research article neuroprotective activity of water soluble...

Research ArticleNeuroprotective Activity of Water SolubleExtract from Chorispora bungeana against Focal CerebralIschemicReperfusion Injury in Mice

Ya-Xuan Sun12 Ting Liu3 Xue-Ling Dai1 Ya-Bo Li4 Yu-Yao Li4

Hua Zhang4 and Li-Zhe An4

1 Beijing Key Laboratory of Bioactive Substances and Functional Foods Beijing Union University Beijing 100191 China2 Cold and Arid Regions Environmental and Engineering Research Institute Chinese Academy of Sciences Lanzhou 730000 China3 School of Pharmacy Shihezi University Shihezi 832002 China4Key Laboratory of Arid and Grassland Agroecology (Ministry Education) School of Life Sciences Lanzhou UniversityLanzhou 730000 China

Correspondence should be addressed to Li-Zhe An lizheanlzueducn

Received 4 April 2014 Revised 3 June 2014 Accepted 4 June 2014 Published 23 June 2014

Academic Editor Qiusheng Zheng

Copyright copy 2014 Ya-Xuan Sun et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The purpose of the present study was to clarify whether the water extract of Chorispora bungeana was an antioxidant agent againstcerebral ischemiareperfusion (IR) Our results showed that water extract of Chorispora bungeana treatment significantly reducedneurological deficit scores infarct size MDA and carbonyl contents and GSHGSSG ratio compared with the model controlgroup After being treated by Chorispora bungeana SOD CAT and GSH-Px activities remarkably increased Chorispora bungeanatreatment also improved 8-OHdG expression and cell apoptosis Our findings indicated that the water extract of Chorisporabungeana possesses neuroprotective effect which is most likely achieved by antioxidant and antiapoptotic activities

1 Introduction

Stroke is one of the leading causes of disability and death withastronomical financial repercussions on health systems andabout 16 million strokes take place and cause a total of 57million deaths in the world ever year The global mortality issupposed to rise to 65 million in 2015 and to 78 million in2030 [1 2] It is therefore urgent to develop effective therapiesfor stroke

Cerebral ischemia is the most common type of strokeaccounting for 88 of cases and it is reported that cerebralischemia results in a cascade of cellular and molecular eventsincluding oxidative stress excitotoxicity calcium overlordneuroinflammation and apoptosis [3] Reperfusion is a goodmeasure for dealing with ischemia but the beneficial effectsof reperfusion may be in part reversed by the occurrence ofreperfusion injury [4]

Oxidative stress was suggested to be implicating in thepathogenesis of cerebral ischemiareperfusion (IR) injuryFormation of reactive oxygen species (ROS) mainly includesthe arachidonic acid pathway the mitochondrial chain res-piratory chain the oxidation of xanthine and hypoxanthineby xanthine oxidase and NADPH-oxidases [5 6] The brainis sensitive to oxidative stress because of relative low level ofantioxidant enzyme and abundance of oxidizable substratesand transition metals [7]

Oxidative stress also induces neuronal apoptosis whichis a feature found in cerebral ischemiareperfusion [8ndash10]Chen et al [11] found that hydrogen peroxide a majoroxidant generated when oxidative stress occurs leads toneuronal apoptosis in a concentration- and time-dependentmanner Wang et al [12] suggested that hydrogen peroxideand nitric oxide synergistically induced neuronal apoptosisinvolving activation of p38 mitogen-activated protein kinase

Hindawi Publishing CorporationJournal of ChemistryVolume 2014 Article ID 373872 9 pageshttpdxdoiorg1011552014373872

2 Journal of Chemistry

and caspase-3 However recovery of blood supply is nec-essary to remedy the compromised ischemic brain tissueAntioxidants may reduce reperfusion-induced injury andextend the therapeutic window for thrombolysis [13ndash16]

Chorispora bungeana Fisch And CA Mey (C bungeana)is a rare alpine subnival plant species that inhabits periglacialregions in a severe low-temperature environment [17 18]The plant is a perennial herb belonging to the Brassicaceae-family [19] Liu et al [20] concluded that brassinosteroidscould play the positive roles in alleviating chilling-inducedoxidative damage by enhancing antioxidant defense systemin suspension cultured cells of Chorispora bungeana Guoet al [21] found that a sequential and synergetic actionbetween antioxidant enzymes such as superoxide dismu-tase dehydroascorbate reductase ascorbate peroxidase andglutathione reductase leading to a low antioxidation ratewhich contributes to retard lipid peroxidation and playsan important role in the resistance of suspension culturedcells of Chorispora bungeana to freezing temperatures Wepresume that Chorispora bungeana may reduce oxidativestress induced by cerebral ischemiareperfusion

The present study was undertaken to evaluate the neu-roprotection of water extract of Chorispora bungeana incerebral ischemiareperfusion with use of middle cerebralartery occlusion (MCAO) model and a series of oxidativestress markers

2 Materials and Methods

21 Chemicals and Reagents 235-Triphenyltetrazoliumchloride hypoxanthine xanthine oxidase catalase 551015840-dithiobis(2-nitrobenzoic acid) oxidized disulfide and re-duced glutathione were purchased from Sigma ChemicalCo (St Louis MO) 1133-Tetramethoxypropane has beenobtained from Fluka Chemical Co (Ronkonkoma NY) Allother chemicals and reagents were of analytical grade

