# Zingiber officinale Mitigates Brain Damage and Improves Memory Impairment in Focal Cerebral Ischemic Rat

Authors: Jintanaporn  Wattanathorn; Jinatta Jittiwat; Terdthai Tongun; Supaporn Muchimapura; Kornkanok Ingkaninan
Journal: Evidence-Based Complementary and Alternative Medicine
 (2011)

---

## Abstract

Cerebral ischemia  is  known to produce brain damage and  related  behavioral deficits  including  memory.  Recently,  accumulating  lines  of  evidence  showed that  dietary  enrichment  with  nutritional  antioxidants  could  reduce  brain  damage and improve cognitive  function.  In this study,  possible  protective  effect  ofZingiber officinale, a medicinal  plant  reputed  for  neuroprotective  effect  against  oxidative  stress-related  brain  damage,  on  brain  damage  and  memory  deficit induced  by  focal cerebral  ischemia was elucidated.  Male  adult  Wistar  rats  were  administrated  an  alcoholic  extract  of  ginger  rhizome orally 14  days  before  and  21  days  after  the permanent  occlusion  of  right  middle  cerebral  artery  (MCAO).  Cognitive function  assessment  was  performed  at  7,  14,  and  21  days  after  MCAO  using the Morris  water  maze  test.  The  brain  infarct volume and density  of  neurons  in  hippocampus were also determined.  Furthermore,  the  level  of malondialdehyde (MDA),  superoxide  dismutase  (SOD),  catalase  (CAT),  and glutathione  peroxidase  (GSH-Px)  in  cerebral  cortex,  striatum,  and  hippocampus was  also  quantified  at  the  end of  experiment.  The  results  showed  that  cognitive function  and  neurons  density  in  hippocampus  of  rats  receiving  ginger rhizome  extract  were  improved  while  the  brain  infarct  volume  was  decreased. The  cognitive enhancing  effect  and  neuroprotective  effect  occurred  partly  via  the antioxidant  activity  of  the  extract.  In  conclusion, our  study  demonstrated  the beneficial  effect  of  ginger  rhizome  to  protect  against  focal  cerebral  ischemia.


---

## Body

## 1. Introduction

Cerebral ischemia  is  known to  produce   brain  damage  and  related  behavioral deficits  including  memory  deficit  and  motor  disorder.  It has been reported  that  the  middle  cerebral  artery  occlusion  occurred  in  10–15%  of stroke  patients [1].  The  main  areas  affected by middle  artery  occlusion are  the cerebral  cortex,  the hippocampus,  and  the striatum [2].  Memory  and  motor  deficits  are  associated  with interruption  of  blood  flow  to the above  areas [3–7].Free radicals  are  produced  in  cells  by  cellular metabolism  and  by  exogenous  agents.  As age advances,  production  of  free  radicals  also  increases.  These  react  with  biomolecules  in  the brain  to produce  neurodegeneration  and  memory  impairment [8].  Accumulating lines  of  evidence  show  that  dietary  enrichment  with  nutritional  antioxidants could  improve  brain  damage  and  cognitive  function [9, 10].  Substances  possessing  antioxidant  properties  improve  the  cognitive  function  not  only  in  normal subjects  but  also  in  cognitive  deficits  after  stroke [11–13].Zingiber officinaleRoscoe  or  ginger,  member of  the  family  of   Zingiberaceae, is widely  used  as a spice.  Moreover,  it  is  used  in  Asian traditional  medicine  for  various  purposes  including  stomach  ache [14],  nausea and  diarrhea,  and joint  and  muscle  pain [15].  In addition  to  the  effects  mentioned above,  ginger  extract  also  possesses  antioxidant  activity [16–20], neuroprotective  effect [21], and   anxiolytic  effect [22].  Because  Z. officinalecould scavenge   free  radicals,  an  important  factor  in producing  brain  damage  induced  by  cerebral  ischemia,  we  hypothesized  that  the  Z. officinaleextract might be  able  to  protect  against  brain  damage  and  memory  impairments  induced by  focal  cerebral  ischemia  via  reduction  of  oxidative  stress.  The objective  of  this  study  is  to determine  possible effects  of Z. officinaleextract  on the  cognitive  deficit,  brain  infarct  volume,  histopathological  changes,  and oxidative  stress  after  occlusion  of the right  middle  cerebral  artery in rats.


## 2. Materials and Methods

### 2.1. Plant  Material  and  the  Preparation  of  the  Extract

Zingiber officinalerhizomes  were  collected  from  Amphoe  Kathum,  Phitsanulok, Thailand,  authenticated  and  prepared  as  analcoholic  extract  by  Associate  Professor Kornkanok  Ingkaninan,  Department  of Pharmaceutical  Chemistry  and Pharmacognosy,  Faculty  of  Pharmaceutical  Sciences,  Naresuan  University, Thailand. A  voucher  specimen  was  also  deposited  at  the  department mentioned above.   26 kg  of  dried  plant  rhizome  powder  was  refluxed  in 45 kg  of   95% ethanol  for  3  hours, and  the  extract    was filtrated.   The residue  was  further  refluxed  with  35 kg  of   95%  ethanol  for  two  times, and  after this  process,  the  extract  was  combined  and  dried  by  a  freeze  dryer.  The  percent yield  of  the  final  product  was  11.54  and  contained  gingerol 6.78  ±  0.13%. The  alcoholic  extract  of Z. officinale was  prepared  as  suspending  agent  with carboxymethyl  cellulose  to  facilitate  administration  via  the  oral  route (gavage).


### 2.2. Animals

Male Wistar  rats  weighing  300–350 gm  (8 weeks old)  were  obtained  from National Laboratory  Animal  Center,  Salaya,  Nakorn  Pathom  and  were  housed in group  of  5  per  cage  in  standard  metal  cages  at  22  ±  2°C   on  10 : 14 h  light-dark  cycle.  All  animals  were  given  free  access  to  food  and  water  ad  libitum. All  efforts  were  made  to  minimize  animal  suffering  in  accordance directives  for the  laboratory  use  and  care  of animals,  issued  by  the European  Community  (EEC directive of 1986; 86/609/EEC).The experimental  protocols  were  approved  by  the  Institutional  Animal  Care  and Use Committee.


