doxorubicin ( dox ) is an important component of multimodality therapy for several antineoplastic combined chemotherapy protocols . it is used to treat a variety of malignacies such as leukemias , hodgkin and non - hodgkin lymphoma , and solid tumors ( 1 ) . however , despite high efficacy , the major side effect of dox is cardio - toxicity , which has dramatically hindered its clinical usage for a prolonged period of time . the mechanisms of dox - induced cardio toxicity are not completely understood , but most evidences indicate the generation of reactive oxygen species ( ros ) involvement ( 2 ) . interestingly , some natural foods have been reported to contain substantial amounts of antioxidants and free radical scavenging agents . these compounds diminish some side effects of chemotherapeutic agents on normal cells by reducing their genotoxicity ( 2 ) . there are many evidences showing the protective effects for different herbs against oxidative injury - related cardio toxicity , and among which n. sativa , zingiber officinale and glycyrrhiza glabra showed the highest protective effect . its seeds have long been used in traditional medicine for a wide range of disorders including bronchial asthma , headache , infections , obesity , back pain , hypertension and gastrointestinal problems ( 4 ) . these effects are mediated via different mechanism such as antioxidant ( 5 ) , anti - inflammatory ( 6 ) , anticancer ( 7 ) , and antihistaminic effects ( 8) . it has been shown that n. sativa attenuates isoproterenol induced myocardial infarction ( mi ) ( 9 ) . also , n. sativa oil supplementation reduces lead - induced cardio - toxicity by mechanisms related to its ability to decrease the pro - inflammatory cytokines ; oxidative stress and cardiac tissue damage , and preserve the activity of antioxidant enzymes ( 10 ) . ebru et al ( 2008 ) showed that pretreatment with n. sativa oil decreased the subsequent cyclosporine - a injury in rat heart ( 11 ) , recent studies revealed a protective effect of thymoquinone against doxorubicin - induced cardio toxicity ( 12 - 14 ) . it is consumed worldwide as a spice and flavoring agent , and is attributed to have many medicinal properties ( 15 ) . it is used in traditional medicine as carminative , and antipyretic , and in the treatment of pain , rheumatism and bronchitis ( 15 ) . it has different pharmacological activities such as hepatoprotective ( 16 ) , antiparasitic ( 17 ) , antimalarial ( 18 ) , antimicrobial ( 19 ) , antidiabetic ( 20 ) , and radioprotective effects ( 21 ) . ginger also is a potential remedy for cardiovascular diseases ( 22 ) , and can prevent the development of morphine analgesic tolerance and physical dependence in rats ( 23 ) . it is also used for the treatment of gastrointestinal disorders including gastric ulcerogenesis ( 24 ) . the plant contains high level of phenolic and flavonoid compounds , responsible for its high antioxidant activities ( 25 ) . g. glabra ( licorice ) is used widely as a flavoring and sweetening agent in tobacco products , chewing gum , candy , toothpaste and beverages . the therapeutic potential of g. glabra as a hypoglycemic ( 27 ) , hypocholesteremic ( 28 ) , anti - ulcer ( 29 ) , anti - inflammatory ( 30 ) , renoprotective ( 31 ) , and antiatherogenic ( 32 ) have been demonstrated . in traditional medicine , it is useful in the treatment of agent renovascular and cardiovascular diseases , and hence constitutes as the major ingredient of polyherbal formulations indicated for cardiovascular diseases ( 31 ) . the investigations have shown that g. glabra has protective effects against ischemic damages of the several body organs due to its potent antioxidant and free radical scavenging activity ( 27 , 28 , 30 - 32 ) . also , a recent study has shown that pretreatment with g. glabra significantly attenuates ischemic  reperfusion induced myocardial injury by improving antioxidant status in heart ( 33 ) . in addition , other species of g. uralensis , suppressed doxorubicin - induced apoptosis in h9c2 , rat cardiac myoblast cells ( 34 ) . treatment with licorice extract significantly protected the mice against dox - induced cardio toxicity ( 35 ) , and improved cardiac performance ( 36 ) . based on these findings , we hypothesized that the combination of these medicinal plants may produce a higher protective effect against doxorubicin - induced cardiomyopathy . therefore , the present study was designed to investigate the potential effects of nigella sativa with glycyrrhiza glabra and zingiber officinale ( ngz ) extracts on cell viability , lipid peroxidation level , ros content and apoptotic induction in h9c2 cardiomyocytes . 