22 Animals Male ICR (26 plusmn 2 g) were obtained from VitalRiver Laboratories (Peking China) for this study Animalswere housed in a room at a temperature of 24 plusmn 1∘C and12-h dark and light cycle with free access to standard foodand water The experimental protocol was approved by theInstitutional Animal Care and Use Committee of NationalInstitute Pharmaceutical Education and Research

23 Chorispora bungeana Water Extract Preparation A wildspecies of Chorispora bungeana was obtained by the methoddescribed by Fu et al [22]

Freshly collected Chorispora bungeana whole plant wasdried under shade and the driedmaterial wasmilled to obtaina coarse powder The powder was immersed in water for 1 hand then was extracted at boiling temperature for 15 h andrepeated twiceThe whole water extract was freeze-dried andredissolved in water at a concentration of 05 gmL

24 Drug Administration Mice were randomly divided intosix groups each consisting of eight animals Water extract

of Chorispora bungeana diluted with distilled water was fedby oral gavage every day at a fixed time for 5 d in 5 25and 125 gkg (equivalent to the amount of crude drug) threedifferent doses The model control group and sham groupwere treated with vehicle orally for 5 d Edaravone groupwas treated with Edaravone at a dose of 3mgkg for 5 dand served as positive control On day 5 1 h after the abovetreatments the mice were subjected to the middle cerebralartery occlusion

25 Transient Focal Cerebral IschemicReperfusionModel Allmice were subjected to 2 h transient focal cerebral ischemiafollowed by 22 h reperfusion using an intraluminal suturetechnique described by Longa et al [23] and Ha et al [24]with little modification In brief mice were anesthetized withchloral hydrate (400mgkg i p) Midline incision was madeon ventral side of mouse neck The left common carotidartery (CCA) the external carotid artery (ECA) and theinternal carotid artery (ICA) were carefully exposed anddissected away from adjacent muscles and nerves Microvas-cular aneurysm clips were applied to the left CCA and theICA A coated filament was introduced into an arteriotomyhole fed distally into the ICA and advanced about 12mmfrom the carotid bifurcation The ICA clamp was removedand focal cerebral ischemia started After occlusion for 2 hthe filament was gently pulled out The collar suture at thebase of the ECA stump was tightened The skin was closedanesthesia discontinued and the animalswere returned to theprewarmed cages Body temperature was maintained at 37 plusmn05∘C by lamp during the whole surgical procedures Animals

in sham control group underwent a neck dissection andcoagulation of the external carotid artery but no occlusionof middle cerebral artery

26 Neurological Deficit Scores After 22 h of reperfusionan observer who was unaware of the identity of the groupsevaluated the neurological deficits by using the methoddescribed by Longa et al [23] 0 no neurological deficit 1failure to extend the right forepaw fully 2 circling to theright 3 falling to the right 4 no spontaneous walking with adepressed level of consciousness

27 Infarct Size Mice were decapitated after 22 h of reperfu-sion and these brains were quickly removed and sectionedcoronally into five 15-mm-thick coronal sections The sec-tions were stained with 05 TTC at 37∘C for 20min Thestained brain sections were postfixed in 10 formalin solu-tion and photographed with a digital camera and the infarctareas of each section were determined with the analysis ofpixel counting by a computer program of Photoshop 60

28 Biochemical Assays

281 Tissue Preparation Tomeasure of the contents ofMDAand carbonyl the activities of SOD CAT and GSH-Pxthe ratio of GSHGSSG these mouse brains were weighed

Journal of Chemistry 3

00

05

10

15

20

25

30

35

12525Chorispora bungeana (gkg)

Edaravone5Sham

Neu

rolo

gica

l defi

cit s

core

s

Model(3mgkg)

lowastlowast

lowastlowast

lowastlowast

(a)

0

5

10

15

20

25

30

35

40

12525 Edaravone5Sham

Infa

rct s

ize (

)

ModelChorispora bungeana (gkg)(3mgkg)

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

Figure 1 Effect of Chorispora bungeana water extract on neurological deficit scores (a) and infarct size (b) in mice subjected to cerebralischemiareperfusion Data were expressed as mean plusmn SME Differences were considered significant at 119875 lt 005 lowast119875 lt 005 versus modelgroup lowastlowast119875 lt 001 versus model group

and homogenized with ice-cold normal saline and thencentrifuged at 4624timesg for 15min at 4∘C The supernatantwas used for bioassays Protein content of the supernatantwas determined by the method of Bradford [25] using bovineserum albumin as the protein standard

282 GSHGSSG Ratio The ratio of GSHGSSG was deter-mined by a previously describedmethod [26] Briefly T-GSHwas determined basing on the 55-dithiobis (2-nitrobenzoic)acid (DTNB)-GSSG reductase recycling GSSG was assayedby measuring 5-thio-2-nitrobenzoic acid (TNB) which wasproduced from the reaction of reduced GSH with DTNBGSH level in brain tissue was calculated as the differencebetween T-GSH and GSSG

283 Measurement of MDA Content TheMDA content wasassayed in the brain tissue using amethod as described byCaoet al [27] 1133-Tetramethoxypropane was used as standardand the content of MDA was expressed as nmolmg protein

284 Measurement of Carbonyl Content Carbonyl contentwas evaluated using the method according to Levine etal [28] The content of carbonyl was calculated by usingthe extinction coefficient of 22000Mminus1cmminus1mg protein andexpressed as nmolmg protein

285 Measurement of Antioxidant Enzyme Activities SODactivity was measured as described by Jung et al [29] Oneunit of SOD activity was defined as the amount that shows50 inhibition SOD activity was expressed as Umg pro-tein

