Research Article Protective Effect of Diospyros kaki...

Research ArticleProtective Effect of Diospyros kaki againstGlucose-Oxygen-Serum Deprivation-Induced PC12 Cells Injury

Fatemeh Forouzanfar1 Shaghayegh Torabi2 Vahid R Askari3

Elham Asadpour4 and Hamid R Sadeghnia123

1Neurocognitive Research Center School of Medicine Mashhad University of Medical Sciences Mashhad 917794-8564 Iran2Pharmacological Research Center of Medicinal Plants School of Medicine Mashhad University of Medical SciencesMashhad 917794-8564 Iran3Department of Pharmacology School of Medicine Mashhad University of Medical Sciences Mashhad 917794-8564 Iran4Anesthesiology and Critical Care Research Center Shiraz University of Medical Sciences Shiraz Iran

Correspondence should be addressed to Hamid R Sadeghnia sadeghniahrmumsacir

Received 21 November 2015 Accepted 10 January 2016

Academic Editor Masahiro Oike

Copyright copy 2016 Fatemeh Forouzanfar et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Ischemic cerebrovascular disease is one of the most common causes of death in the world Recent interests have been focusedon natural antioxidants and anti-inflammatory agents as potentially useful neuroprotective agents Diospyros kaki (persimmon)has been shown to exert anti-inflammatory antioxidant and antineoplastic effects However its effects on ischemic damage havenot been evaluated Here we used an in vitro model of cerebral ischemia and studied the effects of hydroalcoholic extract of peel(PeHE) and fruit pulp (PuHE) of persimmon on cell viability andmarkers of oxidative damagemainly intracellular reactive oxygenspecies (ROS) induced by glucose-oxygen-serum deprivation (GOSD) in PC12 cells GOSD for 6 h produced significant cell deathwhich was accompanied by increased levels of ROS Pretreatment with different concentrations of PeHE and PuHE (0ndash500120583gmL)for 2 and 24 h markedly restored these changes only at high concentrations However no significant differences were seen inthe protection against ischemic insult between different extracts and the time of exposure The experimental results suggest thatpersimmonprotects the PC12 cells fromGOSD-induced injury via antioxidantmechanismsOur findingsmight raise the possibilityof potential therapeutic application of persimmon for managing cerebral ischemic and other neurodegenerative disorders

1 Introduction

Oxidative stress plays an important role in nerve cell damageand is closely related to the pathogenesis of many central ner-vous system diseases such as cerebral ischemia Alzheimerrsquosdisease and Parkinsonrsquos disease During neuropathologicalstates excessive reactive oxygen species (ROS) generationcaused significant damage to cellular macromolecules (iecellular lipids proteins or DNA) leading to cellular death [1]Deprivation of neurons from glucose-oxygen-serum depri-vation (GOSD) is a reliable in vitromodel for understandingof the molecular mechanisms of ischemia-induced neuronaldamage and also for the discovery and development of novelcompounds for better treatment of cerebral ischemia [2]

Recently there are intensive interests towards neuropro-tective properties of herbal products in ischemic brain injurybecause of relatively high therapeutic value and less seriousside effects [3] Persimmon (Diospyros kaki) which belongsto the Ebenaceae family is a deciduous small tree native toEastern Asia and has also been cultivated in NortheasternIndia Middle East Spain and many other regions The dryresidue of persimmon fruit is known to have many bioactivecompounds such as flavonoids polyphenol (especially tan-nins) carotenoids dietary fibers andminerals [4 5] Inmanytraditional medicinal systems the extract of persimmonfruit is used as antitussive carminative and sedative agentand to heal bronchial complaints and hypertension [4 5]Recently it has been shown that persimmon possesses several

Hindawi Publishing CorporationAdvances in Pharmacological SciencesVolume 2016 Article ID 3073078 5 pageshttpdxdoiorg10115520163073078

2 Advances in Pharmacological Sciences

pharmacological activities such as strong radical scavengingand antioxidant properties [6] and antigenotoxic [6] andanticarcinogenic [7 8] and anti-inflammatory [9] and anti-hypertensive [10] and antidiabetic [11] effects

