LT5643B - SILVER / WATER, SILVER GEL AND SILVER BASIC COMPOSITIONS - Google Patents

Abstract

The invention relates to a colorless composition comprising metal particles (e.g., silver nanoparticles) and water, wherein said particles have an exterior of elemental metal (e.g., silver) and metal oxide (e.g., one or more silver oxides), wherein the metal nanoparticles are present in the water at about 5 -40 ppm (ppm) and where the composition clearly shows important antimicrobial properties. Methods of using the composition are described. The composition may be incorporated into the hydrogel without substantially losing its antimicrobial properties. Various metal-containing compositions with unexpected biological efficacy are also described.

Description

The present invention relates essentially to novel silver / water mixtures (sometimes referred to as silver particles dispersed in water) and more particularly to novel compositions and / or morphological silver / water mixtures, silver hydrogels, novel silver compositions compatible with modern antibiotics and various ligands bound with silver ions, silver gels based on certain starting silver / water mixtures, silver ions and / or metal (s) bound to certain clathrates such as clays and / or zeolite materials in the preparation and use of compositions as agents against various organisms (including certain viruses) which are detrimental to health or to the health of humans and / or animals or other organisms. In addition, other metals in silver impurities are also described herein and may used in alternation with silver various combinations and concentrations of the compositions of the invention.

TECHNICAL LEVEL

It is well known that certain silver preparations have bactericidal properties. Silver is used as a bactericide and was used as an antibiotic before the advent of modern antibiotics. In earlier ages, consumers carved silver particles into drinking water or immersed silver in drinking water to swallow silver in drinking water. It seems realistic that eating silverware (eg table silver) came from a belief in the healing properties of silver.

These may be the reasons why the use of silver in solution has improved the health of the individual. It is possible that such a solution has the effect of inhibiting the growth of bacteria, viruses, and other undesirable organisms, as well as eradicating existing bacteria, viruses, and other organisms. It is possible that the silver composition may have anti-inflammatory effects sufficient to reduce, for example, swelling, burn complications and certain asthma symptoms.

A first embodiment of the present invention describes the use of a silver composition in water for the treatment of certain human (or, for example, certain animal) disorders. One embodiment of the invention comprises silver nanoparticles comprising a silver composition (e.g., most of which are 10-50 nanometers in diameter) and which, in the present embodiment, may have an inner metallic silver part and an outer sheath or part different from said inner (e.g., ionic silver). a coating, one or more silver oxide coatings (e.g., different compositions and / or different phases, etc.) wherein the particles are suspended in water (e.g., purified water) .In another embodiment, at least 90% of such particles are 10-50 nanometers in diameter An embodiment of the present invention provides a silver composition comprising silver particles (including certain silver oxide coated silver particles) wherein more than 50% of the particles are less than 0.015 micrometre in size and the particles are colloidally suspended (i.e., not precipitated) in water. variant p produces similar particles, with about 95% of the particles being 10-40 nanometers in diameter. In yet another embodiment, about 95% of the particles are 10-30 nanometers in diameter.

THE SUBSTANCE OF THE INVENTION

The present invention is primarily directed to the use of silver at a level of 5 to 40 ppm in water (but in some cases less than 5 ppm) for killing or rendering inactive microorganisms (including certain viruses) that are hazardous to humans and / or animals or other living organisms. . Further, the present invention is specifically directed to compositions comprising silver nanoparticles, wherein said particles in the present embodiment include, for example, an inner part and an outer coat or part coat of elemental silver or, for example, one or more silver oxides (e.g., ionic silver oxide). , silver oxides such as Ag ₂ O, AgO, Ag ₄ O ₄ etc.), wherein said oxide layers are in various phase states (e.g., Ag ₂ O is monoclinic and / or tetragonal) and water where silver particles are deposited. in suspension (e.g., colloidal suspension) in water at a total level of 5 to 40 ppm. One embodiment of the present invention encompasses silver nanoparticles (hereinafter, as used herein, it is to be understood that the term "silver particle (s)" or the like, when subjected to the electrochemical methods described herein, includes not only elemental silver but also elemental silver). particles which can then partially or completely form a coating of one or more compositions, wherein the coating (s) comprise one or more oxides of silver in the lowest dose thereof) in water (preferably pure water as described below) 5-40 ppm concentrations where more than 50% of the silver particles have a maximum size of less than 0.015 micrometers. In a preferred embodiment, most particles have a diameter of 10-40 nm. In an even better embodiment, most particles have a diameter of 10-30 nm. The silver in water composition (also the silver particles are extracted as substantially single particles from the silver / water mixtures produced by the present invention) as well as the silver / water mixtures prepared according to the present invention and subsequently formed into gels, powders, clays or zeolites ( as described in the embodiments below) are, for example, highly effective antimicrobial agent (s) and antiviral agent (s) (and in many cases also anti-parasitic) in accordance with the present invention. The present invention is also directed to silver compositions of 5 to 40 ppm silver in water and, based on the uses of the silver / water compositions described herein, are highly effective as antimicrobial agents in the use of said compositions for: (1) internal use by living organisms; (2) externally living organisms as well as external or internal use on a variety of surfaces combined with solid and porous surfaces (eg stalls, surfaces for cooking, cooking equipment, hospitals, medical instruments, plumbing (metal and / or plastic), air filtration devices, etc.); and (3) for mixing silver or silver-water compositions with contaminated water (e.g., treatment of contaminated water effluent, water storage facilities, contaminated water tanks, plumbing pipelines, etc., which preferably contain a large amount of solids to be removed prior to mixing). , to carry out the water purification process.

One embodiment of the present invention is directed to silver-in-water compositions prepared using the device and / or methods described in U.S. Pat. 6214299 ("Patent '299"), a modification specifically introduced herein with preference. In addition, other metals such as copper (and copper alloys), zinc, platinum and titanium and their alloys and alloys may be used to form other desired metal compositions according to the methods of the present invention, which are also surprisingly effective.

The apparatus and method of the '299 patent have been modified and improved to prepare a silver composition of the present invention, the method being described in much more detail below. Essentially, the eight silver / one common electrode device as described in '299 patent has been modified and proportionally enlarged to accommodate a large (e.g., 284.25-322.15 L (75-85 gallon)) water chamber. To start the silver / water formulation in a 284.25-322.15L (75-85 gallon) container, approximately 265.3-284.25L (70-75 gallons) of relatively high purity water (e.g., filtered) is placed in the chamber. water, reverse osmosis water, or water that is free of high levels of contaminants, etc.), typically having less than 2 ppm total soluble solids, or even better less than 1 ppm total soluble solids. In the present embodiment, about 18.95 l (five gallons) of the silver / water composition obtained in the previous process is added to all of this. This "fill" of about 18.95 l (5 gallons) is useful but not essential. The filling essentially provides a sufficient amount of conductive silver particles in the container to allow the amount present to flow between the various electrodes when a suitable voltage / current is reached in a relatively short period of time. This "filling" also allows for the formation of slightly smaller Taylor cones, described here later. The water chamber is equipped with an air inlet (typically located near the top of the water chamber), which allows a stream of air bubbles to flow through the water silver liquid during manufacture. It has been found that this path, which obviously improves the mixing as compared to the rotary mixer described in '299, which is undoubtedly obvious, increases the efficiency.

The electrode device (s) operates (at least initially) at approximately ten thousand volts, alternating current (with each group of silver electrodes having a separate voltage supply) as described in the '299 patent. Voltages well above ten thousand volts encourage the obtaining of a solution which may contain significant quantities of ionic silver dissolved in it. This composition comprises more than 97% of metallic silver particles in the range of 5 to 40 ppm with substantially low captive ionic silver in the silver / water solution.

The silver concentration is determined as follows. So,

The 284.25 l (75 gallon) silver / water production unit operates essentially continuously and samples from the unit are analyzed until the desired silver ppm concentration in water is achieved. It has been found that under the operating conditions described herein, the 10 ppm silver / water formulation requires approximately one and a half days of production; A 22 ppm silver / water composition requires approximately three production days and a 32 ppm silver / water composition requires approximately six production days. The rate of silver particle formation in silver / water compositions slows down to a higher concentration of silver particles. When the concentration of silver in silver / water compositions is desired above 50 ppm, it takes relatively long time to produce them, with the process parameters described herein, with the highest concentration achieved to date within a reasonable time of about 50 ppm. If desired, high concentrations of silver particles are possible. However, the effectiveness of lower concentrations of silver particles against various pathogens was so inconclusive that high concentrations of silver particles were not necessary until now.

Silver nanoparticles in silver / water compositions have very similar overall particle size and shape characteristics as detailed below in the Characteristics section, and unlike many known "colloidal silver" compositions, these silver / water compositions are completely colorless and essentially constant in low light and temperature. changes that do not require the use of any additives that maintain stability (required and / or used by many known colloidal silver). It is believed that the components used and the steps of the commercial process make it possible to produce a silver / water composition different from other products known as "colloidal silver" in a way that makes the silver / water compositions much more effective. Some of the most striking physical differences between the silver water compositions of the present invention are described below.

The silver / water compositions of the invention are also substantially unreactive due to the addition of many substances, including, for example, one or, in combination, (1) hydrogen peroxide, (2) disodium EDTA (disodium ethylene diamine tetraacetic acid), which may actually act as silver. / water formulator enhancer (e.g., may even increase the effectiveness of silver / water formulations), (3) iodine (e.g., povidone iodine which may in some cases exhibit low reactivity) which can render silver / water formulations more pathogenic against various pathogens, and (4) various commercially available antibiotics (which may in fact provide some synergistic effects between silver / water formulations and antibiotics, thereby enabling the realization of new and highly desirable combination therapies. (e.g. added to or provided with) novel silver / water compositions of the present invention to synergistically enhance the desired effects that each substance may have. Specifically, in many cases (eg antibiotic combinations), the combined effects obtained are synergistic and outweigh the individual complementary effects of each single substance when combined (eg, 2 + 2 = 6). Of course, some of the possible additives would render the novel compositions suitable for topical or topical treatment only because of their potential for internal toxicity in biological organisms (e.g., human or animal). The amount of additive required may vary depending on many circumstances, including the specific cause of the malaise (eg virus, bacterium, parasite, etc.) or infection, the amount of other substances present in the admixture, etc. However, the exact amount of additive required must be determined by testing in comparison with what is conventionally known in the art. In addition, silver / water concentrations may also affect the amount of additive required, as well as determinations by testing, compared to those commonly known in the art.

One example of a preferred additive is hydrogen peroxide. Hydrogen peroxide is a known disinfectant. It was discovered that hydrogen peroxide has a synergistic interaction with the silver / water compositions of the invention. Hydrogen peroxide is suitable, for example, in a concentration of 30% by weight (% by weight or by volume) or even higher. As higher concentrations are used, more suitable silver / water concentrations used in the present invention have been found to be 30% or less, and more preferably, have fallen to about 1-5% by weight.

One aspect of the present invention is directed to compositions comprising 5 to 40 ppm silver particles, 1 to 3 wt% hydrogen. peroxide and the remainder of the water (e.g. filtered or substantially purified water). Another aspect of the present invention is the use and use of a composition comprising 10-40 ppm silver particles and 1-3 wt% hydrogen peroxide in water as antimicrobial agents.

Another example of an additive of the present invention that works favorably with silver / water compositions is disodium ethylene diammonium acetic acid, also known as "Sodium EDTA" or "Disodium EDTA" (both of which are sometimes referred to in the literature), which may have the following formula: : (CH 2 N (CH 2 COOH) CH 2 COONa) 22 H 2 O. In another aspect of the present invention, a small amount (e.g., 0.5-10 ppm, or more preferably 0.5-5, or even more preferably 0.5 ppm) of EDTA disodium is added or mixed with the silver / water compositions of the present invention. In this embodiment, it has been observed that addition of small amounts of disodium EDTA increases the potency of the silver / water compositions (e.g., improves bactericidal, disinfectant and / or antimicrobial properties). Without being bound by any particular theory or interpretation, it is possible that disodium EDTA can increase the permeability of the cell wall, which may increase the overall efficiency of the silver / water compositions of the present invention. Another aspect of the present invention is the use and use of compositions comprising 10-40 ppm silver and 0.5-10 ppm disodium EDTA in water as an antimicrobial agent, bactericidal agent, antiviral agent and / or disinfectant.

Another example of an additive of the present invention that works favorably with silver / water compositions is povidone iodine. Iodine is a well-known prophylactic remedy in medicine for treatment against a broad spectrum of pathogens. Iodine is commercially available in various concentrations but is commonly used and is preferred at 10% concentration. In this aspect of the present invention, the synergistic combination involves replacing about 25-50 volume percent of a silver / water mixture with a 10% iodine solution. Because some reactions between the silver / water mixture and iodine are possible, it follows from the results of the studies discussed here that the synergistic combination of silver / water with povidone iodine may act as a surface disinfectant (e.g., grease) and / or as a preventive agent against wounds, burns or scratches, etc. infection. Another aspect of the present invention is the use of compositions comprising 10-40 ppm silver and povidone iodine in water as an antimicrobial agent, bactericidal agent, antiviral agent and / or disinfectant.

Another aspect of the present invention is the use of the silver / water compositions of the present invention in combination with various industrially available antibiotics known in some respects as a therapeutic combination. The therapeutic combination has attracted considerable interest, as antibiotic resistance has spread very widely over the last two decades and has thus caused widespread global concern. Infections caused by gram-negative bacteria such as

Escherichia coli, Klebsiella, Proteus, Shigella, and Pseudomonas, have become the subject of great interest as these organisms have developed multi-drug antibiotic resistance. Recent studies to investigate the resistance modulus of Gram-negative clinical strains causing clinical infections showed that most of the strains were resistant to known antibiotics such as ampicillin, gentamycin, chloramphenicol, co-trimoxazole and first arva second generation cephalosporins. Also, approximately 70% of these strains were resistant to ciprofloxacin. In this aspect of the invention, silver / water mixtures (whereby they are combined as a liquid or dried and added as a solid to form, for example, a powder, sometimes referred to as "Sildust"), when combined with various antibiotics, exhibit synergism more often than merely additives. properties. Spreading chess studies have shown that certain antibiotics when combined with antibiotic-formed silver / water blends are several times more effective than silver alone (e.g., silver / water blends combined with amikacin and cefoperazone with a FIX index of approximately 0.1875). , as compared to two antibiotics used in combination with one another, which results in a FIC index of 0.625 when both combinations are used against, for example, MRSA (methicillin resistance to Staphylococcus aureus) described in great detail hereinbelow. the use and compositions of compositions comprising 10-40 ppm silver and various antibiotics as an antimicrobial agent and / or bactericidal agent and / or antiviral agent for the treatment of a therapeutic combination The exact amount (and concentration) of the silver / water mixtures of the present invention, which can be added to known antibiotics yr a is the result of long experimental work. Specifically, the specific disease being treated with a specific course of antibiotics (as well as the effectiveness of the antibiotic against the pathogen) affects the amount and concentration of the silver / water mixture required.

Although a large number of tests using silver / water solutions alone or in combination with various additives are given below, they also reveal that certain carriers can significantly improve the results obtained with silver / water mixtures under various conditions. Specifically, it has been discovered that the formation of an aqueous silver / water composition as a semi-hydrogel (sometimes presented later as Silgel or other version as Silderm) or even thin layers of such materials significantly improves the performance of certain target materials. Hydrogels are standard hydrophilic gels obtained by adding certain hydrophilic organic polymers to an aqueous solution, in this case a solution containing the silver / water solution of the invention. However, it is to be expected that other colloidal silver solutions may also be converted to hydrogels according to the teachings herein and such hydrogels may not be as effective as the present invention where these hydrogels may nevertheless have some desirable application. Accordingly, the present invention is also intended to disclose certain aspects of these hydrogels. As expected, the hydrogel improves the retention of silver on a surface area such as a wound on the skin surface. For wound care, hydrogel or rag materials are also significantly superior to protecting wound tissue and preventing drying, where these factors improve wound healing. Most importantly, the hydrogel does not substantially affect the antimicrobial properties of the silver particles of the present invention. In addition, these hydrogels act as excellent cleansers for the hands or skin, as well as skin protection products (eg, by applying the hydrogels on the hand (s) so that the hands are in contact with pathogens, the skin protection gel can help prevent infections, such as cuts or scratches, thus acting as a preventive measure), which promotes the widespread use of gels in healthcare or wellness.

Specifically, clean hands are one of the most important factors in preventing the spread of dangerous microbes and antibiotic resistance in health care. The most hygienic hand cleaners used in modern medicine are alcohol based and have several disadvantages. The most important of these disadvantages is skin damage due to alcohol-based reusable products. In some cases, (1) irritant contact dermatitis and also (2) allergic contact dermatitis have been reported. This reduces the need for many health care workers to be clean when using hand hygiene products.

Another factor contributing to the failure to comply with effective hand hygiene practices is the fact that, when liquid, hand hygiene products are usually permanently anchored above washbasins or washbasins. As a result, healthcare staff must move from the patient's bed to the washbasin and back to the next patient. If the handwashing could be "erased", this problem would be eliminated, thus enabling better ordering. The hydrogel products of the present invention significantly reduce bacterial counts of demonstrable organisms over extended periods of time as described herein, thereby providing a viable alternative to the hand hygiene product. Accordingly, the hydrogel products of the present invention may also be successfully used as protecting healthy skin from various pathogenic agents for prophylactic purposes.

In another embodiment, the silver-based products may be replaced, at least in part, or in some cases substantially completely, with the silver / water compositions of the present invention. Most importantly, silver EDTA (or AgEDTA) in itself has been found to have very moderate antimicrobial properties. Specifically, as described above, disodium EDTA is a suitable additive for use with the silver / water compositions of the present invention. However, EDTA (Edetic Acid) is an excellent synthetic chelating agent. EDTA (C10-H16-N2-O8) is approved for use in human foods and is often added to soft drinks as a preservative. EDTA is also used in heavy metal chelation therapy for tumors. However, what has not been considered is the use of AgEDTA as an antimicrobial agent (e.g., as such or in combination with other drugs such as those described herein). Consumer products such as meat or protein production and processing, the soap industry, the detergent industry (such as personal or household products), the agricultural or agricultural crop industry, and the health industry can successfully use stable silver that can provide powerful health or wellness sensations (eg, both therapeutically and prophylactically), in powder form. Specifically, AgEDTA is easy to apply and relatively easy to manufacture, store and transport. This aspect of the present invention discloses a novel use of AgEDTA, namely, the use of AgEDTA powder for human, plant and / or animal health or wellness and / or treatment of certain animal and human disorders (e.g., can be used for therapeutic and / or prophylactic treatment). Akzo-Nobel is currently manufacturing the right AgEDTA. Other silver chelating or complexing agents, such as silver EDDS, silver curcuminate, silver berberine and silver tetracycline, also improve antimicrobial properties, and the use of these substances in human or animal health and wellness is also new in the art. various other organic structures may be utilized to transport and / or deliver silver and / or silver ions to various effective allocations or to biological structures. With repeated use, the amount of AgEDTA required will vary depending on the specific biological causes and surrounding needs (eg treatment requirements and / or prophylaxis).

In another embodiment of the invention, the additional silver-based inorganic products may be at least partially, in some cases substantially completely, replaced by the silver / water compositions of the present invention. Specifically, silver (e.g., silver ions, silver metal, Ag +) can be bonded or attached, for example, on or between clay layers and / or inside zeolite cages. Such attachment can occur by controlling, for example, the silicate layer, the charge of the zeolite cage, as well as the distances between the layers or the size of the zeolite cage. In this regard, silver may be bound or bound, firmly or relatively loosely, depending on the particular application to health or wellness and the point of interaction between silver and the biological material (e.g., on the surface of the biological material or in its interior or combination). Accordingly, the resulting products may include products that are completely liquid and therefore are taken orally or sprayed; as well as products in the form of gels or pastes and sprayed on surfaces such as gels or pastes.

Any of the metals discussed herein may be stored in a crystalline or amorphous clathrate with one or more oxygen layers or oxygen-containing molecules. Certain metal / clathrate structures showed unexpected efficacy. In addition, silicates, phosphates and oxides, such as hydrocalcites, incorporated into the oxide layer (e.g. clays) and lattice structures (e.g. zeolites) can also be used. Further preferred clays or mica species that can be used with the present invention (and which may have different surface charges and / or different spacing between layers) include, for example, ilites, montmorillonites, chlorites, and vermiculites.

Moles or mica, like zeolites, are highly acceptable as metal ion carrier for a number of reasons, including the fact that many are naturally occurring and readily obtainable, and particles can be retained in the desired colloidal size, which makes them suitable, for example. suspended in liquids (such as water) and are typically very close in biological terms (e.g., with little or no side effects), In this respect, when silver is deposited, for example, in clay or zeolite clathrate, then the molecules are heated to moderate temperatures ( e.g., 100 ° C to 200 ° C) to bind silver to or introduce it into the clathrate. All of these materials can be made in a very wide range of viscosities, from very liquid to very viscous.

In general, the electronic levels of any given valence elements, such as cations, may change when such an element cation is coordinated by various anions. Specifically, the more covalent the bond, the more energy levels can be replaced. It is possible that small to medium changes will occur in the electronic structure of silver when silver is surrounded (or coordinated) by different numbers of oxide ions. In the electronic structure of cations, such as silver cations, such a change can occur in any of the various silver oxide structures. In addition, there is a more common way in which silver can be placed in an oxygen clathrate or cage. In this respect, reinforcing, for example, the sodium cation in the structure with the silver cation, can then result in the formation of sodium ions present in interchangeable cavities or locations (e.g., either on or between clay sheets or inside zeolite grids). Collectively, the ability of a single substance to replace cations is known as its "CEC" or "cation exchange capacity". CEC standard units are as meq / 100 grams' or milliequivalents per hundred grams. In general, the higher the number of CECs, the greater the ability of the material to accept cations (e.g., silver cations). Accordingly, oxygen-coordinated silver compounds may play a role as a carrier of silver (or other metals) and thus act as therapeutic agents alone or in combination. therapeutic agents.

Furthermore, the introduction of silver metal or silver ions into silica gel by diffusion and drying are also suitable mechanisms for carrying metals of the present invention.

In another embodiment of the present invention, combinations of the above-mentioned particles, organic and / or inorganic structures, can be used to positively affect human and animal health and wellness. In particular, the metal particles of the present invention may be used alone as discussed above. In addition, metal particles may be combined with, for example, organic compounds such as AgEDTA discussed above. In addition, the metal ions of the present invention may be combined with any of the organic compounds (e.g., moles or zeolites). In addition, the metal ions of the present invention can be coupled with both organic molecules (e.g., AgEDTA) and inorganic molecules (e.g., moles and zeolites). This combination of silver metal or silver ion delivery systems may be designed such that, for example, the internal consumption of any of the above silver delivery systems may result in the presentation of silver to different parts of the body, for example. Specifically, for example, in humans, certain silver may be absorbed in the mouth, through the gastrointestinal tract, and through the small or large intestine, and so forth. In addition, depending on, for example, the ratio of the amount of clay (s) or zeolite (s) to water (as well as the various gelling compounds described herein), the resulting product (s) of the present invention can range from highly liquid (low viscosity) to very thick (high viscosity). In this respect, in general, the more clay or zeolite is fed relatively to the water (including the gelling agent), the more viscous the final product.

Detailed Description of the Invention

This description is provided to any person skilled in the art who is familiar with and practicing the invention and provides the best methods devised to carry out the inventor's invention. Thus, various modifications will remain readily apparent to those skilled in the art, but the basic principles of the present invention have been specifically disclosed herein to provide an improved silver / water composition (sometimes referred to as silver nanoparticles dispersed in water) that can be used as such or in combination with (e.g., admixed or substantially supplied in close proximity to this) with the other materials indicated, and which may be formulated into a variety of gel or paste formulations, all of which have a high potential for killing human and / or animal pathogens both in vivo and in vitro. .

In general, the present invention provides a novel approach for the destruction and inactivation of microorganisms that are hazardous to humans and / or animals by using silver nanoparticles in water at concentrations of 5 to 40 ppm silver; or active silver particles such as AgEDTA and the other compounds described herein. Depending on the application and the additives present, the silver / water composition can be used internally and externally. Depending on the application, the silver / water composition may also contain various desired additives, many of which are not specifically listed herein, but are obvious to those of ordinary skill in the art.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1-6 show TEM photomicrographs obtained by varying magnification of silver particles formed in the silver / water compositions of the present invention.

Figures 7a-7d show TEM photomicrographs of various TEMs using a different technique from that used to obtain Figures 1-6; and Figure 7e shows the EDS spectrum of (EDAX) silver particles obtained from the silver / water compositions of the present invention produced by the present invention.

Figure 8 shows an electron diffraction module obtained from silver particles from silver / water compositions made in accordance with the present invention.

Figure 9 comprises three SEM photomicrographs which together show the possible damage of the electron beam to silver particles obtained from the silver / water compositions of the present invention.

Figure 10 shows a SEM foromicrograph of a new silver electron previously used in the method of the present invention.

Figures 11, 12 and 13 show EDS core analyzes of the portions 1, 2 and 3, respectively, shown in Figure 10.

Figure 14 shows a SEM photomicrograph of an electrode tip used to make silver / water compositions of the present invention.

Figures 15 and 16 show the EDS core analyzes of the portions 1 and 2 shown in Figure 14, respectively.

Figure 17 shows a SEM foromicrograph on the tip of a silver electrode used at approximately 3500Χ.

Figures 18a and 18b are TEM photomicrographs of silver particles obtained from the GNC Liquid Silver Dietary Supplement (25ppm).

Figures 19a and 19b are TEM photomicrographs of silver particles derived from a colloidal silver product known as Silverado.

Figures 20a and 20b are TEM photomicrographs of silver particles derived from a colloidal silver product known as the Vitamin World Bioorganic Advanced Colloidal Minerals (3ppm).

Figure 21 is a comparison of five TEM photomicrographs of silver particles, two of which are silver particles of the present invention and three of which are derived from commercially available colloidal silver.

Figures 22a and 22b show seven different Raman spectra, three of which correspond to the silver / water compositions of the present invention, one corresponds to pure water, one corresponds to deionized water and two corresponds to commercially available colloidal silver products.

Figure 23a shows two Raman spectra corresponding to the silver / water compositions of the invention; and Figure 23b shows three Raman spectra corresponding to three commercially available colloidal silver products.

Figure 23c shows another Raman spectrum corresponding to the silver / water compositions of the invention.

Figure 24a shows the Raman spectrum of the silver / water composition of the present invention; Figure 24b shows three Raman spectra of silver / water, zinc / water, and copper water compositions.

Figure 25 shows a graph of bacterial synergy potential interactions in the disk function assay.

Figure 26 shows curves, synergistic and antagonistic combinations of chromatic titres and impurities in therapy.

Figure 27 shows the excitability of MDR releases up to 10 ppm silver / water mixtures.

Figure 28 shows pictures of combinations of MRSA antibiotics.

Figure 29 shows photographs of antibiotic combinations of E. coli.

Figure 30 shows photographs of Pseudomonas antibiotic combinations; Figure 31 shows a graph of instantaneous applied voltage and instantaneous silver concentration as a function of process time through the process of forming a silver / water composition.

Figure 32 shows a graph of instantaneous silver concentration as a function of process time using absorption spectroscopy and electrical specific conductivity, respectively. This Figure also shows the silver concentration after 32 hours of production and after homogenization.

Figure 33 shows a snapshot of applied voltage, force factor, and silver concentration as a function of process time during the process of forming the silver / water composition of the invention.

Figure 34 is a diagram showing SILDERM moisture loss.

Figure 35 is a diagram showing SILDERM moisture absorption.

Figure 36 is a photograph showing the antimicrobial activity of silver chelates (Ag EDTA produced by Akzo-Nobel) against Pseudomonas aeruginosa (MRD).

Figure 37 is a photograph showing the antimicrobial activity of silver chelates (Ag EDTA produced by Alpha Chemicals) against Pseudomonas aeruginosa (MRD).

Figure 38 is a photograph showing SILDUST sensitivity to E. coli (MDR).

Figure 39 is a photograph showing the antiviral activity of SILDUST as a function of demonstration time.

Figure 40 is a photograph of a central test plate showing growth of the plates.

Figure 41 is a photograph of a test plate showing the absence of plaques after three hours and thus showing the antibacterial activity of SILDUST.

Figure 42 shows X-ray diffraction modules of the silver / water composition of the invention at 200 ppm; and the four presented X-ray diffraction sequences (streaks) adhere rapidly thereafter (e.g., AgO, Ag ₂ CO ₃ , Ag, and Ag ₂ O).

Figure 43 shows Ag ₄ O ₄ TGA analysis, as well as Ag ₄ O ₄ DTA analysis.

Figures 44a and 44b are SEM micrographs corresponding to the kaolinite / silver blends of the present invention.

Figures 45a and 45b are EDS (EDAX) analyzes corresponding to photomicrographs 44a and 44b, respectively.

Figure 46 is a SEM foromicrograph of a new zeolite / silver blend obtained according to the present invention.

Figure 47 is an EDS (EDAX) analysis of the zeolite Linde 4A containing substituted silver and obtained according to the present invention.

Figure 48a shows the ultraviolet spectrum of a 10ppm silver / water solution and 32ppm silver / water solution at a wavelength of 190nm to 400nm (both obtained according to the present invention) and Figure 48b shows an ultraviolet spectrum of the same samples at 190nm to 250nm.

BEST IMPLEMENTATION OPTIONS

The following non-limiting embodiments are provided:

A composition comprising silver nanoparticles colloidally suspended in water, wherein the total amount of silver is between 5 and 40 ppm, wherein the composition kills or destroys microorganisms that are hazardous to humans and / or animals.