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium ( mtt ) , thiobarbituric acid ( tba ) , 2,7-dichlorofluorescin diacetate ( dcfh - da ) , propidium iodide ( pi ) , sodium citrate and triton x-100 were purchased from sigma ( st . louis , mo , usa ) . high glucose dulbecco s modified eagles medium ( dmem ) , penicillin - streptomycin and fetal bovine serum were purchased from gibco . trichloroacetic acid ( tca ) and malondialdehyde bis-(dimethyl acetal ) ( mda ) were obtained from merck ( darmstadt , germany ) . n. sativa seeds were collected from gonabad region ( northeast of iran ) and authenticated by herbarium of the ferdowsi university of mashhad ( fumh ) ( voucher specimen = 293 - 0303 - 1 ) . g. glabra rhizomes was purchased from local market and authenticated by fumh ( voucher specimen number = 25947 ) . whereas z. officinale is not native to iran , afforded from a local herb market in mashhad and confirmed by plant specialist of fumh . the seeds of n. sativa were washed , dried , and crushed to a powder with an electric micronizer . the g. glabra and z. officinale rhizomes were peeled chopped into tiny bits , air - dried and were ground with a mechanical grinder . each herbal sample was extracted separately in a soxhlet extractor with ethanol ( 70% ) and the resulting extract was dried and kept at -20c until use . the cells were cultured in dulbecco s modified eagles medium ( dmem ) supplemented with 10% fetal bovine serum , 100 units / ml penicillin and 100 g / ml streptomycin . for the experiments , they were seeded in 96-well and 24-well culture plates for mtt / ros and mda assays , respectively . for apoptosis assay , cells were seeded at 100,000 cell / well in a 24-well plate . the cells were pretreated with each herbal extract alone ( 6 to 200 g / ml ) for 4 hr and then incubation was continued in the presence of the herbal extract with or without 5 m doxorubicin for 24 hr . for assaying the effect of three extract in combination , the minimum effective concentration of each extract was chosen ( n=12.5 , g=25 and z=25 g / ml sum=62.5 g / ml ) as the basis and one ( 62.5 g / ml ) , two ( 125 g / ml ) , four ( 250 g / ml ) and eight ( 500 g / ml ) folds of this concentration were applied . cell viability was determined using a modified mtt assay as described previously ( 37 ) . briefly , mtt solution in phosphate - buffered saline ( 5 mg / ml ) was added to each well at final concentration of 0.05% . after 3 hr , the formazan precipitate was dissolved in dmso . the absorbance at 570 and 620 nm ( background ) was measured using a statfax303 plate reader . the level of lipid peroxidation was estimated by measuring mda , which is the end product of lipid peroxidation ( 38 ) . at the end of incubation , then , 400 l of tca ( 15% ) and 800 l of tba ( 0.7% ) were added to 500 l of cell samples . the mixture was vortexed and heated for 40 min in a boiling water bath . then , 200 l of the sample was transferred to 96-well plate and the fluorescence intensity was read with excitation / emission of 480/530 nm . intracellular ros level was evaluated using a fluorescent probe , dcf - da . at the end of incubation , the cells were treated ( 30 min ) with dcfh - da ( 10 m ) at 4c in the dark . apoptotic cells were detected using pi staining of small dna fragments followed by flow cytometry . it has been reported that a sub - g1 peak that is reflective of dna fragmentation can be observed following the incubation of cells in a hypotonic phosphate - citrate buffer containing a quantitative