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    Antiviral Colloidal Silver Composition

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    2) United States Patent (10) Patent No: US 8,753,691 B2 Holladay et al. (45) Date of Patent: Jun. 17, 2014 (S8) ANTIVIRAL COLLOIDAL SILVER FOREIGN PATENT DOCUMENTS Composirios, a rouge A sams (75) Inventors: Robert J. Holladay. Logan, UT (US); n aaa eae ADIN S16 Willa D. Moeller. Alpin, UT(US) fy. g gzLOd A Wo Woannesss at (73) Asignwe: American iver LLC. Alpine, UT(US) WO woSnesumos4 42 Wo Woomenmily a2 (*) Notice: Subject to any disclaimer, the term of this. WO WO-2007004987 AL 7/2007 patent is extended or adjusted under 35 WO WO2008147427 122008 USC, 154(b) by 67 days. OTHER PUBLICATIONS (21) App. Nos 11838262 Laan, "Srinath atinisoil ft and sayin tse Caren Proms in Demasiogy (000), 33, 1734 (22) Filed: Oct. 3, 2006 (abstract). Tain of Korean ae 200679388 (206) * (65) Prier Publication Data ‘Translation of Korean Patent No. 2006-079388 (Cho ea, 2006).* coe ae Nochntastatn 1200823198 (ng 209," Z Metical Uses of iver, Wikis. pc kia or iki Related US. Appleaton Data Modal uf sve asd May 26200 Giang ctl “Snty of Soomro avi Aton” (68) Contimatoninpar of aplication No, 116.938, Vien Sta Teh DS, 20) pp. 1. fled on Avg 15-2008, now Pat NO-7135,195 and “Amin Me” Google Seach sce Ma 19,2012! contimationinnpat of application No, OOS46834, wwnegapecom exclu omei228ts-com fedon Sep. 42001, now Put. 6743348. whichis nomena ‘contnsation of pplication No, 08/3230, ldon Gar, Stina “iver Nsppaicesas Pt ati Jun 1, 1585, now Fat No. 6.214.299 ‘get Maes, 2011 I 120508 pp BOLT ‘Wipta~Aatl Dg. p11 pe ip (@) Provisional application No, 60475687, filed on Jun, Rana ane tacoma W201 ne 3, 2008. International Search Report of PCT/US2007/080278, (mailed Sep, 230) (51) Int. Cl. Office Action dated May 13, 2011 in Malaysian application No, AGIK 33/38 (2006.01) P120091359. . aa sib 18 (200601) (2) US. * ced by examiner CPC. AIK 3738201301); AIL 2718 201301) see ee “24/618 Primary Examiner —Daniel Salvin (8) Field of Classification Search, Assistant Examiner — Barbara Frazier Nowe (18) torn, Agen, oF Firm — Venable LLP; Stn So opplition fe fr complet search sary Kirtansi 66) References Cit 6 ansTRact . We dale a colorless composton sompising sve par US. PATENT DOCUMENTS ticles and water, wherein said particles comprise an interior of 624420 B1* 42401 Hadeyetal.. 2919621 semen sive anda cero icnic ever oxi erin SPA RES Ot Hy Sat STRGAL serps present water a ee of boa Sh0098 mS Ann 8 and wherein he comnoaition mane spit BIOS 11186 ly a 2461 trl pers inland popes Mt aooziosisa3 Al¢ $2002 Yanetal aa4ols Fro ip snlival prope aaah aaah eaa SESS cof ofthe composition are desea Dooseatseos Als 112008 ns Sika: dourniaes Ale 102007 Topo inbeae Claims, No Drawings US 8,753,691 B2 1 ANTIVIRAL COLLOIDAL SILVER ‘COMPOSITION ‘The present application isa continuaton-in-part of appli- cation Ser No. 10/64], 938, filed Aug, 15,2003, now U.S. Pat No. 7,135,195 which in tumnisa continustion-in-par of appli- cation Ser. No, 09/946,854, filed Sep. 4, 2001, now US, Pat, No. 6,743,348 and anon-rovisional of and claiming peorty from provisional application 60/475,657, filed Jun. 3, 2003, hich application i incorporated by reference herein: appli cation Set. No, 09/946,84, filed Sep. 4, 2001, now US, Pat, No. 6,743.MB is itself a continuation of application Set No, (00/328,310 ied Jun. 1, 1999, now US. Pat No. 6214209. AREA OF THE ART ‘The present invention generally relates to colloidal iver, and more particularly to composition of colloidal silver and a method for using said composition as an agent against ‘organisms harmfil o the health of humans—in particular avin influenza views (*ind "Th DESCRIPTION OF THE PRIOR ART 11s well knowa that certain preparations of silver have ‘germicidal properties, Silver was employed as a germicide and an antibiotic before modem antibities wore developed, Inprevious centuries, users would shave silver particles into theie drinking water, or submerge whole silver pieces ia the inking water, for the purpose of ingesting the silver by ‘drinking the wate. It seems likely that the practice of eating ‘with silver utensil (Le, silverware) resulted ftom a belief in the healthful propertis of silver. ‘There may be many reasons why administering silver sus pend insoluion would enhance an individual's health Its possible that such a solution operates to inibit the growth of bacteria, virwses, and other unwanted organisms, as well as ‘eradicating such existing bacteria, viruses, and other organ isms, Is also possible that a solution of silver can have an ant-inflammatory effet, suficient to reduce symptoms of asthma. “The present invention describes the use ofa silver compo: sition in water to treat certain human ailments. An embodi- ment of the invention isa silver composition comprising small particles of silver which compriseaninteror of metallic silver and aa exterior of ionic silver which particles ae sus- pended in water. A prefered embodiment of the inventions a silver composition comprising particles of silver wherein more than $0% of the particles are less than 0.015 microme- ters in size and the particles are colloidlly suspended in water ‘SUMMARY OF THE INVENTION “The present invention is generally directed to the use of silver, ata level of $ to 40 ppm in water, oki ort disable microorganisms, such as avian influenza vim, which are hazardous to lnman beings. The present invention specii- ‘ally is diected to compositions comprising silver particles, said particles comprising an interior of elemental silver and nexterior of one silver oxide, and water, wherein he silver particles are placed in colloidal suspeasion in the water at a level of 5-40 ppa total silver. An embodies ofthe present invention comprises silver particles in water, a concentra tion of $-40 ppm, wherein more than 50% of the silver pae- ticles havea maximum dimension less than 0.015 microme- ters. The composition of silver in water ofthis invention isan 2 elective antimicrobial agent. This invention is dicted to siker compositions, of $40 ppm silver in water, which are effective atinicrbial agen, and to mad of using sid siver compositions s antimicrobial agent AV prefered emidiment of the present invention is direted to compositions of sive in water made using a notification ofthe devi and methods descrbodin US. Pat. No.6214.299, which sa parent ofthe instant appicasonand ‘8 incorporated herein hy eeerence ‘The device and process of US. Pat. No. 6,214,299 have boon modified and improved o provide the silver eompon- tion ofthe present invention Essentially the eight-silverione common electrode devie as dsslosein the patent has been modified and scaled tft a 75-gallon water chamber To stert the process approximately 70 gallon of high pry waterare placed in the chanber. To this is added approximately five Ballons of slvr composition podced ina peoeprodction fun. Thisis necessary because th igh purty water is insuf- ficiently conductive forthe process to occur propery. The water chamber is equipped wth an ai input that allows 3 stream of ir bubbles to be steamed through te liquid dring