USOO6896907B2 (12)UnitedStatesPatent (10)PatentNo.: US6,896,907B2
Khanujaetal.
(54) USE OF BOACTIVE FRACTION FROM COW URINEDISTILLATE(“GO-MUTRA)AS A BIO-ENHANCER OF ANTI-INFECTIVE, ANT-CANCERAGENTS AND NUTRIENTS
(75) Inventors: SumanPreetSinghKhanuja,
(45)DateofPatent: May24,2005
(60) ProvisionalaplicationNo.60/241,842,filedonOct.20, 2000.
(51) Int.Cl.”.A61K35/24;A61K35/23 (52) U.S.Cl..424/558;424/537 (58) FieldofSearch.424/537,558
Lucknow(IN);SushilKumar, Lucknow(IN);AjitKumarShasany, Lucknow(IN);JaiShankarArya, Lucknow(IN);MahendraPandurang Darokar,Lucknow(IN);Monika Singh,Lucknow(IN);PrachiSinha, Lucknow(IN);SoumyaAwasthi, Lucknow(IN);SubhashChandra Gupta,Lucknow(IN);VivekKumar Gupta,Lucknow(IN);MadanMohan Gupta,Lucknow(IN);Ram Kishore Verma,Lucknow(IN);SwetaAgarwal, Lucknow(IN);SunilBalkrishna Mansinghka,Maharashtra(IN);Suresh HaribhauDawle,Maharashtra(IN)
(73) ASSignee: Council ofScientificand Industrial Research,NewDelhi(IN)
(*)Notice: Subjecttoanydisclaimer,thetermofthis patentisextendedoradjustedunder35
U.S.C.154(b)by119days. (21) Appl.No.:10/135,763
(56)
References Cited U.S. PATENT DOCUMENTS
(2) Filed:
May1,2002 PriorPublication Data
5,462,726A *10/1995 Lodge 5,496.846A * 3/1996 Wilsonetal. 5,504,102A * 4/1996 Agharkaretal.
OTHER PUBLICATIONS
Lafont,Rev.Hist.Pharm(Paris),1999,47(323),343–6.* Oyebola,Afr.J.Med.med.Sci.1982,1,183-189.* Iyunetal.,Soc.Sci.Med.,vol.42,No.3,437–45,1996.* Hawley’sCondensedChemicalDictionary,13″Edition, Lewis,Sr.,1997,pp.418-419.*
*citedbyexaminer
PrimaryExaminerMichaelMeller (74)Atorney,Agent,orFirm-Burns,Doane,Swecker& Mathis,L.L.P.
(57) ABSTRACT
Theinventionrelatestoanovelpharmaceuticalcomposition comprisinganefectiveamountofbio-activefractionfrom cowurinedistilateasabioavailabilityfacilitatorandphar maceuticallyacceptableaditivesSelectedfromanticancer compounds,antibiotics,drugs,therapeuticandnutraceutic agents,ionsandSimilarmoleculeswhicharetargetedtothe livingSystems.
6Claims,1DrawingSheet
(65)
US 2002/0164378A1 Nov.7,2002
RelatedU.S.ApplicationData
(62) DivisionofaplicationNo.09/726,307,filedonDec.1, 2000,nowPat.No.6,410,059.

U.S. Patent
May24,2005
US 6,896,907B2
O
r 4N
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Sample D
Type
Detector
Operator
Method Name :
* Chromatogram” mAbs
al 5nm .
40
5. 20
a. ccy WWSC2 A:
53 & Yt d
S.
Unknown
: SPD-M10Avp
ANAL.MET
rt Sd-
cys Y Yd
min
PKNOChNO TIME AREA MK PURITYUP PURITYDOWN IDNO CONC
*Peak Report’
1 1 0.953 9552712 2 1 1438 2027186 3 1 1909 7380 4 1 2.432 2312 5 1 2.733 26772 6 1 3.002 11050 7 3.459 5526
8 1 4,039 9699
9 1 4.336 9040 10 5.334 59563 11 5.917 11104 12 1 6.438 2800 13 10.665 4544 14 1 11310 265970 15 1 11712 2676 16 1 15.098 94810
12093144
78.9928 16.7631 0.0610 O.O.191 0.2214 0.0914 0.0457 0.0802 0.0748
0.4925 . 0.0918 0.0232 0.0376 2.1993 0.0221 0.7840
100.0000

US 6,896,907B2 12
USE OF BOACTIVE FRACTION FROM COW URINEDISTILLATE(“GO-MUTRA)AS A BIO-ENHANCER OF ANTI-INFECTIVE, ANT-CANCERAGENTS AND NUTRIENTS
ThisaplicationisadivisionofSer.No.09/726,307filed Dec.1,2000nowU.S.Pat.No.6,410,059,whichclaims benefitofSer.No.60/241,842filedOct.20,2000.
