Mixture to increase the effectiveness of antiseptics and/or disinfectants, an agent containing the mixture, and the use of this mixture

Abstract

The invention relates to the increase (improvement) of the effectiveness of antiseptics and/or disinfectants administered in a mixture with hydrolytic or proteolytic enzymes to a mucous membrane, a skin of a mammal or inanimate surfaces in the form of a solution, gel, paste, capsules or other as an agent. The use of the mixture is also stated.

Claims

1. An antiseptic or a disinfectant composition comprising chlorhexidine digluconate in an amount of 0.001% to 1% by weight and proteolytic or hydrolytic enzyme in an amount of 0.01% to 2% by weight based on the total weight of the composition and further comprising buffered saline and water.

2. The antiseptic or a disinfectant composition according to claim 1, wherein the amount of chlorhexidine digluconate is 0.01% to 0.12% by weight and the amount of proteolytic or hydrolytic enzyme is 0.01% to 0.4% by weight based on the total weight of the composition.

3. The antiseptic or a disinfectant composition according to claim 1, comprising two different enzymes.

4. The antiseptic or a disinfectant composition according to claim 3, wherein the two different enzymes are each a hydrolytic enzyme.

5. The antiseptic or a disinfectant composition according to claim 4, wherein the hydrolytic enzyme is selected from protease, lipase or amylase.

6. The antiseptic or a disinfectant composition according to claim 1, comprising two hydrolytic enzymes, wherein the two hydrolytic enzymes are both proteases, lipases or amylases.

7. The antiseptic or a disinfectant composition according to claim 6, wherein the protease is selected from the group consisting of trypsin, chymotrypsin, bromelain and papain.

8. The antiseptic or a disinfectant composition according to claim 7, wherein the amount of chlorhexidine digluconate is 0.001% to 1% by weight, the amount of trypsin or chymotrypsin is 0.01% to 0.2% by weight and the content of bromelain is 0.01% to 0.4% by weight based on the total weight of the composition.

9. The antiseptic or a disinfectant composition according to claim 8, wherein the amount of chlorhexidine digluconate is 0.035% by weight, the amount of trypsin or chymotrypsin is 0.2% by weight and the content of bromelain is 0.4% by weight based on the total weight of the composition.

10. The antiseptic or a disinfectant composition according to claim 1, further comprising a natural extract from a plant with anti-inflammatory, antibacterial or calming effect.

11. The antiseptic or a disinfectant composition according to claim 10, wherein the extract is selected from the group consisting of cranberry, Agrimonia eupatoria, peppermint, Matricaria chamomile, clove, fennel, Salvia officinalis, Potentilla erecta, and a mixtures thereof.

12. The antiseptic or a disinfectant composition according to claim 1, further comprising humectant, filler, whitener, aroma, preservative, thickener, pH regulator, enzyme stabilizer, sweetener, anticaries substance, abrasive substance, viscosity regulator, or mucoadhesive polymer.

13. A method of treating microbial, viral or mycotic illnesses or reducing discoloration of tooth enamel or tongue, the method comprising administering the composition according to claim 1 to a subject in need thereof.

14. The method of claim 13, wherein the microbial illness is related to plaque formation, and subsequent formation of caries and periodontitis.

15. The method of claim 13, wherein the composition is administered orally.

16. The method of claim 13, wherein the composition is in the form of toothpaste, tooth gel, mouthwash, lozenge, chewing gum, gargle, mouthwash, solution or paste for the gums, oral suspension, drops, spray, glaze, oral gel, sublingual, buccal, or chewable tablet, oral capsule, emulsion, cream, mucoadhesive film or disk.

17. A method of treating inanimate surface comprising the step of contacting the composition according to claim 1 with the inanimate surface.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1Shows the different sizes of zones of inhibition 1-6 of the mixture according to the invention compared to the art

(2) FIG. 2Shows the size of zones of inhibition 1-3 depending on the concentration of chlorhexidine digluconate according to art, and with mixture according to the invention

(3) FIG. 3Shows the size of zones of inhibition 1-3 of the mixture according to the invention compared to the art

(4) FIG. 4Shows the effect of proteolytic enzymes on the amount of chlorhexidine digluconate required to inhibit the growth of a mixed bacterial culture.