thoprocessng, thas ben discovered tht his approach gives improved mixing. as compared to the impeller mixer deseribed inthe patent. ‘Theclecirode devices operated at approximately tenthou- sand vllsalterating current wth each sikereectode v= ing an individual voltage supply) as desceibed inthe patent It tas boon found tht voltages significa lower tan his produce composition with larger patiles aot having the ‘ptm properties described. herein. Voges sgnficany higher tnd to producea solution with significant oie silver dissolved therein, The present composition comprises in exces of 97% metallic silver with essentially no fre onic silver in solton The silver concentration is determined according to the methods explained below. Essential the device is operated continuously andsaples ae analyzed the desired silver concentration s attained. The 10 ppm composition requires approximately one and one half days of operation. The 22 rpm soliton requires approximately three day, and the 32 hm composition requires appeoximatly six day, Te rate the eoces ppearstslow athe higher concentration are attained, Higher eonentatons ake a prohibitively longtime with the ulimste highest concentatio being about $0 ppm atlas within the cient parameter. ‘The compositions all have the size characterises deseribed below and unlike conventional iver compositions are completely colorless and sabe to ight and temperature changes without use of any additives. The compositions are unreactive towards aed ydrogen peroxide Hydrogen peroxide, a known dsinfoctng agent, hasbeen found to have a synergistic interaction with the inventive silver composition. Hydrogen peroxide is avaiable in con- cenitation of MP6 waht weiht er volume or weight percent [abt %]) or higher. Although the higher ouceata- tions are usable the prefered concentrations arin the range of Lo S% wghv ‘A prefer embodiment of the present invention is directed to compositions competing 5 to 40 ppm silver sid silver being primarily elemeata silver | to 3 wight % byo- gen peroxide, and water. A prefered embodiment of the presen inventions the se, andl method of use, of compos tions comprising 10 to 40 ppm silver and Ito 3 waht % hydrogen pewxide in wate a antimicrobial agents DETAILED DESCRIPTION OF THE INVENTION “The following description is provided to enableany person skilled in the art to make and use the invention and sets forth US 8,753,691 B2 3 the best modes contemplated by the inventor of carrying out his invention. Various modifications, however, will remain readily apparent to those skilled in the at, since the general Principles ofthe preseat invention have been defined herein specifically to provide an improved colloidal silver product with significant bilities to kill human pathogens bth in vivo and invite ‘Generally, the present invention represents a aovel approach to killing or disabling microorganisms which are hazardous to human beings by the use of silver partes in Water, at a concentration of § to 40 ppm silver. Depending upon the application, the silver composition may be used internally or extemally. Depending on the application, the silver composition may also contain hydrogen peroxide PREFERRED EMBODIMENTS. Non-limiting prefered embodiments axe presente inthe following Acompositon comprising silver paricles, coolly sus- pede in water, wherein he oa content fiver isbetween 5 and 40 pp, which composition ills or disables microoe janis Which ae hazardous to the human body Acompositon comprising silver paricles coolly sus- pode in water, wherein the tot content of sve is about 1022 ppm, which composition kills or disables migroorgan- isms which are hazardous othe hurnaa Body composition comprising silver paricles, coolly ss- pended in water, wherein the total content of sive is about 7222 pp, which composition kills or disibles micoorgan- isms which are hazardous othe human Bad composition comprising stver pricks, colodally sus- pended in water, wherein the total content of ives about 3233 ppm, which composition kills oe disables miroorgan- isms which ae hazardous tothe human body. Ie il be appreciated thet specifying the total amount of silver ia 8 composition of partiles does not completly Speci the material. Asthe particles comprising the compo ston are made smaller, given concentration of iver will represent a ager numberof partici, In ation, the total surlace ara for a given silver concentration will increase ‘Therefore, articles sizeandrange of parilesizeisniempor- tant parameter for defining an effete inventive composi ton, ‘A further class of embodiments 8 any of the shovede sere compositions, wherein more than $0% of the iver paricles have «maximum dimension Jess than 0.018 inicometer. ‘A further class of embodiments is any ofthe shovede- sero compositions, wherein more than 75% of the siver particles have maximum dimension less than 0013 Inicomete. A further class of embodiments is any of the abovede serio compositions, wherein more than 50% ofthe iver particles haves maninum dimensionless than 0.02 mierome- tes. A farther clas of embosments s any of the abovede- scribd compositions, wherein moze than 75% of the silver panicles have 2 minimum dimension greater than 0005 Imirometer. A faker cas of embostments is any of the abovede- scribd compositions, wherein moze than 0% of the silver particles have 2 minimum dimension greater than 0005 Imirometen. ‘A farther class of embodiments is any ofthe abovesde- scribd compositions, wherein the silver particles comprise bos siverin the zero-valnt, tha is, metalic, oxidation tate 4 [Ag(0) and a coating of silver in a ionic oxidation selected Jom the group consisting of Ag), Ag(), and Ag), ‘A further class of embodiments is any of the above-de- scribed compositions, wherein the silver particles comprise both silver inthe ero-valent, that is, metallic, oxidation state [Ag(0)| and « coating of silver in an ionic oxidation selected Jom the group consisting of Ag(1), Ag(), andl Ag( Experimental evidence shows that AgO in the particles of the present invention is at leas partially in the form of Ag,0,—that is, silver oxide. Inaamolecule of ths materia ‘wo ofthe silver atoms are in the I* state (silver I) while the other two silver molecules are in the 3 state (ver IID, Under certain conditions these molecules can give rise to silver atoms in the 2° (silver 11) state ‘A further class of embodiments i the combination of ay ofthe above-deseribed embodiments with hydrogen perox- ie, ta level of 1-3 wat % hydrogen peroxide in the final product. EXAMPLES, 1, Formation of Composition ‘Compositions of silver in water can be made aceon to procedures set forh in US. Pat No. 6.214.299, incorporate by reference herewith ‘A prefered method for producing a composition compris: ing silver according to this invention utilizes a eleetocheeni= cal cell comprising electrodes and comprises the steps (a) placing a silver electrode in coniaet with a quantity of high purity waters (b) conveying eletrical curent through the silver elec= trode to thereby separate panicles of silver from said silver electrode in a manner sufficient to cause prodution of sus- pended silver particles within the water, and {agitating the water