail vein of all the rats: control (Group I), diabetic (Group II), CAEt (Group III), CAEt (Group IV) and tolbutamide (Group V). The rats of all groups were given glucose (2 g/kg) 30 min after dosing and blood samples were collected at 30th and 90th min for the measurement of glucose levels by single touch glucometer after the administration of glucose.
2.8.
Serum insulin level
Serum insulin was measured10 using ELISA kit from Boehringer Mannheim Diagnostic, Mannheim, Germany. The intraassay variation was 4.9%. As the samples were run at a time there was no inter-assay variation. The insulin level in serum was expressed in mIU/ml.
2.9.
Antioxidant activities
Lipid peroxidation in liver and kidney were estimated colorimetrically by thiobarbituric acid reactive substances (TBRAS)11 and hydroperoxides.12 Glutathione (GSH) was estimated using Beutler method,13 glutathione reductase (GSH-R) was estimated using the method of Horn.14 Superoxide dismutase (SOD) was measured by using Kakkar’s15 method. Catalase (CAT) activity was measured by using the rate of decomposition of H2O2 by method of Aebi.16 All these estimations were made in both liver and kidney.
2.10.
Estimation of lipid profile
Total cholesterol (TC), high density lipoproteins (HDL) cholesterol, Triglyceride (TG) levels in serum were measured spectrophometrically by Allian Buccolo method.17 Lowdensity lipoprotein (LDL) cholesterol was calculated by Friedewald’s method.18 Free fatty acid content was estimated by Hron’s method19 and phospholipids were estimated by Folch’s method.20
2.11.
Statistical analysis
All the data was presented as mean S.E.M and analyzed by paired-t-test using SPSS software package (SPSS, Cary, NC, USA).
3.
259
Results
The present investigation highlights the antidiabetogenic and antioxidant efficacy of C. attenuata extract. The antidiabetic potency has been evaluated by the measurement of parameters like body weight, water and fluid intake, fasting blood glucose level, intravenous glucose tolerance along with serum insulin level. It was concluded that there was a significant decrease ( p < 0.01) in body weight, food and liquid intake of diabetic group as compared to the control group. After administration of CAEt there was a significant recovery of these parameters toward the control level. Treatment of CAEt to streptozotocin diabetic animals resulted in a complete recovery of fasting blood glucose level and the animals were able to tolerate the exogenous glucose load compared with normal controls (Table 1). There was a significant increase in blood glucose level ( p < 0.05) in diabetic rats when compared with normal controls. CAEt also showed significant reduction ( p < 0.01) in serum glucose level in STZ diabetic rats (Table 1). The antioxidant efficacy was, contrary, based on the measurement of free radical scavenging enzymes viz. TBARS, GSH, GSH-R, SOD and CAT. Table 2 shows the levels of TBARS, GSH and GSH-R in liver and kidney of control and experimental animals ( p < 0.001). A significant elevation in tissues TBARS and significant reduction in GSH, and GSH-R was observed in the diabetic control rats as compared to the normal control rats. Oral administration of CAEt (100 and 250 mg/kg bw) for three weeks shows significant reduction in TBARS and increase in GSH-R in both liver and kidney ( p < 0.001). With respect to GSH there was a significant increase in the glutathione in the liver and kidney. Table 2 also cite the activities of the enzymatic antioxidants SOD and CAT in liver and kidney ( p < 0.001). Activities of these enzymes decreased significantly in the diabetic control rats as compared to the normal control ( p < 0.001). Oral administration of CAEt (100 mg and 250 mg/kg) for 3 weeks significantly reversed these enzymes to near normal values. The various parameters of blood lipid profile of severely diabetic rats were tested before and after treatment. The effect of CAEt 100 and 250 mg/kg on TC, TG and LDL levels are shown in Table 2. A significant increase in TG ( p < 0.01), TC ( p < 0.05) and LDL ( p < 0.05) levels was observed in diabetic controls as compared to normal controls. Treatment by CAEt significantly reduced TC ( p < 0.05), TG ( p < 0.01), LDL (p < 0.05), free fatty acids ( p < 0.05) and phospholipids ( p < 0.05) levels as well as significantly increased HDL levels.
4.
