they act via mechanisms classified as central or peripheral ; though some act through both pathways
. the antitussive effect of an ethanolic seed extract of picralima nitida ( pne ) has previously been established by the authors .
however , the mode of activity of cough medicines could be attributed to at least five reasons : pharmacological , physiological , true placebo , psychological and non - specific action .
plant / plant products exhibiting antitussive activity may not have one mode of expressing this pharmacological effect.3 , 4 the purport of this study was to establish the possible mode of antitussive and expectorant activity of an ethanolic seed extract of p. nitida .
this will be ascertained by ; evaluating muco - suppression effect , assessing mast cell stabilization effect , and establishing antibacterial , anxiolytic , antioxidant activity in various rodent models .
the pods of p.  nitida were collected from the knust botanical garden in kumasi , ghana , in february , 2013 .
the pods were opened , the seeds removed , air - dried , and milled into powder .
the powder weighing four ( 4 ) kg was extracted by cold maceration with 70% ethanol over a period of 72  h. the resulting extract was then concentrated at a temperature of 40  c and under low pressure to a syrupy mass in a rotary evaporator ( rotavapor r-210 , buchi , switzerland ) .
the syrupy mass obtained was then dried in a hot air oven ( gallenkamp , uk ) maintained at 40  c to obtain 0.532  kg ( % yield : 13.3% ) of a solid mass of p. nitida extract ( pne ) .
macau ) ; ammonium chloride ( philip harris , hyde - cheshire ; uk ) ; phenol red and sodium chloride ( bdh chemicals ltd , poole , england ) ; ketotifen fumarate ( novartis pharma ag , basle , switzerland ) ; compound 48/80 and toluidine blue ( sigma chemical co. , st .
louis , mo , usa ) ; sodium hydroxide ( avondale , england ) ; acetic acid , diazepam ( sigma  aldrich inc . , st .
louis , mo , usa ) , caffeine ( sigma  aldrich inc . , st .
balb / c mice ( 2030  g ) and a sprague  dawley rat ( 130  g ) , obtained from the animal house of the department of pharmacology , knust , kumasi , ghana were used in this study .
they were fed on standard rodent pellet diet ( agricare ltd , tanoso , kumasi , ghana ) and water ad libitum .
the animals were kept in the experimental area of the departmental animal house at ambient conditions of light , temperature and humidity for seven ( 7 ) days prior to experimentation and during experimentation .
the muco - suppressant effect of pne was determined using the ammonium chloride - induced tracheal phenol red secretion model ; described previously .
v , ( n  =  5 ) and pre - treated , for 30  min , as follows : group i , two ( 2 ) ml / kg normal saline ( p.o ) ; group ii , 100  mg / kg sodium cromoglycate ( i.p ) ; groups iii  v , 100 , 300 , and 500  mg / kg of pne ( p.o ) respectively .
tracheal mucus secretion was then induced with five ( 5 ) mg / kg ammonium chloride per os .
animals were then injected with 500  mg / kg phenol red , intraperitoneally , 30  min later .
the trachea was excised from each mouse and cleared of adhering tissues , after sacrificing it by cervical dislocation ( 30  min after the phenol red injection ) .
each excised trachea was washed in three ( 3 ) ml physiological saline ; sodium hydroxide ( 0.3  ml naoh , one ( 1 ) m ) was then added to the washing to stabilize the ph of the lavage fluid .
the absorbance of the mixture was then taken at a wavelength of 460  nm using a spectrophotometer ( t90  +  uv / vis spectrometer  pg instruments ltd ) . a calibration curve for phenol red
was made ; from which concentrations of phenol red secreted by mice tracheae were extrapolated .
mast cell stabilizing effect was ascertained using compound 48/80-induced mesenteric mast cell degranulation as described previously .
dawley rat intestinal mesenteries was excised into several pieces and put into five groups , g1g5 ( n  =  5 ) , in petri dishes containing tyrode solution .
the mesenteries were then subjected to the following treatment : g1 , normal saline ; g2 , 20  g / ml ketotifen fumarate ; g3g5 , 100 , 300 , and 500  g / ml pne respectively .
the petri dishes were then incubated at 37  c for 15  min after which one ( 1 ) ml of compound 48/80 solution ( 10  g / ml ) was added and incubated at 37  c for ten ( 10 ) min .
