Cow urine has been granted US Patents (No. 6,896,907 and 6,410,059)

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
O 5 1O 15 20

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

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