CAT activity was evaluated according to the method ofCampo et al [30] One unit of CAT activity was defined as theamount of CAT required to decompose 1120583molL of hydrogen

peroxide in min CAT activity was expressed as Umg pro-tein

GSH-Px activity was determined by themethod of Jagetiaet al [31] One unit of GSH-Px activity was defined as theGSH-Px in 1mg protein that led to the decrease of 1 120583molLGSH in the reactive system per minute GSH-Px activity wasexpressed as Umg protein

29 Immunohistochemistry Assay Animals were overdosedwith anesthetic and perfused with 4 paraformaldehyde in01M phosphate buffer solution (PBS pH = 74) after 22 h ofreperfusion Brains were removed and further fixed in 4paraformaldehyde at 4∘C for 24 h and then cut into equallyspaced blocks Paraffin-embedded blocks were cut into aseries of 5-120583m-thick slices

8-Hydroxyl-deoxyguanosine (8-OHdG) immunohisto-chemistry was used to identify oxidized DNA and performedby the method of Wang et al [32] In brief deparaffinizedbrain sections were immunostained with 5120583gmL mouseanti-8-OHdGantibody followedby 05goat anti-mouse IgGlabeled with horseradish peroxidase

Cell apoptosis was observed by the terminal deoxynu-cleotidyl transferase-mediated Dutp-biotin nick end labeling(TUNEL) assay In brief brain sections were deparaffinizedand rehydrated followed by incubating with 20120583gmL pro-teinase K in 001M Tris-HCl (pH = 74) and permeabilized ina solutionwhich contains 01 Triton-X 100 and 01 sodiumcitrate Then these sections were incubated in TUNEL-reactionmixture containing terminal deoxynucleotidy trans-ferase

210 Statistical Analysis Data were expressed as mean plusmnSME Statistical analysis was evaluated with one way


0

10

20

30

40

50

60


GSH

GSS

G ra

tio

ModelChorispora bungeana (gkg) (3mgkg)

lowastlowastlowastlowast

lowastlowast

lowastlowast

Figure 2 Effect of Chorispora bungeana water extract on GSHGSSG ratio in mice subjected to cerebral ischemiareperfusionData were expressed as mean plusmn SME Differences were consideredsignificant at 119875 lt 005 lowast119875 lt 005 versus model group lowastlowast119875 lt 001versus model group

analysis of variance (ANOVA) and followed by a Student-Newman-Keuls (SNK) test for multiple comparisons 119875 lt005 was regarded as statistically significant

3 Results

31 Effect of Chorispora bungeana Water Extract on Neu-rological Deficit Scores and Infarct Size After 22 h of re-perfusion neurological deficit scores and infarct size inischemicreperfusion mice were obviously higher than thoseof the sham control mice indicating that we had successfullyestablished the cerebral ischemicreperfusion model Treat-ment with Chorispora bungeana water extract significantlydecreased neurological deficit scores and infarct size ascompared to the model control group (Figures 1(a) and 1(b))

32 Effect of Chorispora bungeana Water Extract onGSHGSSG TheGSHGSSG ratio in the mouse brain tissuea typically of oxidative stress was markedly lower in themodel control group as compared to the sham control groupAs shown in Figure 2 there was a significant increase in theratio of GSHGSSG in the Chorispora bungeanawater extracttreatment groups (5 and 25 gkg) compared with the modelcontrol group (119875 lt 001)

33 Effect of Chorispora bungeana Water Extract on MDAand Carbonyl Contents MDA and carbonyl are the twomainproducts of oxidative damage Figure 3 showed that MDAand carbonyl contents weremuch higher in themodel controlgroup than those in the sham control group The treatmentwith Chorispora bungeana water extract evidently reducedthe contents of MDA and carbonyl in brain tissue comparedwith the model control group

0

1

2

3

4

5

6

7


MD

A co

nten

t (nm

olm

g pr

otei

n)


lowastlowast


lowast

(a)

00

05

10

15

20

25

30


Carb

onyl

cont

ent (

nmol

mg

prot

ein)


lowastlowast


lowast

(b)

Figure 3 Effect of Chorispora bungeana water extract on MDA (a)and carbonyl (b) inmice subjected to cerebral ischemiareperfusionData were expressed as mean plusmn SME Differences were consideredsignificant at 119875 lt 005 lowast119875 lt 005 versus model group lowastlowast119875 lt 001versus model group

34 Effect of Chorispora bungeana Water Extract on Antioxi-dant Enzyme Activities Table 1 showed that the activities ofSOD CAT and GSH-Px were obviously decreased in modelcontrol group as compared to the sham control group andattenuated significantly by treatment of Chorispora bungeanawater extract (5 and 25 gkg) compared with the controlgroup (119875 lt 005)

35 Effect of Chorispora bungeana Water Extract on 8-OHdGExpression The expression of 8-OHdG obviously increasedin the model control group as compared to the sham controlgroup Treatment of Chorispora bungeana water extractmarkedly weakened 8-OHdG compared with the modelcontrol group (Figure 4 Table 2)

36 Effect of Chorispora bungeanaWater Extract on ApoptosisVery few TUNEL positive cells were found in normal mousebrain tissue and the number of TUNEL positive cells was


Table 1 Effect of Chorispora bungeana water extract on antioxidant enzyme activities in mice subjected to cerebral ischemiareperfusion

