trimethyltin ( tmt ) , a short - chain trialkyltin , is used as a stabilizing agent for certain plastics and is a neurotoxin that induces neuronal damage in both humans and experimental animals [ 1 - 3 ] .
tmt causes a selective degeneration of pyramidal neurons in the hippocampus and the cortical areas connected to the hippocampus [ 4 , 5 ] .
proposals have been made that tmt produces excess generation of cellular oxidative species , which leads to apoptotic cell death .
therefore , oxidative stress and apoptosis resulting in cognitive dysfunction may be a hallmark feature of neurodegeneration . in this case , drugs , including herbal - based drugs , that can inhibit tmt - induced oxidative damage may represent a new generation of therapeutics for neurological diseases .
administration of saffron extract and crocin improved spatial cognitive abilities following chronic cerebral hypoperfusion in rats in part through their antioxidant activities . additionally , treatment with rosmarinus officinalis
aerial parts essential oil was found , through performance on the morris water maze task , to enhance both the memory function in the intact memory and the scopolamine - induced learning deficits in rats .
lipophilic extracts of the gum resin of boswellia species have been used in herbal medicine since ancient times for various indications in folk medicine to treat inflammatory and infectious diseases .
a rich unique group of pentacyclic triterpenes , termed boswellic acids ( bas ) , accounts for the active substances in the extract
. some of the chemical compounds present in frankincense are acid resin ( 56% ) , gum , which is similar to gum arabic ( 30% - 36% ) , 3-acetyl - beta- boswellic acid ( boswellia sacra ) , alpha - boswellic acid ( boswellia sacra ) , 4-o - methyl - glucuronic acid ( boswellia sacra ) , incensole acetate and phellandrene .
olibanum gum resin , as a medicinal plant , is traditionally used in india to treat various diseases , including inflammatory ailments , arthritis , cardiac disorders and pain [ 7 , 14 ] .
several studies have shown that bas are the major constituents in olibanum gum resin and that they have anti - inflammatory , anti - cancerous and anti - ulcerous activities [ 8 - 11 ] . moreover , in in vivo and in vitro studies , bas have been found to inhibit the synthesis of pro - inflammatory enzymes and 5-lipoxygenase such as leukotrine b4 ( ltb4 ) , which cause inflammatory reactions , bronchoconstriction , and increased vascular permeability [ 12 , 13 , 15 ] . also , the administration of ethyl acetate ( 0.1 mg/ kg ) and n - butanol ( 0.1 mg / kg ) fractions obtained from gum resin of boswellia carterii ( b. carterii ) has been reported to improve memory function in intact rats and in animals with hyoscine - induced memory impairment .
several studies have demonstrated that excessive production of reactive oxygen species ( ros ) , which occurs in tmt - induced neurotoxicity , leads to oxidative damage in the brain ; therefore , pursuing antioxidant therapies to reduce that damage could be supportive [ 16 - 19 ] .
the present study was designed to evaluate the possible neuroprotective or ameliorative effects of ba on tmt - induced learning and memory deficits and oxidative stress . for this purpose
furthermore , acetylcholinesterase ( ache ) activity , glutathione ( gsh ) content and malondialdehyde ( mda ) level in the cerebral cortex were measured .
because of the important role of the cerebral cortex in memory function and easy separation of total brain , this part of brain has often been used for biochemical analysis .
tmt chloride , mda tetrabutylammonium , reduced gsh and dtnb [ 5,5-dithiobis-(2-nitrobenzoic acid ) ] were obtained from sigma - aldrich , and ba was purchased from shanghai research institute of the chemical industry testing center .
male wistar rats weighting 230 - 260 g were purchased from the animal facilities of the pharmaceutical research center , bu - ali research institute , mashhad university of medical sciences .
all animal experiments were approved and were carried out in accordance with the ethical committee acts of mashhad university of medical sciences .
the animals were housed in standard cages under hygienic conditions and were provided standard animal feed and water ad libitum throughout the treatment period . for acclimatization , the rats were kept in the animal houses for one week prior to the beginning of the study .
forty rats were randomly divided into five groups ( n = 8 per group ) : the control group and four tmt - treated groups . the rats with tmt - induced lesions were treated with saline [ ( tmt - lesioned + saline group , 1.6 ml / kg ) which means rats received saline instead of ba ] , ba at 40 mg / kg ( b40 group ) , ba at 80 mg / kg ( b80 group ) , or ba at 160 mg / kg ( b160 group ) .
