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Journal of Ethnopharmacology 112 (2007) 13–18

Experimental diabetes treated with Achillea santolina:Effect on pancreatic oxidative parameters

Razieh Yazdanparast ∗, Amin Ardestani, Shirin JamshidiInstitute of Biochemistry and Biophysics, P.O. Box 13145-1384, University of Tehran, Tehran, Iran

Received 5 April 2006; received in revised form 22 January 2007; accepted 25 January 2007Available online 31 January 2007

bstract

Oxidative stress is produced under diabetic condition and is likely involved in progression of pancreatic damage found in diabetes. In the presenttudy, we examined possible protective effect of Achillea santolina L. (Compositae) against pancreatic damage in streptozotocin (STZ)-treatediabetic rats. Achillea santolina extract (ASE) is used by the traditional healers in many part of Iraq, as a hypoglycaemic agent. We evaluated theffect of ASE on blood glucose level, serum nitric oxide (NO) concentration and the oxidative stress status in rat pancreatic tissue. STZ was injectedntraperitonealy at a single dose of 40 mg kg−1 to induce diabetes. ASE (0.1 g/kg day) was orally administered to a group of diabetic rats for 30onsecutive days. Results showed significant reduction in the activities of superoxide dismutase (SOD), catalase (CAT) and pancreatic glutathioneGSH) levels in the diabetic rats compared to the control subjects. On the other hand, blood glucose level, serum NO, malondialdehyde (MDA), aarker of lipid peroxidation, protein oxidation indices including protein carbonyl (PCO) and advanced oxidation protein products (AOPP) were

ignificantly elevated in pancreas of the diabetic group. Treatment with ASE reduced blood glucose level, serum NO, pancreatic MDA, PCO and

OPP. In addition, the content of GSH was restored to the normal level of the control group. Furthermore, ASE significantly increased CAT andOD activities in ASE-treated rats. Based on our data, it can be concluded that Achillea santolina have a high hypoglycaemic activity and this maye attributed to its antioxidative potential.

2007 Elsevier Ireland Ltd. All rights reserved.

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eywords: Diabetes; Oxidative stress; Antioxidant; Achillea santolina

. Introduction

Oxidative stress depicts the existence of products called freeadicals and reactive oxygen species (ROS) which are formednder normal physiological condition but become deleterioushen not being quenched by the antioxidant systems (Fang et al.,002). There are convincing experimental and clinical evidenceshat the generation of reactive oxygen species is increased in bothype of diabetes and that the onset of diabetes is closely asso-iated with oxidative stress (Rosen et al., 2001; Johansen et al.,005). Free radicals are formed disproportionately in diabetes bylucose autoxidation, polyol pathway and non-enzymatic gly-

ation of proteins (Wolff and Dean, 1987; Brownlee et al., 1988;brosova et al., 2002). Abnormally high levels of free radicals

nd simultaneous decline of antioxidant defense systems can

∗ Corresponding author. Tel.: +98 21 66956976; fax: +98 21 66404680.E-mail address: yazdan@ibb.ut.ac.ir (R. Yazdanparast).

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ead to damage of cellular organelles and enzymes, increasedipid peroxidation and development of complication of diabetes

ellitus (Maritim et al., 2003).Streptozotocin (STZ) is a monofunctional nitrosurea deriva-

ive, one of the most commonly used substances to induceiabetes in the experimental animals (Szkudelski, 2001).lthough it is generally accepted that the cytotoxicity producedy STZ depends on DNA alkylation and subsequent activation ofoly ADP-ribose synthetase that causes rapid and lethal deple-ion of NAD in pancreatic islets (Bennett and Pegg, 1981; Bolzannd Bianchi, 2002), several lines of evidences indicate that freeadicals may play an essential role in the mechanism of �-cellamage and diabetogenic effect of STZ (Takasu et al., 1991;hkuwa et al., 1995).Several Achillea species are used for their pharmaceutical,

osmetic, and fragrance properties. Their extracts exhibit phar-acological activities such as anti-inflammatory, analgesic, and

ntipyretic. Among them, Achillea millefolium L. is used forwide range of disorders such as treating wounds, stopping

