sleep can be disturbed by several factors such as illness , stress , and noise . over time
these include poor memory , slower reaction time , emotional disturbances , and changes in the immune response ( 1 , 2 ) .
however , administration of these drugs is accompanied by side effects including psychomotor impairment , drug dependence , tolerance , amnesia , rebound insomnia , and the potentiating effects of other central depressant drugs ( 3 ) . moreover , some patients with sleep disorders do not respond effectively to current therapeutics .
therefore , studies to find new hypnotic agents with fewer side effects and more efficacy have continued .
herbal agents have long been a valuable source for developing new therapeutics for disease treatment .
ocimum basilicum ( o. basilicum ) is an annual herb belonging to the lamiaceae family and grows mostly in tropical regions such as india , africa , and south asia ( 4 ) . in traditional medicine ,
o. basilicum is used to treat many disorders such as anxiety , diabetes , cardiovascular diseases , headaches , neurological pain , and seizures ( 5 , 6 ) .
it has been demonstrated that the ethyl acetate fraction ( eaf ) of o. basilicum decreases ischemia - induced oxidative stress in the brain and improves short - term memory and motor coordination ( 5 ) .
it was reported in some traditional medical texts that o. basilicum leads to sleep and a sedative state .
also , some biological activities of o. basilicum were screened by ismail , and it was found that the essential oil of o. basilicum induces anticonvulsant and hypnotic activities ( 7 ) . in folk medicine , maceration is the most widely used form of o. basilicum preparation
. however , there is no pharmacological evidence for the sedative - hypnotic effect of o. basilicum macerated extract .
the present study was designed to evaluate the sleep - prolonging effects of hydro - alcoholic extract ( hae ) of o. basilicum ) and its fractions .
dimethyl sulfoxide ( dmso , code d4540 ) , penicillin - streptomycin ( code p4333 ) , sodium pentobarbital ( code p3761 ) , and 3-(4 , 5-dimethyl-2-thiazolyl)-2 , 5-diphenyl-2h - tetrazolium bromide ( mtt , code m-5655 ) were bought from sigma ( st .
dulbeccos modified eagles medium ( dmem , code 12800 - 082 ) and fetal bovine serum ( fbs , code 10270 - 106 ) were purchased from gibco life technologies ( grand island , ny , usa ) .
the leaves of o. basilicum were collected from mashhad , iran in the month of july and dried in the shade .
a voucher sample was preserved for reference in the herbarium of the department of pharmacognosy , school of pharmacy , mashhad university of medical sciences ( herbarium number : 12937 obl ) .
hae was prepared using the maceration method ( 8) . the powder of the leaves of o. basilicum ( 100 g ) was macerated in 800 ml of 70% ethanol for 48 hours .
the extract was then filtered through a 0.106 mm filter and dried in a water bath .
the yield of the dried extract as related to the weight of the dried leaves was 21% . for the preparation of different fractions from hae ,
10 g of dried extract were suspended in 200 ml of distilled water and transferred to a separator funnel . using solvent - solvent extraction ,
the eaf and n - butanol fraction ( nbf ) were separated to obtain water fraction ( wf ) ( 9 , 10 ) .
all the fractions were dried in a water bath ( memmert , germany ) and stored at -20c until used .
then , the wf was dissolved in saline , and the eaf and nbf were dissolved in distilled water containing 10% dmso .
male albino mice weighting 20 - 30 g were maintained at a controlled temperature ( 22  1c ) with a 12 hours light / dark cycle and free access to water and food .
the study was carried out in accordance with the ethical guidelines of mashhad university of medical sciences ( code 910240 , 2012.07.05 ) .
first , to determine if hae has a sleep - prolonging effect , the animals received saline ( control group ) and diazepam ( as positive control ) or different doses of hae . in the second experiment , to determine the most effective fraction of hae , animals were treated with wf , eaf , nbf , or 10% dmso ( vehicle for eaf and nbf ) .
