Bioflavonoid Compositions and Their Use

Abstract

The present invention relates to pharmaceutical compositions and their use in oral hygiene. More particularly, the compositions comprise one or more flavonoids, such as naringin and neohesperidine and polylysine and/or caprylic acid and/or a zinc salt. Such compositions may be in the form of, for example, a solution, gel, spray, chewing gum or paste suitable for use in the oral cavity. The composition may be used in reducing bacterial numbers on teeth, gums or other surfaces in the oral cavity.

Claims

1. A composition suitable for use in the oral cavity which comprises polylysine and/or caprylic acid, and/or a zinc salt, and one or more flavonoids of Formula (I) ##STR00003## wherein R.sub.1 is hydroxyl or methoxyl and R.sub.2 is hydrogen, hydroxyl, methoxyl and X is hydrogen or a saccharide.

2. A composition as claimed in claim 1 wherein the composition includes a mixture of flavonoids comprising naringin and neohesperidin and other flavonoids of Formula I.

3. A composition as claimed in claim 1 which comprises a mixture of flavonoids comprising 40% to 65% of naringin (wt/wt of flavonoids) and 20% to 35% of neohesperidin (wt/wt of flavonoids present).

4. A composition as claimed in claim 1 which comprises ε-polylysine of molecular weight of 3000 Da to 5000 Da.

5. A composition as claimed in claim 1 which comprises bioflavonoids and ε-polylysine in a ratio (wt/wt) of 50:1 to 1:20, for example 25:1 to 3:1.

6. A composition as claimed in claim 1 wherein the composition comprises 0.06% to 4% of bioflavonoids and 30 mg/L to 240 mg/L of ε-polylysine.

7. A composition as claimed in claim 4, which further comprises caprylic acid.

8. A composition as claimed in claim 4, which further comprises a zinc salt.

9. A composition as claimed in claim 1 for use in reducing bacterial numbers on teeth, gums or other surfaces within the oral cavity.

10. A composition for use as claimed in claim 9 in the form of a solution, gel, spray, chewing gum or paste.

11. A composition for use as claimed in claim 9 in the form of a toothpaste or mouthwash.

12. A toothpaste or mouthwash which comprises a composition as set forth in claim 1.

13. A toothpaste or mouthwash as claimed in claim 12 for use in treating gum disease or reducing plaque formation.

14. A gel, a spray or a chewing gum which comprises a composition as set forth in claim 1.

15. A gel, a spray or a chewing gum as claimed in claim 14 for use in treating gum disease or reducing plaque formation.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0058] FIG. 1 shows the inhibitory effect on Escherichia coli growth of pretreatment at different concentrations of Citrox and/or ε-polylysine in a high binding microtiter plate.

[0059] FIG. 2 shows the inhibitory effect on bacterial growth (E. coli) of direct exposure to Citrox or ε-polylysine compared to other antibacterial substances.

[0060] FIG. 3 shows that ε-polylysine promotes immobilization of Glucose oxidase onto silicate as assessed by measurement of residual activity in solution.

EXAMPLES

Example 1

[0061] Gel

[0062] Water (481.5 g; 96.3%) was added to a beaker and stirring commenced. Keltrol CG-SFT (9.0 g; 1.8%) was added and stirring continued until dissolved. Citrox powder (2.5 g; 0.5%) was added and stirring continued until dissolved. White willow bark extract (2.0 g; 0.4%) was added and stirring continued until dissolved. Glycerol (5.0 g; 1.0%) was added and stirring continued until dissolved.

[0063] The resulting viscous gel was de-aerated. The pH was 4-5. The viscosity 7000-10000 cp at 20° C. (spindle 4/0 rpm). The pH may be adjusted with citric acid if required to bring it within the stated range.

[0064] The Willow Bark extract contains 90% of salicylic acid.

[0065] The Citrox powder (Citrox Biosciences), hereinafter Citrox, comprises 7.5% of residues of extraction from bitter oranges together with the following mixture of bioflavonoids:

TABLE-US-00001 % bioflavonoid Bioflavonoid (component in mixture 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 Total 45%

Example 1 is Repeated in the Presence of Zinc Gluconate (0.85 wt-%)

Example 2

[0066] Foam Composition

[0067] This can be prepared by mixing ingredients as described in Example 1.

TABLE-US-00002 Salicylic acid 0.25% Citric acid 0.15% Bioflavonoid mixture 0.0375% (Example 1) ε-Polylysine or caprylic acid 0.015% Betaine BP20 1.0% Glycerine 0.5% Dermosoft GMCY 1.0% Water 97.0%

[0068] ε-Polylysine is from Everguard PL, Impag AG.

[0069] When tested against spores of Clostridium difficile (NCTC 11209) according to BS EN 13704, satisfactory sporicidal activity was found with a 15 minutes contact time at 20° C.

