| US20180333384A1 | 2018-11-22 | |||
| US7582418B2 | 2009-09-01 | |||
| US4565693A | 1986-01-21 | |||
| US4206236A | 1980-06-03 | |||
| US7582418B2 | 2009-09-01 | |||
| US20140179645A1 | 2014-06-26 | |||
| US4565693A | 1986-01-21 | |||
| GB770381A | 1957-03-20 |
N. HALLAI. P. FERNANDESS. A. HELENOP. COSTAZ. BOUCHERIT-OTMANIK. BOUCHERITA. E. RODRIGUESI. C. F. R. FERREIRAM. F. BARREIRO: "Cosmetic Preservation: A Review on Present Strategies", MOLECULES, vol. 23, 2018, pages 1571, XP055588453, DOI: 10.3390/molecules23071571
R. SMARTD. F. SPOONER: "Microbiological spoilage in pharmaceuticals and cosmetics", J. SOC. COSMET. CHEM., vol. 23, 1972, pages 721 - 737
F. R. LA ROSAE. CRISTINE GIESER. F. H. DEKKERJ. SANCHEZ PELAYOA. DE MELO BARBOSA: "Microbiological contamination of water-based paints from an industry in the state of Parana, Brazil", SEMINA: CIENCIAS EXATAS E DA TERRA, LONDRINA, vol. 29, 2008, pages 85 - 92
F. DAL BELLOC. I. CLARKEL. A. M. RYANH. ULMERT. J. SCHOBERK. STROMJ. SJOGREND. VAN SINDERENJ. SCHNURERE. K. ARENDT: "Improvement of the quality and shelf life of wheat bread by fermentation with the antifungal strain Lactobacillus plantarum FST 1.7", J. CEREAL SCI., vol. 45, 2007, pages 309 - 318, XP022021096, DOI: 10.1016/j.jcs.2006.09.004
P. SARAVANA KUMARN. A. AL-DHABIV. DURAIPANDIYANC. BALACHANDRANP. PRAVEEN KUMARS. IGNACIMUTHU: "In vitro antimicrobial, antioxidant and cytotoxic properties of Streptomyces lavendulae strain SCA5", BMC MICROBIOLOGY, vol. 14, 2014, pages 291, XP021205772, DOI: 10.1186/s12866-014-0291-6
| CLAIMS 1. A preservative composition consisting of: A. active ingredients from 10-100% w/w, selected to be: (i) zinc bis-glycinate <1> or its monohydrate as the principal active ingredient; from 0.5-20% w/w; and one or more secondary active ingredients selected from the following groups: (ii) polyols of general formula II with linear- or branched-chain aliphatic hydrocarbon skeleton R with 5-18 carbon atoms and 4-8 hydroxyl <OH> groups in the molecule; from 0.5-90% w/w; R (OH)4-g II (iii) 3,6-di(substituted)piperazine-2,5-diones of general formula <III> ; from 0.5-90% w/w; wherein the substitution groups X, X' are the same or different, and selected from the group consisting of: hydrogen <H->, methyl <CH3->, hydroxymethyl <HOCH2- >, ethyl <C2H5->, 1-hydroxyethyl <CH3CH<OH>->, 2- hydroxy ethyl <HOCH2CH2->, 1,2-dihydroxyethyl <HOCH2CH<OH>->, n-propyl <CH3CH2CH2->, 3- hydroxypropyl <HOCH2CH2CH2->, isopropyl <<CH3>2CH->, 2-hydroxyisopropyl <HOCH<CH3>CH2->, 1,3- dihydroxypropan-2-yl <<HOCH2>2CH->, 2-methylpropyl <CH3CH<CH3>CH2->, 1-hydroxy-2-methylpropyl <CH3CH<CH3>CH<OH>->, 2-butyl <CH3CH2CH<CH3>->, 1- methyl-2-hydroxypropyl <HOCH<CH3>CH<CH3>->, mercaptomethyl <HSCH2->, selenomethyl <HSeCH2->, 2- methylthioethyl <CH3SCH2CH2->, carboxymethyl <HOOCCH2->, carboxy-hydroxymethyl <HOOCCH<OH>->, 2- carboxyethyl <HOOCCH2CH2->, 2-carboxy-2-hydroxyethyl <HOOCCH<OH>CH2->, carboxamido methyl <H2NCOCH2->, carboxamido-hydroxymethyl <H2NCOCH<OH>->, 2- carboxamidoethyl <H2NCOCH2CH2->, 2-carboxamido-2- hydroxyethyl <H2NCOCH<OH>CH2>, 3-aminopropyl <H2NCH2CH2CH2->, 1-hydroxy-3-aminopropyl <H2NCH2CH2CH<OH>->, 2-hydroxy-3-aminopropyl <H2NCH2CH<OH>CH2->, 4-aminobutyl <H2NCH2CH2CH2CH2->, 3- hydroxy-4-aminobutyl <H2NCH2CH<OH>CH2CH2->, 4- acetamidobutyl <CH3CONHCH2CH2CH2CH2->, 3-guanidino propyl <H2NC<=NH>NHCH2CH2CH2->, 2-hydroxy-3-guanidino propyl <H2NC<=NH>NHCH2CH<OH>CH2->, phenyl <C6H5->, 4- hydroxyphenylmethyl <4-HOC6H4CH2->, benzyl <C6H5CH2->, l/i-imidazole-4-yl-methyl <C3H3N2-4-CH2->, 1H- imidazole-4-yl-hydroxymethyl <C3H3N2-4-CH<OH>->, 