mycobacterium tuberculosis is the world 's largest cause of death from a single microorganism after human immunodeficiency virus ( hiv ) .
globally , there are almost nine million new cases of tuberculosis ( tb ) each year , two million of which results in death .
it is estimated that about 1/3 of the current global population is infected asymptomatically with m. tuberculosis of whom 5%10% will develop clinical disease during their lifetime and approximately two million deaths are attributable annually .
tb continues to be a leading cause of mortality in spite of the availability of an effective chemotherapeutic regimen .
chemotherapy of tb consists of three or four drug regimen , administered for 69 months .
the long duration of therapy results in poor compliance , produces unwanted side effects and therapeutic failure leading to emergence of multidrug resistant strains of m. tuberculosis.[46 ] association with noncompliance , hiv , and increasing multidrug resistant tuberculosis ( mdr - tb ) appears to be a serious issue , especially for the developing nations . extensive drug resistant tuberculosis ( xdr - tb ) spreading all over the world
is defined as a resistance to all first - line drugs and to fluoroquinolones and all of the injectables .
the xdr - tb strain is mixing with the acquired immuno deficiency syndrome ( aids ) , causing nearly100% mortality .
development and evaluation of new chemical entity ( nce ) against tb is a challenging task .
8-[(4-chloro phenyl ) sulfonyl]-7-hydroxy-4-methyl-2h - chromen-2-one ( cshmc ) used in the present study is derivative of coumarin [ figure 1a ] and heterocyclic analogue of dapsone ( diaminodiphenyl sulfone [ dads ] ) .
coumarin has a wide range of pharmacological properties , such as anticoagulant , antibacterial , diuretic , vasodilator and antiallergic .
several coumarin substitutions have shown antitubercular activity.[1214 ] dapsone [ figure 1b ] was found to be effective in suppressing experimental infections with tubercle bacilli.[1518 ] minimum inhibitory concentration ( mic ) of dapsone against m. tuberculosis , m. avium complex ( mac ) , and m. leprae are 50250 mg / l , 2100 mg / l , and 1.54 mg / l , respectively.[1921 ]
metabolism of dapsone by hepatic oxidation to hydroxylamine and its subsequent reduction to amine within red cells leads to persistence of drugs in red cells and which is suspected for its hematological toxicity .
an attempt was made to reduce its toxicity by replacing free amino ( nh2 ) group with chloro group . due to antibacterial activity against a variety of microorganisms by sulfones ,
it has been shown to have an antitubercular activity in vitro on culture of sensitive strain of m. tuberculosis in lowenstein 
the present study was planned to evaluate antitubercular efficacy and safety of cshmc in isoniazid and rifampicin - sensitive strain in guinea pigs .
if infected with m. tuberculosis , they show striking similarities to natural infections in humans , thus making it a good model for testing the effects of drug therapy.[2325 ] chemical structure of ( a ) coumarin ; ( b ) dapsone ( diaminodiphenyl sulfone [ dads ] ) ; ( c ) 8-[(4-chloro phenyl ) sulfonyl]-7-hydroxy-4-methyl-2h - chromen-2-one
05/2007 ) was approved by the institutional animal ethics committee , government medical college , bhavnagar , gujarat , india .
isoniazid ( alfa aesar , a johnson mathey company , lancaster ) , rifampicin ( tokyo chemical industry co. ltd . ,
japan ) , purified protein derivative ( ppd rt 23 tween 80 ; radiant parenterals ltd . ,
cshmc was a kind free gift sample from the department of chemistry , bhavnagar university , bhavnagar , india .
m . tuberculosis h37rv in middle brook 7h9 broth media was obtained from state tuberculosis demonstration centre ( stdc ) , ahmedabad , gujarat , india , as a free gift sample .
hartley guinea pigs ( 250300 g ) and swiss albino mice ( 2030 g ) of both genders were obtained from central animal house of the institute .
male and female guinea pigs were housed in separate stainless steel cages and mice in polypropylene cages under standard condition ; temperature - controlled room ( 24c  2c ) with 12 h light : dark cycles and were given standard laboratory diet and water ad libitum .
