Bioflavonoid coated materials

Abstract

Polymeric materials are described which have a bioflavonoid coating, the bioflavonoid content of the coating comprising at least naringin and neohesperidin. The use of such coated polymeric materials is also described as well as the process for making the coated polymeric materials.

Claims

1. A respiratory mask comprising a synthetic polymeric material coated with a bioflavonoid coating, wherein a bioflavonoid content of the bioflavonoid coating comprises at least 50% wt/wt of a mixture of naringin and neohesperidin, and wherein the bioflavonoid coating comprises a thickness of between 700 nm and 1300 nm.

2. The respiratory mask of claim 1, wherein the mixture of naringin and neohesperidin comprises at least 70% wt/wt of the bioflavonoid content of the bioflavonoid coating.

3. The respiratory mask of claim 1, wherein the mixture of naringin and neohesperidin comprises between 75% to 80% wt/wt of the bioflavonoid content of the bioflavonoid coating.

4. The respiratory mask of claim 1, wherein the bioflavonoid content of the bioflavonoid coating further comprises one or more compounds selected from the group consisting of neoeriocitrin, isonaringin, hesperidin, neodiosmin, naringenin, poncirin and rhiofolin.

5. The respiratory mask of claim 1, wherein the bioflavonoid coating further comprises one or more fruit acids selected from the group consisting of salicylic acid, citric acid, lactic acid, ascorbic acid and malic acid.

6. An antimicrobial respiratory mask comprising a synthetic polymeric material coated with a bioflavonoid coating, wherein the average surface roughness of the bioflavonoid coating is between 600 nm and 1500 nm and wherein a bioflavonoid content of the bioflavonoid coating comprises at least 50% wt/wt of a mixture of naringin and neohesperidin and wherein said mask enhances protection of the user against inhaling bacteria and viruses.

7. The antimicrobial respiratory mask according to claim 6, wherein the synthetic polymer material comprises a film wherein said film comprises at least one synthetic polymer selected from the group comprising polyethylene terephthalate, polystyrene, polyethylene, polypropylene, polyvinylchloride, polyamide, polyvinylidene chloride, ethylene-vinyl alcohol co-polymer, polyethylene vinyl acetate, neoprene, polyurethane, nylon, latex, nitrile rubber and silicone.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1—Comparison between the Citrox coating thicknesses with water contact angle (WCA) obtained after PET treatment with either the PlasmaTreat PT (air) and PlasmaStream™ PS (helium/oxygen) plasmas.

(2) FIG. 2—Comparison between the Citrox coating roughness with water contact angle (WCA) obtained after PET treatment with either the PlasmaTreat PT (air) and PlasmaStream™ PS (helium/oxygen) plasmas.

(3) FIG. 3—Effect of Citrox and vitamin E coatings, onto non He/O.sub.2 pre-treated PET trays, after 2 passes of flow rate 100 μl/min, onto turkey oxidation, using the PlasmaStream™ system.

(4) FIG. 4—Characteristic profilometry image of Citrox coating onto a non He/O.sub.2 pre-treated silicon substrate, after 100 passes using the Labline™ system, one day after treatment (Magnification 2.6×).

(5) FIG. 5—Characteristic profilometry image of Citrox coating onto a He/O.sub.2 pre-treated silicon substrate, after 100 passes using the Labline™ system, one day after treatment (Magnification 2.6×).

(6) FIG. 6—Effect of ageing time on the average surface roughness (R.sub.a) of Citrox (15%) coatings onto the He/O.sub.2 and non pre-treated silicon substrates, after 100 passes using the Labline™ system.

(7) FIG. 7—Effect of ageing time on the thickness of Citrox (15%) coatings onto the He/O.sub.2 and non pre-treated silicon substrates, after 100 passes using the Labline™ system.

(8) FIG. 8—Characteristic profilometry image of Citrox coating onto a non He/O.sub.2 pre-treated silicon substrate after 100 passes using the Labline™ system, twenty one days after treatment (Magnification 2.6×).

(9) FIG. 9—Characteristic profilometry image of Citrox coating onto a He/O.sub.2 pre-treated silicon substrate after 100 passes using the Labline™ system, twenty one days after treatment (Magnification 2.6×).