A composition comprising silver nanoparticles colloidally suspended in water, wherein the total amount of silver is about 10 + 2 ppm, wherein the composition kills or destroys microorganisms that are hazardous to humans and / or animals.

A composition comprising silver nanoparticles colloidally suspended in water, wherein the total amount of silver is about 22 + 2 ppm, wherein the composition kills or destroys microorganisms that are hazardous to humans and / or animals. A composition comprising silver nanoparticles colloidally suspended in water, wherein the total amount of silver is about 32 + 3 ppm, wherein the composition kills or destroys microorganisms that are hazardous to humans and / or animals.

A hydrogel composition derived from a primary silver / water composition containing silver nanoparticles colloidally suspended in water, wherein the total amount of silver in the parent material is preferably about 32 + 3 ppm (but may be significantly less) where the hydrogel composition kills or destroys microorganisms that are hazardous human body and acts as a skin cleanser, wound remover and / or skin protector or skin disinfectant. It should be recognized that accurate determination of the total amount of silver particles in the silver / water composition does not accurately describe the material. Because the nanoparticles in the composition are smaller, the given silver concentration will result in a larger number of particles. In addition, the total surface area of a given silver concentration will increase. Thus, particle sizes and particle size variations are important parameters for characterizing the effectiveness of the silver / water composition of the invention. In addition, coating (s) such as oxide coatings (e.g., partially or fully formed) on said silver particles may also affect the effectiveness of the silver / water compositions of the invention where these coatings naturally occur during the process of the present invention. However, similar coatings on silver particles are obtained by other means (including metals other than silver such as zinc, copper, copper alloys, titanium, platinum and alloys of these alloys) are also described as measuring and limiting the present invention. Accordingly, although silver is mentioned herein, the use of various other alternative metals mentioned herein is also described as demonstrating potential efficacy depending on specific biological conditions (e.g., specific pathogens).

Another type of embodiment is any of the compositions described above wherein more than 50% of the silver nanoparticles have a maximum size of less than 0.015 micrometers.

Another type of embodiment is any of the compositions described above, wherein more than 75% of the silver nanoparticles have a maximum size of less than 0.015 micrometers.

Another type of embodiment is any of the compositions described above, wherein more than 90% of the silver nanoparticles have a maximum size of less than 0.02 micrometers.

Another type of embodiment is any of the compositions described above, wherein more than 75% of the silver nanoparticles have a minimum size greater than 0.005 micrometers.

Another type of embodiment is any of the compositions described above, wherein more than 90% of the silver nanoparticles have a minimum size greater than 0.005 micrometers and less than 0.040 micrometers.

Another type of embodiment is any one of the compositions described above wherein the silver particles have a dual silver zero-valency which, being metallic, has at least one silver coating in ionic oxidation in the core or central portion thereof in an oxidation state (Ag (O)). groups consisting of Ag (l), Ag (II) and Ag (III), with a coating of AgO, Ag ₂ O and / or Ag ₄ O ₄ , are usually at least (or substantially completely) on the metallic silver moiety.

Another type of embodiment is any of the compositions described above, wherein the silver particles have a dual silver zero metal, oxidation state (Ag (O)) and silver oxide coating with stoichiometric AgO or Ag ₂ O or other known stoichiometric remains constant in the process conditions used to make the new silver / water compositions of the invention Ag ₂ O.

Another experimental feature shows that the silver oxide coatings naturally occurring on the smallest part of the particles of the present invention are, for example, in the form of, for example, Ag ₄ O ₄ , that is, silver II oxide. In this molecule, two silver atoms may be in the Γ state (silver I), while the other two silver molecules may be in the 3 ⁺ state (silver III). In addition, under certain conditions, the silver may be 2+ (silver II) forming at least part of the coating, for example Ag ₂ O. These coatings occur naturally in the process conditions of the present invention (e.g., these conditions are created at and around the electrode / water interface) and can be critical to the overall effectiveness of the silver / water compositions of the present invention. It was difficult to determine the composition of the coatings so far, but here the details of the experiment were presented later in the descriptions.

Another type of embodiment is the combination of any of the silver / water embodiments described above with hydrogen peroxide at a level of 1-3 wt% hydrogen peroxide in the final product.

Another type of embodiment is the combination of any of the silver / water embodiments described above with a level of 0.5-10 ppm disodium EDTA in the final product.

Another type of embodiment is a combination of any of the silver / water embodiments described above with about 50-75 vol% replacement of 10% povidone iodine, replacing about 25-50% silver / water mixture in the final product. Another type of embodiment is the combination of any of the silver / water embodiments described above with various commercially available antibiotics (either liquid or powder form) to provide synergistically effective combination therapies.

Another type of embodiment are methods for using all of the above compositions against human or animal pathogens, either: (1) for internal use, (2) for external use, or (3) for both internal use and externally.

Another type of embodiment includes the use of AgEDTA for human and / or animal health or well-being.

Another type of embodiment includes the use of other silver agents such as silver EDDS, silver curcuminate, silver berberim and silver tetracycline.

Another type of embodiment includes the interchangeability of other metals, such as zinc, copper, copper alloys, titanium, platinum and their alloys or alloys, in both the preparation and manufacturing applications described herein. In short, silver is commonly referred to here, but it should be understood that other metals disclosed herein may be equally useful.

In another embodiment of the present invention, the additional silver-based inorganic products may be at least partially, or in some cases, substantially, complete silver / water compositions of the present invention. Specifically, silver (e.g., silver ions, Ag +, silver metal) can be bound or fixed in a controlled manner, for example, between clay layers and / or inside zeolite cages. Such fixation can occur by controlling, for example, the charge on the silicate layer, the charge on the zeolite cage, as well as the distances between the layers or the size of the zeolite cage. In this regard, silver may be elastic or relatively loosely attached or bound, depending on the point of interaction between the application of the particular health or wellness program between silver and the biological material (e.g., on the surface or inside of the biological material or in combination). Accordingly, the resulting products may include products that are completely liquid and therefore are taken orally or sprayed; as well as products in the form of gels or pastes which are applied to the surfaces as gels or pastes. The crystalline or amorphous clathrate may contain any of the metals discussed herein from one or more atomic layers of oxygen or oxygen-containing molecules. Certain metal / clathrate structures demonstrated unexpected efficacy. In addition to silicates, silicates, phosphates and oxides such as hydrotalcites introduced into or on the oxide layer (e.g., clay) and on the crosslinked structures (e.g., zeolites) can also be used. In addition, preferred clays or mica species that can be used with the present invention (and which may have different surface charges and / or different spacing between layers) include, for example, ilites, montmorillonites, chlorites, and vermiculites.

Clay or mica, as well as zeolites, are highly desirable as carriers of metal ions for a number of reasons, including the fact that many are naturally occurring or readily obtainable, particles can be considered to be a desired variation in colloidal size. they are, for example, suspended in a liquid (such as water) and are usually very bioavailable (eg small and without side effects). In this regard, when silver is deposited, for example, on or on clay or zeolite, the molecules are then heated to medium temperatures (e.g., 100-200 ° C) to attach the silver to or on the clathrate. All of these materials can be made in a very wide range of viscosities from very liquid to very viscous.

In addition, silver metal or silver ions introduced into silica gel by scattering and drying are also desirable mechanisms for delivering the metal ions of the present invention.

In another embodiment of the above-mentioned particles of the combinations of the invention, organic and / or inorganic structures can be used for positive effects on human and animal health and wellness. More particularly, the metal particles of the present invention may be combined with, for example, the organic compounds discussed above (e.g., AgEDTA). In addition, the metal ions of the present invention may be combined with any of the organic compounds (e.g., moles or zeolites). In addition, the metal ions of the present invention can be combined with both organic molecules (e.g., AgEDTA) and inorganic molecules (e.g., moles or zeolites). This combination of silver metal or silver ion delivery systems may be configured such that, for example, the internal consumption of any of the above silver supply systems may result in the supply of silver to different parts of the body, for example. Specifically, for example, in humans, certain silver may be absorbed orally, through the gastrointestinal tract, as well as through the large intestine and / or small intestine. In addition, depending on, for example, the amount of clay (s) or zeolite (s) corresponding to water (as well as the various gelling compounds discussed herein), the resulting product (s) can range from highly liquid (low viscosity) to very viscous (high viscosity). In general, the more clay or zeolite is present in relation to water (as well as the gelling agent), the more viscous the final product.

EXAMPLES

COMPOSITION COMPOSITION

Silver / water compositions may be prepared according to the process described in U.S. Pat. 6214299, the disclosure of which is herein incorporated by reference.

A method for preparing a composition comprising silver according to the present invention provides for the use of an electrochemical cell, including electrodes, and comprises the steps of:

(a) placing at least two silver electrodes in contact with high-purity water;

(b) transmitting an electric current through the silver electrodes and thereby separating the silver particles from said silver electrode by a method sufficient to cause the production of the suspended silver particles in water; and (c) mixing the water during said production of the suspended silver particles, thereby dispersing the silver particles in a more constant concentration in water so that each batch can obtain a high quality substantially uniform distribution of the suspended silver particles.

Another method of preparing a composition comprising silver / water compositions suggests using an electrochemical cell and comprising the steps of:

(a) providing an electrical circuit comprising an electrical source and electrical connection of the first conductor to said electrical source and a second conductor to said electrical source, wherein said first conductor is located at a distance from the second conductor and wherein at least one of the conductors is a silver element or alternatively zinc, copper, copper alloys, titanium, platinum and their alloys or mixtures;

(b) closing the circuit by placing the first conductor and the second conductor in contact with the liquid resistor;

(c) controlling the electrical source to generate alternating current simultaneously at the first and second conductors such that the voltage increases and decreases at the first and second conductors and alternates operations successively, thereby causing the silver (or other metal) particles to separate from the first electrode and introducing a liquid resistor and contacting them in suspension with the liquid resistor; and (d) selectively adjusting the electrodes by pushing them toward the liquid resistor to compensate for the reduction in electrode length due to the gradual separation of the silver particles, thereby preventing sparking between the electrodes and said liquid resistor and maintaining the desired current density at the electrode ends.

Each water chamber or reservoir producing silver / water compositions has a power supply consisting of eight transformers (an acceptable transformer for use in the present invention is Franceformer, Serial No. 48765), rated at 120 VAC and having a maximum output of 10500 VAC at 30 milliamperes. Each transformer was preferably equipped with a 45 microfarad capacitor (such as Aerovox, Serial No. M24P3745MP2), wire mounted parallel to the transformer power supply cable.

The combination of transformer and capacitor can be useful in some cases and highly desirable in other cases. Specifically, the transformer assists in transmitting AC power voltage and current sine waves to each other in phase. The degree to which voltage and currents are in phase is known as the power factor. The more the power factor is 1.0, the more the phases are matched between volts and amps and the more power is fed to the electrodes (eg power is usually described by multiplying volts by amps).

Each reservoir is equipped with a transparent cover made, for example, of suitable polymer and is constructed with eight electrode locations. At each electrode site is placed an electrode made, for example, of a size 18 silver plate, surrounded by two wasteful electrodes made, for example, of size 18 silver wire (9999 purity). The electrodes are preferably halved in the middle and the ends twisted into each other in a double helix to obtain the desired voltage and power for the complex combination. a single transformer is supplied to each electrode site.

When each tank is ready for production, the electrodes are adjusted so that the built-in electrodes have good contact with water (eg, at least 1/3 aA submerged plates) and the etched electrodes are above the water surface. When power is applied, the water rises and forms a conical structure around each etched electrode. This cone structure is known in the literature as the "Taylor cone". Initially, the water is very pure and acts as a high electrical resistance. Accordingly, when using, for example, a fixed-current 10,000-volt transformer, the voltage applied to the electrodes can be very high, e.g., about 6500-8500 volts, and the corroded electrodes can be 5-10 mm above the water surface, thereby achieving the desired voltage current density at the corroded electrodes. This yields a relatively large Taylor cone due to its low water specific conductivity compared to a high electrode specific conductivity (e.g., generates a large field). A product of silver nanoparticles is formed as silver particles are removed from the etched electrodes at the air-water-silver electrode contact. As water absorbs more and more silver particles, the electrical resistance of the water / silver mixture droplets occurs. In fixed current or current limiting equipment, the applied voltage will then drop or decrease as a function of time (see, for example, Figure 31). Correspondingly, the corrosive electrodes are typically lowered to be closer to the water surface, for example, only about 1-2 mm above the water surface. In simple terms, the Taylor cones will then be much tapered due to the smaller specific conductivity difference between the electrodes and water (e.g., a smaller field exists). Basically, the level of corrosive electrodes and / or water could be properly adjusted during the manufacturing process to maintain the initial geometry. Even if Taylor cones become smaller during this process (as expressed, for example, by the transition of metal particles into solution), small Taylor cones would still exist at the end of the process. Throughout the process, the water in each tank is mixed with air to maintain homogeneity.

Once the desired or expected ppm of silver in the silver / water solution is achieved, the product can be omitted, if desired or required, through a 1 micron filter into one or more very large storage tanks, for example, 2300 to 6500 gallons, and assayed before pouring into bottles for transportation. The analysis is carried out by systematization using heating and nitric acid and is performed using a 300 atomic absorption spectrophotometer of Perkin-Elmer analysis. The resulting silver / water composition can then be mixed with other ingredients to produce hydrogen, sheet material, or bottled as it is or can be mixed (e.g., as a liquid or as dried and added as a powder) with other attachments as described elsewhere herein.

Referring to Figure 32, 2 silver concentration fields are shown in the same experiment, as well as several values for the additional concentration data. The gray line to the squares represents the instantaneous silver concentration (as determined by atomic absorption spectroscopy) based on a 60 ml sample taken from the reservoir at about mid-depth and about halfway between the center of the reservoir and the walls. The black line with the diamonds represents the instantaneous concentration of silver, roughly predicted by measuring the electrical resistivity of the above-mentioned 60 mL liquid sample on the device. Based on the raw expression of the specific impedance, the initial (e.g. time = zero) electrical resistivity of the water is approximately 175 kilo-ohm centimeters. By comparison, the specific resistance of the water / silver mixture at the 31 hour mark is approximately 62.7 kilo-ohm centimeters.

Immediately below the concentration / specific impedance, the data point at the 32 hour mark is a single data point represented as a "square". This data point represents the concentration of silver as defined by atomic absorption spectroscopy after high-voltage release but allowing the bubbling liquid / mixture to continue working for 20 hours to homogenize the mixture.

One conclusion that can be drawn from Fig. 32 is that initially, the silver may not be homogeneously distributed throughout the reservoir where the silver is formed, regardless of the action of the bubble solution / mixture during the course of the product. In addition, there may be a delay in the addition of silver additives to the bath and before the bubble solution / mixture can "catch" and homogeneously distribute the silver in the water.

Figure 33 is another graph of instantaneous voltage and silver concentration as a function of time during the course of silver / water production. In addition, this diagram shows the power factor of the instantaneous power transformer. Thus, the force factor starts from about 0.8, increases to a peak of about 0.98 for about 6 hours, and decreases to a low of about 0.6 after about 30 hours, in addition, the y / 2 equation has been mathematically applied to the voltage / time data, 1333 Ln (x) + 8.7057 where y stands for voltage and x stands for time. Silver ppm in water is expressed as "squares" and starts at about 1 ppm and reaches a peak at about (e.g. because the water is not completely pure after filtration) at 11 ppm after about 30 hours.

PHYSICAL CHARACTERISTICS

Analysis of the silver content of the present invention in silver compositions may be performed by (acetylene) electrothermal atomic absorption spectroscopy (FAAS), inductively coupled plasma (ICP), atomic emission spectroscopy (AES), or by other methods known to those skilled in the art. . If the silver composition particles are small and of a constant size (e.g. 0.01 millimeter or less), a reasonably accurate sample can be obtained by directing the colloid directly to atomic absorption or ICP / AES. This is because the Atomic Absorption Spectroscopy sample essentially ionizes all silver, enabling its rapid detection.

If the compositions comprise particles of 0.2 micrometers, a systemic procedure is suggested. The systemic procedure is not necessarily ideal for silver compositions, which may be prepared and stored in contact with halides or other anionic sources which may react with a finely divided silver or combined with a protein or other gelatinous material. The implementation of the systematic procedure is as follows:

1. For analysis, take 10 ml of a well-mixed or shaken silver composition and place it in a clean polycarbonate bottle or other suitable container (usually a bottle) with a fitted lid. Available in sizes from 30 to 100 ml.

2. Add 0.1 ml of nitric acid with a micropipette or dropper and gradually add the reagent to the silver composition in the bottle.

3. When the bottle cap is closed tightly, the silver composition is heated to at least about 80 ° C and is offered to about 90 ° C to 100 ° C with gentle agitation sufficient to dissolve the silver - the dissolution is essentially instantaneous.

4. Allow the reaction mixture to cool to room temperature with the stopper closed. Shake the bottle thoroughly. This systematic procedure also dissolves any silver oxide surface layer that may appear on the silver particles.

5. The use of atomic absorption spectroscopy, ICP / AES, or equivalent means analysis of the silver content of the silver mixture. The use of a freshly prepared standard or standards, better prepared with equipment according to the manufacturer's instructions, with appropriate dilution as needed will be preferable.

6. All dilutions during the manufacturing process, including the 1% dilution caused by the addition of nitric acid, should be taken into account when adding up the results.

The silver / water compositions of the present invention have a silver concentration corresponding to that of Figures 31, 32, 33 and so forth. data were determined using a Perkin Elmer AAnalyst 300 Atomic Absorption (AA) spectrometer. Examples of silver / water compositions of the invention were systematized using the procedure described above.

PRINCIPLE

The Perkin Elmer AAnalyst 300 system consists of a highly efficient combustible system with a universal gem-type atomizer and atomic absorption spectrometer. The burner system supplies the heat energy necessary to break down the chemical compounds by supplying the free atoms to be analyzed with absorption of the atoms. The spectrometer measures the amount of light absorbed at a characteristic wavelength using a hollow cathode lamp as the primary light source, a monochromator and a detector. A deuterium arc lamp corrects secondary absorbances caused by non-atomic sources in the atomic cloud.

PHYSICAL / CHEMICAL FORMS OF SILVER AND SILVER / ANDEN COMPOSITIONS

ANALYSIS

a. introduction

A composition sample formally containing 22 ppm silver in water was airborne secondary ion mass spectrometry (TOF-SIMS) to determine the shape of silver in the composition. It was concluded that the masses of silver exist as silver (O) (that is, metallic silver) and that there is a surface that moderately covers the composition, such as silver (II) oxide (AgO). as noted above, silver (II) oxide is usually a stoichiometric combination of silver (I) and silver (III).

B. Experimental Procedure Several drops of the ppm silver composition of the invention were evaporated to dryness on a silica substrate at room temperature. The residue was analyzed by TOF-SIMS and labeled as a specimen. The said silver (II) oxide (AgO) material was assayed by depositing several particles of said powder obtained from a commercially available silicon substrate, and is hereby incorporated by reference.

Flying time secondary ion mass spectrometry (TOFSIMS) is based on the principle of solid-state bombardment of a solid sample with a pulsed, well-focused stream of primary ions, followed by analysis of secondary ions obtained from the sample surface by flight time mass spectroscopy. This analytical technique is a surface sensitivity that receives its information from a layer that extends from about 20 to 40 A (one Angstrom = 1x40-4 micrometers) below the surface. The TOF-SIMS technique is commonly used as a test tool to identify the composition of unknown samples. It may have quantifications provided that suitable microanalytical standards are comparable. This analysis is performed using standard high mass degradation conditions.

C. Results

The negative ion mass of the substance Ag (II) O and the product sample were obtained. The mass spectral region of both spectra showed the presence of more than one species of silver oxide, which is predominantly present in the smallest part of the silver moiety. The data indicate that silver (II) is in the middle state of oxidation of silver on the surface of the sample particles. Signs displayed on silver oxide (e.g., AgO) show a much higher intensity in the given sample compared to the product sample, which is probably because metallic silver is dominant in the sample. It will be appreciated that the particle size in the sample is reduced, and the ratio of silver to silver oxide will also decrease as more silver oxide is present.

SIZE / MORPHOLOGICAL / COMPOSITION TEST

The remarkable effectiveness of the silver / water formulations described herein appears to be due to the relationship between the surface properties / intrinsic properties of the particles (e.g., oxide / metal) and / or the size distribution of the silver nanoparticles and / or the morphology of the silver nanoparticles. The smaller the average particle size, the greater the surface area and the greater the contribution to the specific surface chemistry. However, loss of stability and / or other interactions that can adversely affect the product may occur if the particles are too small. The silver / water compositions of the present invention are excellent because they are stable in substantially pure water without surfactants, and so forth. (e.g., many colloidal silver known in the art require protein to keep the silver particles in suspension). Also, the silver / water compositions are essentially colorless, whereas other colloidal silver preparations (specifically large particle sizes) generally have a color. These properties are the result of production conditions as described hereinbefore.

Numerical composition study showed an average particle diameter of 0.0106 micrometers with a 0.005 oscillation to 0.0851 micrometers. However, size distribution analysis shows that more than 95% of the particles are from about 0.005 micrometres to about 0.015 micrometres in diameter.

Additional particle analysis was performed on SEM, EDS (EDAX), and TEM. Specifically, the silver / water compositions were dried and placed on EM plates and examined with SEM (i.e., scanning electron microscope) and two different TEM (i.e., transmission electron microscopes). These analyzers found particle size distributions in the range of 10-30 nm. However, some particle size estimation was necessary in some generating foromicrographs because the particles tended to fall to pieces or agglomerate during drying. The size of the dried agglomerates was between 50-100 nm. Figures 1-6 show various TEM photomicrographs of silver particles dried from the silver / water compositions of the present invention. Figures 7a-7d show TEM photomicrographs of silver particles of the present invention, where these photomicrographs were obtained by different techniques. Specifically, the silver / water compositions of the present invention were placed on a C-film and assayed for cryo-TEM (i.e., different TEMs than the TEMs used to obtain Figures 1-6) at about -100 ° C. Therefore, the silver / water compositions of the present invention were substantially immediately frozen. Cryo-TEM was operated at a power level of about -100 ° C and about 10OkV, and the resulting photomicrographs are shown in Figures 7a, 7b and 7c. These figures 7a-7c clearly show that the average particle size is less than 20 nanometers. In addition, Figure 7d shows TEM analysis in SAD mode. In essence, these TEM photomicrographs (Figures 7a-7c) show a maximum particle size of ungrouped silver particles of 15 nanometers or less and some smaller particles in the range of 3.5 to 5 nanometers; , are multiplied pairs and are essentially pure. These foromograms contain a hint of a possible cover or layer. Figure 7e shows the EDAX spectrum (i.e., energy dispersion spectrum or "EDS") of silver particles taken from the silver / water compositions of the present invention. Figure 7e shows total non-metallic impurities in silver (e.g., Au, Pt, etc.). The copper present is from the necessary microscope equipment. There is a significant amount of oxygen present which may be present in the distillate as well as the coating (s) on the smallest particle of silver.

Figure 8 shows an electron diffraction module obtained from a silver particle of the present invention. These data indicate the presence of at least one species of silver oxides. However, this data is partially interpreted, as Figure 9 shows, for example, a possible electron beam damage to silver particles during the data collection process. This electron beam damage is not as obvious when the colloidal silver under investigation is manufactured by other manufacturers (described here later). In this way, data acquisition using SEM and TEM techniques is clearly difficult because the electron beams of any of the surface compositions of interest can be vulnerable (and therefore variable). In this way, these results were carefully obtained and analyzed.

Figure 42 shows the results of yet another characterizing means. In this case, powder X-ray diffraction techniques have been employed to further demonstrate the existence of the oxide phase (s). Specifically, Figure 42 shows four X-ray diffraction modules obtained from four different locations on a dried 200 ppm silver / water composition prepared in accordance with the present invention. Moreover, the four X-ray diffraction modules are particularly attached to the four diffraction modules of the non-pure silver metal species. Specifically, the 32 ppm silver / water composition prepared according to the present invention was concentrated to about 200 ppm by standard reverse osmosis water filtration. Specifically, the silver / water composition of the invention was passed through a reverse osmosis filtration system wherein the "effluent" water from the reverse osmosis filtration system contains a much more concentrated silver component. Once a 200 ppm solution was obtained, this solution was dried by passing through a nitrogen medium to obtain a powder that could be redirected to X-ray diffraction Specifically, the silver / water mixture was placed in a bowl, the bowl was covered with a plastic sheet and nitrogen was introduced at the back of the sheet unit; and nitrogen is released at the other end of the bowl / plastic sheet unit. The apparatus temperature did not exceed 75-80 ° C to maintain the integrity of all components in the silver / water mixture Sufficient amount of dried powder (eg, made from 200 ppm solution) was then suitable for X-ray diffraction analysis.

The resulting X-ray diffraction modules clearly show the presence of at least four separate species. In this regard, it is clear that the determination of silver carbonate peaks occurs at approximately 18 to 22 degrees. These peaks are most likely due to the drying procedure. In this regard, the presence of CO ₂ in the air is most likely to occur, even if considerable effort has been made to develop a nitrogen coating at 200 ppm during the drying process. in addition, the pitch setting occurs at approximately 33 degrees. However, each of these peaks can be attributed to silver oxide (AgO), silver carbonate (AgCOs) and / or silver oxide (Ag ₂ O). Therefore, it is not entirely clear which species exist. In addition, a strong silver metal peak occurs at around 38 degrees. This strong peak can be seen in each of the X-ray diffraction modules. However, it should be noted that a small peak of silver oxide (Ag ₂ O) also occurs at approximately 38 degrees. In addition, a strong silver oxide (AgO) peak occurs at about 37 degrees in combination with a relatively strong silver carbonate (Ag ₂ CO ₃ ) peak as well. In addition, it is noted that the silver oxide (AgO) peak corresponds to one of the phases of the silver oxide quadrilateral. What is clear from reviewing the resulting X-ray diffraction data and comparing it with existing database records is that one or more of the silver oxide phases are present in the silver / water compositions of the present invention. It is possible that the oxide combination exists due to new manufacturing techniques according to the present invention. It should be noted that the X-ray diffraction modules are suitable for Ag ₄ O ₄ compared to the X-ray diffraction modules of the present invention.

However, since Ag ₄ O ₄ does not exist commercially, in this respect the Ag ₄ O ₄ sample was obtained industrially and subjected to TGA and DTA analysis of this powder. Specifically, Figure 43 corresponds to TGA analysis and DTA analysis, respectively. From the DTA curve in Figure 43, it is clear that the Ag ₄ O ₄ endotherm exists at about 181 ° C. This endotherm also corresponds to the weight loss depicted in the TGA curve in Fig. 43. These experimental measurements correspond to Ag ₄ O ₄ decomposed to Ag ₂ O. A second very strong endotherm is shown at about 403 ° C, as well as a second corresponding weight loss. These two experimental points correspond to Ag ₂ O degradation to Ag metal.

Figure 10 shows a SEM photomicrograph of a new silver electrode before being used in the process of the present invention. The EDS elemental analysis was performed with the electrode parts designated 1, 2, and 3. These three separate analyzes are shown in Figures 11, 12, and 13, respectively. These analyzes showed the existence of substantially pure silver.

Figure 14 shows a SEM photomicrograph of the end of the spent silver electrode after being used in the process of the present invention. The EDS elemental analysis was performed with the electrode sections labeled 1 and 2. These two separate analyzes are shown in Figures 15 and 16, respectively. Figure 17 shows a SEM photomicrograph of the used electrode tip at a higher magnification (approximately 3500Χ). Parts 4 and 5 were also examined by EDS elemental analysis and also found in substantially pure silver.

Comparison of silver particles of colloidal silver used in industry

In an attempt to understand the differences in the production of the silver / water compositions of the present invention (e.g., bioavailability), the differences in physical properties compared to known colloidal silver have been investigated. Figures 18a and 18b are TEM foromicrographs of silver particles corresponding to the first colloidal silver obtained from the General Nutrition Center in 2004 and marketed as the GNC Liquid Colloidal Silver Dietary Supplement (25 pmm) ("GNC"). Figures 19a and 19b are TEM foromicrographs of silver particles that correspond to a second colloidal silver known at the marketplace as Silverado. Figures 20a and 20b are TEM photomicrographs of silver particles that correspond to the third colloidal silver, commercially known as Vitamin World Bioorganic Advanced Colloidal Minerals. Figure 21 is a summary of silver particles from two silver / water inventive compositions (designated as "ASAP 20" and "ASAP 10") and three commercially available colloidal silver known as "GNC", "Silverado" and "Bioorganic" described above. , Comparison of TEM photomicrographs. Clear differences in particle sizes and shapes are evident from these photomicrographs, indicating that there are physical, structural and potential chemical differences between the different colloidal silver, which may partly help explain the differences in bioavailability between products of a similar overall chemical structure.

SPECTROSCOPIC DESCRIPTION

RAMAN SPECTROSCOPY

Additional analysis of silver / water mixtures was performed by Raman spectroscopy. A series of analytical studies have been conducted with three different Raman spectrometers. The reason for using Raman and resonant Raman spectroscopy was the view that different vibration states (and / or amplitudes) could be observed in different colloidal silver as compared to the silver / water compositions of the present invention, as well as by comparing "pure" and deionized water. In addition, the different observed vibration modes in water molecules may help to better characterize colloidal systems and explain the different biological performance of different silver-based products.

