Patent Abstract:
the present invention deals with the pharmaceutical composition comprising the therapeutically effective amount of a compound oenothein c obtained from the bioactive fraction of plant woodfordia fruticosa optionally along with one or more pharmaceutically acceptable carriers , additives , lubricants and diluents . further it also provides a method of treating peptic ulcer diseases in a subject using the said pharmaceutical composition . it also relates to the use of the compound oenothein c in the treatment of peptic ulcer related diseases and a process for the isolation of the said compound .

Detailed Description:
the molecule oenothein c has been isolated from the flowers of the plant woodfordia fruticosa , abundantly available in the indian subcontinent . dried flowers of woodfordia fruticosa were extracted by soaking overnight with 1 : 1 methanol - water at room temperature . the process was repeated thrice . the combined extract was evaporated at 45 ° c . in a rotary evaporator to get the crude extract . this was partitioned between n - butanol and water . the layers were separately evaporated at 45 ° c . to obtain two fractions . the n - butanol soluble fraction was chromatographed repeatedly over diaion hp - 20 and sephadex lh - 20 columns following bioassay guided fractionation technique to obtain pure oenothein c as an off - white solid , amorphous in nature . it is soluble in methanol , acetone and dmso . in hplc , oenothein c showed a single peak corresponding to one of the major peaks in the crude extract . the hplc profile of the crude extract is shown in fig1 ( a ). fig1 ( c ) shows the hplc profile of the isolated single molecule , oenothein c . fig2 , 3 and 4 represent nmr and mass spectra of the compound oenothein c . during fractionation by chromatography , another compound , methyl gallate was isolated . its structure elucidated by 1 h nmr and esi - mass , then confirmed by comparing with a standard sample prepared in the laboratory . fig1 ( b ) shows its hplc profile . the relative abundance ( yield percent ) of methyl gallate with respect to bioactive fraction was determined to 0 . 003 %. fig5 and 6 respectively represent nmr and mass spectra of the compound methyl gallate . the compound methyl gallate however , did not show any activity , in any of the tests , described below . however this along with oenothein c will serve as the two fingerprint components in hplc profile , in the final pharmaceutical preparation , from which one could judge the authenticity of the bioactive fraction . the two molecules , methyl gallate and oenthein c have been identified to delineate as the fingerprint component of the pharmaceutical preparation made from the bioactive fraction . the following examples are given by way of illustration of the present invention and should not be construed to limit the scope of the present invention . isolation of compounds oenothein c and methyl gallate from the plant woodfordia fructicosa the molecules oenothein c and methyl gallate have been isolated from the flowers of the plant woodfordia fruticosa . dried flowers of woodfordia fruticosa were extracted by soaking overnight with 1 : 1 methanol - water at room temperature . the process was repeated thrice . the combined extract was evaporated at 45 ° c . in a rotary evaporator to get the crude extract . this was partitioned between n - butanol and water . the layers were separately evaporated at 45 ° c . to obtain two fractions . the n - butanol soluble fraction was chromatographed repeatedly over diaion hp - 20 and sephadex lh - 20 columns following bioassay guided fractionation technique to obtain pure oenothein c as an off - white solid , amorphous in nature ( yield 0 . 008 %). it is soluble in methanol , acetone and dmso . the respective yield of methyl gallate was 0 . 003 %. pig gastric membranes enriched in apical and tubulovesicular membranes were prepared according to [ 59 ]. such membranes show k + - stimulated h + - transporting atpase activity , which could be specifically blocked by omeprazole [ 60 ]. the h + , k + - atpase activity was measured at 37 ° c . in a 1 - ml reaction mixture containing 2 mm mgcl 2 , 2 mm atp , 10 mm pipes ( ph 6 . 8 ), 10 mm kcl and 10 - 12 μg membrane protein . an otherwise complete assay mixture was preincubated for 10 min in presence and absence of different concentrations of oenothein c or omeprazole before initiating the reaction with atp . after incubation for 10 min , the reaction was terminated by the addition of 1 ml ice - chilled tca ( 14 %). k + - stimulated activity , referred to as h + , k + - atpase , was calculated as the difference between the activity in presence of mg + plus k + and the basal activity ( mg + - atpase ) in presence of mg + alone . the membrane showed high k + - stimulated activity of around 40 - 50 μmoles p i / mg / h with basal activity of only around 10 μmoles p i / mg / h . following enzymatic assay method [ 61 ], the inhibitory activity of oenothein c was assessed . the effects are quantitated in terms of percent inhibition of h + , k + - atpase , and compared with that of omeprazole , a specific inhibitor of gastric h + , k + - atpase . the molecule oenothein c showed extremely strong inhibition of gastric proton pump activity . at 1 . 3 μm , it produced about 70 - 90 % inhibition of h + , k + - atpase ( table 1 ) which is higher than the standard drug omeprazole ( about 20 % inhibition at 3 μm ) when compared under identical experimental conditions . thus , the degree of inhibition ( ranging 20 - 90 % at 0 . 13 - 1 . 30 μm ) of the gastric proton pump with oenothein c is about 20 - fold higher as compared with the standard medicine omeprazole ( 10 - 90 % inhibition at 3 - 30 μm ). the effect of oenothein c for anti - helicobacter pylori activity was examined using disc diffusion sensitivity assay as well as by determination of mic / mbc values by microbroth dilution assay . six different strains of h . pylori , of which 2 are clinical strains [ 80a ( avirulent ) and 121a ( virulent )] and four standard strains [ atcc 43504 , atcc 49503 , nctc 26695 and atcc 43629 ], were maintained at 37 ° c . incubator under appropriate conditions ( 10 % co 2 , 5 % o 2 , 85 % n 2 and & gt ; 95 % relative humidity ). the molecule oenothein c was dissolved in dmso . for disc diffusion sensitivity assay [ 62 ], the culture plates were made with brain heart infusion agar supplemented with 10 % fetal calf serum ( fcs ), isovitalex ( 0 . 