recent estimates suggest that several thousands of plants have been known to have the potential of medicinal applications in various cultures ( 2 ) . for 80% of the world population in asia , latin america , and africa , use of herbal medicine as traditional health remedies is the most popular and is reported to have minimal side effect ( 3 ) . many studies indicate that in some plants , there are many substances such as peptides , some essential oils , phenols and water , ethanol , chloroform , methanol , and butanol soluble compounds ( 4 ) . in khuzestan , the southwest province of iran , use of plants as folklore remedies for infections is a widespread culture . one of these plants , urtica dioica , is commonly used in traditional medicine for curing different diseases . it has a long history of application as a medicine as well as a food source . this plant is widely scattered in khuzestan fauna and is used by natives as medicinal plant . concerning infectious diseases that are endemic in khuzestan or occur as epidemics , finding alternate and natural resources for antibacterial agents is of great importance . therefore , the present study was conducted in order to examine in vitro antibacterial potential of methanolic and ethanolic extracts of u. dioica . the results of this study can be helpful for treating infectious disease with natural products and fighting against drug resistant bacteria . the aim of the present study was to examine the antibacterial activity of u. dioica , its time - kill kinetics assay , and the structural changes caused by the extracts of this plant in target bacteria . the understudy plant was collected from shahid chamran university farmland in khuzestan province , iran . these samples were identified and confirmed through comparison with existing herbarium in department of biology , shahid chamran university , ahvaz , iran . the aerial parts of u. dioica were shade - dried at room temperature for ten days and then ground to a fine powder using electronic blender . extraction was done from one gram of obtained powder using 10 ml of alcohol ( ethanol or methanol , merck , germany ) and distilled water solution ( 8:2 v / v ) , centrifugation at 3000 rpm for 15 minutes , and harvesting the supernatant . this process was repeated three times and solvents were evaporated by placing the yielded materials at room temperature for seven days ( 4 , 5 ) . a total of eight bacterial strains were tested including bacillus cereus , staphylococcus aureus , and staphylococcus epidermidis as gram - positive and salmonella typhi , klebsiella pneumoniae , escherichia coli , pseudomonas aeruginosa , and proteus mirabilis as gram - negative species . these species were originally isolated from clinical specimens and identified according to standard phenotypic test . the antibiotic sensitivity patterns of these isolates were investigated according to kirby - bauer standard disc diffusion method . antibacterial activity of the ethanolic and methanolic extracts of u. dioica was surveyed by kirby - bauer disc diffusion method ( 6 ) . a cell concentration of 10 cfu / ml of test bacteria , with reference to the mcfarland turbidometry , were prepared by culturing in muller hinton broth ( mhb , merck , germany ) medium and incubation at 37 for 22 hours ( 7 ) . a lawn culture was prepared on muller - hinton agar ( mha , merck , germany ) using sterile cotton swab and allowed to remain in contact for one minute . sterile filter paper discs ( 6.4 mm ) were saturated by different effective doses of 1.5 , 3 , 6 , 12 , and 18 mg were placed on lawn cultures after solvent evaporation ( 2 , 8) . a saturated disc with ethanol and methanol was also prepared simultaneously and regarded as negative control . the plates were left at room temperature for one hour to allow the diffusion of extracts from the discs into the medium , and subsequently , the petri dishes were incubated at 37 for 24 hours . the inhibition zone around each disc the minimum inhibitory concentration ( mic ) of the extracts was determined for the most sensitive bacterial species . for this purpose , twofold dilutions of extract were prepared in tubes containing 1 ml muller - hinton broth and bacterial suspension equal to 0.5 mcfarland turbidity . the first tube in above series with no sign of visible growth was considered as the mic ( 8) . this process was repeated three times . to determine the minimum bactericidal concentration  ( mbc ) , a loop full from those tubes , which did not show any visible growth in mic assay , was cultured on mha and incubated at 37 for 18 to 24 hours . the highest dilution that did not yield colony formation on mha was considered as mbc ( 9 ) . the time - kill kinetic was studied by culturing one standard loop of the bacterial suspension ( 0.5 mcfarland turbidity ) , that was treated with mbc concentration of extract on mha , from zero through 36th hour after incubation . this was performed at one hour intervals for the first 18 hours and then at two hours intervals for the next 18 hours ( 8) . in order to find possible effect of active antibacterial substances of u. dioica alcoholic extracts , sem ( scanning electron microscopy ) analysis was used . for this purpose , an antibacterial susceptibility test has been done with 12 mg effective dose from methanolic extract against s. epidermidis . after 24 hours , when halo of inhibition zone appeared around the disc , a sample was taken from the margin of the halo and was analyzed by sem ( hitachi japan s4160 ) after coating with gold . the understudy plant was collected from shahid chamran university farmland in khuzestan province , iran . these