To our knowledge no study has previously investigatedthe effect of persimmon fruit against cerebral ischemia invitro or in vivo Considering that persimmon has antioxidantand anti-inflammatory properties the aim of the presentstudy was to investigate the effects of hydroalcoholic extractsof persimmon fruit peel and pulp on GOSD-induced PC12cells injury Role of intracellular ROS was also investigated

2 Material and Methods

21 Cell Line and Reagents A PC12 cell line was obtainedfrom Pasteur Institute (Tehran Iran) High glucose Dul-beccorsquos Modified Eagles Medium (DMEM 45 gL) and fetalcalf serum (FCS) were purchased from Gibco (CarlsbadCA) Glucose-free DMEM 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium (MTT) 2101584071015840-dichlorodihydrofluores-cein diacetate (H

2DCF-DA) and other cell culture materials

were purchased from Sigma (St Louis MO)

22 Preparation of PuHE (Pulp Hydroalcoholic Extract) andPeHE (Peel Hydroalcoholic Extract) Fresh fruits of a com-mercial cultivar in Khorasan province (Iran) were harvestedand authenticated by herbarium of Ferdowsi University ofMashhad (Mashhad Iran voucher specimen number 11-0203-1)

The pulps and peels of whole seedless fruits were sep-arated washed and then shaken with ethanol (70) andwater for 2 days The resulting extracts were then filtered andconcentrated under reduced pressure to get pulp hydroalco-holic extract (PuHE) and peel hydroalcoholic extract (PeHE)respectively The yields were found to be about 27ww

Stock solutions of PuHE and PeHE were prepared indeionized water and desired working concentrations weremade from the stock using complete medium

23 Cell Culture PC12 cells were cultured in DMEMsupplemented with 10 FCS and 100 unitsmL of peni-cillinstreptomycin All cells weremaintained in a humidifiedatmosphere containing 5 CO

2at 37∘C [12]

24 Induction of Cell Injury by GOSD Tomimic the ischemiccondition PC12 cells were placed into in an incubationchamber containing ldquo37∘C 5 CO

2 and 95 N

2rdquo and

cultured in glucose- and serum-free DMEM supplementedwith 100UmL penicillin and 100UmL streptomycin for 6 h[2]

25 Cell Proliferation (MTT) Assay MTT was used to iden-tify viable cells which reduce it to a violet formazan dye[13] PC12 cells (5000well) were seeded out in 96-well tissueculture plates and after 24 h the cells were pretreated withPuHE and PeHE (0ndash500120583gmL) for 2 and 24 h and thensubjected to GOSD insult for 6 h respectively The concen-trations and times were chosen based on earlier experimentsControls were treated identically At 6 h after GOSD insultMTT was added to each well to achieve a final concentration

of 05mgmL After incubation at 37∘C for 4 h the mediumwas removed and 100 120583L of dimethyl sulfoxide was addedto each well and kept for 10min and the absorbance at 570and 620 nm (background) was measured using StatFAX303plate reader All experiments were carried out in triplicatethe percentage of viable cells was calculated as the mean plusmnSEM with controls set to 100

26 Measurement of Intracellular Reactive Oxygen Species(ROS) The determination of intracellular ROS levels wasaccomplished with a fluorescent probe H

2DCF-DA as

described previously with minor modifications [12] H2DCF-

DA readily diffuses through the cell membrane and isenzymatically hydrolyzed by intracellular esterases to non-fluorescent H

2DCF which is then rapidly oxidized to highly

fluorescent DCF (2101584071015840-dichlorofluorescin) in the presence ofROS The DCF fluorescence intensity is believed to parallelthe amount of intracellular ROS [14] In brief PC12 cells (104cellswell) were pretreated with different concentrations ofPuHE and PeHE (0ndash500120583gmL) for 2 h and then subjectedto GOSD for 6 h in which the same treatments were appliedAt 6 h after ischemic insult the cells were incubated with20120583M H

2DCF-DA at 37∘C for 30min in the dark The DCF

fluorescence intensity was detected using a FLUO-star galaxyfluorescence plate reader (Perkin Elmer 2030 Multilabelreader Finland) with excitation wavelength set at 485 nm andemission wavelength set at 530 nm