The first series of Raman spectroscopy measurements used a Confocal Raman microscope from Vitek (Ulm, Germany). The model number was CRM200. The spectrum was obtained using Nikon 6Ox immersion lenses (NA = 1) with a 15 second integration time for the spectrum (i.e., three separate 5 second run times).

The CCDs were concentrated to about 1,799 waves. A drop of the solution was placed in a small cavity in a petri dish and immersion lenses were directed there.

The Raman laser source was 532 nm with approximately 10 mW. A co-focusing detection system with a co-focal volume of about 0.3x0.3x0.75 micrometers (approximately 7x10 E-8 picoliters) was used.

Figures 22a and 22b show graphical data of data collected in 7 samples. Two of the samples were identical, even differently labeled (10PR and 10PSU), and corresponded to the aforementioned ASAP 10 (i.e., 10 ppm silver from the silver / water composition of the invention). "HPLC" corresponded to high purity (ultra high purity HPLC) water from Alfa Aesar. Dl corresponded to deionized water. GNC was consistent with GNC Liquid Colloidal Silver Dietary Supplement (25 ppm). AGX-32 corresponded to a 32 ppm silver / water composition of the invention. VW conformed to Vitamins Global Bioorganic Advanced Colloidal Minerals (3 ppm) (previously designated as Bioorganic). There are clear differences between the different samples. For example, the first extension module (e.g., wave numbers around 3400-3500 1 / cm) shows significant differences in these several water / water based solutions. In addition, vibrational / rotational functioning below 1 / cm also shows clear differences between samples. Also, some differences can be seen in bend types of about 1600 1 / cm. Without wishing to be bound by any particular theory or explanation, it has been discovered that the various functionalities of the silver / water compositions of the present invention may, for example, influence or at least assist in explaining the effectiveness of such compositions relative to other samples tested.

The second series of Raman measurements was obtained from different spectrograph systems. Because the numbers obtained between the two series are different (which strongly demonstrates that the Raman water spectroscopy data is a function of the analytical tool used), the data in the data series also show significant differences between the silver / water compositions of the present invention compared to other colloidal silver and other waters. This series of Raman spectroscopy measurements uses a reflection Raman microscope. The spectrum is obtained using Olympus 2Ox lenses (NA = 0.4). The CCD detector was focused on four different wave numbers, namely, 1600, 2500, 3400 and 4400 1 / cm. The source of the Raman laser was

514.5 nm with approximately 11.5 mW. Additional spectrum-related information can be found in each of Figures 23a, 23b, and 23c. The sample designation in these figures corresponds to the text above. Both Raman spectroscopy data series strongly confirm the existence of different molecular motions in these different samples, which can contribute (or at least make it obvious) to the biological effectiveness of the silver / water compositions of the present invention.

The third series of Raman data was obtained using a third multiple Ramishow co-focal Raman micro spectrograph of a laser line. This system was configured to allow measurements both above and below the sample. The series was designed to examine a sample volume of 100x to 1000x as described in the first series of measurements. The reflection micro-spectrograph with a Leica DL DM microscope was equipped with either 2Ox (NA = 0.5) immersed in water or 5x (NA = 12) dry lenses. The aperture size of the posterior portion of each lens was set equal to or greater than the expanded diameter of the laser beam. Two laser frequencies were used, which are multiple 50mW Argon laser ^Λ Α power 514.5nm and 20mW HeNe laser 633 nm. Large scattering lattices were mounted on a monochrometer optical path, allowing scanning from 50 to 4000 waves (1 / cm). Integration times of 10 to 20 seconds were used. The sample liquid was placed below the lenses in a 50 mL beaker. Both lenses were used to study the resonant ranges, whereas the previous laser was initially used to obtain the Raman spectrum. The sample size was approximately 25 ml. Measurements made with 5x dry lenses were made with a lens positioned approximately 5 mm above the liquid to obtain volume information approximately 7 mm below the water meniscus. Immersion measurements were made with 2Ox immersion lenses positioned approximately 4 mm into the sample, which allowed the same volume of the environment to be examined. The CCD detector detection areas were adjusted for each lens to maximize signal intensity and signal-to-noise ratio. The spectrum expressing the silver / water compositions of the present invention is shown in Figure 24a. Figure 24b shows the Raman spectrum of three different metal / water solutions prepared according to the present invention. Field 1 corresponds to a 13 ppm silver / water solution; field 2 corresponds to 10 ppm zinc / water solution and field 3 corresponds to 11 ppm copper / water solution.

Because the numbers from the three data series are slightly different (which strongly suggests that the Raman spectroscopy water data is a function of the analytical instrument and this instrument series), the data comprising the data series exhibit significant differences between the silver / water compositions of the present invention. , when compared to lithium silver or other waters. All Raman spectroscopy series data strongly confirm that different molecular motions and junctions exist in these different samples, which may be a factor (or least obvious measure) in the efficiency of the silver / water compositions of the present invention. In addition, the differences in Raman modules of the three different metal / water solutions shown in Figure 24b also confirm the possibility of different efficiencies.

UV-VIS SPECTROSCOPY

Further analysis of silver / water mixtures was performed by UV-Vis spectroscopy. In addition to Raman spectroscopy, UV-Vis spectroscopy has also been used to determine additional distinctions and / or vibration amplitudes in different parts of the spectrum. A separate UV-Vis spectrometer was used for data acquisition. In this regard, energy absorption spectra were obtained using UV-Vis micro-spectrophotometry. This information was collected using a dual-beam scanning monochrometric system that enables scanning of wavelength fluctuations between about 190 nm and about 1100 nm. The UV-Vis spectrometer that was used to collect the absorption spectrum was the Jasco MSV350. The instrument was used to measure low concentration liquid samples using a 10 mm x 10 mm burnt quartz cuvette. Data were collected at wavelength variation using both a photo multiple camera (PMT) and a photodiode detector with the following operating parameters: bandwidth capture 2 nm, scattering 0.5 nm; and the water baseline background is isolated from the resulting spectrum. In this regard, the UV-Vis pure water tag was isolated from the resulting spectrum so as to show the more characteristic silver / water mixture spectrum tags.

Both tungsten "halogen" and hydrogen "D2" energy sources were used as primary sources of MSV350. An optical spectrometer track was set up to allow the energy beam to pass through the samples, further focusing on the center of the sample cuvette. Sample production was limited to filling and sealing the cuvettes and physically placing them on the cuvette holder inside a fully enclosed sample chamber. Data values were measured and plotted as absorbance units (Beer-Lambert's Law) before wavelength and frequency. The primary difference between the samples corresponding to the two spectra depicted in each of Figures 48a and 48b is the concentration of silver from each of the samples. Specifically, the higher amplitude curve of each of Figures 48a and 48b corresponds to a 32 ppm silver / water solution; and lower amplitude curve corresponds to 10 ppm silver / water solution. The wavelength and frequency positions of the peaks (i.e., the pitch distributions and falls) are very similar.

As described above, silver (e.g., silver ions, silver metals, Ag +, etc.) can be controlled or attached, for example, between and / or clay layers and / or inside zeolite lattices. One way of achieving attachment, for example, of silver ions to or on moles, mica, or zeolites, is accomplished by supplying types of ion of suitable charge to silver and introducing said types into a composition or mixture of clay or zeolite. The concept of a replacement silver ion, such as another positive charge ion, is sometimes referred to as "BEC" or "CEC" (both of which are abbreviated terminology indicating the "system" susceptibility to cation exchange). In this regard, the most widely known is kaolinite, which is susceptible to cation exchange in the range of 2-5 (i.e. 2-5 meq / 100 grams). Montmorillonite moles, for example, are susceptible to cation exchange at about 100 meq / 100 grams. Meanwhile, the zeolite susceptibility to cation exchange can be several hundred meqs / 100 grams. For example, the well-known BEC and CEC of a zeolite known as Linde 4A zeolite is 400-500 meq / 100 grams. In essence, the higher the BEC or CEC number, the greater the material's ability to obtain cations.

Reporting systems have been introduced for experimental procedures to determine the potential of kaolites or zeolites to become silver (or other metal cation (s) holder). Specifically, the following steps were used to obtain and study silver-mole samples as well as silver-zeolite samples.

Generally speaking, typical kaolinite and zeolite Linde 4A materials were first washed three times with deionized water to remove possible chlorine impurities that could cause the deposition (or undesirable reaction) of certain silver precursors (e.g., silver ions) against the silver precursors. possible desired attachment to either kaolinite and / or zeolite structures. These washed materials are then mixed with a solution of silver nitrate (AgNO ₃ ) at appropriate concentrations corresponding to the expected or known "CEC" of each respective substance. The resulting treated materials are then washed again with deionized water to remove unused silver nitrate. The samples are dried overnight in an electrically resistant oven at approximately 120 ° C. specifically, the washing procedure was carried out as follows:

About two grams of each sample of kaolinite or zeolinite is placed in a centrifuge chamber. Then deionized water is added. The mixture of sample and deionized water was then whipped in a hand-held whisk for approximately 40 minutes. The mixture was then centrifuged for about 30 minutes at about 1000 RPM. The excess liquid was then decanted from the sample tube. The deionized water addition, shaking, centrifugation and decanting steps were repeated for all three washes.

Once the initial 2 gram sample was properly washed, silver nitrate was added to the purified kaolinite and zeolite materials to remove any possible chlorine impurities. Specifically, about 0.09 silver nitrate was introduced into the kaolinite mixture and about 4.25 grams of AgNO ₃ was introduced into zeolite Linde 4A. Specifically, measurable amounts of silver nitrate were added to each tube, then deionized water was added to fill the tube, the mixture was shaken with a hand mixer for about 40 minutes, and then centrifuged for about 30 minutes at about 1000 RPM. The liquid was then decanted. This procedure of addition of silver nitrate, addition of deionized water with a hand mixer, centrifugation and decantation was repeated three times in total. After the washing and silver nitrate addition procedures were completed, the samples were removed from the centrifuge chamber and placed in an aluminum (AI2O3) crucible and dried overnight at about 120 ° C in an electrically resistant oven. The resulting kaolinite / silver and zeolite / silver materials were then characterized by SEM photomicrographs and SEM EDS (EDAX) techniques. Figures 44a and 44b show SEM photomicrographs of kaolinite samples obtained by the techniques described above. From these microdiagrams, it is clear what the book-type or sheet-like kaolinite structures (e.g., SiO ₂ and AIO ₂ O ₃ layers identified as "X" and "Y" in Figure 44a) clearly indicate that the silver cations are located around the edges of the clay material. There are obviously some types of silver attachment or exchange, as evident in the lighter page-type portions of these photomicrographs (note: the "X"and>, Y "portions refer to various other book-like structures in the sample). Figures 45a-45b show EDS (EDAX) analysis of the samples shown in Figures 45a and 45b, respectively. These studies clearly show the presence of aluminum and silicon, which may be probable for kaolinite, as well as some titanium (assuming the presence of rutile). Very small silver peaks can also be seen, which corresponds to BEC kaolin numbers, which are relatively low between 2 and 5.

Figure 46 shows a SEM photomicrograph corresponding to zeolites obtained according to the procedures described above. Due to the large number of zeolite CECs (in particular, about 500), the zeolite cube-like structures in Figure 46 begin to glow in the microdiagram (see, for example, part A in Figure 46). This glow confirms that there was a significant uniform distribution of silver within or through the zeolite structures. In this respect, bright dots of silver metal were observed, which themselves shone brightly, after which silver was not included in or on the zeolite. Figure 47 is an EDS (EDAX) analysis of the sample shown in Figure 46. Again, relatively high amplitude aluminum and silicon peaks are observed, but there are extremely large silver peaks (i.e., compared to the Ag peak kaolinite shown in Figures 45a-45b). these very high silver peaks correspond to the significantly higher ability of the zeolite to attract silver in its structure (i.e. high BEC) relative to the kaolite structure (i.e. low BEC) depicted in Figures 44a and 44b.

PROOF OF EFFECTIVENESS OF PPM SILVER COMPOSITION AGAINST BACILLUS SUBTILIS

A. Purpose of the test

The purpose of this test is to demonstrate the antimicrobial activity of the silver-based composition of the present invention against bacterial endospores from the test organism Bacillus subtilis. This was accomplished in a standard time-kill assay using a suspension of B. subtilis endospores. Normally, bacterial endospores are resistant to killing.

B. Materials and Methods

The organism being tested. A test suspension containing endospores from Bacillus subtilis (ATTC # 19659) from a culture grown on nutrient agar was added with additional spore-forming ingredients. The plates were enriched with sterile water and the endospores were purified by repeated centrifugation and water resuspensions. The final wash was performed in 70% ethanol for 30 min to induce destruction of the vegetative bacterium. The spores were resuspended in water containing 0.1 Tween 80 (polysorbate surfactant label) to prevent lumping.

Neutralizer. The neutralizer mixture consisted of 12.7% Tvveen® 80 (polysorbate label), 6.0% Tamol® SN (naphthalene formaldehyde condensate sodium salt), 1.7% lecithin, 1% peptone, and 0.1% cystine. This solution was designed to neutralize any chemicals so that they would not subsequently affect the growth of the bacterium.

Time-kill procedure;

a) 9.9 ml of the disinfectant sample (22 ppm silver composition of water of the invention) was placed in a sterile 20 mm x 150 mm tube. The tube was equilibrated in a 20 ° C water bath.

b) 9.9 ml of disinfectant sample (22 ppm silver composition of water of the invention) was placed in a sterile 20 mm x 150 mm tube. The tube was equilibrated in a 20 ° C water bath.

c) One ml of organism / disinfectant suspension was transferred to a tube containing nine ml of neutralizer after 30 minutes, 1 hour and 4 hours. The tube was thoroughly stirred.

d) After two minutes, the neutralized suspension was gradually diluted 1:10 in saline (PPS).

(e) The row of viable organisms in the selected dilution tubes was examined by membrane filtration. One ml of samples was placed in duplicate. Membranes were washed with approximately 100 mL of sterile PPS and transferred to nutrient agar plates. The plates were incubated at 37 ° C for 20 hours.

(f) The number of columns for each filter was calculated and the logarithmic reductions were evaluated.

Controls:

(a) The titres of the test suspensions were evaluated by membrane filtration tests selected from 1:10 dilutions of test suspensions in PPS.

b) Neutralizer control was performed by adding 9 ml of neutralizer and 1 ml of disinfectant to 100 ml of titre dilution containing 100 cfu. This is done in a tube of approximately 10 cfu / ml, which has been left to stand for 20 minutes, prior to membrane filtration analysis using duplicate 1 ml samples.

C. Results

Titer of Bacillus subtilis

Dilution

1: 1xn: 1: 1 of 1: 1x10x10 ₇ -1 41

TNT 75 7 Colony Count: C TNT 58 8C

TNTC = Too many to count

B. Subtle dilution of spore / disinfectant suspension:

Time 1: 1x10 ¹ , 1: 1x10 ² , 1: 1x10 ³ , 1: 1x10 ⁴ 1: 1x10 ⁵ , 1: 1x10 ⁶ 30 min-TNTC TNTC 57 10 • TNTC TNTC 51 7 or — TNTC TNTC 28 3 • TNTC TNTC 55 3, 2 hours — TNTC TNTC 126 23 • TNTC TNTC 183 17, 4 hours — TNTC TNTC 88 12 - TNTC TNTC 69 12 TNTC = too many to count

Neutralization counter: 1: 1x10 ⁸

D. Discussion

The titre results showed a viable S. subtilis spore concentration of 6.65x10 ⁸ spores per ml in the original suspension. Inoculation of 9.9 ml disinfectant with 100 ml of this suspension gave an initial concentration of 6.65x10 ⁵ spores per ml in the test tube.

The results of these procedures allowed the log values (LR) and percent kill (PK) values to be calculated. They are listed in the table below. Values were calculated using the formulas: LR = -Log (S / SO) and PK = (1- (S / SO)) x 100; where S = concentration of organisms in the defined time; and So = initial concentration of the organisms at zero time.

LOG reduction percentage kill time:

min 0.090; 18.8 or. - 0.205; Or. 0.634; 76.8 or 1.992; 98.8

Neutralization control data indicated that the disinfectant was sufficiently neutralized, with actual calculations consistent with those obtained from dilution without appreciable killing.

The disinfectant tested here exhibited good spore activity against B. subtilis spores. B. subtilis are common spores and are used in spore testing and belong to the same species as the organisms that cause anthrax. Because of their genetic similarities, B. subtilis spores have been used as a lesser surrogate for anthrax bacillus, anthrax bacterium. Therefore, these results are applicable to anthrax. Prolonged exposure is likely to result in additional killing.

PROOF OF EFFECTIVENESS OF SILVER AND 0.1% H ₂ O ₂ COMPOSITION AND 14 PPM SILVER AND 1.5% H ₂ O ₂ COMPOSITION AGAINST BACILLUS SUBTILIS

a. Purpose of the test

The purpose of this test is to demonstrate the efficacy of two silver-based compositions of the present invention against bacterial endospores from the test organism Bacillus suntilis. This was done in a standard time-kill assay using a suspension of B. subtilis endospores. Comparing the example previously reviewed (using 22 ppm silver), this example produces the hydrogen peroxide (H ₂ O ₂ ) promoting effects on the antimicrobial properties of silver compositions. Hydrogen peroxide is stable in the present silver compositions of the present invention. Because silver hydroxide itself has pronounced antimicrobial properties, it is often degraded by catalysis or other microbial enzymes. However, hydrogen peroxide can weaken bacterial cell walls and enhance the entry of silver particles before enzymatic destruction of hydrogen occurs.

B. Materials and Methods.

1. Test organism. A test suspension containing B. subtilis endospores (ATCC # 19659) from culture on nutrient agar was prepared. to which additional spore-forming agents have been added. The plates were enriched with sterile water and the endospores were purified by repeated centrifugations and re-suspensions in water. The final wash was performed in 70% ethanol for 30 min to confirm the death of the vegetative bacterium. Spores were resuspended in water containing 0.1 Tween® (polysorbate label) to prevent clumping.

2. Neutralizer. Neutralizer blend of 12.7 Tween 80, 6.0 Tamol® SN (Naphthalene-formaldehyde condensate sodium salt), 1.7 Lecithin, 1 Peptone and 1 Cystine. This solution was designed to neutralize any chemicals so that they would not affect further bacterial growth.

3- Killing time procedure:

a) 9.9 ml of each disinfectant sample (colloidal silver compositions of the invention: one containing 14 ppm silver and 1.5% H2O2; the other containing 10 ppm silver and 1.0% H ₂ O ₂ ) was placed in sterile 20 mm x 150 mm tube. The tube was equilibrated in a 20 ° C water bath.

(b) 100 ml of the test organism suspension at zero time was introduced into each tube of disinfectant.

c) One ml of organism / disinfectant suspension was transferred to a tube containing 9 ml of neutralizer after 10 min, 30 min, 1 hr, 4 hr, 6 hr. is 8 or. The tube was thoroughly stirred.

d) After two minutes, the neutralized suspension was gradually diluted 1:10 in saline (PPS).

(e) The row of viable organisms in the selected dilution tubes was examined by membrane filtration. One ml of samples was placed in duplicate. Membranes were washed with approximately 100 mL of sterile PPS and transferred to nutrient agar plates. The plates were incubated at 37 ° C for 20 hours.

(f) The number of columns for each filter was calculated and the logarithmic reductions were evaluated.

4. Controls:

(a) The titres of the test suspensions were evaluated by membrane filtration tests selected from 1:10 dilutions of test suspensions in PPS.

b) A neutralizer control was performed by introducing into 9 ml of neutralizer and 1 ml of disinfectant in a 100 ml ^three 1:10 dilution of the titer. This is done in a tube of approximately 2000 cfu / ml, which has been left to stand for 20 minutes before being diluted 1: 1. Both tubes were subjected to membrane filtration using duplicate 1 mL samples. All results are shown in Tables 1a and 1b.

C. Results

Titer of spores of Bacillus subtilis

1: 1x10 ⁶

Dilution n: 1; 1x10 ⁷

1: 1x10 ⁸

Number of Columns: TNTC TNTC

TNTC = Too many to count.

Table 1a

A solution containing 14 ppm silver and 1.5% H2O2;

Dilution of B. subtilis spore / disinfectant suspension:

Time 1: 1x10 ¹ 1: 1x10 ² 1: 1x10 ³ 1: 1x10 ⁴ 1: 1x10 ⁵ 10 min - - TNTC TNTC 227 - - TNTC TNTC 265 30 min - - TNTC TNTC 258 - - TNTC TNTC 273 1 or. - - TNTC TNTC 55 - - TNTC TNTC 33 2 or. - TNTC 207 29th - - TNTC 237 24th - 4 or. 59 3 1 29th - 57 5 1 6 or. 0 0 0 3 0 0 8 or. 1 0 0 1 0 0

TNTC = Too many to count.

Neutralization control:

Undiluted 1: 1x10 ¹

TNTC 195

TNTC 210

TNTC = Too many to count

Table 1b

A solution containing 14 ppm silver and 1.0% H ₂ O ₂ ;

Dilution of B. subtilis spore / disinfectant suspension:

Time 1: 1x10 ¹ 1: 1x10 ² 1: 1x10 ³ 1: 1x10 ⁴ 1: 1x10 ^! 10 min - TNTC TNTC 230 - TNTC TNTC 287 30 min - TNTC TNTC 254 TNTC TNTC 260

1 or. - - TNTC TNTC 146 - - TNTC TNTC 124 2 or. - TNTC TNTC 64 - - TNTC TNTC 71 - 4 or. TNTC 72 5 TNTC 77 5 6 or. 0 0 0 2 0 0 8 or. 0 0 0 0 0 0

TNTG = Too many to count.

Neutralization control:

Undiluted 1: 1x10

TNTC 200

TNTC 184

TNTC = Too many to count

D. Discussion

The titre results showed a viable spore concentration of B. subtilis of 62.59 x ^{10 8} spores of the original suspension. Inoculation of 9.9 ml disinfectant with 100 ml of this suspension gave an initial concentration of 2.59x10 ⁵ spores in ml test tube.

The results of these procedures allowed the log values (LR) and percent kill (PK) values to be calculated. They are listed in the table below. Values were calculated using the formulas: LR = -Log (S / So) and PK = (1- (S / So)) x 100; where S = concentration of organisms in the defined time; and So = initial concentration of the organisms at zero time.

In the absence of significant killing for 30 min, data were used for So values over a 10 min period. 6 or. is 8 or. retention time did not show high enough reliable numbers. Thus, these data were not used in linear regressions. Linear regressions were derived with logarithmic reduction values using customized linear chart programs in the minilab statistical software package. The resulting regression equations and the time required to perform 6-log reduction are plotted along with log reduction and percent kill values in Table 2 below.

table ppm silver + 1.5% H ₂ O ₂ 10 ppm silver + 1.0% H ₂ O ₂

Time log reduction Percent killing log reduction Percent killing

30 min -0.03 -7.9 0.003 0.6 1 or. 0.66 78.0 0.28 47.8 2 or. 2.05 99.1 1.58 97.4 4 or. 4.63 99.998 3.54 99.97

Regression analysis ppm invented linear equation: Y = -0.66704 + 1.32936x. 10 ppm invented linear equation: Y = -0.59690 + 1.03933x. These equations assume a 6-log reduction time of 5.02 hours for a 14 ppm composition and 6.35 hours for a 10 ppm composition.

Neutralization control data indicated that the disinfectant was sufficiently neutralized. The expected numbers correspond to those expected for dilution.

Experimental disinfectants tested The strain of B. subtilis used in these assays is the same as defined in the AOAC spore assay. Spores from this organism show the highest resistance to many disinfectants, the time required to accomplish 6-log reduction is in line with the spore tag approval of most sterilizers.

DEMONSTRATION OF THE EFFECTIVENESS OF PPM SILVER COMPOSITION AS A WIDE-SPECIFIC ANTIMICROBIAL

a. Ways

MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) assays were performed according to the standard broth dilution method. MIC is defined as the lowest concentration of antibiotic that would inhibit the growth of infectious organisms (in vitro). The results are given in micrograms per ml.

Interpretation of in vitro data for medical antibiotics is based on achievable serum concentrations, which vary with dose, route of administration, degree of protein binding, proportion of infection, patient age and weight, and other factors. MBC is described as the lowest concentration of antimicrobial agent required to kill 99.9% of the original organism's infection.

The assay was performed by culturing pure cultures of each of the test organisms in liquid medium. Repeated dilutions of each test antibiotic were made in the broth. Dilutions were calculated to include the fluctuations of the agent within each organism. A standard volume of test culture was added to each tube and the tube transferred to an incubator (37 ± 2 ° C) for growth. Tubes were inspected turbinometrically for bacterial growth. Below MIC concentration tubes showed an increase in optical density with time, detecting bacterial growth. The lowest concentration of antibiotic that does not show growth is MIC. Non-growth tubes were then subcultured in fresh medium. The non-growth tube with the lowest antibiotic concentration showed no growth in the subculture MBC. The results are shown in Table 3.

B. Results Table

Antimicrobial (ppm)

The organism S.pyogenes S. mutans Tetracycline 0.62505 0.62505 Ofyloxacin 1.25 / 2.5 2.505.0 Penicillin G Cefaperazone Erythromycin Silver 2.5 / 5.0 2.5110.0 > 5.0 0.52105 0.313 / 1.25 1.2505 0.003 / 0.019 0.009 / 0.019 S gordonll 0.156 / 0.625 2.5 / 5.0 0.009 / 0.039 1.25 / 1.25 0.005 / 0.019 2: 5 / 10.0 S. pneumoniae 0.078 / 0.625 2.512.5 0.019 / 0.019 0.313 / 0.313 0.00210.004 2.5 / 2.5 S. fascs / is 0.3131> 5 1.25 / 5.0 5.005.0 > 5.0 0.009 / 1.25 10.0 / 10.0 S. aureus 0.31305 0.417 / 0.625 2.505.0 5.0 / 5.0 0.03905.0 5.0 / 5.0 P. aeruginosa 0.078 / 5 0.156 / 0.313 0.131> 5.0 2.5 / 5.0 2.505.0 1.67 / 5 E. coli 1.67 / 05 0.10410.156 > 5.0 0.6251> 5.0 5.005.0 2.5 / 2.5 E. aerogenes > 5 0.078 / 0.156 > 5.0 2.9205.0 > 5.0 2.5 / 2.5 E. c / oacae 1.6705 0.156 / 0.156 > 5.0 > 5.0 > 5.0 2.5 / 5.0 S. typhimurium 1.2505 0.078 / 0.156 > 5.0 1.25 / 2.5 5.005.0 2.5 / 5.0 S arizona 0.62505 0.078 / 0.078 > 5.0 0.83305.0 4.1705.0 2.5 / 5.0 S.boydii 1.2505 0.078 / 0.156 > 5.0 0.625 / 0.625 5.005.0 1.25 / 1.25 K. pneumoniae 2.505 0.417 / 0.625 > 5.0 > 5.0 > 5.0 2.5 / 2.5 K. oxytoca 1.2505 10.104 / 0.156 > 5.0 1.2505.0 > 5.0 1.25 / 1.25

Data are presented as MIC / MBC (Minimum Inhibitory Concentration / Minimum Bacterial Concentration) in parts per million (ppm); “>” Indicates that the concentration required to obtain the MIC or MBC was higher than the assay parameters measured by the assay. For example, the maximum concentration of tetracycline used in S.pyogene was 5 ppm. At this concentration, growth continued after subculture in non-growth tubes. Thus, the MBC should be> (greater than) 5 ppm.

The MIC / MBC of E. coli strain O 157.7, which was associated with outbreaks of hemorrhagic diarrhea and colitis, was determined in a subsequent study. MIC was set to 2.5 ppm and MBC was set to 5 ppm.

C. Conclusion

The 10 ppm silver composition of the present invention was tested and found to be both bacteriostatic and bactericidal from the test organisms. Other studies have compared this formulation to other commercially available colloidal silver products and found it to be more active than all other formulations tested (data not shown). the most interesting observation was the broad spectrum that the 10 ppm silver composition yields. The antimicrobial activity observed was sufficiently constant irrespective of the particular organism tested. With the exception of Streptococcus faecalis and Streptococcus aureus (with MIC values of 10 ppm and 5 ppm, respectively), MIC values ranged between 1.25 ppm and 2.5 ppm for both gram positive and gram negative. MBC values are similar to values ranging from 1.25 ppm to 5 ppm except for Streptococcus mutans, Streptococcus gordonii and Streptococcus faecalis (where all MBC values are 10 ppm). The data show that the implementation of the present invention with 10 ppm silver provides an equal or broader spectrum of activity than any of the antibiotics tested. Antibiotics substantially narrow the antibacterial spectrum limited to susceptible organisms, but as shown by the data, the silver composition of the present invention is equally effective for both Gram-positive and Gram-negative organisms. The data indicate that, overall, with low silver-related toxicity and a broad antimicrobial spectrum of this silver formulation, this formulation can be effective when used as an alternative to antibiotics.

D. References to the example above:

1. US EPA IRIS Report for Silver CASRN 7440-22-4;

2. Fox CL, Modak SM. Mechanism of Silver Sulphadiazine Action on Bum VVound. Infections. Antimicrobial Agents Chemoter. 5: 582-588, 1974;

3. Furchner, JE Richmond CR and GA Drake.Comparative Metabolism of Radionuclides in Mammals. IV.Retention of Silver-110m in the Mouse, rat, Monkey and Dog. Health Phys. 15: 505-514.1968.

