Patent Application: US-40381403-A

Abstract:
the present invention relates to a novel nitrile glycoside of formula i named niaziridin and to analogues and derivatives thereof . the present invention also relates to a process for the isolation of a novel nitrile glycoside of formula i below named niaziridin and its derivatives and analogues by bioactivity - guided fractionation from the pods of moringa oleifera . the present invention particularly relates to the bioenhancing activity of the novel nitrile glycoside of formula i below named niaziridin and its derivatives and analogues in enhancing bioactivity of commonly used antibiotics such as rifampicin , tetracycline and ampicillin against gram and bacteria . the biomolecule also enhances the absorption of drugs , vitamins and nutrients through the gastro - intestinal membrane increasing their bio - availability . therefore niaziridin can be used in combination therapy with drugs and nutrients resulting in reduced drug associated toxicity , reduced cost and duration of chemotherapy .

Description:
in our efforts to discover new biomolecules from plant sources which could function as bioactivity enhancer of commonly used but toxic antibiotics , nutraceuticals etc . we found that the ethanolic extract of the fruit pods with seeds of moringa ( moringa oleifera ) was able to enhance the activity of rifampicin , ampicillin and tetracycline against gram (+) and gram (−) bacteria including mycobacterium tuberculosis the causal organism of tuberculosis . the active ethanolic extracts of the pods were then subjected to bioactivity - guided fractionation to yield a pure compound , which on chemical characterization was found to be niaziridin of the formula i . the compound i was able to increase the activity of antibiotics such as rifampicin , ampicillin and tetracycline against escherichia coli ( kumar et al 1976 ; j . bacteriology 125 : 545 ), bacillus subtilis ( equivalent of atcc 6015 ), mycobacterium smegmatis ( equivalent of atcc 10231 ) and m . tuberculosis h37rv . the compound was also able to enhance the activity of antifungal azole drugs such as clotrimazole against c . albicans , the causal organism of candidiasis . the compound was also able to facilitate the uptake of nutrients like vitamine b12 through the intestinal gut membrane in combination , thus also functioning as bioavailability facilitator . [ 0037 ] m . oleifera lam . is a small and medium size tree , about 10 mt . height , belongs to the family moringaceae and it is also known as ‘ drumstick ’. it is indigenous in the sub - himalayan tract and cultivated throughout india and burma . the tree is indigenous to north west india and is plentiful on recent alluvial land in or near sandy beds of rivers and streams . it is often cultivated in hedges and homeyards . it grows in all types of soils extract stiff clays and thrives best under the tropical peninsular climate of south india . the tree is valuated mainly for the tender pods , which are esteemed as vegetable . they are cut into slices and used in culinary preparations ; they are also pickled . flowers and tender leaves are eaten as potherb . seeds are consumed after frying and are reported to taste like peanuts . all parts of the tree are considered medicinal and used traditionally in the treatment of ascitcs , rheumatism , venomous bites and as cardiac and circulatory stimulants . practitioners of ayurveda prescribe roots of m . oleifera as stimulant , in treatment of intermittent fever , epilepsy and chronic rheumatism 1 . roots are also useful as carminative , anthelmintic and diuretic . bark is used as abortifacient , cardiac stimulant and antifungal . leaves are utilized in inflammation , helmintheses and scurvy . seeds are useful as purgative , antipyretic and anti - inflammatory 20 . flowers are used as diuretic and cholagogue 21 . the leaves are rich in vitamins a and c and are considered useful in scurvy and catarrhal affection ; they are also used as emetic . a paste of the leaves is used as an external application for wounds . flowers are used as tonic , diuretic and cholagogue . the seeds are considered antipyretic , acrid and bitter . the seed oil is applied in rheumatism and gout . number of biomolecules has been isolated from different parts of moringa tree , which are detailed in table - 1 . b ) antifertility activity : the aqueous extract of root and bark of m . oleifera at a dose of 200 mg / kg respectively showed post coital antifertility effect in rat and also induced foetal resorption at late pregnaney 14 . c ) anti - inflammatory activity : crude ethanolic extract of dried seeds was tested for anti - inflammatory activity using carrageenan induced inflammation in hind paw of mice 14 , 15 . d ) anti - ulcer activity : the methanolic extract of leaves of m oleifera inhibited gastric lesion formation induced by aspirin serotonin or indomethacin in