Group Dose(gkg)

SOD(Umg protein)

CAT(Umg protein)

GSH-Px(Umg protein)

Model 6295 plusmn 672 611 plusmn 061 68453 plusmn 8357

Sham 8183 plusmn 409lowastlowast

832 plusmn 047lowastlowast


Chorispora bungeana 5 7904 plusmn 684lowastlowast



Chorispora bungeana 25 7151 plusmn 536lowast

695 plusmn 053lowast


Chorispora bungeana 125 6341 plusmn 697 673 plusmn 058 72403 plusmn 6032

Edaravone 0003 7888 plusmn 523lowastlowast



Data were expressed as mean plusmn SME Differences were considered significant at 119875 lt 005 lowast119875 lt 005 versus model group lowastlowast119875 lt 001 versus model group

(a) (b) (c)

(d) (e) (f)

Figure 4 Effect of Chorispora bungeana water extract on 8-OHdG expression in mice subjected to cerebral ischemiareperfusion Thenucleus of positive expressed cell is brown colored (a) Model group (b) Sham group (c) Edaravone-treatment group (3mgkg) (d)Chorispora bungeana-treatment group (5 gkg) (e) Chorispora bungeana-treatment group (25 gkg) (f) Chorispora bungeana-treatmentgroup (125 gkg)

significantly increased by ischemiareperfusion Administra-tion of Chorispora bungeana water extract markedly reducedTUNELpositive cells as compared to themodel control group(Figure 5 Table 2)

4 Discussion

The present study reported the neuroprotective effect ofChorispora bungeanawater extract in terms of oxidative stress

markers and apoptosis in cerebral ischemicreperfusionmouse brain for the first time We found that water ex-tract of Chorispora bungeana prevents brain from ischem-icreperfusion damage as indicated by the improved recoveryof neurological function decreases in infarct size and oxida-tive stress and increases in antioxidant enzyme activities andreduction in apoptosis

We set up cerebral ischemicreperfusion model withintraluminal thread insertion method in mice and tested


Table 2 Effect of Chorispora bungeana water extract on 8-OHdG expression and cell apoptosis in mice subjected to cerebralischemiareperfusion

Group Dose (gkg) 8-OHdG positive cells TUNEL positive cellsModel 2088 plusmn 331 2163 plusmn 421






1825 plusmn 399

Chorispora bungeana 125 1988 plusmn 383 1950 plusmn 441




(a) (b) (c)

(d) (e) (f)

Figure 5 Effect ofChorispora bungeanawater extract on cell apoptosis inmice subjected to cerebral ischemiareperfusionNucleus has browngranules were positive cell (a) Model group (b) sham group (c) Edaravone-treatment group (3mgkg) (d) Chorispora bungeana-treatmentgroup (5 gkg) (e) Chorispora bungeana-treatment group (25 gkg) (f) Chorispora bungeana-treatment group (125 gkg)

whether the model was successfully established by means ofassaying the neurological deficit scores and infarct size Themice showed visible neurological deficits and brain infarctionin model control group Treatment of Chorispora bungeanawater extract reducing the neurological deficit scores andinfarct size in a dose-dependent manner demonstrated thatwater extract ofChorispora bungeanawas provided with neu-roprotective activity against cerebral ischemicreperfusioninjury

Thenwe determinedwhether brainwas in oxidative stressstatus after 22 h of reperfusion The ratio of GSHGSSG isproposed to be a sensitive index of oxidative stress [33 34]

Our data showed that the ratio of GSHGSSG was decreasedin model control group but the decrease was attenuated bytreatment of Chorispora bungeana water extract That indi-cated that Chorispora bungeana water extract could releaseoxidative stress in ischemicreperfusion brain tissue

Reactive oxygen species (ROS) contain one or moreunpaired electrons in their outer orbit and are highly reactiveplay key roles in organism physiology and pathophysiology[35ndash38] Excessive production of ROSmay depress cell mem-brane properties and cause oxidative damage to lipids pro-teins and DNA that may make them nonfunctional becauseof brain tissue contains large amount of unsaturated fatty


acids and catecholamines after cerebral ischemia and par-ticularly reperfusion [6 39 40] Malondialdehyde (MDA)carbonyl and 8-hydroxy-21015840-deoxyguanosine (8-OHdG) arerepresentative markers of oxidative damage of brain [39]We measured the MDA and carbonyl contents by usingof a spectrocolorimetric assay and detected the 8-OHdGexpression by immunohistochemistry The water extractof Chorispora bungeana effectively reduced the content ofMDA and carbonyl the expression of 8-OHdG revealedthat it could lighten oxidative damage induced by cerebralischemiareperfusion

The ROS is maintained in an appropriate level by theendogenous antioxidant enzymes such as superoxide dis-mutase (SOD) catalase (CAT) and glutathione peroxidase(GSH-Px) under normal physiological conditions [6 41]SOD catalyzes the dismutation of superoxide anions tohydrogen peroxide and molecular oxygen and CAT togetherwith GSH-Px eliminates hydrogen and molecular oxygen[42] But these antioxidant enzyme activities were reported todecrease during cerebral ischemiareperfusion [43 44] Thepresent study showed that the reduced activities of SODCATand GSH-Px in ischemicreperfusion mice were significantlyattenuated in Chorispora bungeana water extract-treatedgroup when compared with the model control group