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lood flow, treating colds, fevers, kidney diseases, and men-trual pain (Conforti et al., 2005). Achillea santolina L. (localame: Gasium) is also used for anti-inflammatory purposesnd treatment of diabetes (Al-Hindawi et al., 1989). Herbaledicines have long been used for treatment of diabetes and

hey are currently accepted as alternative therapy for diabeticreatment. So far more than 1200 plants have been introducedith hypoglycemic activities (Sezik et al., 2005). Achillea san-

olina extract (ASE) is commonly used by the traditional healern many parts of Iraq as a hypoglycemic agent. In this study welanned to evaluate this property in a systematic and scientificpproach. In addition, we evaluated whether ASE is capable ofhanging the antioxidant status of pancreatic tissue of the STZiabetic rats.

. Materials and methods

.1. Plant material

Aerial parts of the plant were collected from Tikrit in May,005. The plant was characterized as: Achillea santolina L. byr. Khalil I. Al-Shemmary (Biology Department, Faculty ofciences, Tikrit University, Iraq) and a voucher specimen (No.625) was deposited at the herbarium of the Faculty of sciences,ikrit University.

.2. Extraction

The powdered plant material (50 g) was extracted three timesith ethanol–water (7:3, v/v), at room temperature. The com-ined extracts were concentrated under reduced pressure and theolume was adjusted to 500 ml (equivalent to 0.1 g plant powderer ml). The concentrated extract was divided into 25 ml aliquotsnd kept at −20 ◦C for further investigation. The (w/w) yield inerms of dried starting plant material was 8%.

.3. Animals

Male wistar albino rats (n = 30), 5–7 months old with a weightf 200–250 g were purchased from Pasteur institute, Tehran,ran. They were housed under conventional conditions and werellowed free access to food and water ad libitum.

.4. Induction of experimental diabetes

Streptozotocin (STZ; Sigma, USA; 40 mg/kg body weight)as dissolved in 0.1 M sodium citrate buffer at pH 4.5 just beforese and injected intraperitoneally (i.p.) to 22 rats. Control ratsgroup1, n = 8) received with the same route of administration anquivalent volume of citrate buffer. One week after STZ admin-stration, the diabetic rats with blood glucose levels higher than5 mmol/l were selected and distributed in two groups (2 and 3).

.5. Oral administration of the plant extract

The plant extract was administered by gavages (i.g.) to sevenats of group 3 in a dose of 1 ml/rat (equivalent to 0.1 g plantowder/kg body weight) for 30 consecutive days. The control

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pharmacology 112 (2007) 13–18

ealthy rats (group 1, n = 8) and the control diabetic rats (group, n = 7) received the same volume of distilled water i.g. Bloodamples for glucose determination were obtained from the rats’earts, after mild ether anesthesia. The blood glucose levels ofats in each group were determined every 10 days, using glu-ose oxidase kit according to manufacture’s instructions (Parszmoon, Tehran, Iran).

.6. Tissue preparation

Each pancreas was quickly removed from the sacrificed rat,laced in ice cold saline solution and trimmed of adipose tis-ue. Each pancreas was finely minced and homogenized in0 mM phosphate buffer, pH 7.4 and centrifuged at 10,000 × gor 15 min at 4 ◦C (Beckman refrigerated, Ultracentrifuge). Theupernatant was used for all the assays. The protein concentra-ion was determined by the method of Lowry et al. (1951) usingovine serum albumin as the standard.

.7. MDA determination

Malondialdehyde (MDA) levels, an index of lipid peroxida-ion, were measured by the double heating method of Drapernd Hadley (1990). The method is based on spectrometriceasurement of the purple color generated by the reaction of

hiobarbituric acid (TBA) with MDA. For this purpose, 2.5 mlf trichloroacetic acid solution (10%, w/v) was added to 0.5 mlupernatant of the tissue preparation in each centrifuge tube andubes were placed in a boiling water bath for 15 min. After cool-ng to room temperature, the tubes were centrifuged at 1000 × gor 10 min and 2 ml of each sample supernatant was trans-erred to a test tube containing 1 ml of TBA solution (0.67%,/v). Each tube was then placed in a boiling water bath for5 min. After cooling to room temperature, the absorbance waseasured at 532 nm. The concentration of MDA was calcu-

ated based on the absorbance coefficient of the TBA–MDAomplex (ε = 1.56 × 105 cm−1 M−1) and it was expressed asmol/mg protein.