during sad production of suspended silver particles to thereby disperse the silver particles into a ‘more uniform coacentation Within suid water such that 8 higher quatityof suspends silver particles can be produced per batch ‘Another preferred method for producing a composition comprising silver uslzes an eletrochemical cell and com- prises the steps of: (a) establishing an electrical circuit comprising a current source, and a fist conductor electrically conected to said curreat source anda second conductor electrically connected to said curent source, wherein said first conductor is disposed spaced apart om said second conductor, and whereinat least one ofthe conductors is made of elemental silver, (closing the circuithy placing the first conductor and the second corsletor in commnication with a fui resistor, (6) operating the eurent source to supply altemating eur reat simultaneously to the fist conductor and the second conductor such that voltage is increasing and decreasing within the fst and second conductors in altemating tandem, to thereby cause silver particles to separate from the first clectnode and eater the fide resistor und become disposed in suspension within sid fide resistor; and (q) selectively adjusting the electrodes by moving them ‘ovard he Muidic resistor to compensate for decrease inelec- trode length defo gradual separation of silver particles there= from to thereby prevent arcing. from oecurring between the electrodes and sad fide resistor, “The analysis the silver content ia the silvercompositions of this invention may be done by atomic absorption (AA), inductively coupled plasma/atomic emission (ICPYAES), ot other techniques known tone of ordinary skilin the art to be sensitive to silver inthe appropriate concentration range. If US 8,753,691 B2 5 thepantclesofthesilvercompositionaresmalland uniformly sized (for example, 001 micrometers or less), reasonably ‘accurate assay may be obtained by running the colloid direlly by AA orICPYAES, Thisis because the sample prepa- ration for AA jonzes essentially all ofthe silver allowing its ready detection If the compositions comprise particles as large as 0.2 imjerometes, its prefered to use a digestion procedure. The digestion procedures not necessarily idea for silver compo sitions that may have been manufactured or stored in contact With halides or other anionic species that may reat with finely divided silver, or combined with poten or other get nous material. An embodiment ofthe digestion procedure is 8 follows Take @ 10 ml aliquot of a thoroughly mixed or shaken silver composition wo be analyzed, and plae it ina clean polyearbonate botle or other container of suitable material (gonerlly, dhe bottle) with tight fiting i. size of 30-100 lis protec. 2 With a mieropipete or dropper, dd 0.1 ml of tic acid, reagent grade tothe silver composition in the baste 3 With the lid ofthe bot tightly in place, heat the silver ‘composition o 80° C with mild agitation foratimesuficient to dissolve the silver—s0aases9 Sos 258 umenise 30 “Boapso Sen” 8as fssnise 230 4 Cuneniss 35012525 saos Gnnenime 380 okS9 Au funmeniss 80 snazs “susan oweuse 38088028 Data are presented as MICIMBC (inion inhibitory concenraton/minimum bactericidal concenteation) in parts permillion(ppm));">" denotes that the concentration needed ‘wobvainthe MIC orthe MBC was higher than est parameters reasued forthe test, For example, the highest concentation of tetracycline used on S. progene was 5 ppm. At that con centration there was still growth upon subculturing ofthe “no growth” tubes. Therefore, the MBC must be (eater than) 5 pin ‘The MICIMBC of Ecol strain 0 157:HT, which has been associated with outbreaks of hemorshagic diarea and coli- tis, was determined in a subsequent study. The MIC was determined to be 25 ppm andthe MEIC was determined to be Spa. C.Conelusion ‘The 10 ppm silver composition of the present invention was tested and found o be both bacteriostatic and bactericidal fo all organisms tested. In other studies, this composition ‘was compared fo other commercially available colloidal sil- ver produts and found to havea superior activity tall other preparations tested (data not shown. The most interesting observation was the broad spectrum that the 10 ppm silver US 8,753,691 B2 13 ‘composition possesses. The antimicrobial activity that was ‘observed was fairly constat independent of the particular ‘organism tested. Withthe exception of Srepiococeus faecalis and Sreprococcus aureus (which had MIC values of 10 ppm and 5 ppm, respectively), MIC values ranged between 1.25 ppm and 2.5 ppm for both gram postive and gram negative ‘organisms. The MBC values behaved similarly with values angg from 1.25 ppanto 5 ppm withthe exception of Sep ‘ococcus mutans, Streptococcus gordon, and Sirepiococcus faecalis (which all hod MBC values of 10 ppm). The data ‘suggest that 10 ppm silver embodiment of this invention ‘exhibits an equal or broader spoctrum of activity than any one autibiotie tested. Antbioies generally have restricted anti- bacterial spectra limited to susceptible organisms, but athe data demonstrat, the silver composition ofthe present iven- tion is equally’ effective against bath gram positive and gram, negative onganisms. The data suggest that withthe low tox icity associated with silver, in general, and the broad spec trum of antimicrobial setivity ofthis silver composition, this, preparation can be effectively used as an alternative to ant biotics. D, Reference for Preceding Example 1 US. EPA IRIS Repor for Slver-CASRN 7440-22-4 2 Fox CL, Modak S M. Mechanism of Silver Sulphadiazine Action on Burn Wound, Infections. Antimicrobial Agents ‘Chemother 5:582-588.1974, 5. Furchner,1E,Richmond R.and GA Drake. Comparative “Metabolism of Radionuclides in Mammals. IV. Retention of Silver-110 m in the Mouse, Rat, Monkey, and Dog Health Phys, 15:505-514.1968, 4. Grier, N Silver and its Compounals in Disinfection, Ster- ization, and Preservation. (Seymour S. Block, ed) 2" Bn, pp 395-407, 1977, 5. Hindle, and J H Jorgensen, Procedure in Antincrobial Testing in Diagnostie Microbiology. (C R Mabon and G Manuselis, ed) pp 63-91.1995 6. Evidence of Efficacy of 32 PPM Silver Composition Against Pseudomonas Aeruginosa, Salmonella Chleraesuis and Staphylococcus Aureus A. Methods Pseudomonas aeruginosa STC #15442, Salmonella choleraesuis ATCC P0708 aad Staphylococcus aureus ATCC #6538 were tested using the AOAC (Association of Official Analytical Chemists AOC Methods, vol 1, [Sth edi- tion, 1990, AOAC Adington, Va.) oficial methods 955.14, 9515 and 96402. Nutrient broth (NBAOAC) tubes were inoculated from the stock culture, and the tubes incubated at 3742°C. Transfers to fresh ubesof nutrient both were made for tree successive days with the final transfer being inca hated at 3722° C. for 48-54 hy. The Pseudomonas culture was ‘decanted into afresh tube to remove the pellicle. The other ‘ultures were vortexed for 3-4 seconds and allowed to stand for 10 min at room temperature. Finally the cultures were dlls 1100 in peptone water (PEPW) to whieh equine serum Was sda to Yield a 5% total organic challenge. Test ‘arrers (10 mum long polished 304 stainless steleylinders ‘with