Broughton et al [45] pointed out that cerebral ischemiatriggers twomain pathways of apoptosis One originates frommitochondrial release of cytochrome c and another originatesfrom the activation of cell surface death receptors Fujimuraet al [46] agreed that ROS mediated the mitochondrialsignaling pathway that may result in apoptosis followingcerebral ischemia In order to determine the antiapoptoticactivity of Chorispora bungeana water extract we evaluatedthe apoptosis with terminal deoxynucleotidyl transferasemediated d UTP-biotin nick end labeling (TUNEL) Cho-rispora bungeana water extract was proved to be able toinhibit cell apoptosis in our study

In summary our study demonstrated that the waterextractof Chorispora bungeana could effectively attenuatebrain injury in focal cerebral ischemiareperfusion The neu-roprotective activity was associated with inhibiting oxidativestress and apoptosis Chorispora bungeana may have thepotential to cure nervous systemdiseases as an unusual alpinesubnival plant and more pharmacodynamics research workneeds to be done

Conflict of Interests

The authors declare that there is no conflict of interests re-garding the publication of this paper

Acknowledgments

This study was supported by the National OutstandingYouth Foundation of China (30625008) the National HighTechnology Research and Development Program (863 Pro-gram 2007AA021401) National Basic Research Programof China (973 2007CB108902) International CooperationProgram of the Ministry of Science and Technology of

China (2009DFA61060) the Major Project of CultivatingNewVarieties of Transgenic Organisms (2008ZX08009-003)and the National Natural Science Foundation (30900172)This work was supported by Grants the National BasicResearch Program of China (973 Program 2013CB429904)the Key Program of National Nature Science Foundationof China (31230014) the Funding Project for AcademicHuman Resources Development in Institutions of HigherLearning Under the Jurisdiction of Beijing Municipality[PHR201107150 PHR201108422]

References

[1] K Strong CMathers and R Bonita ldquoPreventing stroke savinglives around the worldrdquoThe Lancet Neurology vol 6 no 2 pp182ndash187 2007

[2] C L Allen and U Bayraktutan ldquoOxidative stress and its role inthe pathogenesis of ischaemic strokerdquo International Journal ofStroke vol 4 no 6 pp 461ndash470 2009

[3] E Candelario-Jalil ldquoInjury and repair mechanisms in ischemicstroke considerations for the development of novel neurother-apeuticsrdquo Current Opinion in Investigational Drugs vol 10 no7 pp 644ndash654 2009

[4] N Tapuria Y Kumar M M Habib M A Amara A MSeifalian and B R Davidson ldquoRemote ischemic precondi-tioning a novel protective method from ischemia reperfusioninjuryrdquo Journal of Surgical Research vol 150 no 2 pp 304ndash3302008

[5] G FiskumANMurphy andM F Beal ldquoMitochondria in neu-rodegeneration acute ischemia and chronic neurodegenerativediseasesrdquo Journal of Cerebral Blood Flow and Metabolism vol19 no 4 pp 351ndash369 1999

[6] I Margaill M Plotkine and D Lerouet ldquoAntioxidant strategiesin the treatment of strokerdquo Free Radical Biology and Medicinevol 39 no 4 pp 429ndash443 2005

[7] J Lehotsky R Murın A Strapkova A Urıkova Z Tatarkovaand P Kaplan ldquoTime course of ischemiareperfusion-inducedoxidative modification of neural proteins in rat forebrainrdquoGeneral Physiology and Biophysics vol 23 no 4 pp 401ndash4152004

[8] C Cheng S Su N Tang T Ho S Chiang and C HsiehldquoFerulic acid provides neuroprotection against oxidative stress-related apoptosis after cerebral ischemiareperfusion injury byinhibiting ICAM-1 mRNA expression in ratsrdquo Brain Researchvol 1209 pp 136ndash150 2008

[9] C Pekcetin M Kiray B U Ergur et al ldquoCarnosine attenuatesoxidative stress and apoptosis in transient cerebral ischemia inratsrdquo Acta Biologica Hungarica vol 60 no 2 pp 137ndash148 2009

[10] K Niizuma H Yoshioka H Chen et al ldquoMitochondrialand apoptotic neuronal death signaling pathways in cerebralischemiardquo Biochimica et Biophysica Acta vol 1802 no 1 pp 92ndash99 2010

[11] L Chen L Liu J Yin Y Luo and S Huang ldquoHydrogen per-oxide-induced neuronal apoptosis is associated with inhibitionof protein phosphatase 2A and 5 leading to activation ofMAPKpathwayrdquo International Journal of Biochemistry and Cell Biologyvol 41 no 6 pp 1284ndash1295 2009

[12] J Wang A Y C Shum and Y Ho ldquoOxidative neurotoxicityin rat cerebral cortex neurons synergistic effects of H

2O2and

NO on apoptosis involving activation of p38 mitogen-activated


protein kinase and caspase-3rdquo Journal of Neuroscience Researchvol 72 no 4 pp 508ndash519 2003

[13] Y Gursoy-Ozdemir A Can and T Dalkara ldquoReperfusion-induced oxidativenitrativie injury to neurovascular unit afterfocal cerebral ischemiardquo Stroke vol 35 no 6 pp 1449ndash14532004

[14] V M Chandrashekhar V L Ranpariya S Ganapaty A Par-ashar and A A Muchandi ldquoNeuroprotective activity of Matri-caria recutita Linn against global model of ischemia in ratsrdquoJournal of Ethnopharmacology vol 127 no 3 pp 645ndash651 2010