(10) FIG. 10—Antibacterial activity, against S. aureus, of Citrox coating onto the He/O.sub.2 and non pre-treated PET substrates after 100 passes using the Labline™ system, as a function of time.

(11) FIG. 11—Antibacterial activity, against E. coli, of Citrox coating onto the He/O.sub.2 and non pretreated PET substrates after 100 passes using the Labline™ system, as a function of time.

(12) FIG. 12—Antibacterial activity, against Salmonella, of Citrox coating onto the He/O.sub.2 and non pre-treated PET substrates after 100 passes using the Labline™ system, as a function of time.

(13) FIG. 13—Antibacterial activity of Citrox against bacteria found on the chicken, the Citrox being sprayed either i) on a PET tray and then placing the chicken on top or ii) directly on the chicken, in comparison to the control that is chicken placed on a PET tray without any treatment. The antibacterial effect was measured on day 6 after spraying.

(14) FIG. 14—FTIR ATR spectra in the range of 900 to 1900 cm.sup.−1 showing the presence of Citrox on plasma treated and untreated polystyrene sheets after incubation with Citrox and subsequent washing with water. The blue line (or black line) represents untreated polystyrene. The red line (or grey line) represents plasma immersion ion implantation (PIII).

(15) FIG. 15—FTIR ATR spectra in the range of 2400 to 3900 cm.sup.−1 showing the presence of Citrox on plasma treated and untreated polystyrene sheets after incubation with Citrox and subsequent washing with water. The blue line (or black line) represents untreated polystyrene. The red line (or grey line) represents plasma immersion ion implantation (PIII).

(16) The mixture of bioflavonoids employs bioflavonoids in admixture with biomass residues of extraction from bitter oranges. The bioflavonoid components comprise 40-50%, for example about 45% wt/wt of this mixture (HPLC-45). HPLC-45 is available from Exquim (the food arm of Grupo Ferrer) as Citrus Bioflavonoid Complex 45% HPLC.

(17) Citrox HXT powder comprises on a wt/wt basis 7.5% HPLC 45, citric acid 30%, willow bark extract 50% and Olea europaea extract 12.5%. Citrox HXT powder is available from Citrox Biosciences, Limited, Huntington, UK.

(18) The willow bark extract contains 90% of salicylic acid.

(19) The Olea europaea extract contains 20% of oleuropein.

(20) HPLC-45 contains 45% by weight of a mix of bioflavonoids together with residues of extraction from bitter oranges.

(21) TABLE-US-00001 TABLE 1 The mixture of bioflavonoids in HPLC-45: % bioflavonoid in Bioflavonoid mixture 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

Example 1

(22) Experimental Methodology

(23) Preparation of Citrox Precursor: Citrox HXT powder was dissolved in methanol. 10% or 15% (w/w) of Citrox in methanol was used to formulate the precursor.

(24) Pre-treatment of Samples: the Polyethylene Terephthalate (PET) films and the silicon wafers were or were not initially He/O.sub.2 plasma pre-treated.

(25) In the case of the atmospheric Plasma Jet treatment systems (such as PlasmaStream™), the substrates were activated by two passes of a plasma formed with 5% O.sub.2 in He. The applied plasma power was 80%, the CNC speed was 7 mm/sec and the substrate to plasma jet orifice distance was 10 mm.

(26) In the case of a reel-to-reel atmospheric plasma (such as the Labline™ system), the substrates were activated by passing them three times through the treatment chamber containing a 5% O.sub.2 in a He plasma formed between two dielectric plates. The applied plasma power was 1000 W and the samples passed through the chamber at a speed of 1.5 m/min.

(27) Citrox coatings were deposited onto the plasma pre-treated or non pre-treated PET films and silicon wafers, using the nebulizers incorporated in the PlasmaStream™, Labline™ and

(28) PlasmaTreat systems. The use of a Citrox immersion technique was also evaluated as an alternative to spraying however the immersion method did not lead to a homogeneous coating. The spray technique yields coatings with the highest surface roughness and homogeneity.

