Antioxidant and anti-inflammatory effects of Urtica pilulifera extracts in type2 diabetic rats

Journal of Ethnopharmacology 145 (2013) 269–277

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Antioxidant and anti-inflammatory effects of Urtica pilulifera extracts in type2 diabetic rats Dina M. Abo-elmatty a, Soha S. Essawy b,n, Jihan M. Badr c, Olov Sterner d a

Department of Pharmacology, Faculty of Medicine, Suez Canal University, Ismailia, 41522, Egypt Department of Biochemistry, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt c Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt d Department of Organic Chemistry 2, Lund University, P.O. Box 124, S-21100 Lund, Sweden b

a r t i c l e i n f o

abstract

Article history: Received 28 May 2012 Received in revised form 27 October 2012 Accepted 2 November 2012 Available online 16 November 2012

Ethnopharmacological relevance: ’’Urtica pilulifera has been traditionally used in Egyptian system as an herbal remedy to be a diuretic, antiasthmatic, anti-inflammatory, hypoglycemic, hemostatic, antidandruff and astringent’’ Aim of the study: To evaluate the potential effects of ethyl acetate (EA), chloroform (CHLOR) and hexane (HEXA) extracts of Urtica piluliferaas oral anti-diabetic agents as well as to evaluate their possible antioxidant and anti-inflammatory effects in type2 diabetic rat model. Material and methods: Type2 diabetes was induced by a high fat diet and low dose streptozotocin (STZ). Diabetic adult male albino rats were allocated into groups and treated according to the following schedule; Pioglitazone HCL (PIO), EA, CHLOR and HEXA extracts of Urtica pilulifera at two doses of 250 and 500 mg/kg were used. In addition, a normal control group and a diabetic control one were used for comparison. Blood glucose, insulin resistance, antioxidant enzymes, 8-hydroxy-2-deoxyguanosine (8-OHdG) as well as C-reactive protein and tumor necrosis factor-a levels were evaluated. Results: EA and CHLOR extracts of Urtica pilulifera exhibited a significant hypoglycemia associated with antioxidant and anti-inflammatory effects in diabetic rats; however, HEXA extract showed no beneficial effect. These activities are responsible, at least partly, for improvements that have been seen in hyperglycemia and insulin resistance of diabetic rats. Conclusion: Our results encourage the traditional use of Urtica pilulifera extract as an antioxidant and anti-inflammatory agent as an additional therapy of diabetes. & 2012 Elsevier Ireland Ltd. All rights reserved.

Keywords: Urtica pilulifera Diabetes Streptozotocin Rat

1. Introduction Urtica pilulifera which is classified as popular plant found in Palestinian and in Sinai areas (Ali-Shtayeh et al., 2000), belongs to family Urticaceae and it is characterized morphologically by the stinging hairs carried by its leaves and flowers which cause irritation to the skin (Fu et al., 2006). A tea made from the leaves of Urtica pilulifera has traditionally used as a stimulating tonic, blood purifier and hemostatic and for enhancement of hemoglobin concentration (Chrubasik et al., 2007) while the whole plant is a diuretic, antiasthmatic, anti-inflammatory, hypoglycemic, hemostatic, antidandruff and astringent (Delcourt et al., 1996; Abudoleh et al., 2011). In addition, extracts of Urtica pilulifera are useful to relief symptoms of lower urinary tract infection (Lopatkin et al., 2007).

n

Corresponding author. Tel.: þ2 011 27586084; fax: þ 2 0643230741. E-mail address: [email protected] (S.S. Essawy).

0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.11.002

Lectins isolated from seeds of this plant exhibit pronounced hypoglycemic activity and b-cell regenerative potency (Kavalali et al., 2003). Urtica pilulifera is considered non-toxic and does not have any mutagenic and embyrogenic effects (Graf et al., 1994). Type2 diabetic rat model can be induced by combining highfat diet (HFD) with a low dose of STZ (Srinivasan et al., 2004). There is evidence that the generation of reactive oxygen species (ROS) – due to glucose auto-oxidation – plays a critical role in the cytotoxicity of STZ (Matsumoto et al., 2003). ROS can in turn damage cell proteins and DNA and promote non-enzymatic glycoxidation of proteins and lipids (Robertson et al., 2003). Increased oxygen radicals enter into reaction with DNA to permit the formation of 8-OHdG, a product of DNA damage. Previous studies have shown that 8-OHdG is one of the commonly used markers for evaluation of oxidative DNA damage (Marnett, 2000). Artificial and natural agents possessing radical scavenging properties have been proposed to prevent and to treat oxidative damage induced by ROS developed pathological states (Kucharska´ et al., 2004).