the rats were injected intraperitoneally ( i.p . ) with tmt ( 8.0 mg / kg body weight ) dissolved in 0.9% saline .
the morris water maze task   was used to determine spatial memory impairment induced by tmt administration .
the apparatus was a circular pool of 136 cm in diameter and 60 cm in depth .
the pool was filled with water to a depth of 25 cm , and the water temperature remained between 20 - 22c .
several visual cues were placed on the walls of the experiment room . the experiment consisted of five days of training . on the training days , the rats were subjected to 4 sessions ( on days 1 - 5 ) with 10-seconds intervals for each of 2 trials to find a hidden , transparent platform submerged 1 cm below the water s surface . in every trial , the rat was placed in the water facing the wall of the pool in one of the four possible starting locations .
the rat was allowed to search for the platform for 60 seconds and was gently guided if it could not reach it .
rats were allowed to remain on the platform for 15 seconds to learn extra maze cues .
two days after the last training day , each rat was subjected to a probe trial , in which the platform was removed from the pool , and the animals were tested in a 60-second spatial probe trial . the time spent in the quadrant where the platform had been located during the training days was measured , and the amount of time spent in that quadrant was calculated .
this parameter was taken as an indicator of spatial memory . at the end of the experiment ,
the samples were snap - frozen in liquid nitrogen and stored at - 80c until use .
the evaluation of lipid peroxidation was done by determining the ( mda ) level , as a marker of lipid peroxidation , in the cerebral cortex .
mda reacts with thiobarbituric acid ( tba ) as a tba reactive substance ( tbars ) to produce a pink - colored complex , which has a maximum absorbance at 532 nm . in this method , 3 ml of phosphoric acid ( 1% ) and 1 ml of tba ( 0.6% ) were added to brain tissue homogenate ( 10% ) in kcl , after which the mixtures were heated for 45 minutes in boiling water .
after the mixture had been cooled , 4 ml of n - butanol was added and vortex - mixed for 1 minutes , followed by centrifugation at 3,000 g for 10 minutes .
then , the organic layers were separated and transferred to a fresh tube , and the absorbance was recorded at 532 nm .
mda levels were expressed as nmol / g tissue . the gsh level in rat brain tissue
the nitromercaptobenzoic acid anion released has an intense yellow color and can be used to determine the presence of -sh groups .
the absorbance of this yellow component was measured at 412 nm and compared with the standard curve to determine the gsh concentration .
the ache assay was performed according to the colorimetric method of ellman   by using acetylthiocholine iodide as a substrate .the reaction of the thiol compound with dtnb forms a complex with absorbance at 405 nm .
for this test , 20 l of sample serum and 100 l of 5% ( w / v ) acetylthiocholine iodide solution were added to 3 ml of elman reagent ( 0.02% dtnb in 0.1-m sodium dihydrogen phosphate buffer solution , ph = 7.4 ) .
the cholinesterase activity was calculated as follows : cholinesterase ( mu/ ml , at 25c ) = change in absorbance in 30 s  23400 .
statistical differences were evaluated by using the one - way analysis of variance ( anova ) , followed by the tukey post - hoc test .
additionally , for the morris water maze test , statistical analyses were performed using the two - way anova , followed by the bonferroni test . a p - value < 0.05 was considered as significant .
to evaluate spatial memory impairment induced by tmt administration , we used the morris water maze task .
animals were treated with different doses of ba ( 40 , 80 and 160 mg/ kg ) after treatment with tmt at 8 mg / kg . as shown in ( fig .
1)a , the escape latency time ( elt ) , the time spent to find the platform during five days was measured .
administration of tmt at 8 mg / kg significantly increased the elt in five days compared with the control group , and the administration of ba at 160 mg / kg to the tmt - treated rats significantly decreased the elt on days 4 and 5 ( p < 0.01 and p < 0.001 ) .
the results for the swimming paths indicated that rats in the tmt - treated group and the ba treatment groups often searched for the platform in a wrong way .
thus , the rats took a longer time to locate the platform and had fewer excursions into the target region , thereby making the elt longer . a statistically significant decrease in the mean elt
administration of tmt at 8 mg / kg significantly decreased the distance compared with the control group ( p < 0.01 ) , and the administration of ba at 160 mg / kg significantly increased the distance as compared with the tmt - lesioned + saline group ( p < 0.05 ) . during the test trial , the percentage of distance traveled in quadrant 1 ( q1 ) in the control group was higher than it was in the tmt - treated groups .
moreover , the percentage of distance traveled in q1 for the b160 group was increased significantly .