(5) FIG. 5Shows the effect of proteolytic enzymes on the amount of chlorhexidine digluconate required to inhibit the growth of collection strain Streptococcus mutans.

(6) FIG. 6Shows the effect of proteolytic enzymes on the amount of chlorhexidine digluconate required to inhibit the growth of collection strain Streptococcus salivarius.

(7) FIG. 7Shows the effect of proteolytic enzymes on the amount of chlorhexidine digluconate required to inhibit the growth of collection strain Streptococcus sanguinis.

(8) FIG. 8Shows the effect of proteolytic enzymes on the amount of chlorhexidine digluconate required to inhibit the growth of a bacterial culture.

EXAMPLES

Example 1

(9) Example 1 tested the hypothesis that an aqueous solution of chlorhexidine digluconate with variable concentration, with proteolytic enzymes with a constant concentration (fixed concentration) will form the same zone of inhibition of a mixed bacterial culture growth isolated from the saliva as a chlorhexidine solution on its own about twice the concentration. In view of the fact that cosmetic products, designed for hygiene of mucous membranes and the soft tissue of a mammal, i.e. a human or an animal, frequently use chlorhexidine in a concentration of 0.06% by weight and 0.12% by weight, these two concentrations were considered to be the reference mixtures (Sample 1, Sample 3). Mixtures which contained besides chlorhexidine digluconate also proteolytic enzymes were considered to be the tested mixtures (Sample 2, Sample 4).

(10) The inhibitory potentials of chlorhexidine digluconate on its own (Biomedica s.r.o, Czech Republic) and the mixture of chlorhexidine digluconate and proteolytic enzymes of trypsin (2500 IU/mg, Biomedica s.r.o, Czech Republic) and bromelain (2000 GDU/g, Biomedica s.r.o, Czech Republic) were tested by a disk diffusion test.

(11) Stock solutions were prepared of each substance; 1% by weight solution of chlorhexidine digluconate in H.sub.2O; 1% by weight solution of trypsin in phosphate buffered saline (PBS); 1% by weight solution of bromelain in PBS. Mixtures (Table 1) of 1 ml were prepared from these stock solutions. Diagnostic discs (I-TEST PLUS s.r.o., Czech Republic) were immersed in the mixture for 3 minutes. Mixed bacterial culture isolated from saliva was aseptically triturated on the Petri dish with Brain Heart Infusion Agar (Oxoid; Thermo Fisher Scientific, UK) and individual diagnostic disks impregnated with the mixture were placed there. After incubation, which lasted 48 hours in 5% CO.sub.2 atmosphere at 37 C., zones of inhibition were deducted using a ruler.

(12) The results are shown in FIG. 4. During the experiment one concentration of trypsin (0.2% by weight) and bromelain (0.4% by weight) was tested.

(13) TABLE-US-00001 TABLE 1 Tested mixtures of chlorhexidine digluconate and proteolytic enzymes. % (wt.) Mixture 1 Chlorhexidine 0.12 digluconate Trypsin 0 Bromelain 0 H.sub.2O 99.88 Mixture 2 Chlorhexidine 0.06 digluconate Trypsin 0.2 Bromelain 0.4 H.sub.2O 99.34 Mixture 3 Chlorhexidine 0.06 digluconate Trypsin 0 Bromelain 0 H.sub.2O 99.94 Mixture 4 Chlorhexidine 0.03 digluconate Trypsin 0.2 Bromelain 0.4 H.sub.2O 99.37 Mixture 5 Chlorhexidine 0 digluconate Trypsin 0 Bromelain 0 H.sub.2O 100

(14) The mixture of 0.06% by weight of chlorhexidine digluconate and proteolytic enzymes of trypsin and bromelain at a concentration of 0.2% by weight, or rather 0.4% by weight is able to inhibit the growth of the mixed culture isolated from the saliva equally well as the solution of chlorhexidine digluconate alone at a concentration of 0.12% by weight. The use of the given concentration of proteolytic enzymes in the mixture of chlorhexidine digluconate can reduce the concentration of chlorhexidine active substance 2 with the same bacteriostatic effect.