FIELD OF THE INVENTION
Theinventionrelatestoanabsolutelynoveluseofcow urinedistilateasactivityenhancerandavailabilityfacilita torforbioactivemoleculesincludinganti-infectiveand anti-canceragents.Themoleculeswhichexpressanyactiv15 ityinformofeitherinhibitingorpromotingabiological function have been referred in this invention as bioactive molecule e.g. antibiotics, drugs, nutraceuticals, cardiovascular, hepatoprotective, neuro-tonics etc. The presentinventionhasdirectimplicationindrasticalyreduc ingthedosageofantibiotics,drugsandanti-canceragent whileincreasingtheeficiencyofabsorptionofbioactive molecules.
resistanceinparasitesandtheirpredominance,ultimately leadingtofailureofantibioticsagainstresistantinfections.
ThisalsoisresponsibleforSideefects,ilnesandreduction inlifeexpectancybeingmoreacuteintheolderpopulation. Oneoftheways,whichhasbeenfeasibletoreducedrug dosage,hasbeenSynergismbetweentwotherapeuticagents. However,ifbothhavetheantibioticproperty,stilthe problem of continued Selection presure on microbes is likelytocontinue.So,theaplicantsthoughtofutilizing cowurine,whichisnotmicrobicidalbutwhenpresentwith adrugoractivemolecule,enhanceitsactivityandavail ability(bioenhancers).Thisway,theSelectionpresurewil becounter-balancedSimultaneouslyreducingthedosageof antibioticsordrugsforminimizingtheSideefects,which hasalsohighcommercialimportance.
Thepresentinventionwastheresultofplannedexperi mentstoprovideanovelmethodforimprovingactivityand bioavailability of antibiotics, drugs and other molecules using cow urinedistilate indiferentformulations.
Thebioavailabilityofnutrientsandenhancementantibi oticsefectisrelevanttohuman,plantaswellasanimal healthandthusthecompositionsandmethodsoftheinven tionarealsointendedtobeusedinagricultureandVeterinary practice.
DESCRIPTION OF RELATED ART
BACKGROUND OF THE INVENTION
25
InAyurvedacowsurineisSuggestedforimprovinggen
eralhealth.Butitisneverscientificalytestedforanyutility
alone.Theaplicantshavedevelopedthecuriosityaboutthis
componentinthepreparationsandaskedmanyquestionsto
them;whetherthecomponentcowurineishavingany
activitybyitselforitdoesnothaveanyactivitybutenhance
theactivityofothercomponentsinthepreparations?What
arethecomponentspresentintheurineofcow’?Whetherthe urinecontainsmicroorganism,whicharebeneficial?35
Whetherthedegradationproductsfromtheurineareben
eficial?Toanswerthesequestionstheaplicantobtained
“KamadhenuArka”theurinedistilatefrom“Go-Vigyana
AnusandhanaKendraNagpur,India.Thisistheurine distilateSuggestedfordrinkingtoimprovethegeneral40
health and sold and distributed in different size bottles. The
aplicantstestedtheurineonLuriaagarandbrothinSterile
conditionanddidnotnoticeanygrowthofmicroorganism.
