the periodontium is a topographically complex organ consisting of epithelial tissue and 
 soft and mineralized connective tissue .
the structures comprising the periodontium 
 include gingiva , cementum , bone and periodontal ligament .
several diseases affect the composition and integrity of 
 periodontal structures causing the destruction of the connective tissue matrix and 
 cells , the loss of fibrous attachment and the resorption of alveolar bone following an 
 intense inflammatory response to bacteria .
such diseases are highly prevalent among different world 
 populations , being an important impact factor in health oral programs .
the main bacterial pathogens of 
 periodontal diseases are gram - negative anaerobic species that express a number of 
 potential virulence factors that stimulate an unbalanced production of several molecules 
 and pro - inflammatory factors , being an important determinant in the disease 
 outcome .
innumerous studies have shown that , although largely nonspecific , basic treatment 
 consisting of oral hygiene instruction and subgingival instrumentation is able to keep 
 the microbial load at low levels 
 and , if followed by daily self - performed oral hygiene and regular professional 
 periodontal maintenance care , leads to periodontal health in most cases . however , due to its unique patient and 
 site - specific characteristics , a small proportion of the population suffers from 
 ' ' refractory '' types of periodontitis , showing inadequate resolution of inflammation and 
 failure to return tissue to homeostasis . since knowledge of the pathways and processes underlying resolution 
 of periodontal tissue inflammation have grown significantly , an increased interest in 
 substances and/or drugs that may contribute to the restoration of tissue homeostasis has 
 occurred as a more sophisticated biological treatment modality for recurrent types of 
 periodontal disease .
several biological systems are candidates to modulate the inflammatory process involved 
 in periodontal disease .
evidence from animal experiments and short - term clinical trials 
 demonstrate that anti - inflammatory drugs are able to decrease the rate of periodontal 
 bone destruction by local inhibition of pro - inflammatory molecules . in brazil , the leaves of species of 
 mikania laevigata schultz bip .
ex baker , popularly known as " guaco " , 
 have been widely used as infusions or plasters , while the crude extract of this species 
 is commonly commercialized as phytomedicine .
guaco is also popular in brazil as an 
 anti - inflammatory , antispasmodic and pain - reliever for rheumatism , arthritis , intestinal 
 inflammation and ulcers . among the few pharmacological and phytochemical studies 
 published , preparations obtained from aerial parts of mikania laevigata 
 have been described as anti - ulcerogenic , antimicrobial , 
 bronchodilatory and 
 anti - inflammatory , possibly 
 accounted for by the presence of several chemical constituents .
our research group has recently assessed the pharmacological properties and the 
 underlying molecular mechanisms of mikania laevigata , demonstrating 
 that , in animals , its hydroalcoholic extract was able to reduce neutrophil migration and 
 vascular permeability , and prevent the release of tnf- and il-1. since such findings could promote a 
 protective effect in periodontal tissues against damage , the aim of this study was to 
 evaluate the immunomodulatory action of systemic injection of a " guaco " extract in a 
 model of experimental periodontitis in rats .
the extract used in this study was previously obtained and tested as described 
 elsewhere .
briefly , the 
 leaves of mikania laevigata were collected at the farm school of the 
 university of uberaba ( tringulo mineiro , mg , brazil ) . a voucher specimen (
hufu 
 54.748 ) has been deposited at the herbarium of the federal university of uberlndia , 
 brazil .
, between days 15 and 17 of 
 november and allowed to dry at 30c in an oven with air renovation for 15 days .
the 
 dry plant , triturated by knife mill , was extracted by maceration with 70% 
 ethanol : water under continuous agitation ( shaker ) for three times during 7 days each 
 totalizing 21 days [ the end ratio between plant and solvent was 1:8 ( w / v ) ] , obtaining 
 a crude extract .
the crude extract was dried and filtered using filter paper , 
 concentrated in air - forced chamber at 30c until dry crude extract was obtained . 

