dental caries , periodontal disease , and opportunistic infections are among the common diseases of the oral cavity caused by the accumulation of pathogenic microorganisms , inappropriate nutritional habits , and improper oral hygiene [ 14 ] .
oral streptococci are the first isolated species to play a role in the formation of dental plaque and development of caries [ 57 ] .
measures taken to clinically inhibit plaque accumulation , namely , tooth brushing , dental flossing , and use of mouth rinses ( as an adjunct ) , are highly effective for preventing the supragingival plaque and gingivitis and decreasing the number of microorganisms .
however , despite extensive antimicrobial activity , it has side effects such as altered sense of taste and staining of tooth surfaces and restorations [ 911 ] .
many individuals residing in developing countries strongly believe in the efficacy of herbal medications for primary care [ 12 , 13 ] . considering the increasing resistance of microorganisms to chemical synthetic drugs and their side effects , researchers are becoming more and more interested in finding alternative herbal medications and their active components [ 1418 ] .
the pomegranate with the botanical name of punica granatum linn . is a fruit - bearing deciduous shrub or small asian tree from the family of punicaceae growing between 5 to 8 meters tall .
it is native to iran and northern india and has long been cultivated in the asian mediterranean region , europe , and africa .
punica granatum is rich in bioactive compounds , which are used for treatment of cancer , cardiovascular diseases , diabetes mellitus , dental diseases , bacterial infection , antibiotic resistance , skin conditions due to uv radiation , diarrhea , bloody diarrhea , and hemorrhoids .
moreover , p. granatum is used as a mouth rinse for treatment of some types of sore throat .
parts of the plant used for medicinal purposes include its flowers , trunk skin , fruits , roots , and seeds [ 1921 ] .
the punica granatum flowers are odorless but colorful red or reddish , 3.5 to 7  cm in length , and campanulate or cylindrical .
the unfertilized flower is with smaller , barren , and short - styled , short - stamened petals , in which the stigma is far below the anthers .
trunk skin and roots are used for parasitic infections , dried flowers are beneficial for treatment of bronchitis , diarrhea , and bloody diarrhea , and the brewed pomegranate is used for treatment of inflammation of the throat and oral cavity .
pomegranate has long been recommended as a hemostatic agent and for treatment of diabetes mellitus in ancient greek medicine [ 1921 ] .
the antimicrobial properties of p. granatum have been recently noticed [ 19 , 2330 ] .
the ethanol , water , methanol , and acetone extracts of p. granatum have shown strong antimicrobial properties against gram - positive and gram - negative nonoral microorganisms [ 14 , 19 , 22 , 24 , 31 ] . however , a few studies have evaluated the antibacterial properties of this plant on oral bacteria [ 14 , 3133 ] . however , the mentioned studies are very limited and mostly lack an appropriate microbiological method .
search of the literature only yielded one previous study on the effect of water extract of p. granatum flower ( petal ) on oral microbial pathogens , which showed its greatest antimicrobial effect on s. sanguinis , although menezes et al . reported that hydroalcoholic extract of p. granatum was very effective against biofilm forming microorganisms in the mouth of patients . considering the high prevalence of oral and dental diseases due to oral pathogens and the recent public interest in medicinal plants , this study aimed to assess the effect of hydroalcoholic extract of p. granatum petal on s. mutans , s. sanguinis , s. salivarius , s. sobrinus , and e. faecalis in vitro .

 p. granatum flowers were obtained from the last harvest in darab city and its purity was confirmed in a pharmacognosy laboratory .
powdered petals were precisely measured by a digital scale and poured into an erlenmeyer flask .
the erlenmeyer flasks were capped with aluminum foil and stored in the dark for 10 days .
next , the flasks were placed on a shaker ( gfl 3017 ) operating at 90  rpm for 24 hours .
the filtered solution was poured into a sterile glass container and capped by aluminum foil .
a few holes were perforated in the foil and the glass container was placed in bain - marie at 90c to dry .
standard strains of s. mutans ( atcc 35668 , ptcc 1683 ) , s. sanguinis ( atcc 10556 , ptcc 1449 ) , s. salivarius ( atcc 9222 , ptcc 1448 ) , s. sobrinus ( atcc 27607 and ptcc 1601 ) , and e. faecalis ( atcc 11700 , ptcc 1393 ) were obtained in lyophilized form from the persian type culture collection center .
bacteria were activated by inoculation in the brain heart infusion agar ( bhia , merck , germany ) culture medium followed by 24 hours of incubation at 37c . for preparation of microbial suspension , a 24-hour culture was used .
the concentration of microorganisms in the microbial suspension was adjusted to 0.5 mcfarland standard using a spectrophotometer at a wavelength of 625  nm ( a mcfarland standard is a chemical solution with a turbidity comparable to that of microbial suspension . using this suspension , number of bacteria per each milliliter of the suspension
can be estimated , which is equal to 1.5  10  cfu / ml ) .
primary assessment of the antimicrobial effect of extracts was done using the cup - plate technique .