### 2.3. Focal Cerebral Ischemia Induction

All  rats  were  fasted  for  12 hr  but  were  allowed  free  access to  water  before surgery.   Anesthesia   was   induced   with   intraperitoneal   injection   of   thiopental sodium at  dose  of  50 mg/kg body weight.  Focal cerebral  ischemia  was  induced as  described  by  Longa et al. [23].  Briefly,  the right  common  carotid  artery  and  the  right  external carotid  artery  were  exposed through a  ventral  midline  neck  incision and  were  ligated  proximally. A  silicone-coated   nylon   monofilament (4-0) suture (USS DG sutures;  Tyco   Healthcare group LP, Connecticut, USA)  with  its tip  rounded  by  heating  near  a  flame  was inserted through  an arteriectomy  in  the  common  carotid  artery  just  below  the carotid bifurcation  and advanced  into the  internal  carotid  artery  approximately 17 to 18 mm  distal to the carotid   bifurcation  until  a mild resistance was  felt. Occlusion  of  the  origins  of the  anterior  cerebral  artery,  the middle   cerebral   artery,   and  the   posterior   communicating   artery  was   thereby achieved.  The  wound  was  sutured,  and the  rats  were  returned to  their  cages with  free  access  to  food and  water.  The  incision sites  were  infiltrated  with 10% povidone-iodine  solution  for  antiseptic  postoperative  care.


### 2.4. Morris Water Maze Test

The water  maze  consisted  of  a  metal  pool (170 cm in diameter  ×  58 cm tall) filled with  tap  water  (25°C, 40 cm deep)  divided  into  4  quadrants.  In  the  center of  1  quadrant  was  a  removable  escape  platform  below  the  water  level  and covered  with  a  nontoxic  milk  powder.  The  pool  was  divided  into  4  quadrants (NE, NW, SE, and SW)  by  two  imaginary  lines  crossing  the  center  of  the  pool. For  each  animal,  the  location  of  invisible  platform  was  placed  at  the  center of one  quadrant  and  remained  there  throughout  training.  The rats  must  remember location  of  the  platform  in  relation  to  various  environmental  cues.  Each  rat  was  gently  placed  in  the  water  facing the  wall  of  the  pool  from  one  of  the  four  starting  points  (N, E, S, or  W)  along the  perimeter  of  the pool,  and  the  animal  was  allowed  to  swim  until  it  found and  climbed  onto  the  platform.  During  the training  sessions,  the  rat was  gently placed  on  the  platform  by  the  experimenter  when  it  could not  reach  the platform in  60 s.  In  either  case,  the  rat  was  left  on  the  platform  for  15 s and  removed  from  the  pool.  The  time  for  the  animals  to  climb  on  the  hidden  platform was recorded  as  escape  latency.  Retention  memory  was  also determined  on  the  next  day.  The  platform  was  removed  and  the  animals  were  placed  into  the  water maze  for  60 s.  The  retention  of  the memory  or  the  time  that  the  animal  spent  to  swim  around  the location  of  the  platform   before  it  was removed  was  recorded.If  the  spatial  memory  of  the  rat  for the  trained  platform location  is  accurate,  the  rat  will  swim  to  the  platform  location  and  search  around the  exact  location.  Therefore,  the  more  accurate  the  spatial  memory   is, the   greater  the  number  of  times  the  rat  will  swim  across  precious  location  of  platform.  In  each trial,  the  animal  was  quickly  dried  with  towel  before  being  returned  to  the  cage [24].  All  Morris  water  maze  tests   were  carried  out  within  30 minutes  after  the  oral  administration  of  the  test  substances.


### 2.5. Determination of Infarct Volume

All animals  were  killed  24  hours  after  middle  cerebral  artery  occlusion,  and  the  brain  was  removed and  sectioned  at 2-mm  thickness. Sections  were  immersed  in  2% TTC (2, 3, 5-triphenyltetrazolium chloride)  for  30  minutes  at  37°C.   Images  of  stained sections  were  digitized,  and  infarction  volumes  were  determined  using  an indirect  method.


### 2.6. Histological Procedure

Following  anesthesia  with  sodium  pentobarbital  (60 mg/kg  body weight),  the  rats  were  transcardially  perfused with  fixative  containing  4% paraformaldehyde  in  0.1 M  phosphate  buffer  pH 7.3. The  brains  were  removed   and  stored  over   night in  the  same  fixative.  They  were  infiltrated  with  30% sucrose  solution  and  kept  at 4°C.  The  specimens  were frozen  rapidly  and  30μM  thick sections  were  cut  on  a  cryostat. The sections  were  rinsed  in   phosphate  buffer  and  picked  up  on slides  coated  with  0.01% of  poly L-lysine.


### 2.7. Cresyl Violet  Staining

Coronal  sections  of  the  brains  were  stained  with  0.75% cresyl  violet, dehydrated  through  graded alcohols (70, 95, 100% 2x)  and  xylene,  and coverslipped  using  DPX mountant.


### 2.8. Morphological Analysis

Five coronal  sections  of  each  rat  in  each  group  were  studied  quantitatively. Neuronal  counts  in  hippocampus  were  performed  by  eye  using a 40X objective with final field  255μm2  according  to  the  following  stereotaxic  coordinates:  AP-4.8 mm,  lateral ±2.4–6 mm,  and depth 3–8 mm.  The  observer was  blind  to  the treatment at  the  time  of  analysis.  Viable  stained  neurons  were  identified  on  the basis  of  a  stained  soma  with  at  least  two  visible  processes.  Counts  were  made in  five adjacent  fields  and  the  mean  number  extrapolated  to  give  the  total  number of neurons  per  255 μm2. All  data  were  represented  as  number  of  neurons  per  255 μm2.


