Research Article
Antioxidant Activities of Caralluma tuberculata on Streptozotocin-Induced Diabetic Rats
DDR
DRUG DEVELOPMENT RESEARCH 119 : 00–00 (2014)
Jafar Poodineh,1,3 Abdurrashid Khazaei Feizabad,2 and Alireza Nakhaee1,3* Cellular and Molecular Research Center, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran 2 English Department, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran 3 Department of Clinical Biochemistry, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran
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ABSTRACT The aim of this study was to elucidate the antioxidant effects of Caralluma tuberculata (C. tuberculata) in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in male Wistar rats with an intraperitoneal injection of STZ at dose of 60 mg/kg body weight. Three days after diabetes induction, powdered aerial part of plant at doses of 100 and 200 mg/kg body weight were gavaged orally for a period of 45 days. The diabetes significantly decreased the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and level of total thiol in liver, kidney, and heart of animals (P < 0.05). In contrast, a significant increase in the levels of protein carbonyl was observed in diabetic rats compared with control animals (P < 0.05). Oral treatment of diabetic rats with C. tuberculata showed ameliorative effects on blood glucose and markers of oxidative stress in a dose-dependent manner. Altered levels of all oxidative stress parameters in tissues of diabetic rats reverted back to those normal animals after the treatment with dose of 200 mg/kg /day of plant materials. It seems that the appropriate dose of C. tuberculata has both antihyperglycemic and antioxidant activities in STZ-induced diabetic rats. Therefore, it can have preventive properties on oxidative stressC 2014 Wiley Periodicals, Inc. V induced diabetic complications. Drug Dev Res 00 : 00–00, 2014. Key words: Caralluma tuberculata; antioxidant; streptozotocin; diabetes
INTRODUCTION
Diabetes mellitus (DM) is a common metabolic disorder associated with an increased risk of nephropathy, cardiovascular diseases, retinopathy, neuropathy, and foot problems [Giacco and Brownlee, 2010]. DM is associated with increased production of free radicals and a decrease in antioxidant effectiveness, a situation that is defined as oxidative stress [Maritim et al., 2003]. Elevated production of free radicals and oxidative stress play a central role in the development of diabetic complications in various tissues [Baynes and Thorpe, 1999; Brownlee, 2001]. C 2014 Wiley Periodicals, Inc. V
Conflict of interests: The authors declare that they have no conflict of interest in the research. *Correspondence to: Alireza Nakhaee, Department of Clinical Biochemistry, Faculty of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran E-mail: [email protected] Received 3 December 2014; Accepted 14 December 2014 Published online in Wiley Online Library (wileyonlinelibrary. com). DOI: 10.1111/ddr.21239
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Fig. 1. (a) Fresh aerial parts of Caralluma tubeculata washed with tap water. (b) Aerial parts of Caralluma tubeculata is shriveling in the open air and away from the light. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
With appropriate treatment, many people with DM are able to prevent or delay the onset of complications. Although different types of oral synthetic antidiabetic agents are available for the treatment of DM, their side effects have driven an increasing demand by patients to use natural products with antidiabetic activities [Day, 1998]. Because of effectiveness, fewer side effects and relatively low cost, herbal drugs are utilized widely for diabetes control [Vishwakarma et al., 2010]. The antidiabetic potential of plants is mainly related to their ability to regenerate pancreatic b-cells or insulin-mimetic effect of their component compounds. Glycosides, alkaloids, terpenoids, flavonoids, carotenoids, and other plant derived compounds are involved with antihyperglycemic effect of herbs [Malviya et al., 2010]. Plants material can also be effective in preventing diabetes complications due to their antioxidant activities [Nasri and Rafieian-Kopaei, 2014]. Caralluma is a genus of the Asclepiadaceae family with more than 100 species and is distributed in Africa, Spain, Saudi Arabia, Middle East, Pakistan, and India [Venkatesh et al., 2003]. Extracts of Caralluma species are used in traditional medicine as antidiabetic, antipyretic, and antirheumatic agents and possess significant anti-inflammatory and cancer preventive activities [Ahmed et al., 1993]. The species, Caralluma tuberculata (C. tuberculata) is a leafless and succulent plant that grows in some regions of Sistan and Baluchestan province in Iran, where it is known as “Marmootak” and its aerial powder is utilized as a herbal antidiabetic agent. Drug Dev. Res.
However, there is a little scientific information to support the effectiveness of C. tuberculata in diabetes. This study was carried out to evaluate the antidiabetic and in vivo antioxidant activities of Iranian species of C. tuberculata in streptozotocin (STZ)induced diabetic rats. METHODS AND MATERIALS
Plant Material The aerial part of plant was collected in April 2012 from a rural area of Baluchestan, Iran. The plant was authenticated at the biology department of Sistan and Baluchestan University. A voucher specimen was deposited in the Herbarium of this department. Plant materials were washed thoroughly with water to remove soil (Fig. 1a) and were dried in open air away from light (Fig. 1b). The dried materials were crushed, then pulverized in an electrical grinder and the obtained powder was stored at 0– 4 C until use. Animals Male Wistar rats weighing (200–230 g) bred from a stock obtained from Pasteur Institute of Iran were used in this study. The animals were maintained under controlled conditions of humidity, temperature (21–23 C) and 12 h light and dark cycle and had free access to standard food and water. The study was approved by the Institutional Animal Ethics Committee and all the experimental tasks with
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the animals were carried out in accordance with “Guide for the Care and Use of Laboratory Animals”.
tein content measured by the Bradford method using bovine serum albumin as standard [Bradford, 1976].
Induction of Diabetes
Antioxidant Assay
After overnight fasting, diabetes was induced by a single intraperitoneal injection of fresh STZ dissolved in 100 mM cold sodium citrate buffer, pH 4.5 at a dose of 60 mg/kg body weight. Three days after STZ injection, rats with glycosuria and fasting blood glucose over 200 mg/dl were considered as diabetic and used for the study.
Catalase assay Catalase (CAT) activity was measured according to Goth’s method [Goth, 1991]. This assay is based on the ability of hydrogen peroxide to form a stable stained complex with ammonium molybdate that is measured spectrophometically at 405 nm. Superoxide dismutase assay
Experimental Design In this study five following groups of rats with eight rats per group were used: Group I, Normal control rats; Group II, STZinduced diabetic control rats; Group III, Diabetic rats receiving 500 ll distilled water daily via intragastric gavage for 45 days; Group IV, Diabetic rats receiving 100 mg/kg/day of C. tuberculata powder in 500 ll aqueous suspension via intragastric gavage for 45 days; Group V, Diabetic rats receiving 200 mg/kg/ day of C. tuberculata powder in 500 ll aqueous suspension via intragastric gavage for 45 days. Body weight, fluid, and food intake of all the animals were measured every week. Weekly blood glucose levels were mesaured using an Accu-Check Active glucometer on samples collected from the tail vein. Preparation of Tissue Homogenate At the end of 45 days, overnight-fasted rats were anesthetized with ether and blood samples collected from the heart for serum preparation. Thereafter, the animals were killed by decapitation and their kidneys, hearts, and livers removed and placed in an ice-cold Petri dish maintained in finely crushed ice and washed with normal saline. A portion of each organ was homogenized in 10 volumes cold phosphate buffer (50 mmol/L; pH 7.0) containing 1 mM EDTA and 0.2 lM phenylmethylsulfonyl fluoride with Silent Crusher S homogenizer (Heidolph homogenizer, Germany). For enzyme assays and total thiol measurement, homogenates were centrifuged at 20,0003g for 10 min at 4 C and the resulting supernatant used for various measurements. To do the protein carbonyl (PC) assay, streptomycin sulfate with final concentration of 1% was added to the tissue homogenate and left at room temperature for 15 min. The mixture was centrifuged at above conditions to precipitate the extracted DNA which could react with carbonyl reagent. The resulting supernatant was used for PC measurement [Evans et al., 1999]. Pro-
Superoxide dismutase (SOD) activity was measured according to the method of Kakkar et al. [1984], that is based on inhibition of nitroblue tetrazolium (NBT) formazan formation in the reaction mixture of NADH–phenazinemethosulphate (PMS)–NBT. The assay mixture contained 1.2 ml of sodium pyrophosphate buffer (pH 8.3; 52 mM), 0.1 ml of PMS (186 mM), 0.3 ml of NBT (300 lM), and 0.1 ml of supernatant. Blank tube contained 1.3 ml of sodium pyrophosphate buffer (pH 8.3; 52 mM), 0.1 ml of PMS (186 lM), and 0.3 ml of NBT (300 lM). The reaction was started by the addition 0.2 ml of NADH solution (750 lM). After incubation at 30 C for 90 s, the reaction was stopped using 0.2 ml of glacial acetic acid. The reaction mixture was stirred vigorously with 2.0 ml of n-butanol and was centrifuged 4 min at 2000 rpm. After 5–10 min color intensity of the chromogen in butanol was measured spectrophotometrically at 560 nm. One unit of enzyme activity was defined as the amount of enzyme that caused 50% inhibition of NBT reduction/mg protein. Glutathione peroxidase assay Glutathione peroxidase (GPx) activity was assayed in a mixture containing 0.89 ml of 100 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 1 mM NaN3, 0.2 mM NADPH, 1 U/ml glutathione reductase (GR) (Sigma, #G3664), and 1 mM reduced glutathione (GSH). Tissue supernatant was added to a total volume of 0.9 ml. The reaction was initiated by the addition of 100 ll of 2.5 mM H2O2 and the oxidation of NADPH to NADP was measured spectrophotometrically at 340 nm for 3 min. GPx activity was expressed as nmoles of NADPH oxidized to NADP1/min mg protein, using a molar extinction coefficient of 6.22 3 103 cm21 M21 for NADPH [Amini et al., 2009]. Glutathione reductase assay GR was assayed in a reaction mixture containing 0.495 ml of 100 mM potassium phosphate buffer Drug Dev. Res.