4. Grier, N. Silver on its Compounds in Disinfection, Sterilization and Preservation. (Seymour S. Block, ed.) 2nd Edn, pp. 395-407, 1977;

5. Hindler, JA, and JH Jorgensen. Procedure in Antimicrobial Testing in Diagnostic Microbiology. (CR Mahon and G Manusel, eds.) Pp. 63-91.1995

PROOF OF EFFECTIVENESS OF 32PPM SILVER COMPOSITION AGAINST PSEUDOMONAS AERUGINOSA, SALMONELLA CHOLERAESUIS AND STAPHYLOCOCCUS AUREUS

A. Ways

Pseudomonas aeruginosa ATTCC # 15442, Salmonella choleraesults ATTCC #, and Staphylococcus aureus ATCC # 6538 were assayed using AOAC ((Official Journal of the Association of Analytical Chemists AOC Methods Vol. 15, 1990, AOAC Arlington, VA) officially 955.14 and 964.02. (NBAOAC) tubes were inoculated from the raw culture and tubes were incubated at 37 ± 2 ° C. The other cultures were vortexed for 3-4 seconds and allowed to stand for 10 minutes at room temperature, and finally the cultures were diluted 1: 100 in peptone water (PEPVV) to which horse serum was added to give 5 total organic samples. Test carriers (10 mm polished 304 stainless steel cylinders with 8 mm outer diameter and 6 mm v immersed in sample solution for 15 min, removed, dried and dried at 37 ± 2 ° C for 40 ± 2 min. to recovery.

Phenol resistance. Five to one ml samples of each dilution of phenol under study were placed in sterile test tubes and equilibrated in a water bath of 20 ± 2 ° C. At 30-second intervals, 0.5 ml of each sample culture was added to appropriate phenol solutions, shaken and transferred to a water bath. After suitable holding times of 5, 10 and 15 minutes, the crop suspension is removed from the sample tubes and transferred to letheen broth tubes (LETH). LETH tubes were incubated at 37 ± 2 ° C for 2 days.

Carrier titration. 10 ml of peptone Tween® (polysorbate label) blanks were prepared for carrier titration. Two carriers were placed in separate tubes with a first 1:10 dilution. The tubes were stirred vigorously enough to obtain bacteria in solution and re-diluted in 9 ml LETH media. Dilution blanks were incubated at 37 ± 2 ° C. Last tube with growth showed organisms log titer _and the carrier. AOACs require carriers to have a minimum population of 1x10 ⁴ cfu / carriers.

Silver Composition Test. 10 ml of prepared disinfectant samples were placed in sterile test tubes using sterile glass pipettes and allowed to equilibrate in a cold water bath maintained at 20 ± 2 ° C. Without touching the test tube slides, an inoculated dry carrier was added to each silver composition tube at 30-second intervals and returned to the water bath. The disinfectant was assayed with 60 infected carriers per organism at maintenance intervals of 5 and 10 minutes. Following exposure, carriers were removed from the disinfectant and transferred to a LETH tube. Culture tubes were incubated at 37 ± 2 ° C for 2 days and marked as positive (+) or negative (0) growths of test organisms.

Controls. Dry contaminated vehicle was added to each organism as a positive control in the LETH tube. Uninoculated tube medium is considered as a negative control. After incubation, all negative tubes were amplified with 1 to 100 colony forming units (cfu) of the respective organisms to demonstrate the effectiveness of the neutralization. To demonstrate growth promotion of the medium, the negative control tubes were also incubated with the same 1-100 cfu for all three organisms. Inoculation volumes were placed in triplicate on soybean casein assay agar (SCDA) to verify the inoculated titers. Tubes and plates were incubated at 37 ± 2 ° C until growth was observed in all tubes.

During P. aeruginosa neutralization, the initial inoculum titer was set to> 100 cfu, which is quite high by protocol. Because all primary tubes were disrupted, a simulation test was performed with the same medium on vehicles loaded with disinfectant tubes on all three parts of the silver formulation for 10 minutes. Carriers were sub-transferred to LETH voids. These tubes were then disrupted by 1-100 cfu of the organism. Tubes were incubated as before and for growth or non-growth. New sterile tubes from the same section were also incubated as a growth promotion screening.

B. Results

The initial study using S. aureus showed random results for study # 1 and # 2, but study # 3 failed. Based on the study, it was decided what the # 3 sample was damaged during transport, the new vial was obtained from the same part as the # 3 sample, and the new vial was labeled as the # 4 sample. The S. aureus test was repeated using sample # 3. AOAC readings found that for any given time point and organism, only 1 carrier would allow growth in each test portion.

Positive controls showed growth and negative controls showed growth in all parts, time points, and organisms.

Carrier titration was performed twice for all organisms. The titre given is the mean of replications. For all three organisms, the mean titre found on the carriers ranges from 5.5x10 ⁴ to 5.5x10 ⁶ cfu / vehicle. AOAC requires the carrier to have a minimum of 1.0x10 ⁴ cfu / carrier.

P. aeruginosa 3/180 carriers showed growth at 5 min. test point and 2/180 carriers showed growth at 10 min. test point. S. aureaus 16/180 carriers showed growth at 5 min. test point and 2/180 carriers showed growth at 10 min. test point. S. choleraesuis 6/180 carriers showed growth at 5 min. test point and 1/180 carriers showed growth at 10 min. test point.

Pseudomonas culture tested showed growth at 5, 10 or 15 min. with phenol 1:90 and showed growth at 5 or 10 min. with phenol at 1:80 but showed no growth at 15 min with phenol at 1:80. Staphylococcus culture showed growth at 5, 10 or 15 min. with phenol 1:70 and showed growth at 5 or 10 min. with phenol at 1:60, but did not show growth at 15 min, with phenol at 1:60. Salmonella culture showed growth at 5, 10 or 15 min. with phenol at 1: 100 but showed no growth at 5.10 or 15 min. effect with 1:90 phenol.

32, 22 and 10 PPM SILVER AND 22 PPM SILVER IR1.5 H ₂ O ₂ AND 10 PPM SILVER AND 10 PPM K ₂ S ₂ O ₈ EFFECTIVENESS AGAINST SALMONELLA AND ESCHERICHIA COLI IN A NEW INOCULATED BEEF SAMPLE

A. Purpose of the Test The purpose of the test is to demonstrate embodiments of the antimicrobial activity of the silver-based compositions of the present invention in beef bacon samples inoculated on the outer surface with five mixed strains of Salmonella species. Alternatively, Escherichia coli O157: H7 is infected at a high solution level (1x10 ⁶ cfu.cm ³ ) and separately at a low solution level (1x10 ⁴ cfu / cm ² ) (cfu = colony forming unit).

B. Materials and Methods

Beef samples. Taurus tissue samples were taken from the shed horses for 8 hours after dressing. The muscle of Rectus abdominus was shaved off the skeleton, stored in a cool refrigerator, making incisions between the 11th and 12th ribs, and then peeling the muscle along its natural layers. Properly prepared samples were placed in plastic bags of ice and transported the same day to the laboratory, where samples were quickly packed in Multi-Vac (A-300) and placed in a 4 ° C refrigerator. Samples used for testing had a pH between 5.8 and 6.0 and were maintained for up to 36 hours after application. From randomized rectus abdominus muscles, 13x8 samples were dissected and tested. After treatment with 3.5 cm ² fire sterilized stainless steel backing instrument and surgical scalpel, a sterile two-meat layer sample was used to determine the interval for each sample. Tissue layers were placed in a sterile narrowing bladder bag with 25 mL of 0.1% peptone and mixed in a bladder bag for 2 minutes (Lab Bender 400). Repeated dilutions were prepared and incrementally placed at 0 minutes, 20 minutes, 1 hour, 4 hours, and 24 hours after treatment in a selective and reconstituted medium.

Bacterial cultures. Bacterial cultures were obtained from the Kansas State University (KSU) Protected Culture Collection and were stored using a Meat Storage system. The following cultures were used for Salmonella species: S. lille (UGA), S. Montevideo (UGA), S. typhimurium (UGA), S. agona (KSU 05 from CDS outbreak isolate) and S. nevvport (KSU 06 from CDS outbreak) breakthrough isolate). The following cultures were used for Escherichia coli specimen: E. coli O157: H7 (CDC 01.03), E coli 0157: H7 (USDA-FSIS 011-82 Rif resistant 100 ppm), E. coli O157: H7 (ATCC) 43895 Rif Ref. Of HUS-Associated Type I and Type II Toxins) and E. coli ATCC # 23740 (Genotype K-12prothrombotic lobe).

Raw cultures were cultured by placing one impregnated bead in 5 ml Difco® three soy broth (TBS) solution and incubating for 24 hours at 35 ° C. Subsequently, 0.05 ml of the culture of the present culture was inoculated into 5 ml of TBS solution and incubated for 24 hours at 35 ° C to obtain a pure culture. After incubation, 1 mL of the appropriate culture was inoculated into 49 mL of TBS and incubated for 24 h. At 35 ° C, with continued incubation, samples were centrifuged (15300Xg at 4oC) and the precipitated material was decanted and the pellet was resuspended in 50ml of 0.1% peptone and centrifuged (15300Xg at 4oC). The peptone was decanted and the remaining lump was resuspended in 10 mL of 0.1% peptone. Five 10 ml vials of the respective culture were mixed together to obtain a cocktail containing 10 ⁹ cfu / ml Salmonella species. The cocktail was diluted to 10 ⁶ cfu / ml or 10 ⁴ cfu / ml using 0.1% peptone. Cultures were fixed by culturing in selective or differential media and biochemical analysis of potential colonies using API 20E kits.

Method of inoculation. Bull flanks (rectus abdominus muscle) were compared to 13 x 8 cm (104 cm ² ) and inoculated with five strains of Salmonella or Escherichia coli O157: H7 strains at high inoculum level (10 ⁶ log cfu / cm ² ) and individually. at low inoculum level (10 ⁴ log cfu / cm ² ). This inoculum was sprayed onto the tissue surface using a plastic spray bottle with specimens fitted with inoculum chambers. Actual concentrations of Salmonella species on the surface of the meat were approximately 5.0 and 3.4 log cfu / cm ² in high and low level inoculum solution, respectively. Meat surface inoculation levels for E. coli O157: H7 were 4.2 and 3.9 log cfu / cm ² ', respectively

The beef samples were then hung vertically on stainless steel hooks attached to a motorized track that pushed the samples through the sample spray booth (Kansas State University, Food Storage Laboratory) until the spraying procedures were completed. The procedures, either with the silver compositions of the present invention or with deionized water, were applied to the beef at 20 psi from a 13 cm sample for 20 seconds in cabin pressurization. The nozzle orifice (ΒΕΤΕ NF0580 303) sprayed approximately 20 ml of the solution onto the surface of the beef sample. The temperature of the solutions and the treatment room was approximately 14 ° C. After treatment, double strip samples were randomly stripped off the horizontal surface of the beef at 0, 20, 60, and 240 minutes. Samples were cultured and plated on selective deferential and reconstituted media. Log reductions were counted by subtracting the inoculated / treated log cfu / cm ² samples at defined test times from the inoculated / treated log cfu / cm ² samples at 0 min. 32 ppm silver, 22 ppm silver and 10 ppm silver compositions of the present invention were used for sample treatment. Combinations of 22 ppm AG with 1.5 wt% hydrogen peroxide and 10 ppm Ag with potassium peroxide sulfate (K ₂ S ₂ O ₈ ) were tested separately.

C. Results with 32 ppm silver composition

The use of the 32 ppm silver composition of the present invention demonstrated the reduction of bacteria on a piece of beef. Further, this reduction was expressed in the log-proportional control time of bacterial number 0 with bacterial counts during treatment periods (i.e., treatment).

With Salmonella, at a low baseline bacterial level (10 ⁴ ), the following log reductions were found; 0.78 at 0 minutes, 1.11 at 20 minutes, 1.08 at 60 minutes, and 1.23 at 240 minutes. Thus, at 4 hours (240 minutes), the initial amount of bacteria in the sample treated with 32 ppm silver is 10 ^1.23 . At higher bacterial levels (10 ⁶ ), the following log reductions were observed: 0.86 at 0 minutes, 0.95 at 20 minutes, 0.98 at 60 minutes, and 1.17 at 240 minutes. The results showed that the 32 ppm silver embodiment of the present invention showed an effective bactericidal effect on Salmonella on a piece of beef. It could be recognized that disinfecting the surface of meat is the ultimate test of any disinfectant.

The following log reductions were found with E. coli at a low baseline bacterial level (10 ⁴ ); 1.03 at 0 minutes, 1.28 at 20 minutes, 1.42 at 60 minutes, and 1.58 at 240 minutes. At higher bacterial levels (10 ⁶ ), the following log reductions were observed: 0.65 at 0 minutes, 0.60 at 20 minutes, 0.83 at 60 minutes, and 0.87 at 240 minutes. The results showed that the 32 ppm silver embodiment of the present invention exhibited effective bactericidal activity against E. coli on a piece of beef.

D. Results with 22 ppm silver compositions

Results with silver in water. With Salmonella at low baseline bacterial levels (10 ⁴ ), the following log reductions were observed: 0.41 at 0 minutes, 0.43 at 20 minutes, 0.48 at 60 minutes, and 0.68 at 240 minutes. At higher bacterial levels (10 ⁶ ), the following log reductions were observed: 0.24 at 0 minutes, 0.24 at 20 minutes, 0.42 at 60 minutes, and 0.61 at 240 minutes. The results showed that the 22 ppm silver embodiment of the present invention showed an effective bactericidal effect on Salmonella on a piece of beef.

Results with silver in water and 1.5 wt% hydrogen peroxide. With Salmonella at low baseline bacterial levels (10 ⁴ ), the following log reductions were observed: 0.34 at 0 minutes, 0.33 at 20 minutes, 0.36 at 60 minutes, and 0.62 at 240 minutes. At higher bacterial levels (10 ⁶ ), the following log reductions were observed: 0.28 at 0 minutes, 0.14 at 20 minutes, 0.30 at 60 minutes, and 0.69 at 240 minutes. The results showed that an embodiment of the present invention of 22 ppm silver with 1.5 wt% hydrogen peroxide showed an effective bactericidal effect on Salmonella on a piece of beef.

E. Results with 10 ppm silver composition

Results with a silver composition in water. With Salmonella at low baseline bacterial levels (10 ⁴ ), the following log reductions were observed: 0.38 at 0 minutes, 0.41 at 20 minutes, 0.39 at 60 minutes, and 0.61 at 240 minutes. At higher bacterial levels (10 ⁶ ), the following log reductions were observed: 0.24 at 0 minutes, 0.21 at 20 minutes, 0.41 at 60 minutes, and 0.54 at 240 minutes. The results showed that the 10 ppm silver embodiment of the present invention showed an effective bactericidal effect on Salmonella on a piece of beef.

Results with a silver composition in water with 10 ppm KySgOg. With Salmonella at low baseline bacterial levels (10 ⁴ ), the following log reductions were observed: 0.26 at 0 minutes, 0.28 at 20 minutes, 0.35 at 60 minutes, and 0.58 at 240 minutes. At higher bacterial levels (10 ⁶ ), the following log reductions were observed: 0.03 at 0 minutes, 0.16 at 20 minutes, 0.21 at 60 minutes, and 0.36 at 240 minutes. The results showed that an embodiment of the present invention in 10 ppm silver with 10 ppm potassium peroxide sulfate (K2S2O8) showed an effective bactericidal effect on Salmonella on a piece of beef.

PROOF OF EFFECTIVENESS OF PPM SILVER FOR HUMAN DISEASE

a. Purpose of the test

The purpose of this test is to demonstrate the use of silver-based compositions in the treatment of various human ailments. In this series, tests were conducted at the Atmospheric Movement Research Station in Ghana, West Africa, under the direction of Director Dr. Kwabiah, at Korie-Bu Training Hospital under the direction of Sr. At Sackey and Justab Clinic / Maternity Hospital, under the direction of Dr. Abraham, A total of fifty-eight (58) patients were treated using the silver / water compositions of the present invention comprising 10 ppm silver. The formulations have been used both externally and internally as an alternative to conventional antibiotics. Treated ailments included: malaria, upper respiratory tract infections, urinary tract infections, sinusitis, vaginal myeloma, eye, nose and ear infections, cuts, fungal skin diseases and sexually transmitted diseases such as gonorrhea.

B. Treatment modalities and outcomes

Abdominal pain and diarrhea. The method comprises the step of administering about 5 to 25 ml of the silver composition, one to five times daily, orally, until a response is obtained. One patient was treated with approximately 10 ml (about two teaspoons) of the composition of the invention three times daily. The patient fully recovered within one day.

Bronchitis. The method comprises the step of administering about 5 to 25 ml of the silver composition, one to five times daily, orally, until a response is obtained. Two patients were treated with approximately 5 ml (approximately one teaspoon) of each composition of the invention three times daily. The patient fully recovered within three days.

Vaginal yeast (Candida). The method comprises the step of administering about 5 to 25 ml of the silver composition, one to five times daily, orally, until a response is obtained. Five patients were treated with approximately 10 ml (about two teaspoons) of the composition of the invention twice daily. The patient fully recovered within six days.

Conjunctivitis. The method comprises the step of administering a few drops of silver composition, once to five times a day, to the infected eye until a response is obtained. Two patients were treated with several drops of the composition of the invention to each infected eye twice daily. The patient fully recovered within one day.

External incisions and infection (excluding Staphylococcus skin infections, septic ulcers and infected abscesses). The method comprises the step of administering a silver composition, one to five times daily, to the infected area until a response is obtained. Six patients were treated with approximately 5 ml (approximately one teaspoon) of each of the compositions of the invention to the infected sites twice daily. The patient fully recovered within three days.

Outer otites. The method comprises the step of administering a silver composition, one to five times a day, to the infected ear until a response is obtained, six patients being treated with approximately two drops of the composition of the present invention three times a day. The patient fully recovered after approximately four days.

Otite medium. The method comprises the step of administering a silver composition, one to five times a day, to the infected ear until a response is obtained. One patient was treated with about two drops of the composition of the invention to the infected ear three times a day. The patient fully recovered after approximately four days.

Fungal skin infection. The method comprises the step of administering a silver composition, once to five times daily, locally to the infected area until a response is obtained. Two patients were treated with approximately 10 ml (about two teaspoons) of each of the compositions of the invention three times daily. The patient fully recovered within eight days.

Gonorrhea. The method comprises the step of administering a silver composition, one to five times daily, to the infected area until a response is obtained. Two patients were treated with approximately 10 ml (about two teaspoons) of the composition of the invention three times daily. In patients, symptoms disappeared within six days.

Malaria. The method comprises the step of administering a silver composition, one to five times daily, orally, until a response is obtained. Eleven patients were treated in the first stage with approximately 10 ml (about two teaspoons) of each composition of the invention three times daily. In patients, symptoms disappeared within five days. The detailed treatment conditions for malaria are discussed here later.

Halitosis and gingivitis. The method comprises the step of administering a silver composition once to five times per day for mouth rinsing until a response is obtained. Two patients were treated with this composition as a mouthwash. The symptoms disappeared completely within three days (gingivitis) and within one day (halitosis).

Pelvic inflammatory disease. The method comprises the step of administering about 5 to 25 ml of a silver composition, once to five times a day, as a vaginal wash prior to a response. One patient was treated with approximately 5 ml (approximately one teaspoon) of the composition of the invention to the infected sites twice daily. The patient disappeared within five days.

Pharyngitis. The method comprises the step of administering a silver composition, one to five times daily, to the infected area until a response is obtained. Four patients were treated with approximately 10 ml (two teaspoons) of the composition of the invention three times daily. Patients were fully recovered within six days.

Retroviral infection (HIV). The method comprises the step of administering a silver composition comprising 5 to 40 ppm silver, one to five times daily, orally, until a response is obtained. One patient carrying HIV (human immunodeficiency virus) was treated with about 5 ml (about one teaspoon) of the composition of the invention twice daily. The patient disappeared within five days.

Sinusitis and rhinitis. The method comprises the step of administering a silver composition, one to five times daily, to the nose until a response is obtained. Six patients with nasal infections (four with sinusitis and two with rhinitis) were treated with approximately two drops of formulation into the nasal passages three times daily. The patient disappeared within four days.

Tonsillitis. The method comprises the step of administering a silver composition, once to five times a day, as a throat rinse until a response is obtained. One patient was treated with the composition of the invention three times daily. The patient disappeared within seven days.

Upper respiratory tract infection. The method comprises the step of administering a silver composition, one to five times daily, orally, until a response is obtained. Two patients were treated with approximately 5 ml (approximately one teaspoon) of the composition of the invention three times daily. Patients were fully recovered within six days.

Urinary tract infections. The method comprises the step of administering a silver composition once to five times per day orally prior to receiving a response. Three patients were treated with approximately 10 ml (two teaspoons) of the composition of the invention two to three times daily. Patients were fully recovered within six days.

C. Discussion

The results here consist of various in vitro assays. In general, the silver composition is particularly effective against a large number of microbes in vitro. However, tests have shown that this efficacy persists even at high levels of organic matter. Silver formulations are highly effective in vivo, where the organic basis is remarkably high. Many other disinfectants are ineffective at high levels of organic matter and / or are too sharp and toxic for in vivo use.

Supplemental Malaria Study in Ghana, Africa

Another group study was also conducted in Ghana. The purpose of this study was to utilize very specific conditions and to focus solely on the therapeutic properties of the 10 ppm silver / water composition of the present invention in a patient suffering from malaria.

The aim of this study was to find a method in which 10 ppm silver / water composition obtained by the methods described herein could be tested for its potential therapeutic properties in the treatment of patients with malaria infection in any of the four (4) species of Plasmodium. The general description of the test is as follows;

Medical doctors who are very involved with the disease and its outbursts have taken the tests to medical clinics or hospitals. Here, a total of 16 patients were examined by one physician and patients were required to use silver products twice daily for five days, as well as their blood tests the day before the start of the test, thus demonstrating that the parasite was eliminated daily before the blood test. for at least two days. Patients were only trained if they fully adhered to the silver usage schedule and daily blood test.

Detailed description of the test

Number of Medical Doctors (MDs) involved in testing: 2

Number of patients tested for physician: 16-8 males and 8 females.

Total number of test days: 15.

A dose of 10 ppm silver / water formulation was administered to the treated patient. The total daily dose of 28.35 g was divided into two equal doses; One half of 28.35 g (3 teaspoons) was taken in the morning and the other half 28.35 g (3 teaspoons) was taken in the evening. Patients were treated with Silver / water solution for the first five (5) days on a total 15-day cycle, or if the parasite was not completely killed within five days, silver / water treatment was continued until the parasite was killed or up to 15 days, whichever came first.

In the case of killing a patient's parasites within two or three days, the silver / water treatment was continued for up to five days and records indicate when the parasite was completely eradicated.

In the case of a patient who still has parasites after using silver / water for 15 days, the trial is terminated as usual and this patient is unmarked in the records as not responding.

The patient who had been treated for less than five days had a marked date for complete parasite disappearance, and the patient was treated with the silver / water formulation for up to five days and continued for up to 15 days in the trial.

Blood test.

Blood tests were used: The presence (or absence) of parasites in the patient's blood was determined by either an Acridine Orange strain test or by thin or thick blood smears in each patient. Patients' blood, tested on day zero (0), actually had active malaria to ascertain its condition. If the blood test confirmed active malaria, then the patients were tested for eligibility. Sampling included essential data such as name, age, patient-reported onset of disease, record of patient claims during the trial, how they would be reimbursed on full agreement, and finding that non-compliance results in discharge from the trial without the right to return. Patients who agreed to participate in the study were given written instructions on how to use the silver product every day, where to go for blood tests, emphasizing the need for a full study protocol to be reimbursed on any given day.

The above protocol was strictly adhered to in a recent study in Ghana, Africa. All patients received the same dose and their blood was checked daily for the presence of Plasmodia parasites. The following Table 4 presents portions of the previous studies described above (Study 1 and Study 2) as well as the new study 3, followed immediately by the protocol.

Summary table

Investigation 1 2 3 4 Number of patients 11th 16th 16th 13th Age variation 8-75 2-90 3-61 15-57 Men / Women NA NA 8/8 6/7 Average daily dose 10 ml 5 ml 15 ml 15 ml The shortest recovery time 3 days 3 days 2 days 3 days The longest recovery time 7 days 10 days 8 days 6 days Average recovery time 5.0 days 6.3 days 4.3 days 4.0 days # plasmodia patients 0 7th 16th 13th # patients with plasmodia 0 0 0 * 0 * # unsuccessful treatments 0 0 0 0

+ Generation of recovery refers to those when the patient has resolved symptoms as determined by doctors.

* Each of these patients was checked daily for 14 days. Six days later, each of the blood samples showed negative plasmodia.

The clear 10 ppm silver / water solution of the present invention has pronounced beneficial effects against malaria parasites.

INVITRO PROOF OF PPM SILVER AGAINST MALARIA (IN VITRO)

INTRODUCTION

Globally, malaria has been and remains a major public health problem. The disease is caused by protozoan parasites of the Plasmodium gene. The organism's life cycle is complex with parasite variations from invertebrate (mosquito) host sexual reproduction and asexual reproduction of vertebrates, In addition to mammals as vertebrate hosts, birds and reptiles as malaria parasite hosts are also suitable. As part of the life cycle, mosquitoes are a sporadic phase that allows the formation of sporadic animals that are vector-infected to the vertebrate host during feeding. Sporagiae occur in the schizogonal phase, multiplying by parasites in erythrocytic and exoerythrocytic media. Parasites are intracellular during the sporal phase transition to the extracellular medium during the developmental schizogonal phases. In vitro growth of the parasite requires adapted conditions in the sporogonal phase of the life cycle in the mosquito vector and conditions in the schizogonic phase to ensure growth at the sites of vertebrate exoerythrocytes and erythrocytes.

Malaria is one of the most prevalent diseases in the world and is ranked no less than the third largest infectious disease in the world in terms of mortality. The protozoan parasites that cause malaria are of the Plasmodium species. Four Plasmodium protozoa cause malaria: Plasmodium falciparum, Plasmodium vivax, Plasmodium malaiiae and Plasmodium ovale. In principle, malaria infections transmitted through the Anopheles mosquito can also occur through contact with infected blood, such as blood transfusions.

Classic symptoms of malaria include fever, shaking headache, shivering vomiting and convulsions. Some rare forms of sickle malaria, such as tremors and fever, may not occur and patients may start to fall or fall into coma Remission periods can last from a few weeks to several months. Severe anemia is often attributed to the causes of death from malaria infection.

Plasmodium falciparum:

This parasite has several important features. They include the gametocyte sickle shape, the latter's low growth rate, and the localization of the pigment around the nucleus (perimeter nucleation), which is typical of gametocytes of other primary malaria parasites.

P. falciparum also differs from other human species in its higher virulence and lethal activity, although the schizogonia of erythrocyte phases is largely targeted at the capillaries and sinusoids of internal organs. A popular name for this disease, derived from P. falciparum, is malignant butterfly malaria. Materials and Ways

Citrate salt:

Sodium chloride -9 gm;

Sodium citrate - 20 gm;

Distilled water -1000 ml.

Giemsa strains:

Giemsa velvet powder - 75 gm;

Pure alcohol - 75 ml;

Glycerol - 25 ml.

Field strain:

Field'0 Solution # 1

Dissolve 1.1,6 g of methylene blue in 1 liter of distilled water.

2. 2.6 g of Na2HPO ₄ (anhydrous) was dissolved in the solution of step 1.

Dissolve 3.1 g of the solution in the Tier 2 solution.

4. Dissolve 2.6 g of KH2PO4 in a step 3 solution.

5. Place with soft meat, stirring and shaking for 45 minutes to 1 hour.

6. Store at room temperature for 24 hours.

7. Filter.

Field Solution # 2

1. Dissolve 2 g of eosin Y in 1 liter of distilled water.

2. 2.6 g of Na2HPO ₄ dissolved in one step solution.

3. Dissolve 2.6 g of KH ₂ PO ₄ in the step 3 solution.

4. Filter.

Vright strain:

VrightO strain powder - 6.0 g;

Giemsa strain powder - 0.6 g;

Methanol - 1000 ml;

Stir overnight and filter before use.

Blood group AB + human serum / plasma

Blood Group A + Human Serum / Plasma.

RPMI -1640 Partial Media (in person with Dr. Sutar, Haffkie Institute, Parel)

RPMI - 1640 Full Media (in person with Dr. Sutar, Haffkie Institute, Parel)

SAMPLING AND EXAMINATION OF INFECTED BLOOD

Infected erythrocytes were obtained by taking 60 ml of blood samples with 1 ml of intravenous citrate salt from clinically diagnosed cases of Plasmodium vivax and Plasmodium falciparum malaria from Kasturba Infectious Diseases Hospital, Bombay. Blood samples were collected in 10 ml sterile dishes. Samples were examined by preparing thin smears and staining smears with 10% Giemsa strain / Field strain / Wright strain for identification and confirmation of pp malaria parasite. The percentage of sample contamination was determined.

Infected blood cells were washed twice in incomplete medium and once in complete medium and a 6% cell suspension in complete medium was prepared. Cultures were plated by distributing 0.5 ml of suspension in each petri dish. To these 1.5 ml complete media was added and the plates were incubated in 5% CO ₂ and 14-17% O ₂ . The medium was changed daily by aspirating the old medium with a sterile pipette and adding 1.5 ml. complete media. Cultures were maintained by the addition of new cells (from blood type A + or AB +; washed and cell suspension prepared in the same manner) after one week, washing twice a week until a controlled parasite index reached> 1%. If the initial parasite index was greater than 1%, then blood medium mixture (BMM) was used directly for drug sensitivity (Thanh, 2001) and (Tasanor, 2002).

