antimicrobial agents are usually incorporated into hygiene products for the treatment and prevention of plaque and gingivitis . dental caries is a public oral health problem and an infectious - contagious disease that implies an imbalance of normal molecular interactions between the tooth surface / subsurface and the adjacent bacterial biofilm thus , antimicrobial mouth rinses that augment daily home care may provide an efficient income of remove or controlling bacterial plaque to limit gingivitis and periodontitis . here is a continuous need of new antimicrobial components due to rapid appearance of multiple drug - resistance bacteria . the genus juglans ( family juglandaceae ) comprises several species and is widely dispersed throughout the world . many parts of green walnuts such as shells , kernel and seed , bark , and leaves are used in the pharmaceutical and beauty industry [ 6 , 7 ] . juglans regia l. bark is used in some countries as a toothbrush and as a dye for coloring the lips for makeup purpose . walnut ( juglans regia l. ) bark has been claimed to own anti - inflammatory , blood purify , anticancer , depurative , diuretic , and laxative activities . juglans regia stem bark contains chemical constituents , namely , -sitosterol , ascorbic acid5 , juglone , folic acid , gallic acid , regiolone , and quercetin-3--l - arabinoside [ 10 , 11 ] . antifungal , antibacterial , and antioxidant activities of this plant have been described [ 1216 ] . its extract of juglans regia bark showed a broad spectrum antimicrobial activity in a dose - dependent manner . it inhibited the growth of several pathogenic microorganisms such as gram - positive bacteria ( staphylococcus aureus and streptococcus mutans ) , gram - negative bacteria ( escherichia coli and pseudomonas aeruginosa ) , and pathogenic yeast ( candida albicans ) . the extract has either synergistic or additive act when tested with a broad variety of antibacterial drugs . rahul tested the stem bark extracts of j. regia l. for antimicrobial activity against the microbes present in the saliva specimens of patients suffering from dental caries . acetone extract was found to be more effective of the extracts as antimicrobial against the oral microflora . since many of currently used natural products are either detrimental or ineffective , natural folk medicines with antimicrobial effects have been under investigation during the few past decades . growing emphasis of plant studies in the field of dentistry is due to the antibiotic - resistant bacteria , side effects of chemical antibiotics , and their high cost in developing countries . juglans regia bark species have been analyzed chemically adequately in several studies , and in agreement among most of them , with respect to the essential oil composition , the major components are phenolic compounds , terpenoids , alkaloids , flavonoids , and steroids . it is reported that leaves from j. regia l. contain monoterpenes and sesquiterpenes , and the bark contains ketones like juglone , regiolone , sterol , and flavonoid . juglans regia bark is a medicinal plant used in iranian folk medicine as antimicrobial medicine [ 19 , 20 ] . the aim of the present study was to evaluate the antimicrobial activities of ethanolic and aqueous extracts of juglans regia bark against four species of oral bacteria . the plant material ( stem bark ) of the species was collected from local market from kordestan , iran , and confirmed at department of botany , school of agriculture , shahid chamran university , ahvaz , iran . juglans regia bark powdered ( 100  g ) was extracted by 500  ml of ethanol ( for preparing the ethanolic extract ) and 500  cc of water ( for preparing the aqueous one ) consecutively using soxhlet extractor with not exceeding the boiling point of the solvent . the extracts were filtered by whatman filter paper and then concentrated in vacuum at 40c by means of a rotary evaporator . the bacteria strains were used for antibiogram pattern including streptococcus mutans ptcc1683 , streptococcus salivarius ptcc1448 , streptococcus sanguis ptcc1449 , and staphylococcus aureus ptcc : 1112 , were provided by the iranian microbial type culture collection . the strains were inoculated in blood agar and incubated at 37c at least for 24  h , until emergent adequate colonies . the bacteria strains were touching to 4 - 5 colonies raised from pure microorganism culture and inoculated at the concentration in order to attain the mc . farland no : 0.5 density and then streptococci species and staphylococcus aureus incubated in muller - hinton and blood agar , respectively . extracts were diluted in water , and thus 1  gr of each extract inoculated in 1  ml distil water and diluted it to obtain different concentrations as 1000 , 500 , 250 , 125 , and 62.5  mg / ml for aqueous and ethanolic extracts . the blank discs ( padtan teb co , iran ) were inoculated with 20  l of every concentration extracts and placed on the muller - hinton and blood agar were cultivated with bacterial strains . negative controls used the same solvents to dissolve the extracts , and tetracycline ( 30  g ) and erythromycin ( 15  g ) were used as positive references . antimicrobial pattern was evaluated by measuring the zone of inhibition against the test bacteria based on millimeters . the mic values were read as the antibacterial concentration at the point where dense colonial growth intersected the disc [ 22 , 23 ] . analysis