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(pH 7.0), 1.1 mM MgCl2, 5 mM oxidized glutathione (GSSG), and 0.1 mM NADPH. Tissue supernatant was added to start the NADPH conversion reaction. Changes in absorbance were followed up at 340 nm for 5 min at 25 C. The specific enzyme activity was expressed as nmol NADPH oxidized to NADP1/min/ mg protein with 6.22 3 103 cm21 M21as the molar extinction coefficient of NADPH [Amini et al., 2009]. Total thiol assay Tissue total thiol was measured in 96 wellmicrotiter plate according to Hu’s method [Hu, 1994], with slight modification. This assay is based on reaction of thiol groups with 5, 50 -dithiobis-(2-nitrobenzoic acid) (DTNB) and forming a highly colored anion with maximum peak at 412 nm. The assay mixture contained 25 lL fresh supernatant mixed with 1 ml TE buffer (0.25 mM Tris base, 20 mM EDTA, pH 8.2) and absorbance at 412 nm was read (A1). Next, a 25 ll of DTNB stock solution (10 mM in absolute methanol) was added to the solution. After 15 min at room temperature, the absorbance was read again (A2) together with a DTNB blank (B). The concentration of sulfhydryl groups was calculated by using reduced glutathione as thiol group standard and the result was expressed in nM/mg protein. Protein carbonyl assay This assay was performed using the protocol described by Buss and coworkers [Buss et al., 1997]. This method is based on derivitization of the carbonyl groups in proteins with dinitrophenylhydrazine (DNPH) followed by ELISA. Derivatization was achieved in a mixture composed of supernatant containing 4 mg/ml protein and three volumes 10 mM DNPH in 6 M guanidine hydrochloride, pH 2.5 for 20 min at room temperature. The derivatized sample was diluted 200 fold with coating buffer (10 mM Naphosphate, 140 mM NaCl, pH 7.0). Triplicate 200 ll of the diluted sample containing 1 lg protein were added to wells of a Nunc Maxisorp microplate (Nalge Nunc International). The plates were incubated overnight at 4 C and then washed four times with PBS (300 lL/well). The wells were blocked with 250 lL/ well PBS containing 0.1 % Tween20 (blocking buffer) for 1.5 h at room temperature and washed five times with PBS. Next, 200 ll of primary rabbit anti-DNP antibody (Sigma, #D9656) 1/20,000 diluted in blocking buffer was added to each well and incubated at 37 C for 1 h. Then plates were washed five times with PBS and incubated with 200 ll of 1/30,000 diluted HRP conjugated Goat anti-Rabbit IgG antibody (Sigma, #A6154) for 1 h at room temperature. Drug Dev. Res.
TABLE 1. Effect of C. tuberculata Powder Supplementation on Blood Glucose of Rats in Different Experimental Groups Blood glucose (mg/dl) Groups
Day 3
Control STZ diabetic STZ 1 Veh STZ 1 100 CT STZ 1 200 CT
87.00 343.00 369.03 356.78 349.67
6 6 6 6 6
4.72 26.07* 28.84* 33.28* 44.96*
Day 45 97.75 402.29 416.22 275.33 213.00
6 6 6 6 6
11.86 60.89* 40.85* 74.31** 40.7**,†
STZ-veh - diabetic rats gavaged with distilled water; STZ 1 100 CT diabetic rats receiving 100 mg/kg/day of C. tuberculata; STZ 1 200 CT diabetic rats receiving 200 mg/kg/day of C. tuberculata. *P < 0.01 compared with control rats. **P < 0.01 compared with diabetic rats. † P < 0.05 compared with diabetic rats receiving 100mg/kg/day of suspension. Data are mean 6 SD for eight animals in each group.
After five wash steps, 200 ll chromogen substrate was added to the wells and after 3–5 min the reaction was stopped by adding 100 ll H2SO4 2.5 M. The absorbance was read at 450 nm using ELISA reader. The concentration of carbonyl group was calculated by a curve prepared using standard of PC assay kit (Biocell company kit) and the result was expressed in nM/mg protein. Statistical Analysis Results were expressed as mean 6 SD for eight rats in each experimental group. Statistical analysis was performed using SPSS 21 software. One-way analysis of variance followed by turkey’s post hoc test was used to compare differences between experimental groups. A P value less than 0.05 was considered statistically significant. RESULTS
Fasting Blood Glucose Levels As shown in Table 1, fasting blood glucose level in STZ-induced diabetic rats was higher compared to that of normal control rats. Treatment of diabetic rats with C. tuberculata decreased blood glucose levels compared to those of untreated and vehicle receiving diabetic rats in a dose-dependent manner (P < 0.01). There was a relative difference in blood glucose level between the rats receiving 100 and those receiving 200 mg/kg/day of plant powder (P 5 0.04). Body Weight, Fluid, and Food Intake Table 2 shows body weight, food and water intake in the experimental groups after 45 days of C. tuberculata administration. There were no significant differences in body weight between groups in the onset of the
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TABLE 2. Effect of C. tuberculata Powder Supplementation on Body Weight, Fluid and Food Intake of Rats in Different Experimental Groups Body weight (g) Groups
Day 1
Control STZ diabetic STZ 1 Veh STZ 1 100 CT STZ 1 200 CT
229.5 217.0 222.5 221.7 222.8
6 6 6 6 6
Water intake after 45 days (ml)
Day 45
22.1 15.6 19.8 13.7 17.2
268.1 191.8 189.7 230.8 232.0
6 6 6 6 6
18.3 7.1* 9.6* 21.4** 35.3**
56.2 179.4 177.8 123.2 85.7
6 6 6 6 6
Food intake after 45 days (g)
11.5 14.5* 7.4* 7.4** 6.2**
14.2 33.4 32.2 23.0 17.4
6 6 6 6 6
3.1 4.0* 2.7* 2.9** 1.4**
STZ-veh; diabetic rats gavaged with distilled water; STZ 1 100 CT diabetic rats receiving 100 mg/kg/day of C. tuberculata; STZ 1 200 CT diabetic rats receiving 200 mg/kg/day of C. tuberculata. *P < 0.01 compared with control rats (group I). **P < 0.01 compared with diabetic rats. Data are mean 6 SD for eight animals in each group
study. There was a gradual increase in body weight in normal controls while the diabetic controls and diabetic rats gavaged with water continued to lose weight compared to controls (P < 0.05). The C. tuberculata treated rats showed significant improvement in body weight as compared with diabetic control group (P < 0.01). There were no differences in body weight gain between groups receiving 100 and 200 mg/kg/day of C. tuberculata in (P 5 0.5). There was an increase in water and food intake in diabetic control rats compared to normal control rats after 45 days of experimental period (P < 0.01). Treatment with C. tuberculata decreased fluid and food intake in diabetic animals (P < 0.01). There was a difference in water and fluid intake between groups receiving 100 and 200 mg/kg/day of C. tuberculata at the end of study (P < 0.01). Biochemical Assays The effect of C. tuberculata on antioxidant enzymes activities, total thiol, and PC in liver, kidney, and heart of different experimental groups of rats are shown in Tables (3–5). Each of the three tissues
from diabetic rats showed decreases in total thiol along with increased levels of PC compared to normal control rats (P < 0.05). Oral administration of C. tuberculata to diabetic rats increased total thiol and decreased PC in tissues. The effects of C. tuberculata were more pronounced in diabetic rats treated with 200 mg/kg/ day (P < 0.05) and not significant in liver and kidney of diabetic animals gavaged with 100 mg/kg/day of plant compared to the iabetic control group (P > 0.05). The dose of 100 mg/kg/day of C. tuberculata significantly reversed diabetes-induced changes in cardiac total thiol and PC (P < 0.05). As also shown in Tables (3–5), there were reductions in SOD, CAT, GPx, and GR activities in liver (26, 32, 33, and 44%, respectively), kidney (43, 40, 30, and 33%, respectively), and heart (40, 35, 30, and 40%, respectively) of diabetic rats compared with the control group. Treatment with 200 mg/ kg/day C. tuberculata powder increased SOD,CAT, GPx, and GR activities in all three tissues from diabetic rats (P < 0.05). The dose of 100 mg/kg/day of C. tuberculata reversed diabetes-induced changes only in hepatic GPx and GR, renal and cardiac CAT and cardiac GPx activities of diabetic rats (P < 0.05).