forty male wistar rats ( 250 - 350 g ) were used in the study .
the animals were kept in 
 plastic cages with access to food and water ad libitum . prior to the 
 surgical procedures ,
all animals were allowed to acclimatize to the laboratory 
 environment for a period of 5 days .
all experiments were conducted in accordance with 
 national health guidelines for the welfare of experimental animals and were approved 
 by the ethics committee of the university of uberaba ( protocol # 001/2008 ) .
more specifically , 
 under general anesthesia obtained by intramuscular administration of ketamine ( 1.0 
 ml / kg ) , ligature was placed and immobilized around both mandible first molars of each 
 animal .
the ligature was left in position for the whole experimental period so that 
 inflammation could be constantly induced by the colonization of bacteria inside of 
 it .
one day following ligature placement , the animals were randomly assigned to one 
 of the following groups ( n=10 ) : 1 ) animals without ligature placement receiving 
 administration of empty vehicle ( control ) ; 2 ) animals without ligature placement 
 receiving administration of mle ; 3 ) animals with ligature receiving administration of 
 saline ; 4 ) animals with ligature receiving administration of subcutaneous mle ( 10 
 mg / kg / day ; 200 l . mle or vehicle was administered daily for 30 days . twenty - four 
 hours after the last injection
the current dose was chosen based on a previous study showing that it was effective 
 to diminish the carrageenan - induced peritonitis .
neutrophil infiltration to the gingival tissues of rats was evaluated by mpo 
 kinetic - colorimetric assay as previously described .
samples of gingival tissue were collected in 50 mm 
 k2hpo4 buffer ( ph 6.0 ) containing 0.5% hexadecyl 
 trimethylammonium bromide ( htab ) and kept at -80c until use .
samples were 
 homogenized using a polytron ( pt3100 ) and centrifuged at 13,000 g for 4 min .
the 
 resulting supernatant devoid of debris was subjected to mpo activity assay determined 
 by spectrophotometer at 450 nm ( spectra max ; molecular devices inc . , 

briefly , 10 l of sample was 
 mixed with 200 l of 50 mm phosphate buffer ph 6.0 , containing 0.167 mg / ml 
 o - dianisidine dihydrochloride and 0.0005% hydrogen peroxide .
the results were presented 
 as mpo activity ( number of neutrophils x mg gingival tissue ) .
the right and left jaws were dissected , fixed in 10% buffered neutral formalin for 48 
 h and decalcified in a decalcifying solution of ethylenediaminetetraacetic acid 
 ( edta ) 10% for 3 months .
after that , briefly washed in running tap water , dehydrated 
 and embedded in paraffin wax .
sections were mounted on glass slides and stained with hematoxylin and 
 eosin ( he ) for the evaluation of bone resorption . using an image analysis system 
 ( image j - national institute of health ) , the area of bone loss in the furcation 
 region was histometrically determined as previously described .
additional sections were mounted on glass slides pre - treated with 
 3-aminopropyltriethoxy - silane ( sigma chemical co. , st .
sections were treated with 3% hydrogen peroxide 
 ( h2o2 ) for 30 min to block endogenous peroxidase . to block 
 the nonspecific binding of antibody , the sections
were treated with phosphate 
 buffered saline ( pbs)-1% bovine serum albumin ( vector laboratories , burlingame , ca , 
 usa ) for 30 min at room temperature before incubation with the primary antibody 
 ( polyclonal antibodies against rankl ( 1:50 ) ( santa cruz biotechnology , santa cruz , 
 ca , usa ) for 24 h at 4c .
biotinylated secondary antibody was used ( sigma;1:100 ) for 
 60 min at room temperature .
the slides were treated with vectastain abc - ap kit 
 ( diluted at 1:100 ; vector laboratories ) for 60 min at room temperature , and the 
 specific reaction by each antibody was visualized using 3 , 3'diaminobenzidine ( dab ) . 

the slides were then counterstained with mayer 's hematoxylin , dehydrated through 
 graded ethanol , cleared in xylene , and mounted in slides with the help of permount 
 mounting media ( fisher scientific ) .
at least 10 representative sections of each specimen were analyzed under a light 
 microscope ( olympus bx50 , tokyo , japan ) .
immunohistochemical analysis was performed 
 individually by two previous calibrated examiners ( jcc and vjs - f ; kappa index=0.97 ) 
 that were blind to the treatment conditions .
the number of stained cells was counted 
 and normalized in the area where bone resorption was taking place .
statistical comparisons between 
 groups were made using anova analysis of variance followed by bonferroni test . 