dna - binding dye such as pi . apoptotic cells that have lost dna will take up less stain and appear on the left side of the g1 peak in the histogram . briefly , h9c2 cells were seeded in wells of a 24-well plate and treated according to mentioned protocol . floating and adherent cells were then harvested and incubated at 4 c overnight in the dark with 750 l of a hypotonic buffer ( 50 g / ml pi in 0.1% sodium citrate with 0.1% triton x-100 ) . next , flow cytometry was carried out using a facscan flow cytometer ( becton dickinson ) . statistical analysis was performed using one way analysis of variance ( anova ) followed by tamhane s t2 post - hoc test . 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium ( mtt ) , thiobarbituric acid ( tba ) , 2,7-dichlorofluorescin diacetate ( dcfh - da ) , propidium iodide ( pi ) , sodium citrate and triton x-100 were purchased from sigma ( st . louis , mo , usa ) . high glucose dulbecco s modified eagles medium ( dmem ) , penicillin - streptomycin and fetal bovine serum were purchased from gibco . trichloroacetic acid ( tca ) and malondialdehyde bis-(dimethyl acetal ) ( mda ) were obtained from merck ( darmstadt , germany ) . n. sativa seeds were collected from gonabad region ( northeast of iran ) and authenticated by herbarium of the ferdowsi university of mashhad ( fumh ) ( voucher specimen = 293 - 0303 - 1 ) . g. glabra rhizomes was purchased from local market and authenticated by fumh ( voucher specimen number = 25947 ) . whereas z. officinale is not native to iran , afforded from a local herb market in mashhad and confirmed by plant specialist of fumh . the seeds of n. sativa were washed , dried , and crushed to a powder with an electric micronizer . the g. glabra and z. officinale rhizomes were peeled chopped into tiny bits , air - dried and were ground with a mechanical grinder . each herbal sample was extracted separately in a soxhlet extractor with ethanol ( 70% ) and the resulting extract was dried and kept at -20c until use . the cells were cultured in dulbecco s modified eagles medium ( dmem ) supplemented with 10% fetal bovine serum , 100 units / ml penicillin and 100 g / ml streptomycin . for the experiments , they were seeded in 96-well and 24-well culture plates for mtt / ros and mda assays , respectively . for apoptosis assay , the cells were pretreated with each herbal extract alone ( 6 to 200 g / ml ) for 4 hr and then incubation was continued in the presence of the herbal extract with or without 5 m doxorubicin for 24 hr . for assaying the effect of three extract in combination , the minimum effective concentration of each extract was chosen ( n=12.5 , g=25 and z=25 g / ml sum=62.5 g / ml ) as the basis and one ( 62.5 g / ml ) , two ( 125 g / ml ) , four ( 250 g / ml ) and eight ( 500 g / ml ) folds of this concentration were applied . cell viability was determined using a modified mtt assay as described previously ( 37 ) . briefly , mtt solution in phosphate - buffered saline ( 5 mg / ml ) was added to each well at final concentration of 0.05% . after 3 hr , the formazan precipitate was dissolved in dmso . the absorbance at 570 and 620 nm ( background ) was measured using a statfax303 plate reader . the level of lipid peroxidation was estimated by measuring mda , which is the end product of lipid peroxidation ( 38 ) . at the end of incubation , then , 400 l of tca ( 15% ) and 800 l of tba ( 0.7% ) were added to 500 l of cell samples . the mixture was vortexed and heated for 40 min in a boiling water bath . then , 200 l of the sample was transferred to 96-well plate and the fluorescence intensity was read with excitation / emission of 480/530 nm . intracellular ros level was evaluated using a fluorescent probe , dcf - da . at the end of incubation , the cells were treated ( 30 min ) with dcfh - da ( 10 m ) at 4c in the dark . apoptotic cells were detected using pi staining of small dna fragments followed by flow cytometry . it has been reported that a sub - g1 peak that is reflective of dna fragmentation can be observed following the incubation of cells in a hypotonic phosphate - citrate buffer containing a quantitative dna - binding dye such as pi . apoptotic cells that have lost dna will take up less stain and appear on the left side of the g1 peak in the histogram . briefly , h9c2 cells were seeded in wells of a 24-well plate and treated according to mentioned protocol . floating and adherent cells were then harvested and incubated at 4 c overnight in the dark with 750 l of a hypotonic buffer ( 50 g / ml pi in 0.1% sodium citrate with 0.1% triton x-100 ) . next , flow cytometry was carried out using a facscan flow cytometer ( becton dickinson ) . statistical analysis was performed using one way analysis of variance ( anova ) followed by tamhane s t2 post - hoc test . incubation with doxorubicin significantly decreased cell viability to 622% compared to control of control ( p<0.001 ) . cell viability was assayed to determine the optimum concentrations necessary for the three extracts ( n , g and z ) to protect h9c2 cells against dox - induced cytotoxicity . the results demonstrated that , in comparison with dox , n. sativa increased cell viability at doses of 25 to 200 g / ml dose dependently that maximum effect was produced at effect at concentration of 50 g / ml ( 75.40.84 , p<0.01 ) , and z. officinale increased cell viability at doses of 50 to 200 g / ml , its maximum effect was at dose of 100 g / ml ( 752.5 , p<0.01 ) , while g. glabra increased cell viability at dose of 100 g / ml ( 73.51.5 , p<0.05 ) ( figure 1a ) . effect of extracts alone ( figure 1a ) or ngz ( figure 1b ) on percentage of protection against dox - induced cytotoxicity in h9c2 cells . cells were pretreated with different concentrations of ngz for 4 hr before exposure to 5 m of dox for 24 hr . dox decreased cell viability to 62.22 p<0.001 versus control.*p<0.05 , * * p<0.01 and * * * p<0.001 versus dox results of experiment with combination extract ( ngz ) showed that all doses ( 62.5 , 125 , 250 and 500 g / ml ) protected h9c2 cells against dox and increased cell viability significantly . the best protection was obtained at dose of 250 g / ml ( 90.291.2 , p<0.001 , 70% protection ) . the results showed that dox significantly increased mda ( p<0.001 ) and ros ( p<0.001 ) levels in h9c2 compared with control cells . in the presence of dox , treatment with 125 g / ml ( p<0.05 ) , 250 g / ml ( p<0.001 ) and 500 g / ml ( p<0.01 ) doses of ngz significantly reduced the level of mda . the most reduction of mda was observed at the dose of 250 g / ml ( control 1001.4 , dox 2357.9 and 250 g / ml ngz 1195.18 ) . also this combination diminished significantly ros production in comparison with dox , at dose of 125 g / ml ( p<0.01 ) , 250 g / ml ( p<0.001 ) and 500 g / ml ( p<0.01 ) ( figures 2a and 2b , respectively ) . the lowest production of ros was at the dose of 250 g / ml ( control 274 , dox 1208 and 250 g / ml ngz 454 ) . effect of ngz on dox - induced mda production ( figure 2a ) and reactive oxygen species ( ros ) generation ( figure 2b ) in h9c2 cells . cells were pretreated with different concentrations of ngz for 4 hr before exposure to 5 m of dox for 24 hr . data are expressed as mean  sem of three separate experiments p<0.001 versus control , * p<0.05 , * * p<0.01 and * * * p<0.001 versus dox apoptosis in h9c2 cell line was detected with flow cytometry using pi staining . cells were pretreated for 4 hr with various concentrations of the ngz and exposed to dox for 24 hr . analysis of the subg1 peak in flow cytometry histograms revealed the induction of apoptosis in cells treated with dox ( p<0.001 ) . ngz decreased apoptotic induction significantly at the doses of 62.5 g / ml ( p<0.05 ) , 125 g / ml ( p<0.01 ) , 250 g / ml ( p<0.001 ) and 500 g / ml ( p<0.01 ) ( figure 3 ) . the most reduction of apoptotic rate was at the dose of 250 g / ml ( control 121.4 , dox 714 and 250 g / ml ngz 37.52.5 ) the effects of the ngz on apoptosis in h9c2 cells using pi staining and flow cytometry . p<0.001 versus control , * p<0.05 , * * p<0.01 and * * * p<0.001 versus dox incubation with doxorubicin significantly decreased cell viability to 622% compared to control