UTILIZATION of PLASMA ACTIVATED WATER in Biotechnology, Pharmacology and Medicine. JSC TECHNOSYSTEM-ECO Moscow, Russia April, 2009

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1 UTILIZATION of PLASMA ACTIVATED WATER in Biotechnology, Pharmacology and Medicine JSC TECHNOSYSTEM-ECO Moscow, Russia April, 2009

2 METHOD of WATER ACTIVATION with PLASMA of GAS DISCHARGE ANODE VACUUM WATER VAPOUR CATHODE WATER FILM

3 THE ESSENCE of WATER ACTIVATION METHOD with PLASMA of GAS DISCHARGE Simultaneous impact on water of all the factors of gas discharge plasma: Light irradiation in UV,IR and visible band, RF irradiation; Flow of charged particles; Shock acoustic waves; Steady and/or Alternative Magnetic field. That provides for creation and maintenance of primary conditions for behavior of physical and chemical processes such as : Oxidation, Hydration, Reduction; Chemical interaction of water and water admixtures with products of gas discharge; Interaction with light and RF irradiation; Interaction with flow of charged particles and shock acoustic waves; Generation of reaction capable particles and components ( Oxygen, Hydrogen and Hydroxyl ions, Peroxide ions, Hydrogen Peroxide, Oxygen atoms mainly in form of radicals). As the result of this processes water becomes activated, that is: The water structure changes i.e. cluster size is decreased or water becomes monomolecular; The energy structure (state) of water is transferred into another stable state ; Water becomes physically, chemically and biologically active. All the acquired features are stable in time and toward external influence.

4 FEATURES of PLASMA ACTIVATED WATER (PAW) Changed water structure: decrease of size of water clusters down to 2-4 molecules per cluster or even monomolecular character of water; strengthening of proton bonds. Changes in Water Energy Structure: strong and long lasting changes in light absorption spectrums ( visible IR and visible UV spectrum range) and fluorescence spectrums; changes in NMR spectrums; Physical, Chemical and Biological Activity. Other features: Changed ph and ORP; Generation of active components (chemically and bio) in water (admixtures, Oxygen etc.) encapsulated in PAW structure. The acquired features of PAW have long shelf life ( PAW keeps acquired features up to 24 month and are relatively stable towards external influence.

5 BIOLOGICAL ACTIVITY of PLASMA ACTIVATED WATER Strong antioxidant features( at microbiological level). Comments: PAW works complimentary with wide spectrum of other anti oxidants and facilitates their impact. PAW improves intercellular metabolism. Property to facilitate impact (effect) of pharmaceuticals when applied in combination with PAW or even terminating antibiotic resistivity. PAW is in symbiosis with wide spectrum of enzymes. Strong bactericide( bacteria static) even with spore forming Bacteria,fungicide (fungi static) and anti virus effects. Comments: there are modes of activation when PAW acquires properties of Bacteria, virus and Fungi growth stimulation feature.

6 Effect of Plasma activated water (level 3)on the viability of E. coli over time Escherichia coli is the dominant air-tolerant species of bacteria in the human intestine, but some strains can cause severe disease and diarrhea. A lab strain of E. coli (JM109) was incubated with activated water (or normal water as a control) for various lengths of time, and then samples were taken to evaluate the number of viable bacteria after treatment. (April 9, 2002) Active Water = Bottled, drinking spring water activated to level 3 (220 mg H2O2/liter) on April 8, Standard Culture Preparation: The bacterial culture was grown overnight in L broth (10 g Tryptone, 5 g Yeast Extract, 5 g NaCl / liter) at 37oC with aeration. To obtain a fresh culture, the overnight culture was diluted 1:100 in 10 ml L broth (125 ml flask) and grown with aeration for 3-4 hr at 37oC (to OD600 = ~ ). Method: A 100 ml sample of Standard Culture of E. coli JM109 (OD = 1.0) was added to 9.9 ml activated water (or normal water), mixed, and incubated at room temperature (22oC). Samples of 100 ml were taken after various lengths of time, and serial 1:10 dilutions were made in sterile saline. 100 ml samples of each dilution were spread onto L agar plates, incubated overnight, and then colonies were counted to determine colony forming units (CFUs). Results: After just 15 min incubation in activated water, the number of bacteria was reduced by 99.5% compared to controls. No bacteria were viable after 24 hr incubation in activated water. Thus, activated water was highly effective at killing this E. coli strain.

7 Effect on Plasma activated water (level 4)on the viability of P. aeruginosa over time Pseudomonas aeruginosa PAO1 was incubated with level 4 activated water (or sterile saline as a control) for various lengths of time, and then samples were taken to evaluate the number of viable bacteria after treatment. (April 11, 2002) Active Water = Bottled, drinking spring water activated to level 4 (? mg H2O2/liter), activated on 4/10/02. Standard Culture Preparation: The bacterial culture was grown overnight in L broth overnight at 37oC with aeration. This culture was diluted 1:100 in 10 ml L broth (125 ml flask) and grown with aeration to for 3-4 hr at 37oC (OD600 = ~ ). Method: A 50 ml sample of Standard Culture of P. aeruginosa PAO1 (OD = 1.0) was added to 5.0 ml activated water (or sterile saline), mixed, and incubated at room temperature (22oC). Samples of 100 ml were taken after various lengths of time, and serial 1:10 dilutions were made in sterile saline. 100 ml samples of each dilution were spread onto L agar plates, incubated overnight, and then colonies were counted to determine colony-forming units (CFUs). Results: This level 4 sample of activated water had no obvious effect on P. aeruginosa after 60 min of incubation. However, in 24 hours, it did kill 90% of the bacteria. After 24 h, the control sample contained 5 x 107 cells/ml and the activated water sample contained 5 x 106 cells/ml.