the extract used in this study was previously obtained and tested as described 
 elsewhere .
briefly , the 
 leaves of mikania laevigata were collected at the farm school of the 
 university of uberaba ( tringulo mineiro , mg , brazil ) . a voucher specimen (
hufu 
 54.748 ) has been deposited at the herbarium of the federal university of uberlndia , 
 brazil .
, between days 15 and 17 of 
 november and allowed to dry at 30c in an oven with air renovation for 15 days .
the 
 dry plant , triturated by knife mill , was extracted by maceration with 70% 
 ethanol : water under continuous agitation ( shaker ) for three times during 7 days each 
 totalizing 21 days [ the end ratio between plant and solvent was 1:8 ( w / v ) ] , obtaining 
 a crude extract .
the crude extract was dried and filtered using filter paper , 
 concentrated in air - forced chamber at 30c until dry crude extract was obtained . 

forty male wistar rats ( 250 - 350 g ) were used in the study .
the animals were kept in 
 plastic cages with access to food and water ad libitum .
prior to the 
 surgical procedures , all animals were allowed to acclimatize to the laboratory 
 environment for a period of 5 days .
all experiments were conducted in accordance with 
 national health guidelines for the welfare of experimental animals and were approved 
 by the ethics committee of the university of uberaba ( protocol # 001/2008 ) .
more specifically , 
 under general anesthesia obtained by intramuscular administration of ketamine ( 1.0 
 ml / kg ) , ligature was placed and immobilized around both mandible first molars of each 
 animal .
the ligature was left in position for the whole experimental period so that 
 inflammation could be constantly induced by the colonization of bacteria inside of 
 it .
one day following ligature placement , the animals were randomly assigned to one 
 of the following groups ( n=10 ) : 1 ) animals without ligature placement receiving 
 administration of empty vehicle ( control ) ; 2 ) animals without ligature placement 
 receiving administration of mle ; 3 ) animals with ligature receiving administration of 
 saline ; 4 ) animals with ligature receiving administration of subcutaneous mle ( 10 
 mg / kg / day ; 200 l . mle or vehicle was administered daily for 30 days . twenty - four 
 hours after the last injection ,
the current dose was chosen based on a previous study showing that it was effective 
 to diminish the carrageenan - induced peritonitis .
neutrophil infiltration to the gingival tissues of rats was evaluated by mpo 
 kinetic - colorimetric assay as previously described .
samples of gingival tissue were collected in 50 mm 
 k2hpo4 buffer ( ph 6.0 ) containing 0.5% hexadecyl 
 trimethylammonium bromide ( htab ) and kept at -80c until use .
samples were 
 homogenized using a polytron ( pt3100 ) and centrifuged at 13,000 g for 4 min .
the 
 resulting supernatant devoid of debris was subjected to mpo activity assay determined 
 by spectrophotometer at 450 nm ( spectra max ; molecular devices inc . , 
 sunnyvale , ca , usa ) with three readings within 1 min .
briefly , 10 l of sample was 
 mixed with 200 l of 50 mm phosphate buffer ph 6.0 , containing 0.167 mg / ml 
 o - dianisidine dihydrochloride and 0.0005% hydrogen peroxide .
the results were presented 
 as mpo activity ( number of neutrophils x mg gingival tissue ) .
the right and left jaws were dissected , fixed in 10% buffered neutral formalin for 48 
 h and decalcified in a decalcifying solution of ethylenediaminetetraacetic acid 
 ( edta ) 10% for 3 months .
after that , briefly washed in running tap water , dehydrated 
 and embedded in paraffin wax .
sections were mounted on glass slides and stained with hematoxylin and 
 eosin ( he ) for the evaluation of bone resorption . using an image analysis system 
 ( image j - national institute of health ) , the area of bone loss in the furcation 
 region was histometrically determined as previously described .
additional sections were mounted on glass slides pre - treated with 
 3-aminopropyltriethoxy - silane ( sigma chemical co. , st .
sections were treated with 3% hydrogen peroxide 
 ( h2o2 ) for 30 min to block endogenous peroxidase . to block 
 the nonspecific binding of antibody , the sections were treated with phosphate 
 buffered saline ( pbs)-1% bovine serum albumin ( vector laboratories , burlingame , ca , 
 usa ) for 30 min at room temperature before incubation with the primary antibody 
 ( polyclonal antibodies against rankl ( 1:50 ) ( santa cruz biotechnology , santa cruz , 
 ca , usa ) for 24 h at 4c .
biotinylated secondary antibody was used ( sigma;1:100 ) for 
 60 min at room temperature .
the slides were treated with vectastain abc - ap kit 
 ( diluted at 1:100 ; vector laboratories ) for 60 min at room temperature , and the 
 specific reaction by each antibody was visualized using 3 , 3'diaminobenzidine ( dab ) . 