1c shows the spent time in the target area ( probe ) ( s ) .
administration of tmt at 8 mg / kg decreased the time the rats spent in the target area compared with the control group ( p < 0.01 ) , and the treatment of rats with ba at 160 mg / kg significantly increased that time compared with the tmt - lesioned + saline group ( p < 0.01 ) . in the final test trials , the swimming time spent in q1 ( the target quadrant ) was used to estimate retention . in this case , the tmt - lesioned + saline group spent more time swimming in the target quadrant compared with the control group .
moreover , the rats in the b160 group spent more time swimming in the target quadrant than ( fig .
1)e show the rats in the tmt - lesioned + saline group . finally , ( fig . 1)d and ( fig .
1)e shows the measured swimming speeds of the test ( cm / s ) on the probe day and during the 5 days of the test respectively .
as fig . 2 shows , exposure to tmt increased the level of mda in the cortex of brain tissue compared with the control group ( p < 0.01 ) .
remarkably , in the b80 ( p < 0.05 ) and the b160 ( p < 0.01 ) groups , the levels of mda in brain tissue were significantly decreased in comparison with that in the tmt - lesioned + saline group . however , the mda levels in the rats in the b40 groups were not significantly different from those in the tmt - lesioned + saline group . as fig .
3 shows , a significant decrease in the level of gsh was observed in tmt - lesioned + saline group compared with the control group ( p < 0.01 ) .
furthermore , the gsh levels for the rats in the b40 and b80 groups were not significantly different from the gsh levels in the tmt - lesioned + saline group . however , the rats in the b160 group showed significantly increased gsh levels compared with the gsh levels in the rats in the tmt - lesioned + saline group ( p < 0.01 ) .
the activity of ache was significantly increased in the rats in the tmt - lesioned + saline group compared to the control group ( p < 0.01 ) ( fig .
4 ) whereas all groups treated with ba at any of the three doses used in this study exhibited a significant inhibitory effect against ache .
thus , treatments with any of the three different doses of ba ( 40 , 80 and 160 mg / kg ) used in this research led to significant reductions in ache activity compared to that in the tmt - lesioned + saline group .
to evaluate spatial memory impairment induced by tmt administration , we used the morris water maze task .
animals were treated with different doses of ba ( 40 , 80 and 160 mg/ kg ) after treatment with tmt at 8 mg / kg . as shown in ( fig .
1)a , the escape latency time ( elt ) , the time spent to find the platform during five days was measured .
administration of tmt at 8 mg / kg significantly increased the elt in five days compared with the control group , and the administration of ba at 160 mg / kg to the tmt - treated rats significantly decreased the elt on days 4 and 5 ( p < 0.01 and p < 0.001 ) .
the results for the swimming paths indicated that rats in the tmt - treated group and the ba treatment groups often searched for the platform in a wrong way .
thus , the rats took a longer time to locate the platform and had fewer excursions into the target region , thereby making the elt longer . a statistically significant decrease in the mean elt
administration of tmt at 8 mg / kg significantly decreased the distance compared with the control group ( p < 0.01 ) , and the administration of ba at 160 mg / kg significantly increased the distance as compared with the tmt - lesioned + saline group ( p < 0.05 ) . during the test trial , the percentage of distance traveled in quadrant 1 ( q1 ) in the control group was higher than it was in the tmt - treated groups .
moreover , the percentage of distance traveled in q1 for the b160 group was increased significantly .
1c shows the spent time in the target area ( probe ) ( s ) .
administration of tmt at 8 mg / kg decreased the time the rats spent in the target area compared with the control group ( p < 0.01 ) , and the treatment of rats with ba at 160 mg / kg significantly increased that time compared with the tmt - lesioned + saline group ( p < 0.01 ) . in the final test trials , the swimming time spent in q1 ( the target quadrant ) was used to estimate retention . in this case , the tmt - lesioned + saline group spent more time swimming in the target quadrant compared with the control group .
moreover , the rats in the b160 group spent more time swimming in the target quadrant than ( fig .
1)e show the rats in the tmt - lesioned + saline group . finally , ( fig . 1)d and ( fig .
1)e shows the measured swimming speeds of the test ( cm / s ) on the probe day and during the 5 days of the test respectively .
as fig . 2 shows , exposure to tmt increased the level of mda in the cortex of brain tissue compared with the control group ( p < 0.01 ) . remarkably , in the b80 ( p < 0.05 ) and the b160 ( p < 0.01 ) groups , the levels of mda in brain tissue
were significantly decreased in comparison with that in the tmt - lesioned + saline group .
however , the mda levels in the rats in the b40 groups were not significantly different from those in the tmt - lesioned + saline group . as fig .