samples were identified and confirmed through comparison with existing herbarium in department of biology , shahid chamran university , ahvaz , iran . the aerial parts of u. dioica were shade - dried at room temperature for ten days and then ground to a fine powder using electronic blender . extraction was done from one gram of obtained powder using 10 ml of alcohol ( ethanol or methanol , merck , germany ) and distilled water solution ( 8:2 v / v ) , centrifugation at 3000 rpm for 15 minutes , and harvesting the supernatant . this process was repeated three times and solvents were evaporated by placing the yielded materials at room temperature for seven days ( 4 , 5 ) . a total of eight bacterial strains were tested including bacillus cereus , staphylococcus aureus , and staphylococcus epidermidis as gram - positive and salmonella typhi , klebsiella pneumoniae , escherichia coli , pseudomonas aeruginosa , and proteus mirabilis as gram - negative species . these species were originally isolated from clinical specimens and identified according to standard phenotypic test . the antibiotic sensitivity patterns of these isolates were investigated according to kirby - bauer standard disc diffusion method . antibacterial activity of the ethanolic and methanolic extracts of u. dioica was surveyed by kirby - bauer disc diffusion method ( 6 ) . a cell concentration of 10 cfu / ml of test bacteria , with reference to the mcfarland turbidometry , were prepared by culturing in muller hinton broth ( mhb , merck , germany ) medium and incubation at 37 for 22 hours ( 7 ) . a lawn culture was prepared on muller - hinton agar ( mha , merck , germany ) using sterile cotton swab and allowed to remain in contact for one minute . sterile filter paper discs ( 6.4 mm ) were saturated by different effective doses of 1.5 , 3 , 6 , 12 , and 18 mg were placed on lawn cultures after solvent evaporation ( 2 , 8) . a saturated disc with ethanol and methanol was also prepared simultaneously and regarded as negative control . the plates were left at room temperature for one hour to allow the diffusion of extracts from the discs into the medium , and subsequently , the petri dishes were incubated at 37 for 24 hours . the inhibition zone around each disc the minimum inhibitory concentration ( mic ) of the extracts was determined for the most sensitive bacterial species . for this purpose , twofold dilutions of extract were prepared in tubes containing 1 ml muller - hinton broth and bacterial suspension equal to 0.5 mcfarland turbidity . the first tube in above series with no sign of visible growth was considered as the mic ( 8) . this process was repeated three times . to determine the minimum bactericidal concentration  ( mbc ) , a loop full from those tubes , which did not show any visible growth in mic assay , was cultured on mha and incubated at 37 for 18 to 24 hours . the highest dilution that did not yield colony formation on mha was considered as mbc ( 9 ) . the time - kill kinetic was studied by culturing one standard loop of the bacterial suspension ( 0.5 mcfarland turbidity ) , that was treated with mbc concentration of extract on mha , from zero through 36th hour after incubation . this was performed at one hour intervals for the first 18 hours and then at two hours intervals for the next 18 hours ( 8) . in order to find possible effect of active antibacterial substances of u. dioica alcoholic extracts , sem ( scanning electron microscopy ) analysis was used . for this purpose , an antibacterial susceptibility test has been done with 12 mg effective dose from methanolic extract against s. epidermidis . after 24 hours , when halo of inhibition zone appeared around the disc , a sample was taken from the margin of the halo and was analyzed by sem ( hitachi japan s4160 ) after coating with gold . the antibacterial activity of ethanolic and methanolic extracts of u. dioica appeared as growth inhibition of tested bacteria . these results showed that these extracts were effective against four tested organisms including e. coli , s. epidermidis , b. cereus , and s. aureus . the highest activity ( inhibition zone diameter about 18 mm ) was demonstrated by the methanolic extract of u. dioica leaves against s. epidermidis while the lowest activity ( inhibition zone diameter about 7 mm ) was demonstrated by the ethanolic extract against b. cereus and s. aureus and by methanolic extract against b. cereus . on the other hand , the ethanolic and methanolic extracts were not active against s. typhi , p. aeruginosa , k. pneumoniae , and p. mirabilis . the results of mic and mbc of ethanolic and methanolic extracts for three bacterial species are shown in table 3 . time - kill kinetic for methanolic extract of u. dioica was nine hours ( table 4 ) . . as we can found the round shape of this bacterium was changed to ovoid or irregular forms . some of them were collapsed and appear to be somewhat indented . abbreviations : nf , nafcillin ( 1 g ) ; cb , carbenicillin ( 100 g ) ; nb , novobiocin ( 30 g ) ; dx , doxycycline ( 30 g ) ; cl , colistin ( 10 g ) ; mt , methicillin ( 5 g ) ; ox , oxacycline ( 1 g ) ; and r , resistance . abbreviations : mic , minimum inhibitory concentration ; and mbc , minimum bactericidal concentration . ( + ) , growth ; and ( - ) , growth inhibition . the concentration of extracts was 20 mg / ml . the arrows show deformed bacteria . many researchers have reported the antibacterial activities of different plants ( 10 , 11 ) . in traditional therapy , u. dioica l. ( urticaceae family ) have been known for some medicinal properties such as anti - inflammatory and antirheumatic ( 5 , 12 ) , cardiovascular ( 9 ) , natriuretic , hypotensive , and acute diuretic effects ( 13 ) , and stimulation of proliferation of human lymphocytes ( 14 ) . the effects of its nettle are also kindle in the therapy of the prostatic hyperplasia ( 16 - 18 ) . in this study , a variety of bacterial strains was selected for investigating the antibacterial effects of ethanolic and methanolic extracts of u. dioica leaves . the results of this study showed that the hydroalcoholic extracts of this plant have wide range of antimicrobial activity against both tested gram - positive and gram - negative bacteria , which is comparable to the effect of standard antibiotics . the results demonstrated that gram - negative bacteria including s. typhi , k. pneumoniae , p. aeruginosa , and p. mirabilis were resistant to all of the concentrations of both extracts . they have an outer membrane envelope that act as a barrier to the entrance of some substances and their porins determine the type and size of those substances that can reach to their cytoplasm . furthermore , some of these resistant species can produce polysaccharide capsule that prevent the entrance of some substances to the cytoplasm . considerable antibacterial activity against gram - positive species suggests that the extracts of u. dioica can be selective agents for treatment of infections caused by these organisms . notably , excellent inhibition against staphylococcus species can be a hope for discovering new natural antibacterial agent against coagulase - positive and coagulase - negative staphylococci , especially methicillin - resistant s. aureus ( mrsa ) and vancomycin - intermediate s. aureus ( visa ) strains . today , resistant s. aureus ( mrsa , visa , and vancomycin - resistant s. aureus [ vrsa ] ) strains are a serious threat to humans , especially for nosocomial infections . these results showed that the target site of active constituents of u. dioica was bacterial cell wall . as the peptidoglycan synthesis and cell shape of bacteria is dependent to penicillin - binding proteins ( pbps ) , it can be concluded that these antibacterial substances inhibited these proteins and consequently could disrupt the integrity of peptidoglycans . first , they have bactericidal effect and recurrence of the infection due to the persistence of pathogen will be prevented . second and most importantly , these agents affect cell wall and with regard to the uniqueness of this structure , these compounds can be regarded as safe antibacterial agent with no or less side effects on eukaryotic cells . furthermore , the cell wall has a significant role in maintaining high internal pressure of bacterium . those antibacterial agents that can affect this structure are suitable for killing bacteria ( 19 ) . therefore , we believe that the bacterial inhibition or killing can vary based on the plant extract , the used solvent , and the tested organisms . however , to get maximum antibacterial activity , both the solvent and the extraction system influence the final results . the results of this study suggest that the extracts of u. dioica can be used as antibacterial substance against some bacterial species such as s. aureus , e. coli , and s. epidermidis infections surveyed antibacterial activity of ten aqueous and one ethanolic extracts from medicinal plants used in turkey . they found that u. dioica had no significant effect on e. coli , s. epidermidis , s. aureus , p. aeruginosa , and k. pneumoniae ( 20 ) . discovered the antioxidant and antimicrobial properties of aqueous extract of nettle ( u. dioica l. ) . as a result , it showed antimicrobial activity against nine microorganisms such as e. coli , p. mirabilis , and s. aureus ( 21 ) . finally , based on these results , it can be suggested that u. dioica alcoholic extracts target bacterial cell wall and can kill their target bacteria . therefore , this plant is a favorable option in discovering new bactericide antibiotics , especially against staphylococcus species .
background : urtica dioica is a flowering plant with long history of use in folk medicine and as a food source.objectives:this study examined in vitro antibacterial potential of alcoholic extracts of u. dioica.materials and methods : hydroalcoholic extracts from aerial parts were prepared using aqueous solution of ethanol and methanol and their inhibitory effects against clinical isolates was examined by disc diffusion method at different doses . 
 minimum inhibitory concentrations ( mic ) and minimum bactericidal concentration ( mbc ) indexes were also investigated . 
 the scanning electron microscopy ( sem ) analysis was also performed to find structural changes of affected bacteria consequent to exposing with extracts.results:both extracts were active against bacillus cereus , staphylococcus aureus , staphylococcus epidermidis , and escherichia coli with respectively 16 , 10 , 18 , and 14 mm ( methanolic ) and 11 , 9 , 17 , and 16 mm ( ethanolic ) inhibition zone . 
 the mic of ethanolic extract against s. epidermidis and e. coli was respectively 10 and 40 mg / ml . 
 the mic of methanolic extract against s. aureus and s. epidermidis was 40 and 10 mg / ml , respectively . 
 the mbc was found only for s. epidermidis ( 20 mg / ml ) . in sem analysis 
 the round shape of s. epidermidis was changed and irregular shapes were appeared , which suggest that the main target of these extracts was cell wall.conclusions:extracts of u. dioica showed significant antibacterial effect against some clinically important pathogenic bacteria . based on the obtained results it can be concluded that u. dioica is useful as antibacterial and bactericidal agent in treating infectious diseases .