the slides were then counterstained with mayer 's hematoxylin , dehydrated through 
 graded ethanol , cleared in xylene , and mounted in slides with the help of permount 
 mounting media ( fisher scientific ) .
at least 10 representative sections of each specimen were analyzed under a light 
 microscope ( olympus bx50 , tokyo , japan ) .
immunohistochemical analysis was performed 
 individually by two previous calibrated examiners ( jcc and vjs - f ; kappa index=0.97 ) 
 that were blind to the treatment conditions .
the number of stained cells was counted 
 and normalized in the area where bone resorption was taking place .
statistical comparisons between 
 groups were made using anova analysis of variance followed by bonferroni test . 

to assess whether mle would affect bone loss in the furcation region , he sections 
 were histometrically analyzed . the resorption area measured after the experimental 
 period demonstrated that ligature induced experimental periodontitis
significantly 
 increased bone loss ( p<0.05 ) ( figure 1c ) 
 when compared with both vehicle ( 0.310.03 mm ) and mle ( 0.350.06 
 mm ) non - ligated groups , which were not different when compared with 
 each other ( p>0.05 ) ( figures 1a and 1b ) .
additionally , the results demonstrated that 
 mle administration ( 10 mg / kg , daily ) was able to significantly inhibit ( 0.560.13 
 mm ; p<0.05 ) the volume of bone loss in ligated teeth ( figure 1d ) , however still higher when compared 
 with the non - ligated teeth ( p<0.05 ) .
the values of the resorption area of the 
 three groups were represented in figure 1 .
histology at the furcation of first molars sampled from the rats 
 euthanized after 30 days of experiments is shown [ hematoxylin and eosin(he ) 
 staining ] ; ( a ) non - ligated animals treated with vehicle , ( b ) non - ligated 
 animals treated with ( mle ) ( 10 mg / kg / day for 30 consecutive days ) , ( c ) 
 ligature - induced periodontitis treated with vehicle for 30 days , ( d ) 
 ligature - induced periodontitis treated with subcutaneous mle ( 10 mg / kg/ day for 
 30 consecutive days ) .
( e ) results are expressed as mean area ( mm )  
 standard deviation of 10 animals in each group .
different symbols indicate 
 intergroup statistical significance ( anova followed by bonferroni s test ) . 