(15) The mixture of 0.03% by weight of chlorhexidine digluconate and the proteolytic enzymes of trypsin and bromelain at a concentration of 0.2% by weight, or rather 0.4% (wt.) is able to inhibit the growth of the mixed culture isolated from the saliva roughly as well as the solution of chlorhexidine digluconate at a concentration of 0.06% by weight. Using proteolytic enzymes in a mixture with the active ingredient of chlorhexidine digluconate can reduce the concentration of chlorhexidine active substance 2 with the same bacteriostatic effect.

Example 2

(16) Example 2 verified the minimum concentration of chlorhexidine digluconate (variable concentration) in combination with proteolytic enzymes with a constant concentration (fixed concentration) in aqueous solution which still has inhibitory effect on collection strains. Chlorhexidine digluconate in a concentration of 1% by weight (Sample 1) was taken as a reference. Mixtures which contained besides chlorhexidine digluconate also proteolytic enzymes were considered to be the tested mixtures (Sample 2 to Sample 5).

(17) The inhibitory potentials of chlorhexidine digluconate on its own (Biomedica s.r.o, Czech Republic) and a mixture of chlorhexidine digluconate and proteolytic enzymes of trypsin ((2500 IU/mg, Biomedica s.r.o, Czech Republic) and bromelain (2000 GDU/g, Biomedica s.r.o, Czech Republic) were tested by a disk diffusion test.

(18) Stock solutions were prepared of each substance; 1% by weight solution of chlorhexidine digluconate in H.sub.2O; 1% by weight solution of trypsin in phosphate buffered saline (PBS); 1% by weight solution of bromelain in PBS. Mixtures (Table 2) of 1 ml were prepared from these stock solutions. Diagnostic discs (I-TEST PLUS s.r.o., Czech Republic) were immersed in the mixture for 3 minutes.

(19) Suspensions of Streptococcus mutans (strain 6699, SZU, CZ), Streptococcus salivarius (strain 6713, SZU, CZ) or Streptococcus sanguinis (strain 6714, SZU, CZ) was aseptically triturated on the Petri dish with Brain Heart Infusion Agar (Oxoid; Thermo Fisher Scientific, UK) and individual diagnostic disks impregnated with the mixture were placed there. After incubation, which lasted 48 hours in 5% CO.sub.2 atmosphere at 37 C., zones of inhibition were deducted using a ruler.

(20) The results are summarized in FIGS. 5-7.

(21) TABLE-US-00002 TABLE 2 Tested mixtures of chlorhexidine digluconate and proteolytic enzymes % (wt.) Mixture 1 Chlorhexidine 1.0 digluconate Trypsin 0 Bromelain 0 H.sub.2O 99 Mixture 2 Chlorhexidine 0.12 digluconate Trypsin 0.2 Bromelain 0.4 H.sub.2O 99.28 Mixture 3 Chlorhexidine 0.06 digluconate Trypsin 0.2 Bromelain 0.4 H.sub.2O 99.34 Mixture 4 Chlorhexidine 0.03 digluconate Trypsin 0.2 Bromelain 0.4 H.sub.2O 99.37 Mixture 5 Chlorhexidine 0.01 digluconate Trypsin 0.2 Bromelain 0.4 H.sub.2O 99.39 Mixture 6 Chlorhexidine 0 digluconate Trypsin 0 Bromelain 0 H.sub.2O 100

(22) The results show that the mixture of chlorhexidine digluconate in a concentration of 0.12% by weight used in the mixture with proteolytic enzymes retains about 70% inhibition effectiveness compared with the solution of chlorhexidine at a concentration of 1% by weight alone. In the case of collection strains of Streptococcus mutans and Streptococcus salivarius, growth inhibition was observed even at a concentration of 0.01% by weight of chlorhexidine digluconate in combination with the proteolytic enzymes.