(29) In the case of the PlasmaStream™ system, the depositions were carried out using the 10% of Citrox in a methanol precursor under three flow rates: 25, 50 and 100 μl/min and the number of passes varied between 2 and 16. Deposition parameters such as He flow rate, CNC speed and substrate to plasma jet orifice distance were kept constant at 5 l/min, 7 mm/sec and 2 mm respectively.

(30) In the case of the Labline™ system, the depositions were carried out using the 10% or 15% of Citrox in methanol and the aeroneb pro micropumb nebulizer. The flow rate was constant: 0.2 ml/min. The number of passes varied between 5 and 150. Deposition parameters such as N.sub.2 flow rate and speed were kept constant at 5 l/min and 1.5 m/min respectively.

(31) Finally in the case of the PlasmaTreat system, the plasma treatment parameters were chosen so as to replicate the degree of activation achieved previously using the PlasmaStream™ system. Following an iterative study, the following PlasmaTreat parameters were chosen to activate the PET samples: 90% Voltage, 50% PCT, 20 kHz, 3000 mbar, 250 mm/sec cnc speed, and the substrate to jet nozzle distance was set to 15.5 mm.

(32) Materials Characterization: The coatings were examined using optical microscopy, scanning electron microscopy (SEM), optical profilometry, water contact angle measurements, and Fourier transform infrared spectroscopy (FTIR).

(33) Antibacterial Activity of the Citrox deposited onto PET films, either plasma activated or not plasma activated, was examined against three bacterial species: S. aureus, E. coli and Salmonella.

(34) Antioxidant Activity of the Citrox deposited onto PET, either plasma activated or not plasma activated, was examined against turkey oxidation.

(35) Results

(36) 1. Plasma Jet Systems—Influence of Flow Rate/Number of Passes on Citrox Layer Thickness and Morphology

(37) From earlier studies it has been concluded that the activity of Citrox is dependent on the thickness of the deposited layer and possibly the surface roughness. The objective of this study was to determine how Citrox flow rate through the nebulizer influences the roughness and thickness of the Citrox coatings deposited onto silicon wafer substrates. In this study no pre-plasma activation was carried out. It is concluded that at flow rates of 25, 50 and 100 μl/min, where the overall quantity of Citrox deposited is constant, that the R.sub.a values are broadly similar. The thickness results also indicate that when the same total concentration is deposited it does not matter with respect to thickness if the layer is deposited for example from 8×25 μl/min or 2×100 μl/min.

(38) When pre-activation of the substrate is carried out using a plasma, substantially increased levels of Citrox coating adhesion is obtained when all other deposition parameters are fixed. As demonstrated in FIG. 1, nearly a threefold increase in the level of adherent Citrox is obtained after plasma activation (substrate contact angle 15°). This figure also illustrates the minimal effect of changing the helium/oxygen plasma (PlasmaStream™ (PS) system) for an air plasma (PlasmaTreat (PT) system) for the activation of PET. The level of Citrox attachment was found to be dependent on the water contact angle (WCA). At lower water contact angles (higher surface energy), higher levels of Citrox was found to adhere. The surface roughness results (FIG. 2) were found as expected to increase with an increase in the Citrox coating thickness. Note the very similar Citrox coating thickness and roughness values obtained for a given contact angle achieved with both the air and helium jet plasmas. The advantage of using the former is the significantly reduced processing costs in using an air plasma.

(39) Water Contact Angle Measurements and Fourier Transform Infrared Spectroscopy:

(40) The water contact angle and FTIR spectra were obtained for the Citrox layers deposited at the different flow rates. In all cases the water contact angle of the citrox coatings were highly hydrophilic with a contact angle lower than 5°. FTIR confirmed the presence of bioflavonoids in the samples. The characteristic bands (aromatic between 1480 and 1637 cm.sup.−1, —OH—phenolic at 1205, 1293, 1439 and 3476 cm.sup.−1, methoxylic at 1248 cm.sup.−1 and carbonylic at 1657 cm.sup.−1) were in good agreement with the FTIR spectra of naringin and neohesperidin, showing that the chemical functionality is preserved during the deposition process.

