BIOFLAVONOID IMPREGNATED MATERIALS

Abstract

Cellulosic fibrous materials are described which are impregnated with a bioflavonoid composition, the bioflavonoid content of the composition comprising at least naringin and neohesperidin. The use of such impregnated materials is also described, for example as paper or bamboo towels and cardboard, as well as the process for impregnating the materials.

Claims

1-17. (canceled)

18. A dry respiratory mask for use in reducing viral transmission by air, which comprises cellulosic fibres impregnated with a mixture of bioflavonoids comprising at least 70% naringin and neohesperidin.

19. The dry respiratory mask of claim 18, wherein the naringin and neohesperidin together form at least 75% of the bioflavonoid content.

20. The dry respiratory mask of claim 18, wherein the naringin and neohesperidin together form between 75% and 80% of the bioflavonoid content.

21. The dry respiratory mask of claim 18, wherein the mixture of bioflavonoids further comprises one or more compounds selected from the group of neoeriocitrin, isonaringin, hesperidin, neodiosmin, naringenin, poncirin and rhiofolin.

22. The dry respiratory mask of claim 18, wherein the mixture of bioflavonoids comprises neoeriocitrin, isonaringin, hesperidin, neodiosmin, naringenin, poncirin and rhiofolin.

23. The dry respiratory mask of claim 18, wherein the mixture of bioflavonoids is uniform throughout the mask.

24. A method for preparing a dry respiratory mask suitable for reduction of viral transmission by air, which comprises preparing a respiratory mask from cellulosic fibres and impregnating said mask with a mixture of flavonoids which comprises at least 70% of naringin and neohesperidine, and drying said mask.

25. The method as claimed in claim 24, wherein said impregnating comprises immersing or spraying said mask of cellulosic fibres.

26. The method as claimed in claim 24, wherein drying comprises mechanically drying.

27. The method as claimed in claim 24, wherein the mixture of bioflavonoids further comprises one or more compounds selected from the group of neoeriocitrin, isonaringin, hesperidin, neodiosmin, naringenin, poncirin and rhiofolin.

28. The method as claimed in claim 24, wherein the mixture of bioflavonoids is uniform throughout the mask.

29. A method of reducing aerial viral transmission which comprises use of a dry respiratory mask, as claimed in claim 18.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order that the invention may be more fully understood it will now be described, by way of example only, and with reference to the following Figure(s), in which:

[0057] FIG. 1 is a graph showing the results of the effects of different dilutions of the Citrox BC active dried onto Bounty® brand paper towels on S. aureus activity.

[0058] FIG. 2 is a graph showing the results of the effects of different dilutions of the Citrox BC active dried onto Bounty® brand paper towels on E. coli activity.

DETAILED DESCRIPTION

[0059] The bioflavonoid content may comprise 40-50%, for example about 45% wt/wt of the bioflavonoid composition. A suitable source of a bioflavonoid composition is herein referred to as “HPLC 45” or “Citrox BC” of which about 45% (of the total composition of HPLC 45/Citrox BC) comprises bioflavonoids. The bioflavonoids are in admixture with biomass residues of extraction from bitter oranges, such as pectins, sugars and minor organic acids, which make up the remaining 55%. HPLC 45 is available from Exquim (a company of Grupo Ferrer) as Citrus Bioflavonoid Complex 45% HPLC.

TABLE-US-00001 TABLE 1 The mixture of bioflavonoids in HPLC 45 % bioflavonoid in mixture Bioflavonoid with biomass Neoeriocitrin 1.1 Isonaringin 1.2 Naringin 23.4 Hesperidin 1.4 Neohesperidin 12.5 Neodiosmin 1.4 Naringenin 1.5 Poncirin 2.0 Other (Rhiofolin) 0.5

EXAMPLES

[0060] Staphylococcus aureus was chosen as a representative gram positive organism. This organism is found on mammalian skin and is, therefore, shed into the surrounding environment. E. coli was chosen as the representative of the gram negative enteric bacteria. This organism is found in the digestive tract of birds, mammals and reptiles. Its presence in the environment signals fecal contamination. Pseudomonas aeruginosa was chosen to represent the non-enteric gram-negative bacteria. This genera of bacteria is present in water with related species representing major plant pathogens and human opportunistic pathogens. Bacillus subtilis was chosen as the representative gram positive spore-formers. This bacterium is found in soil and water but is also ubiquitous in the environment. This species forms endospores as a survival mechanism. Bacterial endospores are the most resistant form of life on Earth and, therefore, represent an ongoing concern for sanitation, disinfection and sterilisation processes. Endospores represent the “ultimate” challenge for any antimicrobial agent.