of control ( p<0.001 ) . cell viability was assayed to determine the optimum concentrations necessary for the three extracts ( n , g and z ) to protect h9c2 cells against dox - induced cytotoxicity . the results demonstrated that , in comparison with dox , n. sativa increased cell viability at doses of 25 to 200 g / ml dose dependently that maximum effect was produced at effect at concentration of 50 g / ml ( 75.40.84 , p<0.01 ) , and z. officinale increased cell viability at doses of 50 to 200 g / ml , its maximum effect was at dose of 100 g / ml ( 752.5 , p<0.01 ) , while g. glabra increased cell viability at dose of 100 g / ml ( 73.51.5 , p<0.05 ) ( figure 1a ) . effect of extracts alone ( figure 1a ) or ngz ( figure 1b ) on percentage of protection against dox - induced cytotoxicity in h9c2 cells . cells were pretreated with different concentrations of ngz for 4 hr before exposure to 5 m of dox for 24 hr . dox decreased cell viability to 62.22 p<0.001 versus control.*p<0.05 , * * p<0.01 and * * * p<0.001 versus dox results of experiment with combination extract ( ngz ) showed that all doses ( 62.5 , 125 , 250 and 500 g / ml ) protected h9c2 cells against dox and increased cell viability significantly . the best protection was obtained at dose of 250 g / ml ( 90.291.2 , p<0.001 , 70% protection ) . the results showed that dox significantly increased mda ( p<0.001 ) and ros ( p<0.001 ) levels in h9c2 compared with control cells . in the presence of dox , treatment with 125 g / ml ( p<0.05 ) , 250 g / ml ( p<0.001 ) and 500 g / ml ( p<0.01 ) doses of ngz significantly reduced the level of mda . the most reduction of mda was observed at the dose of 250 g / ml ( control 1001.4 , dox 2357.9 and 250 g / ml ngz 1195.18 ) . also this combination diminished significantly ros production in comparison with dox , at dose of 125 g / ml ( p<0.01 ) , 250 g / ml ( p<0.001 ) and 500 g / ml ( p<0.01 ) ( figures 2a and 2b , respectively ) . the lowest production of ros was at the dose of 250 g / ml ( control 274 , dox 1208 and 250 g / ml ngz 454 ) . effect of ngz on dox - induced mda production ( figure 2a ) and reactive oxygen species ( ros ) generation ( figure 2b ) in h9c2 cells . cells were pretreated with different concentrations of ngz for 4 hr before exposure to 5 m of dox for 24 hr . data are expressed as mean  sem of three separate experiments p<0.001 versus control , * p<0.05 , * * p<0.01 and * * * p<0.001 versus dox cells were pretreated for 4 hr with various concentrations of the ngz and exposed to dox for 24 hr . analysis of the subg1 peak in flow cytometry histograms revealed the induction of apoptosis in cells treated with dox ( p<0.001 ) . ngz decreased apoptotic induction significantly at the doses of 62.5 g / ml ( p<0.05 ) , 125 g / ml ( p<0.01 ) , 250 g / ml ( p<0.001 ) and 500 g / ml ( p<0.01 ) ( figure 3 ) . the most reduction of apoptotic rate was at the dose of 250 g / ml ( control 121.4 , dox 714 and 250 g / ml ngz 37.52.5 ) the effects of the ngz on apoptosis in h9c2 cells using pi staining and flow cytometry . p<0.001 versus control , * p<0.05 , * * p<0.01 and * * * p<0.001 versus dox cardiovascular risk factors enhance the production of ros generated by mitochondrial electron - transport chain , xanthine oxidase , nadph oxidase and uncoupled nitric oxide synthases in cardiomyocytes ( 39 ) . oxidative stress occurs when production of ros exceeds the capacity of antioxidant defense systems ( catalase , sod and glutathione peroxidase ) ( 40 ) . because of its deleterious effects weak antioxidant capacity in cardiomyocytes may be a risk factor responsible for their high sensitivity to oxidative damage ( 42 ) , and a promising approach to cardioprotection is the use of pharmacological tools to reduce oxidative stress in the heart ( 37 ) . in the present study , we used h9c2 cells as a pharmacological model to evaluate the potential cardioprotective effects of three different prevalent medicinal plants , n. sativa , glycyrrhizin glabra and zingiber officinale alone and in combination with all three ( ngz ) . the results showed that the