To testwhetherthisisinhibitorytogrowthofdiferent
microorganism,EschenichiacoliandMycobacteriumSmeg 45
matisweregrownatdiferenttemperaturesrangingfrom20
to 40° C. in presence and in absence of the cow urine
distilate,nosignificantdiferenceinthecolonycountis
noticed.Surprisingly,thesamedistilateenhancedtheanti bioticactiononthesebacterialeadingtothisinvention.The50
noveltyoftheinventionliesinthefactrevealedthrough
preciseexperimentationthattheenhancementactionandits
efectivenesisachievableonlyintherangeofconcentration
whichisliteralyinnanotomicromolarlevels.Andwhen
ahigherconcentration/dosageisusedintheformulationor 55
combinationstheactivity(ies)donotapear.Thatshouldbe
thereasonfornon-detectionSuchavaluablepotentialofcow
urine(Go-mutra).Frommiliondosesofannualantibiotics Theinventionrelatestonewuseofaknownabundantly
consumptiongoeswasteasthesecouldnotbeutilizedor targetedtotheinfectiveorganismsefectivelyduetovarious60 factorslikeeficientabsorption,transportationtothetarget Site,retentiontime,operationofefluxpump,metabolism etc.Thus,largeportionsofthedrugsweapplyarewasted andonlyaminisculepercentageisbeingtargetedtothe infectivemicrobes.Also,theunutilizeddrug/antibiotic65 amountremainsasaloadinthebodyandenvironment actingasaSelectionpressuretofacilitateemergenceofdrug
SUMMARY OF THE INVENTION
available cow urine distillate as an enhancer of antibiotic actiononthetarget.Themoleculeofinventionhelpsinthe
absorptionofantibioticsacroSSthecelmembraneinanimal cels,grampositiveandgramnegativebacteriaSimilar activitiescanalsobeobtainedbyusingthedistilateofthe urineofcowat40-50C.andfromtheconcentrate,which islyophilizedanddissolvedforfurtheruse.Furthertheurine
distilatefrombufalo,camel,deerprovidesSimilaractivity ofbioavailability.
Cow’surine(Go-mutra)canbeconsideredasthemost efectiveanimaloriginSubstance/Secretionwiththecapacity ofgeneralhealthimprovementbutitdoesneedSubstantia tionthroughScientificexperimentation.Thus,theaplicants considereditworthwhiletoscientificalylookatthisand definethemolecularbasisofthevaluesthroughinvitroand inVivoasays.Theaplicantsinthefirstinstanceprobed whetheritcontainedanydrugfacilitatorelementsSinceSuch apropertywouldmakeitahighlyusefulnaturalSubstance. Inrecentdays,useofpiperineasabioavailabilityenhancer hasbeendescribed(U.S.Pat.Nos.5,616,593and5,972, 382).Tiltodaythus,theknownbioavailabilityenhancer documentedispiperineandaSeriesofinventionsrelatedto thiscompoundhavebeendescribedinthefollowingprior artS.
OBJECTS OF THE INVENTION
Inanotherobjectiveoftheinventionistoprovidemethod
forimprovingactivityandbioavailabilityofantibiotics,
Themainobjectiveistoprovidenewuseofthebio-active fractionasabio-enhancerandasabioavailabilityfacilitator.
drugsandothermoleculesusingactivefractionfromcow urine distillate.
Stilanotherobjectiveoftheinventionistoprovidea processfortheextractionoftheactivefractionformthecow urine.

US 6,896,907B2 34
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING
FIG.1representsHPLCcharactersofcowurine(Go mutra)distilate
DETAILED DESCRIPTION OF THE INVENTION
OuremphasisherewastoSearchaplentifulyavailable material with bioenhancing action of higher potency. Aditionaly,apropertythattheaplicantsSearchedwasthe bioenhancementofaScarcelyavailableanti-cancernatural agenttaxol(peclitaxel)whichisproducedinmicroscopic amountsbytheYewtre(Taxussp.S.)andhenceisalways 15 alimitedmoleculeinavailability.Cowurinedistilate enhancedthekilingactivitiesofdiferentantibioticson bacteria.Moreimportantwastheobviousenhancementin theceldivisioninhibitoryactivityagainstthebreastcancer cell line MCF-7.
Inanembodimentofthepresentinventionapharmaceu ticalcompositioncomprisinganefectiveamountofcow urinedistilateasabioavailabilityfacilitatorandapharma ceuticaly acceptable aditives Selected from anticancer compounds,antibiotics,drugs,therapeuticandnutraceutic agents,ionsandSimilarmoleculeswhicharetargetedtothe25 livingSystems.
In yet another embodiment, the bioactive fraction enhancestheactivityofanti-bacterialagentsfrom2to80 folds.
Inyetanotherembodiment,theantibacterialagentisan anti-tuberculosis agent Selected from isoniazid, pyraZina mideandothersimilarcompounds.
