A Study of Antidiabetic and Antioxidant Effects of Helichrysum Graveolens Capitulums In Streptozotocin-Induced Diabetic Rats

A Study of Antidiabetic and Antioxidant Effects of Helichrysum Graveolens Capitulums In Streptozotocin-Induced Diabetic Rats
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  See discussions, stats, and author profiles for this publication at: A Study of Antidiabetic and Antioxidant Effectsof Helichrysum graveolens Capitulums inStreptozotocin-Induced...  Article   in  Journal of Medicinal Food · July 2007 DOI: 10.1089/jmf.2006.293 · Source: PubMed CITATIONS 39 READS 125 5 authors , including: Some of the authors of this publication are also working on these related projects: Pharmacognosic Studies on Some Bidens L. Taxons Growing In Turkey (2011) TÜB İ TAK   View projectStudies on Biological Activities of Vitis vinifera Leaves   View projectDidem Deliorman OrhanGazi University 80   PUBLICATIONS   1,260   CITATIONS   SEE PROFILE Nilüfer OrhanGazi University 34   PUBLICATIONS   604   CITATIONS   SEE PROFILE Ekrem SezikGazi University 81   PUBLICATIONS   3,375   CITATIONS   SEE PROFILE Erdem YesiladaYeditepe University 291   PUBLICATIONS   8,097   CITATIONS   SEE PROFILE All content following this page was uploaded by Nilüfer Orhan on 30 January 2014. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  JOURNAL OF MEDICINAL FOOD  J Med Food  10 (2) 2007, 396–400©Mary Ann Liebert, Inc. and Korean Society of Food Science and NutritionDOI: 10.1089/jmf.2006.293 Short CommunicationA Study of Antidiabetic and Antioxidant Effects of  Helichrysum graveolens Capitulums in Streptozotocin-Induced Diabetic Rats Mustafa Aslan, 1 Didem Deliorman Orhan, 1 Nilüfer Orhan, 1 Ekrem Sezik, 1 and Erdem Yes¸ilada 2 1  Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Ankara; and 2 Faculty of Pharmacy,Yeditepe University, Istanbul, Turkey ABSTRACT  Helichrysum graveolens (Bieb.) Sweet (Asteraceae) grows widely in Turkey. Capitulums of  H. graveolens areused in the treatment of many diseases such as jaundice and wound healing and as a diuretic in the rural areas of Anatolia.The decoction from the capitulums of the plant is consumed for the symptoms of diabetes mellitus in folk medicine. In thepresent study, the hypoglycemic, antihyperglycemic, and antioxidant potentials of water and ethanol extracts of  H. graveolens were evaluated by using in vivo methods in normal and streptozotocin-induced diabetic rats. Blood glucose levels of animalswere measured by the glucose oxidase method. The antioxidant activity of these extracts was also studied in liver, kidney,and heart tissues. In order to determine antioxidant activity, tissue malondialdehyde and reduced glutathione levels were mea-sured by using spectrophotometric methods. The experimental data obtained from water and ethanol extracts of capitulumsconfirmed the folkloric utilization. In order to discuss the role of polyphenolic components in the relevant activity, total phe-nol and flavonoid contents of each extract were also determined using the Folin-Ciocalteu reagent, and a positive correlationwas observed. KEY WORDS:  •  antidiabetic  •  antioxidant  •  Asteraceae  • Helichrysum graveolens  L.  •  hypoglycemic 396 INTRODUCTION D IABETES MELLITUS is a common incretion disease causedby the absolute or relative absence of insulin, and it re-mains an acute disease and danger to human health. Theprevalence of diabetes is increasing constantly and theWorld Health Organization has estimated that diabetes willaffect 221 million people worldwide by the year 2010. 1 Plants are the most common form of medicine for manypeople, and plants used by various people throughout theworld for diabetes have been studied for hypoglycemic ac-tion. 2,3 Eighty-one percent of 295 plants used traditionallyfor diabetes-related conditions had the ability to lower bloodglucose, 3 thereby offering a degree of scientific validity totraditional practices.Nineteen  Helichrysum species (Asteraceae) grow inTurkey, 4 and many of them have been widely used as di-uretics, as lithagogues, for stomachache, for anti-asthmaticproperties, against kidney stones, and as herbal tea in Turk-ish folk medicine. 