combination of ngz improves their protective effects against dox - induced oxidative stress in h9c2 cells . h9c2 cells are morphologically similar to immature embryonic cardiomyocytes . considering that these cells preserve electrical and hormonal signal pathways found in adult cardiac cells ( 43 ) , they are a useful model for studying oxidative stress - induced cardiomyocyte damage ( 44 ) . in this model , dox significantly increased the level of ros and lipid peroxidation and induced the rate of apoptosis . these changes are similar to the dox - induced deleterious effects on normal cardiac cells , which lead to the loss of cardiomyocytes viability ( 2 ) . in this study , pretreatment with each of the three herb extracts alone produced some degree of protection in a concentration dependent manner . these findings are in accordance with previous studies which show the capability of cadioprotective effects of n. sativa , g. glabra and z. officinale ( 10 - 14 ) . in mice , thymoquinone , the pharmacologically active component of n. sativa , reduces dox - induced cardio toxicity without reducing the serum concentration of dox ( 12 ) . also , studies have shown that thymoquinone potentiates cisplatin antitumor activity and protects against cisplatin - induced nephrotoxicity in mice and rats ( 45 ) . a recent investigation has shown that n. sativa oil supplementation attenuates lead - induced cardio toxicity by mechanisms related to its ability to decrease the pro - inflammatory cytokines ; oxidative stress and cardiac tissue damage , and preserve the activity of antioxidant enzymes ( 10 ) . ebru et al ( 2008 ) showed that pretreatment with n. sativa oil decreased cyclosporine - a injury in rat heart ( 11 ) , and later studies revealed a protective effect of thymoquinone against doxorubicin - induced cardio toxicity ( 12 - 14 ) . recently , it was shown that gingerol protected cardiomyocytes against dox - induced toxicity through its antioxidative effect and modulation of nf-b as well as apoptosis ( 46 ) . glycyrrhizin , the chief sweet - tasting constituent of g. glabra ( liquorice ) root and its metabolites ( glycyrrhizic acid or glycyrrhizinic acid ) induce apoptosis and cell growth inhibition in various cancer cells , including human stomach cancer cells , promyelotic leukemia hl-60 cells and hepatoma cells ( 47 , 48 ) . moreover , experimental results showed that the combination of antibiotic anti - cancer drug with 18-glycyrrhetinic acid ( derivative of the beta - amyrin type obtained from the hydrolysis of glycyrrhizic acid ) exhibits a synergistic toxic effect on cancer cell lines ( 49 ) . the extract of glycyrrhiza 
 uralensis suppresses doxorubicin - induced apoptosis in h9c2 rat cardiac myoblasts ( 34 ) . recent experimental observations reported that z. officinale is an effective anticancer agent ( 50 ) . also , in a polyherbal preparation , ginger was one of the components and reported to be effective against dox - induced cardio toxicity without interfering its antineoplastic activity ( 51 ) . thus , it is of great interest to investigate the effect of combination of these extracts on dox - induced disorders . in this study , the combination of extracts ( ngz ) augmented their protective effects , suggesting the presence of a positive interaction between components . in this experiment , however , the maximum protective effect was achieved with combination of the threshold concentrations of the extracts and the protection increased in concentration dependent manner to a maximum protection of more than 2.5 fold . these observations do not show an additive effect , but implicates the presence of a positive interaction between extracts against dox - induced apoptosis in h9c2 cells . in high concentrations , n. sativa did not produce any further protective effect , while the effects of the two other extracts were decreased in high concentrations ( figure 1a ) . the decreasing effect of g. glabra or z. officinale at concentration of 200 g / ml could not be explained by a non - specific effect such as ionic disturbances . however , at higher