Inyetanotherpreferedembodiment,thebioactivefrac tionenhancestheactivityofanti-tuberculosisagentsfrom2 to 20 folds.
Inyetanotherembodimentoftheinvention,theanti canceragentisSelectedfromgroupconsistingofPaclitaxel (Taxol).
Inyetanotherpreferedembodimentoftheinvention,the bioactivefractionenhancestheactivityofanti-canceragents from 2 to 20 folds.
The methodology followed by us for this screening includedSpecificalydesignedbioassaysasdescribedbelow. ThebacterialandfungalStrainsusedinthisinventionwere acquiredcommerciallyfromInstituteofMicrobialTechnol ogy(IMTECH),Chandigarhwhichpossessedcorrespond ingpropertiesoftheATCCStrainmentioned. 1.ASSayforBio-enhancementofAnti-infectiveAgents a)Theminimuminhibitoryconcentration(MIC)ofantibi
oticisdeterminedagainstEscherichiacoli(equivalentof ATCC 10536),Bacilussubtilis(equivalentofATCC 6015) and Mycobacterium Smegmatis (equivalent of
ATCC10231)inbrothanddiscdifusionasay. b)Theantibioticsagentsatconcentrations/4,1/3,1/2and equaltoMICareaddedaloneandincombinationwiththe testcompoundatvaryingconcentrationsondiscandin
brothtoevaluatethecomparativeinhibition. c)Thesecombinationshowingsignificantadvantageor higheractivitythanantibioticaloneintermsofenhanced inhibitionofbacterialgrowth(largeinhibitionZonein discdifusionandafectivityoflowerconcentrationin
Inanotherembodiment,thecowurinedistilateisusedas bioavailabilityfacilitatorforanticancertherapydirectlyorin combination with anticancer molecules.
Instilanotherembodiment, thecow urinedistilateis usedinantifungaltherapyforfungalinfections.
Inyetanotherembodiment,theantifungalsareazoles, clotrimazole,myStatin,amphotericinandSimilarmaterials.
Inyetanotherembodiment,fungicoveringinfectionsare mycetial,candida,yeastorotherfungicidalcompounds.
35
Instilanotherembodiment, thecow urinedistilateis usedinTBtherapyincludingmultidrugresistanttubercu brothasay)arepickedupforfuturetesting.
losisincombinationwithisoniazidandotheranti-tubercular agents.
Inyetanotherembodiment,thebioavailabilityfacilitator helpsintransferingthecompoundacroSSthemembraneand forbeterefectivityonthetargetsite.
40
45
d)Inbrothasaytheactivityisquantifiedbycounting numberofViablecelsinagiventreatmentandconverted in fold enhancement by combination compared to antibiotic/drugaloneinthekilingpercentageofcels.
e)Thepre-treatmentasayfollowedtodeterminewhether thecompoundisrequiredalongwithantibiotictoenhance itsactivityorevenitswithdrawalaftertreatmentorprior toantibiotictreatmentwouldbenefit.Forthis,thecelsare
In yet another embodiment, the antibiotics are
Quinolones,fluoroquinoloneslikeNalidixicacidandothers
likeRifampicin,Tetracycline,amplicilinandSimilarcom treatedwithcompoundfor4to8hoursandthenwashed
pounds.
In yet another embodiment, the antibiotics, ions and Similarcompoundsareisoniazidandhydrogenperoxide.
Inyetanotherembodiment,thebioavailabilityfacilitator helpstheantibioticsandothermoleculestoactbeteronthe targetbyincreasingtheefectivity.
In yet another embodiment, the living System may be bacteria,fungioranylivingcels.
Inyetanotherembodiment,theantibacterialagentsare Selected from the group comprising Quinolones, Rifampicin,Tetracyclineandamplicilin.
50
55
freofitbycentrifugationandwashinginSterilewater.
Thiswasfollowedbytreatmentwithantibioticasinsteps b to d.