1H- imidazole-3-hydroxy-4-yl-methyl <3-HOC3H2N2-4-CH2->, l/i-indol-3-yl-methyl <C8H6N>-3-CH2->, 5-hydroxy-l/i- indol-3-yl-methyl <5-HOC8H5N>-3-CH2->; unsymmetrical compounds of general formula Ilia which are derived from amino acid L-proline <4a; Z = H> or 4-hydroxy-L-proline <4b; Z = OH> and amino acid derivatives IV with the group X defined above: symmetrical L-proline-derived compound 3b and 4- hydroxy-L-proline 3c : 3b: Z = H 3c: Z = OH and, optionally, B. one or more excipients selected from the groups comprising: (iv) liquid diluents; (v) suspending agents; (vi) wetting agents; (vii) pH adjusting agents; (viii) solid diluents; and, (ix) anti-caking agents; or mixtures of these substances; from 0-90% w/w. 2. A preservative composition according to claim 1, wherein the excipients B. are selected from the following groups: (iv) liquid diluents: water, glycerol, mixtures of water and glycerol; (v) suspending agents: methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methycellulose, xanthan gum; (vi) wetting agents: polysorbates, poloxamers, lecithins, sorbitan esters; (vii) pH adjusting agents: citric acid, malic acid, sodium hydroxide, trisodium citrate or its dihydrate; (viii) solid diluents: sorbitol, sodium chloride; and, (ix) anti-caking agents: colloidal silicon dioxide, sodium aluminium silicate. 3. A preservative composition according to claims 1 or 2, wherein principal and secondary active ingredients selected to be: (i) zinc bis-glycinate <1> or its monohydrate; from 0.5-20% w/w; (ii) polyol: 5-hydroxymethyl-l,5,6,7,11-undecane-pentol <2>; from 20-50% w/w; and, (iii) 3,6-di(2-hydroxyethyl)piperazine-2,5-dione <3a>; from 20-50% w/w 4. A preservative composition according to any of claims 1-3, wherein the secondary active ingredient is selected to be 5- hydroxymethyl-1,5,6,7,11-undecane-pentol <2>, from 0.5-90% w/w. 5. A preservative composition according to any of claims 1-3, wherein the secondary active ingredient is selected to be 3,6- di(2-hydroxy ethyl)piperazine-2,5-dione <3a>, from 0.5-90% w/w. 6. A preservative composition according to any of claims 1-3, wherein the secondary active ingredients are selected to be 5- hydroxy methyl-1,5,6,7,11-undecane-pentol <2>; from 0.5-90% w/w; and, 3,6-di(hydroxyethyl)piperazine-2,5-dione <3a>; from 0.5-90% w/w. 7. A preservative composition according to claim 1, wherein it consists of principal and secondary active ingredients only, without excipients; up to 100% w/w; said composition is selected to be: (i) zinc bis-glycinate <1> or its monohydrate; from 0.5-20% w/w; (ii) 5-hydroxymethyl-l,5,6,7,11-undecane-pentol <2>; from 20- 50% w/w; and, (iii) 3,6-di(2-hydroxyethyl)piperazine-2,5-dione <3a>; from 20-50% w/w. 8. Process of the preparation of the composition according to any of claims 1-7, wherein it includes homogenization of: A. active ingredients (i)-(iii); with or without, B. one or more excipients selected from (iv)-(ix); at 10-100°C for 1-60 minutes. 9. Use of the preservative composition according to any of claims 1-7, as an industrial product within formulations of cosmetic, pharmaceutical, household, water-based-paints, or chemical products as a preservative against their microbial spoilage. 10. Use of the preservative composition according to claims 9, in concentrations from 0.1-20% w/w within the formulation of the final product. |
DESCRIPTION
Technical Field
The present invention relates to a preservative composition used as an industrial product for antimicrobial preservation of various water-based cosmetic, pharmaceutical, and chemical formulations.