.8. Determination of protein carbonyl content

Protein carbonyls (PCO) were measured by using the methodf Reznick and Packer (1994). Briefly, 1 ml of supernatantas placed in two glass tubes. Then 2 ml of 10 mM 2,4-initrophenylhydrazine (DNPH) in 2.5 M HCl was added to onef the tubes, while 2 ml HCl (2.5 mM) was added to the secondube. Tubes were incubated for 1 h at room temperature. Samplesere vortexed every 15 min. Then 2.5 ml TCA (20%, w/v) was

dded and the tubes were left on ice for 5 min followed by cen-rifugation for 5 min to collect the protein precipitates. The pelletas then washed three times with 2 ml ethanol–ethyl acetate

1:1, v/v). The final precipitate was dissolved in 1 ml 6 M guani-ine hydrochloride solution and it was incubated for 10 min at

7 ◦C while mixing. The absorbance of the sample was measuredt 370 nm. The carbonyl content was calculated based on theolar extinction coefficient of DNPH (ε = 2.2 × 104 cm−1 M−1)

nd expressed as nmol/mg protein.

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.9. Determination of advanced oxidation protein productsevels

Advanced oxidation protein products (AOPP) levels wereetermined according to Kayali et al. method (2006). Briefly,.4 ml of pancreatic supernatant was treated with 0.8 ml phos-hate buffer saline (PBS) solution. After 2 min, 0.1 ml 1.16 Motassium iodide (KI) was added to the tube followed by 0.2 mlf acetic acid. The absorbance of the reaction mixture was imme-iately recorded at 340 nm against the blank solution containing.2 ml PBS, 0.1 ml of KI, and 0.2 ml of acetic acid. The concen-ration of AOPP for each sample was calculated by using thextinction coefficient of 26 l mM−1 cm−1and the results werexpressed as nmol/mg protein.

.10. Antioxidant defense system assays

Catalase (CAT) activity was measured according to theethod of Aebi (1984) by following the decrease in absorbance

f H2O2 at 240 nm for 1 min. The enzyme activity was expresseds k (s mg protein)−1, where k is the rate constant of the first ordereaction of CAT.

Superoxide dismutase (SOD) activity was measured based onnhibition of the formation of amino blue tetrazolium formazann nicotineamide adenine dinucleotide, phenazine methosul-ate and nitroblue tetrazolium (NADH-PMS-NBT) system,ccording to method of Kakkar et al. (1984). A one unit ofnzyme activity was expressed as 50% inhibition of NBT reduc-ion/(min mg protein).

Reduced glutathione (GSH) was determined by the method ofllman (1959). The supernatant (0.5 ml) was treated with 0.5 mlllman’s reagent (19.8 mg of 5,5′-dithiobisnitro benzoic acid,TNB, in 100 ml of 0.1% sodium nitrate) and 3 ml of phosphateuffer (0.2 M, pH 8). The absorbance was read at 412 nm. GSHas expressed as mg/100 g tissue.

.11. Determination of serum nitric oxide

Nitric oxide (NO) level can be determined spectrophotomet-ically by measuring the accumulation of its stable degradation

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ormal ratsBlood glucose (mmol/l) 4.80 ± 1.10 5.20 ± 1.01Body weight (g) 236 ± 14 240 ± 11

iabetic ratsBlood glucose (mmol/l) 4.70 ± 0.55 15.70 ± 0.50a

Body weight (g) 210 ± 6 200 ± 8

SE-treated ratsBlood glucose (mmol/l) 4.60 ± 0.44 16.90 ± 1.10a

Body weight (g) 224 ± 8 215 ± 7

ach measurement has been done at least in triplicate and the values are the means ±a Significantly different from normal rats (P < 0.05).b Significantly different from STZ-treated rats (P < 0.05).

pharmacology 112 (2007) 13–18 15

roducts, nitrite and nitrate. The serum nitrite level was deter-ined by the Griess reagent according to Hortelano et al. (1995).he Griess reagent, a mixture (1:1) of 1% sulfanilamide in 5%hosphoric acid and 0.1% 1-naphtylethylenediamine gives a red-iolent diazo color in the presence of nitrite. The color intensityas measured at 540 nm. Results were expressed as �mol/l usingNaNO2 calibration graph.