the nonpregnant , nulliparous female albino mice of 812 weeks old and 2025 g body weight were selected to find out the acute oral toxicity of the cshmc . the mice were fasted for 3 h prior to the experiment and were administered with cshmc in the range of doses 300 , 1000 , and 2000 mg / kg and observed for mortality up to 14 days according to oecd guidelines .
equivalent dose for guinea pig ( 200 mg / kg ) was calculated as per ghosh .
all the guinea pigs were tested with mantoux test using 0.1 ml of purified protein derivatives ( ppd rt 23 tween 80 ) injected intradermally into the lower left side of abdomen .
all the guinea pigs examined ( up to 72 h ) were mantoux negative and hence used for experiment .
antitubercular study was carried out on guinea pigs and they were divided into 6 groups of either gender with 6 in each group .
cultures of m. tuberculosis h37rv were harvested from a 2-week culture in middle brooke 7h9 broth media .
all the guinea pigs were infected with 0.1 ml ( 1.5  10 cfu / guinea pig ) suspension via intramuscular route in the left thigh muscle .
have produced a tuberculosis infection in guinea pigs via intramuscular route.[2931 ] this suspension was matched with mcfarland standard .
after 30 days , infections were confirmed in 6 guinea pigs by histopathology of spleen , lung , and liver .
vi ) by random allocation software version 1.0 , may 2004 , by simple randomization . drugs and cshmc were administered by mouth once a day for 30 days in groups iii  vi .
human equivalent dose of isoniazid ( 30 mg / kg ) and rifampicin ( 60 mg / kg ) was calculated by extrapolation as mentioned by ghosh .
took a human - equipotent dose of isoniazid and rifampicin in guinea pigs as 30 and 50 mg / kg , respectively .
group ii received vehicle ( 10% ethanol ) by mouth once a day for 30 days .
group i : normal control group : did not receive the injection of m. tuberculosis h37rv and received vehicle ( 10% ethanol ) by mouth once a day for 30 days .
group ii : disease control group : received vehicle by mouth once a day for 30 days .
group iii : standard control group : received isoniazid ( 30 mg / kg ) by mouth once a day for 30 days .
group iv : standard control group : received rifampicin ( 60 mg / kg ) by mouth once a day for 30 days .
group v : test compound group : received cshmc ( 20 mg / kg ) by mouth once a day for 30 days .
group vi : test compound group : received cshmc ( 5 mg / kg ) by mouth once a day 30 for days .
blood samples were collected in ethylene diamino tetra acetic acid ( edta ) and plain bulb from the carotid artery under pentobarbitone anesthesia intraperitoneally ( 30 mg / kg ) 24 h after the last dose of drug .
edta sample was used for estimation of hemoglobin ( hb ) , total white blood cell ( wbc ) count , total red blood cell ( rbc ) count , total platelet count , differential count ( polymorphs , lymphocytes , eosinophils , monocytes ) , blood indices ( packed cell volume , mean corpuscle volume , mean corpuscle hemoglobin , mean corpuscle hemoglobin concentration , red cell distribution width , and erythrocyte sedimentation rate ) . complete blood count was analyzed by using celltac alpha cell counter ( nihontohdem , japan ) .
serum was separated for estimation of biochemical parameters : serum glutamic oxaloacetic transaminase ( sgot ) , serum glutamic pyruvic transaminase ( sgpt ) , alkaline phosphatase ( alp ) by enzyme kinetic method , bilirubin by jendrassik  grof method ( total , direct , and indirect ) and serum creatinine by jaff creatinine method in a fully automated analyzer .
their gross appearance was noted and they were placed in 10% formalin for 24 h. then 5 mm thick pieces of the lung , spleen , and liver were embedded in paraffin , cut into 5 mm thick sections , stained using hematoxylin - eosin dye ( h and e ) , and finally mounted in dibutyl diesterate parathylate xylene .
each slide was coded and observer masked to evaluate histopathological changes in the lung , spleen , and liver .
the guinea pig lungs were scored based on the following three criteria : ( 1 ) primary lesion ( epithelioid cells and other inflammatory cells ) : 0 , no primary lesions present ; 1 , single primary lesion ; 2 , two or more primary lesions ; multifocal ; 3 , two or more primary lesions ; multifocal to coalescing ; 4 , multiple primary lesions , coalescing , and extensive .