magnification at 40 objective ( scale bar=100 m ) next , the possible interference of mle on neutrophil migration was investigated . 
 according to mpo assay
, mle administration did not exert any significant effect on 
 neutrophil accumulation on non - ligated teeth ( 55.7425.84 versus 53.1415.21 
 neutrophils x 10 ; p>0.05 ) by measuring gingival tissue mpo .
on the 
 other hand , the increased levels of neutrophil accumulation observed on ligated teeth 
 ( 113.8238.96 neutrophils x 106 ; p<0.05 ) were significantly decreased by mle 
 administration ( 61.2310.12 neutrophils x 10 ; p<0.05 ) ( figure 2 ) .
thus , mle was able to reduce neutrophil 
 accumulation and consequently inflammation in the gingival tissue .
mikania laevigata extract ( mle ) decreases neutrophil migration 
 to the gingival tissue . in order to estimate
the relative numbers of 
 infiltrating neutrophils in the gingival tissue , myeloperoxidase ( mpo ) activity 
 present in the gingival tissue homogenates was measured .
# p<0.05 compared with control 
 animals ; * p<0.05 compared with ligature - induced periodontitis treated with 
 vehicle ( anova followed by bonferroni 's test ) on the other hand , since a reduced bone resorption was observed in the mle - treated 
 group , we tested the hypothesis that mle decreases the release of osteoclast 
 regulatory factors . in this way , we used an immunohistochemistry assay to visualize 
 the expression of rankl on periodontal tissues .
as shown in figure 3 , animals with ligated - induced periodontitis treated with 
 mle presented significantly less rankl - immunostained cells ( 76.2564.40 immunostained 
 cells ; p<0.05 ) in the periodontal tissue when compared with ligated - animals 
 treated with vehicle ( 242.0046.23 immunostained cells ) .
non - ligated animals 
 ( 49.6618.23 immunostained cells ) were not affected by mle administration 
 ( 45.7522.42 immunostained cells ; p>0.05 ) .
expression patterns of rankl in the 
 periodontal tissue at the furcation of first molars sampled from the rats 
 euthanized at 30 days are shown .
low number of positive staining cells for 
 rankl was observed in the furcation of non - ligated animals treated with vehicle 
 ( a ) as well the non - ligated animals treated with mikania laevigata 
 extract ( mle ) ( b ) . increased number of rankl positive cells
were 
 found in the furcation of the ligated - animals treated with vehicle ( c ) .
lower 
 number of rankl stained cells were found in the furcation of the 
 ligated - animals treated with mle ( 10 mg / kg / day for 30 consecutive days ) .
( e ) 
 data are presented as median  standard deviation of the average rankl positive 
 cells / per area of furcation .
negative control staining was carried out by the 
 incubation with secondary antibody alone that showed lack of immunostaining 
 pattern in all experimental groups ( data not shown )
. # p<0.05 compared with 
 control animals ; * p<0.05 compared with ligature - induced periodontitis 
 treated with vehicle ( anova followed by bonferroni s test ) .
the figure is 
 representative of at least 4 different sections obtained from each group .
scale 
 bar at lower ( 40x ) magnification=100 m , and scale bar at higher ( 400x ) 
 magnification=20 m
to assess whether mle would affect bone loss in the furcation region , he sections 
 were histometrically analyzed . the resorption area measured after the experimental 
 period demonstrated that ligature induced experimental periodontitis
significantly 
 increased bone loss ( p<0.05 ) ( figure 1c ) 
 when compared with both vehicle ( 0.310.03 mm ) and mle ( 0.350.06 
 mm ) non - ligated groups , which were not different when compared with 
 each other ( p>0.05 ) ( figures 1a and 1b ) .
additionally , the results demonstrated that 
 mle administration ( 10 mg / kg , daily ) was able to significantly inhibit ( 0.560.13 
 mm ; p<0.05 ) the volume of bone loss in ligated teeth ( figure 1d ) , however still higher when compared 
 with the non - ligated teeth ( p<0.05 ) .
the values of the resorption area of the 
 three groups were represented in figure 1 .
histology at the furcation of first molars sampled from the rats 
 euthanized after 30 days of experiments is shown [ hematoxylin and eosin(he ) 
 staining ] ; ( a ) non - ligated animals treated with vehicle , ( b ) non - ligated 
 animals treated with ( mle ) ( 10 mg / kg / day for 30 consecutive days ) , ( c ) 
 ligature - induced periodontitis treated with vehicle for 30 days , ( d ) 
 ligature - induced periodontitis treated with subcutaneous mle ( 10 mg / kg/ day for 
 30 consecutive days ) .
( e ) results are expressed as mean area ( mm )  
 standard deviation of 10 animals in each group .
different symbols indicate 
 intergroup statistical significance ( anova followed by bonferroni s test ) . 