PERFORMANCE OF LUBRICANTS AND PAINTING PROCEDURE

Cultures were washed twice a week. For washing, the cultures were removed from the plates and transferred to centrifuge tubes. About 5 ml of crude medium was added and each centrifuge tube was thoroughly mixed. The tubes were centrifuged at about 1000 to 1500 rpm for about 10 minutes. After about 10 minutes, the tubes were removed and the supernatant fluid was drained. Subsequently, the cultures were washed twice more, one with RPMI-1640 incomplete and the other with RPMI-1640 complete. After three washes, the cultures were transferred to separate Petri dishes. A smear stained from each culture was stained with the Giemsa / Field / Wright strain. Approximately 1.5 ml of medium was added to each plate and the smears were examined under the optical microscope and the parasite index or% of parasites per culture was determined. New erythrocytes were added to each plate each week (Pradhan, 1984).

PREPARING THE LUBRICANT

The drop of culture from the dish was applied to a microscope slide. A thin smear was applied and air dried. This smear was fixed by immersing the slide in a sealable glass container containing pure alcohol. A 10% solution of the Giemsa strain and the staining used were prepared. Slides were kept soaked in 10% Giemsa strain solution for approximately 30-40 minutes and then washed with water.

Parasite Index

The parasite index was determined by counting the number of parasites per 100 erythrocytes in thin blood samples. 100 areas or 10000RBCS were reviewed for this purpose.

Cultural systems

Plate cultures were prepared with 5% hematocrit and approximately 1% parasite. The lower the baseline parasite level, the faster the increase in parasite numbers that occur during in vitro growth.

DRUG SENSITIVITY

16 mm flat-bottomed sterile microtube plates were used to determine drug sensitivity. One was for one sample. The first two tubes were used for control and resulted in 50 µl patient BMM or culture and 50 µl RPMI complete medium without drug. For the assay, 50 µl of culture or patient BMM was mixed in a tube containing various concentrations of 50 µl of prepared silver nanoparticles (ESNPs). The microtube plates were covered and incubated in a translucent beaker at about 37 ° C for approximately 48 hours. Most parasites entered the Schizont stage at the end of 48 hours of incubation. After incubation, the surface medium was removed using a micropipette; blood from each tube was drawn for smear preparation and schizont development was monitored. The assay was evaluated by counting the number of parasites in smear-stained pre-incubation and post-incubation plates and relative suppression of ESNP schizontation. In a numerical study, the control tube showed> schizont maturity. (Wernerdorfer and Wernerdorfer, 1995)

Results

Species % Reduction of parasites Plasmodium falciparum 94% Plasmodium vivax 92% Plasmodium berghei 90%

An in vitro assay used as an indicator of antimalarial efficacy convincingly demonstrated that ESNP-100 ppm can reduce parasite levels in vitro. This is very important because the parasites collected were from patients with high fever and classic symptoms of malaria infection.

EVIDENCE OF 10PPM SILVER AGAINST TUBERCULOSIS BACTERIA

A. Purpose

The purpose of this test was to demonstrate the effectiveness of the silver compositions of the present invention against the bacterium which causes tuberculosis. The following examples describe the evaluation of the efficacy of the present invention in tuberculosis. The methodology is based on the Tuberculocidal Activity Assay as accepted by EPA on December 11, 1985. (Reference: U.S. Environmental Protection Agency, 1986. Pesticide and Toxic Substances Service. Report on the Use of the Tuberculocidal Efficacy Data of All Microbial Pesticides with Tuberculocidal Effects (Received June 13, 1986).

B. Materials and Methods

Materials. The silver compositions of the present invention consist of 10 ppm silver and water. The silver composition was evaluated using a liquid matrix against Mycobacterium bovis BCG (TMC 1028). The organism caused tuberculosis in animals and was able to cause tuberculosis in humans. It is used as a "duplicate" of M. tuberculosis, the major causative agent of human tuberculosis, and has been shown to have similar susceptibility to M. tuberculosis as tested. The test organism was exposed to the silver formulation twice for four exposure times and quantified using membrane filtration.

The way. The vial containing frozen raw culture was left for storage and thawed. An equal volume of buffer-gelatin (BUGE) was added to the cell suspension and homogenized with a Teflon® (polytetrafluoroethylene brand) tissue shredder for 1 minute while maintaining the culture in an ice bath at 0-4 ° C. The homogenized cell suspension was diluted with saline Tween ® 80 (polysorbate label) solution (ST80) to approximately 10 ⁷ cfu / ml.

Test titration. Ten-fold repeated culture dilutions were prepared in dilution vessels containing 9 mL of Neutralizing Broth (NEUB) per 10 ⁶ dilutions. Three suitable dilutions of 1 mL of samples were membrane-filtered by first adding 10-20 mL of physiological saline (PHSS) to the filter housing and then adding 1 mL of a properly diluted sample. The filter was then rinsed with approximately 100 mL PHSS. The filters were sterile removed from the filter housing and placed on 7H11 agar plates. The plates were incubated in an irrigation chamber at 37 ± 2 ° C for 21 days.

Positive control. A tube containing 9 ml of ST80 was prepared and equilibrated to 20 ± 0.5 ° C. At time 0, 1 ml of test organism culture was added to the tube (1:10 dilution). The sample was held for 60 minutes. Ten-fold repeated dilutions were prepared in dilution vessels containing 9 mL of Neutralizing Broth (NEUB) at 10 ^-6 dilutions. Three suitable dilutions of 1 mL of samples were membrane-filtered, first by adding 10-20 mL of physiological saline (PHSS) to the filter housing and then adding 1 mL of a properly diluted sample. The filter was then rinsed with approximately 100 mL PHSS. The filters were sterile removed from the filter housing and placed on 7H11 agar plates. The plates were incubated in an irrigation chamber at 37 ± 2 ° C for 21 days.

Assays, two 25 x 30 mm tubes containing 9 ml of test sample and equilibrated to 20 ± 0.5 ° C. One ml of culture of the test organism was added to each tube containing the disinfectant to be tested (i.e., silver composition). The tube was stirred by rotation and placed back in the water bath. At 15, 30, 45, and 60 minutes, 1 mL of the disinfecting cell suspension sample was transferred to 9 mL of NEUB and mixed thoroughly. Ten-fold dilutions were prepared in dilution vessel having 9 ml of neutralizer broth (NEUB) per 10 ^"6 dilution. Three suitable dilutions of 1 mL of samples were membrane-filtered, first by adding 10-20 mL of physiological saline (PHSS) to the filter housing and then adding 1 mL of a properly diluted sample. The filter was then rinsed with approximately 100 mL PHSS. The filters were sterile removed from the filter housing and placed on 7H11 agar plates. The plates were incubated in an irrigation chamber at 37 ± 2 ° C for 21 days.

Phenol control. The culture was assayed against a 0.8% phenol solution to demonstrate minimal culture viability and resistance. 1 ml of the test organism culture sample was added to 9 ml of phenol solution equilibrated to 20 ± 0.5 ° C and incubated for 20 minutes. After exposure time, 1 mL of phenol / body solution was removed and 9 mL of NEUB was added. Ten-fold repeated dilutions were prepared in dilution vessels containing 9 ml of Neutralizing Broth (NEUB) per 10 ⁶ dilutions. Three suitable dilutions of 1 mL of samples were membrane-filtered by first adding 10-20 mL of physiological saline (PHSS) to the filter housing and then adding 1 mL of a properly diluted sample. The filter was then rinsed with approximately 100 mL PHSS. The filters were sterile removed from the filter housing and placed on 7H11 agar plates. The plates were incubated in an irrigation chamber at 37 ± 2 ° C for 21 days.

Verification of Neutralization. 1 ml of disinfectant sample was added to 8 ml NEUB. The disinfectant / neutralizer broth was left to equilibrate to the same temperature as the test samples. One culture of the test organisms was added to the mixture and mixed well. The incubation was continued for an appropriate period of time which would approximately occupy the sample filtration. An additional 1 ml sample of the test organism was added to 9 ml NEUB and mixed well (1:10 dilutions). Ten-fold dilutions of both tubes were prepared in dilution vessels containing 9 mL of Neutralizing Broth (NEUB) at 10 ' ⁶ dilution. Three suitable dilutions of 1 mL of samples were membrane-filtered by first adding 10-20 mL of physiological saline (PHSS) to the filter housing and then adding 1 mL of a properly diluted sample. The filter was then rinsed with approximately 100 mL PHSS. The filters were sterile removed from the filter housing and placed on 7H11 agar plates. The plates were incubated in an irrigation chamber at 37 ± 2 ° C for 21 days

C. Results

Initial experimental culture titer was 4.7 χ 10 ⁷ cfu / ml. The positive control titer was 6.5 x 10 ® cfu / ml. the medium used in this step effectively showed neutralization with a recovery of 92.2% in disinfectant / neutralization broth compared to the medium preparation.

Expected calculations for the test slides were underestimated and therefore the figures given exceeded, indicating that the calculation is approximate and that the exact figures are outside the detection limit.

When calculating the log and percentage reductions of the disinfectant against M. bovis, calculations that are larger than the numbers obtained are less than the log and percentage reductions ("<"). The purpose of this is to demonstrate that the results of the dilutions introduced are approximate and beyond the limits of detection. All reductions were calculated using the positive control as the initial start titer of the organism. The log and percentage reduction results are summarized below. As a measure of test culture resistance, M. bovis showed phenol resistance of <1.81 log over 20 minutes at 0.8% phenol.

First replication: Exposure time Log reduction Percent reduction 15 minutes <0.12 <12.3% 30 minutes <0.22 <40.0% 45 minutes <1.57 <97.2% 60 minutes <1.56 <97.2% Second Replication: Exposure time Log reduction Percent reduction 15 minutes <0.26 <44.8% 30 minutes <0.20 <36.9% 45 minutes <1.58 <97.3% 60 minutes <1.53 <97.1% D. Conclusions The silver of the present invention the compositions are effective against tuberculosis

bacterium. The method comprising the step of administering the silver compositions of the present invention is effective against tuberculosis organisms.

PPM SILVER AGAINST CANDIDA ALBINANTS ATCC # 10231, TRICHONOMAS VAGINAL ATTC # 20235 AND MRSA STAPHYLOCCOCUS AURELIS ATTC # BAA44 PROOF OF EFFECTIVENESS

The purpose of this test was to illustrate the efficacy of the silver composition of the present invention against Candida albicans ATCC 10231, Trichomonas vaginalis ATCC20235 and drug-resistant Staphylococcus aureus ATCC BAA-44.

Candida albicans, yeast, and Trichomonas vaginalis, protozoa, can cause a number of health problems, including vaginal infections, rhizomes and thrush. The results below show that the silver compositions of the present invention produce 100% killing of both organisms. The results have shown the use of the silver compositions of the present invention in female hygiene products and in a product for the treatment of rhombic rash.

Staphylococcus aureus can cause a series of blood poisonings when it enters the wound. He was usually tried for penicillin treatment, but the body did not usually change to the point where penicillin resistance develops. The next successful antibiotic protection was methicillin, but methicillin-resistant strains have become increasingly common, especially in hospitals. These strains are known as MRSA (methicillin-resistant Staphylococcus aureus) and have been termed "superinfection". People who contacted MRSA could die within a few days. Based on the results of this test, it has been found that the silver composition of the present invention can kill 91.6% MRSA in minutes and 99.5% per hour. The results showed the use of the silver composition of the present invention in the treatment of MRSA, a known infection.

B. Methods and Results.

Using a protective rapid test assay with a composition of the present invention comprising 10 ppm silver in water, the following results were obtained. The results showed that the silver compositions of the present invention may be effective against yeast infections, protozoa infections and drug resistant bacterial infections.

Candida albicans ATCC # 10231. The initial concentration of Candida albicans yeast was 6.8x10 ⁵ cfu / ml. After 10 minutes 30 minutes, 1 hour or one day of contact with the silver composition, no colonies were detected.

Trichomonas vaginalis ATCC # 30235. The initial concentration of protozoa in Trichomonas vaginalis was 6.0 x 10 ⁴ cfu / ml. After 10 minutes, 30 minutes, 1 hour, or one day, the silver composition had 0 inactivity per 100 organisms. That is, one hundred (100) parasites of Trichomonas vaginalis were analyzed microscopically to determine the incapacity of the gill. None of the one hundred (100) parasites showed activity after as much as ten (10) minutes after contact with the silver composition, after detecting the lethal effect of the silver composition on the parasites. The other twenty-five (25) percent of the outer membranes of the parasite cut off contact within one (1) day.

Staphylococcus aureus MRSA ATCC # BAA-44. The initial concentration of methicillin-resistant Staphylococcus aureus (MRSA) was 6.0x10 ⁶ cfu / ml. After contact with the silver composition, determined 500000 cfu / ml after 10 minutes of contact (91.6% killed), 700000 cfu / ml after 30 minutes of contact (98.8% killed), 30,000 cfu / ml after 1 hour of contact (99, 5% killed) and a few more than 10 cfu / ml after one day of contact (virtually total killing).

DEMONSTRATION OF THE EFFECTIVENESS OF PPM SILVER, 14 PPM SILVER + 1.5% H ₂ O _2, AND 22 PPM SILVER IN DEGRADING DNA POLYMERASE AND RETRACTING TRANSCRIPTASE IN HEPATITIS B

A. Purpose of the test

The purpose of this test was to illustrate the efficacy of the silver composition of the present invention against hepatitis B. This test demonstrates that the silver compositions of the present invention have antiviral properties. Any agent used in antiviral therapy is sparse or non-toxic, which is why the silver compositions were analyzed for toxicity.

Hepatitis B is caused by a DNA virus from the hepadnaviridae virus family. Hepatitis B virus (HBV) is a 3.2 kb DNA virus that is almost exclusively reproduced in liver cells (hepatocytes). Replication involves two major enzymes: DNA polymerase and reverse transcriptase. The results of this assay indicate that the silver compositions of the present invention interact with replications, including DNA polymerase or reverse transcriptase. The results of this test show that the silver compositions of the present invention have antiviral properties. The results of this test indicate that the silver compositions of the present invention may be effective against hepatitis B.

In more detail, when hepatitis B enters the body of a new host, it infects the liver if it passes the immune system. When infected, the virus attaches to the liver cell membrane and the main part of the virus enters the liver cell. The principal then releases its amounts of DNA and DNA polymerase into the nucleus of the liver cell. Within the liver cell, the virus multiplies by reverse transcription and translation, which include reverse transcriptase and DNA polymerase enzymes. DNA polymerase stimulates liver cells to make copies of hepatitis B DNA. These copies of the virus are released from the liver cell membrane into the bloodstream. From here they can infect other liver cells and thus reproduce effectively. The incubation period for hepatitis B virus is approximately 6 to 25 weeks (i.e., the time before physical or general detection of histologic or physical symptoms), but there are some biochemical and histological abnormalities that occur at an early stage following infection with hepatitis B virus.

B. Materials

Solutions containing 10 ppm, 14 ppm, 22 ppm and 32 ppm silver compositions according to the present invention were used. Nucleotides dATP, dGTP, dCTP and [ ³ H] -dTTP were obtained from standard industrial sources as lamivudine (synthetic antiretroviral agent) and zidovudine (AZT) compounds. Isolated hepatitis B virus was newly obtained from a person with hepatitis B infection and was taken at Haffine Institute, Mumbal, India (WH0 approved testing laboratory). Test cell cultures (Vero and Hep2) were grown as confluent monolayers by typical cell culture techniques.

C. Ways

1) Method of assaying for DNA polymerase inhibition.

Common Approach. Hepatitis B virus, extracted from a human subject, is incubated with the labeled nucleotides and the active inhibitor. Percent inhibition is calculated as a de novo viral nucleic acid synthesized for lamivudine as a positive control and a phosphate buffer salt as a negative control.

Specific procedure. The isolated hepatitis B virus was lysed into a polymerase-free enzyme extract free of contaminating enzymes. Viral extract (25 mL) was added to the reaction mixture comprising dATP, dGTP, dCTP and [ ³ H] -dTTP nucleotides (25 mL). an active inhibitor (3 mL) was added to the mixture comprising the virus extract and nucleotides. The resulting mixture was incubated at 37 ° C for 24 hours.

A separate negative control experiment was performed using phosphate buffer salt (PBS, 3 mL) instead of inhibitor (3 mL).

A separate positive control experiment was performed using a known DNA polymerase inhibitor (3 ml lamivudine at a concentration of 3 mg / ml) in place of the test inhibitor (3 ml).

The reaction was stopped by the addition of 25 mL of EDTA and 25 mL of TCA (trichloroacetic acid). The paper was washed three times with TCA and then with ethyl alcohol. The filter paper was air dried and placed in a scintillator with a glowing cocktail. Radioactivity was measured with a liquid scintillation counter (Blue Star). After counting control, a clean silver formulation was omitted during the complete virus-free procedure to control potential interference in the scintillation counter method.

The reference for this way is P.S. Venkateswaran, I. Millman, and B.S. Blumberg, "Effect of extract from Phyllanthus niruri on hepatitis B and woodchuck hepatitis virus: in vitro and in vivo studies", Proc. Nati. Acad. Sci. USA, 1987, 84, 274-278, which is incorporated herein by reference.

2) Reverse Transcriptase Inhibition Assay Moloney murine leukemia virus reverse transcriptase (MoMuLV) industrial virus enzyme preparation was used. 50 ml of MoMuLV preparation was mixed with a mixture of dATP, dGTP, dCTP and [ ³ H] -dTTP nucleotides.

The mixture was mixed with 3 ml of the test inhibitor and the resulting mixture was incubated at 37 ° C for 24 hours.

A negative control experiment was performed using phosphate buffer salt (PBS, 3 mL) in place of the inhibitor.

A positive control experiment was performed in which a known reverse transcriptase inhibitor (3 ml AZT 0.625 mg / ml) was used in place of the test inhibitor.

The reaction was stopped by the addition of 25 mL of EDTA and 25 mL of TCA. The reaction mixture was then applied onto ionic paper (DEAE paper). The paper was washed three times with TCA and then with ethyl alcohol. The filter paper was air dried and placed in a scintillator with a glowing cocktail. Radioactivity was measured with a liquid scintillation counter (Blue Star).

3) Toxicity testing procedure

Cells were harvested from healthy, confluent Vera and Hep2 cell cultures, which were kept under visualization every 3-4 days. One day before the assay, cells were harvested from cultures using standard techniques and suspended in growth medium and suspended in microtitre plate tubes and placed in a 5% CO ₂ incubator at 37 + 2 ° C. A sample (100 mL) of each test substance was injected into a tube (triple) with 100 mL of PBS as a control. The tubes were examined every 24 hours under a high power inverted microscope to check for cytopathic effect (CPE). All results are shown in Table 5.

D. Results

Results of the reverse transcriptase inhibition assay:

Table 5a

Sample% Inhibition Negative Control (PBS) 0 Positive Control (AZT) 31.33 ppm silver 89.52 ppm silver and 1.5% H2O2 86.93 ppm silver 84.46

Results of DNA polymerase inhibition assay:

Table 5b

Sample% Inhibition Negative Control (PBS) 0 Positive Control (Lamivudine) 31.33 ppm silver 77.73 ppm silver and 1.5% H2O2 65.6 ppm silver 60.89

The silver compositions of the present invention are highly effective in inhibiting DNA polymerase.

Results of the reverse transcriptase inhibition assay:

Table 5c Sample % Inhibition negative control (PBS) 0 positive control (AZT) 18.06 10 ppm silver 89.52 14 ppm silver and 1.5% H2O2 86.93 22 ppm silver 84.46

Thus, the silver compositions of the present invention inhibit reverse transcriptase.

The silver compositions of the present invention were expected to be effective against human diseases caused by viruses such as hepatitis B.

Toxicity results:

Sample Tax Hep2 control (AZT) CPE is not available CPE is not available 10 ppm silver CPE is not available CPE is not available 14 ppm silver and 1.5% H2O2 CPE positive CPE positive 22 ppm silver CPE is not available CPE is not available

These results indicate that, in general, silver compositions are not toxic. As expected, hydrogen peroxide, known to be toxic, shows cytotoxic effects. Therefore, silver should be harmless to cells when used in vivo.

12. PROOF OF EFFECTIVENESS OF SILVER COMPOSITION AS A WATER DISINFECTANT

a. Purpose

Tests were conducted to demonstrate the effectiveness of the composition of the present invention in disinfecting drinking water.

B. Ways

Two crops of Klebsiella oxtyoca were added to the natural river water sample. 100 ml of this supplemented aqueous solution sample was introduced into 0.05 ppm, 0.1 ppm, 0.2 ppm, 0.5 ppm or 1.0 ppm silver composition of the invention. After 5 to 60 minutes of incubation, the samples were membrane-filtered. The filter was rinsed with approximately 100 ml sterile water. The filters were sterile removed from the filter housing and placed on coliform nutrient agar plates. Plates were incubated under growth conditions for 24 hours and evaluated.

Sample Silver (ppm) Table 6 Contact (min) Total coliform Cfu / 100 ml Natural water (per ml) 36 TNTC 1 1.00 5.0 0 0 2 1.00 10.0 0 0 3 1.00 15.0 0 0 4 1.00 30.0 0 0 5 0.50 10.0 0 0 6th 0.50 30.0 0 0 7th 0.50 60.0 0 0 8th 0.20 5.00 0 0 9th 0.20 10.0 0 0 10th 0.20 30.0 0 0 11th 0.20 60.0 0 0 12th 0.10 10.0 0 0 13th 0.05 20.0 0 0

. j. TNTC = Too many to calculate.

Silver compositions have proven to be incredibly effective. Even within a short time (20 min) of incubation at the lowest assay concentration (0.05 ppm), the bacterium was killed. At 0.20 ppm and more, complete killing occurred after 5 minutes. It is clear that complete killing takes less than 5 minutes.

PPM SILVER AS A SURFACE DISINFECTANT PERFORMANCE [SHOW]

The 32 ppm silver composition of the present invention has been approved by the Environmental Protection Agency (EPA) as a broad-spectrum surface disinfectant for use in hospitals, medical applications, residential, commercial and commercial applications. It has been approved for use against some of the most lethal pathogens, including: Gram-positive bacteria such as Staphylococcus aureus (currently identified as the deadliest bacterium in US hospitals), Gram-negative bacteria such as Salmonella choleraesius (food poisoning) and nosocomial or hospital acquired pathogens such as Pseudomonas aeruginosa (common in the mouth and incisions).

The silver compositions of the present invention can be sprayed in or around the affected areas without compromising human or animal health and well-being. They may disinfect a surface selected from the group consisting of walls, tables, chairs, light fixtures, baths, glass, porcelain, metal, glazed ceramics, enamelled or painted by the silver composition of the present invention. The disinfectant method provided comprises one or more surface cleaning steps for disinfecting the composition of the present invention using a spray, cloth, sponge, or cloth, continuously moistening the area to allow the wet surface to stand for at least 10 minutes at 20 ° C. temperature interactions can be controlled by the Arrhenius equation or other means known in the art) and wiping the surface with a clean paper or towel. Surface disinfectant compositions include those containing 5-10 ppm silver. The composition provided for disinfecting surfaces of the present invention comprises (32 ± 3) ppm silver. Another composition provided for disinfecting surfaces of the present invention comprises (22 ± 3) ppm silver.

PROOF OF THE EFFECTIVENESS OF SILVER COMPOSITIONS AS A SUPERDISINFECTANT

The purpose of this assay is to demonstrate the antimicrobial activity of the silver composition of the present invention (10 ppm silver, 14 ppm silver with 1.5 wt% hydrogen peroxide and 32 ppm silver) against the target organism. Yersinia pestis, the etiological agent of bubonic plague. By conducting a standard time-kill assay with Y. pestis suspension, the silver compositions of the present invention were shown to be effective against the bubonic plague bacterium.

B. Materials and Methods

Y. Pestis, strain D27, was grown on a Columbia agar plate for approximately 24 hours at 30 ° C in a 5% CO ₂ inhibitor. The product was scraped off from the plate using sterile 3 mL HPLC water. The suspension was transferred to a 50 mL conical centrifuge tube. The plate was then rinsed with an additional 20 mL of HPLC water. This water was poured into a centrifuge tube. The tube was centrifuged at 3500 xg for 5 minutes. The supernatant was drained and the pellet was resuspended in 1 ml of HPLC water to give a final concentration of about ^{10 10} cells / ml.

The method includes the following steps:

1. 9.9 ml of the silver composition sample was placed in a sterile 20 mm x 150 mm tube. The tube was equilibrated in a 20 ° C water bath.

2. 100 ml of the test organism suspension at zero time was introduced into each silver composition tube. The tube was also turned and returned to the tap bath.

3. 1 mL of the organism / silver composition mixture was transferred to 99 mL of neutralizer in a 250 mL Erlenmeyer flask at 2 min, 3 min, 4 min. and 5 min. with 10 ppm silver or at 2 min, 4 min, 6 min, and 8 min. with 14 ppm silver with 1.5 v / v% H2O2. The flask was thoroughly stirred.

4. The neutralized suspension was immediately diluted 1:10 in physiological saline (PPS).

5. The row of viable organisms in the selected dilution tubes was examined using membrane filtration. One ml of samples was placed in duplicate. The membranes were washed in approximately 150 ml (or 250 ml if the sample was taken from a neutralization flask) with sterile phosphate buffer and transferred to Columbia agar plates. All remaining (98 mL) for 4 and 5 min. amounts of neutralizer flasks were also placed on plates. The plates were incubated at 30 ° C in a 5% CO ₂ inhibitor for 72 hours.

6. The number of columns for each filter was counted and the logarithmic reductions were evaluated.

C. Results The ppm silver results are shown in Table 7.

table

Time

Log reduction

Percent killing min.

min 6 min

min

2.63

3.20

3.46

3.68

99.77

99.94

99.97

99.98 the regression equation applied to compute these data is Y = 2.3965 + 0.1696 x

This indicates a 6-log reduction time of 21.2 minutes.

ppm silver results are given in Table 8.

table

Time Log reduction Percent kill 2 min > 7.61 99.999998 4 min > 7.61 99.999998 6 min > 7.61 99.999998 8 min > 7.61 99.999998

14 ppm silver with 1.5 v / v% H ₂ O ₂ are shown in Table 9. Time Table 9 Log reduction Percent kill 2 min 3.27 99.95 3 min 4.72 99,998 4 min 5.36 99,9996 5 min 6.47 99.99997

The regression equation used to compute these data is Y = 1.371 + 1.024 x. This indicates a 6-log reduction time of 4.52 minutes.

The silver compositions of the present invention exhibited pronounced bactericidal activity against Y. pestis, the atiological agent of bubonic plague. 32 ppm formulations yielded more than 7 log reductions (essentially killing) in less than 2 min. The data showed that 10 ppm silver achieves 6 log kill in 20 minutes. Silver and hydrogen peroxide show significant synergism with estimated 6 log to 5 min. This is significantly better than 10 ppm silver alone. A level of 14 ppm silver was chosen because data from other experiments showed that this level of silver in combination with hydrogen peroxide achieves results that are achieved with a 32 ppm silver product.

DATA SHEET

The following Table A includes a summary of the results obtained above, in terms of the effects of the silver composition of the invention on a wide variety of human disease-causing microbes, in some cases the data in the table are not described above. However, the results were obtained using the procedures described above in such a way that each of those skilled in the art readily reproduces the results.

Human diseases are cured and pathogens killed by silver composition

The disease

Furunkulas

Osteomelites Bacterial Dysentery Burn Infections Dental Stones Diarrhea (Blood)

Diarrhea

Ear Infection Ear Infection Typhoid Epiglottitis (Children)

Eye infections

Corneal ulcer-keratitis

Food poisoning

Food poisoning

Food poisoning

Endocarditis

Endocarditis

Meningitis

Meningitis

Meningitis

Meningitis

Nosocomial infections Nosocomial infections Nosocomial infections (from hospitals)

Pneumonia

Pneumonia

Pneumonia

Table A

Pathogen

Staphylococcus aureus Staphylococcus aureus Shigella boyd ;; Streptococcus mutans Pseudomonasaenugmosa boydii Shigella Escherichia coli Haemophilus influenzae Streptococcus pneumonie Salmonella tyhimurium Haemophilus Staphyfococcus aenuginosa aureus Salmonella Salmonella arizona tyhimurium Escherichia ooft streptococcus' aeca / from Streptococcus gordonii HaemophiJus influenzae Enterobacter aerogenes, Pseudomonas and Klebsiella pneumonie aenuginosa Streptococcus pneumoniae, Streptococcus pyogenes Pseudomonasaerugmosa

Staphyfococcus aureus Haemophilus influenzae Pseudomonas aeruginosa

Effective Concentration

Killed @ 5 ppm Killed @ 5 ppm Killed @ 2.5 ppm Killed @ 5 ppm Killed @ 2.5 pp Killed @ 2.5 pp Killed @ 1.25 p Killed @ 2.5 pp Killed @ 2.5 pp Killed @ 5 ppm Killed @ 5 ppm Killed @ 5 ppm Killed @ 2.5 pp Killed @ 2.5 pp Killed @ 2.5 pp Killed @ 5 ppm Killed @ 2.5 pp Killed @ 5 ppm Killed @ 2.5 pp Killed @ 2.5 ppm Killed @ 1.25 in

Killed @ 5 ppm Killed @ 1.25 p | Killed @ 5 ppm

The disease

Pneumonia

Respiratory tract infections Respiratory tract infections Respiratory tract infections Rubella

Septicemia

Fistula infections Sinusitis Impetigo Skin infections Skin infections Streptococcal arthritis Throat infections Dental caries Urethritis (male) Urinary tract infections Urinary tract infections Urinary tract infections Vaginitis (female)

Wound Infections Wound Infections Wound Infections Wound Infections Wound Infections Yeast Infections

Pathogen

Streptococcus pneumonie Streptococcus pyogenes E. coli

Klebsiella pneumoniae Streptococcus pyogenes Enterobacter aerpyogenes Haemophilus influenzae Streptococcus pneumonia Staphylococcus aureus Staphylococcus aureus Streptococcus pyogenes Streptococcus pyogenes Haemophilus influenzae Haemophilus influenzae Streptococcus mutans

Klebsiella pneumoniae Pseudomonas aeruginosa Streptococcus taecalis Enterobacter aerpyogenes Trichomonas vaginatis Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Streptococcus faecalis Candida albicans

Effective Concentration

Killed @ 2.5 pp Killed @ 1.25p Killed @ 2.5 pp Killed @ 2.5 pp Killed @ 1.25p Killed @ 2.5 pp Killed @ 1.25p Killed @ 2.5 pp Killed @ 1.25p Killed @ 5 ppm Killed @ 1.25p Killed @ 1.25 p Killed @ 1.25 p Killed @ 5 ppm Killed @ 10 pp Killed @ 2.5 pp Killed @ 2.5 pp Killed @ 5ppm Killed @ 2.5 pp Killed @ 2.5 pp Killed @ 10 ppt Killed @ 2.5 pp Killed @ 2.5 pp Killed @ 5 ppm Killed @ 2.5 pp Killed @ 10 ppr

EFFECTIVENESS OF SILVER COLLIDE MADE AS HYDROGEL

Advanced wound care recognizes the fact that the optimal wound treatment is to keep it sterile and protected from desiccation and environmental contamination. Conventional binting is effective in protecting against environmental contamination, even very ineffective in preventing desiccation. Bits can be converted to antimocrobial by adding various disinfectants, but these substances are often harsh and kill body cells as well as germs. Currently, wound care has been fundamentally altered, using either hydrogen materials, which can be semi-solid (amorphous material) or as soft sheet-like material. Hydrogen is hydrophilic, thus preventing wound desiccation. The sheet-like material is effective in protecting against environmental contaminants and due to its hydrophilic nature, hydrogen can actually absorb excess fluid released by the wound.

