herbal medicines are valuable and available resources of primary health care for thousands year in the traditional medicine including the thai folk medicine . generally , the bioactive compounds in herbal medicines are secondary metabolites which act as various pharmacological properties.1 , 2 , 3 they are used as the substances of modern drugs e.g. morphine from papaver somniferum for pain treatment,4 , 5 colchicine from colchicum autumnale for treatment in pericardial disease .
some studies have reported antibacterial activity from various herbs7 , 8 , 9 ; however , sufficient evidence to support their active compounds and their effectiveness for antibacteria and antibiofilm , particularly on oral pathogens is limited .
an imbalance of bacteria in the dental biofilm can cause dental caries and periodontitis which are major oral infections .
the high doses of antimicrobial agents are need for removing the biofilm in clinical approach.11 , 12 moreover , in long term use of chlorhexine ( chx ) mouth - rinse can cause side effects ; disturbance in taste sensation , brown discoloration at dorsum of tongue and desquamative lesions of oral mucosa.13 , 14 our previous study has presented anticandidal and antibiofilm of some thai herbs in thailand .
herbal medicines may be an alternative way for being antibacterial agents to prevent oral infectious diseases . this extended study was to evaluate the antibacterial and antibiofilm activities of 12 ethanol extracts of thai traditional herb included alpinia galanga , curcuma longa , curcuma zedoaria , piper betle , piper chaba , piper nigrum , piper sarmentosum , mentha cordifolia , ocimum africanum , ocimum basilicum , ocimum sanctum and zingiber officinale , and to investigate the active compounds of the most effective extract .
twelve herbs were purchased from the local market , and the details were shown in table  1 .
the herbs were identified by an expert in the department of pharmacognosy and pharmaceutical botany , faculty of pharmaceutical sciences , prince of songkla university , songkhla , thailand , where voucher specimens ( table  1 ) were deposited in the herbarium .
dried plants were macerated with ethanol for 3 days , and then filtrated through whatman no . 4 filter paper .
each filtrate was dried using a rotary evaporator at 40  c and kept at  20  c . for testing ,
0.1  mg of each dried extract was initially dissolved with 100  l of dimethyl sulfoxide ( dmso ) and then adjusted to 1  ml by adding sterile distilled water , giving a final concentration of 10% ( w / v ) extract in 10% ( v / v ) dimethyl sulfoxide ( dmso ) .
a total of 7 oral microorganisms were employed in the study including 5 gram positive cariogenic bacteria , enterobacter faecalis atcc 19433 ( ef ) , lactobacillus fermentum atcc 14931 ( lf ) , lactobacillus salivarius atcc 11741 ( ls ) , streptococcus sobrinus atcc 33478 ( ss ) and streptococcus mutans atcc 25175 ( sm ) , and 2 gram negative periodontopathogenic bacteria , aggregatibacter actinomycetemcomitans atcc 33384 ( aa ) and fusobacterium nucleatum atcc 25586 ( fn )
. microorganisms were maintained on either brain heart infusion agar ( bha ) with 5% ( v / v ) blood for facultative bacteria , and supplemented with 0.5% ( w / v ) yeast extract , hemin and vitamin k for anaerobic bacteria .
the strains were grown under aerobic or anaerobic ( 10% h2 , 10% co2 and 80% n2 ) conditions as appropriate .
one hundred microliters of inoculum , equivalent to 10  cfu / ml , was mixed with 20  ml of warm melted bha .
the mixture was then poured into the plate with a 6  mm diameter metal cup . after solidifying of bha
, the metal cups were removed and the well was added with 100  l of each plant extract .
the plate was incubated at 37  c for 24  h. a 10% dmso were taken as a negative control .
the antimicrobial activity of each plant extract was determined by measuring the diameter of the zone of the inhibition in millimeters .
minimum inhibitory concentration ( mic ) and minimum bactericidal concentration ( mbc ) were carried out as recommended instruction of the clinical and laboratory standards institute ( clsi ) .
briefly , 10% stock solution of each extract was diluted in the brain heart infusion broth ( bhi ) in two - fold serial dilutions to obtain concentrations from 0.02 to 25  mg / ml at a total volume of 100  l per well in 96-well microtiter plates .
each tested strain ( 100  l ) at a final concentration of 1    10  cfu / ml was added to each well and incubated at 37  c in appropriate conditions .
