Neuroprotective effect of 5, 7, 3\', 4\', 5\'-pentahydroxy dihdroflavanol-3-O-(2\'\'-O-galloyl)-[beta]-d-glucopyranoside, a polyphenolic compound in focal cerebral ischemia …

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European Journal of Pharmacology 626 (2010) 205–212

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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Neuropharmacology and Analgesia

Neuroprotective effect of 5,7,3′,4′,5′-pentahydroxy dihdroflavanol-3-O-(2″-O-galloyl)-β-D-glucopyranoside, a polyphenolic compound in focal cerebral ischemia in rat Rathinam ArunaDevi a, Suman Lata b, Brijesh K. Bhadoria b, Vinod D. Ramteke a, Saurabh Kumar a, Palanisamy Sankar a, Dinesh Kumar a, Surendra K. Tandan a,⁎ a b

Division of Pharmacology and Toxicology, Indian Veterinary Research Institute, Izatnagar-243 122 (U.P.), India Division of Plant Animal Relationship, Indian Grassland Fodder Research Institute, Jhansi (U.P.), India

a r t i c l e

i n f o

Article history: Received 5 December 2008 Received in revised form 11 September 2009 Accepted 14 September 2009 Available online 25 September 2009 Keywords: Focal cerebral ischemia Ischemia/reperfusion injury Oxidative stress Free radical Polyphenolic compound Apoptosis Cyclooxygenase Nitrite

a b s t r a c t Ischemia/reperfusion injury ends up in the cascade of excitotoxic stimulation of superoxide and nitric oxide formation leading to the generation of highly reactive products, including peroxinitrite and hydroxyl radical, which are capable of damaging lipids, proteins and DNA. Several polyphenolic compounds scavenge the radicals and protect from injury. 5,7,3′,4′,5′-pentahydroxy dihdroflavanol-3-O-(2″-O-galloyl)-β-D-glucopyranoside (AP1), a polyphenolic compound, isolated from Anogeissus pendula Edgew was tested for its neuroprotective effect in transient focal cerebral ischemia in rats. Transient focal cerebral ischemia was produced by middle cerebral artery occlusion for 2 h for studying infarct volume, brain edema, apoptosis and oxidative stress. AP1 was tested for in vitro protection from glutamate and hydrogen peroxide-induced damage to Neuro-2a cells by MTT assay. It was also tested for its in vitro antioxidant, lipid peroxidation inhibition, NO scavenging and cyclooxygenase inhibitory activities. AP1 treatment (30 mg/kg i.p.) before reperfusion injury (0 h) significantly reduced the infarct volume, cerebral edema, number of apoptotic cells in penumbra and neurobehavioural abnormality score and lipid peroxidation, protein carbonyl levels and total thiols in brain. Increased catalase activity and NOx levels in ischemic animals were significantly reduced by AP1 treatment. AP1 (3 µg/ml) protected Neuro-2a cells to H2O2 and glutamate-induced damage. In in vitro studies, AP1 was found to possess reducing and NO scavenging activities. It also reduced lipid peroxidation and inhibited cyclooxygenase activity (cyclooxygenase-1 and cyclooxygenase-2). AP1 can be used as a neuroprotective agent in stroke as it reduced apoptosis and found to be a good antioxidant and anti-inflammatory compound. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Acute ischemic stroke is the third leading cause of death after heart disease and cancer and major cause of disability in ageing adult (Bronner et al., 1995; De Freitas and Bogousslavsky, 2001). The pathophysiological processes in stroke are extensively diverse and dependent on the severity, duration and localization of ischemic damage in the brain. Focal cerebral ischemia can be transient or permanent and usually produced by occlusion of middle cerebral artery in laboratory animals, which lead to rapid cell death in ischemic core. In the periphery of ischemic region, blood flow reduction is less marked and neurons in this area are referred to as penumbra. Polyphenols are the natural substances with variable phenolic structures and enriched in vegetables, fruits, grains, bark, roots, flowers, seeds, tea and wine (Bravo, 1998). They are generally known

⁎ Corresponding author. Fax: +91 581 2303284. E-mail address: [email protected] (S.K. Tandan). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.09.038

to possess potent antioxidant properties (Rice-Evans et al., 1995; 1997; Sun and Chen, 1998). Studies examining the health benefits of polyphenols have been focused on heart diseases and cancers. Polyphenols have been implicated in the prevention of neurodegenerative diseases. Polyphenols protect neurons against oxidative damage and attenuate ischemia/reperfusion injury by interfering with inducible nitric oxide synthase (iNOS) activity, inhibiting the lipid peroxidation, decreasing the number of immobilized leukocytes during reperfusion which result in diminished inflammatory response (Nijveldt et al., 2001). Epidemiological studies have found that a high consumption of fruits and vegetables can lower the risk of ischemia and stroke (Acheson and Williams 1983; Vollset and Bjelke, 1983; Joshipura et al., 1999). More and more studies have started evaluating antioxidant properties of polyphenols from diverse natural products, with focus on their therapeutic actions as neuroprotectants against cerebral ischemia. Despite aggressive research in developing neuroprotective effect in brain injury by use of polyphenols as a dietary substance, no drug proved to be successful in advance clinical stage. No effort has ever been made to test the polyphenols isolated from


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plants used as fodder for animals. 5,7,3′,4′,5′-pentahydroxy dihdroflavanol-3-O-(2″-O-galloyl)-β-D-glucopyranoside (the compound hereafter referred as AP1) (98% purity) with the following structure (C28H26O17) (Fig. 1): is a natural polyphenol isolated from Anogeissus pendula Edgew (family; Combretaceae); local name is Kardhii. The plant is abundant in Bundelkhand region of U.P. (India) and used as fodder and fuel. The present study was undertaken to examine the neuroprotective effect of AP1 in neuronal damage both in vivo and in vitro.