3 shows , a significant decrease in the level of gsh was observed in tmt - lesioned + saline group compared with the control group ( p < 0.01 ) .
furthermore , the gsh levels for the rats in the b40 and b80 groups were not significantly different from the gsh levels in the tmt - lesioned + saline group . however , the rats in the b160 group showed significantly increased gsh levels compared with the gsh levels in the rats in the tmt - lesioned + saline group ( p < 0.01 ) .
the activity of ache was significantly increased in the rats in the tmt - lesioned + saline group compared to the control group ( p < 0.01 ) ( fig .
4 ) whereas all groups treated with ba at any of the three doses used in this study exhibited a significant inhibitory effect against ache .
thus , treatments with any of the three different doses of ba ( 40 , 80 and 160 mg / kg ) used in this research led to significant reductions in ache activity compared to that in the tmt - lesioned + saline group .
treatment with tmt induced learning and memory impairment in wistar rats , and treatment with ba at a dose of 160 mg / kg markedly improved learning and memory abilities on the water maze tasks . additionally , the activity of ache was significantly elevated following exposure to tmt whereas all groups treated with ba presented a significant inhibitory effect against ache .
administration of tmt reduced gsh content and increased the level of mda in the cerebral cortex while treatment with ba at 160 mg / kg increased the gsh content and decreased the tmt - induced lipid peroxidation in rat brain tissue .
the pathogenesis of tmt neurotoxicity is a complex event ; it contains multiple processes such as glutamate excitotoxicity and impairment of neurotransmission with oxidative stress [ 24 , 25 ] .
a suggestion has been made that tmt causes neuronal oxidative stress and increases the free radical production , which is a potential contributor in apoptotic cell death .
several studies have shown that lipid peroxidation and oxidative stress are increased in tmt - induced neurotoxicity [ 7 , 27 ] . moreover , free radial scavengers have an important role in protection against tmt - induced neurotoxicity in experimental animals .
the possibility of ba being an antioxidant and memory enhancer makes this agent an ideal choice as a neuroprotective agent . for manifestation of oxidative damage , mda ,
the end product of lipid peroxidation generated in tissues by free radical injury , is considered to be a sensitive biomarker . in the present experiment ,
many researchers have found lipid peroxidation changes in brain tissue after treatment with tmt [ 7 , 27 ] . as mentioned in the results , lipid peroxidation returned to near control levels in the b80 and the b160 groups . on the other hand , a major antioxidant , which protects cells against oxidative stress ,
exposure to tmt induces oxidative stress , which decreases the gsh levels and reflects the degree of oxidative insult . in this study
, gsh was measured in all groups , and a significant depletion of gsh was observed in the rats treated only with tmt .
several reports have indicated that gsh depletion is a critical factor in the induction of oxidative stress [ 5 , 6 , 29 , 30 ] .
therefore , depletion of endogenous gsh invariably sensitizes an animal to tmt - induced neuronal damage . in the present study ,
rats treated with 160 mg / kg of ba showed increased gsh levels , which is evidence for a compromised antioxidant defense . an elevated level of gsh may be part of a protective mechanism to reduce neuronal damage .
previous studies have reported that ba has an efficient antioxidant activity [ 16 , 17 , 19 ] .
hartmann et al evaluate the antioxidant effect of an extract of the plant boswellia serrata ( b. serrata ) , which consists of ba , in an experimental model of acute ulcerative colitis induced by administration of acetic acid in rats .
assimopoulou et al reported that b. serrata resin could additionally be used as a natural antioxidant in cosmetics and pharmaceutical preparations .
in the research of sabina et al treatment with ba normalized the levels of lipid peroxidation and antioxidant status in mice with inflammation induced by using monosodium - urate crystals .
they suggested that the effect of ba was similar to that of other pentacyclic triterpenes , such as ursolic acid and lutein .
moreover , several researchers have reported the efficacy of agents with antioxidant activity in reducing tmt - induced neurotoxicity .
kuar et al suggested that a prophylactic treatment with ginkgo biloba extract protected against tmt - induced neurodegeneration because of its antioxidant effects .