the hypnotic assessment method was based on the prolongation of sleep induced by pentobarbital . a single dose of hae ( 25 , 50 , 100 mg / kg ) , fractions of hae , diazepam ( 3 mg / kg ) , or other vehicles were injected intraperitoneally ( ip ) into the mice .
after 30 minutes , pentobarbital ( 30 mg / kg ip ) was administrated to induce sleep ( 11 - 13 ) .
flumazenil ( 1 mg / kg ) was administrated 30 minutes before diazepam or hae ( 12 ) .
all materials were injected with a volume of 10 mg / kg ( 11 ) .
the onset of sleep is the time that the mice stayed immobile and lost their righting reflex .
the time interval between administration of pentobarbital and onset of sleep was considered sleep latency .
nine groups , each containing two mice were used for determination of ld50 of hae .
groups 1 - 8 were injected ip with 25 , 50 , 100 , 200 , 400 , 800 , 1,600 , and 3,200 mg / kg of hae and group 9 received normal saline as a vehicle .
the highest dose which did not kill any mice and the lowest dose which led to the death of one animal were recorded .
the mean of these two doses was considered the median lethal dose ( 14 , 15 ) .
the possible cytotoxicity of o. basilicum was tested on rat pheochromocytoma - derived cells ( pc12 ) and murine fibroblast cells ( l929 ) .
pc12 cells have several neuronal characteristics and , therefore , are useful in the in vitro model for evaluating the neuroprotective or neurotoxic activity of drugs and plant extracts .
also , the l929 cell is considered to be a standard cell line for cytotoxicity assays according to the us pharmacopeia ( usp ) and is frequently used for testing the possible toxic effects of materials .
the cells were cultured in 96-well plates for 24 hours in dmem supplemented with 10% fbs , penicillin ( 100 units / ml ) , and streptomycin ( 100 g / ml ) .
then , the culture medium was changed to a fresh one containing the vehicle ( dmso 1% ) or hae ( 50 , 100 , 200 , 400 , and 800 g / ml ) .
the cells were incubated for 24 hours at 37c in an atmosphere of 5% co2 .
then , cell proliferation was evaluated using the mtt assay as previously described ( 16 ) .
briefly , the mtt solution was added to a culture medium to make a final concentration of 0.5 mg / ml and incubated for 2 hours .
statistical analysis was performed using one - way analysis of variance ( anova ) followed by tamhane s t2 post hoc test using the instat software package ( graphpad , san diego , ca ) .
dimethyl sulfoxide ( dmso , code d4540 ) , penicillin - streptomycin ( code p4333 ) , sodium pentobarbital ( code p3761 ) , and 3-(4 , 5-dimethyl-2-thiazolyl)-2 , 5-diphenyl-2h - tetrazolium bromide ( mtt , code m-5655 ) were bought from sigma ( st .
dulbeccos modified eagles medium ( dmem , code 12800 - 082 ) and fetal bovine serum ( fbs , code 10270 - 106 ) were purchased from gibco life technologies ( grand island , ny , usa ) .
the leaves of o. basilicum were collected from mashhad , iran in the month of july and dried in the shade . a voucher sample was preserved for reference in the herbarium of the department of pharmacognosy , school of pharmacy , mashhad university of medical sciences ( herbarium number : 12937 obl ) .
hae was prepared using the maceration method ( 8) . the powder of the leaves of o. basilicum ( 100 g ) was macerated in 800 ml of 70% ethanol for 48 hours .
the extract was then filtered through a 0.106 mm filter and dried in a water bath .
the yield of the dried extract as related to the weight of the dried leaves was 21% .
for the preparation of different fractions from hae , 10 g of dried extract were suspended in 200 ml of distilled water and transferred to a separator funnel . using solvent - solvent extraction ,
the eaf and n - butanol fraction ( nbf ) were separated to obtain water fraction ( wf ) ( 9 , 10 ) .
all the fractions were dried in a water bath ( memmert , germany ) and stored at -20c until used .
then , the wf was dissolved in saline , and the eaf and nbf were dissolved in distilled water containing 10% dmso .