Example 3

[0070] Sanitizing Gel

[0071] This can be prepared by mixing as described in Example 1.

TABLE-US-00003 Keltrol CG-SFT 1.7% Bioflavonoid mixture 0.0375% (Example 1) ε-Polylysine or caprylic acid 0.015% Citric acid 0.15% Salicylic acid 0.25% Dermosoft GMCY 1.0% Glycerine 1.0% Water 95.8%

[0072] Such a gel provides satisfactory sporicidal activity against the spores of C. difficile.

Example 4

[0073] Toothpaste Formulation

TABLE-US-00004 Raw Material wt-% Water 29.499 K2 HPO4 0.290 KH2PO4 0.580 Veegum D 2.000 Stevia 0.080 Sodium Fluoride 950 ppm 0.229 Citric acid 0.200 Sodium benzoate 0.150 Disodiummonohydrogenphosphate 0.500 Sodium chloride 0.330 Hombitan AFDC 170 nm 1.000 Sorbitol 70% solution 26.700 Genuvisco TPC1 0.500 Glycerine 12.650 Mulsifan csa20 4.000 Sident 8 12.000 Sident 22 5 8.077 Aroma 0.700 Citrox 0.500 ε-Polylysine 0.015

Example 4 is Repeated in the Presence of Zinc Gluconate (0.85 wt-%)

Example 5

[0074] Immobilisation and release of antibacterial activity of Citrox in presence of ε-Polylysine

[0075] High Binding plates were pre-treated with 0.1 mL of pure Citrox/ε-Polylysine solutions incubated 60′ at room temperature. The concentrations of Citrox range from 0.06% to 4%. The ε-Polylysine concentrations range from 30 mg/L to 120 mg/L. The wells have been emptied and rinsed with 0.2 mL bi-distilled water. 0.2 mL of a diluted Escherichia coli culture (turbidity: 5 m OD at 600 nm) were given to the pre-treated wells and incubated for 3 hours at 37° C. Turbidity were measured at 600 nm and percentage of growth inhibition in comparison to untreated well calculated (FIG. 1).

Example 6

[0076] Antibacterial Activity of Citrox and ε-Polylysine Against Escherichia coli

[0077] The antibacterial activity of Citrox and ε-Polylysine has been tested by incubating 0.2 mL mini cultures of Escherichia coli (0.1 OD.sub.600nm) with varying concentrations of antibacterial substances. Growth inhibition was monitored by measuring turbidity of mini cultures after 5 hours of aerobe incubation at 37° C. under agitation. Solutions were diluted to the half in cascade. The lowest concentration of the substance yielding less than 2% (6% in case of ethanol) of growth in comparison to an untreated control is considered the minimal inhibitory concentration (MIC).

[0078] Short-Term Antibacterial Activity of Citrox Toward S. mutans (Bactericidal Effect)

TABLE-US-00005 TABLE 1 Summary of MIC (E. coli) extrapolated from FIG. 2 (1% corresponds to 10 g/L). Minimal inhibitory concentration (MIC) ε-Polylysine  4 mg/L Ampicillin 60 mg/L Ethanol 12.5% Citrox 0.16% Papaya extract 1.25%

[0079] The concomitant pretreatment with Citrox and ε-polylysine emphasizes the effectivity of Citrox pre-treatment. The apparent MIC of pretreating Citrox (0.25% pretreating Citrox with ε-polylysine 120 mg/L) approaches MIC of Citrox in directed exposure (0.16%).

[0080] With ε-polylysine 60 mg/L, 0.5% pretreating Citrox is required to reach inhibition, which is still significantly lower than the 2% required in absence ε-polylysine.

[0081] 30 mg/L ε-polylysine corresponds to the highest concentration that does not affect significantly the antibacterial effect of Citrox pretreatment. Therefore, the preferred range of synergy is between 30 mg/L and 240 mg/L of ε-polylysine, and 0.06% to 4% of Citrox.

Example 7

[0082] Immobilisation of Enzymatic Activity in Presence of ε-Polylysine

[0083] 0.5 mg/mL glucose oxidase enzyme was incubated for 15 minutes at room temperature in an artificial saliva buffer composition including:

TABLE-US-00006 Na.sub.2HPO.sub.4 G/L 0.26 NaCL 6.70 KSCN 0.33 KH2PO4 0.20 KCL 1.20 NaHCO3 1.50,

[0084] with 2 mg/mL silicate and 0.25 mg/mL of an amino acid based compound, bovine serum albumin (BSA), lysine or ε-polylysine. The mixture was centrifuged in order to pellet the silicate and the supernatant tested for its glucose oxidase activity in presence of glucose, lactoperoxidase and a chromogen substrate (ABTS). The resulting activities were compared with the enzyme incubated with silicate in absence of an amino acid compound (FIG. 3).