the mesenteric pieces were then fixed in 10 % buffered formalin and processed in xylene and acetone .
they were then stained with 0.1 % toluidine blue and observed under a leica dm 750 microscope ( leica microsystems cm5 gmbh , wetzlar  germany ) for both intact and degranulated cells .
ten ( 10 ) test tubes ( labeled t1t10 ) each containing 20  ml nutrient agar were stabilized .
the molten agars were inoculated with 0.2  ml each of the following organisms : t1 and t2 , staphylococcus  aureus ; t3 and t4 , streptococcus pneumonia ; t5 and t6 , salmonella typhi ; t7 and t8 , escherichia coli ; t9 and t10 , klebsiella pneumonia .
the seeded agars were poured into labeled sterile petri dishes ( p1p10 ) and allowed to set . using a sterile cork borer number seven ( 7 ) ,
various concentrations of pne were prepared and poured in the wells to three - quarter ( 3/4 ) full as follows : p1 , p3 , p5 , p7 and p9 each received 0.05 , 0.5 , 5 , 25  mg / ml pne , while p2 , p4 , p6 , p8 and p10 received 0.1 , 1 , 10 , 50  mg / ml pne , in four of the five wells created .
the petri dishes were covered and left on the bench for 45  min in order to allow effective diffusion of the extract .
they were then incubated at 37  c for 24  h , after which they were examined for zones of growth inhibition around the wells .
amoxicillin ( 1 % ) was used as a positive control in the test against s. aureus , s. pneumonia , e. coli and k. pneumonia .
the apparatus was made of plexiglas and consisted of two open arms and two closed arms of dimensions 30  cm    5  cm    15  cm .
these arms extend from a central square platform ( 5  cm    5  cm ) .
the maze was elevated to a height of 60  cm above the floor and placed in a lit room .
mice were divided into ten groups ( n  =  6 ) and received the following treatment : vehicle - control , pne ( 100 , 300 , 500  mg / kg ) , diazepam ( 0.1 , 0.3 , 1.0  mg / kg ) , and caffeine ( 3 , 10 , 30  mg / kg ) .
the vehicle , pne , caffeine were orally administered to their respective animals ' an hour before the experiment , whiles diazepam was given intraperitoneally 30  min before the experiment . at the start of the experiment ,
animals were individually placed at the center of the maze , facing one of the enclosed arms and their behavior videotaped for 5  min with a digital camera placed 100  cm above the maze .
behavioral parameters were scored from the videotapes as follows:1)number of closed and open arm entries(absolute value and percentage of the total number);2)time spent in exploring the open and closed arms of the maze  absolute time and percentage of the total time of testing3)number of head - dips ( absolute value and percentage of the total number)protruding the head over the ledge of either an open ( unprotected ) or closed ( protected ) arm and down toward the floor;4)number of stretch - attend postures ( absolute value and percentage of the total number)the mouse stretches forward and retracts to original position from a closed ( protected ) or an open ( unprotected ) arm .
number of closed and open arm entries(absolute value and percentage of the total number ) ; time spent in exploring the open and closed arms of the maze  absolute time and percentage of the total time of testing number of head - dips ( absolute value and percentage of the total number)protruding the head over the ledge of either an open ( unprotected ) or closed ( protected ) arm and down toward the floor ; number of stretch - attend postures ( absolute value and percentage of the total number)the mouse stretches forward and retracts to original position from a closed ( protected ) or an open ( unprotected ) arm .
the behavior was tracked by jwatcher version 1.0 ( university of california , los angeles , usa and macquarie university , sidney , australia ; available at http://www.jwatcher.ucla.edu/. ) the open field method has previously been described .
the test was conducted in clear plexiglas boxes ( 40  cm    40  cm    30  cm ) .
the floor of this box was divided by red lines into 16 equal squares . for behavioral analysis ,
the arena of the open field was designated as;1)corner ( one of the four corner squares);2)periphery ( the squares along the walls ) ; or3)center ( the four inner squares ) .
corner ( one of the four corner squares ) ; periphery ( the squares along the walls ) ; or center ( the four inner squares ) .
the animals were divided into ten groups ( n  =  6 ) , and received either the extracts ( 100 , 300 , or 500  mg / kg , p.o . ) , the vehicle or the reference drug diazepam ( 0.1 , 0.3 or 1  mg / kg , i.p . ) .