8 Effect on Plasma activated water (level 4)on the growth of P. aeruginosa. Pseudomonas aeruginosa PAO1 was incubated in L broth made with activated water (or autoclaved water as a control) and culture turbidity was determined as a measurement of growth. (April 16, 2002) Active Water L Broth = Bottled, drinking spring water activated to level 4 (activated on 4/10/02) was used to make L broth (10 g Tryptone, 5 g Yeast Extract, 5 g NaCl / liter) and then sterilized by 0.45 micron filtration. Culture Preparation: The bacterial starter culture, inoculated from a fresh L agar plate culture, was grown in L broth at 37oC with aeration for 3 hours to OD600 = Method: A 100 ml sample of the culture of P. aeruginosa PAO1 (OD = 0.18) was added to 10 ml activated L broth diluted with normal L broth at various concentrations and incubated at 32oC 18 h. Culture turbidity was measured. Results: P. aeruginosa grew to a high cell density (turbidity Optical Density = 4.4) in normal L broth (0% activated water). However, cultures grown in L broth with 100% activated water barely grew, and culture turbidity was 10-fold less (OD = 0.4). An 80% Activated L Broth culture still showed a growth defect (OD 3.0), but a 50% Activated L Broth culture grew as well as the control after 18 hours. % Activated Water in L Broth OD 600 after 18 hr, 32 C

9 Plasma activated water increased the sensitivity of P. aeruginosa to low levels of antibiotics Pseudomonas aeruginosa is not very sensitive to many commonly used antibiotics, including ampicillin and kanamycin. However, these antibiotics are affective against many other bacteria including E. coli. Here, P. aeruginosa strain PAO1 was incubated in Activated L Broth (made with 100% activated water) or Normal L Broth as a control, and low levels of ampicillin or kanamycin were added. After over night incubation, culture turbidity was determined as a measurement of growth (April 16, 2002) Active Water L Broth = Bottled, drinking spring water activated to level 4 activated on 4/10/02 was used to make L broth (10 g Tryptone, 5 g Yeast Extract, 5 g NaCl / liter) and then sterilized by 0.45 micron filtration. Culture Preparation: L broth (10 ml, 125 ml flask) was inoculated from a fresh L agar plate culture and grown about 3 hours at 37oC with aeration to OD600 = Method: A 100 ml sample of the culture of P. aeruginosa PAO1 (OD = 0.18) was added to 10 ml activated L broth or normal L broth. Kanamycin or ampicillin was added from frozen stocks. Cultures incubated at 32oC 18 h with aeration. Culture turbidity (Optical Density at 600 nm) was measured as an indicator of growth. Results: Activated L Broth (i.e., made with level-4 activated water) inhibited the growth of P. aeruginosa when compared to normal L Broth (as in Exper. 5). Activated L Broth + Ampicillin was further reduced in growth even though there was no effect on the growth of P. aeruginosa in normal L Broth + Ampicillin. Activated L Broth + Kanamycin dramatically blocked all growth even though growth in Normal L Broth + Kanamycin was unaffected by the antibiotic. Thus, activated water appears to greatly enhance the effectiveness of certain antibiotics in killing bacteria like P. aeruginosa, which is very difficult to eradicate due to its intrinsic antibiotic resistance. Normal vs. Active L Broth + antibiotics OD 600 after 18 hr 32C Normal 4.4 Active 0.4 Norm mg/ml Active + 30 mg/ml Norm mg/ml Activ mg/ml

10 Ability of Plasma activated water (level 4)to prevent microbial growth in solutions of soluble fiber. This was a test to determine if activated (energized) water could act as a preservative to prevent a non sterilized solution free of bacterial growth (April 23, 2002). A. Growth in L Broth Active Water L Broth = Bottled, drinking spring water activated to level 4 (activated on 4/10/02) was used to make L broth (10 g Tryptone, 5 g Yeast Extract, 5 g NaCl / liter) and then sterilized by 0.45 micron filtration. Stored at 5oC. Method: 1 gm of dry fiber was added to 10 ml activated-water L broth or normal-water L and incubated at 32oC 48 h. Results: - Normal L broth+fiber - a highly turbid culture was observed due to growth of bacteria present in the fiber powder. - Activated L broth+fiber - a clear solution was observed indicating no growth. This remained clear after >7 days at room temperature. B. Growth of Fiber Water Active Water: 1. Bottled, drinking spring water activated to level 4 (activated on 4/10/02) 2. Richmond tap water activated to level 4 (activated on 4/10/02) Method: 1 gm of dry fiber was added to 10 ml water (activated or normal) in capped, plastic, sterile tubes and incubated at 30oC 48 hr (April 23, 2002). After 10 days, 0.1 ml was spread onto L agar plates and incubated at 30oC. Results: - See Table on the right. TYPE of WATER 24 hr 48 hr Normal visible visible Activated, bottled Activated drinking tap None None None None

11 POSSIBLE WAYS AND AREAS OF PLASMA ACTIVATED WATER USAGE As base for physiological (salt) solutions and injections; As activator of wide spectrum of pharmaceuticals, incl. antibiotics; For vaccines preparation; As transport platform for medication; As antibiotic, anti fungi and anti virus pharmaceutical; As pharmaceutical ( in combination with or without other pharmaceuticals) in treatment of: a) Burns of different origin; b) Wounds, ulcers, sores and other skin diseases; c) Inflammation of internal organs. As STIMULATOR of Bacteria and Fungi growth (for BioTech); As DISINFECTANT (in liquid, vapor and frozen state), that does not affect surfaces/materials/tissue under disinfection; As CONSERVANT (preservative).

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