the medium , 0.1% ( w / v ) chx and 10% dmso were used as the non - treated , positive and negative controls , respectively .
mic was defined as the lowest concentration of the extract that completely inhibited growth in comparison with the non - treated control .
mbc was defined as the lowest concentration of wells that did not allow visible growth when 10  l of the well contents was plated on agar and grown at 37  c in appropriate conditions .
as the results of the screening of herb extracts revealed that p.  betle leaves extract gave the strongest antibacterial activity .
the growing cultures ( 10  cfu / ml ) of representative strains , s.  mutans atcc 25175 and a.  actinomycetemcomitans atcc 33384 , were incubated in bhi broth supplemented with the extract at concentrations equivalent to 1   , 2   , and 4    mic at 37  c .
surviving bacteria were observed at 0 , 0.5 , 1 , 2 , 4 , 6 , 8 , 10 , 12 and 24  h by culturing on agar plates . the procedure was repeated in triplicate , and the log10  cfu / ml was plotted against time .
a 0.1% chx and extract free medium were used as positive and non - treated controls , respectively .
betle extract on biofilm formation of each representative strain , s.  mutans atcc 25175 and a.  actinomycetemcomitans atcc 33384 , was examined by using the modified microdilution method .
betle extract were prepared , with final concentration ranged from 0.02 to 25  mg / ml .
a cell suspension of the tested strains was prepared as described in the mic assay , and 100  l ( 1    10  cfu / ml ) were inoculated in each of a 96-well plate .
a 0.1% chx , phosphate buffered saline ( pbs ) and extract free medium were used as the positive controls , non - treated and blank controls , respectively .
after incubation at 37  c for 24  h , supernatants were discarded and washed 3 times with pbs .
biofilm formation was quantified by using a 3-[4,5-dimethyl-2-thiazolyl]-2 , 5-diphenyl-2h - tetrazolium - bromide ( mtt ) assay .
the numbers of surviving bacteria were determined by measuring their ability to reduce the yellow tetrazolium salt to a purple formazan product at 570  nm .
percentage inhibition was calculated by using the equation [ 1    ( a570 of the test / a570 of non - treated control ) ]    100 .
the minimum biofilm inhibition concentration ( mbic ) was defined as the lowest concentration that showed 50% and 90% inhibition of biofilm formation ( mbic50 and mbic90 ) .
betle extract was also examined using the minimum biofilm eradication concentration ( mbec ) assay .
briefly , a 200  l in early log phase ( 10  cfu / ml ) of each representative strain , s.  mutans atcc 25175 and a.  actinomycetemcomitans atcc 33384 , was inoculated into each well of a 96-well plate and incubated for 24  h  at 37  c for the development of a multilayer biofilm . the culture
was then blotted out , and the well carefully washed 3 times with sterile pbs in order to remove non - adherent cells .
these biofilms were then exposed to a 200  l of various concentrations of p.  betle extract ranged from 0.02 to 25  mg / ml , incubated for 24  h  at 37  c in appropriate conditions . at the end - point of the treatment of the biofilms with p. 
percentage eradication was calculated by using the equation [ 1    ( a570 of the test / a570 of non - treated control ) ]    100 .
the mbec value was defined as the concentrations that showed 50% and 90% eradication of biofilm formation .
the 0.1% chx , pbs and the extract free medium were used as the positive , non - treated and blank controls , respectively .
the active antibacterial compounds of p.  betle extract were analyzed using the high performance liquid chromatography ( hplc ) ( agilent 1100 series , palo alto , ca ) .
the reverse column was apollo c18 , 250  mm    4.6  mm , 5  m particle size ( alltech associates inc . ,
the conditions were set as following ; mobile phases ( methanol : water 70:30 v / v ) , flow rate at 0.7  ml / min and uv detector at 280  nm . a injection volume ( 20  l ) of each sample , 1  mg of p.  betle extract , 5  l of pure eugenol ( the agent controller ) and 0.5  mg of 4-chromanol ( the agent controller ) , were prepared in 1  ml of 95% ( v / v ) ethanol .
each prepared sample was taken to analyze by hplc , and curves were prepared using the microsoft excel software package ( excel 2007 ; microsoft corporation , redmond , wa , usa ) .
for tlc - fingerprinting study , 100  mg of p.  betle extract , 5  l of pure eugenol ( the agent controller ) and 1  mg of 4-chromanol ( the agent controller ) were prepared in 1  ml of 95% ethanol .