2.ASSayforBio-enhancementofAnti-cancerAgents
a)MCF-7(Breastcancercommercialcellineobtainedfrom National Center for Cel Sciences (NCCS), Pune) is inoculatedatadensityofabout0.1×10 celsinMEM mediuminthewellsof24wellplate.
b)Thisisreplacedwithfreshmediumafter18hoursineach well.
c)Thetestcomponent(S)isaddedatdesiredconcentrations indiferentwellsjustafterthemediumreplacement.
d)Observationsarerecordedonthecelcountafter36hours forwhichthefollowingStepsarerequired.
i.The medium is removed from the wells.
i.Thewelsarerinsedwith1mlPBS(Phosphatebufer Saline).
Inyetanotherembodiment,thecowurine(Go-mutra)60 distilateisintherangebetween0.001ul/mlto100ul/ml.
Inyetanotherembodiment,thelyophilizedactivefraction usedisintherangebetween0.1lug/mlto100lug/ml.
In stil another embodiment, the bioactive fraction 65 enhancestheactivityofanti-bacterialagents,anti-cancer i.Toeachwel500uloffreshlypreparedtrypsin(0.1% agentsandanti-tuberculosisagentsfrom2to80folds. inPBS)solutionisadded.

Sodium Bicarbonate = Penicillin G = Streptomycin= Gentamycin= Foetal Calf Serum = Foetal Calf Serum = Distilledwater=
0.22 g 1O 2O
5 15 15 85
3.BioavailabilityTestsThroughBiologicalMembrane
ItwascalculatedasSurvivalfractionofviablecelsupon
US 6,896,907B2 S6
iv.TypsinSolutionisremovedafter30secondsandthe plateisgentlytappedtilthecelsarereleasedfromthe plateSurface.
v.Fresh1mlofMEM growthmediumisaddedand agitatedwithapipetetoobtainacelSuspension. vi.10ulofcelSuspensionistakenonthehaemocytom
eterandacoverglasisplacedoverthecounting chamber.
vi.Thenumberofviablecelsiscountedin5bigsquares andthereadingsaretakenfrom5microscopicfieldsto determinetheaverage.
vi.Thecelcount(titerperml)intheoriginalsampleis thencalculatedasaveragecountx10.
CompositionofMinimumEsentialMedium(MEM);100 ml
MEMpowder(Sigma-Aldrich,USA)= O.96 S. HEPESBufer(Sigma-Aldrich,USA)= 0.26g
15
c)Steriledistiledwaterwasthenfiledinboththesidesto equalheight/level.Theantibiotic/compoundwasaddedto
thedonortube(tapered)andthroughspectro-photometer, thetransferofmoleculewasobservedusingUw and Visibleabsorptionmaximaoftherespectivemoleculesby takingtheOD atdefinedwavelengths.
EXAMPLES
Inthenextstepofelucidationoftheenhanceraction,the aplicantsexperimentedwiththekilingactivitiesofdifer entantibioticsagainstthebacteriaSinglyandincombination withthetestcomponent(cowurinedistilate)folowingthe methoddescribedabove.Theseexperimentsarebeing described in the following examples. When the bacteria were grown in presence of the compound as Such no Significantkilingwasobserved.Inaltheexperimentsthe cowurinedistilateconcentrationwaskeptat1ul/ml,unless itisspecificalymentioned.
Example1
Cowurinedistilatemediatedenhancementinthekiling action of antibiotics against Gram-negative bacterium Escherichia coli.
TABLE 1.
Survival fraction Survival fraction of * Fold ofviable viablecelsupon enhancement
Con- cellsupon treatmentwith anti- in centration treatment with biotic + cow urine antibiotic
Antibiotics pig?ml antibioticalone distilatecombination activity
Rifampicin 1O O.86 Rifampicin 3O O.05 Ampicillin 4 1.11 Ampicillin 6 O.09 Ampicillin 8 O.05
0.17 5.0 O.OO7 7.1 O60 1.85 O.O2 4.50 O.O1 S.OO
a)SpecialydesignedU-tubesofglasconsistingoftwo treatmentwithantibioticandcowurinedistillatein components(oposite-Ltype)wereusedinwhichone45 combination/Survivalfractionofviablecelsupontreatment
openendofanL-shapedwastaperedtofitwithinthe untaperedendoftheotherL-tube(FIG.1).
b)Themembraneofgoatgut(initialpart)wasstretchedand 50 fixed to act as the barrier between the two ends such that
byjoiningthetwoL-tubes,aU-tubewasmade.
with antibiotic alone
Example2
Cow urinedistilatemediatedenhancementinthekiling actionofantibioticsagainstGram-positivebacteriumBacil lusSubtilis.