male albino mice weighting 20 - 30 g were maintained at a controlled temperature ( 22  1c ) with a 12 hours light / dark cycle and free access to water and food .
the study was carried out in accordance with the ethical guidelines of mashhad university of medical sciences ( code 910240 , 2012.07.05 ) .
first , to determine if hae has a sleep - prolonging effect , the animals received saline ( control group ) and diazepam ( as positive control ) or different doses of hae . in the second experiment ,
to determine the most effective fraction of hae , animals were treated with wf , eaf , nbf , or 10% dmso ( vehicle for eaf and nbf ) .
the hypnotic assessment method was based on the prolongation of sleep induced by pentobarbital . a single dose of hae ( 25 , 50 , 100 mg / kg ) , fractions of hae , diazepam ( 3 mg / kg ) , or other vehicles were injected intraperitoneally ( ip ) into the mice .
after 30 minutes , pentobarbital ( 30 mg / kg ip ) was administrated to induce sleep ( 11 - 13 ) .
flumazenil ( 1 mg / kg ) was administrated 30 minutes before diazepam or hae ( 12 ) .
all materials were injected with a volume of 10 mg / kg ( 11 ) .
the onset of sleep is the time that the mice stayed immobile and lost their righting reflex .
the time interval between administration of pentobarbital and onset of sleep was considered sleep latency .
nine groups , each containing two mice were used for determination of ld50 of hae .
groups 1 - 8 were injected ip with 25 , 50 , 100 , 200 , 400 , 800 , 1,600 , and 3,200 mg / kg of hae and group 9 received normal saline as a vehicle .
the highest dose which did not kill any mice and the lowest dose which led to the death of one animal were recorded .
the mean of these two doses was considered the median lethal dose ( 14 , 15 ) .
the possible cytotoxicity of o. basilicum was tested on rat pheochromocytoma - derived cells ( pc12 ) and murine fibroblast cells ( l929 ) .
pc12 cells have several neuronal characteristics and , therefore , are useful in the in vitro model for evaluating the neuroprotective or neurotoxic activity of drugs and plant extracts .
also , the l929 cell is considered to be a standard cell line for cytotoxicity assays according to the us pharmacopeia ( usp ) and is frequently used for testing the possible toxic effects of materials .
the cells were cultured in 96-well plates for 24 hours in dmem supplemented with 10% fbs , penicillin ( 100 units / ml ) , and streptomycin ( 100 g / ml ) .
then , the culture medium was changed to a fresh one containing the vehicle ( dmso 1% ) or hae ( 50 , 100 , 200 , 400 , and 800 g / ml ) .
the cells were incubated for 24 hours at 37c in an atmosphere of 5% co2 .
then , cell proliferation was evaluated using the mtt assay as previously described ( 16 ) .
briefly , the mtt solution was added to a culture medium to make a final concentration of 0.5 mg / ml and incubated for 2 hours .
statistical analysis was performed using one - way analysis of variance ( anova ) followed by tamhane s t2 post hoc test using the instat software package ( graphpad , san diego , ca ) .
sleep duration in the negative control group that received normal saline before pentobarbital was 20  2 minutes ( figure 1 ) .
as expected , the reference drug diazepam was able to increase the duration of sleep ( 42  4.6 minutes , p < 0.001 vs. control ) .
the hae at doses of 25 , 50 , and 100 mg / kg could significantly increase sleep duration to 51  3 minutes ( p < 0.001 ) , 79  3 minutes ( p < 0.001 ) , and 46  5 minutes ( p < 0.001 ) , respectively .
the effect produced by 50 mg / kg was significantly greater than that produced by 25 or 100 mg / kg ( p < 0.001 ) .
the animals were treated with saline ( control ) , diazepam ( 3 mg / kg ) , or hydro - alcoholic extract ( hae ) 30 minutes before administration of pentobarbital ( 30 mg / kg ip ) . * * * p < 0.001 vs. control ; # # # p < 0.001 vs. groups of 25 and 100
mg / kg . as expected , pretreatment of mice with flumazenil decreased the sleep - prolonging effect of diazepam ( 42  4.5 minutes and 20  3 minutes for diazepam and flumazenil plus diazepam , respectively , p < 0.001 ) .