Example 3

An Agent Containing a Volume of the Mixture of the Invention

(23) The agent to the invention comprising the mixture is in the form of a toothpaste, a mucoadhesive gel with hydrophilic or hydrophobic properties with a possibility of administration to the oral cavity or the vaginal mucosa or the skin of a mammal, i.e. a human or an animal in the form of capsules or other lozenges, as a solution, or in another form such as a gel, which can be applied to the oral cavity, on other mucous membranes or on non-living materials.

(24) An advantageous embodiment comprises 0.03-1% by weight of chlorhexidine digluconate and proteolytic enzymes mixture of trypsin and bromelain in concentrations of 0.01-2% by weight. This mixture may be enriched by other enzymes from the group of hydrolytic enzymes, directly proteolytic enzymes (e.g. chymotrypsin, papain, etc.). Or by other enzymes from the class of hydrolytic enzymes (e.g. lipase, amylase and others.).

Example 3a

An Agent Containing the Volume of the Mixture of the Invention Toothpaste

(25) TABLE-US-00003 Ingredient % (by weight) Glycerin anhydrous 13 Kelcogel CG LA 0.1 Water 9.175 Sorbit 70% 49.72 Sodium sacharinate 0.05 Sodium fluorid 0.12 Sodium benzoate 0.6 Chlorhexidine 0.175 digluconate (20% wt.) Magnesium sulphate 0.05 Hydrated silica 20.8 Trypsin 0.2 Bromelain 0.4 Menthol 0.04 Mint 0.55 Anethol 0.02 TEGO betain ZF 5

Example 3b I

An Agent Containing the Volume of the Mixture of the Invention Mucoadhesive Gel

(26) TABLE-US-00004 Ingredient % (by weight) H.sub.2O 91.24 Glycerin 3.5 Hydroxyethylcelulose 3.0 Hydroxypropylmethylcelulose 2.0 Trypsin 0.2 Chlorhexidine digluconate 0.06

Example 3b II

An Agent Containing the Volume of the Mixture of the Invention Mucoadhesive Gel

(27) TABLE-US-00005 Ingredient % (by weight) H.sub.2O 91.19 Glycerin 3.5 Hydroxyethylcelulose 3.0 Hydroxypropylmethylcelulose 2.0 Trypsin 0.2 Chlorhexidine digluconate 0.06 Cetylpyridinium chloride 0.05

Example 3b III

An Agent Containing the Volume of the Mixture of the Invention Mucoadhesive Gel

(28) TABLE-US-00006 Ingredient % (by weight) H.sub.2O 90.465 Glycerin 3.5 Hydroxyethylcelulose 3.0 Hydroxypropylmethylcelulose 2.0 Sodium benzoate 0.8 Trypsin 0.2 Chlorhexidine digluconate 0.035

Example 4

(29) Example 4 verified the inhibitory ability of the agent of the invention, a toothpaste according to Example 3a (Sample 1) compared with widely available industrially produced agents (products) (Samples 2-5) containing only chlorhexidine digluconate in different concentrations, and for comparison a toothpaste according to Example 3a, but without the addition of chlorhexidine and proteolytic enzymes (Sample 6) as shown in Table 3.

(30) Suspensions of Escherichia coli (typical example of gram-negative facultative anaerobic microorganism), was aseptically triturated on the Petri dish with Lauria Broth agar (Oxoid; Thermo Fisher Scientific, UK). Immediately 0.4 ml of each sample was placed there. After incubation, which lasted 24 hours at 35 C., zones of inhibition were detected (FIG. 1).

(31) TABLE-US-00007 TABLE 3 List of examined application samples Chlorhexidine digluconate concentration Sample [% by weight] 1 Mixture of an advantageous embodiment of the 0.035 invention - Example 3a 2 Commercially available toothpaste 1 0.06 3 Commercially available toothpaste 2 0.12 4 Commercially available toothpaste 3 0.12 5 Commercially available toothpaste 4 0.2 6 Mixture of an advantageous embodiment of the invention - Example 3a: without addition of 0 chlorhexidine digluconate and proteolytic enzymes

(32) FIG. 1 shows that the mixture of an advantageous embodiment of the inventionExample 3a, which is a toothpaste (Sample 1) forms a wide zone of inhibition. On the contrary, this zone cannot be observed with the mixture of an advantageous embodiment of the inventionExample 3aa toothpaste without the addition of chlorhexidine and proteolytic enzymes (Sample 6), which is fully in line with the assumption.