( 2 ) secondary lesion ( granuloma formation , giant cells , and caseous necrosis ) : 0 , no secondary lesion present ; 1 , up to 25% of lung involved ; 2 , up to 50% of lung involved ; 3 , up to 75% of lung involved ; 4 , above 75% of lung involved . ( 3 ) pneumonitis ( alveolar wall thickness , interstitial , and peribronchial inflammation ) : 0 , alterations in less than 10% of the fields ; 1 , alterations in 10%30% of the fields ; 2 , alterations in 30%50% of the fields ; 3 , alterations in 50%70% of the fields ; 4 , alterations in more than 70% of the fields are scored based on severity as follows : 0 , none ; 1 , minimal ; 2 , mild ; 3 , moderate ; 4 , marked ; and 5 , severe .
the spleen was scored based on two criteria ( same scales as lung ) ; ( 1 ) primary lesion and ( 2 ) secondary lesion .
the liver was scored based on one criterion ; ballooning degeneration : 0 , no ballooning degeneration ; 1 , minimal enlargement in few hepatocytes ; 2 , mild enlargement in many hepatocytes ; 3 , moderate enlargement in most hepatocytes ; 4 , severe enlargement in most hepatocytes .
the nonparametric kruskal  wallis test followed by dunnett 's test was used to assess statistical significance between total lesion score of lung , spleen , and liver .
the hematological , liver function , and renal function parameters are expressed as mean  standard error of mean ( sem ) .
statistical differences between means were determined by one - way analysis of variance ( anova ) followed by dunnett 's multiple comparison tests using graphpad prism software demo version .
isoniazid ( alfa aesar , a johnson mathey company , lancaster ) , rifampicin ( tokyo chemical industry co. ltd . ,
japan ) , purified protein derivative ( ppd rt 23 tween 80 ; radiant parenterals ltd . ,
cshmc was a kind free gift sample from the department of chemistry , bhavnagar university , bhavnagar , india .
m . tuberculosis h37rv in middle brook 7h9 broth media was obtained from state tuberculosis demonstration centre ( stdc ) , ahmedabad , gujarat , india , as a free gift sample .
hartley guinea pigs ( 250300 g ) and swiss albino mice ( 2030 g ) of both genders were obtained from central animal house of the institute .
male and female guinea pigs were housed in separate stainless steel cages and mice in polypropylene cages under standard condition ; temperature - controlled room ( 24c  2c ) with 12 h light : dark cycles and were given standard laboratory diet and water ad libitum .
the nonpregnant , nulliparous female albino mice of 812 weeks old and 2025 g body weight were selected to find out the acute oral toxicity of the cshmc . the mice were fasted for 3 h prior to the experiment and were administered with cshmc in the range of doses 300 , 1000 , and 2000 mg / kg and observed for mortality up to 14 days according to oecd guidelines .
equivalent dose for guinea pig ( 200 mg / kg ) was calculated as per ghosh .
all the guinea pigs were tested with mantoux test using 0.1 ml of purified protein derivatives ( ppd rt 23 tween 80 ) injected intradermally into the lower left side of abdomen .
all the guinea pigs examined ( up to 72 h ) were mantoux negative and hence used for experiment .
antitubercular study was carried out on guinea pigs and they were divided into 6 groups of either gender with 6 in each group .
cultures of m. tuberculosis h37rv were harvested from a 2-week culture in middle brooke 7h9 broth media .
all the guinea pigs were infected with 0.1 ml ( 1.5  10 cfu / guinea pig ) suspension via intramuscular route in the left thigh muscle .
have produced a tuberculosis infection in guinea pigs via intramuscular route.[2931 ] this suspension was matched with mcfarland standard .
after 30 days , infections were confirmed in 6 guinea pigs by histopathology of spleen , lung , and liver .
vi ) by random allocation software version 1.0 , may 2004 , by simple randomization . drugs and cshmc were administered by mouth once a day for 30 days in groups iii  vi .
human equivalent dose of isoniazid ( 30 mg / kg ) and rifampicin ( 60 mg / kg ) was calculated by extrapolation as mentioned by ghosh .
