modulation of oxidative stress by chlorella vulgaris in streptozotocin (stz) induced diabetic...

· Advances in Medical Sciences · Vol. 55(2) · 2010 · pp 281-288 · DOI: 10.2478/v10039-010-0046-z© Medical University of Bialystok, Poland

Modulation of oxidative stress by Chlorella vulgaris in streptozotocin (STZ) induced diabetic Sprague-Dawley rats

1 Universiti Kebangsaan Malaysia Medical Center (UKMMC), Jalan Yaacob Latif, Bandar Tun Razak, Cheras, Kuala Lumpur, Malaysia2 Department of Anatomy, Faculty of Medicine, Univerisiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

3 Department of Biochemistry, Faculty of Medicine, Univerisiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, Kuala Lumpur, Malaysia

Aizzat O1, Yap SW1, Sopiah H1, Madiha MM1, Hazreen M1, Shailah A3, Wan Junizam WY3, Nur Syaidah A3, Srijit Das2, Musalmah M3, Yasmin Anum MY3*

ABSTRACT

Purpose: Chlorella vulgaris (CV), a fresh water alga has been reported to have hypoglycemic effects. However, antioxidant and anti-inflammatory effects of CV in diabetic animals have not been investigated to date. The aim of the present study was to investigate the role of CV in inflammation and oxidative damage in STZ-induced diabetic rats. Materials and methods: Male Sprague-Dawley rats (300 - 400g) were divided into 4 groups: control, CV, STZ-induced diabetic rats, and STZ rats treated with CV (150mg/kg body wt). Blood samples were drawn from orbital sinus at 1 and 4 weeks for determination of oxidative cellular damage (DNA damage and lipid peroxidation [malondialdehyde, MDA]), inflammation (tumour necrosis factor alpha, TNF-α) and antioxidant status (catalase, CAT, and superoxide dismutase, SOD). Results: CV did not have any effects on glucose levels in diabetic rats, over the 4 weeks of treatment. However, it reduced significantly DNA damage and blood MDA levels in STZ-induced diabetic rats compared to the control group. Plasma levels of TNF-α however did not show any significant changes in STZ-induced diabetic rats fed with CV. Antioxidant enzyme SOD showed no significant changes in all groups but CAT activity was reduced in STZ-induced diabetic rats compared to the control. Conclusions: CV did not have hypoglycaemic effect but it has a protective role in STZ-induced diabetic rats by reducing oxidative DNA damage, and lipid peroxidation.

Key words: diabetes mellitus, Chlorella vulgaris, streptozotocin, oxidative damage, hypoglycaemic

* CORRESPONDING AUTHOR:Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia Jalan Raja Muda Abdul Aziz, 50300 Kuala LumpurTel: 603-9289 7297; Fax: 603-2693 8037Email: [email protected] (Mohd Yusof Yasmin Anum )

Received 10.08.2010 Accepted 04.10.2010Advances in Medical SciencesVol. 55(2) · 2010 · pp 281-288DOI: 10.2478/v10039-010-0046-z© Medical University of Bialystok, Poland

INTRODUCTION

In recent years, there is an increase in the prevalence of diabetes mellitus (DM) worldwide. DM is a major public health problem in all countries including Malaysia. According to the last survey performed by the National Diabetes Institute, the prevalence of diabetes amongst rural and semi-urban populations in the Malaysian subcontinent varies between 8-12% [1].

DM is a chronic disease caused by the inability of the pancreas to produce insulin or the inability of our body to use the insulin produced appropriately. According to the World Health Organization (WHO) classification, basically, there are 2 types of diabetes i.e. type 1 and 2 [2]. Type1 diabetes

is a slow progressive autoimmune disease mediated by T cell, whereas type 2 diabetes is a more complex disease due to insulin resistance combined together with impaired β-cell function [3]. Both types of DM caused oxidative stress, which is defined as the production of reactive oxygen species (ROS) or free radicals. ROS is produced in DM mainly by glucose oxidation. Glucose in its enediol form is oxidized in a transition-metal-dependent reaction to an enediol radical anion that is converted into reactive ketoaldehydes and to superoxide anion radicals. Another source of free radicals in DM is the interaction of protein with glucose (glyated proteins) leading to the formation of advanced glycation endproducts (AGEs). [4]. Abnormally high levels of free radicals can lead to damage of cellular organelles, enzymes and caused lipid


peroxidation, protein carbonylation and DNA guanylation [4, 5]. Oxidative stress is reported to play an important role in the pathogenesis of chronic complications in DM [6]. A relationship between diabetic nephropathy and neuropathy and oxidative stress has been reported, suggesting that oxidative stress affects progress of diabetic complications [7]. It has been reported that activation of inflammatory marker, TNF-α induced oxidative stress by stimulating the production of ROS which caused lipid peroxidation and oxidative damage of DNA and proteins. This alters the normal function of the cells which leads to cell damage in DM patients [8].