TABLE 3. Effect of C. tuberculata Powder Supplementation on Antioxidant Enzyme Activities, Total Thiol and PC Level in Liver of Rats in Different Experimental Groups Groups Control STZ diabetic STZ 1 Veh STZ 1 100CT STZ 1 200CT
Total thiol (nM/mg protein) 47.57 34.16 33.11 39.25 45.07
6 6 6 6 6
8.38 8.65* 6.10* 5.48 3.8**
PC (nM/mg protein) 1.01 1.45 1.45 1.2 1.07
6 6 6 6 6
0.27 0.25* 0.32* 0.16 0.05**
SOD (U/mg protein) 1.14 0.85 0.82 0.97 1.15
6 6 6 6 6
0.24 0.10* 0.12* 0.17 0.14**
CAT (U/mg protein) 887.9 597.2 606.9 713.2 826.1
6 6 6 6 6
118.7 110.5* 96.5* 131.9 103.2**
GPx (U/mg protein) 0.34 0.23 0.23 0.30 0.33
6 6 6 6 6
0.07 0.04* 0.03* 0.07** 0.05**
GR (U/mg protein) 0.127 0.071 0.074 0.104 0.125
6 6 6 6 6
0.019 0.011* 0.014* 0.019** 0.02**
STZ-veh - diabetic rats gavaged with distilled water; STZ 1 100 CT diabetic rats receiving 100 mg/kg/day of C. tuberculata; STZ 1 200 CT diabetic rats receiving 200 mg/kg/day of C. tuberculata. *P < 0.05 compared with control rats. **P < 0.05 compared with diabetic rats. Data are mean 6 SD for eight animals in each group.
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TABLE 4. Effect of C. tuberculata Powder Supplementation on Antioxidant Enzyme Activities, Total Thiol and PC Level in Kidney of Rats in Different Experimental Groups Groups
Total thiol (nM/mg protein)
Control STZ diabetic STZ 1 Veh STZ 1 100 CT STZ 1 200 CT
28.42 17.5 17.25 22.62 26.33
6 6 6 6 6
3.40 5.61* 6.06* 6.21 4.33**
PC (nM/mg protein) 1.00 1.45 1.43 1.25 0.98
6 6 6 6 6
0.18 0.11* 0.28* 0.31 0.14**
SOD (U/mg protein)
CAT (U/mg protein)
GPx (U/mg protein)
1.44 0.82 0.8 1.10 1.32
240.8 144.7 142.1 198.2 222.5
0.37 0.26 0.25 0.32 0.35
6 6 6 6 6
0.21 0.16* 0.13* 0.23 0.25**
6 6 6 6 6
40.05 23.1* 18.7* 24.1** 30.4**
6 6 6 6 6
0.05 0.04* 0.03* 0.04 0.05**
GR (U/mg protein) 0.218 0.146 0.149 0.196 0.207
6 6 6 6 6
0.039 0.02* 0.028* 0.033 0.038**
STZ-veh - diabetic rats gavaged with distilled water; STZ 1 100 CT diabetic rats receiving 100 mg/kg/day of C. tuberculata; STZ 1 200 CT diabetic rats receiving 200 mg/kg/day of C. tuberculata. *P < 0.01 compared with control rats. **P < 0.01 compared with diabetic rats. Data presented as mean 6 SD for eight animals in each group.
food intake results that are consistent with previous studies using a methanolic extract of C. tuberculata in STZ-induced diabetic rats [Abdel-Sattar et al., 2011]. Medicinal plant extracts may have insulin-like effects, improving insulin sensitivity, augmenting glucose-dependent insulin secretion, and stimulating islets of Langerhans regeneration in the pancreas of STZ-induced diabetic rats [Mohan et al., 2013]. Diabetes-induced oxidative stress plays a key role in the pathogenesis of diabetic complications, for example, retinopathy, nephropathy, neuropathy, and cardiovascular disease [Pop-Busui et al., 2006; Stephens et al., 2006; Kowluru and Chan, 2007; Coughlan et al., 2008]. The reactive oxygen species produced in hyperglycemic conditions can deplete the nonenzymatic antioxidant defense system resulting in oxidative damage [Laight et al., 2000; Friedman et al., 2003]. In this study, increased PC and decreased total thiol in tissues from diabetic rat indicated the presence of of oxidative stress and protein damage. PC groups, products of oxidative damage to proteins, are sensitive markers in the evaluation of oxidative stress
DISCUSSION
In this study, we observed elevation of blood glucose and oxidative stress in liver, kidney, and heart of STZ-induced diabetic rats and improvements following oral administration of C. tuberculata powder. The study was conducted on liver because of hepatic metabolism alterations in diabetes that can increase the risk of chronic liver disease and hepatocellular carcinoma [El-serag et al., 2004; Moscatiello et al., 2007].The kidney and heart were chosen as these tissues are also impaired in diabetes. The powder of the aerial part of the plant was used so as to resemble the way in which this herb is used as a medication in Sistan and Baluchestan province. The induction of diabetes by STZ has been associated with weight loss due to elevated protein degradation and muscle wasting [Pepato et al., 1996; Rodriguez et al., 1997]. Moreover, increased water and food intake are typically observed in STZ-induced diabetic animals, a result of metabolic changes caused by insulin deficiency [Wei et al., 2003]. In this study, 200 mg/kg/day of C. tuberculata powder in diabetic rats improved blood glucose, body weight, fluid, and
TABLE 5. Effect of C. tuberculata Powder Supplementation on Antioxidant Enzyme Activities, Total Thiol and PC Level in Heart of Rats in Different Experimental Groups Groups
Total thiol (nM/mg protein)
PC (nM/mg protein)
SOD (U/mg protein)
CAT (U/mg protein)
GPx (U/mg protein)
GR (U/mg protein)
Control STZ diabetic STZ 1 Veh STZ 1 100 CT STZ 1 200 CT
26.07 14.66 14.62 19.75 22.66
1.20 1.92 1.90 1.45 1.36
1.11 0.67 0.73 0.98 1.08
81.2 52.75 53.37 73.2 83.3
0.37 0.26 0.27 0.33 0.35
0.058 0.035 0.034 0.047 0.053
6 6 6 6 6
5.26 2.73* 2.92* 3.45** 4.38**,†
6 6 6 6 6
0.35 0.23* 0.21* 0.32b** 0.35**,†
6 6 6 6 6
0.17 0.12* 0.1* 0.13 0.33**
6 6 6 6 6
10.7 7.1* 8.8* 8.8** 8.5**
6 6 6 6 6
0.05 0.03* 0.05* 0.04** 0.04**
6 6 6 6 6
0.013 0.006* 0.005* 0.008 0.01**
STZ-veh - diabetic rats gavaged with distilled water; STZ 1 100 CT diabetic rats receiving 100 mg/kg/day of C. tuberculata; STZ 1 200 CT diabetic rats receiving 200 mg/kg/day of C. tuberculata. *P < 0.01 compared with control rats. **P < 0.05 compared with diabetic rats. † P < 0.05 compared with treated diabetic rats receiving 100mg/kg/day of C. tuberculata suspension. Data presented as mean 6 SD for eight animals in each group.