a 10  l of each sample was applied onto the alumina silica gel gf254 tlc - plate , and was run through a series of solvent systems using a mixture of toluene and ethyl acetate ( 90:10 v / v ) as the mobile phase .
the plate was air dried , and was stained with a 20% ( w / v ) of phosphomolybdic acid in ethanol .
tlc - plates were placed on plate contained 6  ml of bha , and 10  ml of top agar with 100  l of either s.  mutans atcc 25175 or a.  actinomycetemcomitans atcc 33384 inoculum ( 10  cfu / ml ) was covered on tlc plate . after overnight incubation , the inhibited zone was observed as clear zone .
the relative front values ( rf ) was calculated as : rf  =  distance traveled by solute / distance traveled by solvent .
data were expressed as mean and standard deviation ( sd ) by computational analysis from the three experiments with duplicate or triplicate independent experiments .
antibacterial activity of 12 thai traditional herb extracts against various oral bacteria was revealed in table  1 .
betle leaves showed a good result of antibacterial activity against all tested microorganisms using agar well diffusion assay ( table  1 ) .
the mic of p.  betle extract ranged from 1.04 to 5.21  mg / ml and mbc ranged from 2.08 to 8.33  mg / ml , respectively ( table  2 ) .
time kill assay of p.  betle extract against representative strains , s.  mutans atcc 25175 and a.  actinomycetemcomitans atcc 33384 , is demonstrated in fig . 
the completed killing of s.  mutans atcc 25175 treated with 1   , 2   , and 4    mic of the extract occurred within 12 , 8 , and 6  h , respectively . at 1   , 2   , and 4    mic , a.  actinomycetemcomitans
atcc 33384 was killed after 6 , 2 , and 1  h , respectively .
the killing of positive control ( 0.1% chx ) was observed within 30  min .
the present study demonstrated that commonly used thai herbs could exhibit antimicrobial activity against various oral bacteria .
betle extract revealed the best antibacterial activity against both gram positive bacteria and gram negative bacteria .
the inhibition and eradication of the biofilm formation of p.  betle extract against s.  mutans atcc 25175 and a.  actinomycetemcomitans atcc 33384 is demonstrated in fig . 
the concentrations of p.  betle extract required to inhibit for 50% biofilm formation ( mbic50 ) of s.  mutans atcc 25175 and a.  actinomycetemcomitans atcc 33384 were 0.39    0.20 and 0.10    0.03  mg / ml , respectively . and for 90% biofilm inhibitions ( mbic90 ) were 1.56    0.11 and 0.39    0.65  mg / ml , respectively .
the concentrations of p.  betle extract to eradicate for 50% biofilm formation ( mbec50 ) of s.  mutans atcc 25175 and a.  actinomycetemcomitans atcc 33384 were 0.78    0.74 and 0.78    0.11  mg / ml , respectively . and for 90% biofilm eradication ( mbec90 ) were 6.25    0.58 and 3.13    0.28  mg / ml , respectively .
chx could completely ( 100% ) inhibit or remove biofilm formation of both tested strains .
due to its interesting results , the further study focused on the active compounds of p.  betle leaves extract .
3b ) and the mixture of p.  betle extract and the standards ( fig . 
3a  c was identified as 4-chromanol with retention times of 5.486    0.00  min , 5.654    0.04  min and 5.610    0.00  min , respectively .
3a  c was eugenol with retention times of 7.853    0.01  min , 7.966    0.09  min and 7.757    0.01  min , respectively .
it indicated that at least 4-chromanol and eugenol were the components of p.  betle leaves extract .
there were several bands of p.  betle extract components appeared on the tlc - plate ( fig . 
4a ) . one major band with rf at 0.38 matched to the standard 4-chromanol gave the large inhibited clear zone against s. 