Oxidative cellular damage has been reported in DM patients resulting in changes in the activity of endogenous antioxidant enzymes, such as SOD, glutathione peroxidase (GPx), and elevated levels of malondialdehyde level (MDA), DNA damage and carbonylated proteins [9-12].

Microalgae CV has long been a popular foodstuff in Japan, Korea and Taiwan and it is a good source of protein, vitamins, dietary fibers and essential minerals [13]. Our group has reported that CV has anti tumour and antioxidant effects in liver cancer induced rats [14]. Some studies have reported the hypoglycaemic effects of CV in diabetic rats [15,16]. However, to the best of our knowledge, no previous studies have been conducted to observe the antioxidant effect of CV against oxidative stress in DM in experimental animals. Keeping the above facts in mind, we embarked on the present study in order to investigate the protective role of CV on oxidative damage and inflammation in diabetic rats.

MATERIALS AND METHODS

Animals and dietPrior ethical approval was obtained from institutional animal ethics committee (Date of approval 29th October 2009: PP/BIOK.2008/YASMIN). A total of 24 male Sprague Dawley rats (300-400g) were obtained from the Laboratory Animal Resource Unit, Faculty of Medicine, Universiti Kebangsaan Malaysia and were kept in polycarbonated cages in a room with controlled temperature, humidity and 12 h light-dark-cycle. All experiments were conducted according to the guidelines of National Institute of Health for the Care and Use of Laboratory Animals.

The rats were divided into four groups each consisting of control group: fed with normal rat chow (Gold Coin, Malaysia); CV group: fed with normal chow diet and CV; Streptozocin (STZ)-induced diabetic group and STZ plus CV ( 150 mg /kg weight) group. The dose of 150 mg /kg weight was chosen based on previous experiment which gave the best anti-tumour response in hepatocarcinogenesis induced rats. CV being a food supplement gave no lethal effects on rats [14]. Each group comprised of 6 rats. CV was administered via oral gavage beginning 48h after STZ injection. Diabetes was induced by a single intravenous injection of STZ (Sigma Aldrich, St. Louis,

MO, USA) (45 mg/kg body weight: prepared in 0.15 M citric acid and 0.25M sodium phosphate, pH 4.65) to overnight fasted rats. The glucose level was monitored by using glucose oxidase reagent strips on blood samples obtained from tail veins 72 hours after STZ injection. Rats with blood glucose of higher than 8 mmol/L were included as diabetic group. The duration of experiment was four weeks and the blood sample was drawn from orbital sinus at 1 and 4 weeks for the determination of the endogenous antioxidant enzymes (SOD, CAT), oxidative (MDA, DNA damage), and inflammatory biomarkers (TNF-α). The glucose level in control, STZ and STZ + CV groups were measured weekly. All animals were anesthetized briefly prior to being sacrificed.

Chlorella vulgaris (CV)Stock of CV Beijerinck (strain 072) was obtained from the Department of Biochemistry, UKM Medical Center (Kuala Lumpur, Malaysia). CV was cultured outdoors in a tank in Bold Basal Media (BBM) with 12 h sunlight, 12 h dark and sufficient aeration. The algae were centrifuged 3 times at 3000 rpm for 10 minutes at 400C to separate the media. The pelleted algae were then diluted in distilled water at 150 mg/kg body

weight before being used throughout the experiment.

Biochemical determinationsa) Endogenous Antioxidants SOD and CATSuperoxide dismutase activity was measured according to Beyer et al., [17] by adding 1.0 ml of reaction mixture [3 % L-methionine, 0.1 % nitroblutetrazolium , NBT and 1% triton-X ] (Sigma-Aldrich, Poole, UK) with 20 μl of sample.

The reaction was started by adding 10 μl of riboflavin (0.004%). The solution was vortexed and then placed in a box illuminated with 20 W Sylvania grolux fluorescent lamp for 7 minutes.The reduction of NBT was measured at 560 nm in a spectrophotometer (SHIMADZU, Japan).