Con- centration
TABLE 2
Survival fraction Survival fraction of * Fold ofviable viablecelsupon enhancement
cellsupon treatmentwith anti- in treatment with biotic + cow urine antibiotic
Antibiotics pig?ml antibioticalone distilatecombination activity
Rifampicin O.OOS Rifampicin Ampicillin O1 Ampicillin 0.5
1.1 O.28 5.5
1.OO O.3 3.3 O.18 O.O6 3.0

Isoniazid Hydrogen peroxide (HO)
O.99 x 107 7.5 1.2 x 107 5.9
Taxol Concentration pig?ml
O.OO1 O.OOS O.O1
titre ofviable cells
O.9 x 10 0.9 x 10 0.9×10
cellsupon treatmentwith taxolalone
O.O59 x 10° 0.042 x 10 0.036×10
taxol+ cow urine distilate
O.O39 x 10° 0.032 x 10 0.012×10
Example7 65 DevelopmentofPowderForm
Forenhancingtheutilityandconvenienceofaplication ofcowurine(Go-mutra),theaplicantsfurtherfractionated
Concentration
250 tug/ml O.OO3% w/v.
HO, alone
7.5 x 107 7.14 x 107
combination
activity
US 6,896,907B2 78
Example3
Cowurine(Go-mutra)distilatemediatedenhancementin thekilingactionofantibioticsagainstbacteriumMycobac teriumSmegmatis
TheaplicantsobservedSimilarresultsofenhancementin theanimalcelculture(cancerouscellineMCF-7obtained fromNationalCenterforCelSciences(NCCS),Pune),in whichthekilingactionofanticancerouschemical Taxol isincreased.Thecomponentsofcowurinedistilateinour
Con- centration
Survivalfraction Survivalfractionof *Fold ofviable viablecelsupon enhancement
cellsupon treatmentwith anti- in treatment with biotic + cow urine antibiotic
TABLE 3
Antibiotics pig?ml antibioticalone distilatecombination activity
Rifampicin Rifampicin Ampicillin
O.OS O.O08 O.1 OOO6 0.5 O.O7
Example4
O.OOO1 😯 O.OO36 1.5 O.OO6 11.6
Studyhelpintransferingothercompoundsacrossthemem branetherebyincreasingtheabsorptionin,irespectiveof bacteria,animalandplantcel.Thisin-turnhasimmense
Cowurinedistilatemediatedenhancementinthekiling
action of izoniazid and hydrogen peroxide against OxyR 20 importance for absorption of the drugs, pharmaceuticals,
mutant of bacterium Escherichia coli. The cow urine distill lateconcentrationis0.001ul/ml
nutraceuticalandotherrelatedcompoundsandionsbythe cells.
TABLE 4
Survival fraction ofviable
Survival fraction ofviable celsupon
treatment Fold celsupon withaisoniazidf enhancement
treatment HO +cow in withisoniazidf urinedistilate isoniazid/H.O.
TheOxyRgeneisrequiredfortheinductionofaregulon
Example6
ofhydrogenperoxide-induciblegenesinEscherichiacoli40 (ChristmanMF,StorzGandAmesBN(1989).OxyR,a BioenhancementofantifungalagentclotrimaZolbybio
positiveregulatorofhydrogenperoxide-induciblegenesin activeFractionGm-IV.Theconcentrationoftheactive
EscherichiacoliandSalmonellatyphimurium,ishomolo goustoafamilyofbacterialregulatoryproteins(Proc.Natl. Acad.Sci.(USA).86:3484-3488.).Themutantsofthese45 genesareSensitivetodrugslikeisoniazidandhydrogen peroxide,whichproducefreradicals,damagingthecelular Systems.Sothekilingactivitiesofthesecompoundsare increasedby5to8foldsbycowurine.
fractioniskeptat10ug/ml.
Example5
50
55
Treatment
Clotrimazol Clotrimazol+Gm IV
TABLE 6
Minimuminhibitory Concentration(MIC) Fold
intug/ml enhancement
4.40 O.O O.88 5.0
Cowurinedistilatemediatedenhancementintheactivity of anti cancerous compounds. The cow urine distilate concentrationis1ul/ml
Inotherobservationsthecompoundcowurinedistilate enhances the transport of antibiotics e.g. Rifampicin, Tetracycline,Ampicillinacrossthegutaswellasartificial membrane.Theenhancementintransportisapproximately 2 to 7 folds.
