brazil possesses a large biodiversity and has a wealth of traditional knowledge 
 accumulated by local people who have direct access to nature and its products . 
 traditional knowledge related to medicinal plants is the basis of folk medicine in 
 brazil , which is derived from a mixture of brazilian indigenous cultures and european 
 and african influences from the colonization period . myracrodruon urundeuva , popularly known as " aroeira do serto " , is a 
 brazilian medicinal plant traditionally used for treating gingival bleeding and 
 gynecological disorders . ethanolic extracts from myracrodruon urundeuva 
 stem bark exert anti - inflammatory activity in several experimental models of 
 inflammation , such as carrageenan - induced paw edema , neutrophil migration to peritoneal 
 cavity and hemorrhagic cystitis induced by cyclophosphamide . analgesic , 
 antiulcer , hepatoprotective , antidiarrheal and colonic anastomotic wound healing 
 properties have been reported . a chemical study has demonstrated that 
 the ethyl acetate extract has two main fractions . one of them has predominance of 
 substances of a chalcone nature and the other has predominance of catechic 
 tannins . the chalcone 
 demonstrated neuroprotective effects , which are able to reduce oxidative stress and 
 apoptotic injury . based on these findings , the aim of this study 
 was to evaluate the immediate and late biological tissue responses to ethanolic and 
 aqueous aroeira extracts implanted in rat submucosa . the study was approved by ethics committee on animal research of araatuba school of 
 dentistry / unesp ( process # 2007 - 008938 ) . the aroeira(myracrodruon urundeuva)plants were obtained from 
 ecological reserves at carolina city , maranho state , brazil . the leaves were 
 collected if presented with a healthy aspect , no signs of aging and free of disease 
 and pests . after collection , the plants were washed in water , dried at room 
 temperature and in a heater at 37c until becoming dry and friable . subsequently they 
 were pulverized . the ethanolic and aqueous extracts were prepared according to the methodology 
 described by tsuchiya , et al . the flask was agitated vigorously during 3 min , five times a day , during 12 
 days . the solution was filtered in paper filters and sterilized by filtration in 
 millipore membranes ( 0.22 m ) . the aqueous extract was obtained by mixing 20 g of 
 leaf powder with 250 ml of distilled water . the solution was maintained at 100c for 
 5 min , 55c for 1 h and at room temperature for 72 h. agitation was performed every 
 24 h. the extract was filtered and sterilized as mentioned before . eighteen 4 - 6 month - old male wistar rats weighting 250 - 280 g were divided into 6 
 groups of 3 animals each according to the experimental period of analysis ( 3 and 6 h ) 
 and treatment with the ethanolic / aqueous extracts or saline ( control ) . the animals 
 were anesthetized with xylazine ( 10 mg / kg ) and ketamine ( 25 mg / kg ) and received an 
 intravenous injection of 1% evan 's blue ( 0.2 ml/100 g body weight ) ( evan 's blue ; 
 difco lab , detroit , michigan , usa ) . after 30 min , 0.1 ml of the extracts or saline 
 were injected at the dorsum region , near the tail and using the median line as a 
 reference . after 3 and 6 h , the animals were sacrificed by anesthetic overdose and a 23 mm of 
 diameter tissue fragment containing a blue halo in the center was removed . the tissue 
 was fixed in 4 ml of formamide for 72 h at 45c and filtered for spectrophotometric 
 analysis at 630 nm . thirty 4 - 6 month - old male wistar albino rats weighting 250 - 280 g were divided into six 
 groups of 5 animals each according to the experimental period of analysis ( 7 and 28 
 days ) and treatment with the ethanolic / aqueous extracts or saline ( control ) . sixty polyethylene tubes ( 1.0 mm internal diameter x 1.6 mm outer diameter x 10 mm 
 length ) with a paper cone inside ( 2 mm short of the cone length ) had one side filled 
 with 1 mm of gutta - percha , and were sterilized with nitrous oxide . the shaved back received an incision in a head to tail orientation with a 
 # 15 blade . the skin was reflected creating two pockets : one on the right side and the 
 other on the left side . the tubes were implanted into the spaces created with blunt 
 dissection and the skin closed with a 4/0 silk suture . each animal received 2 implants with the same solution totalizing 10 implants 
 per experimental group . after 7 and 28 days after implantation 
 the animals were killed by anesthetic overdose and the tubes and surrounding tissue 
 were explanted , fixed in 10% formalin and processed and stained with hematoxylin 
 eosin . the quantitative analysis of inflammation was performed according to the number of 
 inflammatory cells present and divided in scores . absent = absence of inflammatory cells low=<25 inflammatory cells moderate=25 - 125 inflammatory cells severe=>125 inflammatory cells data from the edemogenic analysis were analyzed by two - way anova and tukey 's test 
 ( p<0.05 ) . data from the histological analysis were analyzed by the mann - whitney 
 and kruskal - wallis tests . the aroeira(myracrodruon urundeuva)plants were obtained from 
 ecological reserves at carolina city , maranho state , brazil . the leaves were 
 collected if presented with a healthy aspect , no signs of aging and free of disease 
 and pests . after collection , the plants were washed in water , dried at room 