2. Materials and methods 2.1. Drugs and chemicals Glutamate and dimethyl sulphoxide DMSO were procured from Merck India Ltd. 2,3,5-Triphenyltetrazolium chloride was procured from Sigma Aldrich USA. TUNEL, kit was procured from Promega USA and cyclooxygenase (COX) inhibitor assay kit from Cayman Chemical Company USA. AP1 was isolated from A. pendula Edgew. Other chemicals used in this study were of analytical grade.

2.2. Experimental animals Adult Wistar rats (250–300 g) were procured from the Laboratory Animal Resource Section of the Indian Veterinary Research Institute, Izatnagar and maintained at room temperature of 22 ± 2 °C. During the period of experimentation, all the animals had free access to balanced feed supplied by the Feed Technology Unit of IVRI, Izatnagar and drinking water. All the experiments were conducted as per the guidelines of the Institute Animal Ethics Committee.

2.3. Drug administration AP1 was dissolved in DMSO. Six animals were used per group unless otherwise indicated for studying the neuroprotective effect of AP1. The compound (30 mg/kg) was administered immediately before reperfusion (0 h) by intraperitoneal route based on the exploratory study. In the exploratory experiment 10 and 30 mg/kg doses of AP1 were employed for studying the effect on infarction volume. Since 10 mg/kg dose did not reduce infarction volume significantly, the remaining studies were conducted with 30 mg/kg dose. Other groups included sham-operated control and vehicletreated ischemic control.

Fig. 1. Chemical structure of 5,7,3′,4′,5′-pentahydroxy dihdroflavanol-3-O-(2″-Ogalloyl)-β-D-glucopyranoside, a natural polyphenol.

2.4. Experimental production of reversible focal cerebral ischemia in rat Middle cerebral artery occlusion method was employed for transient focal cerebral ischemia and reperfusion injury by the method of Smrcka et al. (2001). A brief description is furnished below. Half an hour before surgery, rats were pre-medicated with atropine (1 mg, s.c.) and anaesthetised by injecting ketamine (100 mg/kg i.p.) and xylazine (15 mg/kg i.m.).Transient focal cerebral ischemia was induced by middle cerebral artery occlusion as described by Smrcka et al. (2001). The right common carotid artery was exposed with careful protection of the vagus nerve. The internal and external carotid arteries were separated from adhering tissue and nerve. External carotid artery was ligated close behind carotid bifurcation and temporary clips were applied to common carotid artery and external carotid artery. An incision was made into external carotid artery near bifurcation to insert poly lysine coated nylon monofilament (4-0). Monofilament was advanced up to 18 to 25 mm from bifurcation according to the size of the animal. When 2 h of ischemic phase was terminated, monofilament was pulled out to resume the blood flow in middle cerebral artery and incision was closed in layers. In sham-operated group, the filament was introduced into external carotid artery but not advanced. Rectal temperature was monitored throughout surgery and maintained at 37 ± 1 °C by placing heated electric pad under the rat. After 2 h of occlusion and 72 h of reperfusion injury, rats were evaluated for neurological abnormality score after which they were sacrificed, brains were taken for measurement of infarction volume, water content, TUNEL assay, etc. 2.5. Measurement of experimental cerebral infarction, brain water (cerebral edema) and neurological examination The method of Bederson et al. (1986a) was followed using 2,3,5triphenyltetrazolium chloride. After 72 h of reperfusion, rats were anaesthetised and perfused with ice-cold normal saline intracardially. Within 3 min of sacrificing the rats, brains were removed and 2 mm thick coronal sections were cut. Sections were immersed in 2% 2,3,5triphenyltetrazolium chloride, incubated for 20 min at 20 °C, and then placed in formalin overnight in the dark. The striatal and cortical areas of infarction (unstained tissue) of each section were scanned. The area of infarction was determined by differential weighing of cut out areas of the tracing. Likewise, hemispheric volume was also measured. Infarction and hemispheric areas (mm2) were quantified to provide subsequent determination of infarct character such as, the percentage of volume of cerebral tissue representing infarction (percentage of hemispheric infarction). Total core infarct volume where core injury was defined as brain tissue completely lacking TTC staining, was calculated by sequential integration of the respective surface areas. Whole cerebral volume was also quantified in a similar way. Brain samples were immediately weighed on an electronic analytical balance to obtain the wet weight and then dried in an oven at 100 °C for 24 h to obtain the dry weight and the water content was determined as (Wet weight − Dry weight)/Dry weight. To evaluate the sensory motor function, the postural reflex test developed by Bederson et al. (1986b) was used with little modification. Neurological examination was performed on each rat immediately after recovery from anaesthesia after middle cerebral artery occlusion and at 24, 48 and 72 h of reperfusion, to assess the change in neurobehavioral abnormality and average of the score was taken for comparison between groups. For the present study, those animals showing neurobehavioral abnormality after recovery from anaesthesia were selected for the experiments. In brief, the scoring was done by suspending the rats 20 cm above the ground. Intact animals extended both the forelimbs towards the floor. These animals were scored 0. Abnormal posture included flexing the contra lateral limb towards the body and/or rotating the contra lateral shoulder and limb medially. If the abnormal posture was