similarly , the effect of hae on sleep duration was significantly inhibited by flumazenil ( 79  3 minutes and 30  2 minutes for hae and hae plus diazepam , respectively , p < 0.001 ) .
the animals were treated with saline ( control ) and 3 mg / kg of diazepam or 50 mg / kg of hydro - alcoholic extract ( hae ) before injection of pentobarbital ( 30 mg / kg ip ) .
flumazenil ( 1 mg / kg ) was administrated 30 minutes before diazepam or hae . * * * p < 0.001 vs. control ; # # # p < 0.001 vs. hae ;  p
values are presented as mean  sem ( n = 8) . as shown in figure 3 , all three fractions of hae exhibited sleep - prolonging activity .
duration of sleep in groups receiving 50 mg / kg of wf , eaf , and nbf was 40  4.5 minutes ( p < 0.001 vs. control ) , 30  2 minutes ( p < 0.001 vs. vehicle ) and 42  2 minutes ( p < 0.001 vs. vehicle ) , respectively . among the fractions , nbf induced the maximum prolongation of sleep .
the animals were treated with 10% dmso ( vehicle ) or 50 mg / kg of water fraction ( wf ) , ethyl acetate fraction ( eaf ) or n - butanol fraction ( nbf ) before administration of pentobarbital ( 30 mg / kg , ip ) . * * * p < 0.001 vs. control and vehicle ; # p
< 0.05 vs. eaf group ; values are presented as mean  sem ( n = 8) .
figure 4 shows the time elapsed between the administration of pentobarbital and the onset of sleep . when compared to the control ( 6.7  0.4 minutes ) , diazepam significantly decreased sleep latency to 3.5  0.4 minutes ( p < 0.001 ) .
the latency time in animals receiving 25 , 50 , and 100 mg / kg of hae was 4.2  0.2 minutes ( p < 0.001 ) , 4.5  0.5 minutes ( p < 0.01 ) , and 4.4  0.4 minutes ( p < 0.01 ) , respectively . the effect of diazepam was not statistically different from that observed with 25 , 50 , or 100 mg / kg of hae .
the animals were treated with saline ( control ) and diazepam ( 3 mg / kg ) or hydro - alcoholic extract ( hae ) . * * p < 0.01 vs. control ; * * * p < 0.001 vs. control ; b , mice were treated with 10% dmso ( vehicle ) or 50 mg / kg of water fraction ( wf ) , ethyl acetate fraction ( eaf ) or n - butanol fraction ( nbf ) . * * * p < 0.01 vs. control ; # # # p < 0.01 vs. wf and nbf .
values are presented as mean  sem ( n = 8) . among the different fractions of hae ,
however , nbf significantly decreased the latency time to 4.7  0.3 minutes ( p < 0.001 ) . although wf could decrease sleep latency from the control level ( 6.7  0.4 minutes ) to 5.2  0.2 minutes , this effect was not statistically significant . the highest dose , which did not kill any mice , and the lowest dose , which led to the death of one mouse , were 1.6 and 3.2 g / kg , respectively .
the mean of these two doses ( 2.4 g / kg ) was considered as ld50 .
the possible cytotoxicity of hae of o. basilicum was evaluated on pc12 and l929 cells ( figure 5 ) .
it was found that up to 24 hours none of hae concentrations decreased proliferation of pc12 cells . in the presence of 50 , 100 , 200 , 400 , and 800 g / ml of the extract , cell viability was 100
 3 , 102  2 , 103  2 , 110  3 , and 113  3% , respectively , as compared to the vehicle ( 100  2.5% ) .
regarding l929 cells , the viability was 106  1.5 , 108  2 , 112  2 , 113  1 , and 123  2.5% in the presence of 50 , 100 , 200 , 400 , and 800 mg / ml , respectively .
again , there was no significant difference in the cell viability when compared to the vehicle ( 100  2% ) .
the cells were cultivated for 24 hours in culture media containing hydro - alcoholic extract of o. basilicum .