Hydrogens are formed by combining a hydrophilic polymer with other ingredients in an aqueous solution. Polymers form a gel, followed by a change in pH, temperature and other triggers, and the gel has a regular molecular network polymer surrounding the aqueous solution. Although the composition may be an amorphous semi-solid or a solid sheet type material, a large volume tends to include an aqueous solution, which is the opposite of a hydrophilic polymer. Hydrophilic polymers suitable for the production of hydrogels include gelatin, carboxymethyl cellulose (and other cellulose derivatives), other plant carbohydrate polymers or algae of tea origin such as alginate, carrageenan, xanthan gum, acacia bean gum, tragacanth gum, guar gum, other plant gums, acrylic acid copolymers (such as Carbopol) and combinations of these or similar hydrophilic polymers.

The aqueous component preferably contains a variety of additive materials that enhance the physical properties of hydrogen and / or improve wound healing. These include various vitamins, amino acids and growth factors added to improve healing and reduce scar formation to reduce scarring. conventional anesthetics such as novocaine, lidocaine and their derivatives may also be included as additives to enhance comfort. Since maintaining a sterile wound is a major challenge for the dressing, various antimicrobial and disinfectant agents are usefully included. These include organic acids such as citric acid, acetic acid, benzoic acid, propionic acid and lactic acid.

Alcohols such as isopropanol or ethanol are suitable for use as organic disinfectants, including chlorinated phenols such as TCP (2,4,6 tichlorophenol), biguanides, chlorhexidine (when mixed with cetrimide), chlorhexidine gluconate and chlorhexidine acetate. Disinfectant surfactants, including amphoteric surfactants and aldehydes such as formaldehyde and glutaraldehyde, may be included. Halogen disinfectants include iodine, iodophores and polyvidone iodine, are effective as peroxides and other oxygenators such as hydrogen peroxide. Other therapeutic ingredients include aluminum zinc astringent agents, furan derivatives and quinoline derivatives such as cliquinol. To the extent that all of these antimicrobial agents can be curative, they all have a tendency to damage tissue and / or can easily develop microbial resistance to them.

As widely described above, the silver colloid of the invention is highly antimicrobial, is very gentle to human tissue, and is effective against microbial resistance Both amorphous gels and hydrogen film together favor the introduction of effective levels of colloidal silver in a moist therapeutic environment. On one side, the amorphous hydrogel slowly releases colloidal silver as it slowly softens in the tissue exudate and gradually begins to dissolve. On the other hand, the amorphous hydrogel releases moisture into the fabric and at the same time makes the silver colloid suitable for application. In addition, the small amount of colloidal silver present in the dressing has the advantage over molecular silver, which, over a prolonged period of time, releases silver ions with excellent oligodynamic activity.

After the initial test, carbopol was selected as an effective hydrogel forming agent for use with colloidal silver. A base formulation was prepared which essentially contained the following ingredients as shown in Table 9a.

Table 9a

The Ingredient Function Supplier Colloidal silver solution (22 ppm or 32 ppm) ppm) Active, antimicrobial and diluting American Biotech Labs Carbopol EDT2020 Rheological modifier Neveon Triethanolamine Neutralizer, penetration agent E. Merck Propylene glycol Moisturizer E. Merck

All raw materials were first investigated:

1. Antibacterial activity

2. Physical and chemical properties:

1. Appearance

2. Smell

3. pH

4. Sensitivity

5. density

6. The property of foaming

7. Flowability

Colloidal silver solution (22 ppm or 32 ppm):

In this composition, the silver solution is used as the active component (antimicrobial agent) It is the only diluent in this specific composition.

A. Antibacterial activity:

Culture Diameter of damping zone 22 ppm 32 ppm MRSA 17 mm 18mm E. coli 14 mm NA Ps. aeruginosa 21mm 22mm

B

1.

2.

3.

4.

5.

6.7.

Physical and chemical properties:

Appearance Colorless clear solution

Odor odorless pH 5.0

Sensitivity has not been established

Density 1.0

The foaming property has not been determined

Flow potential not established

Carbopol

Carbopol is chemically known as carboxypolymethylene or carboxyvinyl polymer. It is a copolymer of acrylic acid and is a highly ionic (i.e. hydrophilic) and slightly acidic compound. Carbopol polymers must be neutralized for maximum viscosity. They are used in the pharmaceutical, cosmetic and textile applications as thickening, suspending, dispersing and emulsifying agents. In this composition, carbopol is used as a gelling or thickening agent.

A. Antibacterial activity not detected

B. Physical and chemical properties:

1. Appearance

2. Smell

3. pH

4. Sensitivity

5. Density

6. The property of foaming

7. Flowability dry, white powder odorless unspecified unspecified unspecified

Triethanolamine (TEA) Ο ₆ Η ₁₅ ΝΟ ₃ (mol m: 149.19)

In this composition, triethanolamine, as an alkylating agent, neutralizes carbopol to increase viscosity. It also increases the penetration force of the active agent.

A. Antibacterial activity not detected

B. Physical and chemical properties:

1. Appearance

2. Smell

3. pH

4. Sensitivity

5. Density

6. The property of foaming

7. Flowability colorless viscous liquid mild ammonia not determined 1.1242 g / cc not determined not determined

Propylene Glycol C ^ HaO? (mol m: 76.09)

Propylene glycol is chemically known as 1: 2 propanediol. It is used as a moisturizer and sensory mofdifector in this composition.

A. Antibacterial activity

C. Physical and chemical properties:

unspecified

1. Appearance

2. Smell

3. pH

4. Sensitivity

5. Density colorless viscous liquid odorless undetermined 1.036 g / cc

6. No foaming property has been identified

7. Flow potential not determined

Once the standard formula was established, a number of batches were made to investigate possible variations in the compositions. Since completing the test 19, the following observations have been achieved.

1. Increasing the pH increases the viscosity of the gel.

2. Increasing the carbopol content increases the viscosity of the gel.

3. The higher the percentage of carbopol, the higher the stickiness.

4. From the experiments described above, it can be concluded that commercial success can be achieved between the amount of carbopol and TEA used and the final pH obtained, which should not exceed 8.5. Thus, composition no. 18 was kept as standard and the batch split up to 10 kg.

PRODUCT DEVELOPMENT STAGES (INTRODUCTION

Carbopol-based gel formulations were standardized with pH, sensory, sticky, and consistency. With this in mind, various laboratory batches were used, using water as the aqueous phase to obtain a product of appropriate quality and sensibility prior to selecting the main batch.

Lot # SG / 001 Composition:

Part A: Distilled water 83.50 g Carbopol 00.62 g

NaOH 18% 00.60 g

Part B; distilled water 1.00 g propylene glycol 5.00 g

NaOH 18% 1.50 g

Procedure: Weigh out the quantity of distilled water referred to in Part A and store in a water bath at 70 ° C. Carbopol is added to the distilled water, stirring constantly to avoid lumps. 18% NaOH was added thereto at 70 ° C after 20 minutes. Weigh all ingredients from Part B and store in a water bath at 70 ° C for 15-20 min. Part B is mixed with Part A for 10-15 min. Cool to room temperature and examine.

Results: 1. pH 10.8

2. Flow rate 90 ° C =>> 5 min.

3. Adhesive very sticky

Lot # SG / 002 Composition:

Part A: Distilled water 83.50 g Carbopol 00.62 g

TEA 01.20 g

Part B; distilled water 1.00 g propylene glycol 5.0 g TEA 1.50 gm

Procedure: Weigh out the quantity of distilled water referred to in Part A and store in a water bath at 70 ° C. Carbopol is added to the distilled water with constant stirring to avoid clumps. it is added with TEA at 70 ° C after 20 minutes. Weigh all ingredients from Part B and store in a water bath at 70 ° C for 15-20 min. Part B is mixed with Part A for 10-15 min. Cool to room temperature and examine.

Results: 1. pH 7.9 (SOP-08)

2. Flow rate 90 ° C =>> 5 min.

3. Adhesive very sticky

Lot # SG / 003 Composition:

Part A: Distilled water 86.00 g Carbopol 00.62 g

TEA 01.20 g

Part B; distilled water 2.00 g propylene glycol 5.0 g

TEA 1.50 g

Procedure: Weigh out the quantity of distilled water referred to in Part A and store in a water bath at 70 ° C. Carbopol is added to the distilled water, stirring constantly to avoid lumps. TEA is added to it at 70 ° C after 20 minutes. Weigh all ingredients from Part B and store in a water bath at 70 ° C for 15-20 min. Part B is mixed with Part A for 10-15 min. Cool to room temperature and examine.

Results: 1. pH 8.62

2. Flow rate 90 ° C =>> 5 min.

3. Adhesive very sticky

Lot # SG / 004 Composition:

Part A: Distilled water 86.00 g Carbopol 00.62 g

TEA 01.00 g

Part B; distilled water 2.00 g propylene glycol 5.0 g TEA 1.50 g

Procedure: Weigh out the quantity of distilled water referred to in Part A and store in a water bath at 70 ° C. Carbopol is added to the distilled water with constant stirring to avoid clumps. it is added with TEA at 70 ° C after 20 minutes. Weigh all ingredients from Part B and store in a water bath at 70 ° C for 15-20 min. Part B is mixed with Part A for 10-15 min. Cool to room temperature and examine.

Results: 1. pH 8.5

2. Flow rate 90 ° C =>> 5 min.

45 ° C =>> 5 min.

3. Adhesive very sticky

Lot # SG / 005 Composition:

Part A: distilled water 86.00 g

Carbopol 00.62 g

TEA 1.20 g

Part B: Distilled Water 2.00 g Propylene Glycol 7.0 g TEA 1.50 g

Procedure: Weigh out the quantity of distilled water referred to in Part A and store in a water bath at 70 ° C. Carbopol is added to the distilled water, stirring constantly to avoid lumps. TEA is added to it at 70 ° C after 20 minutes. Weigh all ingredients from Part B and store in a water bath at 70 ° C for 15-20 minutes. Part B is mixed with Part A for 10-15 min. Cool to room temperature and examine.

Results: 1. pH 8.7

2. Flow rate 90 ° C =>> 5 min.

45 ° C =>> 5 min.

3. Adhesive very sticky

Party N r. SG / 006 Composition:

Part A: Distilled Water 85.00 g Carbopol 00.62 g

TEA 01.00 g

Part B: distilled water 1.00 g propylene glycol 5.0 g TEA 1.40 g

Procedure: Weigh out the quantity of distilled water referred to in Part A and store in a water bath at 70 ° C. Carbopol is added to the distilled water, stirring constantly to avoid lumps. TEA is added to it at 70 ° C after 20 minutes. Weigh all ingredients from Part B and store in a water bath at 70 ° C for 15-20 min. Part B is mixed with Part A for 10-15 min. Cool to room temperature and examine.

Results: 1. pH 8.4

2. Flow rate 90 ° C =>> 5 min.

45 ° C =>> 5 min

3. Adhesive very sticky

Lot # SG / 007 Composition:

Part A: distilled water 172 g

Carbopol 1.24 g

TEA 2.40 g

Part B: distilled water 6.00 g propylene glycol 10.0 g

TEA 2.80 g

Procedure: Weigh out the quantity of distilled water referred to in Part A and store in a water bath at 70 ° C. Carbopol is added to the distilled water, stirring constantly to avoid lumps. it is added with TEA at 70 ° C after 20 minutes. Weigh all ingredients from Part B and store in a water bath at 70 ° C for 15-20 min. Part B is mixed with Part A for 10-15 min. Cool to room temperature and examine.

Results: 1. pH 8.28

2. Flow rate 90 ° C =>> 5 min.

45 ° C =>> 5 min

3. Adhesive very sticky

Lot # SG / 008 Composition:

Part A: Silver solution (32 ppm) 86.00 g Carbopol 0.62 g

TEA 1.20 g

Part B: silver solution (32 ppm) 2.00 g propylene glycol 5.0 g TEA 1.50 g

Procedure: Weigh the amount of silver solution referred to in Part A and store in a water bath at 70 ° C. Carbopol is added to the silver solution with constant stirring to prevent clumping. TEA is added to it at 70 ° C after 20 minutes.

Weigh all ingredients from Part B and store in a water bath at 70 ° C for 15-20 min. Part B is mixed with Part A for 10-15 min. Cool to room temperature and examine.

Results: 1. pH 8.65

2. Flow rate 90 ° C =>> 5 min.

45 ° C =>> 5 min

3. Adhesive very sticky

Lot # SG / 009 Composition:

Part A: Silver solution (32 ppm) 172 g Distilled water 12 g Carbopol 1.24 g

TEA 2.40 g

Part B: Silver solution (32 ppm) 6.00 g propylene glycol 10.0 g

TEA 2.80 g

Procedure: Weigh the amount of silver solution referred to in Part A and store in a water bath at 70 ° C. Carbopol is added to the silver solution with constant stirring to prevent clumping. TEA is added to it at 70 ° C after 20 minutes. Weigh all ingredients from Part B and store in a water bath at 70 ° C for 15-20 min. Part B is mixed with Part A for 10-15 min. Cool to room temperature and examine.

Results: 1. pH 8.54

2. Flow rate 90 ° C =>> 5 min.

45 ° C =>> 5 min

3. Adhesive very sticky

Lot # SG / 010 Composition:

Part A: Silver solution (32 ppm) 172 g Distilled water 24 g Carbopol 1.39 g

TEA 2.40 g

Part B: Silver solution (32 ppm) 6.00 g Propylene Glycol 5.0 g

TEA 2.80 g

Procedure: Weigh the amount of silver solution referred to in Part A and store in a water bath at 70 ° C. Carbopol is added to the silver solution with constant stirring to prevent clumping. TEA is added to it at 70 ° C after 20 minutes. Weigh all ingredients from Part B and store in a water bath at 70 ° C for 15-20 min. Part B is mixed with Part A for 10-15 min. Cool to room temperature and examine.

Results: 1. pH 8.43

2. Flow rate 90 ° C =>> 5 min.

45 ° C =>> 5 min

3. Adhesive adhesive

Lot # SG / 011 Composition:

Part A: Distilled water 98 g Carbopol 0.76 g

TEA 0.56 g

Part B: distilled water 3.0 g propylene glycol 5.0 g

TEA 1.40 g

Procedure: Weigh out the quantity of distilled water referred to in Part A and store in a water bath at 70 ° C. Carbopol is added to the distilled water with constant stirring to avoid clumps. it is added with TEA at 70 ° C after 20 minutes. Weigh all ingredients from Part B and store in a water bath at 70 ° C for 15-20 min. Part B is mixed with Part A for 10-15 min. Cool to room temperature and examine.

Results: 1. pH 8.05

2. Flow rate 90 ° C =>> 5 min.

45 ° C =>> 5 min

3. Adhesive very sticky

Lot # SG / 012 Composition:

Part A: Distilled water 98 g

Carbopol 0.76 g

TEA 0.34 g

Part B: Distilled Water 3.0 g Propylene Glycol 5.00 g TEA 0.64 g

Procedure: Weigh out the quantity of distilled water referred to in Part A and store in a water bath at 70 ° C. Carbopol is added to the distilled water, stirring constantly to avoid lumps. it is added with TEA at 70 ° C after 20 minutes. Weigh all ingredients from Part B and store in a water bath at 70 ° C for 15-20 min. Part B is mixed with Part A for 10-15 min. Cool to room temperature and examine.

Results: 1. pH 6.35

2. Flow rate 90 ° C =>> 5 min.

45 ° C =>> 5 min

3. Adhesive very sticky

Lot # SG / 013 Composition:

Part A: Silver solution (32 ppm) 86 g

Distilled water 12 g

Carbopol 0.76 g

TEA 0.32 g

Part B: Silver solution (32 ppm) 3.00 g propylene glycol 5.0 g

TEA 0.64 g

Procedure: Weigh out the silver solution and distilled water in Part A and place in a water bath at 70 ° C. carbopol is added to the solution under constant stirring to prevent clumping. it is added with TEA at 70 ° C after 20 minutes. Weigh all ingredients from Part B and store in a water bath at 70 ° C for 15-20 min. Part B is mixed with Part A for 10-15 min. Cool to room temperature and examine.

Results: 1. pH 6.7

2. Flow rate 90 ° C =>> 5 min.

45 ° C =>> 5 min

3. Adhesive very sticky

Lot # SG / 014 Composition:

Part A: Silver solution (32 ppm) 86 g

Distilled water 12 g

Carbopol

TEA

0.78 g 0.32 g

Part B; silver solution (32 ppm) 3.00 g propylene glycol 5.0 g

TEA

0.64 g

Procedure: Weigh out the silver solution and distilled water in Part A and place in a water bath at 70 ° C. Carbopol is added to the solution under constant stirring to prevent clumping. TEA is added to it at 70 ° C after 20 minutes. Weigh all ingredients from Part B and store in a water bath at 70 ° C for 15-20 min. Part B is mixed with Part A for 10-15 min. Cool to room temperature and examine.

Results; 1. pH 6.6

2. Flow rate 90 ° C =>> 5 min.

45 ° C =>> 5 min

3. Adhesive very sticky

Lot # SG / 015 Composition;

Part A: Silver solution (32 ppm) 86 g

Distilled water

Carbopol

TEA g 0.68 g 0.40 g

Part B: Silver solution (32 ppm) 5.00 g Propylene Glycol 7.0 g

TEA

7.0 g 0.6 g

Procedure: Weigh out the silver solution and distilled water in Part A and place in a water bath at 70 ° C. carbopol is added to the solution under constant stirring to prevent clumping. TEA is added to it at 70 ° C after 20 minutes. Weigh all ingredients from Part B and store in a water bath at 70 ° C for 15-20 min. Part B is mixed with Part A for 10-15 min. Cool to room temperature and examine.

Results: 1. pH 6.72

2. Flow rate 90 ° C =>> 5 min.

45 ° C =>> 5 min

3. Adhesive adhesive

Lot # SG / 016 Composition:

Part A: Silver solution (32 ppm) 86 g Distilled water 12 g

Carbopol 0.64 g

TEA 0.40 g

Part B: Silver solution (32 ppm) 5.00 g Propylene Glycol 7.0 g

TEA 0.6 g

Procedure: Weigh out the silver solution and distilled water in Part A and place in a water bath at 70 ° C. carbopol is added to the solution under constant stirring to prevent clumping. It is added to TEA at 70 ° C after 20 minutes. Weigh all ingredients from Part B and store in a water bath at 70 ° C for 15-20 min. Part B is mixed with Part A for 10-15 min. Cool to room temperature and examine.

Results: 1. pH 6.87

2. Flow rate 90 ° C =>> 5 min.

45 ° C =>> 5 min

3. Adhesive adhesive

Lot # SG / 017 Composition:

Part A: Silver solution (32 ppm) 86 g Distilled water 12 g

Carbopol 0.62 g

TEA 0.40 g

Part B: Silver solution (32 ppm) 5.00 g Propylene Glycol 7.0 g

TEA 0.6 g

Procedure: Weigh out the silver solution and distilled water in Part A and place in a water bath at 70 ° C. Carbopol is added to the solution under constant stirring to prevent clumping. TEA is added to it at 70 ° C after 20 minutes. Weigh all ingredients from Part B and store in a water bath at 70 ° C for 15-20 min. Part B is mixed with Part A for 10-15 min. Cool to room temperature and examine.

Results: 1. pH 7.05

2. Flow rate 90 ° C =>> 5 min.

45 ° C =>> 5 min

3. Adhesive adhesive

Lot # SG / 018 Composition:

Part A: Silver solution (32 ppm) 86 g Distilled water 12 g

Carbopol 0.58 g

TEA 0.40 g

Part B: Silver solution (32 ppm) 5.00 g Propylene Glycol 7.0 g

TEA 0.6 g

Procedure: Weigh out the silver solution and distilled water in Part A and place in a water bath at 70 ° C. Carbopol is added to the solution under constant stirring to prevent clumping. TEA is added to it at 70 ° C after 20 minutes. Weigh all ingredients from Part B and store in a water bath at 70 ° C for 15-20 min. Part B is mixed with Part A for 10-15 min. Cool to room temperature and examine.

Results: 1. pH 7.40

2. Flow rate 90 ° C =>> 5 min.

45 ° C =>> 5 min

3. Adhesive uniformly thick

Lot # SG / 019 Composition:

Part A: Silver solution (32 ppm) 86 g

Distilled water 12 g

Carbopol 0.54 g

TEA 0.40 g

Part B: Silver solution (32 ppm) 5.00 g Propylene Glycol 7.0 g

TEA 0.6 g

Procedure: Weigh out the silver solution and distilled water in Part A and place in a water bath at 70 ° C. Carbopol is added to the solution under constant stirring to prevent clumping. TEA is added to it at 70 ° C after 20 minutes. Weigh all ingredients from Part B and store in a water bath at 70 ° C for 15-20 min. Part B is mixed with Part A for 10-15 min. Allow to cool

room temperature and investigated. Results: 1. pH 7.65 2. Flow capability 90 ° C => > 5 min 45 ° C => > 5 min 3. Stickiness equally thick

Note: Although the gel has improved sensory properties, it does not measure consistently. Based on the results above, the following instructions for one kilogram batch were created.

Part A Silver solution distilled water 860g 100 g Carbopol 5.80 g TEA 4.00 g Part B. ASAP solution 50.0 g Propylene glycol 70.0 g TEA 6.00 g

Capacity 1.0 kg with application of moisture loss

Procedure: Take the required amount of distilled water and silver solution into a clean sterilized container. The solution is stirred at 70 ° C with constant stirring. Add small amounts of carbopol, continuing mixing / homogenization. After all, the carbopol is added for 30 minutes (time adjusted to batch size), followed by the addition of TEA to solution A.

In a separate container, mix all the ingredients from Part B. Raise the temperature to 70 ° C and slowly part B to part A. After homogenization, cool to room temperature.

Precautions: Carbopol dispersion must be made using good homogenizers. With the new carbopol portion, a small test batch is taken. The minimum heating time, the longer the samples are heated to greater water loss.

Results: 1. pH 7.4

2. Flowability> 5 min.

3. Adhesive uniformly thick.

This composition was easily divided up to 10 kg. test portions. There was no problem with tilting. Vacuum ventilation is recommended to prevent air entrainment and to ensure uniform filling.

This composition exhibits the following physical and chemical properties as shown in Table 10.

table

The test Conditions Results 1. Appearance Gold yellow translucent gel Tolerable 2. The smell Odorless Odorless 3. Specific gravity power 1.02 1.02 4. Flowability 45 ° and 90 ° - more than 5 min. passes through 205 cm (1 inch) from the beginning 45 ° and 90 ° - more than 5 min. passage 2.5 cm (1 inch) from the beginning 5. Possibility of foaming <10ml <10ml

100

6th Sensitivity / stickiness 1-uniformly thick 1-uniformly thick 7th Viscosity RT 30 ^u 370 32000 ± 5000 30000 + 5000 34,000 33500 8th PH 6.5-8.0 7.4 9th Freezing and thawing Passes SOP 1-10 Compared to the original 10th Optimal waves length (X max) 22 ppm - 400 +/- 20 nm 32 ppm-450 +/- 20 nm. 400 nm. ** 450 nm. ** 11th Easy maintenance Without additional dislocation Tolerable 12th Compatibility Product I did not lose color when interacting with the container Refer to Table 3 13th Humidity depletion 10.27% 14th Humidity taking 80%

Microbiological evaluation

There is reason to believe that the silver colloidal gel has microbiological properties similar to the original silver colloid that has been intensively studied above. However, the addition of a hydrophilic polymer for gel preparation may directly interfere with the microbiological properties of silver or may inhibit silver diffusion in such a way that efficacy is reduced. Therefore, microbiological tests similar to those performed with silver colloidal solution were also performed with silver colloidal hydrogel.

Initially, the hydrogel was assayed to determine if the composition itself was sterilized. The protocol was as follows:

Flasks containing 100 ml sterile liquid thioglycolate medium (anaerobic bacterium), sterile soybean casein uptake medium (anaerobic bacterium) and potato dextrose broth (fungi) were prepared. Samples containing approximately 100 mg of gel were assayed by aseptic transfer into the flasks. One preparation was incubated at 37 ° C and the other incubated at room temperature for one week. The flasks were then checked and showed no turbidity or microbial growth. Since the gel samples were not produced under sterile conditions, it can be concluded that the composition itself is sterilizing. 100 mg of gel is used for each assay. This corresponds to 2.2 pg in 100 ml medium or 0.02214 or 0.032 pg silver in medium. These concentrations

101 silver has no antimicrobial activity and therefore false negative results can be eliminated.

Subsequently, a plurality of test organisms were used for comparison of the inhibition zone obtained with either a 22 or 32 ppm silver solution or a 22 or 32 silver gel prepared as described above in Table 11. For each micro-organism (approximately 108 cfu / ml), 0.1 ml samples or 18 hrs. cultured cultures were sprayed onto sterile nutrient agar plates. A 10 mm diameter hole with a corkscrew was squeezed in each inoculated plate. A sample amount of product (0.2-0.3 g) was added to each well and the plate was incubated for 24 hours. The plates were then checked and the following inhibition zones (total diameter of each zone) were measured.

table

Culture Silver solution Silver gel 22 ppm 32 ppm 22 ppm 32 ppm E. coil 14mm 14mm 12mm 13mm Ps. Aeruginosa 21mm 22mm 21mm 20mm B. subtilis 15mm 16mm 14mm 14mm MRSA1 17mm 18mm 16mm 17mm MRSA2 16mm 17mm 16mm 17mm S. aureus ATCC 6638 P 14mm 14.5mm 15mm 15mm S.pyogenes 16mm 18mm 16mm 18mm S.typhi 17mm 16mm 16mm 16mm Sh.f1exneri 20mm 21mm 20mm 21mm K. pneumoniae 17mm 18mm 18mm 18mm C. diptheriae 16mm 18mm 16mm 17mm C. albicans 39mm 40mm 39mm 40mm

These results indicate that the gel inhibition effects are substantially equivalent to those of the silver colloid solution, indicating that the gelation polymers do not adversely affect the antimicrobial powers of the silver colloid. Some cultures (S. piogenes, C. diphtheriae, and S. aureus) were also cultured on blood agar. The results confirmed that silver gel could also be effective for a bleeding, urinating wound.

Similar tests were carried out on the same bacterial strains using many antibiotic agents, in some cases the antibiotics were more effective,

102 other compounds than silver were less effective. This indicates that the strains used were not attenuated or repeled (see Tables 12a and 12b).