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The aim of this work is to investigate the potential effects of EA, CHLOR and HEXA extracts of Urtica pilulifera as oral antidiabetic agents as well as to evaluate their possible anti-oxidant and anti-inflammatory effects in type2 diabetic animal model.

rotary evaporator to give finally 22 g of ethyl acetate extract, 35 g of chloroform extract and 64 g of hexane extract. All extracts were stored at 4 1C until use. 2.4. Extract preparation

2. Material and methods 2.1. Plant collection and preparation of extracts ’’Urtical pilulifera L.’’ was collected from Rashid at Alexandria during April to June 2009. The collected plant was identified by Dr. Saneia Kamal, Assistant Professor, Faculty of Science, Alexandria University. A voucher sample (Urtica pilulifera – 1) was kept at the herbarium of the Department of Pharmacoconsy, Faculty of pharmacy, Suez Canal University, Egypt. The whole aerial parts of the plant were thoroughly washed under running tap water and cut into very small pieces (3 kg of the fresh plant), then extracted. Cold maceration technique was used for extraction of the plant. The small pieces of the plant were soaked in methanol at room temperature. After 7 days, the extract was filtered under vacuum through Whatman filter paper No. 1. The residue was again dipped in methanol for additional 7 days and filtered thereafter. The filtrate was combined and methanol was evaporated under vacuum, using rotary evaporator (Buchi Rotavapor R-200) at 45 1C to yield viscous greenish-colored extract. The quantity of extract obtained from Urtica pilulifera was 200 g. From this total extract, 30 g was withdrawn to be investigated of the major chemical components of the plant. 2.2. Isolation of the major constituents of Urtica pilulifera A portion of the total alcohol extract (30 g) was fractionated between 2 l of CHLOR and 2 l of EA. Each extract was concentrated under vacuum using rotary evaporator. The CHLOR extract was absorbed on silica gel, fractionated on an open column packed with silica gel and elution was performed using HEXA: EA: methanol gradient. The resulted fractions were investigated by TLC and revealed by spraying with anisaldehyde/sulfuric acid and similar fractions were combined together. Repeated purification on column chromatography then final crystallization from methanol: CHLOR 1:1 afforded three major compounds each occurred as white powder. Identification of the compounds was achieved by direct comparison on TLC with known reference compounds or by interpretation of data obtained from H NMR and 13C NMR. The EA extract was adsorbed on silica gel, fractionated on an open column packed with silica gel and elution was performed using chloroform:methanol gradient. The resulted fractions were investigated by TLC and revealed by spraying with anisaldehyde/ sulfuric acid and similar fractions were combined together. Two major spots could be detected where one of the compounds was purified by repeated crystallization from methanol (compound 4) and the second compound was purified by PTLC using CHLOR:methanol (8:2) as a developing system (compound 5). Identification of compound 4 was achieved by interpretation of data obtained from 1H NMR and 13C NMR while compound 5 was identified by direct comparison on TLC with known reference samples. 2.3. Fractionation Fractionation was carried out by suspending the crude methanol extract in 200 ml water separately and partitioning with different organic solvents (EA, CHLOR and HEXA) in order of increasing polarity by using separating funnel. All fractions of the plant extracts were dried by evaporating respective solvent using

Two doses of 250 mg/kg and 500 mg/kg were selected and used in this study. The two doses were prepared by dissolving appropriate amount of this vicious extracts in 1 ml Tween 20. This was followed by adding 9 ml of 0.9% NaCl to each mixture. The vehicle was obtained by dissolving 1 ml of Tween 20 in 9 ml of 0.9% NaCl (Irshaid and Mansi, 2009a). 2.5. Animals Ninety adult male albino rats, weighing 150720 g were obtained from Egyptian Organization for Biological Products and Vaccines. The animal chow diet and water were provided ad libitum. Rats were maintained on normal light-dark schedule and temperature 2573 1C throughout the experiment and left 1 week for acclimatization. All experimental protocols were approved by the Institutional Animal Care and Use Committee at the Faculty of Pharmacy, Suez Canal University (Ismailia, Egypt). 2.6. Experimental induction of Type2 diabetes by high fat diet and low-dose STZ The rats were allocated into two dietary regimens; normal pellet diet (NPD; 12% calories as fat) (10 rats) and high fat diet (HFD; 58% fat, 25% protein and 17% carbohydrate, as a percentage of total kcal) (80 rats) (Reed et al., 2000).The composition and preparation of HFD was described by (Srinivasan et al., 2004) as the following components (g/kg): powdered NPD, 365 (Egyptian market); lard, 310 (Egyptian market); casein, 250 (Difco, Becton Dickinson, France); cholesterol, 10 (Oxford Lab, Mumbai, India); vitamin and mineral mix, 60 (Sigma–Aldrich, MO, USA; 8ADWIC Co., Cairo, Egypt); DL-methionine, 3.0 (Sigma–Aldrich, MO, USA; 8ADWIC Co., Cairo, Egypt); Yeast powder, 1.0 (Egyptian market), sodium chloride, 1.0 (Egyptian market). After the 2 weeks of dietary manipulation, 80 rats, fed HFD, were injected intraperitoneally (i.p) with STZ 35 mg/kg (Srinivasan et al., 2005) dissolved in di-sodium citrate buffer (pH 4.5). Ten days following STZ administration, glucose level was measured in blood samples from tail tip using glucometer method (Accutrenr alpha-Boehringer Mannheim). Only rats with fasting blood glucose level 4250–300 mg/dl were enrolled in the study (Algandaby et al., 2010). 2.7. Experimental design Diabetic rats were received PIO or the extracts under study orally and once daily dissolved in the vehicle in a volume of (1 ml/kg) for 4 weeks starting from day 11 after induction of diabetes. Rats were randomly allocated into nine groups of ten animals each. Group I: non-diabetic rats, fed NPD, received only single i.p. injection of citrate buffer (1 ml/kg) and served as normal control group. Group 2: diabetic rats received only vehicle and served as diabetic control group. Group 3 (PIO): diabetic rats received PIO HCL (Sigma Chemical Company, Egypt) at doses of 20 mg/kg (Dong et al., 2004). Group 4 (EA 250): diabetic rats received 250 mg/kg EA extract. Group 5 (EA 500): diabetic rats received 500 mg/kg EA extract.