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observed, the rat was placed on a sheet of soft plastic-backed paper that could be gripped by its claws. Lateral pressure was applied from behind the shoulders, so that the forelimbs slid gently to the left and then to the right. Rats that resisted sliding in both directions were graded as 1, more severely affected animals exhibiting a decreased resistance to the lateral push were scored as 2, and those that circled towards the paretic side consistently were graded as 3. 2.6. Physiological parameters Physiological parameters were measured at 1 h after AP1 (30 mg/kg) or vehicle treatment. Femoral artery blood pressure was measured using invasive blood pressure and heart rate recording technique, with the help of high-sensitivity blood pressure transducer and recorded in PC using Chart V5.4.1 software programme (Powerlab, AD Instruments, Australia). Blood glucose, hemoglobin and haematocrit were measured by standard protocols. 2.7. TUNEL assay Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was done to identify the extent of DNA fragmentation, as previously reported (Kabra et al., 2004). Three groups for AP1 (sham, control and treatment), each containing three rats were used for the TUNEL assay. After 72 h of reperfusion, rat was anaesthetised and 200 ml of ice-cold phosphate-buffered saline was transcardially perfused, followed by 500 ml of 4% buffered paraformaldehyde in saline (pH 7.4) for in situ fixation of the brain. The brain was isolated, dehydrated and embedded in wax. Serial sections of 4 µm thickness were cut using microtome (Leica, Germany) and spread over microscopic slides. The sections at the level of anterior commissure, and temporal lobe (0.26 to 0.51 mm anterior to bregma), were selected for TUNEL assay. Labeled slides bearing sections were deformalinized by treating the slides with the mixture of 50 ml 95% alcohol and 15 ml ammonia overnight. Later, the slides were washed in running tap water for half an hour to 1 h. The DeadEnd™ Colorimetric TUNEL System end labels the fragmented DNA of apoptic cells using a modified TUNEL assay. Biotinylated nucleotide is incorporated at the 3′ –OH DNA ends using the Terminal Deoxynucleotidyl transverase, Recombinant, (rTdT) enzyme. Horse radish peroxidase-labeled streptavidin (Streptavidin HRP) is then bound to these biotinylated nucleotides, which are detected using the peroxidase substrate, hydrogen peroxide and the stable chromogen, diaminobenzidine (DAB). Using this procedure, apoptotic nuclei are stained dark brown and normal nuclei will not take any stain. Manufacturer's standard procedure was followed (DeadEndTM Colorimetric TUNEL System (Promega product Cat. No. G7132). The TUNEL positive cells were observed in cortical and subcortical regions under light microscope in a high power objective in measured areas and photographed using a digital camera. The numbers of TUNEL positive cells were counted using an image analysis software Leica Quin (Leica, Germany) in randomly selected measured areas in five different fields and mean was taken and represented as number of cells/mm2. 2.8. Oxidative stress parameters A 6 mm thick coronal section was dissected out at the level of anterior commissure of the brain. The infarct tissue, including both cortex and striatum was isolated from the right hemisphere. The tissue was frozen and stored at − 20 °C for subsequent analysis (Parmentier et al., 1999). Brain homogenates were prepared by homogenizing the brain tissue in an ice-cold buffer using a homogenizer. Homogenization buffer contained 100 mM Tris–HCl (pH 7.4) and 0.05 mM EDTA. Homogenates were centrifuged at 10,000 ×g at −4 °C for 10 min and used in for biochemical assays or