### 2.9. Determination of  Antioxidant  Enzymes  Activities

In order  to  determine  the  activities  of  antioxidant  enzymes including  superoxide dismutase  (SOD),   catalase  (CAT),  and  glutathione  peroxidase  (GSH-Px),  the ischemic  brains  tissues  were  weighed  and  homogenized  with  a  buffer  consisting of  10 mM  sucrose,  10 mM  Tris–HCl,  and 0.1 mM EDTA (pH 7.4)  and  then centrifuged  at  3000 g  for 15 min  at  4°C.  The  supernatant  was  used  for bioassays.  The  activity  of  SOD  was  determined  using  a  xanthine/xanthine oxidase  system  for  production  of  superoxide  radical  and  subsequent measurement of   cytochromec as  a  scavenger  of  the  radicals.  Optical   density was  determined  using  a  spectrometer  (UV-1601, Shimadzu)  at   550 nm.  One unit  of  enzyme  activity  was  defined  as  the  quantity  of   SOD  required  to inhibit  the  rate  of  reduction  of  cytochrome c  by  50% [25].  SOD  activity  is  presented as  units  per  milligram  of  protein  (U mg−1  protein). Catalase  activity  in  the supernatant was   measured  by  recording  the  rate  of  decrease  in  H2O2 absorbance at  240 nm [26].  The  activity  of catalase  was  expressed  as  μmol H2O2/min/mg protein.  GSH-Px  was  determined  using t-butylhydroperoxide  as  a substrate.  The  optical  density  was  spectrophotometrically  recorded  at  340 nm. One  unit  of the  enzyme  was  defined  as  micromoles (μmol)  of  reduced nicotinamide  adenine  dinucleotide  phosphate  (NADPH)  oxidized  per minute [27]. The  GSH-Px  activity  was  expressed  as  U/mg protein.


### 2.10. Determination of  Malondialdehyde  Level

Brains  regions of  the  rats  including  the  cerebral  cortex,  striatum,  and  hippocampus were  isolated  and  prepared  as  brain  homogenates  as  mentioned  above.  The  level of  malondialdehyde  in  the  brain  homogenates  was  estimated  by  determining the  accumulation  of  thiobarbituric  acid  reactive  substances (TBARSs)  according to  the  method  of  Ohkawa  et  al. [28].


### 2.11. Experimental Protocol

The animals  were  divided  into  7  groups.  Each  group  comprised  of  6  animals. In this  study,  the  doses  of  the  test  substances  were  selected  based  on  our  pilot study  and  earlier  reports.Group I.  This  served  as  the  control  group.  The  animals  were  orally  given carboxymethylcellulose  which  was used  as  vehicle  to  dissolve  the  tested  substance.Group II.  Aricept  (donepezil),  an  acetylcholinesterase  enzyme  activity inhibitor which was used  as  standard  drug to  improve  memory,  was  orally  given  at  dose  of 1 mg/kg  body weight  and  used  as  a positive  control.Group III.  Vitamin  C,  a  well-known  antioxidant  showing  memory  improvement, was orally  administered  at  dose  of  250 mg/kg  body weight  and  served  as positive  control.Group IV.  Piracetam,  a  standard  drug  which increases  cerebral  blood  flow,  was administered to  the  rat  orally  at  dose  of  250 mg/kg  body weight  and  served  as a  positive  control.Group V-VII.  The animals  were  orally  given Z. officinaleat  doses  of  100,  200, and 300 mg/kg  body weight.The animals  in  all  groups  were    given  the  test substances orally once  daily  at  a period of  14  days  before  and  21 days  after  the  occlusion  of  right  middle  cerebral artery  (MCAO).  Spatial  memory  was assessed   at   7,  14,  and  21 days  after  MCAO.  The  brain  infarct  volume  was  also determined  24 hr  after  MCAO.In order  to  determine  the  effect  ofZ. officinaleon  the  alteration  of malondialdehyde  level  and  the  activities  of  antioxidant  enzymes,  the  animals were  divided  as  mentioned  above.  The Z. officinale-treated  group  which  was selected  for  further  study  in  this  part  was  the Z. officinaleat  dose  which produced  optimum  changes  on  learning  memory and  infarct  volume.  The  brains of  all  rats  were  isolated  and  prepared  the  brain  homogenate  at  24 hr  after MCAO.  Then, the  level  of  MDA  and  the  activities of  antioxidant  enzymes  in the  brain  homogenate  were  estimated.


### 2.12. Statistical Analysis

Data were  presented  as  mean  ±  standard  error  of  mean (S.E.M).  The  analysis was  performed  using  one-way  analysis  of  variance (ANOVA),  followed  by Dunnett’s  test.  All  statistical  results  were  considered  significant  ifP-value  <  .05.


## 2.1. Plant  Material  and  the  Preparation  of  the  Extract

Zingiber officinalerhizomes  were  collected  from  Amphoe  Kathum,  Phitsanulok, Thailand,  authenticated  and  prepared  as  analcoholic  extract  by  Associate  Professor Kornkanok  Ingkaninan,  Department  of Pharmaceutical  Chemistry  and Pharmacognosy,  Faculty  of  Pharmaceutical  Sciences,  Naresuan  University, Thailand. A  voucher  specimen  was  also  deposited  at  the  department mentioned above.   26 kg  of  dried  plant  rhizome  powder  was  refluxed  in 45 kg  of   95% ethanol  for  3  hours, and  the  extract    was filtrated.   The residue  was  further  refluxed  with  35 kg  of   95%  ethanol  for  two  times, and  after this  process,  the  extract  was  combined  and  dried  by  a  freeze  dryer.  The  percent yield  of  the  final  product  was  11.54  and  contained  gingerol 6.78  ±  0.13%. The  alcoholic  extract  of Z. officinale was  prepared  as  suspending  agent  with carboxymethyl  cellulose  to  facilitate  administration  via  the  oral  route (gavage).


## 2.2. Animals

Male Wistar  rats  weighing  300–350 gm  (8 weeks old)  were  obtained  from National Laboratory  Animal  Center,  Salaya,  Nakorn  Pathom  and  were  housed in group  of  5  per  cage  in  standard  metal  cages  at  22  ±  2°C   on  10 : 14 h  light-dark  cycle.  All  animals  were  given  free  access  to  food  and  water  ad  libitum. All  efforts  were  made  to  minimize  animal  suffering  in  accordance directives  for the  laboratory  use  and  care  of animals,  issued  by  the European  Community  (EEC directive of 1986; 86/609/EEC).The experimental  protocols  were  approved  by  the  Institutional  Animal  Care  and Use Committee.