5,6 Moreover, the capitulums of  Helichry-sum species are also used to decrease blood glucose level indiabetes mellitus by people living in rural areas of Turkey.Aerial parts of the plant are marketed in herbal stores asherbal tea. 7 A number of scientific studies have been conducted on  He-lichrysum species, and many biological activities have beenfound by researchers. 8–10 However, the antidiabetic effect of   Helichrysum graveolens has not been studied so far. The ob- jective of this study is to evaluate hypoglycemic, antidiabetic,and antioxidant effects of capitulums of  H. graveolens .On the other hand, the importance of antioxidants in di-abetes is very well known. Oxidative stress has been asso-ciated particularly with the development of complications indiabetes. 11 Many of the complications of diabetes, includ-ing retinopathy and atherosclerotic vascular disease, theleading cause of mortality in diabetics, have been linked tooxidative stress, 12 and antioxidants ( i.e ., vitamin E or C)have been considered as treatments. 13,14 Plants often con-tain substantial amounts of antioxidants, including toco-pherols, carotenoids, ascorbic acid, flavonoids, and tan-nins, 15 and antioxidant action may be an important propertyof plants used in medicines associated with diabetes.To this end, in the present study, we assessed antidiabeticand antioxidant effects of  H. graveolens , which is used as afolk remedy in Turkey, in streptozotocin (STZ)-induced di-abetic rats. Manuscript received 24 May 2006. Revision accepted 25 September 2006.  Address reprint requests to: Dr. Mustafa Aslan, Department of Pharmacognosy, Facultyof Pharmacy, Gazi University, 06330 Ankara, Turkey, E-mail:  MATERIALS AND METHODS  Animals Male Wistar albino rats (weighing 150–200 g), purchasedfrom the Animal House of Gulhane Military Medical Acad-emy (Ankara, Turkey), were used in the present study. An-imal experiments were conducted following the interna-tionally accepted ethical guidelines for the care of laboratoryanimals. Prior to the experiments, rats were fed with stan-dard food for 1 week in order to adapt to the laboratory con-ditions. Sixteen hours before the experiments, they werefasted overnight, but allowed free access to water. The 72rats included in the study were divided into 12 groups, eachconsisting of six animals. Body weight and fasting bloodglucose levels of all the rats were determined before the ex-periment was started. Plant material  H. graveolens (Bieb.) Sweet was collected in August 2004from Ilgaz Mountain (Kastamonu, Turkey). Specimens of each plants were deposited at the Herbarium of the Phar-macy Faculty of Gazi University, Ankara. Preparation of the test samples The capitulums were separated and extracted with dis-tilled hot water and 80% ethanol (EtOH) on a shaker for 24hours. Extracts were filtered and evaporated under low pres-sure. The dried water (10.0%) and EtOH (11.5%) extractswere suspended in 0.5% aqueous carboxymethylcellulosesuspension in distilled water prior to oral administration toanimals (5 mL/kg of body weight). Tolbutamide (100 mg/kgof body weight) was used as the reference drug. Animals inthe control group received only the vehicle (5 mL/kg of bodyweight).  Analytical methods The method of Ohkawa et al . 16 as modified by Jamall andSmith 17 was used to determine lipid peroxidation in tissue sam-ples. Nonprotein sulfhydryl groups [cellular reduced glu-tathione (GSH)] in liver, kidney, and heart tissues were mea-sured by the method of Sedlak and Lindsay. 18 Total phenoland total flavonoid contents of water and EtOH extracts wereestimated by the methods of Gao et al . 