Example 8

[0085] Antibacterial Activity of ε-Polylysine Against S. mutans

[0086] The antibacterial activity of ε-polylysine was semi-quantitatively assessed in a inhibition zone assay. 0.1 mL of a confluent Streptococcus mutans culture was spread over a Brain Heart Infusion (BHI) agar plate. Filter platelets (5 mm diameter) soaked with 0.01 mL substance were applied on plate. Plates were incubated 48 h at 37° C. under anaerobe conditions. The diameter of outer limit of the inhibition zones were measured, the diameter of the platelet deduced and the half result considered the radius of inhibition rings. Concentrations were tested in triplicate and their results averaged.

TABLE-US-00007 TABLE 2 Radius of inhibition rings around platelets diffusing the corresponding antibacterial substance across a Streptococcus mutans culture on agar plate. Antibacterial substance Radius ε-Polylysine 1000 mg/L 6.5 mm ε-Polylysine 100 mg/L 2.5 mm ε-Polylysine 10 mg/L 0.5 mm Ethanol 5 mm Water peroxide 0.1% 1.5 mm Water peroxide 1% 5 mm

[0087] Repetition of the preceding test with inclusion of Citrox substantially increases the radius of antibacterial inhibition.

Example 9

[0088] Short-Term Antibacterial Activity of Citrox Against S. mutans (Bactericidal Effect)

[0089] The short-term antibacterial activity of Citrox was semi-quantitatively assessed in a short exposure assay. A S. mutans culture, diluted in order to contain 5000 Colony Forming Units, was incubated for 10 minutes in presence of the antibacterial substance and extemporarily plated onto a BHI agar plate. Plates were incubated at 37° C. under anaerobe conditions and colony counted. Citrox at final concentrations of 5% resulted in complete absence of colony formation, as did water peroxide 0.05% under the same conditions. Citrox 0.5% reduced the count of CFU to the half (<3000 CFU).

Example 10

[0090] Antibacterial Effect Upon Delayed Release of Citrox is Enhanced by ε-Polylysine

[0091] The tests were performed on a plate upon which ε-polylysine strongly absorbs onto its solid surface and is not significantly released in solution. It therefore does not affect the growth of bacteria in suspension, although it prevents the surface from being colonized by bacteria.

[0092] The tests results demonstrate that the antibacterial activity of Citrox is retained by the high binding plates but also allows its release in solution, thus allowing to affect the growth of the bacteria in suspension, as well as on the solid surface. Quantitatively, pre-treating a high binding plate with Citrox 2% reaches the same inhibitory effect of a direct exposure with Citrox 0.16% (MIC Citrox).

Example 11

[0093] Tested Concentrations of Citrox and ε-Polylysine Employed are as Follows:

TABLE-US-00008 Sample No. Citrox wt-% Polylysine wt-% 1 4 0.012 2 4 0.06 3 4 0.03 4 4 0 5 2 0.012 6 2 0.06 7 2 0.03 8 2 0 9 1 0.012 10 1 0.06 11 1 0.03 12 1 0 13 0.5 0.012 14 0.5 0.06 15 0.5 0.03 16 0.5 0 17 0.25 0.012 18 0.25 0.06 19 0.25 0.03 20 0.25 0 21 0.06 0.012 22 0.06 0.06 23 0.06 0.03 24 0.06 0 25 0 0.012 26 0 0.06 27 0 0.0 28 0.06 0

[0094] These demonstrated that using both agents proved anti-bacterially more effective than the control tests employing singular composition comprising either Citrox or ε-Polylysine (sample No 4, 8, 12, 20, 24-28).

Example 12

[0095] Sterile deionised water was inoculated with a range of different microorganisms of concern in drinking water. The inoculated water was held at 20° C. overnight to allow the cells to acclimatise. The water was then treated with different concentrations of Citrox or Citrox with caprylic acid. The concentrations used were 0.05, 0.1, 0.25, 0.5 and 1.0%. The inoculated water containing the antimicrobial was held at 20° C. for three hours. The same inoculated water, without added antimicrobial, but still held at 20° C. for three hours, was used as a control. The inoculated microorganisms were enumerated after the three-hour hold time to determine the level of inactivation. The entire experiment was repeated on two separate occasions. Further details for each type of microorganism are given below.

[0096] Escherichia coli

[0097] Sterile deionised water was inoculated with a 5-strain cocktail of these pathogenic strains:

[0098] NCTC 9706

[0099] NCTC 9707

[0100] NCTC 11601

[0101] NCTC 11602

[0102] NCTC 11603

[0103] The strains were grown for 18 h at 37° C. in tryptone soya broth+0.6% yeast extract (TSBYE). Cells in stationary phase were harvested by centrifugation, washed in PBS and diluted in an appropriately sterile deionised water to give an initial inoculum level of approximately 105-106 CFU/ml. Enumeration was by spread plating on tryptone soya agar+0.6% yeast extract (TSAYE), with incubation at 37° C. for 24 h.