## 2.3. Focal Cerebral Ischemia Induction

All  rats  were  fasted  for  12 hr  but  were  allowed  free  access to  water  before surgery.   Anesthesia   was   induced   with   intraperitoneal   injection   of   thiopental sodium at  dose  of  50 mg/kg body weight.  Focal cerebral  ischemia  was  induced as  described  by  Longa et al. [23].  Briefly,  the right  common  carotid  artery  and  the  right  external carotid  artery  were  exposed through a  ventral  midline  neck  incision and  were  ligated  proximally. A  silicone-coated   nylon   monofilament (4-0) suture (USS DG sutures;  Tyco   Healthcare group LP, Connecticut, USA)  with  its tip  rounded  by  heating  near  a  flame  was inserted through  an arteriectomy  in  the  common  carotid  artery  just  below  the carotid bifurcation  and advanced  into the  internal  carotid  artery  approximately 17 to 18 mm  distal to the carotid   bifurcation  until  a mild resistance was  felt. Occlusion  of  the  origins  of the  anterior  cerebral  artery,  the middle   cerebral   artery,   and  the   posterior   communicating   artery  was   thereby achieved.  The  wound  was  sutured,  and the  rats  were  returned to  their  cages with  free  access  to  food and  water.  The  incision sites  were  infiltrated  with 10% povidone-iodine  solution  for  antiseptic  postoperative  care.


## 2.4. Morris Water Maze Test

The water  maze  consisted  of  a  metal  pool (170 cm in diameter  ×  58 cm tall) filled with  tap  water  (25°C, 40 cm deep)  divided  into  4  quadrants.  In  the  center of  1  quadrant  was  a  removable  escape  platform  below  the  water  level  and covered  with  a  nontoxic  milk  powder.  The  pool  was  divided  into  4  quadrants (NE, NW, SE, and SW)  by  two  imaginary  lines  crossing  the  center  of  the  pool. For  each  animal,  the  location  of  invisible  platform  was  placed  at  the  center of one  quadrant  and  remained  there  throughout  training.  The rats  must  remember location  of  the  platform  in  relation  to  various  environmental  cues.  Each  rat  was  gently  placed  in  the  water  facing the  wall  of  the  pool  from  one  of  the  four  starting  points  (N, E, S, or  W)  along the  perimeter  of  the pool,  and  the  animal  was  allowed  to  swim  until  it  found and  climbed  onto  the  platform.  During  the training  sessions,  the  rat was  gently placed  on  the  platform  by  the  experimenter  when  it  could not  reach  the platform in  60 s.  In  either  case,  the  rat  was  left  on  the  platform  for  15 s and  removed  from  the  pool.  The  time  for  the  animals  to  climb  on  the  hidden  platform was recorded  as  escape  latency.  Retention  memory  was  also determined  on  the  next  day.  The  platform  was  removed  and  the  animals  were  placed  into  the  water maze  for  60 s.  The  retention  of  the memory  or  the  time  that  the  animal  spent  to  swim  around  the location  of  the  platform   before  it  was removed  was  recorded.If  the  spatial  memory  of  the  rat  for the  trained  platform location  is  accurate,  the  rat  will  swim  to  the  platform  location  and  search  around the  exact  location.  Therefore,  the  more  accurate  the  spatial  memory   is, the   greater  the  number  of  times  the  rat  will  swim  across  precious  location  of  platform.  In  each trial,  the  animal  was  quickly  dried  with  towel  before  being  returned  to  the  cage [24].  All  Morris  water  maze  tests   were  carried  out  within  30 minutes  after  the  oral  administration  of  the  test  substances.


## 2.5. Determination of Infarct Volume

All animals  were  killed  24  hours  after  middle  cerebral  artery  occlusion,  and  the  brain  was  removed and  sectioned  at 2-mm  thickness. Sections  were  immersed  in  2% TTC (2, 3, 5-triphenyltetrazolium chloride)  for  30  minutes  at  37°C.   Images  of  stained sections  were  digitized,  and  infarction  volumes  were  determined  using  an indirect  method.


## 2.6. Histological Procedure

Following  anesthesia  with  sodium  pentobarbital  (60 mg/kg  body weight),  the  rats  were  transcardially  perfused with  fixative  containing  4% paraformaldehyde  in  0.1 M  phosphate  buffer  pH 7.3. The  brains  were  removed   and  stored  over   night in  the  same  fixative.  They  were  infiltrated  with  30% sucrose  solution  and  kept  at 4°C.  The  specimens  were frozen  rapidly  and  30μM  thick sections  were  cut  on  a  cryostat. The sections  were  rinsed  in   phosphate  buffer  and  picked  up  on slides  coated  with  0.01% of  poly L-lysine.


## 2.7. Cresyl Violet  Staining

Coronal  sections  of  the  brains  were  stained  with  0.75% cresyl  violet, dehydrated  through  graded alcohols (70, 95, 100% 2x)  and  xylene,  and coverslipped  using  DPX mountant.


## 2.8. Morphological Analysis

Five coronal  sections  of  each  rat  in  each  group  were  studied  quantitatively. Neuronal  counts  in  hippocampus  were  performed  by  eye  using a 40X objective with final field  255μm2  according  to  the  following  stereotaxic  coordinates:  AP-4.8 mm,  lateral ±2.4–6 mm,  and depth 3–8 mm.  The  observer was  blind  to  the treatment at  the  time  of  analysis.  Viable  stained  neurons  were  identified  on  the basis  of  a  stained  soma  with  at  least  two  visible  processes.  Counts  were  made in  five adjacent  fields  and  the  mean  number  extrapolated  to  give  the  total  number of neurons  per  255 μm2. All  data  were  represented  as  number  of  neurons  per  255 μm2.


## 2.9. Determination of  Antioxidant  Enzymes  Activities

In order  to  determine  the  activities  of  antioxidant  enzymes including  superoxide dismutase  (SOD),   catalase  (CAT),  and  glutathione  peroxidase  (GSH-Px),  the ischemic  brains  tissues  were  weighed  and  homogenized  with  a  buffer  consisting of  10 mM  sucrose,  10 mM  Tris–HCl,  and 0.1 mM EDTA (pH 7.4)  and  then centrifuged  at  3000 g  for 15 min  at  4°C.  The  supernatant  was  used  for bioassays.  The  activity  of  SOD  was  determined  using  a  xanthine/xanthine oxidase  system  for  production  of  superoxide  radical  and  subsequent measurement of   cytochromec as  a  scavenger  of  the  radicals.  Optical   density was  determined  using  a  spectrometer  (UV-1601, Shimadzu)  at   550 nm.  One unit  of  enzyme  activity  was  defined  as  the  quantity  of   SOD  required  to inhibit  the  rate  of  reduction  of  cytochrome c  by  50% [25].  SOD  activity  is  presented as  units  per  milligram  of  protein  (U mg−1  protein). Catalase  activity  in  the supernatant was   measured  by  recording  the  rate  of  decrease  in  H2O2 absorbance at  240 nm [26].  The  activity  of catalase  was  expressed  as  μmol H2O2/min/mg protein.  GSH-Px  was  determined  using t-butylhydroperoxide  as  a substrate.  The  optical  density  was  spectrophotometrically  recorded  at  340 nm. One  unit  of the  enzyme  was  defined  as  micromoles (μmol)  of  reduced nicotinamide  adenine  dinucleotide  phosphate  (NADPH)  oxidized  per minute [27]. The  GSH-Px  activity  was  expressed  as  U/mg protein.


