mycoplasma pneumoniae is a member of the family mycoplasmataceae and order mycoplasmatales , the smallest self - replicating prokaryotes in both cellular dimensions and genome size [ 24 ] . among human mycoplasmas , mp is an important pathogen in acute respiratory illnesses in children , accounting for as many as 20% of all cases of community - acquired pneumonia every year [ 5 , 6 ] . infections generally lead to tracheobronchitis , bronchiolitis , and interstitial pneumonitis ; however 1015% of such infections progress to primary atypical pneumonia . recently , studies have shown that adherence of mp to respiratory tract by a terminal structure plays an important role in mp infection . this structure is a membrane - bound protein structure that plays a key role in adhesion , gliding motility movement , and cell division [ 7 , 8 ] . they are susceptible to antimicrobial agents that affect dna , rna , and protein synthesis . however , tetracyclines are not approved for use in children younger than 8 years of age due to toxicity to developing cartilage . recently , the use of new macrolides and fluoroquinolones such as roxithromycin , azithromycin , levofloxacin , and moxifloxacin with more favorable pharmacokinetic property , greater tolerability , and higher level of in vitro activity has been proposed for treatment of mycoplasma infections . unfortunately , studies have shown that treatment with antimicrobials can cause adverse consequences , such as toxicity , side effects , and drug resistance , which have been associated with clinical treatment failure [ 11 , 12 ] . recently , a study from shanghai , china , has reported that 39 of 50 mp isolates tested were macrolide resistant , indicating macrolide 's limited use . thus , there is a need to look for a novel clinical therapeutic that not only can against mp , but also can protect respiratory tract . qinbai qingfei concentrated pellet ( qqcp ) is a traditional chinese medicine compound preparation developed by research institute of traditional chinese medicine of heilongjiang province according to clinical experience , used to treat mycoplasma pneumonia for many years in china . the initial results of qqcp in treating mycoplasma pneumonia showed that this drug could effectively alleviate its symptoms . in this study , we authenticate protective effect of qqcp on airway smooth muscle cells from damage by mp . it comprises six drugs : scutellaria baicalensis georgi ( major chemical is baicalin ) , pheretima vulgaris chen ( hypoxanthine is the major active ingredient ) , stemona tuberosa lour . ( major constituents are tuberostemonine , stemonine , and croomine ) , aster tataricus l. f. ( major active ingredient is butyl - d - ribuloside ) , ophiopogon japonicus ( l. f. ) ker - gawl . the hplc system consisted of a lc-10as pump system ( shimadzu co. , tokyo , japan ) equipped with a shimadzu spd - m10avp detector , a shimadzu scl-10avp controller , and an sil-10advp autoinjector . the mobile phase was composed of methanol  :  water  :  glacial acetic acid ( 50  :  49  :  1 ) . a diamonsil - c18 column ( 4.6  mm  250  mm , 5  m , dikma , beijing , china ) was used . the flow rate was 1.0  ml / min with uv absorbance detection of 270  nm . the qqcp and rdt ( harbin sixth pharmaceutical factory , china ) were grinded and diluted into 2% and 0.3% ( 50  mg crude drug / ml and 3  mg / ml ) with deionized water , filtered through a 0.22  m millipore membrane filter , and stored at 4c . for test , they were diluted into eight working concentrations ( 250.195  mg / ml , 1.50.012  mg / ml ) in twofold serial dilutions with mm containing 5% fetal bovine serum . mpstrain was grown at 37c in pplo broth supplemented with yeast extract , glucose , penicillin , and 20% fetal calf serum and harvested at the turning point from red to yellow as indicated by the indicator system glucose - phenol red . dulbecco 's modified eagle 's medium ( dmem ) ( gibco , usa ) supplemented with 10% fetal bovine serum ( fbs ) was used as growth medium for culturing the cells ; for maintenance medium ( mm ) , the serum concentration was reduced to 5% and used for maintaining the cells . trypsin ( gibco , usa ) was dissolved with calcium and magnesium - free phosphate - buffered saline ( cmf - pbs , ph 7.4 ) into the concentration of 0.125% . mtt ( gibco , usa ) , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide , was dissolved with cmf - pbs ( ph 7.4 ) into 5  mg  ml . dmem and mm were stored at 4c , trypsin solution at 20c , and mtt solution at 4c in dark bottles . dimethyl sulfoxide ( dmso ) was purchased from chemical agent company of chinese medicine groups ( nanjing , china ) . male wistar rats ( body weight : 250300  g ) were purchased from experimental animal center of veterinary research institute of harbin ( harbin , china ) . all rats ( four rats per cage ) were fed with water and food ad libitum and were kept in the room at the controlled temperature ( 22  1c ) , humidity ( 5070% ) , and 12  h light / dark cycle in the animal center of the northeast agricultural university . animal welfare and experimental procedures were carried out in accordance with the guide for the care and use of laboratory animals ( national research council of usa , 1996 ) and related ethical regulations of our university . primary cultures of rat asm cells were prepared as described previously with modification . in brief , male wistar rat weighing 300  g was sacrificed under ether anesthesia and the trachea was excised . the trachea was opened longitudinally and the epithelium was removed by gentle rubbing with a sterile cotton - wool probe . the trachealis muscle was dissected free from the cartilage , washed twice in hank 's solution containing penicillin 100  iu  ml , and then minced with scalpel blades into ~1  mm pieces . muscle fragments were placed in culture dishes containing dmem supplemented with 10% fbs in humidified air containing 5% co2 at 37c . the culture medium was changed one week later and twice per week thereafter . upon reaching confluence , cells were passaged by dissociation with 0.125% trypsin and 0.02% edta , centrifugation at 1500  rpm for 5  min , and resuspension in culture medium . asm cells at concentration of 2  10 cfu ml were seeded in 96-well plates ( 100  l / hole ) at 37c in a humid atmosphere of 5% co2 . when asm cells grew into monolayer after cultivation for about 24  h , 100 tcid50 mp solution was added to the asm plate , 100  l per well . after incubation of 24  h at 37c in 5% co2 , cell supernatant and the third time cell - flushing fluid were centrifuged at 120 00  rpm for 20  min and the supernatant was removed ; then 20  l stet ( 100  mm nacl , 10  mm tris - hcl ph 8.0 , 1  mm edta ph 8.0 , and 5% triton x-100 ) was added to dna extraction . the dna was subjected to pcr . according to mp sequences described by bernet et al . , the primers for detection of mp ( mp-1 : 5-ttagcaggtaatggctagag-3 ; mp-2 : 5-ctcggttaacctcaattatg-3 ) were designed by primer premier 5.0 . the pcr mixture included 2.8  l of 10  pcr buffer , 200  mmol l of dntps , 0.5  mol l of each primer , 1 u of taq dna polymerase , and 8  l of dna solution . amplification conditions were pre - denaturation at 95c for 5  min , followed by denature at 95c for 40  s , annealing at 55c for 1  min , extension at 72c for 1  min , cycling 35 times , and last extension for 10  min . pcr was performed in a s1000tm thermal cycler ( bio - rad , ny , usa ) . for each pcr pure pcr products were analyzed by agarose gel electrophoresis ( 2% agarose ) and ethidium bromide staining . the safe concentrations of two drugs for asm cells were measured by the cytopathic effect ( cpe ) method . qqcp was diluted twofold serially from 50  mg  ml into 8 concentrations with mm , rdt from 3  mg  ml . asm cells were prepared and diluted into 2  10  cfu  ml with mm and inoculated into 96-well plates ( 100  l / hole ) at 37c in a humid atmosphere of 5% co2 for use . when asm cells grew into monolayer after cultivation for about 24  h , in two groups , qqcp and rdt at series of concentrations were added to the plates , respectively , four wells for each concentration . at the same time , cell control group ( only adding mm ) and blank group ( no cell ) were designed . after cultivation for 72  h at 37c in a humid atmosphere of 5% co2 , cells were observed under inverted microscope , the highest concentration of drug did not display cellular shrinkage , and detachment compared with cell control group was the largest safety concentration of drug . according to the result of cytotoxicity analysis , two drugs at two concentrations , qqcp from 3.125 to 1.563  mg  ml , rdt from 0.047 to 0.024  mg  ml , were used for determination of antimycoplasmal activity by mtt method . when asm cells were cultured into monolayer , the serial twofold dilutions of drugs and mp solution were added to cell plate , respectively , in four manners . drug solutions were added to asm