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Group 6 (CHLOR 250): diabetic rats received 250 mg/kg CHLOR extract. Group 7 (CHLOR 500): diabetic rats that received 500 mg/kg CHLOR extract. Group 8 (HEXA 250): diabetic rats received 250 mg/kg HEXA extract. Group 9 (HEXA 500): diabetic rats that received 500 mg/kg HEXA extract.

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2.9.3. Determination of inflammatory markers Serum C-reactive protein (CRP) was measured according to the principle of (Ben Assayag et al., 2009) by ELISA technique using kits purchased from DIA MED (Belgium). Pancreatic tumor necrosis factor-a (TNF-a) concentration was measured according to the principle of (Mizutani et al., 2003) using ELISA kits (Biosource, USA). 100 mg tissues was homogenized in 0.1 M phosphate buffer (pH 7.2), containing 0.05 w/v% sodium azide at 4 1C. The pancreatic homogenate was centrifuged at 2000  g for 10 min and the supernatant fraction was used for determination of TNF-a.

2.8. Toxicity study The toxicity of three Urtica pilulifera extracts – EA, CHLOR and HEXA – were tested using four doses (250, 500, 1000 and 2000 mg/ kg) (six rats for each dose). Six control rats were kept under the same conditions without any treatments. The animals were observed continuously during the first hour, and then every hour for 6 h, then after 12 and 24 h, and finally after every 24 h, up to 3 weeks, for any physical signs of toxicity such as writhing, gasping, salivation, diarrhea, cyanosis, pupil size, any nervous manifestations, or mortality (Elberry et al., in press). 2.9. Blood sampling and biochemical analysis At the end of the study, rats were fasted overnight, anesthetized with thiopental sodium (50 mg/kg) (Vogler, 2006) and killed by decapitation. Blood samples were collected by cardiac puncture. Whole blood was directed to glycated hemoglobin (HbA1C) assay using STANBIO kit (San Antonio-Texas, USA). The remaining blood was centrifuged at 2000  g for 15 min after 30 min of collection and stored at  80 1C until assayed. Serum glucose level was estimated enzymatically using SPINREACT diagnostics kits (Spain), (TGs), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) were measured colorimetrically using assay kits from (Stanbio, Texas, USA) according to manufactured instructions. 2.9.1. Enzyme-linked immunosorbent assay for insulin Serum insulin was determined using the rat insulin ultrasensitive ELISA kit (Crystal Chem. Inc., Downers Grove, IL 60515, USA) by ELISA reader. 2.9.1.1. Calculation of insulin resistance. Insulin resistance was determined using the homeostasis model assessment index for insulin resistance (HOMA-IR) using the following formula: HOMA-IR index¼[fasting glucose (mg/dl)  fasting insulin (mU/ml)]/ 405) (Matthews et al., 1985). To assess insulin sensitivity, the revised quantitative insulin sensitivity check index (R-QUICKI)¼ 1/[log fasting insulin (mU/ml) þlog fasting glucose (mg/dl)] was used (Katz et al., 2000). 2.9.2. Determination of oxidative stress parameter and antioxidant markers in pancreas The pancreas was isolated, weighed and 100 mg was homogenized using a Teflon homogenizer (Glas Col homogenizer system, Vernon hills, USA). Homogenization was carried out as phosphate-buffered saline (pH 7.4). The homogenate was sonicated and centrifuged at 2000  g for 10 min. The supernatants were kept at –80 1C until the analysis of – oxidative stress parameter – malondialdehyde (MDA) (Preuss et al., 1998) and – antioxidant markers – reduced glutathione (GSH) (Ellman, 1970) levels as well as superoxide dismutase (SOD) (Marklund, 1992) and catalase (CATA) (Aebi, 1984) activities using a UV–visible spectrophotometer (UV-1601PC, Shimadzu, Japan).