## 2.10. Determination of  Malondialdehyde  Level

Brains  regions of  the  rats  including  the  cerebral  cortex,  striatum,  and  hippocampus were  isolated  and  prepared  as  brain  homogenates  as  mentioned  above.  The  level of  malondialdehyde  in  the  brain  homogenates  was  estimated  by  determining the  accumulation  of  thiobarbituric  acid  reactive  substances (TBARSs)  according to  the  method  of  Ohkawa  et  al. [28].


## 2.11. Experimental Protocol

The animals  were  divided  into  7  groups.  Each  group  comprised  of  6  animals. In this  study,  the  doses  of  the  test  substances  were  selected  based  on  our  pilot study  and  earlier  reports.Group I.  This  served  as  the  control  group.  The  animals  were  orally  given carboxymethylcellulose  which  was used  as  vehicle  to  dissolve  the  tested  substance.Group II.  Aricept  (donepezil),  an  acetylcholinesterase  enzyme  activity inhibitor which was used  as  standard  drug to  improve  memory,  was  orally  given  at  dose  of 1 mg/kg  body weight  and  used  as  a positive  control.Group III.  Vitamin  C,  a  well-known  antioxidant  showing  memory  improvement, was orally  administered  at  dose  of  250 mg/kg  body weight  and  served  as positive  control.Group IV.  Piracetam,  a  standard  drug  which increases  cerebral  blood  flow,  was administered to  the  rat  orally  at  dose  of  250 mg/kg  body weight  and  served  as a  positive  control.Group V-VII.  The animals  were  orally  given Z. officinaleat  doses  of  100,  200, and 300 mg/kg  body weight.The animals  in  all  groups  were    given  the  test substances orally once  daily  at  a period of  14  days  before  and  21 days  after  the  occlusion  of  right  middle  cerebral artery  (MCAO).  Spatial  memory  was assessed   at   7,  14,  and  21 days  after  MCAO.  The  brain  infarct  volume  was  also determined  24 hr  after  MCAO.In order  to  determine  the  effect  ofZ. officinaleon  the  alteration  of malondialdehyde  level  and  the  activities  of  antioxidant  enzymes,  the  animals were  divided  as  mentioned  above.  The Z. officinale-treated  group  which  was selected  for  further  study  in  this  part  was  the Z. officinaleat  dose  which produced  optimum  changes  on  learning  memory and  infarct  volume.  The  brains of  all  rats  were  isolated  and  prepared  the  brain  homogenate  at  24 hr  after MCAO.  Then, the  level  of  MDA  and  the  activities of  antioxidant  enzymes  in the  brain  homogenate  were  estimated.


## 2.12. Statistical Analysis

Data were  presented  as  mean  ±  standard  error  of  mean (S.E.M).  The  analysis was  performed  using  one-way  analysis  of  variance (ANOVA),  followed  by Dunnett’s  test.  All  statistical  results  were  considered  significant  ifP-value  <  .05.


## 3. Results

### 3.1.Z. officinale on  Spatial  Memory

Effect ofZ. officinaleon  spatial  memory  was  determined  using  escape  latency and  retention  time  as  indices.  It  was  found  that  all  positive  control  groups  and all  doses  of Z. officinalecould  significantly  decrease  escape  latency  throughout the  experimental  period  as  shown  in  Figure 1 (P-value  <  .05  as compared  with vehicle  plus  MCAO).  However,  they  failed  to  show  the  significant  changes  on retention  time  as  shown  in  Figure 2.Figure 1
Effect  of  Aricept,  Vitamin  C,  Piracetam,  and  various  doses  of  ginger (Zingiber  officinale)  extract  on  escape  latency  in  Morris  water  maze  test. Values  given  are  the  mean  ±  S.E.M. (n=6) *P-value  <  .05  as  compared  with vehicle plus  MCAO.Figure 2
Effect  of  Aricept,  Vitamin  C,  Piracetam,  and  various  doses  of  ginger (Zingiber  officinale)  extract  on  retention  time  in  Morris  water  maze  test.  Value given are  the  mean  ±  S.E.M. (n=6).


### 3.2.Z. officinale on  the  Neuron  Density  in  Hippocampus

Figure3   shows  that  Aricept  significantly  increased  the  neuronal  density in  all subregions  of  the  hippocampus.  Piracetam  also significantly  increased  neurons’ density  in  the  dentate  gyrus  and  CA3  while  Vitamin  C  significantly  increased neuronal density  only  in  CA3 (P-value  <  .05  as  compared  with  vehicle  plus MCAO). Z. officinaleat  dose  of  100 mg/kg  body weight  could  increase  the neuronal density  only  in  CA3,  whereas Z. officinaleextract at  dose of  200 mg/kg body weight could  increase  the  neuronal  density  both  in the  dentate  gyrus  and  CA3  (P-value  <  .05  as  compared  with  vehicle  plus  MCAO).Effect  of  Aricept,  Vitamin  C,  Piracetam,  and  various  doses  of  ginger (Zingiber  officinale)  extract  on  neurons  density  in  various  subregions  of hippocampus.  (a) The  neuron  density  in  various  areas  of  hippocampus  (b).  The photomicrographs  of  coronal  sections  of  CA3  stained  with  cresyl  violet  at 40x magnification.  Values  given  are  the  mean  ±  S.E.M. (n  =  6)  P-value  <  .05  as compared with  vehicle  plus  MCAO.
(a)(b)However, no  significant  change of  this  parameter  was  observed  afterZ. officinaleadministration  at  dose  of  300 mg/kg body weight.