an 8 mm eutside diameter and 6 mm inside dizmeter) ‘were soaked in challenge solution for 15 mia, removed, rained and dried at 3722" C. for 4022 min prior wo use, Phenol Resistance. Five-one ml aliguots of each dilution of the test phenol ‘were placedino sterile test mbes andallewed to equiibratein 182022" C. water bah, At30 second intervals, 0.5 ml ofeach challenge culture was added to the appropriate dilations of pheno, agitated, and replaced into the water bath After the appropiate exposure tines oS 10, and 15 mintes-aoopll ‘of suspension was removed from the assay tubes and trans- s » s 4 fered ones of thee both (LETH). Thebes of LETH were iteubted a 3722" for 2 days Carrie Titration. or tiation of cas, 10m blanks of petone Tweet (eran of poysorbat)(PEPT) solution were prepare. Two cers wee placed int the indivi abe, representing theft 1-10 ition. The tes were aia vigor enogh og bacteria nto solton and serial ditions were tad into 9 blanks of LETH miu, The dition lnk vere incuba 3722°C. The lost abe with growth ini Cet thelog, iter fogaismsonthe carrier AOAC requis Cerner to have nnimam populations of Ix fare, “Tes a iver Compton Using ei ls iets, 10m aligns ofthe prepared disinfectants wereplacd inf seletest tubes andllowedt equate ina effgrte waterbath held ot 202°C. With ov touching th sis of the test toes, one contamina dri cuir was added t 30scond itera cach ube of silver composition and placed eck int the water bth, For coch organ the disinfectant was tested aint 6 rid Contaminated cars t $a 10 mini exposre nerve Following exposure the cars were removed fom te dis: infantandasferedtotibeof LETH. Thecaltire bes ‘ere incubated at 3722° for? days an scores postive (+) or negative (0) for growth of the challenge organism. Cons. reach rgnism dried contaminated core asad ‘watuhe of fFTH sa posive cont Uninc mele tubes served as gate contol. Ar incubation, all ea tvetubes were spiked with 1100 colony foning nits) ofthe conesponding nganisnsto demonstrate peutalzaton eficay. To demonstrate growth promotion ofthe mea te negative contol rbes wee als inoculated withthe same 1-100 efi for ll thse ogi. The inoculating volumes were plated in tic onto soybean casein digest agar (SCDA) to verify the inoculating titers, The tubes and plates ‘ere incubate at 3722" Cun roth wis een inl tubes. ‘On the Paeraginsa netaization, the nial tr of ‘inoculum was found to be >100cfu which was too high forthe procol,Becaseal original besa been spk i= Inted test was performed with sane lotof mia wed in testing by plcing cares into disinfectant tubes Som all thes of siker compositions foe 10 minus. The carers ‘re sub-ransered 0 LETH blanks. These tubes wee ten Spiked with -100cfi oforgnism, The tubes were incubated 2 bofoce and sored for ath oF no roth Nee tobe of Serle media fom the same lot were alo inoclted os {som promotion verison B Ress Intl tsting using Seren demonstrated passing rss for smple an 2. ut sample 3 fale, Upon investiga ton twas decided tat sample #3 may have ben damaged prorte shipment. Anew bore was btn rom the same Iotassample 3, andthe new bows ables sample, The: aeus challenge wes tepestedusingsample 4. AOAC idling state tha foray one ane poi andra oly 1 carer alowed or roth foreach ot tested Positive controls demonstrate growth and negative con- trols demonstrated no gt for al ts, ne points, and orgasms Cari titan was rn in diate forall ngs. The reported eri an aveige ofthe replicates. For all the organs, theaverage ter found onthe carers ranged fom 5x10" to SSO? eflearee AOAC ries camino have minimum of 10x10 fea. For? aerginsa 3/180 carriers sowed growth a the 5 sxe point 2 Scarier showed growth the min US 8,753,691 B2 15 testpoint. For Saweus 16/180cariers showed growin atthe 5 min test point and 2/180 carriers showed grat the 10 min test point. For S choleraesuis 6/180 eariers showed owt atthe 5 min test point and 1/180 eariers showed roth atthe 10 min et point s ‘The test Preudomonas culture showed growth following a 5, 10 0¢ 15 min treatment with 1:90 phenol and showed roth following Sor 10min testment with 1:80 phenol bt no growth fllowing 15 min treatment with 1:80 phenol. The Staphslococcus caltre showed growth following a5, 10 or 15 min treatment with 1:70 phenol and showed growth fle lowing Sor 10min treatment with 1:60 pheno but no growth following #15 min treatment with 1:60 phenol The Saino nella culture showed growth following a8, 10 ot 15 rin treatment with 1:100 phenol bat no growth fellawing aS, 10 1s ‘or 15 min treatment with 1:90 pheno 7 Bvidenee of Effectiveness of 32, 22 and 10 PPM Silver ‘and 22)PPM Silver and 1.5% H,0, and 10 PPM Silver and 10 ppm K,S,0, Against Salmonella and Escherichia Coli in Freshly Inoculated Beef Samples ‘A. Pupose of Example ‘The purpose ofthis example is to demonstrate the antimi- ‘rial activity ofthe iver based composition embodiments ‘ofthe present invention on samples of bef flank steak inocu- lated on the exterior surface with a five stain cocktail of 25 Salmonella species. or Escherichia coli O1ST:K7 at a high inoculum solution level (110° cfuvem*) and separately at a ow inoculum solution level (1x10 efufem®) (efu-colony forming uit), B, Material and Methods Beef Samples Beef tissue samples were obtained from slaughter houses ‘within Shours of evisceration. The rectus abdominus muscle was peeled of carcasses hanging inte cil eooler by mak inganineision betweenthe 11 and 12" ibsandthen peng. the muscle ovt along the natural seam. The aseptically retrieved samples were placedin plastic bags and once packs nud were transported onthe same day tothe laboratory where the samples were promptly packed in a Mult-Vac (4-300) an placed ina" C. cooler Samplesuse for testing hada pH between 5.8 and 60 and were no more than 36 hous post ceviscention. From randomly selected rect alvlomins muscles, 38cm samples were cut and treated. Aer treat- meat, 3.5 em? flame sterilized stainless steel coring device ‘and surgical scalpel were utilized 1 aseptically retieve 160 meat cores per sampling interval from each sample. Tissue ‘cores were place ina sterile stomacher bag with 25 ml of| 10.1% peptone and were mixed fortwo minutes ina stomacher (Cab Bender 400). Serial dilutions were prepared and spiral plated at minutes, 2mintes, 1 hour, 4 hours, and 28 hours posttreatment on selective and recovery medi Bacterial Cultures. Bacterial cultures were obtained fom the Kansas State Univesity (KSU) stock eure collection and were stored using the “Protected Beod” storage system. The following cultures were used forthe Samonella specimen: S file (UGA), 8. montevideo (UGA), S. nphimurium (UGA), S ‘agona (&SU 05 from CDE outbreak isolate), and S.nenport (KSUO6CDC outbreak isolate). The following cultures were used for the Escherichia coli specimen: E.coli OLST:H? (CDC 0168), E.coli O1S7H7 (USDALESIS 11-82 Rit resistant 100 ppm), £ coll O1S7:H7 (ATCC 43895 HUS associated Type 1'& I toxins Rif. Res) and 6. col ATCC#23740 (Genotype K-12 prototrophic lamb). Stockcutures were cultivated by placing one impregnated «3 bead into a ml solution of Dieo® Teyptic Soy Broth (TSB) and incubating for24 hoursat 35°C, Nex,» 0.05 ml lonp of » 16 te respective culture was inoculated