[0104] Enterococcus faecalis

[0105] NCTC 8213

[0106] This strain was grown, inoculated and enumerated as described for E. coli.

[0107] Sulphite-reducing Clostridia

[0108] Sterile deionised water was inoculated with a cocktail containing the following four species:

[0109] Clostridium perfringens ATCC 13124

[0110] Clostridium sporogenes NCIMB 532

[0111] Clostridium tyrobutyricum DSM 663

[0112] Clostridium bifermentans NCTC 506

[0113] Broth cultures were grown in cooked meat medium+1% glucose (steamed to remove oxygen and cooled before use) which was incubated anaerobically for 18 h at 37° C. Cells were harvested by centrifugation, washed in PBS and diluted appropriately in sterile deionised water to give an initial inoculum level of approximately 105-106 CFU/ml. Enumeration was by spread plating on TSAYE, incubated anaerobically at 37° C. for 24 h.

[0114] Yeasts

[0115] Sterile deionised water was inoculated with a cocktail containing the following five species:

[0116] Candida tropicalis NCYC 4

[0117] Candida solani NCYC 2570

[0118] Rhodotorula glutinis NCYC 60

[0119] Metschnikowia pulcherrima NCYC 371

[0120] Debaryomyces hansenii NCYC 9

[0121] Broth cultures were grown in malt extract broth which was incubated in an orbital incubator for 72 h at 25° C. Cells were harvested by centrifugation, washed in PBS and diluted appropriately in sterile deionised water to give an initial inoculum level of approximately 105-106 CFU/ml. Enumeration was by spread plating on malt extract agar, incubated aerobically at 25° C. for 72 h.

[0122] Vibrio parahaemolyticus

[0123] Sterile deionised water was inoculated with a cocktail containing the following four strains:

[0124] Vibrio parahaemolyticus NCTC 1165

[0125] Vibrio parahaemolyticus NCTC 1902

[0126] Vibrio parahaemolyticus AHPND A3

[0127] Vibrio parahaemolyticus AHPND D4

[0128] These strains were grown, inoculated and enumerated as described for E. coli.

[0129] Results (Reduction in Counts after 3 hr Exposure)

[0130] The Citrox reduced counts by from 3.5 log order at 0.1% to 6.5 log orders at 1%. For the formulation also comprising caprylic acid, the reductions were increased by about 1 log order.

[0131] The Citrox reduced counts of Enterococcus faecalis by 4 log orders at 0.1% to 7 log orders at 1%. For the formulation also comprising caprylic acid, the reductions were increased by about 0.7 log orders.

[0132] The Citrox reduced counts of Clostridia by 2 log orders at 0.1% and 3 log orders at 1%. For the formulation also comprising caprylic acid, the reductions were increased by about 0.5 log orders.

[0133] The Citrox reduced counts of yeast by 2 log orders at 0.1% and 6 log orders at 1%. For the composition also comprising caprylic acid, the reductions were increased by about 0.5 log orders.

[0134] The Citrox reduced counts of V. parahaemolyticus by 1.2 log order at 0.1% and 6 log orders at 1%. The composition also comprising caprylic acid increased the reductions by 0.4 log orders at 0.1% and 0.8 log orders at 2%.

Example 13

[0135] Water was sampled from three separate locations on an urban river assumed to have a relatively high microbial load. These water samples were treated with 1% w/w Citrox and 1% w/w Citrox with caprylic acid for three hours at 20° C. The following enumerations were determined in the water with and without added antimicrobials.

[0136] Total count: yeast extract agar incubated at 37° C. for 48 h

[0137] Coliforms: VRB agar with overlay incubated at 37° C. for 24 h

[0138] E. coli: TBX agar incubated at 37° C. for 24 h

[0139] Sulphite-reducing Clostridia: TSC agar with overlay incubated anaerobically at 37° C. for 24 h

[0140] Enterococci: Slanetz and Bartley agar incubated at 37° C. for 4 h and then at 44° C. for 44 h

[0141] Pour plating was used for all enumerations of naturally contaminated water to lower the limit of detection.

[0142] Results

[0143] Inactivation of Microorganisms in Contaminated Water

[0144] Coliforms were present at a level of 2.6 log in untreated water and were significantly reduced with Citrox and Citrox plus caprylic acid, with none detected after treatment. Results for E. coli were similar to coliforms, with initial counts of around 2 log reduced to below the limit of detection (1 CFU/ml) after treatment. Numbers of Enterococci were reduced from 1.6 log to below the limit of detection after treatment with both formulations.