took a human - equipotent dose of isoniazid and rifampicin in guinea pigs as 30 and 50 mg / kg , respectively .
group ii received vehicle ( 10% ethanol ) by mouth once a day for 30 days .
group i : normal control group : did not receive the injection of m. tuberculosis h37rv and received vehicle ( 10% ethanol ) by mouth once a day for 30 days .
group ii : disease control group : received vehicle by mouth once a day for 30 days .
group iii : standard control group : received isoniazid ( 30 mg / kg ) by mouth once a day for 30 days .
group iv : standard control group : received rifampicin ( 60 mg / kg ) by mouth once a day for 30 days .
group v : test compound group : received cshmc ( 20 mg / kg ) by mouth once a day for 30 days .
group vi : test compound group : received cshmc ( 5 mg / kg ) by mouth once a day 30 for days .
blood samples were collected in ethylene diamino tetra acetic acid ( edta ) and plain bulb from the carotid artery under pentobarbitone anesthesia intraperitoneally ( 30 mg / kg ) 24 h after the last dose of drug .
edta sample was used for estimation of hemoglobin ( hb ) , total white blood cell ( wbc ) count , total red blood cell ( rbc ) count , total platelet count , differential count ( polymorphs , lymphocytes , eosinophils , monocytes ) , blood indices ( packed cell volume , mean corpuscle volume , mean corpuscle hemoglobin , mean corpuscle hemoglobin concentration , red cell distribution width , and erythrocyte sedimentation rate ) . complete blood count was analyzed by using celltac alpha cell counter ( nihontohdem , japan ) .
serum was separated for estimation of biochemical parameters : serum glutamic oxaloacetic transaminase ( sgot ) , serum glutamic pyruvic transaminase ( sgpt ) , alkaline phosphatase ( alp ) by enzyme kinetic method , bilirubin by jendrassik  grof method ( total , direct , and indirect ) and serum creatinine by jaff creatinine method in a fully automated analyzer .
their gross appearance was noted and they were placed in 10% formalin for 24 h. then 5 mm thick pieces of the lung , spleen , and liver were embedded in paraffin , cut into 5 mm thick sections , stained using hematoxylin - eosin dye ( h and e ) , and finally mounted in dibutyl diesterate parathylate xylene .
each slide was coded and observer masked to evaluate histopathological changes in the lung , spleen , and liver .
the guinea pig lungs were scored based on the following three criteria : ( 1 ) primary lesion ( epithelioid cells and other inflammatory cells ) : 0 , no primary lesions present ; 1 , single primary lesion ; 2 , two or more primary lesions ; multifocal ; 3 , two or more primary lesions ; multifocal to coalescing ; 4 , multiple primary lesions , coalescing , and extensive .
( 2 ) secondary lesion ( granuloma formation , giant cells , and caseous necrosis ) : 0 , no secondary lesion present ; 1 , up to 25% of lung involved ; 2 , up to 50% of lung involved ; 3 , up to 75% of lung involved ; 4 , above 75% of lung involved .
( 3 ) pneumonitis ( alveolar wall thickness , interstitial , and peribronchial inflammation ) : 0 , alterations in less than 10% of the fields ; 1 , alterations in 10%30% of the fields ; 2 , alterations in 30%50% of the fields ; 3 , alterations in 50%70% of the fields ; 4 , alterations in more than 70% of the fields are scored based on severity as follows : 0 , none ; 1 , minimal ; 2 , mild ; 3 , moderate ; 4 , marked ; and 5 , severe .
the spleen was scored based on two criteria ( same scales as lung ) ; ( 1 ) primary lesion and ( 2 ) secondary lesion .
the liver was scored based on one criterion ; ballooning degeneration : 0 , no ballooning degeneration ; 1 , minimal enlargement in few hepatocytes ; 2 , mild enlargement in many hepatocytes ; 3 , moderate enlargement in most hepatocytes ; 4 , severe enlargement in most hepatocytes .
the histopathologic score of organs are presented as median ( interquartertile range ) . the nonparametric kruskal  wallis test followed by dunnett 's test was used to assess statistical significance between total lesion score of lung , spleen , and liver .
the hematological , liver function , and renal function parameters are expressed as mean  standard error of mean ( sem ) .
statistical differences between means were determined by one - way analysis of variance ( anova ) followed by dunnett 's multiple comparison tests using graphpad prism software demo version .
in normal control group ( group i ) ; gross examination and histology of lung [ figure 2a ] , spleen [ figure 3a ] , and liver [ figure 4a ] of guinea pig showed the normal morphology .