next , the possible interference of mle on neutrophil migration was investigated . 
 according to mpo assay
, mle administration did not exert any significant effect on 
 neutrophil accumulation on non - ligated teeth ( 55.7425.84 versus 53.1415.21 
 neutrophils x 10 ; p>0.05 ) by measuring gingival tissue mpo .
on the 
 other hand , the increased levels of neutrophil accumulation observed on ligated teeth 
 ( 113.8238.96 neutrophils x 106 ; p<0.05 ) were significantly decreased by mle 
 administration ( 61.2310.12 neutrophils x 10 ; p<0.05 ) ( figure 2 ) .
thus , mle was able to reduce neutrophil 
 accumulation and consequently inflammation in the gingival tissue .
mikania laevigata extract ( mle ) decreases neutrophil migration 
 to the gingival tissue . in order to estimate the relative numbers of 
 infiltrating neutrophils in the gingival tissue , myeloperoxidase ( mpo )
# p<0.05 compared with control 
 animals ; * p<0.05 compared with ligature - induced periodontitis treated with 
 vehicle ( anova followed by bonferroni 's test ) on the other hand , since a reduced bone resorption was observed in the mle - treated 
 group , we tested the hypothesis that mle decreases the release of osteoclast 
 regulatory factors . in this way , we used an immunohistochemistry assay to visualize 
 the expression of rankl on periodontal tissues .
as shown in figure 3 , animals with ligated - induced periodontitis treated with 
 mle presented significantly less rankl - immunostained cells ( 76.2564.40 immunostained 
 cells ; p<0.05 ) in the periodontal tissue when compared with ligated - animals 
 treated with vehicle ( 242.0046.23 immunostained cells ) .
non - ligated animals 
 ( 49.6618.23 immunostained cells ) were not affected by mle administration 
 ( 45.7522.42 immunostained cells ; p>0.05 ) .
expression patterns of rankl in the 
 periodontal tissue at the furcation of first molars sampled from the rats 
 euthanized at 30 days are shown .
low number of positive staining cells for 
 rankl was observed in the furcation of non - ligated animals treated with vehicle 
 ( a ) as well the non - ligated animals treated with mikania laevigata 
 extract ( mle ) ( b ) . increased number of rankl positive cells
were 
 found in the furcation of the ligated - animals treated with vehicle ( c ) .
lower 
 number of rankl stained cells were found in the furcation of the 
 ligated - animals treated with mle ( 10 mg / kg / day for 30 consecutive days ) .
( e ) 
 data are presented as median  standard deviation of the average rankl positive 
 cells / per area of furcation .
negative control staining was carried out by the 
 incubation with secondary antibody alone that showed lack of immunostaining 
 pattern in all experimental groups ( data not shown ) .
# p<0.05 compared with 
 control animals ; * p<0.05 compared with ligature - induced periodontitis 
 treated with vehicle ( anova followed by bonferroni s test ) .
the figure is 
 representative of at least 4 different sections obtained from each group .
scale 
 bar at lower ( 40x ) magnification=100 m , and scale bar at higher ( 400x ) 
 magnification=20 m
tooth loss , caused by 
 poor periodontal health ( which affects up to 20% of the adult population worldwide ) can 
 lead to significant morbidity and premature death .
the economic impact of oral diseases is an important 
 consideration with up to 10% of public health expenditure in developed countries related 
 to curative dental care . in cases 

where traditional and basic treatments are not able to arrest periodontal disease 
 progression , the adjunct use of alternative substances aimed at modulating the host 
 immune response is desirable .
however , despite several agents being commercially 
 available , most of these chemicals may present undesirable side effects and limited scientific evidence so far . 

it is recognized that most of the new drugs discovered in the last few decades have 
 originated from nature .
chemical 
 constituents obtained from medicinal plants and other natural products have been 
 increasingly used to treat many inflammatory diseases . in the present study , it was 
 demonstrated that an extract obtained from mikania laevigata , a popular 
 medicinal plant in south american countries , was able to decrease alveolar bone loss , 
 which may be explained , at least in part , by the lower expression of rankl and decreased 
 neutrophil migration observed on mle - treated animals .
a growing body of research suggests that chronic periodontal disease involves a failure 
 of inflammation resolution pathways to restore tissue homeostasis .
therefore , strategies 
 to slow or arrest periodontal disease progression by the modulation of the host immune 
 response have been investigated . 
 among the commercially available substances
earliest evaluated so far to modify 
 resolution pathways , are the nonsteroidal anti - inflammatory drugs ( nsaids ) .
these agents 
 antagonize proinflammatory pathways and/or signaling , preventing the production 
 arachidonic acid metabolites that are proinflammatory mediators implicated in a variety 
 of bone resorptive and tissue degrading processes .
although in vitro and experimental studies have shown promising results , periodontal clinical 
 trials have not reached consistent clinical benefits when combined with conventional 
 therapy besides having 
 undesirable side effects when used in a long - term basis .
hence , the search for 
 alternative products continues and natural phytochemicals isolated from plants used in 
 traditional medicine are considered as good alternatives to synthetic 
 chemicals .
more recently , the number of reports of the use of traditional plants and natural 
 products for the treatment of periodontal disease has increased . in 
 vitro studies evaluating the antimicrobial potential of substances
in addition , 
 in vivo studies in animals using compounds of natural plants such as 
 baicalin and 
 cannabinoids observed a 
 protective role against periodontal tissue breakdown in ligature - induced periodontitis , 
 which was partially explained by their inhibitory effects on prostaglandins ( cox-2 ) , 
 interleukins ( il-1 and tnf- ) , nitric oxide and the bone related molecules rank and 
 rankl .
our results corroborate with such previous findings , with the administration of 
 mle significantly decreasing alveolar bone resorption and promoting an inhibition of 
 neutrophils influx and rankl production . in the progression of inflammatory diseases ,
the interactions of recruited neutrophils 
 in the site of inflammation stimulate the local production of several inflammatory 
 mediators that may further amplify the inflammatory response and injure the surrounding 
 tissues .
in fact , our group 
 recently assessed the pharmacological properties and the underlying molecular mechanisms 
 of the hydroalcoholic mle to confirm the popular wisdom as a putative anti - inflammatory 
 drug .
we could observe that the 
 anti - inflammatory effect of mle was able to reduce neutrophil migration and vascular 
 permeability in a peritonitis model .
mle additionally prevented the release of both 
 tnf- and il-1 and contributed to a reduction in leukocyte adhesion and transmigration 
 across the endothelium as observed by intravital microscopy .
taken together , the results 
 suggest that use of the medicinal guaco extract may be able to suppress the development 
 of inflammatory lesions , which are initiated by neutrophil recruitment including 
 periodontal conditions .
the discovery of the rank - rankl - osteoprotegerin ( opg ) system has brought rapid progress 
 in the understanding of the regulatory mechanisms of osteoclast differentiation and 
 activation exerted by the immune system .
recent studies suggest the involvement of rankl and opg in the 
 pathogenesis of periodontitis . not 
 only osteoblasts but also other resident cells , including cells of the periodontal 
 ligament ,
the findings of the present study , demonstrated that the 
 administration of mle inhibited the expression of rankl . to the best of our knowledge , 