27 Statistical Analysis The results are presented as the meanplusmn standard error (SEM)The values were compared using theone-way analysis of variance (ANOVA) followed by Tukeyrsquospost hoc test for multiple comparisonsThe 119901 values less than005 were considered to be statistically significant

3 Results

31 Effects of PeHEandPuHEonCell Viability followingGOSDInsult in PC12 Cells Exposure to GOSD for 6 h significantlydecreased cell viability as compared with control cells Theaverage survival rate of cells under the GOSD condition wasabout 36

Pretreatment with PeHE for 2 h significantly attenuatedGOSD-induced damage to PC12 cells only at high concen-tration (500120583gmL 1105 plusmn 42 119901 lt 0001) as compared toGOSD group (370 plusmn 40) (Figure 1(a)) Also pretreatmentwith PeHE for 24 h significantly attenuated GOSD-inducedPC12 cells death at concentration of 500120583gmL (273 plusmn 100119901 lt 001) (Figure 1(b))

In the same way pretreatment with PuHE for 2 h signifi-cantly attenuated cell death induced by GOSD at concentra-tion of 500120583gmL (1050 plusmn 53 119901 lt 0001) as compared toGOSD group (350 plusmn 20) (Figure 1(c)) Again pretreatmentwith PuHE for 24 h significantly increased cell survivalfollowing ischemic insult at concentration of 500 120583gmL(575 plusmn 95 119901 lt 0001) and at concentration of 400120583gmL(290 plusmn 130 119901 lt 005) (Figure 1(d))

32 Effects of PeHE and PuHE on ROS Production followingGOSD Insult in PC12 Cells GOSD significantly increased the

Advances in Pharmacological Sciences 3

0 0

0244

0488

097

195

39

78

156

3125

625

125

250

500PeHE conc (120583gmL)

GOSD

lowastlowastlowastlowastlowastlowast

+ + + + + + + + + + + + +minus

0

20

40

60

80

100

Cel

l via

bilit

y (

)

(a)

lowastlowastlowast

lowastlowast

0 0

3125

625

125

250

400


GOSD + + + + + + +minus

0

20

40

60

80

100

Cel

l via

bilit

y (

)

(b)


Cel

l via

bilit

y (

)

GOSD

0

20

40

60

80

100

0 0

0244

0488

097

195

39

78

156

3125

625

125

250

500PuHE conc (120583gmL)

+ + + + + + + + + + + + +minus

(c)

lowastlowastlowast

lowastlowastlowast

lowast

GOSD + + + + + + +minus

PuHE conc (120583gmL)0

20

40

60

80

100

Cel

l via

bilit

y (

)

0 0

3125

625

125

250

400

500

(d)

Figure 1 Effects of hydroalcoholic extracts of fruit peel (PeHE) and pulp (PuHE) of Diospyros kaki on PC12 cells death induced by glucose-oxygen-serum deprivation (GOSD) PC12 cells were treated with PeHE for 2 h (a) and 24 h (b) or PuHE for 2 h (c) and 24 h (d) and thensubjected toGOSD for 6 h the data are presented asmeanplusmn SD from independent experiments performed in triplicate lowast119901 lt 005 lowastlowast119901 lt 001and lowastlowastlowast119901 lt 0001 as compared to ischemic group

20406080

100120140160

0ROS

cont

ent (

o

f con

trol)

PeHE conc (120583gmL) 0 0

0244

0488

097

195

39

78

156

3125

625

125

250

500

GOSD + + + + + + + + + + + + +minus

lowastlowastlowast


(a)

020406080

100120140160

GOSD + + + + + + + + + + + + +minus

ROS

cont

ent (

o

f con

trol)

PuHE conc (120583gmL) 0 0

0244

0488

097

195

39

78

156

3125

625

125

250

500

lowastlowastlowast


(b)