### 3.3.Z. officinale on  Brain  Infarct Volume

Previous study  had  demonstrated  that  the  brain  impairments  were  associated with the  severity  of  brain  infarction.  Hence,  we    determined  the  brain infarct volume  afterZ. officinaleadministration.  The  results  in  Figure 4  show that  both  Aricept  and  Piracetam  markedly  decreased  the  infarct  volume  in the  cortical  area,  but  no  significant  change  was  observed  in  subcortical  areas (P-value  <  .05  as  compared  with  vehicle  plus  MCAO).  No  significant  change  was observed after  Vitamin C  administration.  Surprisingly, Z. officinaleat  a  dose  of  200 mg/kg body weight (dose  which  produced  optimum  change)  could  reduce  the brain  infarct  volume  better than  all  positive  control-treated  groups  in  this study.  The  plant  extract  could decrease  the  brain  infarct  volume  in  both cortical  and  subcortical  areas  (P-value  <  .05  as  compared  with  vehicle  plus MCAO).Effect  of  Aricept,  Vitamin  C,  Piracetam,  and  ginger  (Zingiber officinale; ZO1 200)  extract at  dose  of 200 mg/kg body weight  on brain  infarct volume.  Brain  infarct  volume  was  determined  using  TTC  staining.  Values  given are  the  mean  ±  S.E.M.  (n=6) *P-value  <  .05  as  compared  with  vehicle  plus MCAO.
(a)(b)


### 3.4.Z. officinale on the Level of Malondialdehyde  and Antioxidant  Enzymes

Based  on  the  neuroprotective effect  ofZ. officinaleextract,  we  also  further investigated  the  effect  of  Z. officinaleon  the  malondialdehyde,  an  indirect marker  of  the oxidative damage of various macromolecules as  shown  in  Figure 5.  It  was  found  that  all  positive control  groups  and Z. officinalesignificantly  decreased  malondialdehyde  level in  cerebral  cortex,  striatum,  and  hippocampus  (P-value  <  .05  as  compared  with  vehicle plus  MCAO).Figure 5
Effect  of  Aricept,  Vitamin  C,  Piracetam,  and  ginger  (Zingiber officinale; ZO1 200)  extract at  dose  of 200 mg/kg body weight  on the  level  of malondialdehyde  (MDA),  a  product  of  lipid  peroxidation  in  cerebral  cortex, hippocampus,  and  striatum.  Values  given  are  the  mean  ±  S.E.M.  (n=6)  *P-value  <  .05  as  compared  with  vehicle  plus  MCAO.In addition,  Figure6  shows  that  Aricept  and  Vitamin C  could  increase  the activity  of  SOD  in  all areas  mentioned  earlier  while  Piracetam  significantly increased  the  activity  of  SOD  only  in  the  cerebral  cortex  and  hippocampus. Surprisingly, Z. officinalealso  increased  the  activity of  SOD  in  all  areas mentioned above  at  the  same  magnitude  as  that  induced  by  Aricept  and Vitamin C.  Moreover,  it  appeared  to  show  better  effect  than  Piracetam.  The positive control  group  and Z. officinalealso  increased  the  activity  of  CAT  in both  cerebral  cortex  and  hippocampus  as  shown  in  Figure 7  (P-value  <  .05  as compared  with  vehicle  plus  MCAO).  In  this  study,  we  also  determined  the effect of  Z. officinaleon  the  activity  of  glutathione  peroxidase  as  shown  in Figure 8.  Aricept  and  Piracetam  significantly  increased  the  activity  of  GSH-Px in  cerebral  cortex  and  hippocampus  while  Vitamin  C  could  significantly increase  the  activity  of  this  enzyme  in  all  areas  (P-value  <  .05  as  compared with  vehicle  plus  MCAO).  The Z. officinaleextract also  significantly  increased the  activity  of  GSH-Px  in  cerebral  cortex  and  hippocampus (P-value  <  .05 as compared with vehicle plus MCAO).Figure 6
Effect  of  Aricept,  Vitamin  C,  Piracetam,  and  ginger  (Zingiber  officinale; ZO1 200)  extract  at  dose  of 200 mg/kg body weight on the  activity of superoxide  dismutase (SOD)  in  cerebral  cortex,  hippocampus,  and  striatum. Values given  are  the  mean  ±  S.E.M.  (n=6)  *P-value  <  .05  as  compared  with vehicle plus  MCAO.Figure 7
Effect  of  Aricept,  Vitamin  C,  Piracetam,  and  ginger  (Zingiber  officinale; ZO1 200)  extract  at  dose  of 200 mg/kg body weight on the  activity of catalase (CAT)  in  cerebral  cortex,  hippocampus,  and  striatum.  Values  given  are the  mean  ±  S.E.M.  (n  =  6)  *P-value  <  .05  as  compared  with  vehicle  plus MCAO.Figure 8
Effect  of  Aricept,  vitamin  C,  Piracetam,  and  ginger  (Zingiber officinale; ZO1 200)  extract at  dose  of 200 mg/kg body weight  on the  activity  of glutathione  peroxidase (GSH-Px)  in  cerebral  cortex,  hippocampus,  and  striatum. Values  given  are  the  mean  ±  S.E.M.  (n=6)  *P-value  <  .05  as  compared with vehicle  plus  MCAO.


## 3.1.Z. officinale on  Spatial  Memory

Effect ofZ. officinaleon  spatial  memory  was  determined  using  escape  latency and  retention  time  as  indices.  It  was  found  that  all  positive  control  groups  and all  doses  of Z. officinalecould  significantly  decrease  escape  latency  throughout the  experimental  period  as  shown  in  Figure 1 (P-value  <  .05  as compared  with vehicle  plus  MCAO).  However,  they  failed  to  show  the  significant  changes  on retention  time  as  shown  in  Figure 2.Figure 1
Effect  of  Aricept,  Vitamin  C,  Piracetam,  and  various  doses  of  ginger (Zingiber  officinale)  extract  on  escape  latency  in  Morris  water  maze  test. Values  given  are  the  mean  ±  S.E.M. (n=6) *P-value  <  .05  as  compared  with vehicle plus  MCAO.Figure 2
Effect  of  Aricept,  Vitamin  C,  Piracetam,  and  various  doses  of  ginger (Zingiber  officinale)  extract  on  retention  time  in  Morris  water  maze  test.  Value given are  the  mean  ±  S.E.M. (n=6).