(41) Antibacterial Activity

(42) The Citrox coatings showed bactericidal effects against S. aureus, when Citrox was deposited onto the non He/O.sub.2 pre-treated substrates after either a) 4 passes of flow rate 50 μl/min or b) 2 passes of flow rate 100 μl/min. This test is obviously dependant on the concentration of bacteria exposed to the coated polymer and the ageing effect. In this study the concentration was 1×10.sup.8 Colony Forming Units (CFUs)/ml and the samples were tested one day after deposition. It was concluded therefore that the minimum inhibitory thickness is 60 nm (the associated R.sub.a is 500 nm).

(43) In the case of the PET polymer which had been treated to a He/O.sub.2 plasma, a similar Citrox thickness and roughness is also required but in this case the bactericidal effect was achieved after 2 passes of flow rate 50 μl/min or b) 1 pass of flow rate 100 μl/min.

(44) Antioxidant Activity

(45) The examination of the antioxidant effect of Citrox and Vitamin E coatings deposited onto the non He/O.sub.2 pre-treated PET substrates after 2 passes of flow rate 100 μl/min showed that Citrox is more effective than Vitamin E in reducing turkey oxidation with time (FIG. 3). Lipid oxidation is expressed as mg malonaldehyde (MDA)/kg of meat. Malonaldehyde is the product of polyunsaturated lipids degradation due to oxygen species. The higher the value the higher the oxidation.

(46) 2. Application of Citrox Coatings Using the Labline™ System—Influence of Number of Passes/Concentration on Citrox Layer Thickness and Morphology

(47) The objective of this study was to investigate the effect of using nebulizers mounted in either a reel-to-reel (Labline™) or atmospheric plasma jet (PlasmaStream™) system for the Citrox coating of plasma pre-activated PET polymers. In the case of this reel-to-reel web treatment system it was found that a much larger number of passes were required in order to obtain the same Citrox layer thickness and roughness as obtained using the PlasmaStream™ jet system. In this study the nebulizers used a precursor flow rate of 0.2 ml/min. A layer of thickness 60 nm in the case of the Plasma Jet system was achieved onto the non He/O.sub.2 pre-treated substrates after 4 passes of flow rate 50 μl/min, whereas in the case of the Labline™ system, similar thickness (70 nm) was achieved after 50 passes of flow rate 0.2 ml/min. As far as roughness is concerned, samples produced by the PlasmaStream™ Jet system presented higher average surface roughness (R.sub.a) (˜500 nm) in comparison to the Labline™ system (R.sub.a˜100 nm), for samples that had similar thickness (˜60-70 nm).

(48) Citrox Coating Deposition Using the Labline™ System

(49) The initial study focused on the influence of the number of passes on coating roughness and thickness. There was a broadly linear effect with these two parameters with the number of passes. The surface roughness was considerably lower than coatings with similar thickness deposited using the PlasmaStream™ system. For example in the case of the Labline™ system a thickness of ˜70 nm gave an R.sub.a value of 100 nm while a similar thickness with the PlasmaStream™ system gave a corresponding surface roughness of 500 nm.

(50) These Citrox coatings deposited at a concentration of 10% Citrox in methanol did not exhibit antibacterial activity. Studies were carried out at concentration of 20% but these mixtures could not be nebulized. The focus of the research therefore concentrated on 15% Citrox in methanol solutions. A large number of passes were also required in order for the coating to exhibit anti-bacterial activity against S. aureus. The effect of increasing the number of passes to 150 on both coating thickness and roughness was demonstrated. This study was carried out both with plasma activated and non plasma activated silicon wafers. Coating thicknesses and roughness values of several microns were obtained. An interesting observation is the effect of pre-treating the substrate with He/O.sub.2 plasma prior to the application of the Citrox layer. It was observed that the coating morphology was very different with the plasma activated surfaces exhibiting much larger aggregates of Citrox particles. Surface coverage (as evaluated by using the Image J software) was considerably higher with the plasma activated silicon as detailed in Table 2. This Table compares Citrox (15%) coatings deposited using the Labline™ system after 100 passes. There was a dramatic increase in both coating roughness and thickness with the He/O.sub.2 plasma treatment. This may be associated with the increase in water contact angle. In the case of the un-treated and plasma treated silicon wafers the contact angle values obtained were 68° to 20° respectively. The corresponding contact angles for the PET polymer were 71° to 55° respectively.