Technical Problem
The present invention solves the technical problem of antimicrobial preservation of various water-based cosmetic, pharmaceutical, and chemical products, and is of:
(i) high efficacy in antimicrobial preservation; and
(ii) high safety for end-user due to minimal toxicity
Unlike conventional and widely employed preservative compounds such as esters of 4-hydroxybenzoic acid (parabens), quaternary ammonium surfactants (QAS) like benzalkonium chloride, or formaldehyde releasers such as imidazolidinyl urea, the composition in the present invention provides no known toxicity to humans, while maintaining high efficacy against microbial growth in water-based product formulations.
Previous State of the Art
Cosmetic and pharmaceutical products containing water require preservation against microbial contamination. The preservation ensures the safety of consumers or patients from potentially pathogenic microorganisms and increases the product's shelf-life by preventing biological and physicochemical deterioration. The preservation is achieved by multiple strategies, including the use of antimicrobial agents acting as preservatives against microbial spoilage. Preservatives commonly used are various synthetic or natural compounds or multifunctional ingredients. Typical examples of antimicrobial preservatives are methyl 4-hydroxybenzoate, ethyl 4-hydroxybenzoate, n-propyl 4-hydroxybenzoate collectively known as parabens, methylisothiazolinone, organic acids such as benzoic or sorbic acid, formaldehyde releasers like imidazolidinyl urea, phenols such as triclosan, quaternary ammonium surfactants like benzalkonium chloride, bisbiguanides such as chlorhexidine digluconate and others; see literature references 1 and 2:
1) N. Halla, I. P. Fernandes, S. A. Heleno, P. Costa, Z. Boucherit-
Otmani, K. Boucherit, A. E. Rodrigues, I. C. F. R. Ferreira, M. F. Barreiro: Cosmetic Preservation: A Review on Present
Strategies, Molecules 23 (2018) 1571;
2) R. Smart, D. F. Spooner: Microbiological spoilage in pharmaceuticals and cosmetics, J. Soc. Cosmet. Chem. 23 (1972)
721-737.
Analogously to cosmetic and pharmaceutical products, other water- based chemical formulations such as personal use and household products (e.g., liquid detergents, shampoos, and soaps), as well as water-based paints and adhesives also require preservation against microbial spoilage. For instance, see literature reference 3 describing the technical problem of microbial preservation of water- based acrylic paints:
3) F. R. La Rosa, E. Cristine Giese, R. F. H. Dekker, J. Sanchez
Pelayo, A. de Melo Barbosa: Microbiological contamination of water-based paints from an industry in the state of Parana, Brazil, Semina: Ciencias Exatas e da Terra, Londrina 29 (2008)
85-92.
An existing technical problem in the field of chemical preservation requires the solution that takes into account the use of consumer- and environmentally-friendly preservatives free of unwanted side effects found in common preservatives today, e.g.: (i) higher toxicological safety; lower acute or chronic toxicity potential, inability to induce contact allergies or hormonal disbalance; and,
(ii) lower environmental strain, faster biodegradability, lower toxicity for aquatic organisms, made from renewable and sustainable materials.