Figure 2 Effects of hydroalcoholic extracts of fruit peel (PeHE) and pulp (PuHE) of Diospyros kaki on ROS production in glucose-oxygen-serum deprivation- (GOSD-) treated PC12 cells PC12 cells were preincubated with PeHE (a) or PuHE (b) for 2 h prior to GOSD insultThenPC12 cells were loaded with DCF-DA and intracellular ROS were detected by fluorimetry The data are represented as mean plusmn SD fromindependent experiments performed in triplicate lowastlowastlowast119901 lt 0001 as compared to ischemic group

number of DCF-positive cells illustrating an elevation of ROSproduction to 156 As shown in Figure 2(a) pretreatmentwith PeHE for 2 h resulted in a significant (119901 lt 0001)reduction in ROS production following GOSD (1214 plusmn 20and 1104plusmn20 at concentrations of 250 and 500120583gmL resp)

As illustrated in Figure 2(b) pretreatment with PuHEfor 2 h caused a significant reduction of ROS production

following GOSD (1143 plusmn 42 119901 lt 001 and 1084 plusmn 9119901 lt 0001 at concentrations of 250 and 500120583gmL resp)

4 Discussion

According to our knowledge this is the first report of theneuroprotective effect of D kaki fruit extract In the present


study we demonstrated that pretreatment with peel and pulpfruit extracts of D kaki was able to promote cell survivaland depress ROS increase upon GOSD stress in PC12 cellsRecently substantial efforts have been invested for identifyingoptimal drugs that can reduce ischemic brain damage [2]

It is well known that ischemia increases the formation ofROS in brain tissues and antioxidants and ROS scavengerscan decrease tissue damage following ischemic injury [15ndash17]Excessive production of ROS will act as damaging moleculecausing cell death either directly through interacting anddestroying cellular proteins lipids and DNA or indirectlyby affecting normal cellular signaling pathways and generegulation [15 18]

One possible mechanism underlying the attenuation ofROS generation following ischemic insult is due to antiox-idant and free radical scavenger properties of persimmonPrevious studies have shown that flavonoids frompersimmonpeel and leaf could protect against hydrogen proxide-inducedoxidative damage in vitro [19 20] High molecular weightpersimmon tannin also ameliorates oxidative damage andcognition deficits in D-galactose-induced senescent mice[21] Tian et al showed that administration of highmolecularweight persimmon condensed tannin (HMWPT) signifi-cantly protected serum and liver antioxidant enzymes fromdamage and prevented lipid peroxidation in bromobenzene-treated mice [22] In addition several studies suggest thatpersimmon leaves extract and its flavonoids exhibit neu-roprotective effects and protect rats from both focal andglobal cerebral ischemic injury in vivo as well as corticalneurons from hypoxia-induced injury in vitro [23 24]High-performance liquid chromatography (HPLC) analysisalso revealed that gallic acid and quercetin were the majorphenolic components in the fruit extracts of persimmon [25]

Also antiaging and memory enhancing effects ofoligomeric proanthocyanidins isolated from persimmonfruits have also been described in a senescence-acceleratedmouse prone model [26ndash28] A recent study showedthat ethanol extract of Diospyros kaki leaves significantlyattenuated the upregulation of vascular endothelial growthfactor fibroblast growth factor interleukin-6 and matrixmetalloproteinase-2 (MMP-2) protein levels in alkali burn-induced corneal neovascularization in rats [29] Kim et alalso showed that ethanol extract of Diospyros kaki leavesprevented degenerative retinal diseases in N-methyl-N-nitrosourea- (MNU-) induced retinal degeneration in mice[30]

5 Conclusions

In summary the results of the current study suggest thatpersimmon is able to protect cultured PC12 cells againstdamage induced by GOSD These effects of persimmon areat least in part attributable to its antioxidant property Thisstudy on the neuroprotective effects of persimmon maysuggest the possible application of persimmon in clinicalsetting to prevent and treat the common neurological insults

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgment

This work was supported by the Office of the Vice Chancellorfor Research and Technology of Mashhad University ofMedical Sciences under Grant no 901050 The authors aregratefully acknowledging this financial support

References

[1] V Shukla S K Mishra and H C Pant ldquoOxidative stress inneurodegenerationrdquo Advances in Pharmacological Sciences vol2011 Article ID 572634 13 pages 2011