( a ) normal control group  normal alveolar morphology ; ( b ) disease control group  severe ( score 6 ) epithelioid cells ( e ) , lymphocyte ( l ) , alveolar wall infi ltration ( a ) , caseation necrosis ( cn ) , and giant cells ( g ) ; ( c  e ) isoniazid , rifampicin , and cshmc ( 20 mg / kg ) treated group  minimal ( score 0.51 ) epithelioid cells and alveolar wall infi ltration with no caseation necrosis and giant cell ; ( f ) cshmc ( 5 mg / kg ) treated group  mild ( score 1.5 ) epithelioid cells and alveolar wall infi ltration with no caseation necrosis and giant cell histopathology of spleen ( h and e stain , 40 ) . ( a ) normal control group
 normal morphology ; ( b ) disease control group  severe ( score 6 ) epithelioid cells ( e ) , lymphocyte ( l ) infi ltrate with caseation necrosis ( cn ) , and giant cells ( g ) ; ( c  e ) isoniazid , rifampicin , and cshmc ( 20 mg / kg ) treated group  minimal to mild ( score 11.5 ) epithelioid cells infi ltration with no caseation necrosis and giant cell ; ( f ) cshmc ( 5 mg / kg ) treated group  mild to moderate ( score 2.5 ) epithelioid cells infi ltration with no caseation necrosis and giant cell histopathology of liver ( h and e stain , 40 ) .
liver shows normal morphology ; ( b ) disease control group  a mild ( score 2 ) ballooning degeneration ( bd ) ; ( c  f ) isoniazid , rifampicin , and cshmc ( 20 and 5 mg / kg ) treated group  a minimal or no ( score 0.5 ) ballooning degeneration ( bd ) isoniazid , rifampicin , and cshmc treated groups showed less white caseous granulomatous foci as compared with disease control group in lung and spleen by gross examination .
liver did not show any gross changes in groups ii  vi . as shown in figure 2b , lesion of lung consisted of epithelioid cells , lymphocytic infiltration , multinucleated giant cells , caseation necrosis , granuloma , and pneumonitis .
isoniazid [ figure 2c ] , rifampicin [ figure 2d ] , and cshmc ( 20 mg / kg ) [ figure 2e ] treated group had shown a minimal epithelioid cells and alveolar wall infiltration with no caseation necrosis and giant cell .
the lesion of spleen consisted of epithelioid cells , lymphocytic infiltration , multinucleated giant cells , caseation necrosis , and granuloma [ figure 3b ] .
isoniazid [ figure 3c ] , rifampicin [ figure 3d ] , and cshmc ( 20 mg / kg ) [ figure 3e ] treated group had minimal to mild epithelioid cells infiltration with no caseation necrosis and giant cell . as shown in table 1 , isoniazid , rifampicin , and cshmc ( 20 mg / kg ) significantly reduces the median lesion score in lung and spleen .
reduction in median lesion score of lung and spleen were not statistically significant for cshmc ( 5 mg / kg ) [ figures 2f and 3f ] .
cshmc ( 20 and 5 mg / kg ) treated group had shown a minimal or no ballooning degeneration .
isoniazid [ figure 4c ] , rifampicin [ figure 4d ] , and cshmc [ figure 4e and 4f ] significantly reduce the median lesion scores of liver [ table 1 ]
. the lesion scores of lungs , spleen , and liver were calculated in all the 5 groups after 30 days of the treatment the hemogram [ table 2 ] , liver [ table 3 ] , and renal [ table 3 ] function parameters were monitored after 30 days of isoniazid , rifampicin , and cshmc treatment .
the result indicates that cshmc did not produce any changes in hemogram , liver functions , and renal function parameters with respect to normal values .
effect of isoniazid , rifampicin , and cshmc on hemogram parameters in blood of guinea pigs after 30 days of treatment effect of isoniazid , rifampicin , and cshmc on liver and renal function parameters in blood of guinea pigs after 30 days of treatment
in normal control group ( group i ) ; gross examination and histology of lung [ figure 2a ] , spleen [ figure 3a ] , and liver [ figure 4a ] of guinea pig showed the normal morphology .