Zinc bis-glycinate (1) or its monohydrate form is a known salt and complex of zinc (Zn 2+ ) and amino acid glycine:
Compound 1 is commonly used as a nutritional source of the essential micro mineral zinc. Among its other known roles, it was described as an antimicrobial agent. Thompson disclosed the method for inhibiting microbial growth by the use of zinc chelate consisting of zinc lipoate and zinc bis-glycinate (1). This agent is claimed to be effective against various Gram-negative and Gram-positive bacteria and fungi, more specifically, against Staphylococcus aureus, Streptococcus pyogenes, Salmonella typhimurium, Escherichia coli, and Candida albicans; see literature reference 4:
4) US 7,582,418 B2; Antimicrobial chelates; inventor: R. C.
Thompson; applicant: Albion Laboratories, Inc. (US); priority date: 03. 11. 2014.
Additionally, Arndt described the use of various other zinc salts, including compound 1, as one of the components in a two-component concept of the antibacterial and antifungal composition. One component is a zinc salt, while another one is a ligand which forms a complex with the zinc salt. Such combination provides enhanced antibacterial and antifungal effect; see literature reference 5: 5) US 2014/0179645 A1; Antimicrobial compositions; inventor: D. C. Arndt; applicant: The Pisces Group LLC (US); priority date: 21. 12. 2012.
Also, Marschner disclosed the cosmetic formulation of skin deodorant based on compound 1, for which chemical neutralization of odoriferous compounds in sweat and inhibition of bacterial growth on the skin is claimed; see literature reference 6:
6) US 4,565,693; Deodorant composition; inventor: F. W. Marschner; applicant: Colgate-Palmolive Co. (US); priority date: 09. 11. 1981.
Any of the last three literature references directly suggests the use of compound 1 as MB preservative.
The company Union Carbide & Carbon Corporation (US) disclosed the process of manufacturing polyol 5-hydroxymethyl-l,5,6,7,11-undecane- pentol (2) from acrolein as the starting raw material; see literature reference 7:
7) GB 770381 A; Aldol condensation of acrolein dimer; inventor: not declared; applicant: Union Carbide & Carbon Corporation (US); priority date: 14. 06. 1954.
According to available data, compound 2 is mainly used as a specialty chemical. Its use as an antimicrobial preservative as either a sole ingredient or within complex preservative compositions has not been found in the prior art. It is known from the prior art of organic chemistry that amino acids and its derivatives, upon thermal treatment or chemical cyclization condensation, yield symmetrical (X = X') or unsymmetrical (X A X') diketopiperazine (DKP) derivatives III with their corresponding substitution groups X, X'. In this manner, DKP derivative 3,6-di(2- hydroxyethyl) piperazine-2,5-dione (3a) can be formed from two moles of 2-hydroxy ethyl-glycine (homoserine) or aspartic acid derivative whose terminal carboxyl group (COOH) is previously reduced to alcohol group (CH 2 0H).
III 3a
DKP derivatives are found within various probiotic metabolic products that exhibit antimicrobial effects against some pathogenic microorganisms. For instance, Dal Bello and co-workers disclosed the chemical composition of secondary metabolites from probiotic bacteria Lactobacillus plantarum FST 1.7. They found that, among other metabolites, the bacteria produce and release DKP derivatives („cyclic dipeptides") of L-proline (Pro) and L-leucine (Leu) (DKP L- Pro-L-Leu) or L-phenylalanine (Phe) (DKP L-Pro-L-Phe) . These metabolites exhibit some antifungal effects that can be used for the preservation of food, including bread; see literature reference 8.
DKP L-Pro-L-Leu DKP L-Pro-L-Phe
8) F. Dal Bello, C. I. Clarke, L. A. M. Ryan, H. Ulmer, T. J. Schober, K. Strom, J. Sjogren, D. van Sinderen, J. Schnurer, E. K. Arendt: Improvement of the quality and shelf life of wheat bread by fermentation with the antifungal strain Lactobacillus plantarum FST 1.7, J. Cereal Sci. 45 (2007) 309-318.