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ANTIOXIDANT EFFECTS OF CARALLUMA TUBERCULATA
[Dalle-Donne et al., 2003] and occur in both type 1 and type 2 diabetes [Telci et al., 2000a, 2000b]. Elevated plasma PC levels in diabetic children and adolescents without complications indicate that oxidative damage to protein occurs at diabetes onset [Telci et al., 2000b] probably the result of decreased antioxidant mechanisms [Baynes and Thorpe, 1999]. Additionally, as glucose uptake becomes independent of insulin under hyperglycemic conditions [Cherrington, 1999], excess glucose can enter cells and is shunted to pathways that result in oxidative damage. Total thiol includes reduced protein and nonprotein sulfhydryl groups (-SH) that include reduced glutathione that are involved in free radical scavenging and protect the cell against toxic effects of oxidants. The redox state of protein thiol is also critical in protein structure and function [Wlodek, 2002]. In this study, decreased total thiol in untreated diabetic rats compared with controls indicated depletion of antioxidant defenses due to occurrence of oxidative stress. These results in this regard are consistent with other reports that show a decrease in the concentration of reduced glutathione in liver, kidney and heart of diabetic rats [Matkovics et al., 1997]. C. tuberculata treatment decreased PC and increased total thiol levels in liver, kidney, and heart of STZ-induced diabetic rats suggesting that the plant extarct can reduce diabetes-induced oxidative stress. Polyphenols may also contribute to its antioxidant properties [Ansari et al., 2005]. Antioxidant enzymes (SOD, CAT, GPx, and GR) represent the first line of defense against reactive oxygen specie [Mates et al., 1999]. SOD scavenges superoxide radicals to H2O2, whereas CAT and GPx catalyze the reduction of H2O2 to water and GR participates in the reduction of oxidized glutathione concomitant with the consumption of NADPH [Mates et al., 1999]. Reduced activity of these enzymes can lead to a deleterious effects via the accumulation of free radicals. The results of the present study demonstrated a significant decrease in tissues SOD, CAT, GPx, and GR activities of diabetic rats compared to controls and again are consistent with previous studies reporting decreased activity of SOD, CAT, GPx, and GR in liver, kidney, and heart of diabetic rats [Quine and Raghu, 2005; Kaleem et al., 2006; Kota et al., 2012; Sellamuthu et al., 2013]. This may be the result of oxidative stressinduced inactivation [Mohamadin et al., 2007] and/or glycation of enzymatic proteins, resulting from direct reaction between excess glucose and protein amino groups [Yan and Harding, 1997]. Uncontrolled diabetes in rats is associated with reduced gene expression of antioxidant enzymes [Sindhu et al., 2004; Sadi
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et al., 2008]. Decreased GSH levels in diabetes may also reduce GPx activity. However, some studies have presented contradictory results regarding the activities of antioxidant enzymes in STZ-induced diabetes [Suryanarayana et al., 2007]. Treatment of diabetic rats with C. tuberculata, to increased activities of four antioxidant enzymes in all tissues studies enhancing their free radical scavenging activity. In conclusion, while this study showed that although oral administration of C. tuberculata could not normalize blood glucose in diabetic rats, it had good antioxidant activity and could restore antioxidant enzyme activities and oxidative markers to normal level. Therefore, its use by diabetic patients may prevent oxidative stress-induced complications.
ACKNOWLEDGMENTS
The authors are grateful to Ms Rezaei and Ms Noora (Zahedan University of Medical Sciences, School of Medicine, and Clinical Biochemistry Lab) for technical assistance. This work was supported by dissertion grant (M.Sc. thesis of Jafer Poodineh) from Zahedan University of Medical Sciences, Zahedan, Iran (No: 6160). REFERENCES Abdel-Sattar E, Harraz FM, Ghareib SA, Elberry AA, Gabr S, Suliaman MI. 2011. Antihyperglycaemic and hypolipidaemic effects of the methanolic extract of Caralluma tuberculata in streptozotocin-induced diabetic rats. Nat Prod Res 25: 1171–1179. Ahmed MM, Qureshi S, Al-Bekairi AM, Shah AH, Rao RM, Qazi NS. 1993. Anti-inflammatory activity Caralluma tuberculata alcoholic extract. Fitoterapia 64: 357–360. Amini R, Nosrati N, Yazdanparast R, Molaei M. 2009. Teucrium polium in prevention of steatohepatitis in rats. Liver Int 29: 1216–1221. Ansari NM, Houlihan L, Hussain B, Pieroni A. 2005. Antioxidant activity of five vegetables traditionally consumed by SouthAsian migrants in Bradford, Yorkshire, UK. Phytother Res 19: 907–911. Baynes JW, Thorpe SR. 1999. Role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes 48: 1–9. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254. Brownlee M. 2001. Biochemistry and molecular cell biology of diabetic complications. Nature 414: 813–820. Buss H, Chan TP, Sluis KB, Domigan NM, Winterbourn CC. 1997. Protein carbonyl measurement by a sensitive ELISA method. Free Radic Biol Med 23: 361–366. Cherrington AD. 1999. Banting Lecture 1997. Control of glucose uptake and release by the liver in vivo. Diabetes 48: 1198– 1214.
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Pop-Busui R, Sima A, Stevens M. 2006. Diabetic neuropathy and oxidative stress. Diabetes Metab Res Rev 22: 257–273.
El-Serag HB, Tran T, Everhart JE. 2004. Diabetes increases the risk of chronic liver disease and hepatocellular carcinoma. Gastroenterology 126: 460–468.
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Rodriguez T, Alvarez B, Busquets S, Carbo N, Lopez-Soriano FJ, Argiles JM. 1997. The increased skeletal muscle protein turnover of the streptozotocin diabetic rat is associated with high concentrations of branched-chain amino acids. Biochem Mol Med 61: 87–94.
Friedman J, Peleg E, Kagan T, Shnizer S, Roenthal T. 2003. Oxidative stress in hypertensive,diabetic, and diabetic hypertensive rats. Am J Hypertens 16: 1049–1052. Giacco F, Brownlee M. 2010. Oxidative stress and diabetic complications. Circ Res 107: 1058–1070. Goth L. 1991. A simple method for determination of serum catalase activity and revision of reference range. Clin Chim Acta 196: 143–151. Hu ML. 1994. Measurement of protein thiol groups and glutathione in plasma. Methods Enzymol 233: 380–385. Kakkar P, Das B, Viswanathan PN. 1984. A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys 21: 130–132. Kaleem M, Asif M, Ahmed QU, Bano B. 2006. Antidiabetic and antioxidant activity of Annona squamosa extract in streptozotocininduced diabetic rats. Singapore Med J 47: 670–675. Kota N, Panpatil VV, Kaleb R, Varanasi B, Polasa K. 2012. Dosedependent effect in the inhibition of oxidative stress and anticlastogenic potential of ginger in STZ induced diabetic rats. Food Chem 135: 2954–2959. Kowluru RA, Chan PS. 2007. Oxidative stress and diabetic retinopathy. Exp Diabetes Res 2007: 43603. € Laight D, Carrier M, Anggard E. 2000. Antioxidants, diabetes and endothelial dysfunction. Cardiovasc Res 47: 457–464.