sleep duration in the negative control group that received normal saline before pentobarbital was 20  2 minutes ( figure 1 ) .
as expected , the reference drug diazepam was able to increase the duration of sleep ( 42  4.6 minutes , p < 0.001 vs. control ) .
the hae at doses of 25 , 50 , and 100 mg / kg could significantly increase sleep duration to 51  3 minutes ( p < 0.001 ) , 79  3 minutes ( p < 0.001 ) , and 46  5 minutes ( p < 0.001 ) , respectively .
the effect produced by 50 mg / kg was significantly greater than that produced by 25 or 100 mg / kg ( p < 0.001 ) .
the animals were treated with saline ( control ) , diazepam ( 3 mg / kg ) , or hydro - alcoholic extract ( hae ) 30 minutes before administration of pentobarbital ( 30 mg / kg ip ) . * * * p < 0.001 vs. control ; # # # p < 0.001 vs. groups of 25 and 100
mg / kg . as expected , pretreatment of mice with flumazenil decreased the sleep - prolonging effect of diazepam ( 42  4.5 minutes and 20  3 minutes for diazepam and flumazenil plus diazepam , respectively , p < 0.001 ) .
similarly , the effect of hae on sleep duration was significantly inhibited by flumazenil ( 79  3 minutes and 30  2 minutes for hae and hae plus diazepam , respectively , p < 0.001 ) .
the animals were treated with saline ( control ) and 3 mg / kg of diazepam or 50 mg / kg of hydro - alcoholic extract ( hae ) before injection of pentobarbital ( 30 mg / kg ip ) .
flumazenil ( 1 mg / kg ) was administrated 30 minutes before diazepam or hae . * * * p < 0.001 vs. control ; # # # p < 0.001 vs. hae ;  p
values are presented as mean  sem ( n = 8) . as shown in figure 3 , all three fractions of hae exhibited sleep - prolonging activity .
duration of sleep in groups receiving 50 mg / kg of wf , eaf , and nbf was 40  4.5 minutes ( p < 0.001 vs. control ) , 30  2 minutes ( p < 0.001 vs. vehicle ) and 42  2 minutes ( p < 0.001 vs. vehicle ) , respectively . among the fractions , nbf induced the maximum prolongation of sleep .
the animals were treated with 10% dmso ( vehicle ) or 50 mg / kg of water fraction ( wf ) , ethyl acetate fraction ( eaf ) or n - butanol fraction ( nbf ) before administration of pentobarbital ( 30 mg / kg , ip ) . * * * p < 0.001 vs. control and vehicle ; # p
< 0.05 vs. eaf group ; values are presented as mean  sem ( n = 8) .
figure 4 shows the time elapsed between the administration of pentobarbital and the onset of sleep . when compared to the control ( 6.7  0.4 minutes ) , diazepam significantly decreased sleep latency to 3.5  0.4 minutes ( p < 0.001 ) .
the latency time in animals receiving 25 , 50 , and 100 mg / kg of hae was 4.2  0.2 minutes ( p < 0.001 ) , 4.5  0.5 minutes ( p < 0.01 ) , and 4.4  0.4 minutes ( p < 0.01 ) , respectively .
the effect of diazepam was not statistically different from that observed with 25 , 50 , or 100 mg / kg of hae .
the animals were treated with saline ( control ) and diazepam ( 3 mg / kg ) or hydro - alcoholic extract ( hae ) . * * p < 0.01 vs. control ; * * * p < 0.001 vs. control ; b , mice were treated with 10% dmso ( vehicle ) or 50 mg / kg of water fraction ( wf ) , ethyl acetate fraction ( eaf ) or n - butanol fraction ( nbf ) . * * * p < 0.01 vs. control ; # # # p < 0.01 vs. wf and nbf .
values are presented as mean  sem ( n = 8) . among the different fractions of hae ,
however , nbf significantly decreased the latency time to 4.7  0.3 minutes ( p < 0.001 ) . although wf could decrease sleep latency from the control level ( 6.7  0.4 minutes ) to 5.2  0.2 minutes , this effect was not statistically significant .
