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 .