[15] T Yu Q Chen Z Chen Z Xiong and M Ye ldquoProtectiveeffects of total flavones of rhododendra against global cerebralischemia reperfusion injuryrdquo The American Journal of ChineseMedicine vol 37 no 5 pp 877ndash887 2009

[16] C Elango K S Jayachandaran and S Niranjali DevarajldquoHawthorn extract reduces infarct volume and improves neu-rological score by reducing oxidative stress in rat brain follow-ing middle cerebral artery occlusionrdquo International Journal ofDevelopmental Neuroscience vol 27 no 8 pp 799ndash803 2009

[17] J Wu T Qu S Chen Z Zhao and L An ldquoMolecular cloningand characterization of a 120574-glutamylcysteine synthetase genefrom Chorispora bungeanardquo Protoplasma vol 235 no 1ndash4 pp27ndash36 2009

[18] Y Shi L An M Zhang C Huang H Zhang and S XuldquoRegulation of the plasma membrane during exposure to lowtemperatures in suspension-cultured cells from a cryophyte(Chorispora bungeana)rdquo Protoplasma vol 232 no 3-4 pp 173ndash181 2008

[19] JWang L An RWang et al ldquoPlant regeneration ofChorisporabungeana via somatic embryogenesisrdquo Plant vol 42 no 2 pp148ndash151 2006

[20] Y Liu Z Zhao J Si C Di J Han and L An ldquoBrassinos-teroids alleviate chilling-induced oxidative damage by enhanc-ing antioxidant defense system in suspension cultured cells ofChorispora bungeanardquo Plant Growth Regulation vol 59 no 3pp 207ndash214 2009

[21] F GuoM Zhang Y Chen et al ldquoRelation of several antioxidantenzymes to rapid freezing resistance in suspension culturedcells fromalpineChorispora bungeanardquoCryobiology vol 52 no2 pp 241ndash250 2006

[22] X Fu J Chang L An et al ldquoAssociation of the cold-hardinessofChorispora bungeanawith the distribution and accumulationof calcium in the cells and tissuesrdquo Environmental and Experi-mental Botany vol 55 no 3 pp 282ndash293 2006

[23] E Z Longa P R Weinstein S Carlson and R CumminsldquoReversible middle cerebral artery occlusion without craniec-tomy in ratsrdquo Stroke vol 20 no 1 pp 84ndash91 1989

[24] S K Ha P Lee J A Park et al ldquoApigenin inhibits theproduction of NO and PGE2 inmicroglia and inhibits neuronalcell death in a middle cerebral artery occlusion-induced focalischemiamicemodelrdquoNeurochemistry International vol 52 no4-5 pp 878ndash886 2008

[25] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[26] E Candelario-Jalil N HMhadu S M Al-Dalain G Martınezand O S Leon ldquoTime course of oxidative damage in differentbrain regions following transient cerebral ischemia in gerbilsrdquoNeuroscience Research vol 41 no 3 pp 233ndash241 2001

[27] D Cao J Xu R Xue W Zheng and Z Liu ldquoProtective effect ofchronic ethyl docosahexaenoate administration on brain injury

in ischemic gerbilsrdquo Pharmacology Biochemistry and Behaviorvol 79 no 4 pp 651ndash659 2004

[28] R L Levine D Garland C N Oliver et al ldquoDetermination ofcarbonyl content in oxidatively modified proteinsrdquo Methods inEnzymology vol 186 pp 464ndash478 1990

[29] H Jung J H Nam J Choi K Lee and H Park ldquoAntiinflam-matory effects of chiisanoside and chiisanogenin obtained fromthe leaves of Acanthopanax chiisanensis in the carrageenan-and Freundrsquos complete adjuvant-induced ratsrdquo Journal ofEthnopharmacology vol 97 no 2 pp 359ndash367 2005

[30] G M Campo A Avenoso S Campo et al ldquoHyaluronicacid and chondroitin-4-sulphate treatment reduces damagein carbon tetrachloride-induced acute rat liver injuryrdquo LifeSciences vol 74 no 10 pp 1289ndash1305 2004

[31] G C Jagetia G K Rajanikant S K Rao and M ShrinathBaliga ldquoAlteration in the glutathione glutathione peroxidasesuperoxide dismutase and lipid peroxidation by ascorbic acidin the skin of mice exposed to fractionated 120574 radiationrdquo ClinicaChimica Acta vol 332 no 1-2 pp 111ndash121 2003

[32] Q Wang A Y Sun A Simonyi et al ldquoOral administrationof grape polyphenol extract ameliorates cerebral ischemiareperfusion-induced neuronal damage and behavioral deficitsin gerbils comparison of pre- and post-ischemic administra-tionrdquo Journal of Nutritional Biochemistry vol 20 no 5 pp 369ndash377 2009

[33] D P Jones ldquoRedefining oxidative stressrdquo Antioxidants andRedox Signaling vol 8 no 9-10 pp 1865ndash1879 2006

[34] D P Jones ldquoRedox potential of GSHGSSG couple assay andbiological significancerdquo Methods in Enzymology vol 348 pp93ndash112 2002

[35] N Tuteja and R Tuteja ldquoUnraveling DNA repair in humanmolecular mechanisms and consequences of repair defectrdquoCritical Reviews in Biochemistry and Molecular Biology vol 36no 3 pp 261ndash290 2001