Example 1: Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

[0061] Procedure

[0062] A pure culture of a single microorganism is grown in an appropriate broth. The culture is standardized using standard microbiological techniques to have a concentration of very near 1 million cells per millilitre. The more standard the microbial culture, the more reproducible the test results. The antimicrobial agent is diluted a number of times, 1:1, using sterile diluents. After the antimicrobial agent has been diluted, a volume of the standardised inoculums equal to the volume of the diluted antimicrobial agent is added to each dilution vessel, bringing the microbial concentration to approximately 500,000 cells per millilitre. The inoculated, serially diluted antimicrobial agent is incubated at an appropriate temperature for the test organism for a pre-set period, usually 18 hours. After incubation, the series of dilution vessels is observed for microbial growth, usually indicated by turbidity and/or a pellet of microorganisms in the bottom of the vessel. The last tube in the dilution series that does not demonstrate growth corresponds with the minimum inhibitory concentration (MIC) of the antimicrobial agent.

[0063] In order to differentiate between a microbiostatic agent (bacteria are not killed just inhibited) and a microbiocidal agent (bacteria are killed) an MBC test is performed. When a microbiostatic agent is removed or neutralized, previously inhibited bacteria begin to grow again. Each well showing no growth/turbidity in the MIC test is sub-cultured on media that contains no biocide. Any microbial growth resulting from this test indicates that, at that concentration, the active is microbiostatic. If the subculture results in no bacterial regrowth, then, at that concentration, the active is microbiosidal. The range of concentration of Citrox BC active tested was 0.075-0.75%.

Discussion of Results

[0064] The MIC test is an established “screen” for the biostatic (and possibly also biocidal) activity of liquid antimicrobials. It is often used to find the appropriate concentrations of an antimicrobial active to use for further efficacy testing. Performing both the MIC and MBC test will enable one to differentiate between a biocidal or biostatic mode of action. Depending on the concentration of active used and the contact time an active will often demonstrate both biostatic and biocidal modes of action.

[0065] The range of Citrox BC active tested was 0.075%-0.75%. For P. aeruginosa, no MIC value was obtained as all concentrations of the Citrox BC active tested showed no turbidity (Table 2).

[0066] MCB testing showed that all concentrations were also bactericidal for B. subtilis, there was also no MIC value obtained demonstrating that inhibition of growth took place at all concentrations tested. The MBC value obtained for B. subtilis was 0.315% Citrox BC active. This means that concentrations ranging from 0.075% to 0.315% are bacteristatic and all concentrations of the Citrox BC active greater than or equal to 0.315% are bactericidal.

[0067] These results indicate that gram negatives like P. aeruginosa are more easily killed by the Citrox BC active than the gram positive B. subtilis.

TABLE-US-00002 TABLE 2 MIC/MBC Testing % Citrox MIC(G/NG) MBC (CFU/mL) BC P.a B.s P.a B.s 0 G G 0 0 0.075 NG G 0 4.2 × 10.sup.2 0.095 NG G 0 3.1 × 10.sup.2 0.115 NG G 0 3.3 × 10.sup.2 0.135 NG G 0 3.5 × 10.sup.2 0.155 NG G 0 3.6 × 10.sup.2 0.175 NG G 0 2.4 × 10.sup.2 0.195 NG G 0 1.5 × 10.sup.2 0.215 NG G 0 1.3 × 10.sup.2 0.235 NG G 0 1.6 × 10.sup.2 0.255 NG G 0 40 0.275 NG G 0 40 0.295 NG G 0 1 0.315 NG NG 0 0 0.335 NG NG 0 0 0.355 NG NG 0 0 0.375-0.750 NG NG 0 0 G = Growth, NG = No Growth P.a. = Psuedomonas aeruginosa B.s. = Bacillus subtilis

Example 2: Time Kill Test

[0068] Procedure

[0069] All timed kill tests were performed using a standard viable count procedure. Reference NB X34689.

[0070] The following neutralising solution was used in all kill tests.