concentration ( 500 g / ml ) in mixed extracts , the effect was reduced . this may suggest the presence of some components in crude extracts , which may have toxic effect and reduce the protection . the molecular mechanisms for protective effect of each extract and augmenting effect of their combination ( ngz ) are not clear . however , it has been shown that glycyrrhizate could ameliorate rabbit myocardial ischemia - reperfusion injury through p38mapk pathway ( 52 ) . also , treatment with glycyrrhiza uralensis extract significantly protected mice from dox - induced cardio toxicity , by decreasing levels of serum ldh and creatin kinase - mb improving heart morphology and increasing gsh - p(x ) activity and gsh level ( 35 ) . also , results revealed that glycyrrhizin and glycyrrhetinic acid directly affected cardiac performance ( 36 ) . in this paper , we showed that the protective effects may be mediated by reducing oxidative stress , which could decrease the production of ros , lipid peroxidation and diminished apoptotic induction . it is interesting that in comparison with the effect of each extract alone , the combination produced a stronger protective effect . therefore , it can be concluded that cardioprotective effect of ngz is mediated at least in part by its active ingredients . however , further investigations are needed to reveal molecular mechanisms to increase the possible positive interactions and diminish negative interactions in combination . these must be included in fractionation of the extracts to assess the effect of each fraction in combination . these may lead to find purified active ingredients , which could be used in a clinical trial . it was shown that all of three extract produced protective effect on h9c2 cell against dox - induced toxicity with different potencies , but similar efficacy with a maximum effect of about 30 percent . the effects of extracts in combination showed a positive interaction between components of different extracts , which augments their protective effect . these observations could suggest new experiments that can be performed to test the roles of each extract components in the combination . these may lead to the development of a new class of drugs to prevent cardio toxicity of dox and improve its anticancer effect .
objective(s):the use of doxorubicin ( dox ) is limited by its dose - dependent cardio toxicity in which reactive oxygen species ( ros ) play an important role in the pathological process . 
 the aim of this study was to evaluate the protective effect of three medicinal plants , nigella sativa ( n ) , glycyrrhiza glabra ( g ) and zingiber officinale ( z ) , and their combination ( ngz ) , against dox - induced apoptosis and death in h9c2 cells.materials and methods : the cells were incubated with different concentrations of each extract or ngz for 4 hr which continued in the presence or absence of 5 m doxorubicin for 24 hr . 
 cell viability and the apoptotic rate were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium ( mtt ) and propidium iodide ( pi ) staining assays , respectively . 
 the level of ros and lipid peroxidation were measured by fluorimetric methods.results:treatment with doxorubicin increased ros generation , enhanced malondialdehyde ( mda ) formation , and induced apoptosis . 
 co - treatment of the cells with each herb extract increased viability of cells dose - dependently with a maximum protection effect of about 30% , and their potencies were n > g > z . 
 the combination of the threshold dose of each extract ( ngz ) produced a similar effect , which was increased dose - dependently to a maximum protection of 70% . 
 these effects were correlated with the effects of ngz on ros and mda.conclusion:all of the extracts have some protective effects against dox - induced toxicity in cardiomyocytes with similar efficacies , but with different potencies . however , ngz produced much higher protective effect via reducing oxidative stress and inhibiting of apoptotic induction processes . 
 further investigations are needed to determine the effects of ngz on dox chemotherapy .