the highest dose , which did not kill any mice , and the lowest dose , which led to the death of one mouse , were 1.6 and 3.2 g / kg , respectively .
the mean of these two doses ( 2.4 g / kg ) was considered as ld50 .
the possible cytotoxicity of hae of o. basilicum was evaluated on pc12 and l929 cells ( figure 5 ) .
it was found that up to 24 hours none of hae concentrations decreased proliferation of pc12 cells . in the presence of 50 , 100 , 200 , 400 , and 800 g / ml of the extract ,
cell viability was 100  3 , 102  2 , 103  2 , 110  3 , and 113  3% , respectively , as compared to the vehicle ( 100  2.5% ) .
regarding l929 cells , the viability was 106  1.5 , 108  2 , 112  2 , 113  1 , and 123  2.5% in the presence of 50 , 100 , 200 , 400 , and 800 mg / ml , respectively .
again , there was no significant difference in the cell viability when compared to the vehicle ( 100  2% ) .
the cells were cultivated for 24 hours in culture media containing hydro - alcoholic extract of o. basilicum .
the present study showed that o. basilicum further enhances sleep behavior , confirming that this plant has a hypnotic action as claimed in traditional medicine . to our knowledge , this is the first pharmacological study showing the effects of the macerated extract of this plant on sleep duration and sleep latency . also , this is the first work to determine the ld50 value for hae of o. basilicum and to assess possible cytotoxicity of this extract on neuronal cells .
yet , results of this study are preliminary and need to be confirmed by further clinical trials .
the hypnotic assessment method was based on prolongation of sleep induced by pentobarbital , which is the most commonly used method for screening sedative - hypnotic agents ( 11 - 13 ) . in agreement with the previously published works and as expected , diazepam significantly increased pentobarbital - induced sleeping time , indicating that our study method was optimized ( 17 , 18 ) .
the effect of hae of o. basilicum on sleep latency was lower than that of diazepam .
however , the sleep - prolonging effect of hae was comparable to that of diazepam , and , even at a dose of 50 mg / kg , hae had a greater effect . also , the hypnotic effect of hae of o. basilicum is greater when compared with the effect of essential oil of o. basilicum reported by ismail ( 4-fold and 2-fold increase in sleep duration , respectively ) ( 7 ) . according to our results , the hypnotic effect of hae was not dose - dependent in the range of given doses , and the maximum effect occurred with a dose of 50 mg / kg .
therefore , we used this dose to investigate the effects of different fractions of hae . the fractions of hae were prepared to obtain better insight into the nature of compounds responsible for the hypnotic effect of o. basilicum : 1 , wf which extracts water - soluble constituents ( e.g. , glycosides , quaternary alkaloids , tannins ) ; 2 , eaf which extracts compounds of intermediate polarity ( e.g. , some flavonoids ) ; and 3 , nbf which extracts low polar agents ( e.g. , sterols , alkanes , and some terpenoids ) ( 19 , 20 ) .
although wf , eaf , and nbf at a dose of 50 mg / kg were all able to increase sleep duration , not one of them could enhance sleep duration to the level induced by 50 mg / kg of hae .
because hae contains the active constituents of all the above - mentioned fractions , it can be concluded that an additive effect was caused by the interaction between these constituents when hae was administrated .
therefore , both polar and non - polar constituents in o. basilicum extract are involved in the sleep - prolonging effect of this plant . among wf ,
eaf , and nbf fractions , nbf not only induced the maximum prolongation of sleep duration , but also was the only fraction to induce a significant decrease in sleep latency . on the other hand
therefore , nbf contains a higher concentration of constituents responsible for the hypnotic effect of o. basilicum .
these include terpenoids ( e.g. , linalool , eugenol ) , flavonoids ( e.g. , quercetrin , luteolin ) , alkaloids ( e.g. , rosmarinic acid ) , steroids ( e.g. , beta - sitisterol ) , and saponins ( 21 , 22 ) .