cells plate firstly , 100  l per well and four wells per concentration . after incubation for 24  h at 37c in 5% co2 , the drug solutions were removed , the cell was washed twice with hank 's solution , and the mp solution was added . mp solution was added to asm cells plate firstly , 100  l per well . after incubation of 2  h at 37c in 5% co2 , the mp solution was removed , the cell was washed twice with hank 's solution , and the drug solutions were added , four wells for each concentration . the drug solutions at each concentration were mixed with mp solution , incubated for 2  h at 37c in 5% co2 , and then centrifuged at 50  000  rpm for 1  h and the supernatant was removed . the mp was washed twice , and then the mp solution without drug was added to asm cells plate , 100  l per well and four wells per concentration . mp solution was added to asm cells plate , then drug solutions were added immediately , 100  l per well and four wells per concentration . after incubation for 1  h at 4c , the supernatant was removed and the mm solution was added . at the same time , the cell control group ( only adding mm ) , the mp control group ( only adding mp ) , and blank group ( no cell ) were designed . when the cells displayed cellular shrinkage , and detachment in mp control group , 20  l mtt was added to each well and sequentially incubated for 4  h , the supernatant was removed , and 100  l of dmso was added to each well to dissolve the purple formazan crystals . finally , the absorbance at 570  nm ( a570 value ) of each well was measured by microliter enzyme - linked immunosorbent assay reader ( bio - rad , ny , usa ) . the mp inhibitory rate was calculated based on the following formula : mp inhibitory rate = ( a-570 value of drug group   a-570 value of mp control)/(a-570  value of cell control group -  a-570 value of mp control )  100% ( the  a-570  value was the average of four wells ) . the a570 values and mp inhibitory rate were considered as the indicator of antimycoplasmal activity . values were expressed as mean  sd . differences between groups were determined by analysis of variance ( anova ) using the sigma stat statistical software ( spss science , chicago , usa ) . after incubation of 3 d , spindle cells appeared around the muscle fragments . on the 7th day , cells reached confluence and displayed the typical  hill and valley asm cells grew into monolayer at long shuttle - type , identical shape and size and arranged closely ( figure 2 ) after cultivation for 3 d. the third time cell - flushing fluid and supernatant were collected after cultivation for 24  h and were subject to pcr analysis . mp was detected in supernatant , but not in the cell - flushing fluid ( figure 3 ) . the asm cells became round firstly , then distinct lesion with some cells collapsing and falling off ( figure 4 ) was observed after incubation for 7 d with mp ; the normal cells with clear cell structure arranged closely and each cell was shuttle - type . the cells at 6.25  mg  ml qqcp group and 0.094  mg  mlrdt group did not show collapsing and falling off compared with cell control group . the a570 values and mp inhibitory rates of every group were listed in table 1 . the a570 values of qqcp at 3.125 and 1.563  mg  ml groups were significantly higher than those of the corresponding mp control group ( p < 0.01 ) . the a570 values of rdt were not significantly higher than those of the corresponding mp control group ( p > 0.05 ) . qqcp ( 44.08% ) at 3.125  mg  ml and rdt ( 15.59% ) at 0.024  mg  ml group presented the highest mp inhibitory rate . the a570 values and mp inhibitory rates of every group were listed in table 2 . the a570 values of qqcp at 3.125 and 1.563  mg  ml group and rdt at 0.047 and 0.024  mg  ml were significantly higher than those of the corresponding mp control group ( p < 0.01 ) . qqcp ( 89.36% ) at 3.125  mg / ml and rdt ( 95.80% ) at 0.024  mg  ml group presented the highest mp inhibitory rate . the a570 values and mp inhibitory rates of every group were listed in table 3 . the a570 values of qqcp at 3.125 and 1.563  mg  ml group and rdt at 0.047 and 0.024  mg  ml were significantly higher than those of the corresponding mp control group ( p < 0.01 ) . qqcp ( 86.66% ) at 3.125  mg  ml and rdt ( 95.50% ) at 0.047  mg  ml group presented the highest mp inhibitory rate . the a570 values and mp inhibitory rates of every group were listed in table 4 . the a570 values of qqcp at 3.125 and 1.563  mg  ml group and rdt at 0.047 