5 %) and dent ( 0 . 0025 %). the plates were flooded with 1 × 10 6 cfu / ml of liquid cultures of h . pylori , the discs were placed on the plate and the sample was impregnated directly on the disc at a dose of 5 μl / disc or 10 μl / disc so as to give 100 μg / disc and 200 μg / disc . after 3 days of incubation , the inhibition zone diameter was measured and compared to that with standard antibiotic clarithromycin . the molecule oenothein c produced inhibition zone diameter ranging 1 . 7 - 2 . 5 cm at the dose range of 100 - 200 μg / disc when examined against 6 different strains of h . pylori . the activities with clarithromycin , standard antibiotics , were determined under similar experimental conditions ( table 2 ). for the determination of mic and mbc values by microbroth dilution assay [ 63 ], a two - fold serial dilution ( ranging between 1 - 50 μg / ml ) of oenothein c was prepared in a 96 - well microtitre plate containing a total volume of 100 μl of brucella broth supplemented with 5 % fcs . a 3 - day liquid culture of h . pylori was diluted 10 - times in brucella broth and 100 μl of these cultures were inoculated into each well to give a final concentration of ˜ 10 6 cfu / ml . the plates were incubated for 3 days in a microaerophilic atmosphere at 37 ° c . following incubation , the plates were examined visually , and the lowest concentration showing complete inhibition of growth was recorded as the mic of the respective compound . for mbc determination , aliquots ( 10 μl ) of 72 - h culture in which no growth had been detected were taken from the wells of the above microtitre plates and used to streak on fresh brain heart infusion agar plates . mbcs were determined by visual inspection of such plates after further incubation for 72 h at 37 ° c . and the point where no growth ( less than 10 colony ) appeared was considered as the mbcs . standard antibiotics clarithromycin , amoxycillin and metronidazole were included for comparison with oenothein c employing different strains of h . pylori . the mic and the mbc values of the molecule oenothein c were determined against five strains by microbroth dilution assay . with strains 80a , atcc 43504 , atcc 49503 , nctc 26695 and atcc 43629 , the observed mic values were 25 , 25 , 6 . 25 , 12 . 5 , and 25 μg / ml respectively ( table 3 ). the mic values of clarithromycin under identical experimental conditions have been 20 , 25 , 6 . 25 , 62 . 5 , and 50 ng / ml for the respective strains . likewise , the mic values of amoxycillin employing such strains were observed to be 20 , 25 , 12 . 5 , 100 , and 25 ng / ml respectively . based on mic values against different strains , the molecule oenothein c appears to be about 500 - 1000 fold less potent as compared with clarithromycin and amoxycillin both . however , in respect of metronidazole mic values ( 12 . 5 , 50 , 1 . 56 , 1 . 56 and 1 . 56 μg / ml ), oenothein c is just about 2 - 20 fold less competent as observed in 5 such strains . in terms of mbc values , a measure of bactericidal activity , similar trend in efficacy of oenothein c vis - à - vis the three standard antibiotics was noted . to summarize , the efficacy of the molecule oenothein c against different strains , clinical as well as standard , is evident . further , since some of the strains are toxin producing ( cag - a , vac - a , cytotoxin positive ), the molecule is expected to show efficacy against such strains under pathogenic conditions . further , the antibacterial activity of oenothein c was examined against four aerobic bacteria ( 1 gram positive , b . cereus and 3 gram negative , s . typhi , e . coli & amp ; k . pneumoniae ) by microbroth dilution assay in muller hinton broth essentially by following nccls ( national commmittee for clinical laboratory standards ) guideline [ 64 ]. the molecule oenothein c showed no activity against any of the bacterial strains ( mic & gt ; 400 μg / ml ). hence the compound is specific against h . pylori . the compound oenothein c was checked for mortality of the swiss albino mice . maximum dosage of 250 mg / kg body weight of compound oenothein c was given to 5 swiss albino mice via oral route . after 24 hours , it was seen that there is no mortality in any of the mice . further , the mice were kept under observation for 15 days , during which period they were found to remain healthy , and no behavioral abnormality were noted . 1 . the inhibition of gastric proton pump by the compound oenothein c is much stronger than standard medicine omeprazole ( about 20 times ). 2 . reasonably low mic values ( 6 . 25 - 25 . 0 μg / ml ) of the same molecule oenothein c against clinical and different standard strains of h . pylori has also been provided in the present invention . 3 . the specific anti h . pylori activity of the molecule oenothein c is evident from its high mic values (& gt ; 400 μg / ml ) against a panel of aerobic bacteria , suggesting its potential as a useful therapeutic agent . 4 . since the acid hcl and the bug h . pylori are the two major reasons for the pathogenesis of gastroduodenal ulcers , position of oenothein c as a single molecule anti gastric and duodenal ulcer medicine is unique . this is more because of the failure of the current medical management even after using triple therapy and also quadruple therapy ( clarithromycin , amoxycillin , proton pump inhibitor , h 2 receptor blocker , sucralfate — any 3 or 4 depending on the diagnosis and other criteria ) in terms of ulcer recurrence and antibiotic resistance . lee a . 1996 . in helicobacter pylori : techniques for clinical diagnosis & amp ; basic research ( eds . adrian lee & amp ; francis megraud ), wb saunders co . ltd ., london , pp . xiii - 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