Sadi G, Yilmaz O, Guray T. 2008. Effect of vitamin C and lipoic acid on streptozotocin-induced diabetes gene expression: mRNA and protein expressions of Cu-Zn SOD and catalase. Mol Cell Biochem 309: 109–116. Sellamuthu PS, Arulselvan P, Kamalraj S, Fakurazi I S, Kandasamy M. 2013. Protective nature of mangiferin on oxidative stress and antioxidant status in tissues of streptozotocininduced diabetic rats. ISRN Pharmacol 2013: 750109. Sindhu RK, Koo JR, Roberts CK, Vaziri ND. 2004. Dysregulation of hepatic superoxide dismutase, catalase and glutathione peroxidase in diabetes: response to insulin and antioxidant therapies. Clin Exp Hypertens 26: 43–53. Stephens JW, Gable DR, Hurel SJ, Miller GJ, Cooper JA, Humphries SE. 2006. Increased plasma markers of oxidative stress are associated with coronary heart disease in males with diabetes mellitus and with 10-year risk in a prospective sample of males. Clin Chem 52: 446–452. Suryanarayana P, Satyanarayana A, Balakrishna N, Kumar PU, Reddy GB. 2007. Effect of turmeric and curcumin on oxidative stress and antioxidant enzymes in streptozotocin-induced diabetic rat. Med Sci Monit 13: Br286–Br292.
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Antioxidant Activities of Caralluma tuberculata on Streptozotocin-Induced Diabetic Rats
DDR
DRUG DEVELOPMENT RESEARCH 119 : 00–00 (2014)
Jafar Poodineh,1,3 Abdurrashid Khazaei Feizabad,2 and Alireza Nakhaee1,3* Cellular and Molecular Research Center, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran 2 English Department, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran 3 Department of Clinical Biochemistry, School of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran
1
Strategy, Management and Health Policy Enabling Technology, Genomics, Proteomics
Preclinical Research
Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics
Clinical Development Phases I-III Regulatory, Quality, Manufacturing
Postmarketing Phase IV
ABSTRACT The aim of this study was to elucidate the antioxidant effects of Caralluma tuberculata (C. tuberculata) in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced in male Wistar rats with an intraperitoneal injection of STZ at dose of 60 mg/kg body weight. Three days after diabetes induction, powdered aerial part of plant at doses of 100 and 200 mg/kg body weight were gavaged orally for a period of 45 days. The diabetes significantly decreased the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and level of total thiol in liver, kidney, and heart of animals (P < 0.05). In contrast, a significant increase in the levels of protein carbonyl was observed in diabetic rats compared with control animals (P < 0.05). Oral treatment of diabetic rats with C. tuberculata showed ameliorative effects on blood glucose and markers of oxidative stress in a dose-dependent manner. Altered levels of all oxidative stress parameters in tissues of diabetic rats reverted back to those normal animals after the treatment with dose of 200 mg/kg /day of plant materials. It seems that the appropriate dose of C. tuberculata has both antihyperglycemic and antioxidant activities in STZ-induced diabetic rats. Therefore, it can have preventive properties on oxidative stressC 2014 Wiley Periodicals, Inc. V induced diabetic complications. Drug Dev Res 00 : 00–00, 2014. Key words: Caralluma tuberculata; antioxidant; streptozotocin; diabetes
INTRODUCTION
Diabetes mellitus (DM) is a common metabolic disorder associated with an increased risk of nephropathy, cardiovascular diseases, retinopathy, neuropathy, and foot problems [Giacco and Brownlee, 2010]. DM is associated with increased production of free radicals and a decrease in antioxidant effectiveness, a situation that is defined as oxidative stress [Maritim et al., 2003]. Elevated production of free radicals and oxidative stress play a central role in the development of diabetic complications in various tissues [Baynes and Thorpe, 1999; Brownlee, 2001]. C 2014 Wiley Periodicals, Inc. V
Conflict of interests: The authors declare that they have no conflict of interest in the research. *Correspondence to: Alireza Nakhaee, Department of Clinical Biochemistry, Faculty of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran E-mail: [email protected] Received 3 December 2014; Accepted 14 December 2014 Published online in Wiley Online Library (wileyonlinelibrary. com). DOI: 10.1111/ddr.21239
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Fig. 1. (a) Fresh aerial parts of Caralluma tubeculata washed with tap water. (b) Aerial parts of Caralluma tubeculata is shriveling in the open air and away from the light. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
With appropriate treatment, many people with DM are able to prevent or delay the onset of complications. Although different types of oral synthetic antidiabetic agents are available for the treatment of DM, their side effects have driven an increasing demand by patients to use natural products with antidiabetic activities [Day, 1998]. Because of effectiveness, fewer side effects and relatively low cost, herbal drugs are utilized widely for diabetes control [Vishwakarma et al., 2010]. The antidiabetic potential of plants is mainly related to their ability to regenerate pancreatic b-cells or insulin-mimetic effect of their component compounds. Glycosides, alkaloids, terpenoids, flavonoids, carotenoids, and other plant derived compounds are involved with antihyperglycemic effect of herbs [Malviya et al., 2010]. Plants material can also be effective in preventing diabetes complications due to their antioxidant activities [Nasri and Rafieian-Kopaei, 2014]. Caralluma is a genus of the Asclepiadaceae family with more than 100 species and is distributed in Africa, Spain, Saudi Arabia, Middle East, Pakistan, and India [Venkatesh et al., 2003]. Extracts of Caralluma species are used in traditional medicine as antidiabetic, antipyretic, and antirheumatic agents and possess significant anti-inflammatory and cancer preventive activities [Ahmed et al., 1993]. The species, Caralluma tuberculata (C. tuberculata) is a leafless and succulent plant that grows in some regions of Sistan and Baluchestan province in Iran, where it is known as “Marmootak” and its aerial powder is utilized as a herbal antidiabetic agent. Drug Dev. Res.
However, there is a little scientific information to support the effectiveness of C. tuberculata in diabetes. This study was carried out to evaluate the antidiabetic and in vivo antioxidant activities of Iranian species of C. tuberculata in streptozotocin (STZ)induced diabetic rats. METHODS AND MATERIALS
Plant Material The aerial part of plant was collected in April 2012 from a rural area of Baluchestan, Iran. The plant was authenticated at the biology department of Sistan and Baluchestan University. A voucher specimen was deposited in the Herbarium of this department. Plant materials were washed thoroughly with water to remove soil (Fig. 1a) and were dried in open air away from light (Fig. 1b). The dried materials were crushed, then pulverized in an electrical grinder and the obtained powder was stored at 0– 4 C until use. Animals Male Wistar rats weighing (200–230 g) bred from a stock obtained from Pasteur Institute of Iran were used in this study. The animals were maintained under controlled conditions of humidity, temperature (21–23 C) and 12 h light and dark cycle and had free access to standard food and water. The study was approved by the Institutional Animal Ethics Committee and all the experimental tasks with
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3
the animals were carried out in accordance with “Guide for the Care and Use of Laboratory Animals”.
tein content measured by the Bradford method using bovine serum albumin as standard [Bradford, 1976].
Induction of Diabetes
Antioxidant Assay
After overnight fasting, diabetes was induced by a single intraperitoneal injection of fresh STZ dissolved in 100 mM cold sodium citrate buffer, pH 4.5 at a dose of 60 mg/kg body weight. Three days after STZ injection, rats with glycosuria and fasting blood glucose over 200 mg/dl were considered as diabetic and used for the study.