Discussion
Following hypothesis has been proposed for the mode of action of the C. attenuata extract. Streptozotocin induced diabetes mellitus is associated with the generation of reactive oxygen species (ROS) causing oxidative damage. STZ diabetic animals may exhibit most of the diabetic complications mediated through oxidative stress.21 Lipid peroxidation is a free radical induced process leading to oxidative deterioration of polyunsaturated fatty acids. Under Physiologic condition, low concentrations of lipid peroxides are found in tissues.22 It
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Table 1 e Effect of CAEt on fasting blood glucose level, serum glucose level and glucose tolerance in diabetic rats. Group
*Fasting blood glucose level (mg/dl) 0 day
I II III IV V
81.2 78.6 80.1 81.4 79.0
4.20 5.20 4.20 4.50 4.10
7th day 79.6 338.9 266.1 259.3 170.4
**Serum glucose level (mg/dl)
14th day
4.10 4.60a 4.70y 4.50z 4.10z
76.9 422.3 130.3 134.2 97.6
0 day
4.30 4.90a 4.10y 4.70y 4.40
83.30 241.48 243.31 242.67 235.53
13.21 24.63 22.92 36.16 32.41
7th day 82.33 231.56 190.46 181.35 103.82
11.59 12.26c 13.27x 16.31x 12.67y
14th day 83.36 228.72 158.58 146.37 93.37
12.72 13.09c 19.53y 14.25y 13.67y
***Blood glucose tolerance (mg/100 ml) Basic value 79.25 81.28 84.53 80.23 64.48
I II III IV V
30 min
0.10 0.91 0.70 0.81 1.13
80.23 144.59 131.37 137.56 82.87
90 min
1.53 1.76c 2.92x 1.11x 1.66z
80.91 114.71 106.73 104.93 56.94
1.15 1.60c 1.89y 1.01y 2.40z
*Values are mean S.E.M. for 6 animals in each group. xp < 0.05, yp < 0.01 and zp < 0.001 when compared with diabetic controls ap < 0.05. **The values represent the means S. E. M for six rats per group. xp < 0.05 and yp < 0.01 compared to diabetic control group. cp < 0.001 as compared to normal. ***The values represent the means S. E. M for six rats per group. xp < 0.05, yp < 0.01 and zp < 0.001 compared to diabetic control group. cp < 0.001 as compared to normal.
has been proposed that antioxidants that maintain the concentration of reduced glutathione may restore the cellular defense mechanisms, block lipid peroxidation and thus protect the tissue damage against oxidative damage.23 Our results showed that in diabetic control animals the level of TBARS was high due to increased lipid peroxidation. CAEt reduced the TBARS levels in both liver and kidney, which may be due to the free radical scavenging action of the active ingredients present in CAEt. CAEt inhibited the lipid peroxidation process effectively. The decrease in GSH level in liver during diabetes is probably due to its increased utilization by the hepatic cells which could be the result of decreased synthesis or increased
degradation of GSH by oxidative stress in diabetes.23 We have also observed the decrease in GSH in liver and kidney. The treatment with C. attenuata significantly altered the GSH and GSH-R to be comparable with the control group. SOD and CAT are two major scavenging enzymes that remove the toxic free radical in vivo. SOD scavenges the superoxide ions produced as cellular by-products. SOD is a major defense for aerobic cells in combating the toxic effects of superoxide radicals.24 CAT reduces hydrogen peroxide produced by disputation reaction and preventing generation of hydroxyl radicals thereby protecting the cellular constituents from oxidative damage in peroxisomes. Reduced activities of SOD and CAT in liver and kidney have been observed
Table 2 e Effect of CAEt on various biochemical parameters and antioxidant enzyme activity in diabetic rats. Group I II III IV V
*TC 60.4 122.5 82.0 84.2 82.1
3.4 2.3a 2.0x 1.7x 1.2x
*HDL 29.2 27.5 33.4 30.2 29.9
*LDL
1.6 0.5 1.1y 0.5y 0.4y
14.3 64.2 21.0 23.4 24.1
2.4 1.9a 2.1x 1.9x 0.8x
*TG 93.0 155.4 76.3 81.2 101.9
*Phospholipids 2.8 1.0a 0.8y 1.5y 0.9y
78.3 151 86.1 89.4 98.2
6.3 11.2a 5.3y 7.6 1.6
*Free fatty acids 79.1 154.1 85.2 84.2 84.3
6.2 10.3a 5.6y 5.4 7.4
Effect of CAEt on antioxidant enzyme activity in diabetic rats **TBARS (Nmoles/ mg protein) I II III IV V
6.64 7.45 7.10 6.92 6.79
0.05 0.09c 0.08y 0.08z 0.06z
**Glutathione (Nmoles/ mg protein) 39.68 23.82 32.25 34.50 38.78
1.82 2.10b 1.24y 2.16z 1.55z