500  l of each microbial suspension at 0.5 mcfarland standard concentration was cultured in bhia ( swabbed on the plate ) .
then , wells measuring 8  mm in diameter were created on the agar surface .
different concentrations of the extracts were prepared by serial dilution ( dilution by one - half ) using sterile distilled water solvent ; 100  l of each concentration of extract was poured into each well .
this process was repeated in triplicate and the mean diameter of the growth inhibition zone was calculated for different concentrations of the extract .
the mic is defined as the lowest concentration of an antimicrobial agent that inhibits the growth of a microorganism ( 0.5 mcfarland standard in this study ) .
it is the minimum concentration of the extract that completely prevents visible growth and proliferation of bacteria compared to the negative control group . to determine mic , macrodilution method according to the standard technique described by the clinical and laboratory standards institute ( 2012 )
different concentrations of the extract were prepared by serial dilution ( dilution by one - half ) in bhi broth medium . using this medium , the 0.5 mcfarland standard suspension
was diluted 1 to 150 to obtain a bacterial count of 10  cfu / ml . microbial suspension was then diluted by one - half using the culture medium and 1  ml of it was added to the tubes containing serially diluted extract .
the negative control tube only contained the culture medium and extract with no microbial suspension .
after 24 hours of incubation at 37c , growth and proliferation of microorganisms were evaluated and the mic value of the extract for each bacterial strain was determined .
, 20  l of the suspension in the tube containing mic of the extract and tubes showing no bacterial growth were cultured on plates containing bhia .
after 24 hours of incubation at 37c , the plates were evaluated for growth of microorganisms . the concentration with no bacterial growth
the effect of 0.2% chx ( shahr daru , iran ) and ampicillin on the microorganisms was also evaluated using the cup - plate method and the mic and mbc values of chx and ampicillin for the microorganisms were determined as well .
the tests were repeated in triplicate and the mean and standard deviation ( sd ) of the growth inhibition zone diameter in cup - plate method as well as the mic and mbc of the extract , chx , and ampicillin were determined .
the hydroalcoholic extract of p. granatum showed inhibitory effects on the growth and proliferation of all five bacteria using the cup - plate method . the largest and the smallest diameter of growth inhibition zone belonged to s. sanguinis and e. faecalis , respectively .
the mean ( and sd ) diameter of the growth inhibition zone due to the effect of hydroalcoholic extract of p. granatum on different microorganisms is shown in table 1 .
the mic and mbc of hydroalcoholic extract of p. granatum were determined using serial dilution method .
the highest antibacterial effect of p. granatum extract was on s. mutans with mic and mbc values of 3.9  mg / ml . the mic and mbc values of this extract for different microorganisms are presented in tables 2 and 3 .
the cup - plate method showed that 0.2% chx had antimicrobial effect on all five bacterial strains .
the mean and sd of the diameter of growth inhibition zone due to the effect of 0.2% chx on the five bacterial strains are shown in table 4 .
assessment of the mic and mbc values revealed that chx had the highest effect on s. sanguinis .
tables 2 and 3 show the mic and mbc of chx for the five bacterial strains .
the cup - plate method showed that ampicillin had inhibitory effects on the growth and proliferation of all five bacterial strains .
the greatest diameter of growth inhibition zone belonged to s. mutans and s. sanguinis due to the effect of ampicillin .
the mean and sd of the diameter of growth inhibition zone due to the effect of ampicillin on different microorganisms are shown in table 5 .
assessment of the mic and mbc values revealed that ampicillin had the highest effect on s. sanguinis .
tables 2 and 3 show the mic and mbc of ampicillin for the five bacterial strains .
this study was the first to experimentally assess the antimicrobial effect of hydroalcoholic extract of p. granatum petal on s. mutans , s. sanguinis , s. salivarius , s. sobrinus , and e. faecalis in vitro using the cup - plate method .
the mic , mbc , and growth inhibition zone diameter values of the extract for different microorganisms were also calculated and compared to chx and ampicillin .
based on the results , the hydroalcoholic extract of p. granatum petal had inhibitory effect on the growth and proliferation of all five bacterial strains .
evaluated the mic of pomegranate fruit gel and compared it with miconazole against adhesion of s. mutans , s. mitis , and c. albicans .
they showed that pomegranate fruit gel was more effective than miconazole in preventing the adhesion of streptococci to glass .
dahham et al . assessed the antimicrobial effect of ethanolic extract of seed , fruit , peel , and juice of pomegranate on specific bacteria and reported that the pomegranate peel extract had the greatest antimicrobial activity .
stated that only at concentrations of 8  mg / ml and 12  mg / ml methanolic extract of p. granatum peel ( mepgp ) was effective against l. acidophilus , s. mutans , and s. salivarius .
furthermore , no concentrations of mepgp inhibited a. viscosus and c. albicans . in present study
hydroalcoholic extract of p. granatum petal was effective against s. mutans in lower concentration than mepgp in abdollahzadeh et al .