stored − 20 °C for subsequent analysis. The total protein concentration in the brain homogenate was determined using the modified Lowry protein assay with bovine serum albumin (BSA) as a standard. This supernatant was used for biochemical estimation for oxidative stress. In order to evaluate the pro-oxidant–antioxidant balance, we determined the free radical production by measuring the level of lipid peroxidation (Shafiq-ur-Rehman, 1984) and protein carbonyl (Raghuramulu et al., 2003). Total thiol (Sedlak and Lindsay, 1968) was measured as nonenzymatic antioxidant and activity of some enzymatic antioxidants like catalase (Bergmeyer, 1983) and glutathione peroxidase (Paglia and Valentine, 1967) were also measured in brain homogenate of AP1-treated groups. Nitrate/nitrite estimation (NOx) in brain homogenate was done as described by Cortas and Wakid (1990). 2.9. Neuroprotective studies in neuroblastoma Neuro2a cells Mouse neuroblastoma Neuro2a cells were procured from NCCS Pune, India and maintained in modified Eagle's medium supplemented with 10% (v/v) heat-inactivated fetal calf serum and nonessential amino acids. All cultured cells were maintained at 37 °C in 5% CO2/95% air. In a 96 well plate cells were seeded at the concentration of 2 × 105 cells/ml in growth medium and 100 µl per well. After 48 h of incubation, 10 µl of protective agent (1 and 3 µg/ml) was added. Thirty min after the treatment, 10 µl of damaging agent (10− 4 M H2O2 and/or1mM glutamate) was added to the cells in each well. Then, the cells were incubated for 24 h. Damage assessment for morphological changes was done by ethidium bromide and acridine orange apoptosis assay (Zhi-Jun et al., 1997) and quantified by mitochondrial tetrazolium (MTT) assay (Denizot and Lang, 1986). 2.10. In vitro antioxidant potential The in vitro antioxidant potential, evaluated by iron reduction, ascorbate iron (III) catalyzed phospholipid peroxidation and nitric oxide scavenging activities were also determined. The ability of the AP1 to reduce iron (III) was assessed by the method of Oyaizu (1986) with certain modifications. Different volumes (20, 80, 160 and 320 µl) of AP1 (30 mg/ml) were mixed with 0.980, 0.920, 0.840 and 0.680 ml of phosphate buffer (0.2 M, pH 6.6) to make the final volume up to 1 ml and to this, 0.1 ml of 1% aqueous potassium hexacyanoferrate [K3Fe(CN)6) solution was added. After 30 min of incubation at 50 °C, the aliquot (0.5 ml) was drawn and 0.5 ml water and 0.1 ml of 0.1% aqueous FeCl3 were added. Ascorbic acid was used as a standard (20, 80, 160, and 320 µl). The concentration of stock solution was 2 mM. The absorbance was recorded at 700 nm. Reducing activity was assessed by plotting the graph of absorbance against concentration. An increase in absorbance indicates reducing power. The ability of AP1 to scavenge hydroxyl radicals was determined by the method of Shinde et al. (2006). Mouse liver sample was mixed (1:10) with 10 mM phosphate-buffered saline (PBS, pH 7.4) and sonicated in an ice bath for preparation of homogenate. The homogenates (0.2 ml) were combined with 0.5 ml of PBS buffer, 0.1 ml of 1 mM FeCl3 and 0.5 ml solution of varying concentrations (60, 120, 240 and 480 µg/ml) of AP1. Peroxidation was initiated by adding 0.1 ml of 1 mM ascorbic acid. The mixture was incubated at 37 °C for 60 min. The reaction was stopped by adding 1 ml of 10% trichloroacetic acid in the tube. The tube was centrifuged at 2000 rpm for 10 min. After centrifugation, 1 ml of 0.67% 2-thiobarbituric acid (TBA) in 0.05 M NaOH was added to the supernatant of the tube. The sample was vortexed and heated in a water bath at 100 °C for 20 min. After cooling, 1 ml of distilled water was added. Reading was taken at 532 nm. The percentage inhibition was calculated, where the control contained all the reaction reagents except extract.


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Nitric oxide scavenging activity of AP1 was measured by spectrophotometer (Pandey et al., 2005). Sodium nitroprusside (5 mM) in phosphate-buffered saline was mixed with a control without the test compound, but with an equivalent amount of methanol. AP1 at a concentration of 10, 30 or 100 µg/ml was dissolved in methanol and incubated at 25 °C for 30 min. After 30 min, 1.5 ml of the incubated solution was removed and diluted with 1.5 ml of Griess reagent (1% sulphanilamide, 2% phosphoric acid, and 0.1% naphthylethylenediamine dihydrochloride). The absorbance of the chromophore formed during the diazotization of the nitrite with sulphanilamide and the subsequent coupling with naphthylethylenediamine dihydrochloride was measured at 546 nm. 2.11. Cyclooxygenase assay COX inhibitory screening assay directly measures PGF2α produced by SnCl2 reduction of COX-derived PGH2. The prostanoid product is quantified via ELISA immunosorbent assay (EIA) using a broadly specific antibody that binds to all the major prostaglandin compounds. Assay was done as per the COX inhibitory screening assay manufacturer's instruction (Caymen Chemical Cat. No. S60131). 2.12. Statistical analysis The data obtained from various experiments were compared by two way ANOVA with Bonferroni's correction and one way ANOVA followed by Tukey's multiple comparison's test using graph pad prism. Differences were considered significant if P < 0.05. 3. Results 3.1. Effect of AP1 on infarct volume, brain edema and neurobehavioral abnormality of transient focal cerebral ischemia rats Injection of AP1 (30 mg/kg) at 0 h of reperfusion injury, reduced the % infarct volume (7.22 ± 1.22) significantly (P < 0.01), as compared to vehicle control value of 13.95 ± 1.53 (Fig. 2A). AP1 at 10 mg/kg did not reduce infarction volume. The brain water content of shamoperated rats was 3.47 ± 0.03 g/g dry weight which was significantly increased to 4.54 ± 0.02 in ischemic vehicle control. AP1 treatment significantly reduced the brain water content in comparison to vehicle control (Fig. 2B). AP1 treatment at 0 h of reperfusion injury, significantly (P < 0.05) reduced the neurobehavioral abnormality score from 2.83 ± 0.11 to 2.15 ± 0.23 (Fig. 2C). 3.2. Effect of AP1 on physiological parameters In vehicle-treated control, the blood pressure, heart rate, blood glucose, haematocrit and hemoglobin values were 120 ± 1.2 mmHg, 396 ± 18/min, 140.3 ± 10.3 mg/dl, 42.23 ± 1.36% and 11.38 ± 1.2 g/dl, respectively. AP1 treatment did not result in any significant change in blood pressure (118 ± 2.1 mmHg), heart rate (406 ± 12/Min), blood glucose (136.6 ± 9.8 mg/dl), haematocrit (43.141 ± 1.41%) and hemoglobin (10.58 ± 0.52 g/dl). 3.3. Effect of AP1 on oxidative stress parameters Table 1 summarizes the oxidative stress parameters, following AP1 injection before reperfusion injury (0 h) in brain tissue homogenate. Lipid peroxidation increased significantly in vehicle-treated control animals (0.057 ± 0.006 n mole/mg tissue), than sham-operated animals (0.004 ± 0.0009 n mole/mg tissue). AP1 treatment at 0 h, significantly (P < 0.01) reduced the amount of thiobarbituric acid reactive substance to 0.02 ± 0.003 n mole/mg tissue.