Catalase activity was determined according to Aebi et al., [18] whereby 2 ml of sample solution was mixed with 1 ml of H2O2 solution and the decomposition of hydrogen peroxide was measured spectrophotometrically at 240 nm against a blank containing 2 ml of sample solution and 1 ml of phosphate buffer. The reaction began soon after the addition of H2O2 and the decomposition was detected within 30 seconds.

b) DNA DamageThe lymphocytes’ nuclear DNA fragmentation was assessed using alkaline single cell gel electrophoresis (Comet) assay [19]. All the samples were prepared at room temperature (18ºC) under dark condition, in order to prevent DNA damage. Fully frosted microscope slides (Surgipath Europe, Peterborough, UK) were coated with 100 μL of 0.6% normal melting agarose (Sigma-Aldrich, Poole, UK) and covered immediately with cover slips (24 x 40 mm). Five microliter of whole blood sample was mixed in a microcentrifuge tube with 90 μL of 0.6% low-melting point agarose at 37ºC. The cell suspensions were pipetted over first agarose layer and re-covered with cover

Aizzat O et al.

slips and allowed to solidify at room temperature. The cover slips were removed and immersed in Coplin jar containing fresh cold lysing solution. The lysing solution was prepared using 2.5 M NaCl, 100 mM NA2EDTA.2H2O, 10mM Tris-HCl, sodium sarcosinate, Triton X-100 and dimethylsufoxide (Sigma, USA) and was stored for 1 h in refrigerator at 40C before use. The slides were removed from the lysing solution, drained and placed in horizontal gel electrophoresis platform in the freshly prepared and cooled (4ºC) alkaline electrophoresis buffer (10M NaOH, 1mM EDTA; Sigma, USA). The slides were then placed in the buffer for 20 min for unwinding of DNA. Electrophoresis was then conducted for 20 min using 25 V, with the current adjusted to 300 mA . Following this, the slides were removed from the tank, drained and flooded with three changes of neutralization buffer (0.4 M Tris base; pH 7.5; Sigma Sigma-Aldrich, Poole, UK) to neutralize residual alkali. Slides were then stained with 30μL of 20 μg/ml ethidium bromide (Sigma-Aldrich, Poole, UK) and enclosed with new cover slips. The slides were then stored in humidified container in darkness at 4ºC overnight until the time of analysis. The slides were examined at 200x magnification using a fluorescent microscope (Nikon, Tokyo, Japan). One hundred and fifty randomly selected non-overlapping cells on each slide were analyzed by manual scoring based on estimated comet tail length and relative proportion of DNA in the comet tail (14). The severity of nuclear DNA fragmentation was determined by assigning score on an arbitrary scale of 0 – 4 (i.e. 0 = No DNA damage, 4 = Severe DNA damage) as shown in Figure 4. The scoring was performed by two technicians in a blinded manner, and the results were almost similar between them.

c) Determination of plasma Malondialdehyde (MDA)Lipid peroxidation product, MDA was quantified by the method of Mateos et al. (20). The principle of the test is based on the derivation of MDA with 2,4 dinitrophenylhydrazine (DNPH) and conversion into pyrazole and hydrazone derivatives, which is then, separated using HPLC. This method allows more specific estimation of MDA. HPLC analyses are performed on a LC-10 AT VP® SHIMADZU (Kyoto, Japan) Liquid chromatography system equipped with a diode array detector and an auto injection valve. An Alpha Bond C18 125A column (3.9 x 150 mm) with a 5 μm particle size (Alltech, Deerfield, IL) and a Shidmadzu Class-VP software system for data processing were used. Elution was performed isocratically with a mixture of 0.2 % (v/v) acetic acid in milliQ water and acetonitrile (62:38) (v/v) at a flow rate of 0.6 ml/min at room temperature. Chromatograms were acquired at 310 nm.