 temperature and in a heater at 37c until becoming dry and friable . the ethanolic and aqueous extracts were prepared according to the methodology 
 described by tsuchiya , et al . the flask was agitated vigorously during 3 min , five times a day , during 12 
 days . the solution was filtered in paper filters and sterilized by filtration in 
 millipore membranes ( 0.22 m ) . the aqueous extract was obtained by mixing 20 g of 
 leaf powder with 250 ml of distilled water . the solution was maintained at 100c for 
 5 min , 55c for 1 h and at room temperature for 72 h. agitation was performed every 
 24 h. the extract was filtered and sterilized as mentioned before . eighteen 4 - 6 month - old male wistar rats weighting 250 - 280 g were divided into 6 
 groups of 3 animals each according to the experimental period of analysis ( 3 and 6 h ) 
 and treatment with the ethanolic / aqueous extracts or saline ( control ) . the animals 
 were anesthetized with xylazine ( 10 mg / kg ) and ketamine ( 25 mg / kg ) and received an 
 intravenous injection of 1% evan 's blue ( 0.2 ml/100 g body weight ) ( evan 's blue ; 
 difco lab , detroit , michigan , usa ) . after 30 min , 0.1 ml of the extracts or saline 
 were injected at the dorsum region , near the tail and using the median line as a 
 reference . after 3 and 6 h , the animals were sacrificed by anesthetic overdose and a 23 mm of 
 diameter tissue fragment containing a blue halo in the center was removed . the tissue 
 was fixed in 4 ml of formamide for 72 h at 45c and filtered for spectrophotometric 
 analysis at 630 nm . thirty 4 - 6 month - old male wistar albino rats weighting 250 - 280 g were divided into six 
 groups of 5 animals each according to the experimental period of analysis ( 7 and 28 
 days ) and treatment with the ethanolic / aqueous extracts or saline ( control ) . sixty polyethylene tubes ( 1.0 mm internal diameter x 1.6 mm outer diameter x 10 mm 
 length ) with a paper cone inside ( 2 mm short of the cone length ) had one side filled 
 with 1 mm of gutta - percha , and were sterilized with nitrous oxide . the shaved back received an incision in a head to tail orientation with a 
 # 15 blade . the skin was reflected creating two pockets : one on the right side and the 
 other on the left side . the tubes were implanted into the spaces created with blunt 
 dissection and the skin closed with a 4/0 silk suture . each animal received 2 implants with the same solution totalizing 10 implants 
 per experimental group . after 7 and 28 days after implantation 
 the animals were killed by anesthetic overdose and the tubes and surrounding tissue 
 were explanted , fixed in 10% formalin and processed and stained with hematoxylin 
 eosin . the quantitative analysis of inflammation was performed according to the number of 
 inflammatory cells present and divided in scores . absent = absence of inflammatory cells low=<25 inflammatory cells moderate=25 - 125 inflammatory cells severe=>125 inflammatory cells data from the edemogenic analysis were analyzed by two - way anova and tukey 's test 
 ( p<0.05 ) . data from the histological analysis were analyzed by the mann - whitney 
 and kruskal - wallis tests . the results for edema quantification showed no statistically significant differences 
 between the experiemntal periods ( p>0.05 ) . among the groups , the aqueous extract 
 and saline presented less edema than the ethanolic extract , which showed significant 
 differences from the other groups in both experimental periods ( p<0.05 ) ( figure 1 ) . mean values of edema on both time period and the experimental groups od = optical density ( * p<0.05 ) the histological analysis was performed after 7 and 28 days , according iso 
 10993 - 6 . the analysis of all 
 groups in relation to the experimental period showed a statistically significant 
 difference between the ethanolic extract and the other groups for the 28-day period 
 ( p<0.05 ) ( figure 2 ) . mean scores of inflammatory cells of the three groups at each time period 
 different letters = p<0.05 . same letters = p>0.05 at 7 days , moderate inflammatory infiltrate was present , probably due to surgical 
 trauma ( figures 3 - 5 ) . at 28 days , healing was in an advanced stage presenting lower 
 levels of inflammation when compared with 7 days for the three groups ( figures 3 - 5 ) . b - at 28 
 days , the fibrous tissue present well organized collagen fibers parallel to 
 implant . original 
 magnification=40x aroeira s ethanolic extract group . a  at 7 days , the granulation tissue is 
 thick and presents macrophages , lymphocytes and rare neutrophils . blood vessels 
 are distributed all over the tissue and the fibroblasts are near disorganized 
 collagen fibers at the periphery of the tissue . b - at 28 days , the connective 
 tissue is thin with rare inflammatory cells . fibroblasts are near collagen fibers at the central and periphery 
 regions of granulation tissue . original magnification 40x the ethanolic extract group showed persistency of inflammation with large amounts of 
 inflammatory cells up to 28 days , with no statistically significant differences 
 between 7 and 28 days ( p>0.05 ) ( figure 
 5 ) . the results for edema quantification showed no statistically significant differences 
 between the experiemntal periods ( p>0.05 ) . among the groups , the aqueous extract 
 and saline presented less edema than the ethanolic extract , which showed significant 