Catalase assay Catalase (CAT) activity was measured according to Goth’s method [Goth, 1991]. This assay is based on the ability of hydrogen peroxide to form a stable stained complex with ammonium molybdate that is measured spectrophometically at 405 nm. Superoxide dismutase assay
Experimental Design In this study five following groups of rats with eight rats per group were used: Group I, Normal control rats; Group II, STZinduced diabetic control rats; Group III, Diabetic rats receiving 500 ll distilled water daily via intragastric gavage for 45 days; Group IV, Diabetic rats receiving 100 mg/kg/day of C. tuberculata powder in 500 ll aqueous suspension via intragastric gavage for 45 days; Group V, Diabetic rats receiving 200 mg/kg/ day of C. tuberculata powder in 500 ll aqueous suspension via intragastric gavage for 45 days. Body weight, fluid, and food intake of all the animals were measured every week. Weekly blood glucose levels were mesaured using an Accu-Check Active glucometer on samples collected from the tail vein. Preparation of Tissue Homogenate At the end of 45 days, overnight-fasted rats were anesthetized with ether and blood samples collected from the heart for serum preparation. Thereafter, the animals were killed by decapitation and their kidneys, hearts, and livers removed and placed in an ice-cold Petri dish maintained in finely crushed ice and washed with normal saline. A portion of each organ was homogenized in 10 volumes cold phosphate buffer (50 mmol/L; pH 7.0) containing 1 mM EDTA and 0.2 lM phenylmethylsulfonyl fluoride with Silent Crusher S homogenizer (Heidolph homogenizer, Germany). For enzyme assays and total thiol measurement, homogenates were centrifuged at 20,0003g for 10 min at 4 C and the resulting supernatant used for various measurements. To do the protein carbonyl (PC) assay, streptomycin sulfate with final concentration of 1% was added to the tissue homogenate and left at room temperature for 15 min. The mixture was centrifuged at above conditions to precipitate the extracted DNA which could react with carbonyl reagent. The resulting supernatant was used for PC measurement [Evans et al., 1999]. Pro-
Superoxide dismutase (SOD) activity was measured according to the method of Kakkar et al. [1984], that is based on inhibition of nitroblue tetrazolium (NBT) formazan formation in the reaction mixture of NADH–phenazinemethosulphate (PMS)–NBT. The assay mixture contained 1.2 ml of sodium pyrophosphate buffer (pH 8.3; 52 mM), 0.1 ml of PMS (186 mM), 0.3 ml of NBT (300 lM), and 0.1 ml of supernatant. Blank tube contained 1.3 ml of sodium pyrophosphate buffer (pH 8.3; 52 mM), 0.1 ml of PMS (186 lM), and 0.3 ml of NBT (300 lM). The reaction was started by the addition 0.2 ml of NADH solution (750 lM). After incubation at 30 C for 90 s, the reaction was stopped using 0.2 ml of glacial acetic acid. The reaction mixture was stirred vigorously with 2.0 ml of n-butanol and was centrifuged 4 min at 2000 rpm. After 5–10 min color intensity of the chromogen in butanol was measured spectrophotometrically at 560 nm. One unit of enzyme activity was defined as the amount of enzyme that caused 50% inhibition of NBT reduction/mg protein. Glutathione peroxidase assay Glutathione peroxidase (GPx) activity was assayed in a mixture containing 0.89 ml of 100 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 1 mM NaN3, 0.2 mM NADPH, 1 U/ml glutathione reductase (GR) (Sigma, #G3664), and 1 mM reduced glutathione (GSH). Tissue supernatant was added to a total volume of 0.9 ml. The reaction was initiated by the addition of 100 ll of 2.5 mM H2O2 and the oxidation of NADPH to NADP was measured spectrophotometrically at 340 nm for 3 min. GPx activity was expressed as nmoles of NADPH oxidized to NADP1/min mg protein, using a molar extinction coefficient of 6.22 3 103 cm21 M21 for NADPH [Amini et al., 2009]. Glutathione reductase assay GR was assayed in a reaction mixture containing 0.495 ml of 100 mM potassium phosphate buffer Drug Dev. Res.
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(pH 7.0), 1.1 mM MgCl2, 5 mM oxidized glutathione (GSSG), and 0.1 mM NADPH. Tissue supernatant was added to start the NADPH conversion reaction. Changes in absorbance were followed up at 340 nm for 5 min at 25 C. The specific enzyme activity was expressed as nmol NADPH oxidized to NADP1/min/ mg protein with 6.22 3 103 cm21 M21as the molar extinction coefficient of NADPH [Amini et al., 2009]. Total thiol assay Tissue total thiol was measured in 96 wellmicrotiter plate according to Hu’s method [Hu, 1994], with slight modification. This assay is based on reaction of thiol groups with 5, 50 -dithiobis-(2-nitrobenzoic acid) (DTNB) and forming a highly colored anion with maximum peak at 412 nm. The assay mixture contained 25 lL fresh supernatant mixed with 1 ml TE buffer (0.25 mM Tris base, 20 mM EDTA, pH 8.2) and absorbance at 412 nm was read (A1). Next, a 25 ll of DTNB stock solution (10 mM in absolute methanol) was added to the solution. After 15 min at room temperature, the absorbance was read again (A2) together with a DTNB blank (B). The concentration of sulfhydryl groups was calculated by using reduced glutathione as thiol group standard and the result was expressed in nM/mg protein. Protein carbonyl assay This assay was performed using the protocol described by Buss and coworkers [Buss et al., 1997]. This method is based on derivitization of the carbonyl groups in proteins with dinitrophenylhydrazine (DNPH) followed by ELISA. Derivatization was achieved in a mixture composed of supernatant containing 4 mg/ml protein and three volumes 10 mM DNPH in 6 M guanidine hydrochloride, pH 2.5 for 20 min at room temperature. The derivatized sample was diluted 200 fold with coating buffer (10 mM Naphosphate, 140 mM NaCl, pH 7.0). Triplicate 200 ll of the diluted sample containing 1 lg protein were added to wells of a Nunc Maxisorp microplate (Nalge Nunc International). The plates were incubated overnight at 4 C and then washed four times with PBS (300 lL/well). The wells were blocked with 250 lL/ well PBS containing 0.1 % Tween20 (blocking buffer) for 1.5 h at room temperature and washed five times with PBS. Next, 200 ll of primary rabbit anti-DNP antibody (Sigma, #D9656) 1/20,000 diluted in blocking buffer was added to each well and incubated at 37 C for 1 h. Then plates were washed five times with PBS and incubated with 200 ll of 1/30,000 diluted HRP conjugated Goat anti-Rabbit IgG antibody (Sigma, #A6154) for 1 h at room temperature. Drug Dev. Res.
TABLE 1. Effect of C. tuberculata Powder Supplementation on Blood Glucose of Rats in Different Experimental Groups Blood glucose (mg/dl) Groups
Day 3
Control STZ diabetic STZ 1 Veh STZ 1 100 CT STZ 1 200 CT
87.00 343.00 369.03 356.78 349.67
6 6 6 6 6
4.72 26.07* 28.84* 33.28* 44.96*
Day 45 97.75 402.29 416.22 275.33 213.00
6 6 6 6 6
11.86 60.89* 40.85* 74.31** 40.7**,†
STZ-veh - diabetic rats gavaged with distilled water; STZ 1 100 CT diabetic rats receiving 100 mg/kg/day of C. tuberculata; STZ 1 200 CT diabetic rats receiving 200 mg/kg/day of C. tuberculata. *P < 0.01 compared with control rats. **P < 0.01 compared with diabetic rats. † P < 0.05 compared with diabetic rats receiving 100mg/kg/day of suspension. Data are mean 6 SD for eight animals in each group.