rats 16 . e ) cardiovascular activity : compounds niazinin a and b , niazimicin , niaziminin a and b isolated from the ethanolic extract of the leaves produced hypotensive and bradycardiac effect in anaesthetized rat at a dose of 1 - 10 mg / kg i . v 17 . f ) antimicrobial activity ; the antimicrobial activity of leaves , root , bark and seeds were investigated against — bacteria , yeast , dermatophytes and helminths pathogenic to man . the fresh leaf juice and aqueous extract of seeds inhibited the growth of pseudomonas aeruginosa , staphylococcus aureus 18 . the extract of m . oleifera seeds at 1 - 10 dilution inhibited the growth of fungi like trichophyton rubrum and t . mentagrophytes 19 . however , bioenhancement of antibiotics , antifungal drugs and nutrients by molecules from moringa pods has not been reported anywhere . the present invention provides a pharmaceutical composition comprising an effective amount of niaziridin as a bioactivity and bioavailability facilitator and pharmaceutically acceptable additives selected from antibiotics , drugs , therapeutic and nutraceutic agents and similar molecules which are targeted to the living systems . niaziridin can be used in antifungal therapy for the treatment fungal infection caused by yeasts such as c . albicans . the niaziridin can also be used in tuberculosis therapy in combination with rifampicin , isoniazid and other anti - tubercular agents . niaziridin the bioavailability facilitator helps in transferring the compounds , drugs and nutrients across the membrane and for better effectiveness on the target site . the antibiotics are rifampicin , tetracycline , ampicillin , quinolones , fluoroquinolones , isoniazid and similar compounds ; nutrients are vitamines , aminoacids , nitrogen bases ; antifungal agents are azoles such as clotrimazole , ketaconazole , micanozole and itracanozole . the compound of formula i functions as a bioavailability facilitator and helps the antibiotics and other molecules to act better on the target by increasing the effectiveness . the living system may be bacteria , fungi or any living cells . preferably niaziridin is used in the range between 0 . 1 μg / ml to 10 μg / ml . in one feature , the lyophilized active fraction isolated from the pods of moringa oleifera used is in the range between 0 . 1 μg / ml to 100 μg / ml . the bioactive fraction enhances the activity of anti - bacterial , anti - fungal and anti - tuberculosis agents from 2 to 80 folds . the methodology followed by us for this screening included specifically designed bioassays described below as examples . the bacterial and fungal strains used in this invention were acquired commercially from microbial type culture collection ( mtcc ) at institute of microbial technology ( imtech ), chandigarh , india that possessed corresponding properties of the atcc strain mentioned . 1 . collection and extraction of pod materials : 20 . 0 kg of the moringa oleifera pods were collected from local area of lucknow . pods were separated and air dried at room temperature ( 35 ° c .- 40 ° c .) in shade . after air drying they were ground and sieved to fine powder in a mixer grinder . 8 kg of the powdered pod materials were dipped in absolute ethanol in a percolator for 48 hrs at room temperature ( 35 ° c .- 40 ° c .). ethanol extract was filtered using whatman filter paper no . 1 and concentrated at the 40 ° c . under reduced pressure . the ethanolic extract was then lyophilized to obtain 208 g of crude extract . stock of 100 mg / ml was made in dmso and tested for bio enhancing activity . the results in table - 2 indicate that the crude ethanolic extract prepared from the pods of moringa oleifera was able to enhance the activity of rifampicin by 2 . 5 to 3 . 0 folds against e . coli ca 8000 ( kumar et al 1976 ; j . bacteriology 125 : 545 ). solvent fractionation of the active crude extracts was undertaken to isolate the active principle . 250 g ethanolic extract was dissolved in 500 ml of distilled water . then it was filtered using whatman no . 1 filter paper . the insoluble portion was dissolved in methanol and the soluble portion ( h 2 o ) was extracted with hexane , then chloroform , ethyl acetate and n - butanol ( saturated with h 2 o ) respectively . naso 4 ( anhydrous ) was added to each fraction for removing the moisture . all the solvent fractions were concentrated at 40 ° c . under reduced pressure and tested . the results indicate that ethyl acetate fraction obtained from the crude pod extract possessed the activity enhancement property . column chromatography of the bioactive ethyl acetate fraction ( 7 . 0 g ) was performed using silica gel . different solvent systems were used for the elution of the column sub fractions starting with 100 % hexane , then chloroform and then ethyl acetate . 