2.9.4. Determination of liver enzymes Commercial kit purchased from BioMed Diagnostics (Oregon, USA), was used for determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphates (ALP) using UV–visible spectrophotometer (UV-1601PC, Shimadzu, Japan). 2.9.5. Measurement of Blood urea nitrogen (BUN), creatinine and total protein Creatinine and BUN were determined enzymatically using commercially available kits (Spinreact, Gerona, Spain).Total protein was determined using Diamond Diagnostic Kit (Egypt), according to the manufacturer’s protocol. Serum levels of BUN, creatinine and total protein were determined by colorimetric methods using UV–visible spectrophotometer (UV-1601PC, Shimadzu, Japan). 2.9.6. Detection of 8-OHdG in pancreatic tissue 2.9.6.1. Isolation of mitochondria. Mitochondria were extracted by differential centrifugations following (Chappel and Hansford, 1969). 100 mg of pancreatic tissue was homogenized in 0.25 M sucrose in 0.7 M Tris–HCl Buffer (pH 7.4) at 1 g tissue 9 ml of Tris–sucrose. EDTA was added to aid disruption of cells. Tissue homogenate was spun at 2500  g for 10 min to remove nuclei and unbroken cells. Supernatant fluid was decanted into centrifuge tubes and centrifuged at 10,000  g for 10 min to form primary mitochondrial pellet. Supernatant fluid was decanted and the pellet is gently resuspended in 10 ml Tris–sucrose for washing. The pellet was recentrifuged and supernatant fluid was decanted. This washing cycle was repeated several times to improve the degree of mitochondrial purity. The final mitochondrial pellet is resuspended (1 ml Tris–sucrose/1 g of original sample). 2.9.6.2. Isolation of mitochondrial DNA (mtDNA). Mitochondrial DNA (mtDNA) was isolated by using mtDNA Isolation kit (Bio Vision, USA) (Chang et al., 2002). Furthermore, mtDNA quantity and purity were determined using NanoDropTM 1000 Spectrophotometer V3.7 (Thermo Fisher Scientific Inc, Wilmington, DE, USA). 2.9.6.3. Nuclear DNA (nDNA). Genomic DNA was extracted from the pancreatic tissue using Wizards Genomic DNA Purification kit Jena Bioscience (Germany) according to the manufacturer’s instructions. The absorbance of this fraction was measured at 260 nm. Purification of DNA was determined at A260/280 ration 1:8 by using NanoDropTM 1000 Spectrophotometer V3.7 (Thermo Fisher Scientific Inc, Wilmington, DE, USA). Digest DNA using nuclease P1 (Sigma N8630 or equivalent) following the manufacturer’s instructions. One hundred mg of mtDNA and nDNA was hydrolyzed and dephosphorylated using manufacturer’s instructions. The pH was adjusted to 7.5–8.5 using I.M. Tris, one unit of alkaline phosphatase per 100 mg of DNA was added, incubated at 37 1C for 30 min, boiled for 10 min and placed on ice unit use. Samples were analyzed for detection of 8-hydroxy-2-deoxy guanosine using

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8-hydroxy-2-deoxy guanosine EIA kit (Cayman Chemical Company USA). Enzymatic digests of DNA were analyzed by competitive enzyme-linked (ELISA) using a monoclonal antibody to 8OHdG.

2.10. Statistical analysis All data were expressed as mean 7SEM and analyzed using the Statistical Package of Social Sciences (SPSS) program version 17, (Chicago, IL, USA). For all parameters, comparisons among groups were carried out using one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparisons test (Katz, 2006). All P values reported are two-tailed and Po0.05 was considered significant.

3. Results 3.1. Toxicity study The toxicity study revealed the non-toxic nature of EA, CHLOR and HEXA extracts of Urtica pilulifera at doses up to 2 g/kg. The rats did not show any drug-induced physical signs of toxicity during the whole experimental period, and no deaths were reported.

3.2. Effect of Urtica pilulifera extracts on blood glucose, %HbA1C and insulin resistance As shown in Table 1, injection of STZ after 2 weeks of HFD significantly (Po0.05) increased blood glucose and %HbA1C levels in comparison with normal control group. Although serum insulin did not change significantly in diabetic rats, the calculated HOMA-IR was significantly increased while R-QUICKI was significantly decreased as compared with normal controls (Po0.05, Table 1). Oral administration of PIO and Urtica pilulifera EA and CHLOR extracts showed a significant reduction in serum glucose, %HbA1C as well as insulin resistance in comparison with diabetic control group. The effect of Urtica pilulifera EA and CHLOR extracts on %HbA1C is significantly different from PIO effect, while the improvement in insulin resistance was comparable to the beneficial effect of PIO (Po0.05, Table 1). Notably, the Urtica pilulifera CHLOR extract did not show significant improvement in HOMA-IR compared with diabetic controls. There is no significant difference between the two extracts regarding insulin resistance over the 4-week treatment, while HEXA extract of Urtica pilulifera showed non-significant improvement in blood glucose or insulin resistance. 3.3. Effect of Urtica pilulifera extracts on serum lipid profile Diabetic rats showed a significant increase in the serum levels of TG, TC, and LDL-C (P o0.05, Table 2). On the other hand, HDL-C

Table 1 Effect of Urtica pilulifera extracts on fasting blood glucose, %HbA1C, serum insulin, calculated HOMA-IR index and R-QUICKI in diabetic rats. Groups

Blood glucose (mg/dl)

Normal Diabetic Diabetic þPIO Diabetic þEA 250 mg/kg Diabetic þEA 500 mg/kg Diabetic þCHLOR 250 mg/kg Diabetic þCHLOR 500 mg/kg Diabetic þHEXA 250 mg/kg Diabetic þHEXA 500 mg/kg

100.87 3.0 316.4 7 16.1n 188.5 7 6.6n# 217.9 7 7.9n#f 220.1 7 7.1n#f 233 7 10.5n#$f 230.1 7 11.4n#f 325 7 13.1n$ 324 7 14.8n$

HbA1c (%) 5.1 70.4 10.5 70.5n 6.0 70.4#f 8.0 70.36n#$f 8.4 70.42n#$f 8.3 70.4n#$f 8.3 70.5n#$f 10.1 70.52n$ 10.7 70.5n$

Serum insulin (lU/ml) 12.2 7 0.5 12.4 7 0.7 12.3 7 0.5 12.07 0.6 13.5 7 0.9 13.3 7 0.8 13.8 7 0.5 12.07 0.9 12.7 7 1.05