into a ml solution of TSB and incubated for 24 hour at 35° C. to obtain a pure culture. After incubation, | ml of the respective culture was inoculated into 49 ml "TSB and incubated for 24 hours a 35° C. Following incubation, samples were centrifuged (15,300 at 4° C) andthe supematant material decanted nthe pelet was re-sspended with 0 mi of 0.1% peptone and centrifuged (15,300. at 4 Ca final time. The peptone was decanted andthe remaining pellet was resuspended with 10 ml of 0.1% peptone. The five 10 ml bottles of respective culture were mixed together to crete a 50 ml cocktail con- taining 10° cfu of Saimonetia species. The cocktail was dilute 1o 10° efor 10* ef using 0.1% peptone. Cul- tures were confimed by cultivation on selective and iffer- ati media, and biochemical analysis of presumptive colo- nies using API 20F its. Method of Inoculation. Samples of bef flank steak (rectus abdominus mmusels) were timmed to 3x8em (104em*) and were inoculated with a five tain cocktail of Salmonella species. or Escherichia. coli 157247 ata high inoculum sTution level (10" log ef em) and separately at lo inoculum solution level (0 log efvem), This ioculam was misted onio the tissue surice using plastic spray botle with samples conned within a sealed inocuium chamber. The actual Salmonelta species. concentration onthe meat surlace was approximately 5.0 3.4 logeticm? forthe high and low level inoculum solution, respetively.ForB. coli O1ST-H7. he espectvemestsuriace inoculation levels were 42 and 39 log fwvem? ‘The boof samples were then hung verically on stainless stee hooks attaches 10 a motorized track that pulled the beef samples through model spray cabinet (Kansas State Univer- siy, Food Safety Laboratory while spray treatments were spplied. Treatments witheitherthe silver compositions ofthis invention ordeionied water were applied othe beet a 20 psi Joma distance of 13 cm in the model pressure rinse cabinet for 20 seconds The spay nozzle BETE NFOS80 303) dliv- eredappeoximately 20 mo solution tothe surfceot the beef sample. The temperature of solutions and treatment applica- ion room was approximately 14° C, After treatment, dupli- cate 3.5 em” core sumples were randomly drawn from the lateral surface ofthe be! sample at 0, 2,6 and 240 minutes Samples were cultivated and enumerated on selective differ- ental and recovery mei. Log eduetions were calculated by subtracting the logy of elulen? of the inoeulated/reated samples at the specified sampling times (0,20, 60, and 250 minutes) fom the lg. of efvem™ of the inoculstedun- treated samples at O mites. Sample teatment included the use of 32 ppm silver, 2 ppm silver, and 10 ppm silver com postionsacconing the present invention, Separately com- bination of 22 ppm Ag with 1 waht % hydrogen peroxide snd 10 ppm Ag with 10 ppm peroxydisullate(K.8,0,) were tested Results with 32 pm Silver Composition ‘Theuseof composition of 32 ppmsilveraccontingt this invention proved a reduction in bacteria on bee steak. In the following, this reduction is expressed asthe log ofthe ratio ofthe number of bacteria in the contol at time O to the amount of bacteria in the treated specimen atthe sampling (i, treatment) ime For Salmonella a the lower inital bacteria level (10) the following log reductions were recone: 0:78 at O minute. 111 at 20 minutes, 1.08 at 60 minutes, and 1.23 at 240, minutes: Thus at ours (240 minutes), the rato othe inital bacteria count inthe contrl to bacteria in the sample treated With 32 ppm silver is 10'*, For the hisher initial bacteria iol he llowing eens weer O88 US 8,753,691 B2 17 a0 minutes, 0.95 at 20 min, 0.98 at 60 min and 1.17 at 240 ‘min, Th results indicate that the 32 ppm silver embodiment ‘of this invention shows an effective bactericidal effect for Salmonella on beet steak. It wil be sppreiated that dsin- fecting 2 met surface is an exteme challenge for any disin- fectant, For B. coli forthe lower intial bacteria level (10%), the following log reductions were recorded: 1.03 at 0 minutes, 1.28 at 20 minutes, 142 at 60 minutes, and 1.58 at 240 rinutes. Forth higher inital bacteria level (10%) the follow- ing log eiutions were reconded: 0.65 Ominutes, 0604120 minutes, 0.83 at 60 minutes and 0.87 at 240 minutes. The resuls indicate thatthe 32 ppm silver embodiment ofthis invention shows an effective bactericidal effet for patho genic. coli on bet steak. , Results with 22 ppm Silver Composition Results with Silver in Water For Salmonella atthe lower initial bacteria level (10°, the following log reductions were recoded: 0-41 at O minutes, (043 at 20 minutes, 0.48 at 60 minutes, and 0.68 at 240 rinutes. Forth higher inital bacteria level (10%) the follow ing logeedutions were reconded: 0-24 Ominutes, 0.243120 minutes, 0.42 at 60 minutes and 0.61 at 240 minutes. The resulsinieate thatthe 22 ppm silver embodiment ofthis invention furishes an effective bactericidal effect for Salmo: rela on beet steak, Results with Silver in Water and 5 waht % Hydrogen Peroxide For Salmonella, for the lower inital bacteria level (10*), the following log reductions were record: 0.34 at Omtinuts, (033 at 20 minutes, 0.36 at 60 minutes, and 0.62 at 240 minutes Forte higher inital bacteria level (10°), te follow inglogedutions were reconded: 0-28 Ominues, 0.143120 minutes, 030 at 60 minutes and 0.69 at 240 minutes. The results indicate that the 22 ppm silver with 1S wght% hydro en peroxide embosliment of this invention provides an eflec- lve bactericidal elect for Salmonetla on beet steak, . Results with 10 ppm Silver Composition Results with Silver Composition in Wate. For Salmonella, for the lower inital bacteria level (10*), the following log reductions were records: 0.38 tOminutes, O41 at 20 minutes, 039 at 60 minutes, and O61 at 240 minutes. Forte higher inital bacteria level (10°), the follow- ing log reductions were recorded: 0.24 (tO miutes, 021 at 20 minutes, 0-41 at 60 minutes and 0.54 at 240 minutes. The results indicate thatthe 10 ppm silver embodiment ofthis, invention provides an effective bactericidal elect for Samo: rela on beet steak, Results with Siler Composition ia Water with 10 ppm K8.0, For Salmonella for the lower inital bacteria level (10%) the following log reductions were records: 0.26. O minutes, 0.28 at 20 minutes, 035 at 60 minutes, and 0.58 at 240 minute. Forte higher inital bacteria level (10%), the follow ing logredutions wereeconled: 003 a Ominutes, 0.163120 minutes, 021 at 60 minutes and 0.36 at 240 minutes. The results indicate thatthe 10 ppm silver with 10 ppm potassium, peroxydisullate(K,,O, ) embodiment of this invention pro- Vides an elective bactericidal elfect for Salmonella on beet stoak 8. Evidence of Effectiveness of 10 PPM Silver for Treat- meat of Human Ailments A Purpose of Example ‘The purpose ofthis exampleisto demonstrate the uty oF silver-based composition embodiments ofthe present inven tion for eating a variety of human silmens, The studies in this section were performed in Ghana, West Africa at the Air 18 Force Staton Hospital under the direction of De Kwabiah, at the Korie-u Teaching Hospital under the direction of Se. S.ockey, andt the sab liicMaterity Hospital unde the direction of De. Abraham. a