[36] M Valko D Leibfritz J Moncol M T D Cronin M Mazurand J Telser ldquoFree radicals and antioxidants in normal physi-ological functions and human diseaserdquo International Journal ofBiochemistry and Cell Biology vol 39 no 1 pp 44ndash84 2007

[37] C Kohchi H Inagawa T Nishizawa and G Soma ldquoROS andinnate immunityrdquo Anticancer Research vol 29 no 3 pp 817ndash822 2009

[38] M Hultqvist L M Olsson K A Gelderman and R HolmdahlldquoThe protective role of ROS in autoimmune diseaserdquo Trends inImmunology vol 30 no 5 pp 201ndash208 2009

[39] A Cherubini C Ruggiero M C Polidori and P MecoccildquoPotential markers of oxidative stress in strokerdquo Free RadicalBiology and Medicine vol 39 no 7 pp 841ndash852 2005

[40] N Ogawa ldquoFree radicals and neural cell damagerdquo RinshoShinkeigaku vol 34 no 12 pp 1266ndash1268 1994

[41] C D Kamat S Gadal M Mhatre K S Williamson Q N Pyeand K Hensley ldquoAntioxidants in central nervous system dis-eases preclinical promise and translational challengesrdquo Journalof Alzheimerrsquos Disease vol 15 no 3 pp 473ndash493 2008

[42] N S Dhalla A B Elmoselhi T Hata and N Makino ldquoStatusof myocardial antioxidants in ischemia-reperfusion injuryrdquoCardiovascular Research vol 47 no 3 pp 446ndash456 2000

[43] S Yousuf S Salim M Ahmad A S Ahmed M A Ansariand F Islam ldquoProtective effect of Khamira Abresham UoodMastagiwala against free radical induced damage in focalcerebral ischemiardquo Journal of Ethnopharmacology vol 99 no2 pp 179ndash184 2005


[44] M M Khan A Ahmad T Ishrat et al ldquoRutin protects theneural damage induced by transient focal ischemia in ratsrdquoBrain Research vol 1292 pp 123ndash135 2009

[45] B R S Broughton D C Reutens and C G Sobey ldquoApoptoticmechanisms after cerebral ischemiardquo Stroke vol 40 no 5 pp331ndash339 2009

[46] M Fujimura T Tominaga and P H Chan ldquoNeuroprotectiveeffect of an antioxidant in ischemic brain injury involvementof neuronal apoptosisrdquo Neurocritical Care vol 2 no 1 pp 59ndash66 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


and caspase-3 However recovery of blood supply is nec-essary to remedy the compromised ischemic brain tissueAntioxidants may reduce reperfusion-induced injury andextend the therapeutic window for thrombolysis [13ndash16]

Chorispora bungeana Fisch And CA Mey (C bungeana)is a rare alpine subnival plant species that inhabits periglacialregions in a severe low-temperature environment [17 18]The plant is a perennial herb belonging to the Brassicaceae-family [19] Liu et al [20] concluded that brassinosteroidscould play the positive roles in alleviating chilling-inducedoxidative damage by enhancing antioxidant defense systemin suspension cultured cells of Chorispora bungeana Guoet al [21] found that a sequential and synergetic actionbetween antioxidant enzymes such as superoxide dismu-tase dehydroascorbate reductase ascorbate peroxidase andglutathione reductase leading to a low antioxidation ratewhich contributes to retard lipid peroxidation and playsan important role in the resistance of suspension culturedcells of Chorispora bungeana to freezing temperatures Wepresume that Chorispora bungeana may reduce oxidativestress induced by cerebral ischemiareperfusion

The present study was undertaken to evaluate the neu-roprotection of water extract of Chorispora bungeana incerebral ischemiareperfusion with use of middle cerebralartery occlusion (MCAO) model and a series of oxidativestress markers

2 Materials and Methods

21 Chemicals and Reagents 235-Triphenyltetrazoliumchloride hypoxanthine xanthine oxidase catalase 551015840-dithiobis(2-nitrobenzoic acid) oxidized disulfide and re-duced glutathione were purchased from Sigma ChemicalCo (St Louis MO) 1133-Tetramethoxypropane has beenobtained from Fluka Chemical Co (Ronkonkoma NY) Allother chemicals and reagents were of analytical grade

22 Animals Male ICR (26 plusmn 2 g) were obtained from VitalRiver Laboratories (Peking China) for this study Animalswere housed in a room at a temperature of 24 plusmn 1∘C and12-h dark and light cycle with free access to standard foodand water The experimental protocol was approved by theInstitutional Animal Care and Use Committee of NationalInstitute Pharmaceutical Education and Research

23 Chorispora bungeana Water Extract Preparation A wildspecies of Chorispora bungeana was obtained by the methoddescribed by Fu et al [22]

Freshly collected Chorispora bungeana whole plant wasdried under shade and the driedmaterial wasmilled to obtaina coarse powder The powder was immersed in water for 1 hand then was extracted at boiling temperature for 15 h andrepeated twiceThe whole water extract was freeze-dried andredissolved in water at a concentration of 05 gmL

24 Drug Administration Mice were randomly divided intosix groups each consisting of eight animals Water extract

of Chorispora bungeana diluted with distilled water was fedby oral gavage every day at a fixed time for 5 d in 5 25and 125 gkg (equivalent to the amount of crude drug) threedifferent doses The model control group and sham groupwere treated with vehicle orally for 5 d Edaravone groupwas treated with Edaravone at a dose of 3mgkg for 5 dand served as positive control On day 5 1 h after the abovetreatments the mice were subjected to the middle cerebralartery occlusion