Greatemphasisnowisbeinglaidtowardsqualityassur anceofcrudedrugsfromalternativeSourceswidelyusedin the Indian System of medicine. The present invention enlargestheScopeanduseofthecow urinedistilatein therapeuticalandnutraceuticalaplication.
TABLE 5
Finaltitre celsupon
Initial ofviable treatmentwith 60
Final titre ofviable

Hexanefraction(Gm-I)
Ethylacetatefraction(Gm-II)
Butanolfractioncontaining paleyellowprecipitate(Gm-I)
Aqueousfraction
Extractedwith Ethylacetate
aqueousfractioncontaining whiteprecipitate
Extractedwithbar
aqueousfraction withwhiteprecipitate
Dried Whitepowder(Gm-IV)
US 6,896,907B2
9 10
toreachasolidformwhichisalsofreofthetypicalsmell ofcowurinedistilatethatitismorereadilyacceptabletothe humans.Forthispurposetheurinedistilatewasfraction atedasdescribedbythefollowingprocedure:
FractionationofaWhiteCrystalineSolidfromCowUrine
Step1:Cow urineiscolectedasepticalyintheStainleS Stelcontainerdirectlyfromthecow,whichismaintained inhygienicenvironment.
Step2:Fiftenlitersofcowurineisdistiledcontinuouslyat 40-50C.inglasdistilationapparatustoobtain10-1215 liters of the distillate in 16 to 18 hours.
Step3:ThedistilateispackedinSurfacesterilizedplasticor glascontainerforfurtheruse
Step4:200mlofcowurinedistilatewasmixedwithhalf the Volume of methanol and extracted with hexane.
Step5:Thehexanefraction(Gm-I)waslyophilizedand testedforsimilaractivityasthatofcowurine(Go-mutra). Step 6: The aqueous fraction was extracted with ethyl
acetateandtheethylacetatefraction(Gm-II)waslyo 25 philizedandtestedforSimilaractivityasthatofcowurine (Go-mutra).
Step7:Further,aqueousfractioncontainingwhiteprecipi tate was extracted with butanol and the butanol fraction (Gm-M)havingpaleyellowprecipitatewaslyophilized andtestedforsimilaractivityasthatofcowurine(Go mutra).
Step 8: The remaining aqueous fraction containing white crystalineprecipitate(Gm-IV)wasdriedandtestedfor35 Similaractivityasthatofcowurine(Go-mutra).
Scheme
200 ml cow urine distillate mixed with 100 ml methanol
Thewhitepowder(Gm-IV)thatwasobtainedintherange of10to20gramsper100mlofthedistilateshowedalthe above activities as described for cow urine distillate at
concentration0.001to10ug/ml,withmuchmorestability andbeingdevoidoftheunpleasantSmellandhencewas usedastheadvancedproductoftheinvention.Thenovelty oftheinventionliesinthefactrevealedthroughprecise experimentationthattheenhancementactionanditseffec tivenesisachievableonlyintherangeofconcentration whichisliteralyinnanotomicromolarlevels.Andwhen ahigherconcentration/dosageisusedintheformulationor combinationstheactivity(ies)donotapear.
PhysicalCharactersoftheGm-IVFraction Color: White
Physicalstate:SolidCrystaline Solubility:Water-solubleandmixturecontainingwater Meltingpoint:Above400°C. SpecificGravity:1,006 RFvalueinmethanol:Chloroform(50:50)phase:0.65
HPLCCharacterizationofCowUrine(Go-mutra)Distilate
HPLCwasperformedonLC-8AShimadzuHPLCwith mobile phase water: acetonitrile (80:20), flow rate 1.0 ml/min, UV detection at275 nm and C-18 E MERCK (150x4mm)column.Twomajorpeaks(retentiontime5.334 and 11.310 min) observed in the profile of cow urine
Extractedwith Hexane
40 (Go-mutra)distilate(FIG.1).