Similarly, Saravana Kumar and co-workers described that DKP derivative of the amino acid L-leucine (Leu) is a component of exudate from microorganism Streptomyces lavendulae, strain SCA5, and exhibits antimicrobial activity; see literature reference 9:
DKP derivative of L-Leu
9) P. Saravana Kumar, N. A. Al-Dhabi, V. Duraipandiyan, C. Balachandran, P. Praveen Kumar, S. Ignacimuthu: In vitro antimicrobial, antioxidant and cytotoxic properties of Streptomyces lavendulae strain SCA5, BMC Microbiology 14 (2014)
291.
The latter two literature references demonstrate the presence of DKP derivatives in microbe-derived antimicrobial compositions, thus hinting to the possibility of use of the DKPs as antimicrobial agents in preservatives for water-based cosmetic, pharmaceutical, and chemical products.
The use of the DKP derivative 3,6-di(2-hydroxyethyl)piperazine-2,5- dione (3a) as either sole antimicrobial agent or as an active ingredient in preservative compositions have not been found in the prior art.
To the best of our knowledge, prior art closest to the present invention is disclosed in the literature references 4-6. The preservative composition consisting of:
(i) zinc bis-glycinate (1) or its monohydrate;
(ii) polyols of general formula II with linear- or branched-chain aliphatic hydrocarbon skeleton R with 5-18 carbon atoms and 4-8 hydroxyl (OH) groups in the molecule, e.g., 5-hydroxy methyl-1,5,6,7,11-undecane-pentol (2); and,
(ill) various 3,6-di(substituted)piperazine-2,5-diones III, including, e.g., 3,6-di(2-hydroxyethyl)piperazine-2,5-dione
(3a); has not been shown in the prior art.
The above-defined technical problem of obtaining an improved preservative composition for use in various water-based cosmetic, pharmaceutical, and chemical products, that is of:
(i) high efficacy in antimicrobial preservation; and
(ii) high safety for end-user due to minimal toxicity; is solved by the present invention in a new and inventive manner, as described in the section Detailed Description of the Invention.
Summary of the Invention
The invention describes the antimicrobial preservative composition for use in cosmetic, pharmaceutical, and chemical products. The preservative composition, according to the present invention, consists of:
A. active ingredients from 10-100% w/w, selected to be:
(i) zinc bis-glycinate (1) or its monohydrate as the principal active ingredient; from 0.5-20% w/w; and one or more secondary active ingredients selected from the following groups:
(ii) polyols of general formula II with linear- or branched- chain aliphatic hydrocarbon skeleton R with 5-18 carbon atoms and 4-8 hydroxyl (OH) groups in the molecule, e.g., 5-hydroxymethyl-l,5,6,7,11-undecane-pentol (2 ) ; from 0.5-90% w/w;
(ill) 3,6-di(substituted)piperazine-2,5-diones III ; from 0.5- 90% w/w;
III wherein the substitution groups X, X' are the same or different, and selected from the group consisting of: hydrogen (H-), methyl (CH 3 -), hydroxymethyl (HOCH 2 -), ethyl (C 2 H 5 -), 1-hydroxyethyl [CH 3 CH(OH)-], 2-hydroxy ethyl (HOCH 2 CH 2 -), 1,2-dihydroxyethyl [HOCH 2 CH(OH)-], n- propyl (CH 3 CH 2 CH 2 -), 3-hydroxypropyl (HOCH 2 CH 2 CH 2 -), isopropyl [(CH 3 ) 2 CH—], 2-hydroxyisopropyl [HOCH(CH 3 )CH 2 -], 1,3-dihydroxypropan-2-yl [(HOCH 2 ) 2 CH-], 2-methylpropyl [CH 3 CH(OKs)CH 2 -], l-hydroxy-2-methylpropyl