the findings of qu et al showed that the anti - apoptotic effect of lycopene against tmt - induced neurotoxicity was due to its potential antioxidant activity . in the investigations of shin et al , ascorbate treatment significantly attenuated the early oxidative stress induced by tmt , leading to a persistent deficiency in gsh status .
choi et al reported that quercetin , with its high antioxidant activity , could possess a wide range of beneficial activities for treating neurodegenerative disorders , notably alzheimer 's disease ( ad ) .
another possible mechanism underlying the beneficial effects of ba in our study is its ache inhibitory effects .
additionally , neuronal damage in the cholinergic systems of animal models has been reported following exposure to tmt . in our experiment ,
exposure to tmt led to increased ache activity whereas ba treatment effectively reduced the activity of ache in the serum samples .
therefore , another protective mechanism of ba in tmt - induced memory dysfunction may be mediated through inhibition of ache activity . in another study ,
the ethyl acetate fraction from onion ( allium cepa l. ) flesh and peel significantly reduced tmt - induced learning and memory impairment in mice due to ache inhibition . obviously , tmt is a neurotoxin that induces neural damage , which leads to behavioral and learning deficits and disrupts memory [ 35 , 36 ] .
many researchers have proven that tmt causes a degeneration of the pyramidal neurons in the rat hippocampus and the cortical areas ( pyriform cortex , entorhinal cortex and subiculum ) connected to the hippocampus , as well as neuronal loss in associated areas [ 2 , 4 , 37 ] .
the best way to evaluate the deficits in hippocampal - dependent memory is to use the morris water maze test .
the present study showed impairment in spatial learning in the group treated only with tmt , which agrees with the results in previous reports on spatial learning impairments [ 35 , 39 ] .
moreover , the results of the current study indicated that the spatial memory improved during the training days when the dose of ba in the treatment groups was increased .
this finding is in agreement with the results reported by researcher that high antioxidant agents enhance the learning and memory abilities of rats [ 7 , 32 , 33 ] .
in conclusion , ba effectively ameliorated tmt - induced neurotoxicity through ache inhibition and antioxidant activity . therefore , ba may be used as a potential natural resource for mitigating learning and memory dysfunction caused by aging and neurodegenerative diseases .
ba , boswellic acid ; tmt , trimethyltin ; sd , standard deviation ; b , boswellic acid .
ba , boswellic acid ; gsh , glutathione ; tmt , trimethyltin ; sd , standard deviation .
ba , boswellic acid ; ache , acetylcholinesterase ; tmt , trimethyltin ; sd , standard deviation .
 objectives : boswellic acid ( ba ) , a compound isolated from the gum - resin of boswellia carterii , is a pentacyclic terpenoid that is active against many inflammatory diseases , including cancer , arthritis , chronic colitis , ulcerative colitis , crohn 's disease , and memory impairment , but the mechanism is poorly understood . 
 this study investigated the effects of boswellic acid on spatial learning and memory impairment induced by trimethyltin ( tmt ) in wistar rats.methods:forty male wistar rats were randomly divided into 5 groups : normal group , tmt - administrated rats ( 8.0 mg / kg , intraperitoneally , i.p . ) and tmt + ba ( 40 , 80 and 160 mg / kg , i.p.)-administrated rats . 
 ba was used daily for 21 days . to evaluate the cognitive improving of ba 
 , we performed the morris water maze test . 
 moreover , to investigate the neuroprotective effect of ba , we determined the acetylcholinesterase ( ache ) activity , the malondialdehyde ( mda ) level as a marker of lipid peroxidation , and the glutathione ( gsh ) content in the cerebral cortex.results:treatment with tmt impaired learning and memory , and treatment with ba at a dose of 160 mg / kg produced a significant improvement in learning and memory abilities in the water maze tasks . 
 consistent with behavioral data , the activity of ache was significantly increased in the tmt - injected rats compared to the control group ( p < 0.01 ) whereas all groups treated with ba presented a more significant inhibitory effect against ache than the tmt - injected animals . 
 in addition , tmt reduced the gsh content and increased the mda level in the cerebral cortex as compared to the control group ) p < 0.01 ) . 
 on the other hand , treatment with ba at 160 mg / kg slightly increased the gsh content and reduced the mda level in comparison to the tmt - administered group ( p < 0.01).conclusion : the above results suggest that the effect of ba in improving the cognitive function may be mediated through its antioxidant activity . 
1. Introduction
2. Materials and Methods
3. Results
3.1. Effects of BA on spatial memory impairment induced by TMT
3.2. Biochemical findings
4. Discussion
5. Conclusion