Gram-positive bacterium (Table 12a)

Antibiotic Conc. S. aureus MRSA1 MRSA2 B. subtllls Ampicillin 200 mcg Clear 15mm 18mm 16mm Cefotaxime 30 mcg 26mm Inhibitions there is no Clear 12mm Cephalexin 30 mcg Clear 1.0mm 0.8mm Clear Ciprofloxacin 5mcg 28mm 14mm 14mm 20mm Cloxacillin 1mcg Clear Clear 13mm 18mm Co-Trimoxazole 25 mcg Clear Inhibitions there is no Inhibitions nsrfl 15mm Gentamicin 10 mcg 1 Clear Inhibitions there is no 11 mm 18mm Lincomycin 2mcg 1 Clear Clear Clear 18mm Ofloxacin 5mcg Clear 15mm 16mm 22mm Pefloxloxacin 10 mcg 30mm 11 mm 13mm 21mm Roxithromycin 15 mcg Clear 1.0mm 12mm 20mm Tetracycline 30 mcg 34mm Inhibitions there is no 0.7mm 19mm

Gram-positive bacterium (Table 12b)

Abtibiotics Conc. E.coli K. pneumoniae S.typhi Ps. aeruginosa Amikacin 30 mcg Clear 18mm Clear 10mm Ampicillin 200 mcg 23mm 18mm 20mm 13mm Cefotaxime 30 mcg 21mm 20mm 22mm 19mm Ceftizoxime 30 mcg 18mm 18mm 15mm No inhibition Chloramphenicol 30 mcg 22mm 21mm 23mm No inhibition

103

Ciprofloxacin 5mcg 29mm 22mm 25mm 15mm Co- Trimoxazole 25 mcg 24mm 19mm 27mm Clear Gentamicin 10 mcg Clear 17mm Clear No inhibition Ofloxacin 5mcg Clear 29mm Clear 15mm Pefloxacin 10 mcg Clear 25mm Clear 10mm Piperacillin 100 mcg 22mm 15mm 16mm 10mm Tetracycline 30mcg 19mm 18mm 16mm No inhibition

Hand cleaning test

Because the hydrogel has the potential to increase the adhesion of silver to the skin surface, the effectiveness of the gel as a hand cleaner has been evaluated. For this study, the hands area of 2.5 cm was marked and then cleaned with approximately 1 g of gel. The control area was cleaned with sterile distilled water. Swabs were taken from the areas and smeared with strips of nutrient agar. Swabs were repeated every four hours. The stained sites were incubated for 24 hours. 37 ° C and evaluate results.

As shown in Table 13 below, the control smears produced so many bacteria that too many were counted (TNTC). These areas are treated with a silver gel that remains essentially sterile for three hours and shows easy growth to four hours, which could give higher results to health professionals who need to sterilize their hand surface without the use of coarse or irrigating compounds.

Table

time Control 22 ppm 32 ppm 0 or. TNTC There is no growth There is no growth 1 or. TNTC There is no growth There is no growth 2 or. TNTC There is no growth There is no growth 3 or. TNTC There is no growth There is no growth 4 or. TNTC 3 Cfu 2 Cfu

104

Although gels show exceptional wound healing properties, the disadvantage of a typical hydrogel is that microorganisms can often migrate within the matrix. Therefore, if the wound is covered with a hydrogel and one of the areas becomes infected, the infectious organisms have the ability to move through the hydrogel and infect other areas. The possibility was tested using a hydrogel strip to separate individual areas of the nutrient agar plate. Each agar plate was divided into two areas by placing a 2 cm agar strip along the plate diameter. The interval was set with a 1.5 cm wide hydrogel strip that partially overlapped each end of the agar by approximately 5 mm. One part of the plate was then inoculated with about 0.5 ml of culture and the plate was incubated to see if the microorganisms could cross the hydrogel "bridge". The results in Table 14 show that silver hydrogel completely stops migration.

table

Culture Area of incubation Migration area E. coli Growing hard Not growing B. subtilis Growing hard Not growing MRSA 1 Growing hard Not growing Ps. Aeruginosa Growing hard Not growing Control hydrogel Growing hard Growing up

From the above results a prototype gel blend was selected and variants are shown in the following examples.

Example Components of Part A and Part B taken from an A kg gel batch as shown below:

Part A The silver colloid of the invention is 32 ppm distilled water Carbopol Triethanolamine 860g 100 g 6.8 g 4.0 g Part B. The silver colloid of the invention is 32 ppm 50g Propylene glycol 70g triethanolamine 6.0 g

105

First add the required amount of distilled water and silver solution to the mixer and start mixing. Slowly folded into carbopol (Noveon, USA). The stirring should be energetic enough to disperse the carbopil and prevent the formation of lumps. The temperature of the mixing is 60-70 ° C.

All ingredients A and B are mixed in the censer. heat to 70 ° C and mix into A with vigorous stirring. Stirring is continued and cooled to room temperature. Batch yield is checked. It should be about 1000 g. Triethanolamine helps the carbopol to enter the gel.

Example B:

All ingredients are prepared as in Example A, including the addition of 1% collagen. This makes the gel both antimicrobial and useful for collagen, which has a supportive effect on accelerating wound healing.

Example C:

Prepare all ingredients as in Example A, but including addition of 1-5% aloe vera (powder or solution). It provides additional wound healing properties.

Example D:

Prepare all ingredients as in Example A by adding 1-10% maltodextrin. This forms a gel composition that promotes wound granulation.

Summarization of silver hydrogel results

It was possible to make carbopol-based gels using the silver colloidal solutions of the invention at 22 ppm and 32 ppm. The gels prepared in this way have many advantages over all parts of the solution based on their ability to remain in place while maintaining the properties of the original silver solution. The origin of the drug amorphous hydrogel offers positive benefits for accelerating wet wound healing and also limiting the severity of burn wound healing by limiting thermal shock. In addition, the active ingredient, the colloidal silver solution, was tested in a cell line at the initial stage and found to be non-toxic.

106

Comprehensive physico-chemical evaluation of the gel was performed with various batches of arranged techniques and parts of a series of described techniques were prepared to standardize and control the product and processes during manufacture.

Microbiological studies have been conducted essentially and show that the gel retains its bactericidal origin. Migration studies of silver have been simulated and have convincingly shown that the gel can deliver silver to the wound over time. Composition also prevents microbial migration from outside to inside and vice versa.

These studies show that the hypothetical evaluation of silver gel is based on a publication (Journal of Wound Care Vol 12, No 8 SEPT 2003) where alternative silver-based dressings have been evaluated for the following characteristics:

1. Inhibition antimicrobial domain;

2. Microbial challenge test;

Microbial transmission test; and

The silver content of the material being tested.

In the first test, the silver hydrogel would be placed in group B, and in all remaining tests, the silver hydrogel would be placed in group A, giving it a total of 25 points, equivalent to Calgitrol Ag and Acticot, which have the highest marks for commercial products.

The antibacterial and antiviral properties of the colloidal silver solution provided several significant uses for the silver hydrogel, except for wound dressings, as described above, the hydrogel is an ideal bacterial hand cleaner. In addition, the non-irritating feature of silver colloid and hydrogel has made the combination an ideal personal lubricant for sex life, with or without condoms or diaphragms, where the combination traps bacteria, fungus (marked efficacy in Candida albicans) and dangerous viruses such as HIV, and disinfect reusable bulkheads such as diaphragms. Because the hydrogel needs a little oil, it does not damage condoms or diaphragms like other personal lubricants.

HYDROGEL HAND CLEANER (Note: Hydrogel and SILGEL are the same products of the invention and are used interchangeably)

Clean hands were considered as a single key factor in preventing the spread of dangerous germs and antibiotic resistance to health

107 areas of care. Accordingly, it was decided to test the effectiveness of the hydrogel known as SILGEL as a hand hygiene product based on the MMWR, October 25, 2002A / OL 51 no. RR-16.

The following standard operating procedure was used:

Materials Required (SOP):

Serratia marcescens standard suspension (10 ⁸ cfu), tap water, sterile rubber glove, sterile sample solution, sterile triple soy agar, sterile pipettes, sterile test tubes.

Method:

1. 5 ml of Serratia marcescens suspension was applied to the hands and applied to the surface of the hands.

2. Spray 3 ml of the test substance on the hands and 1/3 of the front of the hands.

3. Apply 2 ml of tap water to the hands and lather (see Fig. 1).

4. Rinse hands and their tips with tap water for 30 seconds at RT.

5. Repeat steps 2 through 4.

6. After 1, 3, 7 and 10 washes, the sterile rubber gloves used for the test are put on your right and left hands.

7. 75 ml of sterile sample solution are added to gloves.

8. The entire surface of the hands is massaged for one minute.

9. Samples are prepared for aseptic quantitative analysis using a scalable sterile triple soy agar.

10. A plate spray technique is used using the initial, 10 ' ¹ , 10' ² and 10 ' ³ as dilutions.

The plates are incubated at 37 ° C for 24 hours.

MATERIALS AND BODIES

The procedures with the above SOP were used.

Media used: Sterile triple soy agar.

Cultures used: 16 hours. old Serratia marcescens culture (density about 10 ⁸ CFU / ml)

Incubation temperature: 37 ° C

108

Incubation time: 24 hours.

Rated products: Silgel 22 and 32 ppm, spitaderm, clean liquid sterile

Left hand

The results are shown in Tables 15a-15e, 16a-16e

Table 15a Spitaderm (Annex II)

Number of washes Dilution CFU / ml Original 10 · ' 10> 10- ^J 1 wash 10th zero zero zero 3 washing zero zero zero zero 5 washing zero zero zero zero 10 washing zero zero zero zero

Table 15b Herringbone 32 ppm

Number of washes Dilution CFU / ml The original 10- * 10> 10 ' ³ 1 wash zero zero zero zero 3 washing zero zero zero zero 7 washing zero zero zero zero 10 washing zero zero zero zero

Table 15c Herringbone 32 ppm

Number of washes Dilution CFU / ml The original 10- ¹ 10- ² 1CT ³ 1 wash zero zero zero zero 3 washing zero zero zero zero 7 washing zero zero zero zero 10 washing zero zero zero zero

Table 15d Sterile (Annex II)

Number of washes Dilution CFUIml Original 10- ¹ 10 * 10 ' ³ 1 wash 30th 30th zero zero 3 washing zero 10th zero zero 7 washing zero zero zero zero 10 washing zero zero zero zero

Table 15e Liquid cleaner (Annex II)

Number of washes Dilution CFUIml Original 10- ¹ 10 '* 10 ' ^a 1 wash zero zero zero zero 3 washing zero zero zero zero 7 washing zero zero zero zero 10 washing zero zero zero zero

109

Right hand

Table 16a Spitaderm (Annex II)

Number of washes Dilution CFU / ml The original 10 ' 10> 10- ³ 1 wash zero zero zero zero 3 washing zero zero zero zero 5 washing zero zero zero zero 10 washing zero zero zero zero

Table 16b Herringbone 32 ppm

Number of washes Dilution CFU / ml The original 10- ' 10- ² 10 * 1 wash zero zero zero zero 3 washing zero zero zero zero 7 washing zero zero zero zero 10 washing zero zero zero zero

Table 16c Herringbone 32 ppm

Number of washes Dilution CFU / ml The original 10- ' 10- ² 10 * 1 wash zero zero zero zero 3 washing zero zero zero zero 7 washing zero zero zero zero 10 washing zero zero zero zero

Table 16d Sterile (Annex II)

Number of washes Dilution CFUIml Original 10- ¹ 10 * 10 * 1 wash zero zero zero zero 3 washing zero zero zero zero 7 washing zero zero zero zero 10 washing zero zero zero zero

Table 16e Liquid cleaner (Annex II)

Number of washes Dilution CFUIml Original 10- ¹ 10 * 10 * 1 wash zero zero zero zero 3 washing zero zero zero zero 7 washing zero zero zero zero 10 washing zero zero zero zero

110

Conclusion: Silgel 22 ppm and Silgel 22 ppm matched the efficacy of TMF (pilot study monograph) as a hygiene product that defines efficacy as a reduction of the 2-log determinant for each arm after 1 use and the 3-log determinant for each arm. 5 minutes after the 10th use reduction, criterion. In addition, SILGEL was more effective as a hand cleaner than Sterillium and Spitader. Finally, SILGEL, rubbed as a hand cleaner, is well tolerated because it eliminates the need for a washbasin and does not dry or irritate the user's hands, but tends to moisturize the surface to be treated.

HYDROGEL AS A IMAGE MATERIAL

introduction

Hydrogel dressings can be used as primary dressings (amorphous and impregnated gauze) or as primary or secondary dressings (canvas) for the complete or partial control of wound dehydration, deep wounds (amorphous and impregnated gauze), wounds with necrosis, or hopeless, minor burns and radiation skin damage.

Today, almost all hydrogels in the market do not contain an antimicrobial agent. This is because antibiotics and antiseptics are potentially cytotoxic and often arrest the skin from contracting.

Since the silver / water solution of the invention is not cytotoxic, it was decided to make the hydrogel using the silver particles activated by the invention.

Recently, a specially formulated hydrogel has been introduced to deal with radiation-induced dermatitis. These dressings have a high specific heat to provide a cooling effect and at least three times absorb water, serum and blood.

Benefits • It is soothing and relieves pain;

• Rehydrates the wound base;

• Improves autolytic removal of nonviable tissue.

• Fill wound volume (amorphous and impregnated gauze);

• Provides minimal moderate absorption;

• Easy application from wound removal;

• Can be used when the infection is accelerating;

• Enables visualization of the wound area;

Disadvantages • Not often recommended for severe exudate wounds;

• Occasionally, additional revision is needed;

• Dehydrated slightly if not burnt;

• Sometimes difficult to monitor;

• Can sometimes cause soaking;

Procedure

The hydrogel of the present invention was prepared in the form of a bandage as an intended wound dressing. The shape of the hydrogel bandage can be variable, depending on the SILDERM.

Results

SILDERM - Loses moisture

Objective • Define the SILDERM moisture loss potential.

Procedure:

Required equipment:

• Analytical balance

Required Materials:

• Plastic tray

Method:

• Adjusts the weight of the empty pallet;

• SILDERM bandage is placed on a pallet;

• Adjusts the weight of the pallet + SILDERM bandage.

• The note is read as t = 0 hours.

• recording is made every 1 hour.

• The recording is made overnight;

• Then plot the time with moisture loss;

• Determines the percentage moisture loss.

112

Results: See Table 17 below and Figure 34.

Table: SILDERM moisture loss potential

Time (or.) Mass 0 68g 1 64g 2 60 g 3 56g 4 51g 5 48g 22nd 40g

Conclusion: One conclusion is that SILDREM bandage can lose 30% moisture by weight.

SILDERM - MOISTURE ABSORPTION Objective • Define the ability of SILDERM to absorb moisture.

Procedure:

Required equipment:

• Analytical Balance Materials Required:

• Censorship Method:

• Determine the weight of the SILDERM bandage in grams;

• The note is read as t = 0 hours.

• The manure is filled with water;

• The SILDERM bandage is placed in the beaker, fully immersed;

• At hourly intervals, the bandage is removed for draining and its weight is determined;

• the recording is made overnight;

• Then plot the time with moisture loss;

113 • Determines the percentage moisture loss.

Results: See Table 18 below and Figure 35.

table

Time (or.) Mass 0 45g 1 48g 2 51g 3 53 g 4 54g 5 56g 6th 57g 22nd 68g

Conclusion: The dehydrated SILDERM bandage can absorb 52% of its mass moisture.

SILDERM - SILVER RELEASE

Objective • To define the long-term release of silver nanoparticles from SILDERM.

Procedure:

• Hydrogel bandages are normally applied to the wound for 48-72 hours. In this situation, it may be desirable to determine the antimicrobial activity of the dressing over a period of time in relation to the release of silver.

Required equipment:

• Incubator, natural cover.

Required Materials:

• Sterile nutrient agar plates, sterile cotton swabs, micropipette (volume 100 μΙ - 1000 μΙ) for 16 h. culture of Pseudomonas aeruginosa (wild type).

Method:

• SILDERM is cut into 4 cm x 3 cm pieces;

• SILDERM is placed on a nutrient agar plate with Ps. Aeruginosa (wild type) smear;

• Incubate at 37 ° C for approximately 18 hours.

• The area of inhibition is checked vertically and horizontally;

• The same piece of SILDERM is then placed on a nutrient agar plate with a new Ps. Aeruginosa (wild type) smear;

• Incubate as above.

This procedure is repeated for a minimum of 7 days.

Results: At the time of entry, SILDERM showed inhibitory activity for 3 translocations as shown in Table 17 below.

Table: SILDERM Sample Test

Transfer No Vertical inhibition Horizontal inhibition 1 transfer 52mm 35 mm 2 transposition 53 mm 35 mm 3 transposition 51mm 34 mm

Conclusion: SILDERM Hydrogel can exert its antimicrobial activity in 3 fresh inoculum assays every 24 hours. absorb 52% of its mass moisture. Further testing is making progress.

For the above embodiments, the average composition is as follows:

Food Agar:

Peptone -10.0 g

Sodium chloride - 5.0 g

Meat extract - 3.0 g

Distilled water - 900 ml

Agar - 2.5 g

PH-7.2 ± 0.2

Further it is possible to enhance the compositions of SILDERM with the following:

Collagen

Collagen, the most abundant protein in the body, is fibrous and insoluble and is derived from fibroplasts. Its fibers are found in connective tissues including

115 skin, bones, ligaments and cartilage. In wound healing, collagen promotes the removal and healing of newly formed collagen fibers and granular tissue in the wound. It also stimulates new tissue formation and removal of non-viable tissue from the wound, creating an environment that promotes healing.

Maltodextrin

Maltodextrin is a wound healing promoter that promotes healing by activation and attraction of macrophages, thereby reducing infection and increasing granulation.

Platelet-derived Growth Factors (PDGF)

PDGF promotes chemotherapy recovery and proliferation of cells involved in wound healing and accelerates granulation tissue formation. It is mainly used for the treatment of lower grade diabetic neuropathic ulcers.

Disodium ECTA as an additive

Disodium EDTA is known to enhance the antibacterial activity of various compounds, both natural and synthetic, by a mechanism believed to result in bacterial cell penetration through the wall, thus improving the delivery of bacterial compounds.

The metal chelator and promoter of bacterial outer membrane permeability, ethylenediammonium tetraacetic acid (EDTA), has shown increased activity of various antimicrobial agents against Pseudomonas aeruginosa. Addition of a subinhibitory concentration of EDTA significantly reduced the MICs of cefprozil against E. coli and Serratia marcescens.

It was found that imipenem, ceftazidinem and cefepime with 150 mcg EDTA could increase the diameter of said inhibition zone of P. aeruginosa. The study found that ethylenediaminetetraacetic acid (EDTA) acts on sensitive sites in P. aeruginosa. When used in combination with AgNO ₃ , EDTA significantly enhances the antibacterial activity of AgNO ₃ , such that Klesbiella pneumoniae and Staphylococcus aureus strains are resistant to 70 micrograms / ml Ag NO3, where a sensitivity of 10 micrograms / ml has been observed.

116

The specific composition and assay tests were designed to determine the suitability of the silver / water compositions of the present invention for wound healing with disodium EDTA. More specifically, disodium EDTA was manufactured in West Coast Laboratories, Mumbai, India. Disodium EDTA is also known as Na ₂ EDTA (disodium ethylenediaminetetraacetic acid) of the formula: (CH ₂ N (CH ₂ COOH) CH ₂ COONa) _{2 2} H ₂ O and has a molecular weight of 372.24.

Materials used for this study were: Food Agar (HiMedia) 1000 ml; B. No. 1G115 exp. Aug 2006; animal gastrointestinal tissue 50.0 g; yeast extract 1.50 g; beef extract 1.50 g; sodium chloride 5.00 g; Type I agar-agar 25 g; pH 7.4 +/- 0.2.

Microbial strains of ppm silver / water composition one, 22 ppm silver / water composition one and 32 ppm silver / water composition as well as 22 ppm silver / water composition were added Na ₂ EDTA and each was tested against a group of microorganisms including:

Escherichia coli (multiple drug-resistant strain) from a stool sample;

Saliva of Pseudomonas aeruginosa (multiple drug-resistant strain); and

Methicillin-resistant Staphylococcus aureus multiple drug-resistant strain) from a stool specimen from the lumbar region.

The above strains were obtained from P.D. Hindu Hospitals (MUMBAI, India).

Shigella flexneri (laboratory strain);

Salmonella typhi (Lab strain)

Bacterial strains were grown for 24 h. 37 ° C on nutrient agar (pH 7.4).

ppm and 22 ppm dilutions were added to Na ₂ EDTA prepared in sterile distilled water. Each microorganism was suspended in sterile saline and diluted to 10 ⁶ colony forming units (cfu / ml). They were applied to a nutrient agar surface (pH 7.4) using sterile cotton swabs. Circles of 10 mm diameter were squeezed out of agar and 0.1 mL of appropriate dilutions were added. After 24 or. At 37 ° C incubation, all plates were assayed at various growth inhibition sites and diameters in mm were measured using a zone reader (Hi Media). The results are shown in Table 20.

117

Results and Comments Table Silver / Water + Na ₂ EDTA

The system E. coli MRSA C. albicans Silver / water 32 ppm-Ctrl 21mm 24mm 27mm Silver / water 32 ppm + 0.5% Na ₂ EDTA 22mm 29 mm 40 mm Silver / water 22 ppm + 20 mm 23mm 29 mm Silver / water 22 ppm + 0.5% Na ₂ EDTA 22mm 31 mm > 40 mm

Disodium EDTA 0.5 ppm greatly enhances the potential of the silver / water compositions of the present invention at both concentration levels of 22 and 32 ppm.

Silver EDTA as a stand alone antibacterial unit

Specific formulations and test samples have been prepared to determine whether silver chelates such as silver EDTA (or AgEDTA) have antibacterial properties. Specifically, commercially available silver EDTA formulations were obtained from AKZO Nobel and Alpha Chemicals.

Required equipment:

• Incubator, natural cover;

Required Materials:

• Sterile food agar plates, sterile cotton swabs, micropipette (100-1000 μ000):

or. culture of the following strains (approximately 10 ⁸ CFU / ml)

118

Escherichia coli (wild type) and Escherichia coli (MDR), Pseudomonas aeruginosa (wild type) and Pseudomonas aeruginosa (MDR), Staphylococcus aureus ATCC 6538P and methicillin-resistant Staphylococcus aureus.

Method:

• 16 h of the test organism. life smear was applied to a sterile plate of nutrient agar.

• Allow the plates to absorb for 15 minutes. • After 15 minutes, aseptically press the spheres into the agar surface using a 10 mm corkscrew.

• Distribute 100 μΙ of the appropriate sample into balls. Leave for 15 minutes for diffusion.

• Incubate plates for 24 hours. 37 ° C and results are being investigated.

• Measure the attenuation area in mm using a HiMedia area reader. Results: See Table 21 below and Figures 36 and 37.

Table: Comparative evaluation of silver chelates

The organism Concentration Area of inhibition AKZO ALPHA E. coli (wild type) 28 ppm 20 mm 22mm 57 ppm 22mm 24mm 114 ppm 22mm 25 mm E. coli (MDR) 28 ppm 20 mm 19mm 57 ppm 21mm 21mm 114 ppm 23mm 22mm Pseudomonas aeruginosa (wild type) 28 ppm 21mm 20 mm 57 ppm 27mm 24mm 114 ppm 28mm 27mm

119

Pseudomonas aeruginosa (MDR) 28 ppm 15 mm 17 mm 57 ppm 21mm 20 mm 114 ppm 25 mm 22mm S. aureus (wild type) 28 ppm 16 mm 15 mm 57 ppm 19mm 18mm 114 ppm 22mm 21mm MRSA 28 ppm 19mm 20 mm 57 ppm 21mm 22mm 114 ppm 26 mm 24mm

Conclusion: Silver chelates such as silver EDTA have antibacterial properties.

THERAPEUTIC COMBINATION OF ANTIBIOTICS

Once discovered, antibiotics were widely promoted as a miracle cure and literally they were. Infections that were fatal before the onset of the century were defeated at this age. But medicine has gone almost full circle. Over-the-counter or antibiotic misuse - these misuses have led to the development of bacterial resistant strains and once again to the health and life of bacterial strains.

Some of the other factors contributing to the development of bacterial resistance are the use of antibiotics for agricultural purposes and nutritional supplements in agriculture (eg poultry, beef, pork, etc.). By definition, antibiotics are very common in US agriculture and very common in other foreign countries. Antibiotic therapy in agriculture often begins with the bacterial species being sent to the laboratory. Avian influenza (such as H5N1 or ΉΡΑΓ) has become highly resistant to antibiotics due to the reluctance of Asian poultry farmers to use antibiotics. Patients are also facilitated in obtaining antibiotics. Incorrect dosing and incomplete treatment time also influence the development of drug resistance of the strain. Clinical rooting of the resistance problem is very important.

120

Antibiotic resistance of a pathogenic bacterium has several effects in the treatment of infectious diseases. Many drugs, such as penicillin, which were considered miracle cures, had a great potential for effective control in the detection, adaptation, and drastic reduction of their effectiveness when first used.

Today, antibiotic resistance is a global problem. Some common and highly pathogenic bacteria, such as Staphylococcus aureus, specifically found in hospitals, are known to be resistant to everything except vantomycin, and it appears that vantomycin will soon become resistant as well. MRSA (methicillin-resistant Staphylococcus aureus) and VRE (vancomycin-resistant Enterococci) are the causative agents of several nosocomial infections and, when found in hospitals, often close and even eliminate hospital wards.

With this problem, an alternative need arose either to use new antibiotics in place of old or to make more effective use of existing antibiotics. Also, the growing threat of drug-resistant bacteria is a great reason to apply the silver / water compositions of the present invention.

One of several ways to combat bacterial resistance to antibiotics is to use combination therapy, where two or more antibiotics with different types of effects are used. There are various in vitro methods for measuring the synergistic effects of antibiotic combinations, but the results can be contradictory when different tests are used and are not a general rule for developing resistance.

OBJECTIVES AND OBJECTIVES

This study was conducted with the following goals and objectives:

1. Identify a multiple drug resistance module in clinical cases.

2. To determine the sensitivity of clinical cases to the silver / water solutions of the present invention.

3. Determine the antibiotic combination (synergistically) by a disk approximation test.

4. Determine the minimum inhibitory concentration of antibiotics and silver / water compositions of the present invention.

5. To investigate the synergistic action between the antibiotics and the silver / water compositions of the present invention by a chess test.

MATERIALS AND WAYS

121

Selection of clinical cases

Clinical cases of multiple drug resistance were selected from P.D.Hinduja Hospital and are as follows:

Cadell road, Mahim, Mumbai-400016, India.

• Escherichia coli (isolated from stool);

• Pseudomonas aeruginosa (isolated from saliva);

• Methicillin-resistant Staphylococcus aureus (MRSA - isolated from pus in the waist).

Medium, solutions and antibiotic disks:

Medium:

• Food broth.

• Food Agar.

• Muller and Hinton agar.

Solutions:

• Antibiotic solutions;

• Silver / water solution (22 ppm).

Compositions and solutions are used for various experiments and are listed in Table 26 (here below).

A realistic antibiotic disk of appropriate concentration was used. The amount of disk for each antibiotic is listed in Table 27 (Here below).

Preparation of inoculum:

A piece of pure cultured culture was inoculated into nutrient broth and incubated overnight at 37 ° C. 500 mL of overnight culture was transferred to 5 mL of fresh broth and incubated for 4-6 hours. about 37 ° C. The culture density is adjusted to about 10 ⁵ -10 ⁶ cfu / ml.

Antibiotic Susceptibility Test - Kirby Bauer Method:

In this assay, antibiotic-impregnated disks are placed on an agar plate pre-incubated with the bacterial suspension. Antobiotics diffuse from the surrounding medium. These are algorithmic reductions in antibiotic concentration as distance

122 from disk increases. The transparent area around the disc indicates the body's sensitivity to the antibiotic. Transparent areas are measured in mm and compared to a standard NCCLS chart.

Method:

1. Sterile cotton swabs were immersed in the aforementioned inoculum broth tubes and used for spraying onto M.H. surface of agar plates to obtain confluent growth.

2. After the medium has been absorbed by the inoculum, antibiotic discs are placed on sprayed surfaces with sterile surgical forceps.

3. Incubate plates at approximately 37 ° C for 24 hours.

4. Transparency around the disc indicates the sensitivity of the organism. The zone is measured in diameters and compared with the standard diagrams in the NCCLS (see Table 27) (Koneman 5th ed. 1997).

Determination of sensitivity of isolates to 10 ppm agar by diffusion:

This was determined by a bead test in which each portion of the isolate is seeded on an agar medium and added 10 ppm silver / water solution to the beads embedded in the solid inoculated medium. The size of the damping zone is then set.

Method:

1. To a 20 ml slice of Muller and Hinton agar, 0.5 ml of inoculum was added to a Petri dish and left to react with salt.

2. The beads were embedded in a layer of agar.

3. Different concentrations of silver / water were then added to each ball.

4. Plates were incubated at 37 ° C for 24 hours.

5. The size of the damping zone is then set.

Determination of antibiotic combination by disk diffusion.

This is a simple qualitative test to determine the interaction between a clinical isolate and a combination of antibiotics. In this assay, antibiotic disks are placed on an agar plate inoculated with the Kirby-Bauer technique. These discs can be separated by a distance equal to or slightly above the average attenuation caused

123 each disk individually, diameters. This resulting form of inhibition zone determines the type of interaction between the clinical isolate and the antibiotic combination.

Method:

1. Sterile cotton swabs were immersed in the aforementioned inoculum broth tubes and used for spraying onto M.H. surface of agar plates to obtain confluent growth.

2. After absorption of the medium by the inoculum, the antibiotic discs (test combination) are placed on sprayed surfaces with sterile surgical forceps at a distance equal to or slightly greater than the sum of the diameters of the inhibition induced by each disc.

3. Incubate plates at approximately 37 ° C for 24 hours.

4. The shape of the inhibition zones indicates interactions, i. y. synergistic, antagonistic or indifferent.

figure is a diagram that shows possible interactions between disks in a bacterial synergy diffusion test.