.12. Statistical analysis

All data are presented as means ± S.D. Comparison betweenroups was made by one-way analysis of variance (ANOVA)ollowed by Tukey test to analyze the difference. Statisticalignificance was achieved when P < 0.05.

. Results

.1. The effect of Achillea santolina extract on the bloodlucose levels and body weights

As shown in Table 1 after 30 days, the blood glucose levels ofhe plant extract-treated rats were significantly lower than that ofhe rats of diabetic group (6.70 ± 0.44 and 16.30 ± 0.55 mmol/l,espectively; P < 0.05). In contrast, the blood glucose levels ofhe untreated diabetic rats remained high throughout this inves-igation.

Table 1 also shows that, in contrast to normal healthy rats,here was a weight loss among the diabetic group. However, noody weight loss was observed among the rats of the treatediabetic group.

.2. The effect of Achillea santolina extract on pancreaticxidative status and serum nitric oxide level

In order to explore the effect of the crude extract on theancreatic function of the diabetic rats, lipid peroxidation,

ntioxidant defense system capabilities and protein oxidationere evaluated according to appropriate methods reported inaterials and methods. Table 2 shows the level of lipid per-

xidation marker, malondialdehyde (MDA), serum nitric oxide

body weight of diabetic rats

10 20 30

5.10 ± 1.02 5.30 ± 0.72 4.80 ± 0.77263 ± 12 286 ± 9 305 ± 24

15.60 ± 0.55a 16.20 ± 0.22a 16.30 ± 0.55a

192 ± 11 174 ± 10 154 ± 7

11.50 ± 0.61b 8.40 ± 1.10b 6.70 ± 0.44b

223 ± 4 241 ± 10 252 ± 7

S.D for six rats in each group. For experimental details, please see Section 2.

16 R. Yazdanparast et al. / Journal of Ethnopharmacology 112 (2007) 13–18

Table 2Changes in the concentration of malondialdehyde (MDA), reduced glutathione (GSH), serum nitric oxide (NO) and the activities of catalase (CAT), superoxidedismutase (SOD) in pancreas of control and experimental animals

Groups Normal Diabetic Diabetic + ASE

MDA (nmol/mg protein) 0.33 ± 0.03 0.67 ± 0.08a 0.46 ± 0.04b

GSH (mg/100 g tissue) 15.90 ± 1.20 6.60 ± 0.59a 12.40 ± 0.56b

CAT (×10−3 k (s mg protein)−1) 47.50 ± 2.20 25.50 ± 2.10a 37.10 ± 1.60b

SOD (U/mg protein) 4.11 ± 0.09 2.21 ± 0.05a 3.28 ± 0.10b

Serum NO (�mol/l) 5.27 ± 0.90 9.15 ± 0.40a 5.82 ± 0.18b

Each measurement has been done at least in triplicate and values are the means ± S.Da Significantly different from normal (P < 0.05).b Significantly different from STZ-treated group (P < 0.05).

Table 3Concentration of protein oxidation markers, protein carbonyl (PCO) andadvanced oxidation protein products (AOPP), in the pancreas of normal andexperimental animals

Group Normal Diabetic Diabetic + ASE

PCO (nmol/mg protein) 1.22 ± 0.05 1.80 ± 0.02a 1.42 ± 0.06b

AOPP (nmol/mg protein) 0.35 ± 0.03 0.70 ± 0.04a 0.49 ± 0.03b

Each measurement has been done at least in triplicate and values are them

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NO) and antioxidant defense system components in normal andxperimental rats. There was a significant elevation in MDAoncentration and serum NO level, while the activity of CATnd SOD and GSH content decreased in diabetes when com-ared with corresponding control group. Treatment of the ratsith ASE significantly decreased lipid peroxidation end prod-ct, MDA (by 60%) and serum NO (by 85%). In addition, SODnd CAT activities and the GSH content in pancreatic tissue sig-ificantly increased (by 49, 48 and 88%, respectively) comparedo the corresponding diabetic group.