(51) TABLE-US-00002 TABLE 2 Influence of He/O.sub.2 plasma pre-treatment of silicon wafer substrates prior to the deposition of Citrox with 100 passes using the Labline ™ system. Untreated wafer Plasma treated wafer Surface coverage (%) 34% 55% Citrox layer 152 ± 17 1179 ± 89 thickness (nm) Citrox layer 619 ± 27 1350 ± 70 roughness R.sub.a (nm)
Antibacterial Activity

(52) The Citrox coatings showed bactericidal effects against S. aureus, when citrox was deposited onto the He/O.sub.2 or non pre-treated substrates after 100 and 150 passes. This means that the minimum inhibitory R.sub.a is 500 nm and the minimum inhibitory thickness is approximately 100 nm.

(53) 3. Ageing Effect Study of Labline™ Deposited Citrox Coatings

(54) The objective of this study was to investigate if there was an ageing effect for Citrox deposited onto silicon wafer substrates. This investigation was carried out by measuring changes in both roughness and thickness with time after Citrox deposition. As illustrated in FIGS. 6 and 7 both these parameters decreased significantly during the 35 day study. Moreover, the surface coverage of the Si wafer by Citrox decreased from 55% to 47.6% in the case of the He/O.sub.2 pre-treated substrate and from 34.4% to 23.3% in the case of the non He/O.sub.2 pre-treated one, 35 days after treatment. This may be due to the loss of a volatile component in the Citrox/methanol layer.

(55) FTIR was used to study changes in the relatively intensity of peaks with time. Overall peak intensity was observed to decrease with time. In particular, a decrease in the absolute intensity of the —OH phenolic band at 1200 cm-1 from 0.641 to 0.508 was observed 35 days after treatment in the case of the non He/O.sub.2 pre-treated substrate, whereas in the case of the He/O.sub.2 plasma pre-treatment the absolute intensity decreased from 0.862 to 0.726.