d) Standard and sample preparation1, 1, 3, 3- Tetraetoxypropane (TEP) was used as the standard. A stock solution of MDA was obtained as follows: 25 μl TEP was dissolved in 100 ml of deionised water to give a 1mM stock solution. MDA was prepared by hydrolysis of 1ml TEP stock solution in 50 ml 1% sulphuric acid and incubation for 2 h at room temperature. The solution was stored at 4 ◦C and

used within 4 weeks. The resulting MDA standard of 20 nmol/ml was further diluted with 1% sulphuric acid to yield different concentrations from 1 to 20 nmol/ml of MDA. An aliquot of 250 μl diluted standard or plasma was placed in a 1.5 ml eppendorf vial and 50 μl of 6M aqueous sodium hydroxide was added. This mixture was incubated in 60 ◦C water bath for 30 min to achieve alkaline hydrolysis of protein bound MDA. Then, protein was precipitated by adding 125 μl of 35 % (v/v) perchloric acid, and the mixture was centrifuged at 6000 × g for 10 min. All volume of supernatant was transferred to an eppendorf vial and mixed with 50 μl DNPH prepared as a 5mM solution in 2M hydrochloric acid. Finally, this reaction mixture was incubated for 30 min at room temperature protected from light. An aliquot of 40 μl of this reaction mixture was injected into the HPLC system. Retention time for MDA was detected at 11 min and the area under the peak represented the amount of MDA in 40 μl of reaction mixture. Serial concentration of standard was generated a reference curve.

e) Tumor Necrosis Factor Alpha (TNF-α)The TNF-alpha protein levels were estimated with ELISA kits (Pierce Biotechnology, Rockfold, IL. USA) according to the manufacturer’s instructions. This assay employed an antibody specific for rat TNF-alpha coated on a 96-well plate. All the reagents and samples were stored at room temperature before conducting the experiment.

Sample preparation and incubation with Biotinylated Antibody ReagentA standard curve of TNF-α was prepared according to the manufacturer’s instructions. Six concentrations selected were 2500 pg/ml, 833 pg/ml, 278 pg/ml, 93 pg/ml, 31 pg/ml and 0 pg/ml. Fifty microliter of the pre-treatment buffer was added to each well followed by 50μl of reconstituted standard added into standard well in duplicate. A 1:1 (a final volume of 50 μl ) dilution of the sample was prepared in each well. The plate was covered with adhesive plate cover and incubated for 1 h at room temperature (i.e. 20-25°C). The plate was then washed with wash buffer for three times followed by 50uL of biotinalyted antibody reagent. Adhesive plate cover was attached and the plate was incubated for 1 h at room temperature. The plate content was discarded and the plate was washed 3 times. One hundred μl of Streptavidin-HRP reagent was added to each well and was incubated for 30 min at room temperature. The plate content was discarded and washed 3 times with wash buffer. One hundred microliter of tetramethyl benzidine (TMB) substrate solution was added to each well. The colour reaction was allowed to occur in the dark for 10 min at room temperature. Finally, 100μl stop solution was added to each well to produce a colour reaction from blue to yellow and the absorbance was measured in a plate reader at 450nm and 550 nm. All measurements were performed in duplicate.


Statistical AnalysisData was analysed using SPSS package (version 9.0). Results refer to mean ± SD. Statistical evaluation was assessed using analysis of variance (ANOVA) and post-hoc test (LSD). A p<0.05 was considered significant.

RESULTS

Blood Glucose and body weightAll rats injected with STZ developed severe diabetes as indicated by increased serum glucose concentrations (>15 mmol/L) over the period of four weeks while control rats showed normal values (5.20-5.95 mmol/L). The glucose level in untreated STZ group showed an increasing trend week by week. However in treated STZ group, CV did not reduce

the increasing blood sugar levels of diabetic rats over the four week duration of treatment. (Tab. 1)Body weights of STZ rats deteriorated significantly over the four weeks (p<0.05) compared to the control group. SODThere were no significant changes in the SOD activity in all groups (Fig. 1).

CATCatalase activity was decreased in STZ diabetic induced rats compared to the control group and CV managed to increase its activity, but it did not reach the control values. (Fig. 2).

DNA DamageThere was an increase in DNA damage in STZ-induced diabetic rats (Fig. 3) with elevated score 4 (Fig. 4) but interestingly,

Groups Week Blood glucose (mM)Mean ± S.E.M

Body weight (kg)Mean ± S.E.M

Control 1 5.20 ± 0.18 365.38 ± 11.07

2 5.41 ± 0.22 413.12 ± 5.60 b

3 5.48 ± 0.21 427.00 ± 7.24 b

4 5.95 ± 0.16 450.10 ± 7.58 b,c

STZ 1 20.58 ± 0.87 a 312.54 ± 5.65 a

2 22.65 ± 0.75 a 303.07 ± 5.39 a

3 23.38 ± 1.05 a 295.88 ± 7.40 a

4 23.13 ± 0.82 a 284.80 ± 8.67 a

STZ + CV 1 20.98 ± 0.87 a 314.30 ± 6.25 a

2 22.95 ± 0.79 a 319.58 ± 10.95 a

3 21.45 ± 0.74 a 315.53 ± 11.32 a

4 22.12 ± 0.89 a 307.22 ± 7.14 a

a Significant different (p<0.05) when compared to control at respec-tive week of treatment b Significant different (p<0.05) when compared to week 1c Significant different (p<0.05) when compared to week 2