Table 3 provides the levels of the PCO and AOPP, indices ofrotein oxidative damage, in pancreatic tissue. Under diabeticondition, there was a significant elevation of PCO and AOPPn the pancreatic tissue. But, ASE-treated diabetic rats exhibitedignificant decrease in their pancreatic PCO and AOPP levelsby 66% and 59%, respectively) compared to untreated diabeticats.

. Discussion

Oxidative stress is produced under diabetic condition and it isikely involved in progression of pancreatic �-cell dysfunctionKajimoto and Kaneto, 2004). Also, because of the relativelyow expression of antioxidant enzymes such as catalase anduperoxide dismutase, pancreatic �-cells may be vulnerableo ROS attack when the system is under oxidative stressituation (Lenzen et al., 1996; Tiedge et al., 1997). Similarly,levated levels of free radicals, due to insufficiency of the

ntioxidant defense system, may lead to disruption of cellularunction, oxidative damages to membranes and enhance theirusceptibility to lipid peroxidation (Baynes, 1991). In recentears dietary plants with antioxidative property have been the

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enter of focus. It is believe that these plants can prevent orrotect tissues against damaging effect of free radicals (Osawand Kato, 2005). In addition, it has been shown that dietaryupplementation with natural antioxidants such as, vitamins Cnd E, melatonin and flavonoids attenuated the oxidative stressnd diabetic state induced by STZ (Kaneto et al., 1999; Coskunt al., 2005; Montilla et al., 1998).

Lipid peroxidation products such as MDA are generatednder high levels of un-scavenged free radicals (Levy et al.,999). These products may be important in the pathogenesisf vascular complication in diabetes mellitus (Halliwell, 2000).he increased MDA level may have an important role inancreatic damage associated with diabetes. Under diabeticondition, the level of lipid peroxidation in the pancreas isnormously higher than non-diabetic rats; however, treatmentf the diabetic rats with ASE significantly decreased MDAontent. In addition, it has been suggested that overproductionf free radical NO under the influence of STZ may play arucial role in destruction of the �-cells during the developmentf type 1 diabetes (Haluzik and Nedvidkova, 2000). Treatmentith ASE strongly decreased serum NO to normal level of theealthy subject. Our data indicated a significant depletion ofoth enzymatic and non-enzymatic antioxidants in the pancre-tic tissue of STZ-induced diabetic rats. The enzymes includeOD and CAT, which can decompose superoxide and hydrogeneroxide in the cells, respectively. A decreased in the activitiesf these enzymes can lead to an excess availability of superoxidend hydrogen proxide in the biological systems, which in turnenerate hydroxyl radicals involved in initiation and propa-ation of lipid peroxidation (Halliwell and Chiricos, 1993).SH, as a non-enzymatic antioxidant, effectively scavenges

ree radicals and other ROS directly and indirectly throughnzymatic reactions (Guoyao et al., 2004). ASE had a potentncreasing effect on GSH content, along with, elevating SODnd CAT activities on pancreatic tissue compared to diabeticroup.

On the other hand, based on present scientific documentsaintenance of protein redox state is fundamental for cell func-

ion, whereas structural changes in proteins are considered toe among the molecular mechanism leading to progression and

evelopment of diabetes and its complication (Altomare et al.,997). An overload of ROS is known to modify proteins and toenerate PCO products that are presently considered as mark-rs of oxidative injury to proteins (Reznick and Packer, 1994).