HOMA-IR

R-QUICKI

3.07 0.1 9.9 7 1.1n 6.9 7 0.3n#f 5.6 7 0.3n#f 7.2 7 0.5n#f 7.6 7 0.6nf 7.4 7 0.2nf 9.4 7 0.89n$ 9.8 7 1.1n$

2.9 7 0.02 1.97 0.01n 3.27 0.03n#f 3.27 0.02n#f 3.27 0.023n#f 3.37 0.027n#f 3.27 0.019n#f 2.2 7 0.05n$ 2.17 0.1n$

HbA1c, glycated hemoglobin; HMOA-IR index, homeostasis model assessment index for insulin resistance index; R-QUICKI, revised quantitative insulin sensitivity check index; PIO, pioglitazone; EA, ethyl acetate extract; CHLOR, chloroform extract. Rats were treated for 4 weeks. Results were expressed as mean 7 SEM and analyzed using one-way ANOVA followed by Bonferroni’s post hoc test. n

# $ f

P o0.05 compared to normal control group. Po 0.05 compared to diabetic control group. P o0.05 compared to pioglitazone group. significant compared to HEXA groups, n¼ 10.

Table 2 Effect of Urtica pilulifera extracts on serum lipid profile in diabetic rats. Groups Normal Diabetic Diabetic þPIO Diabetic þEA 250 mg/kg Diabetic þEA 500 mg/kg Diabetic þCHLOR 250 mg/kg Diabetic þCHLOR 500 mg/kg Diabetic þHEXA 250 mg/kg Diabetic þHEXA 500 mg/kg

TG (mg/dl)

TC (mg/dl)

88.0 75.7 129.5 79.3n 90.6 73.7#f 111.0 73.2n#$f 107.3 72.9n#$f 114.0 72.3n#$f 108 72.6n#$f 134.4 78.8n$ 132.9 79.4n$

98.4 74.7 142.0 710.4n 101.0 75.4#f 123.0 76.3n#f 115.2 73.8#f 125.7 75.2n$f 117.4 75.0#f 144.3 711.1n$ 143.5 710.4n$

LDL-C (mg/dl)

HDL-C (mg/dl)

90.17 4.7 123.8 7 5.0n 96.1 7 3.3#f 103.57 5.3#f 102.17 3.8#f 105.97 4.8#f 1037 3.7#f 128.6 7 5.3n$ 125.1 7 6.8n$

40.9 7 1.6 22.2 7 1.4n 36.0 7 1.4#f 28.2 7 1.7n#$f 31.5 7 1.7n#$f 27.8 7 1.6n#$f 31.0 7 0.8n#$f 23.6 7 1.6n$ 33.5 7 1.19n$

TG, triglycerides; TC, total cholesterol; LDL-C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol; PIO, pioglitazone; EA, ethyl acetate extract; CHLOR, chloroform extract. Rats were treated for 4 weeks. Results were expressed as mean 7SEM and analyzed using one-way ANOVA followed by Bonferroni’s post hoc test. n

# $ f

P o0.05 compared to normal control group. Po 0.05 compared to diabetic control group. P o0.05 compared to pioglitazone group. significant compared to HEXA groups, n¼ 10.

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was significantly reduced when compared with the values of normal control rats. Oral administration of PIO significantly decreased the blood levels of TG, TC and LDL-C while the level of HDL-C significantly increased when compared with diabetic control rats. Treatment with Urtica pilulifera EA and CHLOR extracts significantly ameliorated the abnormalities in lipid profile when compared with diabetic control rats (Po0.05, Table 2). The beneficial effect of Urtica pilulifera EA and CHLOR extracts on LDL-C level was comparable to its level in both normal control and PIO groups. Notably, the lowest dose of the Urtica pilulifera CHLOR extract (250 mg/kg) did not show significant improvement in TC level compared with diabetic controls, while HEXA

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extract of Urtica pilulifera showed non-significant change in serum lipid profile. 3.4. Effect of Urtica pilulifera extracts on pancreatic oxidative stress parameter and antioxidant markers Diabetic control rats showed a significant reduction in cellular antioxidant defense system. Pancreatic tissue – oxidative stress parameter – MAD level was significantly increased while antioxidant markers; GSH level and SOD as well as CATA activities were reduced significantly in pancreatic homogenate (Po0.05, Table 3). Administration of PIO to diabetic rats induced a

Table 3 Effect of Urtica pilulifera extracts on pancreatic MDA and antioxidant markers. Groups

MDA (nmol/mg protein )

GSH (nmol/mg protein)

SOD activity (U/mg protein)

CATA activity (U/mg protein)

Normal Diabetic Diabeticþ PIO Diabeticþ EA 250 mg/kg Diabeticþ EA 500 mg/kg Diabeticþ CHLOR 250 mg/kg Diabeticþ CHLOR 500 mg/kg Diabeticþ HEXA 250 mg/kg Diabeticþ HEXA 500 mg/kg

30.4 7 2.7 89.7 7 4.8n 34.8 7 2.8#f 52.2 7 2.8n#$f 47.4 7 2.4n#$@f 58.0 7 4.9n#$f 50.7 7 2.6n#$f 88.2 7 3.5n$ 99.1 7 8.3n$

66.77 3.6 25.67 2.4n 57.57 2.6n#f 44.57 3.1n#$f 47.77 2.9n#$f 45.07 2.9n#$f 46.77 2.5n#$f 27.07 2.02n$ 25.27 2.1n$