oa, filly-eight (58) patients were tated using & compesition of the present ivention comprising 10 pp silver. The composition was sed both intemaly and extemaly a6 an alternative t teitonlanti- biotis. The silments wete included malaria, upper respira- tor ac nfesions, urna tactinfectons, sinusitis, vail 3yestinfetions, ee, nose andearinfctonscus, nga skin infections, and sexually ransmitd diseases, such 8 gonoe- thea B, Treatment Methods and Outcomes Abdominal Pain and Drea ‘The method comprises the step of administering appeoxi- mately $25 mlof siver compostion, one to five times a day oallyunltherewasaresponse. epatient was teted with about 10 mi ebout 0 teaspoons) a «composition a the peseatinvention tne tines none day. The patent hada fll recovery inane dy. Bronchitis. ‘The method comprises the step of administering a. 2-25 anof ier eompositon orl net fv times day nti there was a response. Two patients were treated with about 5 nl about one teaspoon) each ofa composition ofthe preset inveation for wo umes day for thee day. The patient ha all ecaveryin thre days ‘Vaginal Yeast (Candida) ‘The method comprises the step of adainisterng ca 5:25 nl of silver composition, one to five times a day a vaginal dehes uni there Wasa sponse. Five patents Were rated ihabout 10m (about two teaspoons) each of acompostion of the present invention fortwo times po day. The patients shove fll covery within six days Conjunctivitis, ‘Themethod comprisesthe sep ofaminisering ca. several dips of silver composition, one to five times & day tothe infected eye until thee was a response, Two patients were treated with several drops of composition of the present invention in each othe infected eyes fortwo tines pr day: “The patients ad a ill ecavery afer one da. External Cuts and Infection (Including Saphsfacoceus Skin infeton, Septic Ulcers and Infected Abscesse), “The method comprises the step of administering a sibver composition, ose oie times a day tothe infected are ut thore was a response Six patents Were tested with about S nl about one teaspoon each fs composition ofthe preset invention om the iafected areas fortwo tines per day. The Patients showed a fll ecovery within thre days External Ottis “The method comprises the step of administering a silver compositioa, one to five times a day to the fed ea ut there was response. Sx patents were twatod with approxi- matey to drops of composition of the present invention it the infected ears fo tee tines per day. The patients shoved fll covery after about four days, Otitis Mea The method comprises the step of administering a sibver composition, one to five times a day t the infected eae ut there was a response. One patient was treated with approxi mately two drops ofa composition ofthe preset invention compesing into the infected ear tee times per day. The Patiat shoved fll recovery in four days Fungal Skin Infection. The method comprises the step of administering a sibver composition, one ove times day tpialy othe infected area unl there was a response. Two patents were treated US 8,753,691 B2 19 ‘with about ten ml (Wo teaspoons) each ofa composition of the present invention tree imes per day. The patients showed 8 fll recovery within eight days ‘Gonorrhea “The method comprises the step of administering a silver ‘composition tothe infected area tui there was a response. ‘Two patents wore each teated with about ten ml (wo tea spoons) of a composition ofthe present invention theee times per day. The patients showed an absence of symptoms within six days Malaria, ‘The method comprises the step of administering a silver ‘composition, oneto ive times a day orally to the patient unt there was a response. Eleven patients were treated with about ten ml (two teaspoons) each ofa composition of the present invention three times per day. The patients showed aresolu- tion of symptoms within five days Halitosis and Gingivitis, ‘Te method comprises the step of administering a silver ‘composition, one to five times a day as « mouthwash until there was esponse. Two patients were each rated with the ‘composition asa mouthwash, There was a full resolution of| symptoms within three days (gingivitis) and within one day hlitosis), Pelvic Inflammatory Disease, ‘The method comprises the step of administering about 5.25 ml of silver composition, one to five times a day a8 ‘inal douche until dhere was a response. One patient was treated with about 5 mi (approximately one teaspoon) of ‘composition ofthe present invention two times per day: The Patients symptoms resolved within five day. Pharyngitis, ‘The method comprises the stop of administering a silver ‘composition, oneto fivetimesaday as aganleuni there was ‘response. Four patients were each eated with about en ml (Qwo teaspoons) of a composition of the present invention thzve times pe day. The patents showed full recovery within six days Rotrovirus Infection (HIV), ‘The method comprises the step of administering a silver ‘composition, comprising Sw 40 ppm silverone to fve times 8 day orally area ati there was a response. One patient ‘exhibiting HIV Giuman immunodeficieney virus) was treated With about 5 sal (approximately one teaspoon) ofa cemposi- tion ofthe present invention two times per day. The patent's symptoms resolved within five days Sinusitis and Rhinitis, ‘The method comprises the step of administering a silver ‘composition, onetofivetimesadayto the nose until there was ‘response. Six patents with nasal infections (four with Sinusitis and two with ini) were each eated with ‘approximately two drops of a composition of the present invention comprising in their nasal passeges tree times per clay The patients showed fll recovery within four days, “Tousllis, ‘The method comprises the step of administering a silver ‘composition, oneto fvetimesa day as agarleuntil there was ‘azesponse. One patiet was teated with a composition ofthe presen iaveation theee ies per day The patent showed fll recovery within soven days Upper Respiratory Trat Infection. “The method comprises the step of administering a silver ‘composition, one to five tines a day’ orally until there was a response. Two pationts were each treated with about § ml {approximately one teaspoon) ofa composition ofthe present invention thee times pt day. The patients showed fll recov ‘xy within sx days. Fa 20 Urinary Trat Infections “The method comprises the step of administering a silver composition, one to five times a day orally until there was response. Thee patients were each treated with about ten ml (wo teaspoons) of composition ofthe present invention wo to three times per day. The patients showed full recovery within six days €. Discussion “These results are consistemt with the various in vito tests reported herein. Essentially, the silver composition is extremely effective against 2 lage number of microbes in vitro. However, the tests indicate that this elfctiveness remains even in the presence of lange amount of organic: material. The silver compositions are widely effective in vivo ‘where the organic background is extremely igh, Many other disinfecting agents are ineffective in the presence af large amount of organic material and/or ar too caustic or toxic be used in vivo, 9, Evidence of Efficaey of 10 PPM Silver Against Tuber- culos Bacteria A. Purpose ‘The purpose ofthis example isto demonstrate the elicaey ofa silver composition of the present invention aginst the bacteria that cause tuberculosis. This eximple describes the procedures fr evaluation ofthe present invention for tuber- culocidal efficacy. The methodology is based on the Tuber- culocidal Activity Test Method as accepted by the EPA on Dee. 11, 1985. [Refer (0 United States Environmental Pro- tection Agency, 1986, Office of Pesticides and Toxie Sub- stances, Data Call-In Notice for Tubercuolocidl Effetive- ness Data for AIl_ Antimicrobial Pesticides with Tuberculocidal Claims. (Received Jun. 13,1986). B, Material and Methods Material. ‘The silvercomposition ofthe present invention comprised. 