(33) Furthermore, FIG. 1 shows that sample 1 forms a comparable or wider zone of inhibition than a widely available toothpaste 4 (Sample 5) having a concentration of chlorhexidine digluconate 0.2% by weight. Diffluence was observed in Samples 2 and 3, which can be attributed to the specific composition of the products, while exposing the samples to constant temperature of 35 C.

(34) Therefore, it can be concluded that the proposed mixture of the advantageous embodimenta toothpaste (Sample 1) with chlorhexidine digluconate at a concentration of 0.035% by weight and proteolytic enzymes trypsin at a concentration of 0.2% by weight and bromelain at a concentration of 0.4% by weight inhibits in vitro microbial growth at least as well as a widely available toothpaste containing chlorhexidine digluconate at a concentration of 0.2% by weight. During concomitant administration of proteolytic enzymes and chlorhexidine it is, therefore, possible to reduce the concentration of chlorhexidine in the mixtures 5-6 times while maintaining the same inhibitory activity of the mixture.

Example 5

(35) Example 5 verified inhibitory ability of the agent comprising chlorhexidine and one proteolytic enzyme (trypsin2500 IU/mg) in the form of gel (embodiment example 3b I) in comparison with the agentsgels (products) containing only chlorhexidine digluconate in various concentrations (Table 4a).

(36) Suspensions of Escherichia coli (typical example of gram-negative facultative anaerobic microorganism), was aseptically triturated on the Petri dish with Lauria Broth agar (Oxoid; Thermo Fisher Scientific, UK). Immediately 0.4 ml of each sample was placed there. After incubation, which lasted 24 hours at 35 C., zones of inhibition were detected (FIG. 2).

(37) TABLE-US-00008 TABLE 4a List of examined application samples Chlorhexidine digluconate concentration Sample [% (wt.)] 1 Mixture of an advantageous embodiment of the 0.06 invention - Example 3b I 2 Commercially available gel 1 0.15 3 Commercially available gel 2 0.5

(38) FIG. 2 shows that the mixture of the advantageous embodiment of the inventionExample 3b I: a mucoadhesive gel (Sample 1) forms the wide zone of inhibition wider than that which is observable with widely available gel 1 containing chlorhexidine digluconate at a concentration of 0.15% by weight, and smaller than commonly available gel 2 containing chlorhexidine digluconate at a concentration of 0.5% by weight.

(39) Therefore, it can be concluded that the proposed mixture of an advantageous embodimenta mucoadhesive gel (Sample 1) with chlorhexidine digluconate at a concentration of 0.06% by weight and a proteolytic enzyme trypsin at a concentration of 0.2% by weight inhibits in vitro microbial growth clearly better than a comparable product containing chlorhexidine at a concentration of 0.15% by weight, and worse than a comparable product with chlorhexidine at a concentration of 0.5% by weight. During concomitant administration of one proteolytic enzyme and chlorhexidine digluconate it is, therefore, possible to reduce the concentration of chlorhexidine digluconate in the mixture 2.5-3 times while maintaining the same inhibitory activity of the mixture.

Example 6

(40) Example 6 tested inhibitory ability of the agent comprising chlorhexidine and one proteolytic enzyme (trypsin2500 IU/mg) in the form of gel (embodiment example 3b III) in comparison with the agentsgels containing only chlorhexidine digluconate in different concentrations, or Example 3b IIIan embodiment of the invention, but without the addition of chlorhexidine and proteolytic enzymes (Table 4b).

(41) Suspensions of Escherichia coli (typical example of gram-negative facultative anaerobic microorganism), was aseptically triturated on the Petri dish with Lauria Broth agar (Oxoid; Thermo Fisher Scientific, UK). Immediately 0.4 ml of each sample was placed there. After incubation, which lasted 24 hours at 35 C., zones of inhibition were detected (FIG. 3).