and 0.024  mg  ml were significantly higher than those of the corresponding mp control group ( p < 0.01 ) . qqcp ( 55.02% ) at 3.125  mg  ml and rdt ( 85.91% ) at 0.047  mg  ml group presented the highest mp inhibitory rate . to date , the pathogenesis of mycoplasma pneumonia has not clear yet ; the main pathogenesis tends to be adhesion and penetration of mp , direct injury to cells , and inflammation reaction . adhesion of mp to host cells is a prerequisite for colonization by the parasite and for infection . this close interaction between the mp and host cells protects it from removal by the host 's mucociliary clearance mechanism and allows it to produce local cytotoxin effects . because mp depends on close association with host cells to survive , this attachment organelle consists of a specialized tip structure with a central core of a dense rodlike central filament surrounded by a lucent space that is enveloped by an extension of the organism 's cell membrane . the tip structure is actually a network of adhesins , interactive proteins , and adherence accessory proteins that cooperate structurally and functionally to mobilize and concentrate adhesins at the tip of organism [ 18 , 19 ] . the p1 adhesin is a 169-kda protein concentrated in the attachment tip that is now known to be the major structure responsible for interaction of mp with host cells [ 8 , 20 ] . in addition to p1 , p30 , p116 , and hmw1 - 3 are believed to participate in the establishment of the polar structure [ 7 , 21 , 22 ] . in the present experiment , the third time cell - flushing fluid and supernatant were collected in the following day and were subject to pcr analysis . after cultivation for 7 d , cells became unclear in the structure , atrophy , round , and shedding , even scattered at end . these results showed that mp had strong adherence to asm cells , causing significant damages to the respiratory tract . the preadding drug mode is thought to exert the inhibitory action at a very early stage of mp infection cycle , that is , mp adsorption or penetration into the host cell . in agreement with this idea , our results showed that all only the a570 values of qqcp at 2 concentration groups were significantly higher than those of the corresponding mp control group , which indicated that qqcp could prevent mp infection . this effect may be due to blockade of drug in mp binding to cell receptor during adsorption , the first step of infection . in agreement with this idea , our results showed that all a570 values of two drug groups at 2 concentration groups were significantly higher than those of the corresponding mp control group , which indicated that they could treat mp infection . the simultaneous - adding after mp and drug mixed is thought to be the direct inactivation of mp . our results showed that all a570 values of two drug groups at 2 concentration groups were significantly higher than those of the corresponding mp control group . the simultaneous - adding mp and drug mode is thought to prevent sticking of mp to the host cell . it may affect the protein of tip structure , which plays a key role in adhesion . our results showed that all a570 values of two drug groups at 2 concentration groups were significantly higher than those of the corresponding mp control group . these results confirmed that both qqcp and rdt had significant activity in killing mp , treating mp infection , and antiabsorption . at the same time
aim . to study the effect and mechanism of traditional chinese medicine 
 qinbai qingfei concentrated pellet ( qqcp ) against mycoplasma pneumoniae ( mp ) . methods . 
 rat airway smooth muscle ( asm ) cells were used to examine the antimycoplasmal activity of qqcp via four drug - adding modes : pre- and postadding drugs , simultaneous - adding after drug and mp mixed , and simultaneous - adding drug and mp ; taking roxithromycin dispersive tablets ( rdt ) as positive control , the cellular a570 values were determined by mtt method . 
 results . 
 all of a570 values in qqcp group were significantly higher than those of the corresponding mp control group ( p < 0.01 ) in four drug - adding modes ; there was no significant difference in a570 values between the qqcp group and that of the positive control group ( p > 0.05 ) , confirming that qqcp could significantly inhibit the infectivity of mp to asm cells . 
 conclusion . 
 qqcp had significant activity in preventing and treating mp infection , killing mp , and antiabsorption .