it has been shown that o. basilicum contains high amounts of linalool , eugenol , and rosmarinic acid ( 7 ) .
linck and coworkers demonstrated that linalool increases barbiturate - induced sleeping time in mice ( 23 ) .
similarly , the sleep - prolonging effect of eugenol was reported by sharma et al .
rosmarinic acid is found in methanolic extract of o. basilicum and can be isolated from the nbf of plant extracts ( 24 , 25 ) .
it plays a major role in the sedative - hypnotic actions of some medicinal plants ( 26 ) .
neurons located in the anterior hypothalamus release gamma - aminobutyric acid ( gaba ) to inhibit wake - promoting areas in the hypothalamus and brainstem ( 27 , 28 ) .
barbiturates such as pentobarbital act on the gaba receptor s ionophore complex and favor the binding of gaba .
benzodiazepines such as diazepam increase the affinity of gaba for its receptor and thereby enhance pentobarbital - induced sleeping time ( 29 ) .
consistent with this , we observed that pretreatment of mice with flumazenil decreases the sleep - prolonging effect of diazepam .
in agreement with our finding , awad et al . reported that rosmarinic acid , which is found in the extract of o. basilicum , can act as a gaba transaminase inhibitor and therefore increases the brain level of gaba ( 30 ) . therefore , it is rational to suggest that the sleep - prolonging effect of o. basilicum is mediated , at least in part , through the potentiating of the gabaergic system .
it is now accepted that some natural compounds interact with the gabaergic system to increase sleep behavior ( 31 , 32 ) .
the toxicity assay showed that the ld50 value for hae of o. basilicum is 2.4 g / kg .
this dose is far from its hypnotic doses ( 25 - 100 mg / kg ) .
also , hae , even at high concentrations , did not decrease the viability of neuronal and fibroblast cells .
therefore , it seems that the hypnotic effect of o. basilicum is not accompanied by neurotoxicity . in conclusion
, present data demonstrated that the macerated extract of o. basilicum potentiates sleeping behaviors without any cytotoxicity .
 backgroundsleep disorders are accompanied by several complications , and currently used soporific drugs can induce unwanted effects such as psychomotor impairment , tolerance , amnesia , and rebound insomnia.objectivesthe present study was carried out to investigate if ocimum basilicum has a sleep - prolonging effect.materials and methodsthis work was an experimental study on 72 mice which were randomly divided into 9 groups : saline ( control ) ; diazepam ( 3 mg / kg , positive control ) ; hydro - alcoholic extract ( hae ) of ocimum basilicum ( 25 , 50 , or 100 mg / kg ) ; ethyl acetate fraction ( eaf , 50 mg / kg ) ; n - butanol fraction ( nbf , 50 mg / kg ) ; water fraction ( wf , 50 mg / kg ) ; and saline containing 10% dmso ( vehicle for eaf and nbf ) . 
 all the test compounds were injected intraperitoneally ( ip ) 30 minutes before pentobarbital administration ( 30 mg / kg ) . duration and latency of pentobarbital - induced sleep were recorded . 
 also , ld50 of hae was determined and the cytotoxicity of hae was tested on neural and fibroblast cells using the mtt assay.resultshae increased the duration of pentobarbital - induced sleep at doses of 25 , 50 , and 100 mg / kg ( p < 0.001 ) . the hypnotic effect of hae was comparable to that induced by diazepam . similarly , wf , eaf , and nbf at 50 mg / kg could increase sleep duration . 
 the sleep latency was decreased by hae ( p < 0.01 - p < 0.001 ) and nbf ( p 
 < 0.001 ) , but not by wf and eaf . the ld50 value for hae was found to be 2.4 g / kg . 
 hae had no effect on the viability of neuronal pc12 cells and l929 fibroblast cells.conclusionsthe present data demonstrated that ocimum basilicum potentiates sleeping behaviors without any cytotoxicity . 
 the main component ( s ) responsible for the hypnotic effects of this plant is most likely a non - polar agent ( s ) which is found in nbf . 
 isolation of the active constituents may yield a novel sedative drug . 