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OPP are also considered as novel markers to estimate theegree of oxidant-mediated protein damages (Locckie et al.,999). Some authors have reported the elevation of AOPP iniabetic patients (Gallan et al., 2003; Kalousova et al., 2002).e also found an increased production of PCO and AOPP in

he pancreas of diabetic rats. Treatment with ASE, however,ignificantly reduced PCO and AOPP levels in the treated ratsompared to diabetic subjects. This clearly indicated that ASE,y decreasing oxidative stress, may be effective in preventingxidative protein damages which are thought to be involved in-cell damages under the diabetic condition.

Theoretically, hypoglycemic plants act through a variety ofechanisms such as improving insulin sensitivity, augmenting

lucose-dependent insulin secretion and stimulate the regen-ration of islets of langerhans in pancreas of STZ-inducediabetic rats (Sezik et al., 2005). Moreover, the role of antiox-dant compounds in both protection and therapy of diabetesave been considered in various scientific researches. Forxample, treatment of STZ-injected diabetic animals with N-cetyl-l-cysteine (NAC), a well-known antioxidant, preventsyperglycemia through reduce oxidative stress and restores-cell function (Takatori et al., 2004). In that regard, it is antic-

pated that the crude extract of Achillea santolina acts throughreventing diabetes by decreasing the oxidative damage to pan-reatic tissue and restore the plasma insulin levels. However,y this speculation, we do not exclude the possibilities of otherechanisms by which the plant exerts its effects.The antioxidative property of Achillea santolina certainly

s due to its chemical constituents. Phytochemical investiga-ions of Achillea spp. have demonstrated the presence of terpeneerivatives (Todorova et al., 2000) and in particular, flavonoidsKhafagy et al., 1976; Krenn et al., 2003). Flavonoids are a classf secondary plant phenolics with powerful antioxidant prop-rties (Heim et al., 2002). Independent studies have shown thentidiabetic effects of flavonoids (Coskun et al., 2005; Sezik etl., 2005). Thus, it is anticipated that the same group of com-ounds (e.g. flavonoids) are responsive for the antidiabetic andntioxidative property of Achillea santolina. Further work isequired to disclose this point.

In conclusion, the present data revealed that hypoglycemicffect of Achillea santolina extract may be due its antioxidativeotential which was shown by significant quenching impact onhe extent of lipid peroxidation and protein oxidation along with,nhancement of antioxidant defense systems in rat pancreaticissue. To elucidate the exact mechanism of this modulatoryffect further studies regarding the structure elucidation of thective compound(s) and evaluation of their biological activitiesre essential.

cknowledgments

The authors thank the Research Council of University ofehran for financial support of this investigation. Also, the authorould like to express their appreciation to Mr. Ahmmad Kamalohammad from Takrit University, to kindly provide the plantaterial for this investigation.

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pharmacology 112 (2007) 13–18 17

eferences

ebi, H., 1984. Methods in Enzymology. Academic Press, New York, p. 121.l-Hindawi, M.K., Al-Deen, I.H., Nabi, M.H., Ismail, M.A., 1989. Anti-

inflammatory activity of some Iraqi plants using intact rats. Journal ofEthnopharmacology 26, 163–168.

ltomare, E., Grattagliano, I., Vendemaile, G., Micelli, F.T., Signorile, A., Car-dia, L., 1997. Oxidative protein damage in human diabetic eye: evidence of aretinal participation. European Journal of Clinical Investigation 27, 141–147.

aynes, J.W., 1991. Role of oxidative stress in the development of complicationin diabetes. Diabetes 40, 405–412.

ennett, R.A., Pegg, A.E., 1981. Alkylation of DNA in rat tissues followingadministration of streptozotocin. Cancer Research 41, 2786–2790.

olzan, A.D., Bianchi, M.S., 2002. Genetotoxicity of streptozotocin. MutationResearch 512, 121–134.

rownlee, M., Cerami, A., Vlassura, H., 1988. Advanced glycosylation endproducts in tissue and the biochemical basis of diabetes complication. TheNew England Journal of Medicine 318, 1315–1321.

onforti, F., Loizzo, M.R., Statti, G.A., Menichini, F., 2005. Comparative radicalscavenging and antidiabetic activities of methanolic extract and fractionsfrom Achillea ligustica ALL. Biological and Pharmaceutical Bulletin 28,1791–1794.