64.6 7 2.8 29.5 7 2.4n 59.7 7 2.9#f 46.2 7 2.4n#$f 48.9 7 2.2n#$f 45.2 7 1.9n#$f 48.6 7 1.5n#$f 29.3 7 2.4n$ 30.9 7 2.6n$

8.67 0.4 3.97 0.4n 7.67 0.3#f 5.27 0.3n#$f 5.97 0.4n#$@f 4.77 0.3n$f 5.37 0.4n#$f 4.07 0.33n$ 4.57 0.42n$

MDA, malondialdhyde; GSH, reduced glutathione; SOD, superoxide dismutase; CATA, catalase; PIO, pioglitazone; EA, ethyl acetate extract; CHLOR, chloroform extract. Rats were treated for 4 weeks. Results were expressed as mean 7SEM and analyzed using one-way ANOVA followed by Bonferroni’s post hoc test. P o 0.05 compared to normal control group. P o0.05 compared to diabetic control group. $ P o 0.05 compared to pioglitazone group. @ P o 0.05 compared to CHLOR extract 250 mg/kg. f significant compared to HEXA groups. n¼10. n

#

Fig. 1. Serum CRP (A) and pancreatic TNF-a(B)in experimental groups. Urtica pilulifera extracts ameliorated the increased levels of CRP and TNF-a in diabetic rats at the end of 4-week treatment. CRP, C-reactive protein; TNF-a, tumor necrosis factor-a; N, normal; D, diabetic; PIO, pioglitazone; EA, ethyl acetate extract; CHLOR, chloroform extract; HEXA, hexane extract. Results were expressed as mean 7 SEM and analyzed using one-way ANOVA followed by Bonferroni’s post hoc test.*P o0.05 compared to normal control group, #P o0.05 compared to diabetic control group, $P o0.05 compared to pioglitazone group, fP o 0.05 compared to HEXA groups. n¼ 10.

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significant decrease in MDA level; meanwhile it exhibited a significant increase in pancreatic GSH level, SOD and CATA activities (Po0.05, Table 3). While Urtica pilulifera CHLOR extract (250 mg/kg) had no significant effect on CATA activity, the higher dose as well as EA extract showed a significant improvement in antioxidant markers and MDA compared with diabetic controls. Additionally, Urtica pilulifera EA extract supplementation (500 mg/kg) showed a significant decrement in pancreatic MDA level and increment in CATA activity as compared with Urtica pilulifera CHLOR extract (250 mg/kg). However HEXA extract of Urtica pilulifera showed non-significant improvement in antioxidant markers or MDA.

3.5. Effect of Urtica pilulifera extracts on inflammatory markers The current data revealed that serum level of CRP and pancreatic level of TNF-a were significantly increased by 4.8and 6.2-folds, respectively, in diabetic rats as compared with normal controls. Both PIO and Urtica pilulifera EA and CHLOR extracts showed anti-inflammatory activity, as manifested by a significant decrease in serum CRP and pancreatic TNF-a levels compared with diabetic control rats (Po0.05, Fig. 1A and B). The beneficial effect of Urtica pilulifera EA and CHLOR extracts on CRP level was comparable to the effect of PIO (Po0.05, Fig. 1A and B). Notably, HEXA extract of Urtica pilulifera showed non-significant improvement in inflammatory markers.

3.6. Effect of Urtica pilulifera extracts on liver enzyme activity Feeding of the HFD for 2 weeks followed by a low dose STZ resulted in a non-significant increase in serum levels of ALT, AST and ALP compared with normal control rats at the end of the study. Urtica pilulifera extracts showed a non-significant change in these serum levels (Table 4).

Table 5 Effect of Urtica pilulifera extracts on blood urea nitrogen, serum creatinine and total protein in diabetic rats. Groups

BUN (mg/dl)

Creatinine (mg/dl)

Total protein (g/dl)

Normal Diabetic Diabeticþ PIO Diabeticþ EA 2 250 mg/kg Diabeticþ EA 500 mg/kg Diabeticþ CHLOR 250 mg/ kg Diabeticþ CHLOR 500 mg/ kg Diabeticþ HEXA 250 mg/kg Diabeticþ HEXA 500 mg/kg

23.8 7 1.3 28.8 7 1.8 27.4 7 1.5 28.4 7 1.3 28.5 7 2.2 29.0 7 1.3

1.097 0.08 1.37 0.12 1.15 7 0.08 1.37 0.11 1.16 7 0.1 1.27 0.09

6.5 7 0.5 7.6 7 0.7 6.7 7 0.5 7.2 7 0.4 7.7 7 0.5 7.1 7 0.6

28.8 7 1.7

1.37 0.09

7.6 7 0.6

23.9 7 1.2 23.0 7 1.4

1.12 7 0.1 1.16 7 0.08

6.2 7 0.55 6.7 7 0.42

BUN, blood urea nitrogen; PIO, pioglitazone; EA, ethyl acetate extract; CHLOR, chloroform extract. Rats were treated for 4 weeks. Results were expressed as mean 7SEM and analyzed using one-way ANOVA followed by Bonferroni’s post hoc test. n¼10.