Table 1. Effect of Chlorella vulgaris (CV) on blood glucose level (mmol/L) and body weight in STZ induced diabetic rats. Data are expressed as mean ±SD.

Figure 1. Blood SOD activity (U/mg/Hb) in STZ-induced diabetic rats with or without CV. Data are expressed as mean value of SOD activity ± SD. No significant changes were observed.

Figure 2. Blood CAT activity (U/mg/Hb) in STZ-induced diabetic rats with or without CV. Data are expressed as mean ±SD. a: significant (p < 0.05) compared to control, b: significant compared to CV, c: significant compared to STZ + CV.

Figure 3. Effect of CV on total DNA damage score in STZ-induced diabetic rats. Data are expressed as mean ± SD. a: significant compared to control, b: significant compared to CV, c: significant (p < 0.05) compared to STZ, d: significant compared to STZ + CV.

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CV was able to reduce the severity of DNA damage in STZ group with concomitant reduction in score 4 (Fig. 4).

MDAFig. 5 showed that lipid peroxidation (as measured by MDA levels) was high in diabetic rats but CV was able to reduce its level significantly. CV also managed to reduce baseline MDA level significantly compared to control rats.

TNF-αNo significant changes were observed in all the groups for inflammatory marker, TNF-α (Fig 6).

DISCUSSION

Chlorella vulgaris (CV), a unicellular fresh water green algae, is a popular foodstuff in Japan. Korea and Taiwan. It is credited with high amino acid contents and antioxidants such as vitamin C, beta carotene, vitamin E, and minerals [13, 21]. CV modulates antioxidants status and reduced lipid

Figure 4. Effect of CV on individual DNA damage score in STZ-induced diabetic rats. Data are expressed as mean ± SEM. a: significantly compared to control and CV (p < 0.05), b: significant compared to STZ group.Representative image of a comet generated in the single cell electrophoresis assay (comet assay). 0 – cell without DNA damage. 1 & 2 – mild damage. 3 & 4 – severe damage.

Figure 5. Blood MDA levels in STZ-induced diabetic rats with and without CV. Data are expressed as mean ± SD. a: significant compared to control, b: significant compared to CV, c: significant compared to STZ, d: significant compared to STZ + CV.

Figure 6. Effect of CV on plasma TNF-alpha levels in control and STZ-induced diabetic rats. Data are expressed as mean ± SEM. No significant changes were observed.


peroxidation in naphthalene-induced oxidative stress in the albino rats and enhanced and prolonged the hypoglycemic effects of injected insulin in STZ mice [15, 22]. CV has anti-AGE (advanced glycation end products) effect that may help in the prevention or reduction of complication of age related disorders including diabetes mellitus (DM) [23]. Our previous findings had shown that CV exhibited chemopreventive effect by inducing apoptosis via decreasing the expression of Bcl-2 and increasing the expression of caspase 8 in hepatocarcinogenesis-induced rats [24].

In DM, persistent hyperglycemia had been shown to cause high production of ROS that resulted in cellular oxidative damage including lipids, DNA and protein [4,5,9-12]. Thus, normalizing the glucose level in the blood may decrease the production of ROS and alleviate the deleterious effect on tissue damage. Cherng and Shih [15] had shown that CV enhanced the hypoglycemic effect of exogenous insulin in STZ induced mice while Layam and Reddy [25] had shown similar hypoglycemic effect of spirulina in diabetic rats. Both researchers have noted that CV and spirulina also increased insulin sensitivity in experimental animals. In the present study, however, we did not observe hypoglycaemic effect of CV in the diabetic rats over the four weeks. This may be due to the short term supplementation of CV which may not yet exhibit the hypoglycaemic effect.