25 Transient Focal Cerebral IschemicReperfusionModel Allmice were subjected to 2 h transient focal cerebral ischemiafollowed by 22 h reperfusion using an intraluminal suturetechnique described by Longa et al [23] and Ha et al [24]with little modification In brief mice were anesthetized withchloral hydrate (400mgkg i p) Midline incision was madeon ventral side of mouse neck The left common carotidartery (CCA) the external carotid artery (ECA) and theinternal carotid artery (ICA) were carefully exposed anddissected away from adjacent muscles and nerves Microvas-cular aneurysm clips were applied to the left CCA and theICA A coated filament was introduced into an arteriotomyhole fed distally into the ICA and advanced about 12mmfrom the carotid bifurcation The ICA clamp was removedand focal cerebral ischemia started After occlusion for 2 hthe filament was gently pulled out The collar suture at thebase of the ECA stump was tightened The skin was closedanesthesia discontinued and the animalswere returned to theprewarmed cages Body temperature was maintained at 37 plusmn05∘C by lamp during the whole surgical procedures Animals

in sham control group underwent a neck dissection andcoagulation of the external carotid artery but no occlusionof middle cerebral artery

26 Neurological Deficit Scores After 22 h of reperfusionan observer who was unaware of the identity of the groupsevaluated the neurological deficits by using the methoddescribed by Longa et al [23] 0 no neurological deficit 1failure to extend the right forepaw fully 2 circling to theright 3 falling to the right 4 no spontaneous walking with adepressed level of consciousness

27 Infarct Size Mice were decapitated after 22 h of reperfu-sion and these brains were quickly removed and sectionedcoronally into five 15-mm-thick coronal sections The sec-tions were stained with 05 TTC at 37∘C for 20min Thestained brain sections were postfixed in 10 formalin solu-tion and photographed with a digital camera and the infarctareas of each section were determined with the analysis ofpixel counting by a computer program of Photoshop 60

28 Biochemical Assays

281 Tissue Preparation Tomeasure of the contents ofMDAand carbonyl the activities of SOD CAT and GSH-Pxthe ratio of GSHGSSG these mouse brains were weighed


00

05

10

15

20

25

30

35


Edaravone5Sham

Neu

rolo

gica

l defi

cit s

core

s

Model(3mgkg)

lowastlowast

lowastlowast

lowastlowast

(a)

0

5

10

15

20

25

30

35

40


Infa

rct s

ize (

)


lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)















0

10

20

30

40

50

60


GSH

GSS

G ra

tio



lowastlowast

lowastlowast



3 Results




0

1

2

3

4

5

6

7


MD

A co

nten

t (nm

olm

g pr

otei

n)


lowastlowast


lowast

(a)

00

05

10

15

20

25

30


Carb

onyl

cont

ent (

nmol

mg

prot

ein)


lowastlowast


lowast

(b)







Group Dose(gkg)

SOD(Umg protein)

CAT(Umg protein)

GSH-Px(Umg protein)
















(a) (b) (c)

(d) (e) (f)



4 Discussion












1825 plusmn 399





(a) (b) (c)

(d) (e) (f)













Acknowledgments



References













2O2and

















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


00

05

10

15

20

25

30

35


Edaravone5Sham

Neu

rolo

gica

l defi

cit s

core

s

Model(3mgkg)

lowastlowast

lowastlowast

lowastlowast

(a)

0

5

10

15

20

25

30

35

40


Infa

rct s

ize (

)


lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)















0

10

20

30

40

50

60


GSH

GSS

G ra

tio



lowastlowast

lowastlowast



3 Results




0

1

2

3

4

5

6

7


MD

A co

nten

t (nm

olm

g pr

otei

n)


lowastlowast


lowast

(a)

00

05

10

15

20

25

30


Carb

onyl

cont

ent (

nmol

mg

prot

ein)


lowastlowast


lowast

(b)







Group Dose(gkg)

SOD(Umg protein)

CAT(Umg protein)

GSH-Px(Umg protein)
















(a) (b) (c)

(d) (e) (f)



4 Discussion












1825 plusmn 399





(a) (b) (c)

(d) (e) (f)













Acknowledgments



References













2O2and

















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


0

10

20

30

40

50

60


GSH

GSS

G ra

tio



lowastlowast

lowastlowast



3 Results




0

1

2

3

4

5

6

7


MD

A co

nten

t (nm

olm

g pr

otei

n)


lowastlowast


lowast

(a)

00

05

10

15

20

25

30


Carb

onyl

cont

ent (

nmol

mg

prot

ein)


lowastlowast


lowast

(b)







Group Dose(gkg)

SOD(Umg protein)

CAT(Umg protein)

GSH-Px(Umg protein)
















(a) (b) (c)

(d) (e) (f)



4 Discussion












1825 plusmn 399





(a) (b) (c)

(d) (e) (f)













Acknowledgments



References













2O2and

















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



Group Dose(gkg)

SOD(Umg protein)

CAT(Umg protein)

GSH-Px(Umg protein)
















(a) (b) (c)

(d) (e) (f)



4 Discussion












1825 plusmn 399





(a) (b) (c)

(d) (e) (f)













Acknowledgments



References













2O2and

















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of









1825 plusmn 399





(a) (b) (c)

(d) (e) (f)













Acknowledgments



References













2O2and

















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of








Acknowledgments



References













2O2and

















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of
















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

research article neuroprotective activity of water soluble...

Documents