10pm silver in wate. The silver composition was evaluated employing a liquid to liquid matrix against Mycobacterium bovis BCG (TMC 1028), This organisms causes tuberculosis ‘animals and can cause tuberculosis in humans. Iis used as “stand-in” For M. tuberculosis, dhe major case of human tuberculosis, as tests have shown it to have a similar suscep ibility tL uberewoss. The test onganisea was expose 0 the silver composition in duplicate at fourexposure times and quantified using membraae fiat Procedure A vial of frozen stock culture was removed from storage and thawed. An equal volume of buffered gelatin (BUGE) Was added to the eell suspension and homogenized with 2 Teflon (brand of polyetruluoroethylene) tissue grinder for 1 minute while keeping the cultureat 010 4°C. nance at The homogenized cell suspension was diluted with saline Tween $0 (brand of polysorbate) solution (STSO) to spproximately 10” film (Challenge Titration, “Tenfold serial dilutions of the cultue were prepared in dilution blanks containing 9 ml of neutralizer broth (NEUB) througha 10°°dition. Three 1 ml aliquots ofthe appropriate dilutions were membrane filtered by frst adding 10-20 ml physiological saline solution (PHSS) othe filter housing and then adding a 1 ml aliguot of the appropriate dilution. The filer was then rinsed with approximately 100 ml of PHS, The fers were aseptically removed fom the filter housing sand placed onto 7H] agar plates, The plates were incubated ina humidifiod chamber at 3722° C. for 21 days. Positive Control. ‘A tube containig 9 ml of ST80-was prepared and equli- brated 2020.5°C. AttimeO, 1 ml of test organism culture US 8,753,691 B2 a ‘wasauded tothe tue (1:10 ition) -The sample washed for (0 minutes. Tenfold serial lotions were prepared inciluton blanks containing 9 ml of NEUB through 10 dition Thee 1 mi aliquots ofthe appropiate ditions were membrane filtered by firs ating 10-20 mil PHSS to the filter housing. $ andthenadinga 1 mlaliquot of the appropriate dition, The filter was rinsed with approximately 100m PHSS, The ites were aseptically removed from the fier housing and placed ‘onto 71111 aga plates. The plates were incubated ina humid fied chamber at 3722" C. for 21 days Tess “Two 25150 mm mbes containing 9 ml oF the test sample ver equirate to 2020.5°C. ina waterbath. To each mibe ‘containing te test disinfectant (je. silvercompsiton), ml ‘of test organism culture was added, The abe Was mixed by ssving and pled back int the waterbath. At 1S, 30, 48, and 60 minutes, 1.0 ml aliquots ofthe dsinectan-" to mark that the counts an estimation and that aceurate counts are beyond the Timit of detection forthe dilutions plated. Incaleulating the log and percent reductions ofthe disin- fectant against M. bovis, the estimated counts which have “wear than” counts resultad in “less than log and percent reductions (°°), The purpose ofthis isto demonstrate that the results are an estimation and beyond the accurate limit of detection forte dilutions plated. Al reductions were caleu- Jatedusing the positive contol asthe inital starting ter of the organism. The results for log and percent reductions are sum marized below, As a measure of the resistance ofthe chal- Jenge culture, the phenol resistance ofthe M. bovis showed 1.81 log reduction with 20 minutes of exposure t 0.8% phenol Replicate One Eipoazoline —lapraicion —_Peen cto Sains =n a9 miter 2 srs ‘Saintes a8 am ‘inter 2186 Replicate Two: Exgoazetine ——_Lapraicion Pee ction 1Sainnes =i ‘Sanne tse fmt <3 D. Conclusions ‘The use of silver composins ofthe present invention is elective aginst therculsis bacteria. A method compasing the step of admiistering silver compositions ofthe present invention effective agains tuberculosis onanism 10, Evidence of Elicacy of 10 PPM Silver Against Cay dida Albian ATCC #1023, Trichomonas Vaginas NICC #20035, and MRSA Staph foccocus ureus ATCC PBA ‘A. Purpose of Example ‘The purpose o this examples wo illustrate the ecay of silver compositions af the present inveation gains! Candida albicans ATCCHO231, Trichomonas vaginalis ACC 20285, anu drug sistant Staphylococcus aureas ATCC BAA, Candida albicans, a east and Trichomonas vginalisis, 0 ptozoa, can exuse numerous health problems including *aginal infections, diaper rsh andthmh. The rests Blow sha that silver composition ofthe prseat aveation pro- duced nearly a 100% Kilo bothorgansms. Th sults show the tlt of silver compositions ofthe present iveation ina feminine hypiene product and in a diaper rash roduc. ‘Staphvlococeus aureus can cause serious blood poisoning when it enters a wound tence was easily treated with pei- cil, bu the organism has now mutated to he pint where it is toly resistant to pencil. The next defense onthe at- biotic ladder has bora methicilin, but snebiilin- resistant sins have bcomeinstasingly common especialy in hos- pit, Those stain are known as MRSA (mthiilinresis- tant Staphylococcus aureus) and have been dubbed the “superbug” People whacontrect MRSA can dieinamaterof US 8,753,691 B2 23 ‘lays. Inthe results reported i this example silver compo- sition ofthe present invention was found to kill 91.69% of the MRSA in jst 10 minutes, and 99.5% in an hou. The resus show the utility of silver compositions of te present inven tion in killing MRS. 2 kaown infectious trea BB, Methods and Results Employing the USP Preservative Rapid Challenge Test ‘with composition of the present invention comprising 10 ppm silver in water, the following resus were obtained, ‘These results show that silver compositions of the present invention can be effective against yeast infections, protozoa infections, andl deug resistant bacteria infections Candida albicans ATCC #10231, ‘The initial concentration of Candida albicans yeas was 68510" ef, After contact fr either 10 minutes, 30 min- utes, I hour, or one day withthe silver composition, there were no colonies detected. Trichomonas waginalls NTICC #30238. ‘The initial concentration of Trichomonas vaginalis proto- ‘oa was 6.010" ef. Aler contact with the silver compo- ‘ikon fr either 10 minutes, 30 minutes, 1 hour or one day, there was 0% motility of 100 Organisms. Thats, one hundred (100) Trichomonas vaginalis parasites were analyzed via microscopy for motility of flagella. None ofthe one-hundred (100) parasites demonstated motility after only ten(10)mia- utes of eontat with the silver composition indicating inhibi tory