thirty minutes after i.p and 1  h after oral administration , the animals were placed at the center of the open field and were allowed to explore for 5  min .
this was recorded by a video camera which was suspended 100  cm above the arena .
behavioral parameters for all the tests were scored from videotapes with the aid of the public domain software jwatcher version 1.0 .
the numbers of entries , as well as the duration of stay in individual zones were assessed . the antioxidant property of pne ( 0.130  mg / ml ) was evaluated in 2 , 2-diphenyl-1-picrylhydrazyl hydrate ( dpph ) free radical scavenging , linoleic acid lipid peroxidation , reducing power , and total antioxidant assays by standard procedures previously described by amoateng et  al . , 2011 .
statistical analyses were done by one - way analysis of variance ( anova ) with dunnett 's multiple comparison test ( post hoc test ) using graph - pad prism for windows version 6.0 ( graph - pad software , san diego , ca , usa ) .
some aspects of the open field and elevated plus maze were done with two - way analysis of variance followed by bonferroni 's post - hoc test .
differences between means of treated groups and the control were regarded as statistically significant at p    0.05 .
sodium cromoglycate , and pne ( 100300  mg / kg ) significantly ( p    0.050.001 ) reduced tracheal phenol red secretion compared to the control ( fig . 
treatments with ketotifen fumarate , and pne ( 100500  mg / kg ) were also able to reduce significantly ( p    0.050.001 ) inhibit mast cell degranulation induced by compound 48/80 ( fig . 
1b ) . significant ( p    0.050.0001 ) antibacterial activity was observed e. coli , s. typhi , k. pneumonia , s. pneumonia , and s. aureus from 1050  mg / ml . the lowest effect was against s. typhi at 5  mg / ml with a zone of 13.0    0.00  mm whiles the highest response was observed against s. pneumonia at 50  mg / ml with a zone of 22.3    0.88  mm ( table  1 ) .
pne ( 100 , 300 , 500  mg / kg ) enhanced , dose - independently , activities in the open arm by increasing percentage of entry into open arms ( p    0.0001 ; fig . 
2f ) and percentage time spent in open arms ( p  =  0.1278 ; fig .  2c ) .
there was also reduction in risk assessment by decreasing both the percentage protected head dips ( p  =  0.0030 ; fig . 
3c ) and percentage protected stretch attend postures ( p  =  0.0860 ; fig . 
diazepam ( 0.11  mg / kg ) also dose dependently and significantly increased percentage of open arm entries ( p  =  0.0005 ; fig . 
2e ) and percentage time spent in open arm ( p  =  0.0004 ; fig . 
diazepam also reduced risk assessment by decreasing both the percentage protected head dips ( p  =  0.0002 ; fig . 
3b ) and percentage protected stretch attend postures ( p  =  0.0192 ; fig . 
these effects confirmed the anxiolytic effects of both pne and diazepam . with regard to caffeine , it increased open arm avoidance by reducing percentage of open arm entries ( p  =  0.8997 ; fig . 
2d ) and percentage time spent in open arms ( p  =  0.0285 ; fig . 
also , caffeine increased both the percentage protected head dips ( p  =  0.7205 ; fig .  3a ) and percentage protected stretch attend postures ( p  =  0.0247 ; fig . 
pne ( 100 , 300 , 500  mg / kg ) dose - independently increased the percentage entry into central zone ( p  =  0.0201 ; fig . 
4d ) and percentage time spent in central zones ( p  =  0.0427 ; fig . 
the reference anxiolytic , diazepam ( 0.11  mg / kg ) dose dependently increased the percentage entry into central zone ( p  =  0.0023 ; fig . 
4c ) and percentage time spent in central zones ( p  =  0.0603 ; fig . 
the reference antioxidant , n - propyl gallate ( 0.0010.1  mg / ml ) , and pne ( 0.130  mg / ml ) exhibited dpph free radical scavenging effect ( fig . 
5a ) , and showed concentration dependent ability to inhibit the autoxidation of linoleic acid lipid peroxidation ( fig . 