therefore , it seems that hydroalcoholic extract of petal against s. mutans was more effective than mepgp .
it should be considered that s. mutans is main microorganism of dental plaque and caries . also , in current study , both mic and mbc were considered .
furthermore , both mic and mbc values against s. mutans were similar ( 3.9  mg / ml ) .
the mic and mbc values of p. granatum showed the best antibacterial effect of this extract on s. mutans , while the lowest effect was noticed on e. faecalis .
the only previous study on the antimicrobial effect of methanolic extract p. granatum flower on s. mutans was conducted by haghighati et al . .
also , they showed no significant difference between the efficacy of p. granatum and chx against s. mutans ; these findings show that the method used was not proper and therefore their results are not reliable . in a study by vahid - dastjerdi et al . , the effect of water extract of p. granatum flower ( petal ) on the same microorganisms as in our study was evaluated .
the highest antimicrobial effect was seen on s. sanguinis   compared to the mic and mbc values obtained in the current study .
therefore , it appears that the solvent containing alcohol releases more amount of the antimicrobial agent and subsequently exerts a greater antimicrobial effect on s. mutans .
the composition of extract is influenced by the geographical location of the plant , season of harvesting , age of plant , growth stage , method of drying , and extraction technique .
also , extracts of different parts of the plant have variable level of antimicrobial activity and bacteria have variable sensitivity to different extracts .
also , isolated components of an extract show greater antimicrobial effects than the extract itself . alcoholic extract of p. granatum has well known isolated components such as gamma terpinene , borneol , camphor , carvacrol methyl ether , and methyl palmitate .
a previous study isolated the constituents of rosemary and evaluated their effects on oral pathogenic microorganisms .
camphor had inhibitory effects on s. mutans , s. sanguinis , s. salivarius , s. sobrinus , and e. faecalis .
thymol is an isomer of carvacrol , showing its antimicrobial activity by making the bacterial membrane permeable .
chlorhexidine mouthwash has extensive antimicrobial activity and is more effective on gram - positive than gram - negative bacteria .
it is known as the most effective mouthwash and the gold standard of antibacterial activity .
the mic and mbc values and inhibitory effects obtained in our study for chx were in accord with the results of specific microbiological assessments on chx and ampicillin , which confirmed the accuracy and precision of the laboratory phases in our study .
one limitation of our study was that we only evaluated the p. granatum flowers harvested at a specific time from a specific location .
also , standardized microbial strains were used and microorganisms collected and purified from the oral cavity were not assessed .
future studies are required to confirm the results in normal biological environment ( oral cavity ) .
also , future studies must focus on the isolated constituents of different extracts and the antimicrobial agent in their composition .
given that the effects of these constituents on other microorganisms such as l. acidophilus , a. viscosus , and c. albicans are confirmed , they may be used in the form of pure extract , mouth rinse , or other antimicrobial products in clinical trials .
within the limitations of this study , the following conclusions were drawn : hydroalcoholic extract of p. granatum had a significant antibacterial effect on common oral bacteria , namely , s. sanguinis , s. mutans , s. salivarius , s. sobrinus , and e. faecalis.hydroalcoholic extract of p. granatum had maximum antibacterial effect on s. mutans with mic and mbc of 3.9  mg / ml . hydroalcoholic extract of p. granatum had a significant antibacterial effect on common oral bacteria , namely , s. sanguinis , s. mutans , s. salivarius , s. sobrinus , and e. faecalis .
hydroalcoholic extract of p. granatum had maximum antibacterial effect on s. mutans with mic and mbc of 3.9  mg / ml .
 
 objectives . this study aimed to assess the effect of hydroalcoholic extract of punica granatum linn . 
 ( p. granatum ) petal on streptococcus sanguinis , streptococcus mutans , streptococcus salivarius , streptococcus sobrinus , and enterococcus faecalis . materials and methods . in this in vitro study , p. granatum extract was prepared using powdered petals and water - ethanol solvent . 
 antibacterial effect of the extract , chlorhexidine ( chx ) , and ampicillin was evaluated on brain heart infusion agar ( bhia ) using the cup - plate method . by assessing the diameter of the growth inhibition zone , the minimum inhibitory concentration ( mic ) and minimum bactericidal concentration ( mbc ) of the extract were determined for the above - mentioned bacteria 
 . results . 
 hydroalcoholic extract of p. granatum petal had inhibitory effects on the proliferation of all five bacterial strains with maximum effect on s. mutans with mic and mbc of 3.9  mg / ml . the largest growth inhibition zone diameter belonged to s. sanguinis and the smallest to e. faecalis . 
 ampicillin and chx had the greatest inhibitory effect on s. sanguinis . 
 conclusions . 
 hydroalcoholic extract of p. granatum had a significant antibacterial effect on common oral bacterial pathogens with maximum effect on s. mutans , which is the main microorganism responsible for dental plaque and caries . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion