the method of preparation of eaks was reported previously by hwang et al ( 2004 ) . briefly , for the preparation of ethanol eaks , alpinia katsumadai seeds were dried and ground into fine powder . the powder was dispersed in 75% ethanol and refluxed for 1 hour at 50. this extraction procedure was repeated three times . male mongolian gerbils ( 6 months of age ) were obtained from the experimental animal center , hallym university , chuncheon , korea . the procedures for handling animals and their care conformed to guidelines compliant with current international laws and policies ( nih guide for the care and use of laboratory animals , nih publication no . the animals were divided into the following groups : sham - operated gerbils ( sham group ) , vehicle - treated ischemia gerbils ( vehicle - ischemia group ) and eaks ( 25 and 50 mg / kg)-treated ischemia gerbils ( eaks - ischemia group ) . eaks was orally administered through a feeding needle once a day for 7 days before transient ischemia , and the last treatment was 30 min before ischemia / reperfusion . because in traditional medicine eaks is taken orally and there are no data about the absorption and metabolism of eaks ( yang et al , 2009 ) , we selected oral administration of 50 mg / kg eaks . gerbils underwent transient cerebral ischemia as in our previous study ( ahn et al , 2009 ) . bilateral common carotid arteries were occluded for 5 minutes . the rectal temperature was monitored and maintained ( 370.5 ) before , during and after the surgery . sham - operated animals were subjected to the same surgical procedures except that the common carotid arteries were not occluded . to elucidate the protective effects of eaks , brain sections from each group ( n=7 in each group ) were prepared at 4 days post - ischemia and stained with cresyl violet as we previously described . cresyl violet - positive cells were counted using an image analyzing system ( software : optimas 6.5 , cybermetrics , scottsdale , usa ) ( choi et al , 2009a ) . we also examined the effect of brain - derived neurotrophic factor ( bdnf ) on ischemic damage at sham , 2 and 4 days post - ischemia ( n=7 in each group ) through immunochemistry using rabbit anti - bdnf ( 1:1,000 ; chemicon international , temecula , ca , usa ) ( kim et al , 2007 ) . in addition , we examined bdnf levels in the ischemic ca1 region of animals ( n=5 in each group ) through western blot analysis ( kim et al , 2007 ) . the relative number of positive cells and the relative optical density of the bands of the western blot analysis were shown as % of the sham group . the data were evaluated by one - way anova ( spss program ) , and the means assessed using duncan 's multiple - range test . in the sham group , neurons in the hippocampus were well stained with cresyl violet ( figures 1a and 1e ) . delayed neuronal death in the hippocampal ca1 region was observed in the vehicle - ischemia group 4 days after ischemia / reperfusion ( figures 1b and 1f ) : about 13.8% of ca1 neurons were stained with cresyl violet ( figure 1i ) . in the 25 mg / kg eaks - ischemia group , cresyl violet - positive cells were similar to those in the vehicle - ischemia group ( figures 1c , 1 g and 1i ) . however , many neurons ( about 67% of ca1 neurons ) were well stained with cresyl violet in the 50 mg / kg eaks - treated group ( figures 1d , 1h and 1i ) . change in bdnf immunoreactivity was well detected in the ca1 region of the vehicle - ischemia group ( figures 2a , 2c , 2e , 2 g and 2i ) . in the vehicle - sham group , strong bdnf immunoreactivity was detected in the stratum pyramidale ( figure 1a ) . at 3 hours after ischemic insult , bdnf immunoreactivity was significantly decreased in the vehicle - ischemia group ( figure 2c ) . thereafter , bdnf immuno - reactivity gradually decreased with time and was hardly detected at 4 days post - ischemia ( figures 2e , 2 g and 2i ) . in each eaks - ischemia group , bdnf immunoreactivity was significantly higher than in each vehicle - ischemia group ( figures 2d , 2f , 2h and 2j ) : the bdnf immunoreactivity was maintained in the stratum pyramidale of the ca1 region after 50 mg / kg eaks treatment . western blot analysis also showed a similar change in bdnf levels in the vehicle - ischemia group compared to the immunohistochemical findings after ischemia / reperfusion ( figure 3 ) : bdnf level in the vehicle - ischemia group was lowest at 4 days post - ischemia . in the 50 mg / kg eaks - ischemia group , bdnf levels were higher than in the vehicle - ischemia group , showing levels that were similar to those in the eaks - sham group . interestingly , extracts from natural substances have the ability to protect neurons from ischemic damage ( chun et al , 2008 ; nazam ansari et al , 2008 ; yoo et al , 2010 ) . the administration of 50 mg / kg eaks to gerbil protected the ca1 pyramidal neurons from ischemic damage : about 67% of the ca1 pyramidal neurons survived after transient ischemic insults . this is the first report to show that eaks has neuroprotective effects in a gerbil model of ischemic damage . this finding is supported by a previous study in which eaks inhibited h2o2-induced apoptosis and enhanced the viability of chinese hamster lung fibroblasts ( v79 - 4 ) ( lee et al , 2003 ) . the results of this study show that the eaks - treated group had significantly increased bdnf immunoreactivity in the ca1 region after 12 hours post - ischemia compared to the vehicle - treated group . bdnf , a neurotrophic factor , has been associated with neuronal survival , synaptic plasticity , learning and memory , as well as neuronal plasticity ( miller and kaplan , 2001 ; ploughman et al , 2009 ) . it has been reported that intraventricular treatment with bdnf reduced neuronal apoptosis induced by ischemia , suggesting that its effect is correlated with phosphorylation of akt ( han and holtzman , 2000 ) . in addition , bdnf treatment prevented arabinoside - induced apoptosis in cultured cerebellar granular cells and glutamate - induced apoptosis in hippocampal neurons by increasing akt and bcl-2 levels ( almeida et al , 2005 ; leeds et al , 2005 ) . in conclusion , the findings of the present study demonstrate that repeated oral administration of eaks protects neurons from ischemic damage in the hippocampus in a gerbil model of transient cerebral ischemia , and indicate that the effect might be associated with an upregulation of bdnf , a neurotrophic factor .
the extract of alpinia katsumadai , a member of the family zingiberaceae , shows anti - inflammatory effects and antioxidant activity . 
 we observed the neuroprotective effects of the extract from alpinia katsumadai seed ( eaks ) against ischemic damage in gerbils administered oral eaks ( 25 , and 50 mg / kg ) once a day for 7 days before transient cerebral ischemia . in the 50 mg / kg eaks - treated ischemia group , about 67% of neurons in the hippocampal ca1 region ( ca1 ) survived after ischemia / reperfusion ( i / r ) based on cresyl violet staining . 
 we observed that eaks treatment significantly maintained brain - derived neurotrophic factor ( bdnf ) immunoreactivity in the ischemic ca1 region after i / r . 
 in addition , protein levels of bdnf in the 50 mg / kg eaks - treated ischemia group were much higher than those in the vehicle - treated ischemia group after i / r . 
 these findings indicate that repeated supplementation of eaks protects neurons from ischemic damage , such that bdnf is distinctively maintained in ischemic areas .