(42) TABLE-US-00009 TABLE 4b List of examined application samples Chlorhexidine digluconate concentration Sample [% (wt.)] 1 Mixture of an advantageous embodiment of the 0.035 invention - Example 3b III 2 Commercially available gel 1 0.15 3 Mixture of an advantageous embodiment of the 0 invention - Example 3b III: without addition of chlorhexidine digluconate and proteolytic enzymes

(43) FIG. 3 shows that the mixture of the advantageous embodiment of the inventionExample 3b III: a mucoadhesive gel (Sample 1) forms a similarly wide zone of inhibition which is observable with widely available gel 1 containing chlorhexidine digluconate at a concentration of 0.15% by weight.

(44) On the contrary, this zone cannot be observed with the mixture of an advantageous embodiment of the inventionExample 3b IIIa mucoadhesive gel without the addition of chlorhexidine and proteolytic enzymes (Sample 3), which is fully in line with the assumption.

(45) Therefore, it can be concluded that the proposed mixture of an advantageous embodimenta mucoadhesive gel (Sample 1) with chlorhexidine digluconate at a concentration of 0.035% by weight and a proteolytic enzyme trypsin at a concentration of 0.2% by weight inhibits in vitro microbial growth similar to a comparable product containing chlorhexidine at a concentration of 0.15% by weight. During concomitant administration of one proteolytic enzyme and chlorhexidine digluconate it is therefore possible to reduce the concentration of chlorhexidine digluconate in the mixture 2.5-3 times while maintaining the same inhibitory activity of the mixture.

Example 7

(46) Example 7 tested the hypothesis that an aqueous solution of chlorhexidine digluconate with proteolytic enzymes will form the same zone of inhibition of a bacterial culture growth as a chlorhexidine solution on its own with about twice the concentration. A chlorhexidine solution at a concentration of 0.06% by weight was considered a reference mixture (Sample 1). A mixture which contained besides chlorhexidine digluconate also proteolytic enzymes was considered to be the tested mixture (Sample 2).

(47) The inhibitory potentials of chlorhexidine digluconate on its own (Biomedica s.r.o, Czech Republic) and the mixture of chlorhexidine digluconate and proteolytic enzymes of chymotrypsin (1000 IU/mg, Biomedica s.r.o, Czech republic) a papain (80000 IU/g, Biomedica s.r.o, Czech republic) were tested by a disk diffusion test.

(48) Stock solutions were prepared of each substance; 1% by weight solution of chlorhexidine digluconate in H.sub.2O; 1% by weight solution of chymotrypsin in phosphate buffered saline (PBS); 1% by weight solution of papain in PBS. Mixtures (Table 5) of 1 ml were prepared from these stock solutions. Diagnostic discs (I-TEST PLUS s.r.o., Czech Republic) were immersed in the mixture for 3 minutes. Suspensions of Escherichia coli (typical example of gram-negative facultative anaerobic microorganism), was aseptically triturated on the Petri dish with Lauria Broth agar (Oxoid; Thermo Fisher Scientific, UK) and individual diagnostic disks impregnated with the mixture were placed there. After incubation, which lasted 24 hours at 35 C., zones of inhibition were detected.

(49) The results are shown in FIG. 8.

(50) TABLE-US-00010 TABLE 5 Tested mixtures of chlorhexidine digluconate and proteolytic enzymes. % (wt.) Mixture 1 Chlorhexidine 0.06 digluconate Chymotrypsin 0 Papain 0 H.sub.2O 99.94 Mixture 2 Chlorhexidine 0.03 digluconate Chymotrypsin 0.2 Papain 0.4 H.sub.2O 99.37 Mixture 3 Chlorhexidine 0 digluconate Chymotrypsin 0 Papain 0 H.sub.2O 100

(51) The mixture of 0.03% by weight of chlorhexidine digluconate and the proteolytic enzymes of chymotrypsin and papain at a concentration of 0.2% by weight, or rather 0.4% by weight is able to inhibit the growth of a bacterial culture equally well as the solution of chlorhexidine digluconate alone at a concentration of 0.06% by weight. The use of the given concentration of proteolytic enzymes in the mixture of chlorhexidine digluconate can reduce the concentration of chlorhexidine active substance 2 with the same bacteriostatic effect.