Fig. 2. Effect of AP1 (10 and 30 mg/kg) on infarct volume (A), brain water content (B) and neurobehavioural abnormality score (C) measured 72 h after transient focal cerebral ischemia in rats. Ischemic rats were treated with AP1 or vehicle. The figure (A) represents the % infarct volume of serial brain sections stained by TTC. The figure (B) represents the water content (g/g of dry weight) of the brain tissue. Neurological score was assessed at 24, 48 and 72 h of cerebral ischemia and mean score was calculated. The figure (C) represents the neurobehavioural abnormality score on a scale of 0–3. The data are expressed as mean ± S.E.M.; *P < 0.05, ***P < 0.001 in comparison to vehicle control; #P < 0.001 in comparison to sham. n = 6 animal in each group.

The amount of protein carbonyl formed is the direct indication of oxidative damage to protein and amino acids and was found to be increased in vehicle-treated control animals (60.69 ± 12.47 n mole/ mg protein) from 13.09 ± 5.61 n mole/mg protein in sham-operated animals. AP1 treatment significantly (P < 0.01) decreased the protein carbonyl to 18.26 ± 4.35 n mole/mg protein in comparison to vehicletreated control animals. Total thiol in the brain of sham-operated animals was 13.82 ± 2.61 n mole/mg tissue which reduced in ischemic vehicle-treated animals, to 0.85 ± 0.25 n mole/mg tissue. In AP1-treated animals, the thiol level elevated to 2.75 ± 0.16, but not to that of sham-operated animals. Catalase activity in the brain increased to the level of 137.71 ± 27.59 catalase units/mg protein in vehicle-treated transient focal cerebral ischemia rats significantly, than in sham-operated animals (40.64 ± 10.18 units/mg protein). In AP1-treated animals, the catalase activity decreased significantly (P < 0.05) to 35.71 ± 8.722 catalase units/mg protein. Glutathione peroxidase activity was found to be rich in shamoperated animals (103.51 ± 18.28 units/mg protein) than the transient focal cerebral ischemia rats, where the activity was 49.68 ± 4.94

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Table 1 Effect of AP1 on oxidative stress parameters of transient focal cerebral ischemia ratsn. Treatment

Dose mg/kg (i.p.)

Lipid peroxidation n mole/mg tissue

Protein carbonyl n mole/mg protein

Total thiol n mole/mg tissue

Catalase Units/mg protein

Glutathione peroxidase units/mg protein

Total nitrites (NOx) n mole/mg tissue

Sham-operated control Vehicle control AP1

– – 30

0.004 ± 0.0009 0.057 ± 0.006b 0.02 ± 0.003d

13.09 ± 5.61 60.69 ± 12.47b 18.26 ± 4.35d

13.82 ± 2.61 0.85 ± 0.25b 2.75 ± 0.16c

40.64 ± 10.18 137.71 ± 27.59a 35.71 ± 8.722e

103.51 ± 18.28 49.68 ± 4.94b 39.73 ± 9.16

5.49 ± 0.30 50.82 ± 8.02b 9.31 ± 4.92d

n = 6; aP < 0.05; bP < 0.01 in comparison to sham-operated animals; cP < 0.05; dP < 0.01; eP < 0.001 in comparison to ischemic vehicle-treated control.