Furthercharacterizationtotestthechemicalnatureofthe compoundwasperformedthroughFeigl’stest(In:EStahl, ThinLayerChromatography)whichwaspositiveindicating
45 thepresenceofglycosideorSugar.
The novelty of the invention is that from cow urine
(Go-mutra)distilate,astablesolidfractioncouldbeisolated 50 whichiswaterSolubleanddevoidoftheurineSmellandcan
directlybeusedinanyformulation.
ThefractionGm-IValsoenhancesthetransportofanti 55 bioticsandVitaminsacroSSthemammaliangutmembrane. Theexampledescribingtheenhancedtransportofrifampi
cinbythefractionGm-IVisgivenbelow. 60 Example8
FractionGm-IVofcowurine(Go-mutra)distilatemedi 65 atedenhancementinthebioavailabilityacroSSthebiological membrane(Rifampicin,1mg/mlandfractionGm-IV,1.0
lug/ml).

TABLE 7
Wave ODmeasuredasAbsorbance(specifictothecompound Compound(s)inlength maxima)acrosthemembraneinreceivingtubeafter
the donor tube (nm)
Rifampicin A340 A47s Rifampicin+ A340 Gm-IV A47s
1 hr 2 hr 3 hr 4 hr 5 hr 6 hr
O.OO97 O.O214 O.O334 O.O771 O.O858 O.O910 O.O177 O.O284 O.O.309 O.O412 O.O484 O.O496 O.O525 O.O961. O.1353 O.1639 O.1919 O.1989 O.OSO2 O.O904 O.O793 O.O966 0.1157 0.11.83
US 6,896,907B2
What isclaimed is:
Gm-IV, both shows enhanced membrane permeability by 1.A pharmaceuticalcompositioncomprisingatleastone
enhancingtheantibioticsacrosstheSemi-permeablemem 15 anticanceragent,andacowurinedistilateoradried
AS cow urine (Go-mutra) distilate and the fraction
braneandmammaliangutmembrane,itcanbeassumedthe
aboveSubstancescanbeusedforenhancingintestinaltrans 2.Apharmaceuticalcompositioncomprisingatleastone
portandtransportofmoleculesacroSSmembranesofvarious biologicalfunctionsincludingurinary/renalSystems.
Inaltheexperiments1to5,theenhancingactivityofthe lyophilizedproductoftheinventionisfoundtohavesame enhancingactivitylikethatofthedistilate.
anticanceragentandcowurinedistilateoradriedfraction (GM-IV)obtainedfromcowurinedistilate,whereinthe cowurinedistilateispresentinaconcentrationrangeof 0.001ul/mlto100ul/ml.
3.A pharmaceuticalcompositioncomprisingTaxoland
Theinventioncanfurtherbeexplainedasfolows: 1.Thesampleshowsenhancementofbioavailabilityof25 fromcowurinedistillate.
rifampicin(antibiotic)andVitaminB-12acrosthe mammaliangutmembrane(Goatintestinewasused) within2hoursanditkeepsincreasingupto4hours.
Itshows clear inhibition of ascorbic acid action to preventoxidationofcutappleindicatingthatitprob ablyenhancestheisoniazid(INH)byoxidativemecha nismthatsynergisesthedrugactionofINH.
.The distillateshowedenhancementactionforINH even at 10-50 thousand-fold dilution in the final volume of culture.
.Stilmoreinterestingobservationisthatat1ul/mlthe distilateshowedenhancementintheactivityoftaxol byatleast5folds.Furtherexperimentsinthisdirection havebeentakenuponpriority.
4.Thecompositionofclaim1,whereinthedriedfraction
(GM-IV)ofthecowurinedistilatecanbeobtainedby lyophilization.
5.Thecompositionofclaim4,whereinthedriedfraction (GM-IV) obtainedfrom thecow urinedistilatehasthe followingphysicalcharacteristics:awhitecolor,aSolid crystalineform,watersolubility,ameltingpointabove400 C.,aspecificgravityof1.006andanRFvalueinmetha nol:chloroform (50:50)phase: 0.65.
35 6.Thecompositionofclaim4,whereinthedriedfraction
(GM-IV)obtainedfromthecowurinedistilateisdevoidof a cow urine Smell.
fraction(GM-IV)obtainedfromcowurinedistilate.
cowurinedistilateoradriedfraction(GM-IV)obtained