3.8. Effect of Urtica pilulifera extracts on 8-OHdG contents of mtDNA and nDNA The pancreatic level of 8-OHdG contents of mtDNA and nDNA was significantly increased in diabetic rats as compared with controls (mtDNA; 6.72 70.3 vs 1.5770.1 and nDNA; 0.7470.004 vs 0.1670.008, Po0.05, Fig. 2A and B). Level of 8-OHdG in pancreatic mtDNA and nDNA was increased significantly upon treatment with PIO compared with diabetic control rats which indicates its beneficial effect in oxidative damage. On the other hand, Urtica pilulifera EA and CHLOR extracts significantly decreased mtDNA and nDNA 8-OHdG contents in the pancreas compared with diabetic control rats (Po0.05, Fig.2A and B), while HEXA extract resulted in non-significant decrease in pancreatic 8-OHdG contents of mtDNA and nDNA in diabetic rats.

4. Discussion 3.7. Effect of Urtica pilulifera extracts on BUN, serum creatinine and total protein At the end of study period, type2 diabetic rats showed a nonsignificant deterioration in renal function as manifested by a nonsignificant increase in BUN, serum creatinine and total protein as compared with normal controls. Oral administration of Urtica pilulifera extracts did not show any significant change in these serum levels (Table 5).

Table 4 Effect of Urtica pilulifera extracts on liver enzymes in diabetic rats. Groups

Serum ALT (U/L)

Serum AST (U/L)

Serum ALP (U/L)

Normal Diabetic Diabetic þPIO Diabetic þEA 250 mg/kg Diabetic þEA 500 mg/kg Diabetic þCHLOR 250 mg/kg Diabetic þCHLOR 500 mg/kg Diabetic þHEXA 250 mg/kg Diabetic þHEXA 500 mg/kg

16.5 71.0 18.1 70.5 17.5 70.7 16.8 70.7 17.3 70.8 16.4 70.9 17.6 70.8 18.5 70.34 17.5 70.68

71.2.7 2.0 74.27 2.6 75.97 2.2 78.57 2.9 75.37 2.0 77.07 1.8 71.97 3.5 73.07 2.7 75.57 2.6

72.5 72.0 74.9 73.1 77.9 71.9 76.0 71.9 78.9 72.9 81.0 73.8 78.3 73.3 75.5 73.0 75.1 73.1

ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; PIO, pioglitazone; EA, ethyl acetate extract; CHLOR, chloroform extract. Rats were treated for 4 weeks. Results were expressed as mean 7SEM and analyzed using one-way ANOVA followed by Bonferroni’s post hoc test. n¼ 10.

The current experiment was designed to evaluate the potential effects of Urtica pilulifera extracts as hypoglycemic agents as well as their effects on pancreatic oxidative stress and inflammation in high fat diet, low dose STZ -induced type2 diabetic rats. Firstly, the present results showed non-significant differences in the serum levels of ALT, AST and ALP in all studied groups in comparison to normal control group. Moreover, our data showed non-significant changes in BUN, serum creatinine and total protein. These results go along with the previous report, which indicated that methanol extract of Urtica pilulifera did not induce increase in hepatic enzymes (Irshaid and Mansi, 2009b). Therefore, it is possible to suggest that these extracts are safe and did not have hepatocellular or renal damaging effect in diabetic rats. Our current data indicated that a combination of HFD followed by a low dose of STZ resulted in a significant increase in blood glucose level and insulin resistance in a manner reported in literature (Hininger-Favier et al., 2009). The insulin resistance can be explained mainly through glucose–fatty acid cycle. Briefly, the presence of high level of triglycerides due to excess fat intake could constitute a source of increased fatty acid bioavailability and oxidation which blunts the insulin-mediated reduction of hepatic glucose output and reduces glucose uptake or utilization in skeletal muscle leading to insulin resistance (Tanaka et al., 2007). Apart from glucose, the diabetic rats of the current study showed abnormalities in lipid metabolism as evidenced by significantly increased TG, TC and LDL-C levels associated with

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Fig. 2. Pancreatic 8-OHdG content in mtDNA (A) and nDNA (B) in experimental groups. Urtica pilulifera extracts decreased both pancreatic 8-OHdG content in mtDNA and nDNA significantly compared to diabetic control rats at the end of 4-week treatment. mtDNA, mitochondrial DNA; nDNA, nuclear DNA; 8-OHdG, 8-hydroxy2-deoxyguanosine; N, normal; D, diabetic; PIO, pioglitazone; EA, ethyl acetate extract; CHLOR, chloroform extract; HEXA, hexane extract. Results were expressed as mean 7 SEM and analyzed using one-way ANOVA followed by Bonferroni’s post hoc test.*P o0.05 compared to normal control group, #P o 0.05 compared to diabetic control group, $Po 0.05 compared to pioglitazone group, fPo 0.05 compared to HEXA groups. n¼ 10.