units/mg protein. AP1 treatment did not improve the activity of glutathione peroxidase (39.73 ± 9.16 units/mg protein) in transient focal cerebral ischemia rats. Total nitrites (NOx) increased in transient focal cerebral ischemia rats to 50.82 ± 8.02 n mole/mg brain tissue than in sham animal's brain (5.49 ± 0.30 n mole/mg tissue). In AP1-treated animals, the amount of total nitrite level reduced significantly (9.31 ± 4.92 n mole/ mg tissue) than the vehicle-treated control animals.

produced 30.40 ± 4.7% cell viability. AP1 (1 µg/ml) and (3 µg/ml) improved nonsignificantly and significantly (P < 0.01) the viability percentage to 47.16 ± 9.20 and 79.22 ± 9.84, respectively (Fig. 4E). Sodium glutamate at a concentration of 1 mM reduced the viability of the cells to 16.00 ± 2.74%. Simultaneous treatment with AP1 at 1 and 3 µg/ml doses improved the viability to 47.16 ± 9.20 and 44.01 ± 2.28%, respectively (Fig. 4F). 3.6. In vitro antioxidant activity of AP1

3.4. Effect of AP1 on apoptosis Apoptotic nuclei were confirmed by specific staining pattern (Fig. 3C) of penumbral sections of the brain. The number of apoptotic cells in the penumbral tissue was significantly reduced (P < 0.01) in comparison to vehicle treatment. There was no difference between ischemic control group and vehicle-treated group (Fig. 3D). 3.5. Effect of AP1 on H2O2 and glutamate-induced damage on Neuro-2a cells In vitro cell morphology was studied for damage and protective effect by AP1 by ethidium bromide/acridine orange staining pattern (Fig. 4). Green stained nuclei indicated live cells and red stained nuclei indicated dead cells. Fragmented nuclei indicating apoptosis were stained red or green. Apoptotic and dead cells were more in number in damage induced by H2O2 (Fig. 4A and B) and sodium glutamate (Fig. 4C and D). Hydrogen peroxide at a concentration of 10− 4 M

In vitro reducing activity of AP1 was tested on the ability of the compound to convert iron III to iron II and its spectrometric measurement at 700 nm. AP1 activity is less than that of ascorbic acid as evident from Fig. 5A. Ascorbic acid 2 mM was used as standard for the experiment. AP1 reduced the iron III to iron II in a concentration-dependent manner (Fig. 5A). Lipid peroxidation was induced by treating the liver homogenate with 1% K3[Fe(CN)6] solution and its protection by AP1 was tested. Increasing concentrations of AP1 (30 µg, 60 µg, 120 µg and 240 µg) inhibited the lipid peroxidation in a concentration-dependent manner with the percentage inhibition of 95.21 ± 0.05, 95.74 ± 0.80, 97.07 ± 0.90 and 98.93 ± 0.70%, respectively (Fig. 5B). In vitro NO scavenging activity of AP1 tested at different concentrations (10, 30 and 100 µg/ml) was found to scavenge NO in concentration-dependent manner. In other words, the percentage inhibition of spontaneous NO formation from SNP was calculated as 22.21 ± 0.31, 39.43 ± 0.56 and 52.28 ± 0.39, respectively (Fig. 5C).

Fig. 3. Effect of AP1 (30 mg/kg) on apoptosis in ischemic brain of rats. After 72 h of reperfusion, the brain was removed and serial sections of 4 μm were cut and the TUNEL assay was done to identify the extent of DNA fragmentation. TUNEL stained brain sections of sham-operated (A), transient focal cerebral ischemic vehicle-treated control (B) and AP1 (30 mg/kg, i.p.)-treated (C) groups of rats (40×). Number of TUNEL positive cells/mm2 of brain sections was counted (D). TUNEL positive apoptotic cells stained dark brown nuclei and normal nuclei did not take any stain. Number of TUNEL positive cells/mm2 of brain sections was counted. The data are expressed as mean ± S.E.M.; n = 3 animals in each group. **P < 0.01 in comparison to vehicle control.


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Fig. 5. Effect of AP1 on in vitro reducing activity (A), inhibition of lipid peroxidation (B) and NO scavenging activity (C). Vertical bar represents S.E.M. n = three.

Fig. 4. Ethidium bromide/acridine orange staining of vehicle control (A) and AP1treated (B) Neuro-2a cells. AP1-treated Neuro-2a cells were damaged with H2O2 (C) and sodium glutamate (D). The green fluorescent nuclei indicated live cells and red stained nuclei indicated dead/apoptotic cells. Effect of AP1 on cell viability of H2O2 (E) (10− 4 M) (and sodium glutamate (F) (1 mM)-induced damage in Neuro-2a cells. The cells were seeded in 96 well plates. After 48 h of incubation, AP1 was added at a concentration of 1 and 3 μg/ml and 30 min later H2O2 or sodium glutamate was added to each well. The cells were incubated for 24 h and quantified by MTT assay for % cell viability. The data are expressed as mean ± S.E.M.; **P < 0.01;***P < 0.001 in comparison to vehicle control.