After five wash steps, 200 ll chromogen substrate was added to the wells and after 3–5 min the reaction was stopped by adding 100 ll H2SO4 2.5 M. The absorbance was read at 450 nm using ELISA reader. The concentration of carbonyl group was calculated by a curve prepared using standard of PC assay kit (Biocell company kit) and the result was expressed in nM/mg protein. Statistical Analysis Results were expressed as mean 6 SD for eight rats in each experimental group. Statistical analysis was performed using SPSS 21 software. One-way analysis of variance followed by turkey’s post hoc test was used to compare differences between experimental groups. A P value less than 0.05 was considered statistically significant. RESULTS
Fasting Blood Glucose Levels As shown in Table 1, fasting blood glucose level in STZ-induced diabetic rats was higher compared to that of normal control rats. Treatment of diabetic rats with C. tuberculata decreased blood glucose levels compared to those of untreated and vehicle receiving diabetic rats in a dose-dependent manner (P < 0.01). There was a relative difference in blood glucose level between the rats receiving 100 and those receiving 200 mg/kg/day of plant powder (P 5 0.04). Body Weight, Fluid, and Food Intake Table 2 shows body weight, food and water intake in the experimental groups after 45 days of C. tuberculata administration. There were no significant differences in body weight between groups in the onset of the
ANTIOXIDANT EFFECTS OF CARALLUMA TUBERCULATA
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TABLE 2. Effect of C. tuberculata Powder Supplementation on Body Weight, Fluid and Food Intake of Rats in Different Experimental Groups Body weight (g) Groups
Day 1
Control STZ diabetic STZ 1 Veh STZ 1 100 CT STZ 1 200 CT
229.5 217.0 222.5 221.7 222.8
6 6 6 6 6
Water intake after 45 days (ml)
Day 45
22.1 15.6 19.8 13.7 17.2
268.1 191.8 189.7 230.8 232.0
6 6 6 6 6
18.3 7.1* 9.6* 21.4** 35.3**
56.2 179.4 177.8 123.2 85.7
6 6 6 6 6
Food intake after 45 days (g)
11.5 14.5* 7.4* 7.4** 6.2**
14.2 33.4 32.2 23.0 17.4
6 6 6 6 6
3.1 4.0* 2.7* 2.9** 1.4**
STZ-veh; diabetic rats gavaged with distilled water; STZ 1 100 CT diabetic rats receiving 100 mg/kg/day of C. tuberculata; STZ 1 200 CT diabetic rats receiving 200 mg/kg/day of C. tuberculata. *P < 0.01 compared with control rats (group I). **P < 0.01 compared with diabetic rats. Data are mean 6 SD for eight animals in each group
study. There was a gradual increase in body weight in normal controls while the diabetic controls and diabetic rats gavaged with water continued to lose weight compared to controls (P < 0.05). The C. tuberculata treated rats showed significant improvement in body weight as compared with diabetic control group (P < 0.01). There were no differences in body weight gain between groups receiving 100 and 200 mg/kg/day of C. tuberculata in (P 5 0.5). There was an increase in water and food intake in diabetic control rats compared to normal control rats after 45 days of experimental period (P < 0.01). Treatment with C. tuberculata decreased fluid and food intake in diabetic animals (P < 0.01). There was a difference in water and fluid intake between groups receiving 100 and 200 mg/kg/day of C. tuberculata at the end of study (P < 0.01). Biochemical Assays The effect of C. tuberculata on antioxidant enzymes activities, total thiol, and PC in liver, kidney, and heart of different experimental groups of rats are shown in Tables (3–5). Each of the three tissues
from diabetic rats showed decreases in total thiol along with increased levels of PC compared to normal control rats (P < 0.05). Oral administration of C. tuberculata to diabetic rats increased total thiol and decreased PC in tissues. The effects of C. tuberculata were more pronounced in diabetic rats treated with 200 mg/kg/ day (P < 0.05) and not significant in liver and kidney of diabetic animals gavaged with 100 mg/kg/day of plant compared to the iabetic control group (P > 0.05). The dose of 100 mg/kg/day of C. tuberculata significantly reversed diabetes-induced changes in cardiac total thiol and PC (P < 0.05). As also shown in Tables (3–5), there were reductions in SOD, CAT, GPx, and GR activities in liver (26, 32, 33, and 44%, respectively), kidney (43, 40, 30, and 33%, respectively), and heart (40, 35, 30, and 40%, respectively) of diabetic rats compared with the control group. Treatment with 200 mg/ kg/day C. tuberculata powder increased SOD,CAT, GPx, and GR activities in all three tissues from diabetic rats (P < 0.05). The dose of 100 mg/kg/day of C. tuberculata reversed diabetes-induced changes only in hepatic GPx and GR, renal and cardiac CAT and cardiac GPx activities of diabetic rats (P < 0.05).
TABLE 3. Effect of C. tuberculata Powder Supplementation on Antioxidant Enzyme Activities, Total Thiol and PC Level in Liver of Rats in Different Experimental Groups Groups Control STZ diabetic STZ 1 Veh STZ 1 100CT STZ 1 200CT
Total thiol (nM/mg protein) 47.57 34.16 33.11 39.25 45.07
6 6 6 6 6
8.38 8.65* 6.10* 5.48 3.8**
PC (nM/mg protein) 1.01 1.45 1.45 1.2 1.07
6 6 6 6 6
0.27 0.25* 0.32* 0.16 0.05**
SOD (U/mg protein) 1.14 0.85 0.82 0.97 1.15
6 6 6 6 6
0.24 0.10* 0.12* 0.17 0.14**
CAT (U/mg protein) 887.9 597.2 606.9 713.2 826.1
6 6 6 6 6
118.7 110.5* 96.5* 131.9 103.2**
GPx (U/mg protein) 0.34 0.23 0.23 0.30 0.33
6 6 6 6 6
0.07 0.04* 0.03* 0.07** 0.05**
GR (U/mg protein) 0.127 0.071 0.074 0.104 0.125
6 6 6 6 6
0.019 0.011* 0.014* 0.019** 0.02**
STZ-veh - diabetic rats gavaged with distilled water; STZ 1 100 CT diabetic rats receiving 100 mg/kg/day of C. tuberculata; STZ 1 200 CT diabetic rats receiving 200 mg/kg/day of C. tuberculata. *P < 0.05 compared with control rats. **P < 0.05 compared with diabetic rats. Data are mean 6 SD for eight animals in each group.
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TABLE 4. Effect of C. tuberculata Powder Supplementation on Antioxidant Enzyme Activities, Total Thiol and PC Level in Kidney of Rats in Different Experimental Groups Groups
Total thiol (nM/mg protein)
Control STZ diabetic STZ 1 Veh STZ 1 100 CT STZ 1 200 CT
28.42 17.5 17.25 22.62 26.33
6 6 6 6 6
3.40 5.61* 6.06* 6.21 4.33**
PC (nM/mg protein) 1.00 1.45 1.43 1.25 0.98
6 6 6 6 6
0.18 0.11* 0.28* 0.31 0.14**
SOD (U/mg protein)
CAT (U/mg protein)
GPx (U/mg protein)
1.44 0.82 0.8 1.10 1.32
240.8 144.7 142.1 198.2 222.5
0.37 0.26 0.25 0.32 0.35
6 6 6 6 6
0.21 0.16* 0.13* 0.23 0.25**
6 6 6 6 6
40.05 23.1* 18.7* 24.1** 30.4**
6 6 6 6 6
0.05 0.04* 0.03* 0.04 0.05**
GR (U/mg protein) 0.218 0.146 0.149 0.196 0.207
6 6 6 6 6
0.039 0.02* 0.028* 0.033 0.038**
STZ-veh - diabetic rats gavaged with distilled water; STZ 1 100 CT diabetic rats receiving 100 mg/kg/day of C. tuberculata; STZ 1 200 CT diabetic rats receiving 200 mg/kg/day of C. tuberculata. *P < 0.01 compared with control rats. **P < 0.01 compared with diabetic rats. Data presented as mean 6 SD for eight animals in each group.