5b ) . pne and n - propyl gallate also dose dependently reduced fe to fe in the reducing power assay ( fig . 
pne ( 0.130  mg / ml ) showed a concentration dependent increase in total antioxidant capacity expressed as ascorbic acid equivalent ( aae ) ( fig . 
ec50 values for the reference antioxidants and pne are as shown in table  2 .
this study carried out on pne to determine muco - suppressant , antibacterial , mast cell stabilizing , anxiolytic , and antioxidant properties in a bid to establish its mode of activity in the management of cough evolved from previous work on pne , by the authors , which established antitussive , and expectorant properties of the extract .
tracheal mucus secretion , mast cell degranulation , bacterial infection of the airways , psychogenic factors ( e.g. anxiety ) and oxidative stress could culminate into cough .
sodium cromoglycate and pne significantly reduced tracheal phenol red secretion after ammonium chloride induction , indicating muco - suppressant activity .
sodium cromoglycate is known to exhibit muco - suppressant effect by prevent the release of inflammatory mediators resulting from chronic inflammation of the airways .
the importance of this result lies in the fact that mucus in the airways mechanically stimulates and increases rapidly the activity of rapidly adapting receptor ( rar ) ; a cough sensor .
the presence of excess mucus moves the cough sensor response up the stimulus / response curve and gives the impression of sensitization . by pne 's ability to inhibit mucus secretion from the airway , stimulation of rar
compound 48/80-induced mast cell degranulation is used to test mast cell stabilizing effects of potential therapeutic agents .
ketotifen stabilizes mast cells by inhibiting the release of histamine , serotonin and other inflammatory mediators
. mast cell stabilization could contribute to antitussive effect in that leukotriene and histamine ( which can stimulate c - fibers ) are not release .
apart from such direct effect , leukotrienes and histamine can induce mucus secretion and bronchoconstriction which can in turn stimulate rar receptors .
mast cell stabilization also reduces chronic inflammation of the airway which can lead to hypertrophy and hyperplasia of surface goblet ( mucous ) cells and sub - mucosal glands , which consequently increases mucus production .
pne demonstrated antibacterial activity against two gram positive and three gram negative bacteria , indicating a possible remedy for cough arising from bacterial infection of the airway .
increase in cough sensitivity seems to be the most likely reason why infection contributes to cough .
treatment and/or prevention of airway infection could be an important way of reducing inflammation and hyper - secretion and hence cough sensitivity .
it is worth noting that there could be a potential reduction in c - fiber stimulation as a result of anti - bacterial effect .
bacterial infection is known to reduce the level of endopeptisase especially substance p which gets released from c - fiber .
considering the fact that substance p can augment c - fiber activity and that of the rar through mucus secretion and bronchospasm it induces suppressing bacterial proliferation will enhance the level of endopeptidase , which in turn will reduce substance p mediated events .
anxiety in particular has been found to be an independent risk factor for both productive and non - productive cough .
this may reflect a somatic manifestation of a wide range of psychological problems , an increased awareness of physical symptoms in anxious individuals as well as difficulty in maintaining voluntarily cough suppression.23 , 24 therapeutically , therefore , anxiolytics may help to relieve cough .
psychogenic cough is generally regarded as a respiratory tic and may be precipitated by a variety of emotional stimuli . in an attempt to establish the fact that pne could be useful in managing anxiety - related cough ,
its anxiolytic effect was established using the elevated plus maze and the open field tests .
the open - field test examines anxiety - related behavior characterized by avoidance of an open , brightly lit area by rodents .
animals placed in a novel environment express anxiety and fear , by showing alteration in certain parameters , such as decreases in ambulation and exploration time in the center of the open field with an attendant increased peripheral movement .
the elevated plus maze utilizes the fear of a novel , brightly lit open space and fear of balancing on a relatively narrow , raised platform .
generally , anxiolytics increase the number of entries into and the time spent in the open arms of the epm .
the fact that pne reduced the percentage protected head dips and stretch attend posture in the epm also confirm its ability to reduce fear related behavior .
oxidative stress contributes to cough in that reactive oxygen species ( ros ) have the potential of stimulating c - fibers . in testing the antioxidant effect , dpph , 2 , 2-diphenyl-1-picrylhydrazyl hydrate a stable radical having a characteristic violet color ( and maximum absorption at 517  nm ) and which accepts an electron or hydrogen in the presence of a suitable free radical scavenger ( reducing agent ) was used . from the ic50 values
oxidants can cause damage by interacting with anti - proteases or other processes leading to the development of chronic lung damage .