3.7. Cyclooxygenase (COX) assay The cyclooxygenase-2 inhibitory activity of AP1 was 70.56%, while cyclooxygenase-1 inhibitory activity of AP1 was 34.45%. 4. Discussion AP1 is an acylated glucoside of dihydroflavanol. It has been characterized on the basis of chemical reactions and spectral data of 1 H, 13 C NMR (1D and 2D), FABMS (Protonated Fast Atomic Bombardment Mass spectrum) and its chemical name is (2R,3R)5,7,3′,4′,5′-pentahydroxy dihdroflavanol-3-O-(2″-O-galloyl)-β-D-glucopyranitric oxide.

In the present study, AP1 protected neuronal damage induced by cerebral ischemia by significant reduction in the infarct volume, cerebral edema and neurobehavioral abnormality. In pathogenesis of cerebral ischemia, reperfusion, while critical to limit ischemia, contributes to the cell death by enhancing the production of free radicals, inflammation and blood brain barrier breakdown, each of which has been targeted by therapies (Carden and Granger, 2000; Ovbiagele et al., 2003). Levels of reactive oxygen species are increased particularly, during the reperfusion phase and included accumulation of superoxide anion, hydrogen peroxide, hydroxyl radicals and peroxynitrite (Love, 1999). The brain tissue is most vulnerable to oxidative damage because of its high rate of oxidative metabolic activity, intensive production of reactive oxygen metabolites, relatively low antioxidant capacity, low repair mechanisms, nonreplacing nature of its neuronal cells and the high membrane surface to cytoplasm ratio (Evans, 1993; Reiter, 1995). Mitochondrial dysfunction ensues as a result of oxidative stress, energy failure and disruption of cellular calcium homeostasis resulting in further production of reactive oxygen species which damages cellular proteins, DNA and membrane lipids. Since polyphenolics have been reported to be antioxidants and free radical scavengers (Gao et al., 1999; Shieh et al., 2000), we tested AP1 for these properties in various in vitro models and observed that AP1 possesses all these properties. Based on the fact that TUNEL staining is a sensitive indicator of apoptotic neurons, in our study, the number of TUNEL positive neurons (apoptotic cells) was increased in transient focal cerebral

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ischemia in ischemic penumbra. Brain damage due to transient focal cerebral ischemia is an evolving process. It begins during the insult and extends into the recovery period after the reperfusion (Irmak et al., 2003). Cerebral injury takes the form of selective neuronal necrosis or infarction, the latter with destruction of all cellular elements including neurons, glia and blood vessels. When the infarction occurs, the immediate area surrounding the infarct (in penumbra) consists of neurons undergoing either necrosis or apoptosis. It is the penumbral area that appears most amenable to reversal of cellular injury through therapeutic intervention (Irmak et al., 2003). Our study showed that AP1 treatment significantly reduced the number of apoptotic cells in the representative penumbral area suggesting neuroprotective effect (Fig. 5C) which was further confirmed by in vitro studies with Neuro-2a cells, wherein AP1 improved the percentage cell viability of glutamate, as well as hydrogen peroxide-induced toxicity. In H2O2 and glutamate-induced toxicity, Neuro-2a cells when stained with ethidium bromide/acridine orange staining, the resultant green fluorescence indicates the live cells and the red indicates dead cells. The number of green cells has been increased with AP1 treatment in H2O2 and glutamate-induced toxicity (Fig. 4C and D). It has been reported that glutamate excitotoxicity is mediated by intracellular Ca2+ overload, caspase-3 activation, and reactive oxygen species generation. Our results of glutamate toxicity are supported by the observation that curcumin, tannic acid (TA) and (+)-catechin hydrate (CA) all inhibited glutamate-induced excitotoxicity (Yazawa et al., 2003). The findings of the present study, therefore, suggest that AP1 produces neuroprotection by inhibiting apoptosis and excitotoxicity. Cerebral ischemia/reperfusion injury induced lipid oxidation (Irmak et al., 2003; Cao et al., 2004; Choi-Kwon et al., 2004). In our study, we observed that MDA levels were higher in ischemic control rats which were reduced by AP1 treatment, thereby confirming its antioxidant role in transient focal cerebral ischemia. Lipid peroxidation was also inhibited by AP1 in in vitro observations. Protein carbonyl is most widely used for the assessment of protein oxidation and it seems to be a sensitive indicator of ischemia/reperfusioninduced oxidative stress which is increased in gerbil brain after bilateral carotid occlusion (Olga et al., 2006). Plasma protein carbonyl was significantly reduced by AP1 treatment, thereby suggesting that AP1 protects protein and amino acids from oxidative damage. As such, thiol groups are highly reactive constituents of protein molecule and they participate in important biochemical and metabolic processes, such as cell division, blood coagulation, maintenance of protein system and enzymatic activation including antioxidant enzymes (Jensen, 1959). They are also important scavengers of oxygen free radicals (Dormandy, 1980). SH group is known to be sensitive to oxidative damage and depleted following ischemic insult. Therefore, we studied the effect of AP1 on total thiols following transient focal cerebral ischemia and observed that the total thiol was significantly decreased. There was significant improvement in total thiol levels, in comparison to ischemic control, but not to the level of sham control, suggesting that in this model, more amount of reducing agents are required and/or the endogenous antioxidants were depleted. Excessive production of reactive oxygen species can be partially scavenged by endogenous antioxidants including SOD, catalase and GPx. Catalase metabolizes peroxides including H2O2 and protects the cellular membranes from lipid peroxidation (Choi-Kwon et al., 2004). Increased catalase activity observed in this study following IR injury was reduced by AP1 treatment probably, because of reduction in inflammatory stimulus for the enzyme induction. Increased catalase activity was also observed in rat after cerebral ischemia (Danielisová et al., 2007). The increased activity of catalase in ischemic control animals can be well correlated with decreased glutathione peroxidase (GPx) activity, as the substrate for both enzymes is H2O2. Further, in our study, we observed reduced total thiol and consequently, reduced glutathione culminating in the reduced glutathione peroxidase