food intake results that are consistent with previous studies using a methanolic extract of C. tuberculata in STZ-induced diabetic rats [Abdel-Sattar et al., 2011]. Medicinal plant extracts may have insulin-like effects, improving insulin sensitivity, augmenting glucose-dependent insulin secretion, and stimulating islets of Langerhans regeneration in the pancreas of STZ-induced diabetic rats [Mohan et al., 2013]. Diabetes-induced oxidative stress plays a key role in the pathogenesis of diabetic complications, for example, retinopathy, nephropathy, neuropathy, and cardiovascular disease [Pop-Busui et al., 2006; Stephens et al., 2006; Kowluru and Chan, 2007; Coughlan et al., 2008]. The reactive oxygen species produced in hyperglycemic conditions can deplete the nonenzymatic antioxidant defense system resulting in oxidative damage [Laight et al., 2000; Friedman et al., 2003]. In this study, increased PC and decreased total thiol in tissues from diabetic rat indicated the presence of of oxidative stress and protein damage. PC groups, products of oxidative damage to proteins, are sensitive markers in the evaluation of oxidative stress
DISCUSSION
In this study, we observed elevation of blood glucose and oxidative stress in liver, kidney, and heart of STZ-induced diabetic rats and improvements following oral administration of C. tuberculata powder. The study was conducted on liver because of hepatic metabolism alterations in diabetes that can increase the risk of chronic liver disease and hepatocellular carcinoma [El-serag et al., 2004; Moscatiello et al., 2007].The kidney and heart were chosen as these tissues are also impaired in diabetes. The powder of the aerial part of the plant was used so as to resemble the way in which this herb is used as a medication in Sistan and Baluchestan province. The induction of diabetes by STZ has been associated with weight loss due to elevated protein degradation and muscle wasting [Pepato et al., 1996; Rodriguez et al., 1997]. Moreover, increased water and food intake are typically observed in STZ-induced diabetic animals, a result of metabolic changes caused by insulin deficiency [Wei et al., 2003]. In this study, 200 mg/kg/day of C. tuberculata powder in diabetic rats improved blood glucose, body weight, fluid, and
TABLE 5. Effect of C. tuberculata Powder Supplementation on Antioxidant Enzyme Activities, Total Thiol and PC Level in Heart of Rats in Different Experimental Groups Groups
Total thiol (nM/mg protein)
PC (nM/mg protein)
SOD (U/mg protein)
CAT (U/mg protein)
GPx (U/mg protein)
GR (U/mg protein)
Control STZ diabetic STZ 1 Veh STZ 1 100 CT STZ 1 200 CT
26.07 14.66 14.62 19.75 22.66
1.20 1.92 1.90 1.45 1.36
1.11 0.67 0.73 0.98 1.08
81.2 52.75 53.37 73.2 83.3
0.37 0.26 0.27 0.33 0.35
0.058 0.035 0.034 0.047 0.053
6 6 6 6 6
5.26 2.73* 2.92* 3.45** 4.38**,†
6 6 6 6 6
0.35 0.23* 0.21* 0.32b** 0.35**,†
6 6 6 6 6
0.17 0.12* 0.1* 0.13 0.33**
6 6 6 6 6
10.7 7.1* 8.8* 8.8** 8.5**
6 6 6 6 6
0.05 0.03* 0.05* 0.04** 0.04**
6 6 6 6 6
0.013 0.006* 0.005* 0.008 0.01**
STZ-veh - diabetic rats gavaged with distilled water; STZ 1 100 CT diabetic rats receiving 100 mg/kg/day of C. tuberculata; STZ 1 200 CT diabetic rats receiving 200 mg/kg/day of C. tuberculata. *P < 0.01 compared with control rats. **P < 0.05 compared with diabetic rats. † P < 0.05 compared with treated diabetic rats receiving 100mg/kg/day of C. tuberculata suspension. Data presented as mean 6 SD for eight animals in each group.
Drug Dev. Res.
ANTIOXIDANT EFFECTS OF CARALLUMA TUBERCULATA
[Dalle-Donne et al., 2003] and occur in both type 1 and type 2 diabetes [Telci et al., 2000a, 2000b]. Elevated plasma PC levels in diabetic children and adolescents without complications indicate that oxidative damage to protein occurs at diabetes onset [Telci et al., 2000b] probably the result of decreased antioxidant mechanisms [Baynes and Thorpe, 1999]. Additionally, as glucose uptake becomes independent of insulin under hyperglycemic conditions [Cherrington, 1999], excess glucose can enter cells and is shunted to pathways that result in oxidative damage. Total thiol includes reduced protein and nonprotein sulfhydryl groups (-SH) that include reduced glutathione that are involved in free radical scavenging and protect the cell against toxic effects of oxidants. The redox state of protein thiol is also critical in protein structure and function [Wlodek, 2002]. In this study, decreased total thiol in untreated diabetic rats compared with controls indicated depletion of antioxidant defenses due to occurrence of oxidative stress. These results in this regard are consistent with other reports that show a decrease in the concentration of reduced glutathione in liver, kidney and heart of diabetic rats [Matkovics et al., 1997]. C. tuberculata treatment decreased PC and increased total thiol levels in liver, kidney, and heart of STZ-induced diabetic rats suggesting that the plant extarct can reduce diabetes-induced oxidative stress. Polyphenols may also contribute to its antioxidant properties [Ansari et al., 2005]. Antioxidant enzymes (SOD, CAT, GPx, and GR) represent the first line of defense against reactive oxygen specie [Mates et al., 1999]. SOD scavenges superoxide radicals to H2O2, whereas CAT and GPx catalyze the reduction of H2O2 to water and GR participates in the reduction of oxidized glutathione concomitant with the consumption of NADPH [Mates et al., 1999]. Reduced activity of these enzymes can lead to a deleterious effects via the accumulation of free radicals. The results of the present study demonstrated a significant decrease in tissues SOD, CAT, GPx, and GR activities of diabetic rats compared to controls and again are consistent with previous studies reporting decreased activity of SOD, CAT, GPx, and GR in liver, kidney, and heart of diabetic rats [Quine and Raghu, 2005; Kaleem et al., 2006; Kota et al., 2012; Sellamuthu et al., 2013]. This may be the result of oxidative stressinduced inactivation [Mohamadin et al., 2007] and/or glycation of enzymatic proteins, resulting from direct reaction between excess glucose and protein amino groups [Yan and Harding, 1997]. Uncontrolled diabetes in rats is associated with reduced gene expression of antioxidant enzymes [Sindhu et al., 2004; Sadi
7
et al., 2008]. Decreased GSH levels in diabetes may also reduce GPx activity. However, some studies have presented contradictory results regarding the activities of antioxidant enzymes in STZ-induced diabetes [Suryanarayana et al., 2007]. Treatment of diabetic rats with C. tuberculata, to increased activities of four antioxidant enzymes in all tissues studies enhancing their free radical scavenging activity. In conclusion, while this study showed that although oral administration of C. tuberculata could not normalize blood glucose in diabetic rats, it had good antioxidant activity and could restore antioxidant enzyme activities and oxidative markers to normal level. Therefore, its use by diabetic patients may prevent oxidative stress-induced complications.
ACKNOWLEDGMENTS
The authors are grateful to Ms Rezaei and Ms Noora (Zahedan University of Medical Sciences, School of Medicine, and Clinical Biochemistry Lab) for technical assistance. This work was supported by dissertion grant (M.Sc. thesis of Jafer Poodineh) from Zahedan University of Medical Sciences, Zahedan, Iran (No: 6160). REFERENCES Abdel-Sattar E, Harraz FM, Ghareib SA, Elberry AA, Gabr S, Suliaman MI. 2011. Antihyperglycaemic and hypolipidaemic effects of the methanolic extract of Caralluma tuberculata in streptozotocin-induced diabetic rats. Nat Prod Res 25: 1171–1179. Ahmed MM, Qureshi S, Al-Bekairi AM, Shah AH, Rao RM, Qazi NS. 1993. Anti-inflammatory activity Caralluma tuberculata alcoholic extract. Fitoterapia 64: 357–360. Amini R, Nosrati N, Yazdanparast R, Molaei M. 2009. Teucrium polium in prevention of steatohepatitis in rats. Liver Int 29: 1216–1221. Ansari NM, Houlihan L, Hussain B, Pieroni A. 2005. Antioxidant activity of five vegetables traditionally consumed by SouthAsian migrants in Bradford, Yorkshire, UK. Phytother Res 19: 907–911. Baynes JW, Thorpe SR. 1999. Role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes 48: 1–9. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254. Brownlee M. 2001. Biochemistry and molecular cell biology of diabetic complications. Nature 414: 813–820. Buss H, Chan TP, Sluis KB, Domigan NM, Winterbourn CC. 1997. Protein carbonyl measurement by a sensitive ELISA method. Free Radic Biol Med 23: 361–366. Cherrington AD. 1999. Banting Lecture 1997. Control of glucose uptake and release by the liver in vivo. Diabetes 48: 1198– 1214.
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