 differences from the other groups in both experimental periods ( p<0.05 ) ( figure 1 ) . mean values of edema on both time period and the experimental groups od = optical density ( * p<0.05 ) the histological analysis was performed after 7 and 28 days , according iso 
 10993 - 6 . the analysis of all 
 groups in relation to the experimental period showed a statistically significant 
 difference between the ethanolic extract and the other groups for the 28-day period 
 ( p<0.05 ) ( figure 2 ) . mean scores of inflammatory cells of the three groups at each time period 
 different letters = p<0.05 . same letters = p>0.05 at 7 days , moderate inflammatory infiltrate was present , probably due to surgical 
 trauma ( figures 3 - 5 ) . at 28 days , healing was in an advanced stage presenting lower 
 levels of inflammation when compared with 7 days for the three groups ( figures 3 - 5 ) . b - at 28 
 days , the fibrous tissue present well organized collagen fibers parallel to 
 implant . he  original 
 magnification=40x aroeira s ethanolic extract group . a  at 7 days , the granulation tissue is 
 thick and presents macrophages , lymphocytes and rare neutrophils . blood vessels 
 are distributed all over the tissue and the fibroblasts are near disorganized 
 collagen fibers at the periphery of the tissue . b - at 28 days , the connective 
 tissue is thin with rare inflammatory cells . fibroblasts are near collagen fibers at the central and periphery 
 regions of granulation tissue . original magnification 40x the ethanolic extract group showed persistency of inflammation with large amounts of 
 inflammatory cells up to 28 days , with no statistically significant differences 
 between 7 and 28 days ( p>0.05 ) ( figure 
 5 ) . approximately 85% of the world 's population depends on traditional medical treatments 
 based on herbal medicines , and approximately 25% of the world 's pharmaceutical medicines 
 are derived from plants . myracrodruon urundeuva , called aroeira in brazil , was chosen for this 
 study because of its antibacterial properties demonstrated in previous studies . the present study evaluated the 
 inflammatory tissue response to aqueous and ethanolic aroeira extracts . during the 
 studied period , wound healing was in an advanced stage , presenting diminished levels of 
 inflammation for aqueous and control group . however , the ethanolic extract group 
 demonstrated persistency of inflammation until up to the 28 day . this 
 result may be due to some component released during the preparation of the extract 
 rather than ethanol . although clinical studies revealed 
 an absence of significant toxic effects of the plant elixir , repetto , et al . the edemogenic analysis demonstrated slightly reduction of edema by the aqueous aroeira 
 extracts in relation to the control group , while the ethanolic extract group showed 
 significantly more edema . the experimental periods for histological analysis were based in previous studies , which 
 showed an unaltered tissue response after 30 days . the use of polyethylene tubes with a paper cone was based on 
 pullin and carvalho ( 1984 ) . the histological analysis also showed a significantly worse result for the ethanolic 
 extract in relation to persistency of inflammation up to 28 days . on the other hand , natural 
 resources conservative service ( usa , 
 2012 ) used the same methodology and found better results with ethanolic ara 
 extract . in the present study , the aqueous extract group presented healing evolution similar to 
 that of the saline group and at 28 days was slightly better despite not significant . the 
 moderate inflammatory infiltrate observed at 7 days may be due to surgical trauma rather 
 than a reaction to the extracts . in conclusion , the biological response the aroeira 's aqueous extract is biocompatible 
 and similar the response to saline . the ethanolic extract interfered with the wound 
 healing maintaining the inflammation up to advanced periods . this research is supported by coordenao de aperfeioamento de pessoal de nvel 
 superior ( capes ) . authors declare there are no financial / commercial conflicts of interest of this 
 study .
objectivesthis study evaluated subcutaneous tissue response to aroeira ( myracrodruon 
 urundeuva ) extract employing edemogenic and histological analyses . 
 material and methodstest groups consisted of aqueous and ethanolic aroeira extracts and saline 
 ( control ) . for edema quantification 
 , 18 rats received an intravenous injection of 
 evan 's blue . 
 after 30 min , the extracts and saline were injected on the dorsum of 
 the rats , which were then sacrificed after 3 and 6 h. readings were performed in a 
 spectrophotometer . for subcutaneous implantation , 
 30 rats received a polyethylene 
 tube containing the extracts on their dorsum and then they were killed after 7 and 
 28 days . 
 the samples were processed for histological analysis and evaluated with a 
 light microscope . 
 the inflammatory infiltrate was quantified . 
 resultsthere were no statistically significant differences between aqueous extract and 
 saline groups in relation to edema quantification in the different periods 
 ( p>0.05 ) . 
 ethanolic solution resulted in more edema independently of the 
 experimental period ( p<0.05 ) . 
 histological analysis showed similar results on 
 the 7-day period for the 3 groups . 
 there was a notable reduction on inflammatory 
 cell number for saline and aqueous extract groups at 28 days . 
 conclusionthe aqueous extract showed biocompatible properties similar to those of 
 saline .