oskun, O., Kanter, M., Korkmaz, A., Oter, S., 2005. Quercetin, a flavonoidantioxidant, prevents and protects streptozotocin-induced oxidative stressand �-cell damage in rat pancreas. Pharmacological Research 51, 117–123.

raper, H.H., Hadley, M., 1990. Methods in Enzymology. Academic Press, NewYork, p. 421.

llman, G.L., 1959. Tissue sulfhydryl groups. Archive of Biochemistry andBiophysics 82, 70–77.

ang, Y.Z., Yang, S., Wu, G., 2002. Free radical, antioxidant and nutrition.Nutrition 18, 872–890.

allan, P.M., Carrascosa, A., Gussinye, M., Dominguez, C., 2003. Biomarkers ofdiabetes-associated oxidative stress and antioxidant status in young diabeticpatients with or without subclinical complication. Free Radical Biology andMedicine 34, 1563–1574.

uoyao, W., Fang, Z.Y., Yag, S., Lupton, R.J., Turner, D.N., 2004. Glu-tathione metabolism and implication for health. The Journal of Nutrition134, 489–492.

alliwell, B., 2000. Lipid peroxidation, antioxidants and cardiovascular dis-ease: how should we more forward? Cardiovascular Research 47, 410–448.

alliwell, B., Chiricos, S., 1993. Lipid peroxidation: its mechanism, measure-ments and significance. The American Journal of Clinical Nutrition 57,7155–7255.

aluzik, M., Nedvidkova, J., 2000. The role of nitric oxide in the develop-ment of streptozotocin-induced diabetes mellitus: experimental and clinicalimplication. Physiological Research 49, 37–42.

eim, E.K., Tagliaferro, R.A., Bobilya, J.D., 2002. Flavonoid antioxidants:chemistry, metabolism and structure-activity relationship. Journal of Nutri-tional Biochemistry 13, 572–584.

ortelano, S., Dewer, B., Genaro, A.N., Diaz-Guerra, M.J., Bosca, L., 1995.Nitric oxide is released resulting in regenerating liver after partial hepatec-tomy. Hepatology 21, 776–786.

ohansen, J.S., Harris, A.K., Rychly, D.J., Ergul, A., 2005. Oxidative stress andthe use of antioxidants in diabetes: Linking basic science to clinical practice.Cardiovascular Diabetology 4:5.

ajimoto, Y., Kaneto, H., 2004. Role of oxidative stress in pancreatic �-celldysfunction. Annals of the New York Academy of Sciences 1011, 168–176.

akkar, P., Das, B., Viswanathan, P.N., 1984. A modified spectrophotometricassay of superoxide dismutase. Indian Journal of Biochemistry and Bio-physics 21, 130–132.

alousova, M., Skrha, J., Zima, T., 2002. Advanced glycation end productsand advanced oxidation protein products in patients with diabetes mellitus.

Physiological Research 51, 597–604.

aneto, H., Kajimoto, Y., Miyagawa, J., Matsuoka, T., Fumigant, Y., Umayahara,Y., Hanafusa, T., Matsuzawa, Y., Yamasaki, Y., Hori, M., 1999. Beneficialeffects of antioxidants in diabetes, possible protection of pancreatic �-cellsagainst glucose toxicity. Diabetes 48, 2398–2406.

1 Ethno

K

K

K

L

L

L

L

M

M

O

O

O

R

R

S

S

T

T

T

8 R. Yazdanparast et al. / Journal of

ayali, R., Cakatay, U., Akcay, T., Altug, T., 2006. Effect of alpha-lipoic acidsupplementation on markers of protein oxidation in post-mitotic tissues ofageing rat. Cell Biochemistry and Function 24, 79–85.

hafagy, S.M., Sabri, N.N., Soliman, F.S., Abou-Donia, A.H., Mosandl, A.,1976. Isolation of two flavonoids from Achillea santolina L. growing inEgypt. Pharmazie 31, 894–895.