Example 8

(52) Nine volunteers were divided into 3 groups. Volunteers in Group 1 used for oral hygiene an agent containing the mixture according to Example 3a. Volunteers in Group 2 used for oral hygiene an agent containing chlorhexidine 0.12% by weight. Volunteers in Group 3 used for oral hygiene an agent containing chlorhexidine 0.06% by weight. Papilla Bleeding Index (PBI) was measured in all volunteers and their oral pathogens analysis carried out using PCR chromosomal DNA on day 0 and after 30 days of use of the agent. Characteristics of volunteers: without antibiotic treatment for at least 3 months, non-smokers, without hormonal contraception, age 35-50 years.

(53) Results of PBI values are given in Table 6. The results of genetic analysis of oral pathogens are shown in Table 7.

(54) TABLE-US-00011 TABLE 6 Results of PBI estimation Active Volun- Day Day Improvement Group compounds teer 0 30 (%) 1 Chlorhexidine 1 2.8 1.6 43 digluconate 0.035% 2 2.5 2.3 8 trypsin 0.2% 3 2.1 1.6 25 bromelain 0.4% 2 Chlorhexidine 4 3.1 2.7 12 digluconate 0.12% 5 2.6 2.3 9 6 2.4 2.3 4 3 Chlorhexidine 7 3.0 3.0 3 digluconate 0.06% 8 2.3 2.4 8 9 2.3 2.4 4

(55) TABLE-US-00012 TABLE 7 Results of oral pathogens analysis Active Group compounds Volunteer Day PG TF TD PI AA 1 Chlorhexidine 1 0 + + + + 0 digluconate 0.035% 30 + + + 0 0 trypsin 0.2% 2 0 + + + + 0 bromelain 0.4% 30 + + + + 0 3 0 + + 0 0 0 30 0 0 0 0 0 2 Chlorhexidine 4 0 + + + + + digluconate 0.12% 30 + + + + + 5 0 + + + 0 0 30 + + 0 0 0 6 0 + + 0 0 0 30 0 + 0 0 0 3 Chlorhexidine 7 0 + 0 + 0 + digluconate 0.06% 30 + 0 + 0 + 8 0 + 0 0 0 0 30 0 0 0 0 0 9 0 + + + 0 0 30 + + + 0 0 The abbreviations used in Table 7: Oral pathogens: PG - Porphyromonas gingivalis; TF - Tannerella forsythia; TD - Treponema denticola; PI - Prevotella intermedia; AA - Aggregatibacter actinomycetemcomitans. + - identified; 0 - not identified.

(56) At the end of a 30-day period during which volunteers used an agent containing the mixture according to Example 3a (chlorhexidine digluconate at a concentration of 0.06% by weight, trypsin 0.2% by weight, bromelain 0.4% by weight), all volunteers had their bleeding index (PBI) reduced by 8-43% compared to initial values. Bacteria load count of two volunteers changed for the better. The group of volunteers using during their oral hygiene procedure an agent with chlorhexidine at a concentration of 0.12% had their bleeding index (PBI) decreased by about 4-12% compared to initial values. Bacteria load count of two volunteers changed for the better. The group of volunteers using during their oral hygiene procedure an agent with chlorhexidine at a concentration of 0.06% did not have their bleeding index (PBI) reduced. Bacteria load count of one patient changed for the better.

INDUSTRIAL APPLICATION

(57) It is evident that products containing proteolytic enzymes directly reduce the risk of adverse effects caused by the salts of chlorhexidine. Above all, because it is possible to use significantly smaller amounts of salts of chlorhexidine like the chlorhexidine digluconate whilst achieving the same inhibition of bacterial growth, and thus suppress the negative effects during the prevention or treatment of antimicrobial, antivirotic or antimycotic illnesses.