of variance ( anova ) was used to determine the significance ( p  0.05 ) of the data obtained in all tests . the inhibition zones due to aqueous extract , negative control ( water ) , and positive control ( tetracycline 30  g , erythromycine 15  g ) are showed in table 1 . aqueous extract had significantly antibacterial effect against staphylococcus aureus , s. salivarius ( figure 2 ) , and s. sanguis ( figure 4 ) compared to control and was significant ( p < 0.0001 ) , but it did not show effect on s. mutans when compared with erythromycin . also the results were shown that ethanolic extracts were significant ( p < 0.0001 ) with inhibitory effect on the growth of four tested bacteria , in contrast to negative control ( table 2 ) . the mic was evaluated for the antimicrobial activity of ethanolic and aqueous extracts of juglans regia on bacteria and the results were shown in table 3 . according to the obtained mic values , ethanol extract of juglans regia bark had the lowest mic of 1.25  microg / ml on s. sanguis ( figure 3 ) . ethanol extract mic value for s. salivarius ( figure 1 ) and aqueous extract mic value for s. sanguis and s. salivarius were similar , 2.50  microg / ml . antibacterial activity of the aqueous extract on staphylococcus aureus was the least sensitivity with a highest determined mic of 125  microg / ml , while mic value for the ethanolic extract was 2.00  microg / ml . aqueous extract did not have any inhibitory effect on s. mutans in terms of antimicrobial activity ; however , for the ethanol extract it was 5.00  microg / ml . the most important cause of gingival inflammation and dental caries is bacterial plaque . some of people keep away from chemical mouth rinse because of the presence of alcohol , artificial preservation , or artificial color in mouth rinses the present study showed that juglans regia are potential antimicrobial agents and can be used in oral hygiene products .   according to our finding , we indicated that the high concentrations of ethanolic and aqueous extracts have had antimicrobial effects against s. sanguis , s. mutans , s. salivarius , and staphylococcus aureus with significant difference in contrast to control . we indicated that ethanolic extract exhibited zones of inhibition against all the tested samples , whereas aqueous extract is active with comparatively smaller zones of inhibition ( tables 1 and 2 ) . reported that the acetone extract of j. regia l was found to be more effective of the extracts as antimicrobial against the oral microflora . . showed that walnut leaves could be used as an easily available source of natural compounds to inhibit the growth of different gram - positive bacteria responsible for dental plaques and oral hygiene problems . recently , darmani et al . reported the growth inhibition of various cariogenic bacteria ( streptococcus mutans , streptococcus salivarius , lactobacillus casei , and actinomyces viscosus ) by walnut aqueous extract . the data of this study clearly indicated that ethanolic and aqueous extracts of juglans regia bark significantly inhibited the growth of the tested oral bacteria , and those reports are compatible with our finding . the antibacterial property of the plant material may be due to the presence of phenolic compounds , terpenoid , alkaloids , flavonoids , and steroids . this study has confirmed the antimicrobial potentials of this kind of iranian plant , thus supporting its folklore application as a preventive remedy for various microbial diseases ( caries and periodontal disease ) in the oral cavity in iran . it provides the basis for the present rapidly increasing interest for the use of natural antioxidants and antimicrobials . further studies are required to find these effects in order to replace synthetic medications with natural remedies . we concluded that iranian bark of juglone , regiolane , has the antibacterial effects against the important oral bacteria , and ethanolic extract was of higher effectivity against tested bacteria than aqueous extract .
background . in this study antimicrobial effect of ethanolic and aqueous extracts of juglans regia 
 bark in iran was evaluated on four different oral bacteria , streptococcus mutans , streptococcus salivarius , streptococcus sanguis , and staphylococcus aureus . 
 methods . 
 aqueous and ethanol extracts of juglans regia bark were prepared by using disk diffusion technique and minimal inhibitory concentration ( mic ) methods . tetracycline 30  g and erythromycin 15  g were used as positive control and water as negative control in disk diffusion and mic methods . 
 data were analyzed by anova test . 
 results . 
 the results showed that s. sanguis and s. mutans were the most sensitive and the most resistant bacteria against ethanolic and aqueous extracts , respectively . 
 ethanolic extract had significant antibacterial effect against all tested bacteria . 
 aqueous extract did not show antibacterial effect on s. mutans , in contrast to ethanolic extract . 
 aqueous extract had significantly antibacterial effect against staphylococcus aureus , s. salivarius , and s. sanguis compared to control ( p < 0.0001 ) , but it did not show effect on s. mutans when compared with erythromycin . according to the obtained mic values , ethanol extract of juglans regia bark 
 had the lowest rate . 
 conclusion . 
 the results may provide the basis for using natural antimicrobial substance for oral hygiene prophylaxis purposes .