150 ml of fractions were collected and ten such fractions were pooled for further bio - evaluation . the bioactivity enhancement of antibiotics was detected in fractions starting from 30 % chloroform + 70 % ethyl acetate and upto 95 % ethyl acetate + 5 % methanol fractions . thin layer chromatography ( tlc ) of the active column fractions was then performed by a procedure that is well documented and can be performed by a person skilled in the art of natural product isolation . all the spots developed by iodine treatment were separated , and re - dissolved in the appropriate solvent ( methanol ethyl acetate and chloroform ) for bio - evaluation . the active spot was further purified five times by the same procedure to obtain 20 mg of single active tlc spot . the active spot obtained by tlc was further purified by repetitive column chromatography which can be performed by a person skilled in the art and then analyzed by 1 h & amp ; 13 c nmr , lc - ms to determine the structure of the active pure compound . on the basis of spectroscopic data the new compound isolated from the pods of moringa oleifera was characterized as niaziridin ( formula i ), a new nitrile glycoside from nature . bioactivity enhancement of antibiotics was done by working out killing efficiency of antibiotics alone and in combination with bioenhancer ( extract / pure compounds ). bacterial / yeast culture was grown in 6 ml nutrient broth and sabouraud &# 39 ; s dextrose broth respectively for 48 hours at 37 ° c . by inoculating a single colony . the culture was divided into 3 sections and fresh broth of 2 . 0 ml was added into each tube to made up to 4 ml . test compounds @ 1 . 0 and 0 . 1 μg / ml were added into two of the culture tubes whereas methanol the solvent used for dissolving test compound was added in third tube to serve as a control . the cultures were grown at 37 ° c . for 4 hours in an incubator shaker at 200 rpm and centrifuged to pellet cells . cells were washed once as above in sterile water or pbs . pellet of each tube were then dissolved in 4 ml fresh broth and titre of cells were estimated from each tube by serial dilution and spread plating on na . colony counts obtained after incubation for 48 hours at 37 ° c . gave initial titre of cells in each tube . a series of tube containing 5 ml of nb with antibiotics of different concentration were prepared in three sets . 0 . 1 ml of culture pre - treated with test compounds ( 1 . 0 and 0 . 1 μg / ml ) as well as from control tube was inoculated into series of tubes maintained in three sets . the tubes were incubated at 37 ° c . for 48 hours to obtain growth . 0 . 1 ml of culture was removed from all tubes and titre was estimated as before to give final titre . results were calculated as fold enhancement in antibiotic killing action from initial and final titre of cells . it was evident from table - 4 that pure compound niaziridin enhanced activity of commonly used antibiotics such as rifampicin , tetracycline , nalidixic acid etc against e . coli a gram (−) bacteria . therefore the compound was further tested for its bioenhancing property against representative gram (+) bacterial species mycobacterium smegmatis and b . subtilis . results in table - 5 indicates that the compound enhanced activity of rifampicin , ampicillin , nalidixic acid by 1 . 2 - 19 folds against both the gram (+) strains . [ 0065 ] table 5 enhancemnt of antibiotic activity of niaziridin ( 1 ) @ 1 . 0 μg / ml in combination with different antibiotics against gram (+) bacteria b subtilis and m . smegmatis . b subtilis ( mtcc121 ) m . smegmatis ( mc 2 155 ) folds folds enhancement enhancement % in % in treatment titre survival killing treatment titre survival killing control ( lb ) 4 . 0 × 10 8 100 . 0 19 . 4 control ( lb ) 4 . 0 × 10 7 100 . 0 1 . 9 rif ( 0 . 05 ) 2 . 1 × 10 8 52 . 5 rif ( 0 . 05 ) 1 . 3 × 10 7 32 . 5 rif ( 0 . 05 ) + comp ( 1 ) 1 . 1 × 10 7 2 . 7 rif ( 0 . 05 ) + comp ( 1 ) 6 . 7 × 10 6 16 . 75 control ( lb ) 4 . 0 × 10 8 100 . 0 5 . 1 control ( lb ) 4 . 0 × 10 8 100 . 0 4 . 7 amp ( 0 . 01 ) 2 . 9 × 10 8 72 . 5 amp ( 0 . 1 ) 1 . 0 × 10 8 25 . 0 amp ( 0 . 01 ) + comp ( 1 ) 5 . 7 × 10 7 14 . 2 amp ( 0 . 1 ) + comp ( 1 ) 2 . 1 × 10 7 5 . 25 control ( lb ) 4 . 0 × 10 8 100 . 0 3 . 5 control ( lb ) 4 . 0 × 10 8 100 . 0 4 . 0 tet ( 1 . 0 ) 7 . 0 × 10 7 17 . 5 tet ( 2 . 0 ) 1 . 2 × 10 8 30 . 0 tet ( 1 . 0 ) + comp ( 1 ) 2 . 0 × 10 7 5 . 0 tet ( 2 . 0 ) + comp ( 1 ) 3 . 0 × 10 7 7 . 5 control ( lb ) 4 . 0 × 10 8 100 . 0 7 . 0 control ( lb ) 4 . 0 × 10 8 100 . 0 1 . 2 nal ( 2 . 0 ) 3 . 1 × 10 7 7 . 75 nal ( 1 . 0 ) 3 . 7 × 10 8 92 . 5 nal ( 2 . 0 ) + comp ( 1 ) 4 . 6 × 10 6 1 . 1 nal ( 1 . 0 ) + comp ( 1 ) 3 . 0 × 10 8 75 . 