**Gluthione oxidase (unit/mg protein) 208.82 214.20 217.51 217.86 216.50
0.62 0.32b 0.78y 0.69y 0.72z
**CAT (unit/mg protein) 29.39 14.57 21.24 25.35 27.23
2.48 1.09c 1.74x 2.22y 2.12y
**SOD (unit/mg protein) 3.20 19.16 32.28 37.28 1.10
0.48 0.20a 9.12x 0.10x 8.8x
*Values are mean S.E.M. for 6 animals in each group. ap < 0.05, bp < 0.01 when compared with diabetic controls. xp < 0.001 as compared to normal group. **The values represent the means S. E. M for six rats per group. xp < 0.05, yp < 0.01 and zp < 0.001 compared to diabetic control group. a p < 0.05bp < 0.01cp < 0.001 as compared to normal.
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during diabetes and this may result in a number of deleterious effects due to the accumulation of superoxide radicals and hydrogen peroxide.25 C. attenuata and tolbutamide treated rats showed decreased lipid peroxidation that is associated with increased activity of SOD and CAT. Insulin also plays an important role in the metabolism of lipids. Insulin is a potent inhibitor of lipolysis. Since it inhibits the activity of the hormone sensitive lipases in adipose tissue and suppresses the release of free fatty acids,26 during diabetes, enhanced activity of this enzyme increases lipolysis and releases more free fatty acids in to the circulation. Increased fatty acids concentration also increases the boxidation of fatty acids, producing more acetyl CoA and cholesterol during diabetes. In normal condition, insulin increases the receptor-mediated removal of LDL-cholesterol while the decreased activity of insulin during diabetes causes hypercholesterolemia. Hypercholesterolemia and hypertriglyceridemia have been reported to occur in diabetic rats.27 The increased concentration of cholesterol could result in a relative molecular ordering of the residual phospholipids resulting in a decrease in membrane fluidity. The increased concentration of free fatty acids in liver and kidney may be due to lipid breakdown and this may cause increased generation of NADPH, which results in the activation of NADPH dependent microsomal lipid peroxidation. Liver and kidney phospholipids were increased in diabetic control rats. Phospholipids are present in cell membrane and make up vast majority of the surface lipoprotein forming a lipid bilayer that acts as an interface with both polar plasma environment and non-polar lipoprotein of lipoprotein core.28 Phospholipids are vital part of biomembrane rich in polyunsaturated fatty acids, which are susceptible substrate for free radicals such as O2and OH radicals. Increased phospholipids levels in tissues were reported in streptozotocin diabetic rats.29 Administration of C. attenuata decreased the levels of tissue free fatty acids and phospholipids. Accumulation of triglycerides is one of the risk factors in coronary heart disease. The significant increase in the level of triglycerides in liver and kidney of diabetic control rats may be due to the lack of insulin as under normal condition insulin activates the enzyme lipoprotein lipase and hydrolysis triglycerides.30 CAEt reduces triglycerides in tissues of streptozotocin-induced diabetic rats and hence may prevent the progression of coronary heart disease. It is interesting to note that CAEt brought down the elevated level of TC, LDL and VLDL cholesterol and TG in diabetic animals to nearly normal level.
5.
Conclusion
On the basis of above results, it could be concluded that CAEt has a potent anti-diabetogenic effect in diabetic rats. It may be stated that this composite extract contains the active antihyperglycemic agent (s) that can be used to overcome diabetic complications by pancreatic b cell regeneration or stimulation of insulin secretion or in other ways. These findings could lead identification of novel molecule from C. attenuata, which serves as a good adjuvant in the present armamentarium of diabetic complications.
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Conflicts of interest All authors have none to declare.
Acknowledgments The authors are thankful to the director of NBRI for providing necessary facilities and resources to carry out the research work.
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