[0071] Tween 80—3%

[0072] Saponin—3%

[0073] Histidine—0.1%

[0074] Cysteine—0.1%

[0075] Rationale

[0076] A timed kill test assesses the amount of time it takes to kill a defined population of microorganisms. A wide variety of microorganisms are killed by the Citrox BC active. An important first step in characterising this active for use in an antimicrobial towel is to verify the kill claims. Claims for efficacy are based on the number of bacterial killed within a defined time frame. The most rigorous claims are those made for food contact where the active must affect a 5 log reduction of the challenge organism in 30 seconds.

Discussion of Results

Example 2(a): Timed Kill Test: 10 Minute Contact Time

[0077] Bacterial kill kinetics are affected by bacterial numbers, the concentration of active used and the contact time. In order to determine the most effective range of the Citrox BC active, S. aureus was used in a 10 minute kill test to assess the efficacy of various concentrations of the Citrox BC active. A >6.56 log reduction was observed for all concentrations (0.45-0.65%) of the Citrox BC active tested (Table 3).

[0078] When B. subtilis was used as a challenge organism, 0.7% Citrox BC was required to effect a >5 log reduction in 10 minutes (Table 4). Based on previous tests, 0.5% active is the most effective for general use.

TABLE-US-00003 TABLE 3 Time Kill Test: S. aureus, 10 min. % Citrox Log10 Log BC CFU/mL CFU/mL Reduction 0 7.4 × 10.sup.6 6.86 0 0.45 <2 0.3 6.56 0.5 <2 0.3 6.56 0.55 <2 0.3 6.56 0.6 <2 0.3 6.56 0.65 <2 0.3 6.56 0.65 + 6.6 × 10.sup.6 6.81 0.05 neutralizer

TABLE-US-00004 TABLE 4 Time Kill Test: B. subtilis, 10 min. % Citrox Log10 Log BC CFU/mL CFU/mL Reduction 0 1.1 × 10.sup.6 6.04 NA 0.5 2.9 × 10.sup.4 4.4 1.64 0.7 <2 0.3 5.74

Example 2(b): Timed Kill Test: 30 Second Contact Time

[0079] Timed kill studies using E. coli, P. aeruginosa and S. aureus were performed using 0.5% Citrox BC active with a contact time of 30 seconds. Log reductions of >6.4 were seen for all organisms (Table 5). This confirms that this active would meet the criteria for use in food contact situations.

TABLE-US-00005 TABLE 5 Time Kill Test: 30 seconds E. coli P. aeruginosa S. aureus % Citrox BC 0 0.5 0 0.5 0 0.5 CFU/mL 5.1 × <2 6.4 × <2 7.4 × <2 10.sup.6 10.sup.7 10.sup.6 Log10 CFU/mL 6.7 <0.3 7.8 <0.3 6.8 <0.3 Log Reduction NA 6.5 NA 7.3 NA 6.5

Example 2(c): Timed Kill Test: Sporicidal Activity

[0080] As stated above, the ultimate test for any antimicrobial active is the ability to kill spores. Any chemical or process that kills a bacterial spore is, by definition, a sterilant. In order to assess if the Citrox BC active was sporicidal, a kill test was performed on an actual spore suspension. Citrox BC, over a range 0.5% to 1.5%, was tested over a 1 hour time period. There were some limitations to this test. The spore suspension (B. subtilis, ATCC 6633, 6.4×10.sup.4 CFU/pellet, Microbiologics) in the test was only at ˜2×10.sup.4 CFU/ml, limiting the log reduction calculation. The lyophilized pellets were found to contain charcoal, a substance known to neutralise the bioflavonoid component of the Citrox BC active. With those limitations, approximately a 2 log reduction in spores was demonstrated. This indicates that the Citrox BC active has definite activity against spores. Spore suspensions at a higher titer without a charcoal additive should be used to investigate this activity further.

Example 3: Surface Testing Using Paper Towel Impregnated with Citrox BC Active

[0081] 1) Procedure: Adding Citrox BC to Paper Towel

[0082] Bounty® (“Bounty” is a registered trademark of Procter & Gamble) brand paper towels were used to make the dry antimicrobial towels. Bounty® paper towels are a conventional, commercially available paper towel product. Citrox BC active concentrate was diluted to desired concentrations. One paper towel was immersed completely into the diluted active and then wrung out by hand. The towel was dried overnight.

[0083] 2) Procedure: Weight of Citrox BC Active Dried onto Bounty® Brand Paper Towel

[0084] Bounty® brand paper towels were dried to a constant weight in a 54° C. oven. Various dilutions of the Citrox BC active were dried onto Bounty® brand paper towels as described above. The towels were dried at room temperature overnight. The treated towels were then dried to a constant weight at 54° C. The weight difference between the untreated and treated towels is presumed to be the weight of the Citrox BC active.