Free radicals such as hydroxyl radical (OH∙), superoxide anion (O2-) and hydrogen peroxide (H2O2) are regularly produced in our body during normal metabolism. Our human body has a unique mechanism to safeguard against this damaging effect by having endogenous antioxidants such as SOD and CAT that scavenge the free radicals in order to maintain the balance between oxidant and antioxidant levels [26].

The result of several studies involving antioxidant enzymes in streptozotocin (STZ) induced diabetic animals have been conflicting or equivocal. Decreased SOD and CAT activities have been reported by some authors with others reported increased activities [27-29] or even with no changes [30] with supplementation of antioxidants. In the present study, the CAT activity was reduced in STZ-induced diabetic rats when compared to normal rats, however, CV was not able to improve the status of CAT in diabetic rats. We did not observe any significant changes in the SOD activities in the diabetic rats treated with CV. The CV however reduced both SOD and CAT activities of normal rats. A possible explanation is that oxidative stress was attenuated by the CV intake through inhibiting superoxide radical generation normally found in normal metabolism rather than improving activities of antioxidant enzymes [30].

Oxidative stress plays an important role in the pathogenesis of chronic complications in DM. As a result of hyperglycemia-induced oxidative stress, oxidative DNA damage may occur. The alteration in the oxidative stress has been demonstrated by the degree of nuclear DNA damage. A previous study by Wu et al., showed that there was a significant increase of DNA damage

in diabetic rats which was reduced by the polysaccharide Lycium barbarum (LBP) [31]. A similar pattern of results was also observed in the present study whereby the amount of DNA damage was significantly increased in STZ-induced diabetic rats as shown by Comet assay. The increase in the amount and degree of DNA damage could be explained by high amount of ROS production in hyperglycemic-induced oxidative stress in diabetic rats. The antioxidant effect of CV extract was inevitably proven in which DNA damage was significantly reduced in diabetic rats compared to the untreated diabetic rats as shown by significant reduction in extensive DNA damage (score of 4). The decrease in both quantitative and qualitative oxidative DNA damage after CV supplementation may be due to the neutralizing effect of antioxidants in CV such as beta carotene, vitamin E and vitamin C on excessive ROS production. Thus, the present study showed that CV may play an important role in preventing oxidative DNA damage by scavenging excessive ROS that were generated in hyperglycemic condition such as DM .

Increased MDA level is an indication of lipid peroxidation at cellular level. STZ-diabetic rats were found to exhibit increased MDA level by Shah et al [32] but methanolic extract of Centratherum anthelminticum was able to significantly decrease its level as compared to the normal control rats. We found similar results whereby elevated levels of MDA in STZ rats were significantly reduced by CV.

TNF-α is a pro-inflammatory cytokine secreted by the macrophages. TNF-α has been implicated in many autoimmune diseases, insulin resistance diabetes and cancer. TNF-α plays an important role in the initiation of Type I DM in the non obese diabetic mice by regulating the maturation of dendritic cells and the activation of islet-specific pancreatic lymph node T cells [33]. This promotes inflammatory reaction and contributes to further activation of TNF-α which subsequently produces oxidative stress by stimulating the generation of ROS [7]. All these factors may alter the normal function of the cells which ultimately leads to cell damage as observed in DM patients. To our knowledge, not many studies have shown the inhibitory effect of natural herbs/plants on TNF-α expression in experimental diabetes. An antidiabetic drug troglitazone however, was shown to reduce serum TNF-α activity which was increased in diabetic rats [34]. Our present study did not show any anti-inflammatory effect of CV supplementation on the diabetic rats. Perhaps, four weeks of CV supplementation could be too early to see any anti inflammatory effect on STZ-induced diabetic rats.

CONCLUSIONS

DM is a disease which leads to multiple complications. The oxidative stress is the most important reason behind any organ damage in DM. Thus, there is a need to explore further the relationship between free radicals, oxidative stress, diabetes,

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and its complications. Interventions with antioxidants would elucidate further the mechanisms involved in which increased oxidative stress accelerates the development of diabetic complications. The present study observed the protective effect of CV as evident by the decreased level of the oxidative stress indicated by reduced MDA levels and DNA damage in experimental diabetic animals. Perhaps, this may open the door for further research with CV as an effective antioxidant in preventing complications in DM.

ACKNOWLEDGEMENTS

The study was funded by the special study grant sanctioned by the Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre (UKMMC)( FF-055-2009) and the University Research Grant ( UKM-GUP-SK-07-21-201).

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modulation of oxidative stress by chlorella vulgaris in streptozotocin (stz) induced diabetic...

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