[2] C-H Wang W-J Lee V K Ghanta W-T Wang S-Y Chengand C-M Hsueh ldquoMolecules involve in the self-protection ofneurons against glucose-oxygen-serum deprivation (GOSD)-induced cell damagerdquo Brain Research Bulletin vol 79 no 3-4pp 169ndash176 2009

[3] K Suk ldquoRegulation of neuroinflammation by herbal medicineand its implications for neurodegenerative diseases a focus ontraditional medicines and flavonoidsrdquo NeuroSignals vol 14 no1-2 pp 23ndash33 2005

[4] U V Mallavadhani A K Panda and Y R Rao ldquoPharmacologyand chemotaxonomy of Diospyrosrdquo Phytochemistry vol 49 no4 pp 901ndash951 1998

[5] S Singh and H Joshi ldquoDiospyros kaki (Ebenaceae) a reviewrdquoAsian Journal of Research in Pharmaceutical Sciences vol 1 no3 pp 55ndash58 2011

[6] I-C Jang E-K Jo M-S Bae et al ldquoAntioxidant and antigeno-toxic activities of different parts of persimmon (Diospyros kakicv Fuyu) fruitrdquo Journal of Medicinal Plants Research vol 4 no2 pp 155ndash160 2010

[7] M Kawase N Motohashi K Satoh et al ldquoBiological activityof persimmon (Diospyros kaki) peel extractsrdquo PhytotherapyResearch vol 17 no 5 pp 495ndash500 2003

[8] Y Achiwa H Hibasami H Katsuzaki K Imai and T KomiyaldquoInhibitory effects of persimmon (Diospyros kaki) extract andrelated polyphenol compounds on growth of human lymphoidleukemia cellsrdquo Bioscience Biotechnology and Biochemistry vol61 no 7 pp 1099ndash1101 1997

[9] M Del Carmen Recio R M Giner S Manez et al ldquoInves-tigations on the steroidal anti-inflammatory activity of triter-penoids from Diospyros leucomelasrdquo Planta Medica vol 61 no1 pp 9ndash12 1995

[10] K Kawakami S Aketa H Sakai Y Watanabe H Nishidaand M Hirayama ldquoAntihypertensive and vasorelaxant effectsof water-soluble proanthocyanidins from persimmon leaf tea inspontaneously hypertensive ratsrdquo Bioscience Biotechnology andBiochemistry vol 75 no 8 pp 1435ndash1439 2011

[11] C Li W Bei Y Li and J Lou ldquoEthylacetate extract ofDiospyroskaki leaf for preventing and treating hyperglycemic diabetesand metabolic syndromesrdquo Faming Zhuanli Shenqing GongkaiShuomingshu vol 32 pp 21ndash23 2007

[12] F Forouzanfar A Afkhami Goli E Asadpour A Ghorbaniand H R Sadeghnia ldquoProtective effect of Punica granatum Lagainst serumglucose deprivation-induced PC12 cells injuryrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 716730 9 pages 2013

[13] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983


[14] H Wang and J A Joseph ldquoQuantifying cellular oxidativestress by dichlorofluorescein assay using microplate readerrdquoFree Radical Biology and Medicine vol 27 no 5-6 pp 612ndash6161999

[15] Y Li Y Bao B Jiang et al ldquoCatalpol protects primary culturedastrocytes from in vitro ischemia-induced damagerdquo Interna-tional Journal of Developmental Neuroscience vol 26 no 3-4pp 309ndash317 2008

[16] J Yamada S Yoshimura H Yamakawa et al ldquoCell permeableROS scavengers Tiron and Tempol rescue PC12 cell deathcaused by pyrogallol or hypoxiareoxygenationrdquo NeuroscienceResearch vol 45 no 1 pp 1ndash8 2003

[17] M Tagami K Yamagata K Ikeda et al ldquoVitamin E preventsapoptosis in cortical neurons during hypoxia and oxygenreperfusionrdquo Laboratory Investigation vol 78 no 11 pp 1415ndash1429 1998

[18] P H Chan ldquoReactive oxygen radicals in signaling and damagein the ischemic brainrdquo Journal of Cerebral Blood Flow ampMetabolism vol 21 no 1 pp 2ndash14 2001

[19] Y A Lee E J Cho and T Yokozawa ldquoProtective effect ofpersimmon (Diospyros kaki) peel proanthocyanidin againstoxidative damage under H

2O2-induced cellular senescencerdquo

Biological and Pharmaceutical Bulletin vol 31 no 6 pp 1265ndash1269 2008

[20] W Bei W Peng Y Ma and A Xu ldquoFlavonoids from the leavesof Diospyros kaki reduce hydrogen peroxide-induced injury ofNG108-15 cellsrdquo Life Sciences vol 76 no 17 pp 1975ndash1988 2005

[21] Y Tian B Zou L Yang et al ldquoHigh molecular weight per-simmon tannin ameliorates cognition deficits and attenuatesoxidative damage in senescent mice induced by D-galactoserdquoFood andChemical Toxicology vol 49 no 8 pp 1728ndash1736 2011

[22] Y Tian B Zou C-M Li J Yang S-F Xu and A E HagermanldquoHigh molecular weight persimmon tannin is a potent antioxi-dant both ex vivo and in vivordquo Food Research International vol45 no 1 pp 26ndash30 2012

[23] W Bei W Peng L Zang Z Xie D Hu and A Xu ldquoNeuropro-tective effects of a standardized extract of Diospyros kaki leaveson MCAO transient focal cerebral ischemic rats and culturedneurons injured by glutamate or hypoxiardquo Planta Medica vol73 no 7 pp 636ndash643 2007

[24] W Bei L Zang J Guo et al ldquoNeuroprotective effects ofa standardized flavonoid extract from Diospyros kaki leavesrdquoJournal of Ethnopharmacology vol 126 no 1 pp 134ndash142 2009

[25] F Pu X-L Ren and X-P Zhang ldquoPhenolic compounds andantioxidant activity in fruits of six Diospyros kaki genotypesrdquoEuropean Food Research and Technology vol 237 no 6 pp 923ndash932 2013

[26] Y A Lee E J Cho and T Yokozawa ldquoOligomeric proantho-cyanidins improve memory and enhance phosphorylation ofvascular endothelial growth factor receptor-2 in senescence-acceleratedmouse prone8rdquoBritish Journal ofNutrition vol 103no 4 pp 479ndash489 2010

[27] T Yokozawa Y A Lee E J Cho K Matsumoto C H Parkand N Shibahara ldquoAnti-aging effects of oligomeric proantho-cyanidins isolated from persimmon fruitsrdquo Drug Discoveries ampTherapeutics vol 5 no 3 pp 109ndash118 2011

[28] T Yokozawa Y A Lee Q Zhao K Matsumoto and E J CholdquoPersimmon oligomeric proanthocyanidins extend life span ofsenescence-acceleratedmicerdquo Journal ofMedicinal Food vol 12no 6 pp 1199ndash1205 2009

[29] S J Yang H Jo K-A Kim H R Ahn S W Kang and SH Jung ldquoDiospyros kaki extract inhibits alkali burn-inducedcorneal neovascularizationrdquo Journal of Medicinal Food vol 19no 1 pp 106ndash109 2016

[30] K-A Kim S W Kang H R Ahn Y Song S J Yangand S H Jung ldquoThe leaves of persimmon (Diospyros kakiThunb) ameliorate N-methyl-N-nitrosourea (MNU)-inducedretinal degeneration in micerdquo Journal of Agricultural and FoodChemistry vol 63 no 35 pp 7750ndash7759 2015

Submit your manuscripts athttpwwwhindawicom

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Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


pharmacological activities such as strong radical scavengingand antioxidant properties [6] and antigenotoxic [6] andanticarcinogenic [7 8] and anti-inflammatory [9] and anti-hypertensive [10] and antidiabetic [11] effects

To our knowledge no study has previously investigatedthe effect of persimmon fruit against cerebral ischemia invitro or in vivo Considering that persimmon has antioxidantand anti-inflammatory properties the aim of the presentstudy was to investigate the effects of hydroalcoholic extractsof persimmon fruit peel and pulp on GOSD-induced PC12cells injury Role of intracellular ROS was also investigated