of lethal properies ofthe silver composition on the parasites. Theovter membranes twenty-five(25)pereentof the parasites had ptured after contact of one (1) day. ‘Staphslococeus aureus MRSA ATCC #BAA-44, The ini lial concentration of methicilin-resistant Staphsloooecus ‘aureus (MRSA) was 6,0x10° em, After contact with the silver composition, there were 500,000 cfu/ml detected after 10 minutes contact (91.6% killed), 70,000 fal after 30, minutes contact (98.8% killed), 30,000 efuiml after 1 hour contact (99.5% killed), and fewerthan 10efuvl afer oneday contact (vitally total kill), Evidence ofthe Eficacy and Lack of Cytotoxicity of 10 PPM Silver, 14 PPM Silvers 1.5% HO, and 22 PPM Silver in Inhibiting DNA Polymerase and Reverse Transeriptse in the Context of Hepatitis B A. Purpose of Fxample ‘The purpose ofthe example isto illustrate the eicacy of silver compositions ofthe present aveation against hepatitis B. This example shows that iver compositions of the preset invention have antiviral properties. Any agent used inantvi- ral therapy should exhibit litle or no cytotoxicity so eytotex- icity of he slver compositions was analyzed, Hepatitis Bis caused by a DNA virus of thehepadnaviridae family of viruses. The Hepatitis B Virus (HBV) is 33.2 kb DNA virus, replicating almost exclusively in the Liver cells (hepatocytes). Replication iavolves two main enzymes: DNA, polymerase and reverse transcriptase. The results of this, ‘example show that silver compositions of th present inven- tion interfere with replication involving ether DNA poly- merase or reverse transcriptase, The results ofthis example show that silver compositions of the present invention have antiviral properties. The results of this example show that silver compositions of the present invention can be effective gains hepatit As furterdetil, when hepatitis Benters the body ofa new host, it infects the liver if it gets past the hosts immune stem. In theinfection, the viusattaches tothe membrane of a liver cll, and the core particle ofthe virus entors the liver ‘el, The core particle then releases its coatents of DNA and DNA polymerase into the lve eell nucleus. Within the liver «el the virus replicates via reverse transcription an transla- s Fa 4 tion processes, which involve reverse transcriptase and DNA polymerase enzyaes. The DNA polyaserase causes the iver tell 10 make copies of hepatitis B DNA. These copies ofthe ‘ins are leased ro the iver cell membrane int the blood scam. From thee, they can infet oi lier cells and thus replicate effstvely. The incubation period ofthe hepatitis B Wins is about 6 to 25 weeks (ie, tne before physical and enerlly detectable histological or physical symptoms occur, However there areseeral biochemical andhisologi- cal changes that ocur inthe early stges following infection with the hepatitis B views 3, Materials Soltis comprising 10 ppm, 14 ppm, 22 ppm, and 32 pm silver compositions according to the present disclosure were used. The mclotdes dATP, dGTP. ACT, and (*H]- AETTP were obtained fom standard commercial soures, were the compounds lamivudine (a syathetieanierviral goat) and zidovudine (AZT, Isolated Hepatitis B vias was freshly obtained ftom a person suffering rom Hepatitis B infection and was taken up by Haffie Taste, Mumbai INDIA (a WHO eerie testing labrator). Test ell cul tures (Vero and Hep2) were grown a convent monolayers by typial el cultre methods. C-Metods 1) Procedure for Testof DNA Polymerase Inhibition, Overall Appeoach. Hepatitis vial extras fom human subjects are ine ‘bated wih radiolabelled ncleoides and an sete inhibitor. Percent inhibition is calulated based on the amount of de novo viral ale aid symiesized with espet to laivu- dineasapostve contol and phosphate bufler saline (PBS) 2s a negative cont Specific Proce Innate Hepatitis B virus was lysed to extract fe poly- merase enzyme, which ee fom contaminating ese “Avinsextract (25) was added to a eation mixture com> Prisag AATD dGTP, dCTP and HTTP uclooids (25 ‘ative inhibitor (3p) was aed to the mixture comprising virss extract and nucleotides. The rsuitant mite was incu- bated at 37°C. for? hours ‘A separate negative conc! experiment was perform in whieh phosphate hesaline PHS, 3p) was se instead tho inhibitor @ pb “A separate positive contol experiment was performed in whieh known DNA polymerase ibibo (3 jo lamiva- din ata concentration 3 mg/ml) was used instead ofthe texto inhibitor (3 “The reaction was topped hy ang 25 EDTA ane 25g TCA (tichleroseae acid). The ation mixture Was then spoited on oni pper (DEAF pane) The paper was washed thre times with TCA and then with eth! lel. The filter oper wes ai dried and put ito sinllation vial with a Scintillation celta: Radioactivity was measured by a liguid Scitation counter (Bive Star). Asa counting cont, a blank silver composton was run through te complete pro- ceslure without viral load, to chock any potential nterereace inthe scintillation counter method ‘A reference for this method is PS. Venkateswara, 1 Millman, and B. S. Blumberg, “Elfet of an extract fom Phyllanthusnirri on hepatitis B and woodchuck hepatitis rss: in vitro and in vivo studios.” Pro. Natl. Acad. Sci. USA, 1987, 84, 274-278, whic is incorporated herein by reference. 2) Procedure fo Test of Reverse Transcriptase Inhibition. ‘A commercial vial enzyme preparation of Moloney rrine leukemia virus reverse transcriptase (MoMLV a= ing Poly AQT (primer for RT) was wed. $0 yl of the US 8,753,691 B2 25 MoMuL’ preparation was combined witha mixture of ATR AGT, dCTP and [HTTP nucleotides. ‘This minture was combined wih3 lof the iaibitor tw be tested, nth resin mixture was incubated at 37 Cfor2 hous 5 negative contol experiment was performed in which phosphate buffer saline (PBS, 3 i!) was used instead of the inhbior A postive contol experiment was performed in which a known reverse transcriptase inhibitor (3 jl of AZT at a con- ceitation 0.625 micrograminl) was wed instead ofthe tested inhibitor. “The eaton was topped by adding 25 ylEDTA and 25 ‘TCA. The ection minture was then spotted on ioe paper (DEAE pope). The paper wos washed three times with TCA andthen with ey slob. The ltr paper wassir died and put ina scallion vial wih sinilaton coetal. Rao ‘etvly was measured by gud scan counter Blue Su) 3) Procedure for Testing Cytotoxic ‘ettswere prepared fom healthy, conduen Vero and Hep2 cellule tht were maitsined by passage every 3-4 ds, ‘Onedaypriorothe test cells wer release fom te cles using tala tetnigues adsuspendedina growth medium and dispensed into walls of a meroiter plate and placedin a $%CO} incubator at 372°C. Analiqnt (1004) feachtest substance wasintdicedintoa wel itp) wih 10D ‘OFS as contol. Every 24 hrs the wells were examined unde high power ofan iver microscope wo check forany «ptopaic effet (CPE) D.Resils Results for Test of Revere Transcriptase Inhibition: » Sump ‘kon Tee cot PS) 7 peste stl (AZT) 3133 Sie 0 ppm wn * Sine 1 pp and 18% H202 a Site. 2 9p as Results for Test of DNA Polymerase Inhibition Sup Skin Tee soa PBS) o posite ot ani) a3 0 Eisen pn mn Sie pp with LS% 202 oo Silver compositions of the present invention are highly 3s

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