4b ) , and this band also gave inhibited clear zone against a.  actinomycetemcomitans atcc 33384 ( data not shown ) .
another minor band with rf at 0.74 matched to the standard eugenol did not show any inhibited zone .
p.  betle ( family piperaceae ) is known as  phlu  , which has been extensively used in traditional herbal remedies in thailand and many other countries in tropical asia .
it is reported having various pharmacological activities such as antimicrobial , immunomodulatory , and anti - inflammatory . a significant antimicrobial activity against a wide range of medical bacteria such as streptococcus pyogenes , staphylococcus aureus , proteus vulgaris , escherichia coli , pseudomonas aeruginosa etc .
was found in the leaves . in this study , it has shown that p. 
betle extract also exhibited antimicrobial activity against various oral microorganisms including cariogenic and periodontogenic pathogens e.g. s.  mutans and a.  actinomycetemcomitans .
our results agree with the study of deshpande and kadam who demonstrated the finding of antibacterial activity of ethanol and aqueous crude extract of p.  betle leaves against s.  mutans . in that report ,
zone of inhibition of ethanol extract ( 20.6    1.12  mm ) was found to be larger than aqueous extract ( 18.3    0.53  mm ) .
the mic value for the aqueous and ethanol extract was 10  mg / ml and 5  mg / ml , respectively .
the p.  betle leaves ethanol extract in this study was found to be more potent with mic value of 1.56  mg / ml .
this may be due to ecological and geographical conditions , age of herb and time of harvesting .
however , there was no information of antibiofilm was mentioned in that study . the antibiofilm is a desired property that expected from herbs extracts . in this study ,
betle leaves extract has been revealed having dual actions in preventing and eradicating biofilm formation .
it may be worth for considering the p.  betle leaves extracts to replace chx mouth - rinse .
chx is a chemical - based antimicrobial agent which is used extensively in the mouth - rinse to maintain dental biofilm at a level compatible with oral health .
however , it was reported on some undesired local side effects for long term use.13 , 14 results of both planktonic and biofilm form indicated that p. 
the similar results were also reported in citrus bergamia , 
 woodfordia fruticosa , and artocarpus lakoocha showing that gram negative bacteria were more susceptible to some plant extracts than gram positive .
this contrasted to previous studies showed that plant extracts were more active against gram positive bacteria than gram negative bacteria.24 , 25 the gram negative bacteria are considered to be more resistant due to their outer membrane acting as a barrier to many environmental substances including antimicrobial substances .
moreover , they could stimulate the membrane - associated mechanisms of resistance via the expression of efflux pumps to control the antibacterial agents.26 , 27 deshpande and kadam reported that 4-chromanol was a predominant compound in p.  betle leaves aqueous extract , which responded for antibacterial activity .
our previously study demonstrated that the major constituents of p.  betle leaves extract were 4-chromanol ( 62.33% ) and eugenol ( 17.10% ) in gc - ms analysis , and the 4-chromanol showed good anticandidal activities . in this study ,
the 4-chromanol also exhibited the strong antibacterial activity by observing a clear inhibition zone against oral pathogens in the tlc - bioautography assay .
thus , it indicated that the 4-chromanol could overcome the barrier and membrane - associated resistance of gram negative bacterial membrane .
this study suggested that the ethanol extract of p.  betle leaves is a potential source of natural antibacterial and antibiofilm agents .
the major constituents of p.  betle leaves extract was 4-chromanol exhibiting a good antibacterial and antibiofilm properties that may be used for oral infectious diseases . for further study
, the mechanisms of antibacterial activity of 4-chromanol should be investigated in more detail as well as its activity in  vivo .
rawee teanpaisan : project initiation and design , data analysis , writing and preparing the manuscript , editing the manuscript .
pajaree kawsud : project initiation and design , laboratory performance , data analysis , writing and preparing the manuscript .
 to evaluate the antibacterial activity of 12 ethanol extracts of thai traditional herb against oral pathogens . 
 the antibacterial activities were assessed by agar well diffusion , broth microdilution , and time - kill methods . 
 antibiofilm activity was investigated using a 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2h - tetrazolium - bromide ( mtt ) assay . 
 high performance liquid chromatography ( hplc ) , thin layer chromatography ( tlc ) fingerprinting , and tlc - bioautography were used to determine the active antibacterial compounds . 
 piper betle showed the best antibacterial activities against all tested strains in the minimal inhibitory concentration and minimal bactericidal concentration , ranged from 1.045.21  mg / ml and 2.088.33  mg / ml , respectively . 
 killing ability depended on time and concentrations of the extract . 
 p.  
 betle extract acts as a potent antibiofilm agent with dual actions , preventing and eradicating the biofilm . 
 the major constituent of p.  
 betle extract was 4-chromanol , which responded for antibacteria and antibiofilm against oral pathogens . 
 it suggests that the ethanol p.  betle leaves extract may be used for preventing oral diseases . 
Introduction
Materials and methods
Results
Discussion
Conclusion
Author contribution
Conflict of interest