19 and Kosalec et al ., 20 respectively. Total phenolic content was expressed in mg of gallic acid equivalents/g of extract, and total flavonoid contentwas expressed in mg of quercetin equivalents/g of extract.  Blood collection and determination of blood glucose levels Blood glucose concentration (in mg/100 mL) was deter-mined using an Ascensia ® Elite™commercial test (Bayer,Tarrytown, NY), based on the glucose oxidase method.Blood samples were collected from the tip of tail at the de-fined time patterns.  Effect on normoglycemic plus glucose-hyperglycemicmodel (NG-OGTT) A combined methodology of Kato and Miura 21 is pre-ferred for the activity assessment of extracts in order to avoidusing an excess number of animals with some modificationsin time pattern for blood glucose level determination.Test samples were taken immediately after the collectionof initial blood samples. The blood glucose levels were de-termined at 30 and 60 minutes to assess the effect of the testsamples on normoglycemic animals. After the last mea-surement (at 2 hours) the rats were orally loaded with 2 g/kgof glucose, and the blood glucose measurements were con-tinued to determine to assess the effects on glucose-hyper-glycemic rats at 1, 1.5, and 2.5 hours. Study on diabetic rats (non–insulin-dependent diabetes model)  Induction of diabetes . Diabetes was induced in rats by in-traperitoneal injection of STZ at a dose of 55 mg/kg of bodyweight dissolved in distilled water (1 mL/kg). Seven daysafter the injection, the blood glucose levels were measured.Each animal with a blood glucose concentration level above EFFECT OF  H. GRAVEOLENS  CAPITULUMS IN DIABETES  397 T ABLE 1.E FFECTS OF W ATER AND E T OH E XTRACTS FROM  H. GRAVEOLENS  ON B LOOD G LUCOSE L EVELS IN N ORMAL AND 2 G / KG OF G LUCOSE -L OADED H YPERGLYCEMIC (NG-OGTT) R ATS Blood glucose concentration (% inhibition)Dose60 minTest samples(mg/kg)0 min30 min(glucose load)120 min150 min270 min Control—92.5  1.990.3  3.291.3  2.5127.0  1.5103.7  2.2883.  4.0Tolbutamide10088.0  2.557.8  3.7 (36)***56.7  3.5 (38)***84.7  5.4 (34)***67.9  2.9 (35)***56.5  2.7 (37)***Water extract50097.2  2.193.9  3.197.9  3.4111.3  4.0 (12.4)*103.8  3.892.4  2.9EtOH extract50094.6  3.394.3  2.6102.6  2.1115.7  1.9 (8.9)*121.7  3.0103.8  4.9Data are mean  SEM values (in mg/dL) ( n  6).* P  .05, *** P  .001, significantly different from the control animals.  250 mg/dL was considered to be diabetic and used in theexperiments. To overcome the hypoglycemia that occurredduring the first 24 hours following the STZ administration,5% glucose solution was orally given to the diabetic rats. Inall experiments, rats were fasted for 16 hours prior to STZinjection.  Acute antidiabetic effect  . The test samples (water extract,EtOH extract, and tolbutamide) were administered orally byusing a gastric gavage needle. Blood glucose levels were de-termined at 30, 60, 120, 240, and 360 minutes after admin-istration of the test samples. Subacute antidiabetic effect  . The test samples (water ex-tract, EtOH extract, and tolbutamide) were administered for7 days consecutively. Blood glucose levels were determinedon days 1, 3, 5, and 8 after the administration of test sam-ples. The effect on body weight of each test sample was alsomonitored at the same days. On day 8, all animals were sac-rificed, and then the kidney, liver, and heart of each animalwere removed for measurement of tissue malondialdehyde(MDA) and GSH levels. Statistical analysis Data are presented as means  SEM values. Statisticaldifferences between the treatments and the controls weretested by one-way analysis of variance followed by the Stu-dent-Newman-Keuls test using the INSTAT statistical com-puter program (GraphPad, San Diego, CA). A difference inthe mean values of P  .05 was considered to be statisti-cally significant. RESULTS  Effect on blood glucose level Acute effects of the water and EtOH extracts obtainedfrom  H. graveolens on blood glucose levels of NG-OGTTand STZ-diabetic rats are shown in Tables 1 and 2.As shown in Table 1, while the reference drug, tolbu-tamide, possesses potent activity during the experiment(34–38%), water and EtOH extracts do not show any re-markable effect. However, the extracts do show a slight ac-tivity (8.9–12.4%) that appeared just after the glucose load-ing.According to the data shown in Table 2, tolbutamide, wa-ter, and EtOH extracts exerted continual significant activityon blood glucose levels of STZ-induced diabetic ratsthroughout the experiment. At all measurements, the extractswere found to be more potent than the reference drug. More-over, the water extract exerted its maximum hypoglycemiceffect at between 120 and 360 minutes after the adminis-tration (18–20%).As demonstrated in Table 3, the extracts used in the ex-periments have shown outstanding antidiabetic effects onblood glucose levels of diabetic rats. Although all test sam-ples, i.e ., reference drug and both extracts, remained inef-fective at the first two measurements, significant antidia-betic activity was observed on days 5 and 8 (13.4–31.2%). 398  ASLAN ET AL. T ABLE 2.E FFECTS OF W ATER AND E T OH E XTRACTS FROM  H. GRAVEOLENS  ON B LOOD G LUCOSE L EVELS IN STZ-I NDUCED D IABETIC R ATS Blood glucose concentration (% inhibition)DoseTest sample(mg/kg)0 min30 min60 min120 min240 min360 min Control—374.8  6.9439.0  10.7399.2  6.0391.6  9.8378.6  7.8364.2  4.7Tolbutamide100373.8  7.5388.0  6.7 (12)***377.0  4.0351.0  7.2 (10)**355.0  4.0 (6)*341.8  5.9 (6)**Water extract500374.9  4.1***368.5  9.8 (16)***392.6  4.6316.8  10.2 (19)***304.3  4.4 (20)***298.0  3.3 (18)***EtOH extract500385.5  11.3360.7  5.7 (18)***363.8  12.1 (9)*338.0  7.0 (14)***337.0  6.9 (11)***302.1  7.5 (17)***Data are mean  SEM values (in mg/dL) ( n  6).* P  .05, ** P  .01, *** P  .001, significantly different from the control animals. T ABLE 3.S UBACUTE H YPOGLYCEMIC E FFECT OF  H. GRAVEOLENS E XTRACTS ON STZ-I NDUCED D IABETIC R ATS Blood glucose concentration (% inhibition)Test sampleDose (mg/kg)Day 1Day 3Day 5Day 8 Control—399.4  15.4418.0  9.50347.4  10.6339.2  4.1Tolbutamide100404.6  8.20401.3  10.9317.2  6.3 (8.7)293.0  4.6 (13.7)**Water extract500388.6  14.7420.2  10.4301.0  8.7 (13.4)*240.6  5.9 (29.1)***EtOH extract500390.0  16.5403.8  13.9283.6  6.8 (18.4)**233.4  4.7 (31.2)***Data are mean  SEM values (in mg/dL) ( n  6).* P  .05, ** P  .01, *** P  .001, significantly different from the control group.   Effect on body weight  During the subacute study, the body weights of diabeticanimals that were treated with extracts and reference drugthroughout the 8-day period were also followed, and no sig-nificant change was observed.  Effect on tissue (liver, kidney, and heart) lipid  peroxidation and GSH levels As demonstrated in Table 4, while kidney (  87.8%) andheart (  68.5%) tissue MDA levels of the diabetic controlgroup increased dramatically, water (  12.3%) and EtOH(  9.5%) extracts induced significant alleviation only in kid-ney tissue MDA levels.Moreover, only the EtOH extract (  29.4%) showed a de-creasing effect on liver tissue MDA levels. On the otherhand, the data shown in Table 4 suggest that the water ex-tract restored slightly the GSH levels only in kidney tissueof diabetic rats. Total phenolic and flavonoid contents of H. graveolens In order to establish a relationship between the chemical con-tent and the antidiabetic activity, the total phenol and flavonoidcontents of water and EtOH extracts were determined accord-ing to previously described methods. 19,20 The EtOH extract ex-hibited the highest amount of both total phenols (91.4  2.1mg of gallic acid equivalents/g of extract) and flavonoids(59.5  3.9 mg of gallic acid equivalents/g of extract), whilethe lowest amounts of both total phenols (70.9  0.9 mg of gallic acid equivalents/g of extract) and flavonoids (32.7  1.9mg of gallic acid equivalents/g of extract) were observed in thewater extract. A positive correlation was found between totalphenol and flavonoid contents and antidiabetic activity. Thesame correlation was also observed between the antilipoper-oxidation activity and phenolic contents. DISCUSSION Several investigations have focused on the antioxidantstatus and oxidative stress in type 2 diabetes mellitus. 22–24 Under diabetic conditions, reactive oxygen species are pro-duced via glucose autooxidation 25 and also via nonenzy-matic protein glycation in various tissues. 26,27 Reactive oxy-gen species are associated with microvascular complications( e.g ., eye, kidney, and nerve damage) and, to a lesser ex-tent, with cardiovascular disease in patients with dia-betes. 28,29 Therefore, chemical or natural compounds used in thetreatment of diabetes should possess both antidiabetic andantioxidant effects.Many flavonoids have been isolated from natural sourcesfor their hypoglycemic effects. 30 The antioxidant effect of flavonoids is also very well known. In the light of these find-ings, it can be predicted that  Helichrysum species, includ-ing  H. graveolens , used for diabetes symptoms as a folkremedy, are effective because of the their rich content of flavonoids and phenolic compounds.Indeed, in previous studies, the chemical composition of the  H. graveolens has been investigated, and it was found that capitulums of  H. graveolens contain manyflavonoids, including apigenin, luteolin, galangin-3-methylether, 3,5-dihydroxy-6,7,8-trimethoxy flavone, kaempferol,naringenin, apigenin-7-glucoside, apigenin-4  -glucoside,luteolin-7-glucoside, luteolin-4  -glucoside, kaempferol-3-glucoside, helichrysin B, and naringenin-4  -glucoside asmajor constituents. 8–10 Additionally, some of these com-pounds (especially kaempferol-3-rhamnoside, quercetin,and quercetin-3-rhamnoside) isolated from different plantshave previously been suggested as the active antidiabetic in-gredients of various plant remedies. 30 Indeed, these litera-ture findings support the proposal that antidiabetic and an-tioxidant properties of  H. graveolens may arise from theflavonoids found in its extracts.The recognized benefits of antioxidants in prevention of the complications of diabetes mellitus have been known. Inthis study, an EtOH extract, which was found to have a morepotent antidiabetic effect than the water extract, has alsoshown significantly more antilipoperoxidation activity inkidney and liver tissues.This study is the first report on antidiabetic and antioxi-dant properties of the  H. graveolens varieties growing in EFFECT OF  H. GRAVEOLENS  CAPITULUMS IN DIABETES  399 T ABLE 4.MDA AND GSH L EVELS IN L IVER , K IDNEY , AND H EART OF STZ-I NDUCED D IABETIC R ATS A FTER T REATMENT WITH  H. GRAVEOLENS E XTRACTS Tissue level (% chance)LiverKidneyHeartDoseTest sample(mg/kg)MDAGSHMDAGSHMDAGSH  Control—279.3  11.5124.8  1.9181.2  18.081.2  1.3142.4  19.344.8  1.1Diabetic control—305.9  9.9114.5  1.8 (  8.3)**340.3  7.4 (  87.8)***75.3  2.1 (  7.3)239.9  8.4 (  68.5)***42.5  1.3 (  5.1)Water extract500309.6  7.5 (  1.2)101.6  2.5 (  11.3)**298.4  7.9 (  12.3)*79.2  1.5 (  5.2)252.1  7.6 (  5.1)39.3  1.4 (  7.5)EtOH extract500216.1  3.3 (  29.4)***110.1  3.3 (  3.8)307.9  5.6 (  9.5)*72.8  1.7 (  3.3)286.6  8.4 (  19.5)*42.5  4.0Data are mean  SEM values. For percentage chance, (  ) represents percentage of increase, and (  ) represents decrease in each value. MDA is in units of nmol/g of wet weight, and GSH is in units of  mol/g of tissue. For statistical comparisons, diabetic control values were compared with vehicle control (0.5%carboxymethylcellulose), and water and EtOH extract values were compared with diabetic control values.* P  .05, ** P  .01, *** P  .001, significantly different from the control or diabetic control group.
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