renn, L., Miron, A., Pemp, E., Petr, U., Kopp, B., 2003. Flavonoids fromAchillea nobilis L. Z Naturforsch [C] 58, 11–16.

enzen, S., Drinkgern, J., Tiedge, M., 1996. Low antioxidant enzyme geneexpression in pancreatic islets compared with various other mouse tissues.Free Radical Biology and Medicine 20, 463–466.

evy, U., Zalizber, I., Ben-Amotz, A., Kanter, Y., Aviram, M., 1999. �-Caroteneaffects antioxidant status in non-insulin dependent diabetes mellitus. Patho-physiology 6, 157–161.

occkie, L.Z., Meerman, J.H.N., Commander, J.N.M., Vermeulen, N.P.E., 1999.Biomarkers of free radical damage: application in experimental animals andin humans. Free Radical Biology and Medicine 26, 202–226.

owry, O.H., Rosebrought, N.J., Randell, R.J., 1951. Protein measurement withthe Folin phenol reagent. Journal of Biological Chemistry 193, 265–272.

aritim, A.C., Sanders, R.A., Watkins, J.B., 2003. Diabetes, oxidative stress andantioxidants: a review. Journal of Biochemical and Molecular Toxicology17, 24–38.

ontilla, P., Vargas, J., Tunez, I., Munoz, M.C., Valdelvira, M.E., Cabrera, E.,1998. Oxidative stress in diabetic rats induced by streptozotocin: protectiveeffects of melatonin. Journal of Pineal Research 25, 94–100.

brosova, I.G., Vanlteysen, C., Fathallah, L., Cao, X., Greene, D.A., Stevens,M.J., 2002. An aldose reductase inhibitor reverses early diabetes-inducedchanges in peripheral nerve function. FASEB Journal 16, 123–125.

hkuwa, T., Sato, Y., Naoi, M., 1995. Hydroxyl radical formation in diabeticrat induced by streptozotocin. Life Sciences 56, 1789–1798.

T

W

pharmacology 112 (2007) 13–18

sawa, T., Kato, Y., 2005. Protective role of antioxidative food factors in oxida-tive stress caused by hyperglycemia. Annals of the New York Academy ofSciences 1043, 440–451.

eznick, A.Z., Packer, L., 1994. Methods in Enzymology. Academic Press, NewYork, p. 357.

osen, P., Nawroth, P.P., King, G., Moller, G., Tritschrev, H.J., Packer, L.,2001. The role of oxidative stress in the onset and progression of diabetesand its complication. Diabetes/Metabolism Research and Reviews 17, 189–212.

ezik, E., Aslan, M., Yesilada, E., Ito, S., 2005. Hypoglycemic activity of Gen-tiana olivieri and isolation of the active constituent through bioassay-directedfractionation techniques. Life Sciences 76, 1223–1238.

zkudelski, T., 2001. The mechanism of alloxan and streptozotocin action in Bcells of rat pancreas. Physiological Research 50, 536–546.

akasu, N., Komiya, T., Asawa, T., Nagasawa, Y., Yamada, T., 1991. Strepto-zotocin and alloxan-induced H2O2 generation and DNA fragmentation inpancreatic islets. H2O2 as mediator for DNA fragmentation. Diabetes 40,1141–1145.

akatori, A., Ishii, Y., Itakagi, S., Kyuwa, S., Yoshikawa, Y., 2004. Ameliorationof the �-cell dysfunction in diabetic APA hamsters by antioxidants andAGE inhibitor treatments. Diabetes/Metabolism Research and Reviews 20,211–218.

iedge, M., Lortz, S., Drinkgern, J., Lenzen, S., 1997. Relation between antiox-idant enzyme gene expression and antioxidative defense system status ofinsulin producing cells. Diabetes 46, 1733–1742.

odorova, M., Vogler, B., Tsankova, E., 2000. Terpenoids from Achillea setacea.Z Naturforsch [C] 55, 840–842.

olff, S.P., Dean, R.T., 1987. Glucose autoxidation and protein modification.The potential role of ‘autoxidative glycosylation’ in diabetes. The Biochem-ical Journal 245, 243–250.