no direct effect of mle on the rank / rankl / opg system has been addressed in the 
 literature .
however , several investigations have addressed the close relation between 
 the production of inflammatory mediators and the modulation of rankl / opg balance . during 
 the progression of periodontal disease ,
high levels of pro - inflammatory cytokines have 
 been positively related to rankl expression , suggesting that such inflammatory molecules 
 play a major role in periodontal bone resorption .
interestingly , a recent work demonstrated that 
 lipopolysaccharide ( lps ) , a toll - like receptor 4 ligand , up - regulated the expression of 
 membrane rankl in human blood neutrophils .
besides , lps - activated human and murine 
 neutrophils , cocultured with human monocyte - derived osteoclasts and raw 264.7 cells , 
 respectively , stimulated bone resorption . therefore it is possible to suggest that the potent 
 anti - inflammatory properties of mle may have caused the diminished production of rankl 
 observed in mle - treated animals observed in the present study and indirectly 
 participated in the decreased volume of bone loss during ligature - induced periodontal 
 disease progression .
there is considerable evidence that plant extracts have the potential to be developed 
 into agents that can be used as preventative or treatment therapies for oral diseases . 

the results of the present investigation indicate that " guaco " extract may be useful to 
 control bone resorption during progression of experimental periodontitis in rats in 
 animal studies .
while our results are encouraging , further controlled trial studies will 
 be important to establish whether guaco is able to offer clinical therapeutic 
 benefits .
 objectivesthe mikania laevigata extract ( mle ) ( popularly known in brazil as 
 " guaco " ) possesses anti - inflammatory properties . in the present study we tested 
 the effects of mle in a periodontitis experimental model in rats . 
 we also 
 investigated possible mechanisms underlying such effects . 
 material and methodsperiodontal disease was induced by a ligature placed around the mandibular first 
 molars of each animal . 
 male wistar rats were divided into 4 groups : non - ligated 
 animals treated with vehicle ; non - ligated animals treated with mle ( 10 mg / kg , 
 daily ) ; ligature - induced animals treated with vehicle and ligature - induced animals 
 treated with mle ( 10 mg / kg , daily ) . 
 thirty days after the induction of periodontal 
 disease , the animals were euthanized and mandibles and gingival tissues removed 
 for further analysis . 
 resultsmorphometric analysis of alveolar bone loss demonstrated that mle - treated animals 
 presented a decreased alveolar bone loss and a lower expression of the activator 
 of nuclear factor-b ligand ( rankl ) measured by immunohistochemistry . 
 moreover , 
 gingival tissues from the mle - treated group showed decreased neutrophil migration 
 myeloperoxidase ( mpo ) assay . 
 conclusionsthese results indicate that mle may be useful to control bone resorption during 
 progression of experimental periodontitis in rats . 
INTRODUCTION
MATERIAL AND METHODS
Source of Mikania laevigata and preparation of the "guaco" extract
Animals
Experimental animal design
MPO activity assay
Histological and immunohistochemical analyses
Statistical analysis
RESULTS
Effect of MLE on furcation bone loss
Effect of MLE on neutrophil migration and RANKL expression
DISCUSSION
CONCLUSION
ACKNOWLEDGEMENTS AND FUNDING