2 Material and Methods

21 Cell Line and Reagents A PC12 cell line was obtainedfrom Pasteur Institute (Tehran Iran) High glucose Dul-beccorsquos Modified Eagles Medium (DMEM 45 gL) and fetalcalf serum (FCS) were purchased from Gibco (CarlsbadCA) Glucose-free DMEM 3-(45-dimethylthiazol-2-yl)-25-diphenyl tetrazolium (MTT) 2101584071015840-dichlorodihydrofluores-cein diacetate (H

2DCF-DA) and other cell culture materials

were purchased from Sigma (St Louis MO)

22 Preparation of PuHE (Pulp Hydroalcoholic Extract) andPeHE (Peel Hydroalcoholic Extract) Fresh fruits of a com-mercial cultivar in Khorasan province (Iran) were harvestedand authenticated by herbarium of Ferdowsi University ofMashhad (Mashhad Iran voucher specimen number 11-0203-1)

The pulps and peels of whole seedless fruits were sep-arated washed and then shaken with ethanol (70) andwater for 2 days The resulting extracts were then filtered andconcentrated under reduced pressure to get pulp hydroalco-holic extract (PuHE) and peel hydroalcoholic extract (PeHE)respectively The yields were found to be about 27ww

Stock solutions of PuHE and PeHE were prepared indeionized water and desired working concentrations weremade from the stock using complete medium

23 Cell Culture PC12 cells were cultured in DMEMsupplemented with 10 FCS and 100 unitsmL of peni-cillinstreptomycin All cells weremaintained in a humidifiedatmosphere containing 5 CO

2at 37∘C [12]

24 Induction of Cell Injury by GOSD Tomimic the ischemiccondition PC12 cells were placed into in an incubationchamber containing ldquo37∘C 5 CO

2 and 95 N

2rdquo and

cultured in glucose- and serum-free DMEM supplementedwith 100UmL penicillin and 100UmL streptomycin for 6 h[2]

25 Cell Proliferation (MTT) Assay MTT was used to iden-tify viable cells which reduce it to a violet formazan dye[13] PC12 cells (5000well) were seeded out in 96-well tissueculture plates and after 24 h the cells were pretreated withPuHE and PeHE (0ndash500120583gmL) for 2 and 24 h and thensubjected to GOSD insult for 6 h respectively The concen-trations and times were chosen based on earlier experimentsControls were treated identically At 6 h after GOSD insultMTT was added to each well to achieve a final concentration

of 05mgmL After incubation at 37∘C for 4 h the mediumwas removed and 100 120583L of dimethyl sulfoxide was addedto each well and kept for 10min and the absorbance at 570and 620 nm (background) was measured using StatFAX303plate reader All experiments were carried out in triplicatethe percentage of viable cells was calculated as the mean plusmnSEM with controls set to 100

26 Measurement of Intracellular Reactive Oxygen Species(ROS) The determination of intracellular ROS levels wasaccomplished with a fluorescent probe H

2DCF-DA as

described previously with minor modifications [12] H2DCF-

DA readily diffuses through the cell membrane and isenzymatically hydrolyzed by intracellular esterases to non-fluorescent H

2DCF which is then rapidly oxidized to highly

fluorescent DCF (2101584071015840-dichlorofluorescin) in the presence ofROS The DCF fluorescence intensity is believed to parallelthe amount of intracellular ROS [14] In brief PC12 cells (104cellswell) were pretreated with different concentrations ofPuHE and PeHE (0ndash500120583gmL) for 2 h and then subjectedto GOSD for 6 h in which the same treatments were appliedAt 6 h after ischemic insult the cells were incubated with20120583M H

2DCF-DA at 37∘C for 30min in the dark The DCF

fluorescence intensity was detected using a FLUO-star galaxyfluorescence plate reader (Perkin Elmer 2030 Multilabelreader Finland) with excitation wavelength set at 485 nm andemission wavelength set at 530 nm

27 Statistical Analysis The results are presented as the meanplusmn standard error (SEM)The values were compared using theone-way analysis of variance (ANOVA) followed by Tukeyrsquospost hoc test for multiple comparisonsThe 119901 values less than005 were considered to be statistically significant