intake of fruit and vegetable , which are major sources of antioxidants , have been associated with higher lung function and reduced symptoms of cough with phlegm .
it is to be noted that the mucolytic effect of acetylcysteine have been attributed to their antioxidant activities and that gives an indication of the fact that pne may also have mucolytic activity .
there seems to be notable similarities between mucolytics and expectorants : mucolytics just like expectorants have been found to be effective in productive cough34 , 35 also the model used in testing expectorant effects i.e. the tracheal phenol red secretion has been used in ascertaining mucolytic activity of various extracts . the antioxidant effect established in this work
can then be said to contribute to the extracts ability to manage productive cough ; a property which is typical of expectorants .
however this antioxidant effect could also account for pne 's antitussive activity since reactive oxygen species ( ros ) are known activators of c - fibers .
the mast cell stabilization , muco - suppressant , and antioxidant effects were the most prominent effect of pne even though it shows significant antimicrobial and anxiolytic activity .
preliminary phytochemical screening of pne indicated the presence of alkaloids , tannins , steroids , glycosides , anthraquinones , and terpenoids some of which would have contributed to its properties.37 , 38 , 39 a review of the medicinal uses , phytochemistry and pharmacology of p. nitida has indicated that its alkaloids ( especially from the seed ) have significant antimicrobial , analgesic , and anti - inflammatory properties while its coumestan glycosides have antimicrobial activity ; these which could make it useful as antitussive .
alkaloids , phenolic compounds ( tannins ) , glycosides , saponins , sterols , and terpenoids has been reported to have mast cell stabilizing effect.42 , 43 , 44 it is possible that the anxiolytic action could be mediated by synergistic action of these phytochemicals as seen in several studies
. the lower dose of hie ( i.e. 100  mg / kg ) seemed to be more effective ; giving more consistent results than the higher doses ( i.e. 300 and 500  mg / kg ) .
this may not be surprising since the extract may have several other components , some of which may have antagonist effects to the agonist effect being elicited ; but would only be of significance at higher doses i.e. proportional to the dose administered .
the ethanolic seed extract of p. nitida has demonstrated very significant mast cell stabilizing , mucus suppressant and antioxidant activity as well as substantial antibacterial and anxiolytic properties ; all of which could contribute to its antitussive and expectorant property .
 it has been established that picralima nitida has antitussive effect . 
 this study therefore aimed at determining the possible mode of antitussive and expectorant activity of an ethanolic seed extract of p. nitida ( pne ) . 
 the muco - suppressant , mast cell stabilization , and the anxiolytic effects of pne were ascertained using ammonium chloride - induced phenol red secretion in balb / c mice ; compound 48/80-induced mesenteric mast cell degranulation assay ; and the open field and the elevated plus maze models respectively . 
 antibacterial potential was ascertained by the agar plate diffusion method and its antioxidant potential by the 2,2-diphenyl-1-picrylhydrazyl hydrate ( dpph ) free radical scavenging , linoleic acid lipid peroxidation , reducing power , and total antioxidant assays . 
 data obtained was analyzed using one - way analysis of variance ( anova ) with dunnett 's multiple comparison post hoc test . 
 pne ( 100500  mg / kg ) reduced ( p    0.050.001 ) tracheal phenol red secretion . 
 the extract ( 100500  g / ml ) also dose - dependently ( p    0.050.0001 ) stabilized mast cells . 
 pne ( 100500  mg / kg ) increased open arm activities in the elevated plus maze ( p    0.05 ) as well as central zone exploration ( p    0.05 ) in the open field test . 
 pne ( 1050  mg / ml ) showed activity against staphylococcus aureus , streptococcus pneumonia , escherichia coli , klebsiella pneumonia , and salmonella typhi . by the assays 
 , pne showed significant antioxidant effect . 
 the ethanolic seed extract of p. nitida has demonstrated very significant mast cell stabilizing , mucus suppressant , and antioxidant activity as well as substantial antibacterial and anxiolytic properties ; all of which could contribute to its antitussive and expectorant property . 
Introduction
Materials and methods
Results
Discusssion
Conclusion
Conflict of interest