decreased HDL-C, as in case of human type2 diabetic patients which might contribute to various cardiovascular complications. Hypercholesterolemia may be attributed to increased dietary cholesterol absorption following the intake of HFD in a diabetic condition (Singala et al., 2009). The present study showed also a significant increase in lipid peroxidation and decrease in the antioxidant enzyme activity of GSH, SOD and CATA in agreement with (Coskun et al., 2005) study. These changes are due to functional defects of b-cells in diabetes which are associated with multiple functional and molecular alterations and increased oxidative stress (Obrosova, 2005). Additionally, mtDNA and nDNA 8-OHdG content increased significantly in diabetic group which indicates an increase in the degree of oxidative stress affecting tissue function and integrity, and therefore provides useful information on oxidative stress and disease progression (Al-Aubaidy and Jelinek, 2011). The current data revealed that serum level of CRP and pancreatic level of TNF-a were significantly increased in diabetic rats. It has been reported that TNF-a induces insulin resistance in pancreatic cells by interfering with insulin signaling (Duan et al., 2007). Administration of PIO for 4 weeks was able to reduce the blood glucose and improve insulin resistance and dyslipedemia, indicating its potent anti-hyperglycemic and hypolipidemic activity, without significant alteration in the insulin level. This can be explained according to its mechanism of action through improving whole body insulin sensitivity rather than stimulating the beta cell insulin secretion (Srinivasan et al., 2005). Furthermore, the attenuating effect of PIO on hyperlipidemia might result either from the inhibition of TG synthesis in liver or increased TG clearance in the periphery by stimulating the enzyme

lipoprotein lipase and/or inhibition of dietary cholesterol absorption from the intestine (Srinivasan et al., 2004). Moreover, PIO can reduce oxidative stress, and that it contributes to reduction of susceptibility of LDL-C to oxidation as well as decrease in TNF-a serum level which result in improvement of b-cell insulin resistance (Iida et al., 2003). In agreement with (Golalipour and Khori, 2007), the results of the current study indicated that administration of E.A and CHLOR extracts of Urtica pilulifera aerial parts leaves lowered blood glucose significantly in diabetic rats. This coincides with the traditional use of Urtica pilulifera in folk medicine as an antihyperglycemic agent (Chrubasik et al., 2007). The hypoglycemic effects of Urtica pilulifera confirm the results of (Kavalali et al., 2003), who assumed that Urtica pilulifera seed lectin directly inhibited STZ by competing with STZ for glucose associated receptors on beta cells membrane. Plant lectins, in addition to their metabolic effect, mimic insulin actions by interacting with the glycoprotein residues of the insulin receptor. In addition, (Irshaid and Mansi, 2009a) reported the possible utilization of Urtica pilulifera to prevent the development of diabetes in later life and improved the performance of male reproductive system in animals and humans. Hyperlipidemia is directly linked to insulin resistance as high lipid concentrations in blood secrete humoral factors such as resistin and adiponectin that alter insulin sensitivity, leading to insulin resistance (Trujillo and Scherer, 2006) so the hypolipedemic effect of Urtica pilulifera extracts in the current study may contribute to improvement of insulin resistance. Our study demonstrated also that Urtica pilulifera showed a marked effect on antioxidant enzymes. These findings are

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coincided with (Ozen et al., 2010) who used hydroalcoholic herb extract of Urtica dioica (same species of Urtica pilulifera). In addition, a study reported that methanol extract of Urtica pilulifera induced reduction of lipid peroxidation and increased activity of antioxidant enzyme systems (Mahmoud et al., 2006). So, it is possible to suggest that reduction of islet cell oxidative stress was accompanied by improved secretory capability and this could be a target for therapeutic approaches. Moreover, Urtica pilulifera extracts supplementation exhibits a significant reduction in 8-OHdG content in pancreatic mtDNA and nDNA. Additionally, this can be explained by the improvement action of the Urtica pilulifera extracts on hyperglycemia & and hyperlipidemia (Al-Aubaidy and Jelinek, 2011). These findings could be supported by the previously reported activities proved for the major active compounds isolated from this plant. Investigation of the CHLOR extract led to the isolation and identification of three compounds namely: b-sitosterol, b-amyrin and ursolic acid. Meanwhile, investigation of the EA extract afforded two major compounds identified as b-sitosterol-3-O-glucoside and caffeic acid. All these five compounds are known compounds previously isolated from plants. Reviewing the previously reported activities of these compounds, it was found that all of them exhibit significant anti-inflammatory effect (Holanda et al., 2008; Ikeda et al., 2008; Loizou et al., 2010). Moreover, both ursolic acid and caffeic acid demonstrated also antioxidant activity (Gulcin, 2006; Ramachandran and Prasad, 2008; Chao et al., 2010). These findings can also justify the current results reported with Urtica pilulifera. However, it is also important to note that presence of these bioactive compounds may contribute to the antidiabetic activity of Urtica pilulifera resulting in an increase in glucose utilization and metabolism in peripheral tissues that may lead to resolution of the other down-stream pathologic effects as reported by (Kavalali et al., 2003). However, additional experiments will be needed to characterize the details of the mechanism(s) by which Urtica pilulifera normalizes the glucose level and/or affects the function and growth of pancreas in diabetic rats.

In conclusion The present study indicated that EA and CHLOR extract of Urtica pilulifera exhibited antioxidant and anti-inflammatory effects in diabetic rats. These activities are responsible, at least partly, for improvements that have been seen in hyperglycemia and insulin resistance of diabetic rats. The precise mechanism of these effects remains to be elucidated.

Declaration of interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding Natural products from Egyptian plants with hypoglycemic effect. Principal investigator for the Egyptian side: Dr. Ghada Hadad. Awarded by the MENA – Swedish Research Links Program. References Abudoleh, S., Disi, A., Qunaibi, E., Aburjai, T., 2011. Anti-Arthritic Activity of the Methanolic Leaf Extract of Urtica pilulifera L. on Albino Rats. American Journal of Pharmacology and Toxicology 6, 27–32.

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