3 Results

31 Effects of PeHEandPuHEonCell Viability followingGOSDInsult in PC12 Cells Exposure to GOSD for 6 h significantlydecreased cell viability as compared with control cells Theaverage survival rate of cells under the GOSD condition wasabout 36

Pretreatment with PeHE for 2 h significantly attenuatedGOSD-induced damage to PC12 cells only at high concen-tration (500120583gmL 1105 plusmn 42 119901 lt 0001) as compared toGOSD group (370 plusmn 40) (Figure 1(a)) Also pretreatmentwith PeHE for 24 h significantly attenuated GOSD-inducedPC12 cells death at concentration of 500120583gmL (273 plusmn 100119901 lt 001) (Figure 1(b))

In the same way pretreatment with PuHE for 2 h signifi-cantly attenuated cell death induced by GOSD at concentra-tion of 500120583gmL (1050 plusmn 53 119901 lt 0001) as compared toGOSD group (350 plusmn 20) (Figure 1(c)) Again pretreatmentwith PuHE for 24 h significantly increased cell survivalfollowing ischemic insult at concentration of 500 120583gmL(575 plusmn 95 119901 lt 0001) and at concentration of 400120583gmL(290 plusmn 130 119901 lt 005) (Figure 1(d))

32 Effects of PeHE and PuHE on ROS Production followingGOSD Insult in PC12 Cells GOSD significantly increased the


0 0

0244

0488

097

195

39

78

156

3125

625

125

250


GOSD


+ + + + + + + + + + + + +minus

0

20

40

60

80

100

Cel

l via

bilit

y (

)

(a)

lowastlowastlowast

lowastlowast

0 0

3125

625

125

250

400


GOSD + + + + + + +minus

0

20

40

60

80

100

Cel

l via

bilit

y (

)

(b)


Cel

l via

bilit

y (

)

GOSD

0

20

40

60

80

100

0 0

0244

0488

097

195

39

78

156

3125

625

125

250


+ + + + + + + + + + + + +minus

(c)

lowastlowastlowast

lowastlowastlowast

lowast

GOSD + + + + + + +minus


20

40

60

80

100

Cel

l via

bilit

y (

)

0 0

3125

625

125

250

400

500

(d)


20406080

100120140160

0ROS

cont

ent (

o

f con

trol)


0244

0488

097

195

39

78

156

3125

625

125

250

500

GOSD + + + + + + + + + + + + +minus

lowastlowastlowast


(a)

020406080

100120140160

GOSD + + + + + + + + + + + + +minus

ROS

cont

ent (

o

f con

trol)


0244

0488

097

195

39

78

156

3125

625

125

250

500

lowastlowastlowast


(b)





4 Discussion







5 Conclusions




Acknowledgment


References






































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0 0

0244

0488

097

195

39

78

156

3125

625

125

250


GOSD


+ + + + + + + + + + + + +minus

0

20

40

60

80

100

Cel

l via

bilit

y (

)

(a)

lowastlowastlowast

lowastlowast

0 0

3125

625

125

250

400


GOSD + + + + + + +minus

0

20

40

60

80

100

Cel

l via

bilit

y (

)

(b)


Cel

l via

bilit

y (

)

GOSD

0

20

40

60

80

100

0 0

0244

0488

097

195

39

78

156

3125

625

125

250


+ + + + + + + + + + + + +minus

(c)

lowastlowastlowast

lowastlowastlowast

lowast

GOSD + + + + + + +minus


20

40

60

80

100

Cel

l via

bilit

y (

)

0 0

3125

625

125

250

400

500

(d)


20406080

100120140160

0ROS

cont

ent (

o

f con

trol)


0244

0488

097

195

39

78

156

3125

625

125

250

500

GOSD + + + + + + + + + + + + +minus

lowastlowastlowast


(a)

020406080

100120140160

GOSD + + + + + + + + + + + + +minus

ROS

cont

ent (

o

f con

trol)


0244

0488

097

195

39

78

156

3125

625

125

250

500

lowastlowastlowast


(b)





4 Discussion







5 Conclusions




Acknowledgment


References






































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






5 Conclusions




Acknowledgment


References






































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

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