[0085] 3) Procedure: For Testing Affect of Administration of Impregnated Paper Towels to a Surface

[0086] Using the lab bench top as a representative hard, non-porous surface, a grid was marked off using tape. Cotton-tipped swabs saturated with a broth culture of the challenge organism were used to inoculate the surface and air dried. Paper towels treated with dilutions of the Citrox BC active were wetted and then used to clean the inoculated bench top. The bench top was visibly wet for 3 minutes (contact time) and then allowed to completely air dry. RODAC (Replicate Organism Detection and Counting) plates were used to sample the cleaned surface for surviving bacteria. The plates were incubated overnight at a temperature appropriate to the challenge organism. Colonies were counted and the number used to calculate CFU/cm.sup.2. Results were calculated by averaging the counts from five 3″×3″ “grid squares”.

[0087] 4) Procedure: RODAC Sampling

[0088] A RODAC plate is used to touch the surface to be sampled after which the plate is incubated at an appropriate temperature. There are nutrients in the media that promote the growth of a variety of microbes. Lecithin and Polysorbate 80 are incorporated in the agar and function as disinfectant/sanitizer neutralisers. The type and number of microorganisms is detected by the appearance of colonies on the surface of the agar medium. Collection of samples from the same area before and after cleaning and treatment with a disinfectant permits the evaluation of sanitary procedures.

[0089] Results

[0090] Paper towels wetted with water and containing no Citrox BC active were assessed for the ability to remove bacteria from a contaminated hard surface. The results for this control (i.e. unimpregnated paper towels) are shown by the bar labelled “0” in FIGS. 1 and 2. FIGS. 1 and 2 show the results for both S. aureus and E. coli. Paper towels containing dilutions of the Citrox BC active greater than 1:200 were able to reduce the levels of S. aureus from >50 CFU/cm.sup.2 to <1 CFU/cm.sup.2. The paper towels containing dilutions of the Citrox BC active greater than 1:200 were able to reduce the levels of E. coli from >7 CFU/cm.sup.2 to <1 CFU/cm.sup.2.

[0091] These results show that a dry antimicrobial towel are activated by wetting.

Discussion of Results

[0092] Different dilutions of the Citrox BC active were dried onto Bounty® brand paper towels. These treated towels were used to decontaminate a lab bench heavily inoculated with bacteria. The ability of the treated towels to affect a decrease of contaminants on the lab bench was evaluated using RODAC plates.

[0093] A method was developed to assess ability of a paper towel impregnated with the Citrox BC active to reduce bacterial numbers on a contaminated hard surface. RODAC plates are recommended for the detection and enumeration of microorganisms present on surfaces of sanitary importance. RODAC plates are specially constructed so that an agar medium can be overfilled producing a dome-shaped surface that can be pressed on a surface for sampling its microbial content. RODAC plates are used in a variety of programs to establish and monitor cleaning techniques and schedules.

[0094] When using a paper towel plus an antimicrobial active, one must keep in mind that removal of bacteria from a contaminated surface occurs by two mechanisms: first is the kill achieved by the action of the antimicrobial active and second is mechanical removal of the contaminants by the paper towel itself.

[0095] Lab scale antibacterial towels were used to calculate the weight of the Citrox BC active dried onto the towels. The weight of active present on the towel (Table 6) can be used as a starting point for cost analysis.

TABLE-US-00006 TABLE 6 Weight of Citrox BC active dried onto Bounty ® paper towel Ave. wt. Citrox Pre- Post- of BC BC treatment treatment active Dilution Dry Weight Dry Weight Difference (g/towel) Std Dev 1:150 4.32547 4.31821 −0.00726 0.00388 0.006875 4.31226 4.32089 0.00863 4.31476 4.32400 0.00924 4.32424 4.32607 0.00183 4.32625 4.33321 0.00696 1:100 4.30877 4.35320 0.04443 0.05244 0.010257 4.30530 4.36125 0.05595 4.32380 4.37021 0.04641 4.30800 4.35446 0.04646 4.30435 4.37329 0.06895 1:50  4.31739 4.49804 0.18065 0.20090 0.046566 4.38309 4.51604 0.13295 4.31517 4.54191 0.22674 4.31539 4.52472 0.20933 4.32305 4.57787 0.25482