0 further to determine whether the pure compound isolated from the pods of moringa is able to enhance the activity of antifungal drugs , similar broth assays were done with clotrimazole , a commonly used azole group of antifungal agent against candida albicans . the results indicate that ( table - 6 ) the compound niaziridin was able to enhance the antifungal activity of clotrimazole by 5 - 6 folds against c . albicans when present in combination . however , the antifungal activity enhancement was observed only at a relatively higher concentration ( 10 μg / ml ) of the compound . since , the bioenhancer compound was able to enhance the activity of antibiotics against gram (+) bacteria such as m . smegmatis , our subsequent interest was to test the compound against m . tuberculosis h37rv ( atcc 27294 ) ( philipp et al 1996 ; proc . natl . acad . sci usa 93 , 3132 - 3137 ), the causal organism of tuberculosis . to accomplish this we employed a radiometric assay using the bactec 460 tb system manufactured by m / s becton dickinson ltd ., usa . the antimycobacterial property of the drugs in combination with the bioenhancer was studied as per the recommended drug sensitivity protocol prescribed by the manufacturer ( bactec 460 tb system : product & amp ; procedure manual , ma 0029 , 1996 ; becton dickinson co ., usa ). briefly the procedure is described below . ( a ) the 12 b vials as provided by the manufacture were pre - tested prior to inoculation to establish the recommended co 2 atmosphere . the vials that showed a growth index ( gi ) of 20 or more on the initial test were rejected . ( b ) 100 μl of the mycobacterial suspension indicating a gi value of 500 were inoculated into 12b containing the antibiotics . ( c ) in the control vial 1 : 100 fold diluted inoculum was used by transferring 0 . 1 ml of the suspension into 9 . 9 ml of special diluting fluid . after mixing thoroughly , 0 . 1 ml of this dilution was added into the control 12 b vial ( without a drug ). ( e ) vials were tested daily in bactec 460 tb system for 12 days at approximately the same time each day as recommended by the manufacturer . the mycobacterial growth is expressed as growth index ( gi ) and compared to that of the control vials ( antibiotic alone ). it is evident from fig1 and 2 that the compound niaziridin was able to enhance the antimycobacterial activity of rifampicin and isoniazid against the virulent strains of m . tuberculsis . specially designed u - tubes of glass consisting of two components ( opposite - l type ) were used in which one open end of an l - shaped was tapered to fit within the end of the other l - tube ( as described in u . s . pat . no . 6 , 410 , 059 ). the membrane of goat gut ( initial part ) was stretched and fixed to act as the barrier between the two ends such that by joining the two l - tubes , a u - tube was made . sterile distilled water was then filled in both the sides to equal height / level . the antibiotic / compound was added to the donor tube ( tapered ) and through spectro - photometer , the transfer of molecule was observed using uv and visible absorption maxima of the respective molecules by taking the od at defined wavelengths ( 264 nm for isoniazid ). overnight grown bacterial culture ( escherichia coli , ca 8000 ) was divided into 5 ml of aliquot each in screw cap tubes . one of the tube with the bacterial culture was treated with niaziridin to a final concentration of 1 μg / ml . both the control and the treated tubes were incubated at 37 ° c . for 4 hours . the cultures were then infected with λ phage ( moi = 10 ) for 10 minutes at 37 ° c . ( for adsorption ) followed by centrifugation at 10000 rpm for 10 minutes at 4 ° c . the supernatant and the pellets for both treated and untreated cultures were then compared for the phage titer by plating on top agar containing sensitive bacteria at different dilutions . the phage titer in the supernatant of the treated culture was reduced by 2 . 6 folds indicating increase in adsorption to the bacterial cells harvested in the pellet . overnight grown bacterial e . coli dh5α ( sambrook at al 28 ) cells were diluted to 10 folds in growth medium and treated with niaziridin @ 1 - 10 μg / ml for 4 hours at 37 ° c . in a shaker ( 200 rpm ). these log phase cells were then treated with calcium chloride to induce competence and then transformed with puc18 plasmid dna and plated on luria agar plates containing 100 μg / ml ampicillin . the detailed procedure can be found in sambrook at al 28 . the colonies thus appeared , on the plates for both treated and untreated cultures were scored . an increase of 2 . 3 folds for transformation was observed in case of the strain dh5α □ and 5 . 5 folds in case of ca8000 indicating that the compound enhances the uptake of plasmid dna in e . coli cells . e . coli strains ca8000 dh5α cells pre - fold fold treated with enhancement enhancement niaziridin titre in dna titre in dna at ( 10 3 ) transformation ( 10 4 ) transformation 10 μg / ml 24 . 0 5 . 5 20 . 0 2 . 3 1 μg / ml 8 . 30 1 . 9 16 . 0 1 . 9 untreated 4 . 3 — 8 . 4 control 1 . kirtikar k r , basu b d . indian medicinal plants . 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