## 3.2.Z. officinale on  the  Neuron  Density  in  Hippocampus

Figure3   shows  that  Aricept  significantly  increased  the  neuronal  density in  all subregions  of  the  hippocampus.  Piracetam  also significantly  increased  neurons’ density  in  the  dentate  gyrus  and  CA3  while  Vitamin  C  significantly  increased neuronal density  only  in  CA3 (P-value  <  .05  as  compared  with  vehicle  plus MCAO). Z. officinaleat  dose  of  100 mg/kg  body weight  could  increase  the neuronal density  only  in  CA3,  whereas Z. officinaleextract at  dose of  200 mg/kg body weight could  increase  the  neuronal  density  both  in the  dentate  gyrus  and  CA3  (P-value  <  .05  as  compared  with  vehicle  plus  MCAO).Effect  of  Aricept,  Vitamin  C,  Piracetam,  and  various  doses  of  ginger (Zingiber  officinale)  extract  on  neurons  density  in  various  subregions  of hippocampus.  (a) The  neuron  density  in  various  areas  of  hippocampus  (b).  The photomicrographs  of  coronal  sections  of  CA3  stained  with  cresyl  violet  at 40x magnification.  Values  given  are  the  mean  ±  S.E.M. (n  =  6)  P-value  <  .05  as compared with  vehicle  plus  MCAO.
(a)(b)However, no  significant  change of  this  parameter  was  observed  afterZ. officinaleadministration  at  dose  of  300 mg/kg body weight.


## 3.3.Z. officinale on  Brain  Infarct Volume

Previous study  had  demonstrated  that  the  brain  impairments  were  associated with the  severity  of  brain  infarction.  Hence,  we    determined  the  brain infarct volume  afterZ. officinaleadministration.  The  results  in  Figure 4  show that  both  Aricept  and  Piracetam  markedly  decreased  the  infarct  volume  in the  cortical  area,  but  no  significant  change  was  observed  in  subcortical  areas (P-value  <  .05  as  compared  with  vehicle  plus  MCAO).  No  significant  change  was observed after  Vitamin C  administration.  Surprisingly, Z. officinaleat  a  dose  of  200 mg/kg body weight (dose  which  produced  optimum  change)  could  reduce  the brain  infarct  volume  better than  all  positive  control-treated  groups  in  this study.  The  plant  extract  could decrease  the  brain  infarct  volume  in  both cortical  and  subcortical  areas  (P-value  <  .05  as  compared  with  vehicle  plus MCAO).Effect  of  Aricept,  Vitamin  C,  Piracetam,  and  ginger  (Zingiber officinale; ZO1 200)  extract at  dose  of 200 mg/kg body weight  on brain  infarct volume.  Brain  infarct  volume  was  determined  using  TTC  staining.  Values  given are  the  mean  ±  S.E.M.  (n=6) *P-value  <  .05  as  compared  with  vehicle  plus MCAO.
(a)(b)


## 3.4.Z. officinale on the Level of Malondialdehyde  and Antioxidant  Enzymes

Based  on  the  neuroprotective effect  ofZ. officinaleextract,  we  also  further investigated  the  effect  of  Z. officinaleon  the  malondialdehyde,  an  indirect marker  of  the oxidative damage of various macromolecules as  shown  in  Figure 5.  It  was  found  that  all  positive control  groups  and Z. officinalesignificantly  decreased  malondialdehyde  level in  cerebral  cortex,  striatum,  and  hippocampus  (P-value  <  .05  as  compared  with  vehicle plus  MCAO).Figure 5
Effect  of  Aricept,  Vitamin  C,  Piracetam,  and  ginger  (Zingiber officinale; ZO1 200)  extract at  dose  of 200 mg/kg body weight  on the  level  of malondialdehyde  (MDA),  a  product  of  lipid  peroxidation  in  cerebral  cortex, hippocampus,  and  striatum.  Values  given  are  the  mean  ±  S.E.M.  (n=6)  *P-value  <  .05  as  compared  with  vehicle  plus  MCAO.In addition,  Figure6  shows  that  Aricept  and  Vitamin C  could  increase  the activity  of  SOD  in  all areas  mentioned  earlier  while  Piracetam  significantly increased  the  activity  of  SOD  only  in  the  cerebral  cortex  and  hippocampus. Surprisingly, Z. officinalealso  increased  the  activity of  SOD  in  all  areas mentioned above  at  the  same  magnitude  as  that  induced  by  Aricept  and Vitamin C.  Moreover,  it  appeared  to  show  better  effect  than  Piracetam.  The positive control  group  and Z. officinalealso  increased  the  activity  of  CAT  in both  cerebral  cortex  and  hippocampus  as  shown  in  Figure 7  (P-value  <  .05  as compared  with  vehicle  plus  MCAO).  In  this  study,  we  also  determined  the effect of  Z. officinaleon  the  activity  of  glutathione  peroxidase  as  shown  in Figure 8.  Aricept  and  Piracetam  significantly  increased  the  activity  of  GSH-Px in  cerebral  cortex  and  hippocampus  while  Vitamin  C  could  significantly increase  the  activity  of  this  enzyme  in  all  areas  (P-value  <  .05  as  compared with  vehicle  plus  MCAO).  The Z. officinaleextract also  significantly  increased the  activity  of  GSH-Px  in  cerebral  cortex  and  hippocampus (P-value  <  .05 as compared with vehicle plus MCAO).Figure 6
Effect  of  Aricept,  Vitamin  C,  Piracetam,  and  ginger  (Zingiber  officinale; ZO1 200)  extract  at  dose  of 200 mg/kg body weight on the  activity of superoxide  dismutase (SOD)  in  cerebral  cortex,  hippocampus,  and  striatum. Values given  are  the  mean  ±  S.E.M.  (n=6)  *P-value  <  .05  as  compared  with vehicle plus  MCAO.Figure 7
Effect  of  Aricept,  Vitamin  C,  Piracetam,  and  ginger  (Zingiber  officinale; ZO1 200)  extract  at  dose  of 200 mg/kg body weight on the  activity of catalase (CAT)  in  cerebral  cortex,  hippocampus,  and  striatum.  Values  given  are the  mean  ±  S.E.M.  (n  =  6)  *P-value  <  .05  as  compared  with  vehicle  plus MCAO.Figure 8
Effect  of  Aricept,  vitamin  C,  Piracetam,  and  ginger  (Zingiber officinale; ZO1 200)  extract at  dose  of 200 mg/kg body weight  on the  activity  of glutathione  peroxidase (GSH-Px)  in  cerebral  cortex,  hippocampus,  and  striatum. Values  given  are  the  mean  ±  S.E.M.  (n=6)  *P-value  <  .05  as  compared with vehicle  plus  MCAO.