activity in ischemic control animals. Total thiols, as well as GPx activity were not improved by treatment. The levels of NOx in the brain homogenate were significantly lower in AP1-treated group, than in ischemic control group of rats. NO is involved in the cascade of metabolic events that causes or contributes to ischemic brain damage (Irmak et al., 2003). The results of the present study indicate that transient focal cerebral ischemia induces significant increase in peroxynitrite levels (data not included). In contrast, AP1 might reduce NOx levels in ischemic events which are substantiated by NO scavenging activity of AP1 in in vitro and/or NOx level in brain by inhibiting nitric oxide synthase. It has been clearly shown that reactive oxygen species regulate the expression of many pro-inflammatory genes including COX-2 (Wang et al., 2004; Rodrigo et al., 2005), and iNOS (Floyd, 1999; Rodrigo et al., 2005). The role of inflammation in cerebral ischemic damage has been reported in human and various animal models of stroke (Iadecola and Alexander, 2001) and its importance in stroke has been highlighted by the observation that anti-inflammatory compounds or deletion of pro-inflammatory genes is neuroprotective (Barone and Feuerstein, 1999). AP1 was found to possess good in vitro cyclooxygenase inhibitory activity and could protect the inflammatory damage, thus contributing to neuroprotective effect. In conclusion, the present findings suggest that ischemia/reperfusion injury causes accumulation of oxidation products, such as MDA and NO, alterations in antioxidant enzymes, and induction of apoptosis. AP1 can be used as a neuroprotective agent in stroke as it reduced apoptosis and found to be a good antioxidant and antiinflammatory compound. Acknowledgement The first author is grateful to CSIR, New Delhi (India) for providing financial assistance in the form of JRF to carry out this work. References Acheson, R.M., Williams, D.R.R., 1983. Does consumption of fruit and vegetables protect against stroke? Lancet 1, 1191–1193. Barone, F.C., Feuerstein, G.Z., 1999. Inflammatory mediators and stroke: new opportunities for novel therapeutics. J. Cereb. Blood Flow. Metab. 19, 819–834. Bederson, J.B., Pitts, L.H., Germano, S.M., Nishimura, M.C., Davis, R.L., Bartkowski, H., 1986a. Evaluation of 2, 3, 5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke 17, 1304–1308. Bederson, J.B., Pitts, L.H., Tsuji, M., Nishimura, M.C., Davis, R.L., Bartkowski, H., 1986b. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 17, 472–476. Bergmeyer, H.U., 1983. In: Bergmeyer, H.U. (Ed.), 3rd Ed. Methods of Enzymatic Analysis, Vol. 2, pp. 165–166. Bravo, L., 1998. Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutr. Rev. 56, 317–333. Bronner, L.L., Kanter, D.S., Manson, J.E., 1995. Primary prevention of stroke. N. Engl. J. Med. 333, 1392–1400. Cao, D.H., Xu, J.F., Xue, R.H., Zheng, W.F., Liu, Z.L., 2004. Protective effect of chronic ethyl docosahexaenoate administration on brain injury in ischemic gerbils. Pharmacol. Biochem. Behav. 79, 651–659. Carden, D.L., Granger, D.N., 2000. Pathophysiology of ischemia–reperfusion injury. J. Pathol. 190, 255–266. Choi-Kwon, S., Park, K., Lee, H., Park, M., Lee, J., Jeon, S., Choe, M., Park, K., 2004. Temporal changes in cerebral antioxidant enzyme activities after ischemia and reperfusion in a rat focal brain ischemia model: effect of dietary fish oil. Brain Res. 152, 11–18. Cortas, N.K., Wakid, N.W., 1990. Determination of inorganic nitrate in serum and urine by kinetic cadmium-reduction methods. Chin. Chem. 36, 1440–1443. Danielisová, V., Gottlieb, M., Némethová, M., Burda, J., 2007. Activities of endogenous antioxidant enzymes in the cerebrospinal fluid and the hippocampus after transient forebrain ischemia in rat. J. Neurol. Sci. 253, 61–65. De Freitas, G.R., Bogousslavsky, J., 2001. Primary stroke prevention. Eur. J. Neurol. 8, 1–15. Denizot, F., Lang, R., 1986. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J. Immunol. Methods 22, 271–277. Dormandy, T.L., 1980. An approach to free radicals in medicine and biology. Acta Physiol. Scand. 492, 153–168.


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