Specifically, Part A shows additive or indifferent effects; each antibiotic creates an inhibition zone that is inactive adjacent; Part B shows antagonistic effects where the inhibition zones for each antibiotic are reduced with the existing other antibiotic. Part C shows the manifestations of two possible synergistic interactions. The left area of increased inhibition occurs when two antibiotics meet. To the right, there is no such antibiotic inhibition, but bacterial growth is inhibited, where two antibiotics co-exist

Determination of Minimum Inhibition Concentrations of Antimicrobial Agents. This is a test for the sensitivity of a macro diluted broth. Serial dilutions of the antimicrobial agent were prepared in broth to which standardized bacterial suspensions were added. At the end of the incubation period, growth was visualized in the tubes. The lowest concentration of antimicrobial agent that inhibits visible growth is taken as MIC.

Antibiotics used:

Amikacin: Mycacio inj. (250 mg) Aristo Labs, Mumbai Indja.

124

Example No. 02D054, mfd apr. 2004

Cefoperazone: Cifran (200mg / ml) Ranbaxy labs, Jaipur, India.

Example No. 9042601 ,, mfd 2004.

Method:

1. The amount of antimicrobial agent is gradually diluted in an appropriate amount.

2. Tubes lacking antimicrobial agent show growth control.

3. Each tube was inoculated with a standard bacterial suspension and incubated at about 37 ° C for 24 hours.

4. Tube turbidity was visually observed at the end of the incubation period. The turbidity indicates that bacterial growth was not inhibited by the concentration of the antimicrobial agent present in the medium.

5. MIC is the lowest concentration of antimicrobial agent that inhibits visible growth.

Investigation of synergistic activity by chess test.

The chess technique is used when multiple antibiotics and / or multiple dilutions are tested. Serial dilutions of two types were selected so as to include between one sixteenth and at least double the MIC concentration. Drug A is gradually diluted through the ordinate, while B is gradually diluted through the abscissa. The resulting chess arrangement yields each combination of two antibiotics from the tube having the highest concentration of each of the opposite angles.

Minutes:

125

2x

MIC

MIC

1: 2

MIC

1: 4

MIC

1: 8

MIC

1:16

MIC

+ ve C

1:16

MIC

1: 8

MIC

1: 4

MIC

1: 2

MIC

MIC x MIC

Drug B pg / ml Drug A pg / ml

Performing drug dilutions in a chess assay.

The first row of tubes and column with only one drug was submitted for confirmation of individual MIC values in the test isolates.

One tube without antibiotic is a positive control.

1. In a final volume of 5 ml of each tube, antimicrobial agents diluted in broth are added from suitable strong broth solutions.

2. Add 0.1 ml of culture suspension.

3. Incubate at approximately 37 ° C for 24 hours.

4. The results shall be presented by plotting the isobolograms obtained by combining the dots representing all combinations with the same effect, including the effective concentrations of one of the antibiotics used.

Calculations:

Elion et al (1954) described a method for calculating MIC results obtained by the fractional inhibition concentration (FIC) index obtained as the sum of the FIC values of two drug combinations.

126

FIC index = A drug FIC + B drug FIC.

FIC of drug A = MIC of drug A in combination with drug B

A instinct for MIC

An index lower than 0.5 clearly indicates synergism An index greater than 2.0 clearly demonstrates antagonism.

(Koneman 5th ed. 1997).

figure shows chess titration of antimicrobial synergy.

Each lot represents a tube. Elevated concentrations of antibiotic A are distributed across the horizontal axis and antibiotic B is distributed through the vertical axis. The dashed sites show bacterial growth. In Plate A, antibiotics show an additive effect; The right isobologram is a straight line. Plate B shows synergism where the isobologram is a concave curve. Plate C shows antagonistic results with a convex curve.

Determination of the antibiotic susceptibility module by Kirby-Bauer.

table; the antobiograms (mm of area) of the isolates used in the study.

Antibiotics Area measurements (mm) Organisms Amikacin 20th 9th 22nd Ciprofloxacin - - 20th Kanamycin 14th - - Gentamicin 19th - - Tetracycline - - 27th Nalidixic acid - - - Cefoperazone - 14th 23rd Ceflazidime 10th 16th - Chloramphenicol - - -

127

Key: No inhibition.

Determination of sensitivity of clinical isolates to ASAP-agar by diffusion.

ASAP concentration PPm Area measurements (mm) Organisms E. coli Pseudomonas MRSA 32 16th 16th 13th 16th 15th 14th 11th 8th 11th 11th - 4 - - 2 - - -

Key: No inhibition

See. 27 figure photos.

Determination of antibiotic combination by disk approximation test

Areas of inhibition of tested synergistic or complementary effects of the various antibiotic combinations on isolates were obtained, sensitive to potential synergism only in the presence of MRSA using the combination of amikacin with cefoperazone and amikacin with tetracycline (Figure 28). No inhibition zones were found that affected the synergistic combination in the case of the other two isolates, i.e., E. coli and Pseudomonas (see Figures 29 and 30).

Determination of the minimum inhibitory concentration of antibiotics.

MICs for antibiotics were found to show zones of inhibition affecting potential synergies.

table

MIC of Amikacin

Strong broth: 125 mcg / ml

Solvent: food broth

Kutura: MRSA

Keys:

+ growth

- no growth

The MIC of amikacin MRSA was set at 0.8 mcg / ml.

Cefoperazone MIC Strong Broth: 100 mcg / ml

128

Solvent: food broth

Cutthroat: MRSA

Tube No. Concentration in mcg / ml Growth 1 0.2 + 2 0.4 + 3 0.6 + 4 0.8 - 5 1 - 6th 2 - 7th 3 - 8th 4 - 9th 5 - 10th + ve + 11th -ve -

Key: + Growth

- no growth

The MIC MRSA of cefoperazone was set at 10 mcg / ml.

Silver / Water MIC

Strong broth 20 ppm silver water solution Thinner: edible broth Culture: MRSA

Pipe no. Concentration in ppm Growth 1 5 + 2 10th - 3 15th - 4 20th - 5 25th - 6th 30th - 7th 35 - 8th 40 -

129

9th 45 - 10th 50 - 11th + ve + 12th -ve -

Table a

Pipe no. Concentration in ppm Growth 1 1 + 2 2 + 3 3 + 4 4 + 5 5 + 6th 6th + 7th 7th + 8th 8th - 9th 9th - 10th 10th - 11th + ve + 12th - ve -

Key: + Growth

- no growth

The silver / water MIC MRSA was set at 8 mcg / ml.

Silver / Water MIC

Strong broth 20 ppm

Solvent: food broth

Culture: Table of E. coli

Pipe no. Concentration in ppm Growth 1 1 + 2 2 + 3 3 - 4 4 -

130

5 5 - 6th 6th - 7th 7th - 8th 8th - 9th 9th - 10th + ve + 11th - ve -

Key: + Growth

- no growth

The silver / water MIC in E. coli was set at 3 ppm.

Silver / Water MIC

Strong broth: 20 ppm silver / water

Solvent: food broth

Culture: Pseudomonas Table

Pipe no. Concentration in ppm Growth 1 1 + 2 2 + 3 3 - 4 4 - 5 5 - 6th 6th - 7th 7th - 8th 8th - 9th 9th - 10th + ve + 11th -ve -

Key: + Growth

- no growth

The silver / water MIC for Pseudomonas was set at 3 ppm.

A study of the synergistic effects of chess research

131

I. Combination of Amikacin and Silver / Water.

MIC of amikacin - 0.8 mcg / ml.

Silver / Water MIC - 8 ppm.

Culture: MRSA.

1.6

0.8

0.4

0.2

0.1

0.05

- - - - - - - - - - - - - - + - - - - - - ++ - - - - - - ++ ++ - - - - - ++ ++ - - - - - + ve C ++ ++ ++ + - -

Amikacin 0 0.5 1 2 4 8 12 (mcg / ml) Silver / water (ppm)

Key: + Growth

- no growth

The synergistic concentration of MRSA was found to be 0.05 msg / ml amikacin and 1 ppm silver / water of the present invention.

Calculated FIC index:

Amikacin FIC = MIC of Amikacin in combination

MIC of single Amikacin = 0.05 / 0.8 = 0.0625.

ASAP FIC = Silver / Water MIC in Combination Single Silver / Water MIC = 1/8 = 0.125.

FIC Index = Amikacin FIC + Silver / Water FIC = 0.0625 + 0.125

132 = 0.1875.

The FIC index is a synergistic trait between amikacin and silver / water.

II. A combination of cefoperazone and silver / water. Cefoperazone Mic -10 mcg / ml. Silver / Water MIC - 8 ppm.

Culture: MRSA.

2.5

1.25

0.625

- - - - - - - - - - - - - - + - - - - - - ++ - - - - - - ++ - - - - - - ++ ⁺⁺ - - - - - + ve C ++ ++ ++ + - -

12th

Cefoperazone 0 (mcg / ml)

0.5 1 2 4

Silver / water (ppm)

Key: + Growth

- no growth

The synergistic concentration of MRSA was determined to be 0.625 msg / ml for cefoperazone and 1 ppm for silver / water of the present invention.

Calculated FIC index:

Cefoperazone FIC = Cefoperazone MIC in combination One Cefoperazone MIC = 0.625 / 10 = 0.0625.

ASAP FIC = Silver / Water MIC in Combination Single Silver / Water MIC = 1/8 = 0.125.

133

FIC index = cefoperazone FIC + silver / water FIC = 0.0625 + 0.125 = 0.1875.

The FIC index is a synergistic feature between cefoperazone and silver / water.

III. Combination of cefoperazone and amikacin. Cefoperazone Mic -10 mcg / ml. Amikacin MIC - 8 ppm.

Culture: MRSA.

2.5

1.25

0.625

- - - - - - - - - - - - - - + - - - - - - ++ - - - - - ++ ++ ++ ++ - - - ++ ++ ++ ++ + - - + veC ++ ++ ++ ++ - -

Cefoperazone 0 (mcg / ml)

0.05 0.1 0.2 0.4 0.8 1.6

Amikacin (mcg / ml)

Key: + Growth

- no growth

Additional concentrations of cefoperazone were found at 0.25 and amikacin at 0.4.

Calculated FIC index:

Cefoperazone FIC = Cefoperazone MIC in combination

MIC of Cefoperazone per unit = 1.25 / 10 = 0.125.

Amikacin FIC = MIC of amikacin in combination Silver / water MIC = 0.4 / 0.8 = 0.5.

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FIC index = cefoperazone FIC + amikacin FIC = 0.125 + 0.5 = 0.625.

The FIC index is a synergistic feature between cefoperazone and silver / water. DISCUSSION

In this example of three clinical cases selected from PDHinduja Hospital, Mumbai, India, Gram-negative isolates showed resistance to older antibiotics such as ampicillin, tetracycline, kanamycin and older quinolones such as nalidixic acid, as well as to third-generation cephalosporins. and cefpperazone. The clinical isolates of Pseudomonas used in the study were also resistant to current ciprofloxacin and the semi-synthetic aminoglycoside and amikacin. MRSA Gram-positive isolate was also resistant to other antibiotics and also to third-generation cephalosporins like ceftazimide.

Their sensitivity to the silver / water compositions of the present invention showed that Gram-negative isolates are readily sensitive to about 3 ppm silver / water solutions and MRSA isolate was found to be inhibited by 8 ppm silver / water solutions as determined by agar diffusion and macrosoluble broth.

Interaction between the two antibiotics in combination with isolates was determined by disk diffusion, which revealed synergistic results between cefopoerazone and amigacin against MRSA. A chess test was conducted to confirm this. Gram-negative isolates showed no additional synergies between antibiotics by diffusion.

A chess test was performed and the FIC index of the two antibiotics was set at 0.625, thus determining the additional synergy or absence of amikacin-cefoperazone combination.

A chess test was conducted to investigate the combination of silver / water solutions with amikacin as well as cefpoperazone. The results showed that with the silver / water compositions of the invention, the effective concentration of the antibiotic is reduced by about four points. The FIC index for these combinations was determined

135

0.1875 in each case, to determine the synergy of silver / water with amikacin and silver / water with cefoperazone.

The results showed that silver / water compositions play an important role in combination with antibiotic therapy, especially against multiple drug resistance strains.

table

1. Food Broth Peptone -10.0 g;

Sodium chloride - 5.0 g;

Meat extract - 3.0 g;

Dextrose - 5.0 g;

Phenol red (indicator) - 0.001%;

Distilled water - 900 ml.

2. Food Agar:

Peptone -10.0 g;

Sodium chloride - 5.0 g;

Meat extract - 3.0 g;

Distilled water - 900 ml.

Agar - 2.0%;

pH 7.2.

3. Muller and Hint Agar:

Casein hydrolyzate 29.0 g; Beef starch -10.0 g;

Potato starch - 2.5 g;

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Agar -1.2%;

Distilled water -1000 ml.

pH 7.6.

table

Interpretation zone diameters (NCCLS document, 1988)

Antibiotics Discs Diameter of zone mm Kone. (meg) Resistance Intermediate Sensitive Amikacin (AK) 30th <14 15-16 2:17 Ciprofloxacin (RC) 5 <15 16-20 > 21 Kanamycin (KA) 30th <13 14-17 > 18 Gentamicin (GM) 10th <12 13-14 > 15 Tetracycline (TE) 30th <14 15-18 > 19 Nalidixic acid (NA) 30th <14 14-18 > 19 Cefoperazone (CP) 75 <15 16-20 > 21 Ceftazidime (FG) 30th <14 15-17 > 18 Chloramphenicol (CH) 30th <12 13-17 > 18

COMBINATION OF GENTAMYCIN AND SILVER / ANDENE COMPOSITIONS AS LIGHT ANTISEPTIC POWDER

Wound antiseptic powder is a composition used to prevent or treat wound, burns or abscesses after incision surface bacterial infections.

137

Wound Powder is usually a broad spectrum antibiotic / antiseptic. The use of such a powder does not exclude concomitant, where appropriate, antibiotic therapy.

Wound treatment products found today in the market are based on povidone iodine. Povidone iodine is highly toxic to open wounds specifically contraindicated in diabetic wounds. In addition, iodine sublimates approximately every 6-8 hours. must be reapplied.

Another potential area of application is the veterinary field. Pet animals often get cut, scratched or injured by scratching caused by parasites, as well as other animals. Slight but wide-spectrum antimicrobials can help in this area.

It was decided to produce a wound antiseptic powder consisting of a slow release formulation containing gentamicin and silver nanoparticles excited by the present invention. A talc-based preparation containing about 200 ppm silver nanoparticles and about 100 ppm gentamicin is herein designated as SILDUST.

RESULTS

SILDUST - Sensitivity

Purpose: To determine the susceptibility of SILDUST and its components to microorganisms.

Procedure:

Required equipment:

Incubator, natural cover

Required Materials:

• Sterile food agar plates, sterile cotton swabs, micropipette (100-1000 μΙ), 16 hrs. culture of the following strains (approximately 10 ⁸ CFU / ml) Escherichia coli (MDR), Pseudomonas aeruginosa (MDR) and methicillin-resistant Staphylococcus aureus.

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The way;

• 0.1 ml cultures of the given test organism were sprayed using sterile cotton swabs onto a sterile nutrient agar plate. Hold outside for 15 minutes.

• After 15 minutes, the aseptic beads are pressed onto the agar surface using a 10 mm corkscrew.

• 100 μΙ SILDUST (200 ppm silver talc + 100 ppm gentamicin) is introduced into one ball.

• Introduce 100 μΙ SILDUST + 100 ppm gentamicin into another ball. 200 ppm silver talc + 100 μΙ distilled water is also introduced. Both prepared as controls.

• Incubate plates for 24 hours. 37 ° C and results are being investigated.

• Measure the damping area in mm using a HiMedia area scanner.

Results; see Table 28 below and Figure 38.

Table: SILDUST Sensitivity

Culture Suppression zone 100 ppm gentamicin 200 ppm ASAP work BRIDGE * Escherichia coli (MDR) 24mm 17 mm 26 mm

SILDUST * - 200 ppm ASAP pathway + 100 ppm gentamicin

Conclusion: Synergistic activity of SILDUST (comprising 200 ppm silver talc and 100 ppm gentamicin) is observed.

Key:

BRIDGE 1 - 200 ppm silver talc + 50 ppm gentamicin;

BRIDGE 2 - 200 ppm silver talc + 100 ppm gentamicin;

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SILDUST - antibacterial activity

Objective: To determine the time of killing of SILDUST against microorganisms.

Procedure:

Required equipment:

Incubator, natural cover.

Required Materials:

• Sterile phenol red dextrose broth, 16 hours. culture of the following strains (approximately 10 ⁸ CFU / ml) Escherichia coli (MDR), Pseudomonas aeruginosa (MDR) and methicillin-resistant Staphylococcus aureus.

Method:

• Prepare a 5 ml sample containing 2 g of SILDUST in a sterile test tube.

• Inoculate 0.1 ml of culture into the prepared solution. Stir vigorously.

• At intervals of 0, 5, 10 to 50 minutes, a sample inoculum is added to 5 ml of sterile phenol red dextrose broth for testing. Stir vigorously.

• Incubate for 24 hours. 37 ° C and results are being investigated.

• Observed growth.

• As a negative control, a slice of inoculated SILDUST was suspended in 5 ml of sterile phenol red dextrose broth and incubated at 37 ° C for approximately 24 hours.

• For positive control, the culture slice was inoculated into 5 ml of sterile phenol red dextrose broth and incubated at 37 ° C for approximately 24 hours. Results: See Tables 29, 30 and 31.

Table Escherichia coli (MDR)

Time intervals (min) 10 ppm gentamicin 200 ppm ASAP talk BRIDGE * Wokadin * 0 + + + - 5 + + + - 10th + + + -

140

15th + + + - 20th + + + - 25th + + + - 30th + + + - 25th + + + - 40 + + + - 45 + + + - 50 + + - - Positive control + + + + Negative control

SILDUST * - 200 ppm ASAP pathway + 100 ppm gentamicin

VOCADINE * - 200 ppm of applied iodine

Key: + - Growth

- - no growth

Conclusion: The combination shows synergistic effects. The tube containing the vocadine (described below, at the end of the test) was browned in seconds with the addition of powder to the medium due to the release of iodine. Although Vokadin shows faster killing, such high toxicity is undesirable in wound healing.

Table Pseudomonas aeruginosa (MDR)

Time intervals (min) 10 ppm gentomycin 200 ppm ASAP talk BRIDGE * Wokadin * 0 + + + - 5 + + + - 10th + + - - 15th - + - - 20th - + - - 25th - + - - 30th - - - -

141

25th - - - - 40 - - - - 45 - - - - 50 - - - - Positive control + + + + Negative control

SILDUST * - 200 ppm ASAP Path + 100 ppm gentamicin vocadine * - 200 ppm applied iodine.

Key: + - Growth

- - no growth

Conclusion: The combination shows a synergistic effect table MRSA

Time intervals (min) 10 ppm gentamicin 200 ppm ASAP talk BRIDGE * Wokadin * 0 + + + - 5 + + + - 10th + + + - 15th + + + - 20th + + + - 25th + + + - 30th + + + - 25th + + + - 40 + + - - 45 + + - - 50 - - - Positive control + + + + Negative control

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SILDUST * - 200 ppm ASAP pathway + 100 ppm gentamicin

VOCADINE * - 200 ppm of applied iodine.

Key: + - Growth

- - no growth

Conclusion: The combination shows synergistic effects.

SILDUST - Antibacterial activity

Purpose: To determine the susceptibility of the host bacteriophage to SILDUST.

Principle: A proper mortar was obtained to dispel misconceptions that SILDUST kills host cells.

Procedure:

Principle:

• T-bacteriophage and host Escherichia coli were used as a detection system. The silver concentration in SILDUST was neutralized by dilution so as not to kill host bacteria. The experimental samples were prepared as follows:

1. Test - Phage + SILDUST

2. Control - phage + salt.

Required equipment:

• Weight Balancer, Layer Air Flow Unit, Incubator.

Required Materials:

• Petri dishes, marker, spatula, micropipette.

Method:

• Prepare a 2.5 ml sample containing 1 g of SILDUST (which does not show antibacterial activity) and salt in separate sterile test tubes.

• 0.1 ml of lysate phage (approximately 10 ¹ ° infective phage particles / ml) is added to each.

• Mix well in a vortex mixer and incubate at 37 ° C.

• At t = 0.1 and 0.5 ml samples are withdrawn at hour intervals and diluted to a SILDUST test dilution that does not show bacterial effects.

143 • Apply this mortar on a freshly prepared host surface layer. This must be done for investigation and control.

• Incubate the plate at 37 ° C for approximately 24 hours.

• Mix 0.1 ml of this dilution with 0.5 ml of visibly growing host material and incubate at approximately 37 ° C for 15 minutes.

• Add 7 ml of ground soft agar to it.

• Mix vigorously and place on a nutrient agar plate.

• Incubate the plate at 37 ° C for approximately 24 hours.

• Surface plates are examined and plates forming units of cover are listed.

Results: See Table 32.

Table 32 - SILDUST specimen Dilutions The result + - 10- ⁴ -

Key:

+ contains active phage particles:

- no phage particles.

SILDUST - antiviral activity

Procedure: Same as SILDUST-antibacterial activity, part 2 The results are shown in Tables 33 and 34.

table SILDUST killing time

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Time intervals (or.) Salt BRIDGE * 0 + + 1 + + 2 + - 3 + -

BRIDGE * - 200 ppm ASAp Talc + 100 ppm gentamicin

Key:

+ contains active phage particles;

- no phage particles.

Table Phage list

Time intervals (or.) Salt (pfu / ml) BRIDGE * (pfu / ml) 0 TNTC 1.15 x 10 ⁵ 1 TNTC 1.0 x ^{10 4} 2 TNTC 3.0 x ^{10 3} 3 TNTC NIL

BRIDGE * - 200 ppm ASAp Talc + 100 ppm gentamicin.

Key:

TNTC - Too many to count.

Pfu / ml - Measurement of infectious phage particles.

Conclusion: SILDUST did not show bactericidal activity against host culture at 10 ' ² dilution. The same dilution of SILDUST tested antiviral activity and was effective. Bacterial-forming units were found to be reduced from 10 ⁵ to 0 within 3 hours, demonstrating that SILDUST is likely to have an effect against the animal virus as well.

For the experiments described above, the following composition was used:

145

Food Agar -10.0 g;

Sodium chloride - 5.0 g;

Meat extract - 3.0 g;

Distilled water - 900 ml;

Agar - 2.5 g;

PH-7.2 ± 0.2.

Phenol red dextrose broth

Protease Peptone -10.0 g / lt; Beef extract -1,0 g / lt;

Sodium chloride - 5.0 g / l;

Dextrose - 5,0 g / lt;

Phenol red - 0.018 g / lt;

PH 7.4 ± 0.2

Soft agar:

Agar -1.0%;

Salt:

Sodium chloride - 0.9%.

Vocadin

Mfg. Lic, No. AD / 200-A; Test # WNR 5008; Mfg Date: March 2005; Graduation Date: March 2008;

146

Active ingredients:

Povidone Iodine IP 5 w / w%.

Mfgd. Navketan Research and Lab. Ltd.

SILVER / ANDENS ADDITIVES TO POVIDON IODE 10% SOLUTION

Another example of an additive that is successfully used with the silver / water compositions of the present invention is povidone iodine. Iodine is well known in medicine as a prophylactic agent for the treatment of a broad spectrum of pathogens. Iodine is commercially available in varying concentrations, but is commonly used and offered for use at a concentration of 10%. In this embodiment of the invention, the synergistic combination comprises about 25-50 vol% silver / water exchange, replaced by 10% iodine solution. Because of the variety of reactions possible between the silver / water mixture and iodine, it is clear from the experimental results that the silver / water synergistic mixture with povidone iodine may act as a topical disinfectant (eg ointment) and / or as a prophylactic agent against cuts, burns and burns. scratches, etc. infections.

Specifically, a combination of 32 ppm silver / water formulations with various percentages of povidone iodine (PI) showed a synergistic effect on a number of bacteria. The research methods and results follow below. From these results, it can be concluded that a synergistic relationship exists between the two materials. This synergism can be utilized to obtain a great local disinfectant.

The following definition is intended to be understood, as specifically illustrated and described above, to include what is a conceptual equivalent, what may be obviously altered, and also what is essentially the idea of the present invention. It will be apparent to one skilled in the art that various adaptations and modifications of the embodiment described herein can be configured without departing from the present invention. The illustrated embodiment is provided by way of example only and not by way of limitation of the present invention. It is, therefore, to be understood that, based on the entirety of the appended definition, the invention may be practiced otherwise than as specifically described herein.

Claims (31)

DEFINITION A composition in water of silver comprising a total concentration of silver in the range of about 5 to 40 parts per million, wherein said silver is in the form of silver nanoparticles having an inner silver surface and at least one oxide of silver, wherein most silver particles have a smaller maximum diameter. less than 0.015 micrometres in that most colloidal silver particles have a minimum diameter greater than 0.005 micrometres in that the composition exhibits antimicrobial properties. 2. The composition of claim 1, further comprising hydrogen peroxide. 3. The composition of claim 2, wherein the concentration of hydrogen peroxide is from about 1% w / v to about 3.0% w / v. 4. The composition of claim 1, further comprising EDTA. 5. The composition of claim 4, wherein said EDTA comprises sodium EDTA. 6. The composition of claim 1, wherein the composition comprises a hydrogel obtained by dissolving a hydrophilic polymer in silver water. 7. The composition of claim 6, wherein said composition is in the form of an amorphous gel. 8. The composition of claim 6, wherein said composition is in the form of a solid gel sheet. 148 9. The composition of claim 8, wherein the hydrophilic polymer is selected from the group consisting of gelatin, a copolymer of carbohydrates, and acrylic acid. 10. The composition of claim 9, wherein the carbonate polymer comprises at least one polymer selected from the group consisting of cellulose derivatives, alginate, carrageenan and plant gums. A composition according to any one of claims 1 to 10 for use in the treatment of a disease selected from the group consisting of malaria, skin fungal infections, bacterial skin infections, vaginal infections, urinary tract infections, tonsillitis, pelvic inflammatory diseases, pharyngitis, gonorrhea, conjunctivitis, atitis, respiratory tract infections and nasal infections, characterized in that the composition of said composition is administered to a subject afflicted with the disease. A composition according to any one of claims 1 to 10 for use in the treatment of a disease selected from the group consisting of malaria, skin fungal infections, bacterial skin infections, vaginal infections, urinary tract infections, tonsillitis, pelvic inflammatory diseases, pharyngitis, gonorrhea, conjunctivitis, atitis, respiratory tract infections, and nasal infections, in that it further includes silver EDTA. 13. The activity of microbes selected from the group consisting of Bacillus anthracis, Bacillus subtilis, Candida albicans, Mycobacteria bovis, Mycobacteria tuberculosis, Pseudomonas aeruginosa, Salmonella choleraesius, Staphylococcus aureus, Trichomonas vaginalis, and Yersinia pestis. 14. The method of claim 13, wherein said effect comprises ingestion of silver EDTA. 15. Microbes selected from the group consisting of Bacillus anthracis, Bacillus subtilis, Candida albicans, Mycobacteria bovis, Mycobacteria tuberculosis, Pseudomonas 149 aeruginosa, Salmonella choleraesius, Staphylococcus aureus, Trichomonas vaginalis and Yersinia pestis, the method comprising the step of exposing these microbes to at least one composition selected from the group consisting of silver EDTA, silver EDDS, silver curcuminate, silver of silver tetracycline. 16. A method of introducing at least one metal into a biological organism, wherein said method comprises attaching at least one metal selected from the group consisting of silver, copper, zinc, platinum, titanium and their alloys to at least one clathrate, to form a mat / clathrate structure and the effect of said biological organism on said metal clathrate structure. 17. The method of claim 16, wherein the clathrate comprises at least one kaolinite. 18. The method of claim 16, wherein the clathrate comprises at least one zeolite. 19. The method of claim 16, wherein the at least one metal comprises silver. 20. The use of AgEDTA for the preparation of a composition for the prophylactic treatment of bovine animals, wherein the AgEDTA is added to at least one bovine food and bovine drinking water. 21. The use of AgEDTA for the manufacture of a composition for the prophylactic treatment of a human or animal being characterized in that AgEDTA is added to anything that said humans and animals ingest. 22. The use of claim 21, wherein said AgEDTA is present in an amount sufficient to prevent infections. 150 23. The use according to claim 21, wherein said AgEDTA is present in an amount of less than 20 ppm. Use of AgEDTA in the treatment of human and animal infections, wherein the amount of AgEDTA is sufficient to improve healing of said infection. Use of AgEDTA for the treatment of a human or animal infection, characterized in that at least one element selected from the group consisting of AgEDTA, silver EDDS, silver curcuminate, silver berberine and silver tetracycline is ingested. 26. The use of AgEDTA for treating the surface of a human or animal skin, characterized in that the paste or gel comprises at least one element selected from the group consisting of AgEDTA, silver EDDS, silver curcuminate, silver berberine and silver tetracycline, and said paste or gel comes into contact with the human or animal skin surface. A gel or paste product comprising at least one element selected from the group consisting of AgEDTA, silver EDDS, silver curcuminate, silver berberine and silver tetracycline. A method of increasing the efficacy of an antibiotic dose comprising adding to said antibiotic dose at least one substance selected from EDTA and AgEDTA. 29. The method of claim 28, wherein AgEDTA is added to said dose of antibiotic. 30. Use of a composition according to claim 11, wherein said composition further comprises a selected dose of antibiotic, wherein said selected dose of antibiotic is based on antibiotics having at least some known efficacy against said disease. 151 31. The composition of claim 1, wherein said composition further comprises at least one material selected from the group consisting of AgEDTA, silver EDDS, silver curcuminate, silver berberine, and silver tetracycline.