( a ) normal control group  normal alveolar morphology ; ( b ) disease control group  severe ( score 6 ) epithelioid cells ( e ) , lymphocyte ( l ) , alveolar wall infi ltration ( a ) , caseation necrosis ( cn ) , and giant cells ( g ) ; ( c  e ) isoniazid , rifampicin , and cshmc ( 20 mg / kg ) treated group  minimal ( score 0.51 ) epithelioid cells and alveolar wall infi ltration with no caseation necrosis and giant cell ; ( f ) cshmc ( 5 mg / kg ) treated group  mild ( score 1.5 ) epithelioid cells and alveolar wall infi ltration with no caseation necrosis and giant cell histopathology of spleen ( h and e stain , 40 ) . ( a ) normal control group
 normal morphology ; ( b ) disease control group  severe ( score 6 ) epithelioid cells ( e ) , lymphocyte ( l ) infi ltrate with caseation necrosis ( cn ) , and giant cells ( g ) ; ( c  e ) isoniazid , rifampicin , and cshmc ( 20 mg / kg ) treated group  minimal to mild ( score 11.5 ) epithelioid cells infi ltration with no caseation necrosis and giant cell ; ( f ) cshmc ( 5 mg / kg ) treated group  mild to moderate ( score 2.5 ) epithelioid cells infi ltration with no caseation necrosis and giant cell histopathology of liver ( h and e stain , 40 ) .
liver shows normal morphology ; ( b ) disease control group  a mild ( score 2 ) ballooning degeneration ( bd ) ; ( c  f ) isoniazid , rifampicin , and cshmc ( 20 and 5 mg / kg ) treated group  a minimal or no ( score 0.5 ) ballooning degeneration ( bd ) isoniazid , rifampicin , and cshmc treated groups showed less white caseous granulomatous foci as compared with disease control group in lung and spleen by gross examination .
liver did not show any gross changes in groups ii  vi . as shown in figure 2b , lesion of lung consisted of epithelioid cells , lymphocytic infiltration , multinucleated giant cells , caseation necrosis , granuloma , and pneumonitis .
isoniazid [ figure 2c ] , rifampicin [ figure 2d ] , and cshmc ( 20 mg / kg ) [ figure 2e ] treated group had shown a minimal epithelioid cells and alveolar wall infiltration with no caseation necrosis and giant cell .
the lesion of spleen consisted of epithelioid cells , lymphocytic infiltration , multinucleated giant cells , caseation necrosis , and granuloma [ figure 3b ] .
isoniazid [ figure 3c ] , rifampicin [ figure 3d ] , and cshmc ( 20 mg / kg ) [ figure 3e ] treated group had minimal to mild epithelioid cells infiltration with no caseation necrosis and giant cell . as shown in table 1 , isoniazid , rifampicin , and cshmc ( 20 mg / kg ) significantly reduces the median lesion score in lung and spleen .
reduction in median lesion score of lung and spleen were not statistically significant for cshmc ( 5 mg / kg ) [ figures 2f and 3f ] .
cshmc ( 20 and 5 mg / kg ) treated group had shown a minimal or no ballooning degeneration .
isoniazid [ figure 4c ] , rifampicin [ figure 4d ] , and cshmc [ figure 4e and 4f ] significantly reduce the median lesion scores of liver [ table 1 ]
. the lesion scores of lungs , spleen , and liver were calculated in all the 5 groups after 30 days of the treatment
the hemogram [ table 2 ] , liver [ table 3 ] , and renal [ table 3 ] function parameters were monitored after 30 days of isoniazid , rifampicin , and cshmc treatment .
the result indicates that cshmc did not produce any changes in hemogram , liver functions , and renal function parameters with respect to normal values .
effect of isoniazid , rifampicin , and cshmc on hemogram parameters in blood of guinea pigs after 30 days of treatment effect of isoniazid , rifampicin , and cshmc on liver and renal function parameters in blood of guinea pigs after 30 days of treatment
the main drawback of antitubercular chemotherapy is the lack of patient compliance , therapeutic failure , long duration of therapy , emergence of multidrug and extensive drug resistance tuberculosis , increasing incidence of hiv and toxic side effects , such as hepatotoxicity .
the xdr - tb is virtually untreatable with high mortality and now treatment of it will depend on sensitivity of drugs .
the second - line drugs are less efficacious , less convenient , more toxic , and more expensive .