## 4. Discussion

Our results  demonstrated  for  the  first  time  thatZ. officinalecould  protect ischemic  brain  damage  in  a  rat  model  of  focal  cerebral  ischemia.  Moreover,  it also  reduced  cognitive  deficits  induced  by  focal  cerebral  ischemia.MCAO had  been  reported  to  induce  a  number  of  important  cellular  changes including  increase  in  intracellular  calcium  and  excessive  free  radicals leading  to brain  damage  and  impairments  including  cognitive  deficit [29].  MCAO  has  been previously  reported  to  induce brain damage  and  neuronal  death  in  the  hippocampus [30].  It  was  found  that  the neurodegeneration  in  hippocampus  was  associated  with  spatial  memory  impairment [31–33]. In  addition,  MCAO  also  decreased  acetylcholine [34].  These  findings  were  in  agreement  with  our  data.  Aricept,  an  acetylcholineseterase  inhibitor,  was  previously  shown  to  improve  brain  damage  and memory  impairment  in  MCAO [35].  It  was  found  that  Aricept  inhibited not only  acetylcholinesterase  activity  but  also  oxidative  stress [36].  Therefore, the neuroprotective  effect  of Aricept  might  be  possibly  related  to  its antioxidant activity.  However,  its  effect  to  inhibit  acetylcholinesterase  activity  still  could  not be omitted.  It  was  previously  reported  that  during  a  memory  task,  the  cerebral blood  flow  increased  and  the  disruption  of  cerebral  blood  flow  resulted  in memory  impairment [37].  Our  results  also  demonstrated  that  Piracetam,  a  drug reputed  to  increase  cerebral  blood  flow,   improved  the  cognitive  function induced  by  MCAO.  Therefore,  the  present  results  were  consistent  with  previous study  which  reported  the  neuroprotective  and  nootropic  effect  of  this  drug  and its  ability  to  improve  symptoms  of  acute  stroke [38].  Moreover,  the  current study  also  showed  that  the  interruption  of  cerebral  blood  flow  resulted  in  the insufficient  supply  of  ascorbic  acid  in  the  brain  and  inadequate  delivery  of oxygen  and  glucose [39, 40],  and  finally  leading  to  brain  damage  and  brain impairment.  Vitamin  C  had  been  reported  to  be involved  in  almost  the  important  neurochemical  processes  during  cerebral  ischemia [41];  therefore,  supplementation  of  Vitamin  C  or  ascorbic  could  improve  brain  damage  and impairment  as  shown  in this  study.Surprisingly,Z. officinalecould  increase  the  neurons’  density  in  hippocampus and  improved  the  spatial  memory.  Although  the neurodegeneration  in hippocampus is  reported  to  be  associated  with  the  spatial  memory deficit [32, 33], the  present  findings  showed  that  the  improvement  of  spatial memory  was  not tightly  correlated  with  the  increase  of neuronal  density  in the hippocampus. It  was found  that  all  doses  of Z. officinalecould  improve  spatial  memory while Z. officinaleat  dose  of 300 mg/kg body weight failed  to  show neuroprotective  effect  in  this  area.  This  indicates  that  the  cognitive enhancing effect  of Z. officinaleoccurred  not  only  due  to   increasing  neuronal density in  hippocampus  but also  due  to  other  mechanisms.  Z. officinalehas previously  been  reported  to  induce  vasodilation [42].  Therefore,  it  might  be  possible that  Z. officinalecould  enhance  cerebral  blood  flow  resulting  in  the  improvement of  spatial  memory  as  Piracetam.  A  dose  which  provided  the  most beneficial  effect  was  200 mg/kg  body weight.  The  lower  dose  failed  to  show the  neuroprotective  effect  in  dentate gyrus  while  the  Z. officinaleat  dose of 200 mg/kg  body weight  could  increase  the  neuronal  density  in  this  area.  One possible explanation  for  this  phenomenon  might  be  related  to  the  insufficient level of  Z. officinaleto  reach  the  therapeutic  level.  On the other hand, Z. officinaleat  dose  of  300 mg/kg  body weight  also  failed  to  show  a  neuroprotective  effect  in hippocampus.  This  might  occur  because  the Z. officinaleextract  used  in  this  study  was  the  crude  extract;  therefore,    increasing  the  dose  of  the  extract  might  also increase  the  concentration  of  some  ingredients  which  masked  the  effect  of  the  active ingredient.Our results  also  demonstrated  thatZ. officinaleat  dose  of  200 mg/kg  body weight  could  mitigate the  brain  infarct  volume  and  could  decrease  oxidative stress  by  increasing  the  activity  of  SOD  in  cerebral  cortex,  hippocampus,  and striatum and  increased  the  activities  of  CAT  and  GSH-Px  in  cerebral  cortex and  hippocampus resulting  in  the  decrease  of lipid  peroxidation  level  in  all areas mentioned  earlier.  Therefore,  the  neuroprotective  effect  of  Z. officinaleextract might  be  related  to  its  antioxidant  effect.In conclusion, our data  suggested  thatZ. officinale possessed  the  protective effect against  focal  cerebral  ischemia  induced  by  the  occlusion  of  right  middle cerebral artery.  It  could  attenuate  the  memory  impairment,  neurodegeneration, and  brain  infarct  volume  in  this  condition.  The  cognitive  enhancing  effect  and neuroprotective  effect  of  Z. officinaleappeared  to  show  almost  the  same magnitude  as  the  positive  control  groups  used  in  this  study.  Moreover, Z. officinalealso  showed  multiple  sites  of  action.  Therefore,  Z. officinaleat  the correct dose  is  a potential novel  candidate  for  developing  food  supplement  against  focal  cerebral  ischemia. However,  further  clinical  trial  study  is  still  required.



---

*Source: 429505-2010-12-20.xml*