(56) Antibacterial Activity

(57) The objective of this study was to assess the longevity of the antibacterial effect of Citrox against S. aureus, E. coli and Salmonella. Citrox coatings were applied onto PET samples and the coated polymers were then stored by wrapping them in a polymer roll. The objective of this study was to determine if a roll of the coated polymer continued to exhibit antibacterial activity over time. The test samples were then removed from the roll just prior to the antibacterial study. It was attempted to correlate the level of antimicrobial activity with Citrox roughness and thickness. From FIGS. 10 to 12, which detail the level of anti-bacterial activity with time the following conclusions can be drawn— The Citrox coatings showed bactericidal effects against S. aureus up to a period of 21 days, when Citrox was deposited onto the He/O.sub.2 plasma pre-treated substrates, (100 passes, using the Labline™ system). A very small number of bacteria was observed on the test samples up to the completion of the 35 day test period indicating continued level of activity (FIG. 10). In the case of the Citrox coatings onto the non plasma treated substrates, with the same number of passes, the bactericidal effect was lost 21 days after Citrox deposition. As illustrated in FIG. 11, the Citrox coatings showed a higher bactericidal effect against E. coli with no growth observed after 35 days of deposition, when Citrox was deposited onto the Labline™ He/O.sub.2 plasma pre-treated substrates, after 100 passes. As before, for the Citrox coatings onto the non He/O.sub.2 pre-treated substrates, the bactericidal effect was lost 21 days after Citrox deposition. The Citrox coatings showed low Salmonella growth after 21 days of deposition, when Citrox was deposited onto the He/O.sub.2 pre-treated substrates, after 100 passes using the Labline™ system. As before, for the non treated substrates, the bactericidal effect was lost 21 days after treatment. In the case of the plasma activated surface, in contrast, continued activity was observed up to the 35 day test period (FIG. 12). FIGS. 10, 11 and 12 demonstrated the enhanced antibacterial performance of the Citrox coatings deposited on plasma activated polymers. The explanation for this is the substantial enhancement in thickness and roughness achieved for the coatings deposited on the pre-treated polymers. From these figures it is clear that very few bacteria adhere to the thicker and rougher coatings on the activated surfaces. FIG. 13 demonstrates the effect of spraying Citrox, either i) on a PET tray and then placing the chicken on top or i) on the chicken, on bacterial viability, in comparison to control that is chicken that was placed on a PET tray without any treatment. The antibacterial effect was measured on day 6 after spraying.
Conclusions The use of plasma pre-treatments substantially increases both the thickness and roughness of the Citrox layer deposited by spraying. For a given set of processing conditions up to a 3 fold increase in Citrox thickness was obtained on PET substrates and an 8 fold increase on silicon wafer substrates, which had been plasma pre-treated. This increase may be due to the higher energy surfaces enhancing the adhesion of the nebulized particles. The enhanced coating thickness yielded surfaces exhibiting antimicrobial performance longer periods after Citrox coating deposition. Comparing the anti-microbial performance of Citrox coatings deposited using the Labline™ and PlasmaStream™ systems it is clear that in the case of the Labline™ system a Citrox layer thickness of ˜70 nm is required, in contrast the thickness required using the PlasmaStream™ jet system was only ˜50 nm. The corresponding surface roughness values are approximately 100 and 500 nm respectively. This result indicates that higher surface roughness is required to achieve higher levels of antibacterial activity with Citrox. Use of an immersion technique for the application of Citrox did not lead to a homogeneous coating. The spray technique yields coatings with the highest surface roughness and homogeneity. Citrox coatings deposited onto the He/O.sub.2 plasma pre-treated substrates exhibited bactericidal effect against S. aureus, E. coli and Salmonella for a minimum of 35 days after the application of the Citrox layer (note very low levels of S. aureus and Salmonella observed after 21 days). A significant reduction in the level of turkey meat oxidation was observed after the application of Citrox onto PET trays. The antioxidant results compared favourably with Vitamin E coatings, a known commercial anti-oxidant.

Example 2

(58) Impregnation of Polystyrene Sheets with Citrox

(59) Polystyrene sheets were treated using plasma immersion ion implantation (PIII) with nitrogen ions of 20 keV energy for 800 seconds to create surface embedded radicals capable of covalently binding biologically active molecules (as described in Bilek et al, PNAS, 108(35):14405-14410, 2011, herein incorporated by reference). The PIII treated and untreated sheets were incubated in tubes containing Citrox concentrate (H PLC 45) or deionized water (for control) for 3 hours. In HPLC 45 or “Citrox BC” 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. After incubation the sheets were washed intensively in tubes with deionized water (3 times with shaking for 20 minutes and changing the tubes each time). After washing the sheets were dried overnight.

(60) FTIR ATR spectra were recorded with a Digilab spectrometer. The ATR crystal was Germanium trapezium, 45 degrees incident angle, 25 reflections. Spectral resolution was 4 cm.sup.−1 and the number of scans was 500.

(61) The spectra were analysed with Resolution-pro software. The spectra of the control samples were subtracted from that of the Citrox coated samples. The water vapour spectra and the Germanium crystal glue spectra were also subtracted. Baseline correction was done. The final spectra are presented in FIGS. 14 and 15.

(62) The spectra show that the components of Citrox remain on untreated and PIII treated polystyrene sheets. The clear and intense lines at 910, 1040, 1070, 1460, 1510, 1620-1650, 2860-2980 cm.sup.−1 are observed in the subtracted spectra for both untreated and treated sheets. The line in the range of 1750-1700 cm.sup.−1 is present only in the spectra of the untreated sheets.

(63) As the Germanium crystal was clean after contact with the sheets, the components of Citrox are strongly absorbed on the surface or diffused into bulk layers of the polystyrene sheets. Therefore, the Citrox components remain in the polystyrene sheets after incubation and washing with water.

(64) Conclusions

(65) Plasma treated polystyrene sheets contain higher levels of Citrox.