, we investigated the antitubercular efficacy and safety of coumarin derivatives : cshmc in guinea pig model .
the antitubercular efficacy was evaluated in terms of histopathological score of the organs of infected guinea pigs after daily administration of cshmc for 30 days .
higher reduction of the histopathological score of lesion from the lung , spleen , and liver with cshmc in the dose of 20 mg / kg than with 5 mg / kg suggests clearance of tubercle bacilli from the lesion in a dose - dependent manner . cshmc-20
mg / kg had shown similar reduction in median score lesion of lung , spleen , and liver as compared to isoniazid and rifampicin .
antitubercular efficacy can be better measured by colony counting in the culture of the organs ( lung , spleen , and liver ) of the guinea pigs infected with m. tuberculosis .
the safety of cshmc was evaluated in terms of alteration on hematological , liver and renal function parameters of the infected guinea pigs .
antitubercular drugs , such as isoniazid , rifampicin , and ethambutol have been shown to induce hematological toxicity . in the present study ,
no alteration in hematological parameters with nce in the dose of 20 and 5 mg / kg compared to normal control group .
most of the antitubercular drugs , such as isoniazid , rifampicin , and pyrizinamide have been shown to induce hepatotoxicity particularly when used in combinations .
levels of sgpt , sgot , alp , and total bilirubin were monitored as an index of hepatotoxicity . in the present study , no alteration in the levels of liver function parameters with cshmc in the dose of 20 and 5 mg / kg were observed compared to normal control group .
no alteration in the level of renal function parameters with cshmc in the dose of 20 and 5 mg / kg were observed compared to normal control group .
this study indicates the cshmc in the dose of 20 and 5 mg / kg did not show the hematological toxicity , hepatotoxicity , and nephrotoxicity .
nce is a coumarin derivative and heterocyclic analogue of dapsone ( dads ) was attached to it . in dads free amino group
antimycobacterial effect of this compound may be due to its 8-[(4-chloro phenyl ) sulfonyl ] group and/or due to coumarin ring .
quantitative structure  activity relationship ( qsar ) study remains open for the further studies .
cshmc in the dose of 20 mg / kg had shown more efficacies against the m. tuberculosis infection in guinea pigs compared with a dose of 5 mg / kg , which is comparable to isoniazid and rifampicin by histopathological data . in both doses
 objective : to evaluate the antitubercular efficacy and safety of new chemical entity ( nce ) : 8-[(4-chloro phenyl ) sulfonyl]-7-hydroxy-4-methyl-2h - chromen-2-one ( cshmc ) in guinea pigs.materials and methods : this pilot study was carried out in guinea pigs . 
 they were infected with m. tuberculosis h37rv ( 1.5  104 cfu / guinea pig ) via intramuscular route . 
 after 30 days , infections were confirmed in 6 guinea pigs by histopathology of spleen , lung , and liver . 
 after that cshmc ( 5 and 20 mg / kg ) was administered for 1 month and its effect was compared with vehicle , rifampicin ( 60 mg / kg ) and isoniazid ( 30 mg / kg ) . 
 efficacy of cshmc was evaluated on the basis of histopathologic scoring of lesion in lung , spleen , liver , and safety on the basis of measuring hemogram , liver and renal function parameters.results:isoniazid , rifampicin , and cshmc ( 20 mg / kg ) significantly reduce the median lesion score in lung , spleen , and liver as compared to disease control group . 
 reduction in median lesion score for lung and spleen were not statistically significant for cshmc 5 mg / kg . 
 cshmc ( 20 and 5 mg / kg ) did not produce any changes in hemogram , liver and renal function parameters with respect to normal values.conclusions:cshmc had shown significant antitubercular efficacy comparable to isoniazid and rifampicin and did not show hematological , hepato- and nephrotoxicity . 