[CH 3 CH(CH 3 )CH(OH)-], 2-butyl [CH 3 CH 2 CH(CH 3 )-], l-methyl-2- hydroxypropyl [HOCH(CH 3 )CH(CH 3 )-], mercaptomethyl (HSCH 2 - ), selenomethyl (HSeCH 2 -), 2-methylthioethyl (CH 3 SCH 2 CH 2 - ), carboxymethyl (HOOCCH 2 -), carboxy-hydroxymethyl [HOOCCH(OH)-], 2-carboxyethyl (HOOCCH 2 CH 2 -), 2-carboxy-2- hydroxyethyl [HOOCCH(OH)CH 2 -], carboxamidomethyl
(H 2 NCOCH 2 -), carboxamido-hydroxymethyl [H 2 NCOCH(OH)-], 2- carboxamidoethyl (H 2 NCOCH 2 CH 2 -), 2-carboxamido-2- hydroxyethyl [H 2 NCOCH(OH)CH 2 ], 3-aminopropyl
(H 2 NCH 2 CH 2 CH 2 -), 1-hydroxy-3-aminopropyl [H 2 NCH 2 CH 2 CH(OH)-
], 2-hydroxy-3-aminopropyl [H 2 NCH 2 CH(OH)CH 2 -], 4- aminobutyl (H 2 NCH 2 CH 2 CH 2 CH 2 -), 3-hydroxy-4-aminobutyl [H 2 NCH 2 CH(OH)CH 2 CH 2 -], 4-acetamidobutyl
(CH 3 CONHCH 2 CH 2 CH 2 CH 2 -), 3-guanidino propyl
[H 2 NC(=NH)NHCH 2 CH 2 CH 2 -], 2-hydroxy-3-guanidino propyl [H 2 NC(=NH)NHCH 2 CH(OH)CH 2 -], phenyl (C 6 H 5 -), 4- hydroxyphenyl methyl (4-HOC 6 HCH 2 -), benzyl (C 6 H 5 CH 2 -), lH-imidazole-4-yl-methyl (C 3 H 3 N 2 -4-CH 2 -), lH-imidazole-4- yl-hydroxymethyl [C 3 H 3 N 2 -4-CH(OH)-], lH-imidazole-3- hydroxy-4-yl-methyl (3-HOC 3 H 2 N 2 -4-CH 2 -), lH-indol-3-yl- methyl (C 8 H 6 N-3-CH 2 -), 5-hydroxy-lH-indol-3-yl-methyl (5- HOC 8 H 5 N-3-CH 2 -); unsymmetrical compounds of general formula Ilia which are derived from amino acid L-proline ( 4a; Z = H) or 4 - hydroxy-L-proline ( 4b; Z = OH) and amino acid derivatives IV with the group X defined above:
I a 4b: Z OH IV symmetrical L-proline-derived compound 3b and 4-hydroxy- L-proline 3c :
3b: Z = H
3c : Z = OH and, optionally,
B. one or more excipients selected from the groups comprising:
(iv) liquid diluents;
(v) suspending agents;
(vi) wetting agents;
(vii) pH adjusting agents;
(viii) solid diluents; and,
(ix) anti-caking agents; or mixtures of these substances; from 0-90% w/w.
The composition is used in concentrations from 0.1-20% w/w in water- based cosmetic, pharmaceutical, and chemical products to prevent their microbial spoilage.
Detailed Description of the Invention
The present invention relates to an improved industrial product in the field of antimicrobial preservatives for water-based cosmetic, pharmaceutical, and chemicals products. The preservative composition according to the present invention consists of:
A. active ingredients from 10-100% w/w, selected to be:
(i) zinc bis-glycinate <1> or its monohydrate as the principal active ingredient; from 0.5-20% w/w; and one or more secondary active ingredients selected from the following groups:
(ii) polyols of general formula II with linear- or branched- chain aliphatic hydrocarbon skeleton R with 5-18 carbon atoms and 4-8 hydroxyl <OH> groups in the molecule; from 0.5-90% w/w; R (OH)4-8
II
(iii) 3,6-di(substituted)piperazine-2,5-diones of general formula <III> ; from 0.5-90% w/w;
III wherein the substitution groups X, X' are the same or different, and selected from the group consisting of: hydrogen (H-), methyl (CH 3 -), hydroxymethyl (HOCH 2 -), ethyl (C 2 H 5 -), 1-hydroxyethyl [CH 3 CH(OH)-], 2-hydroxy ethyl (HOCH 2 CH 2 -), 1,2-dihydroxyethyl [HOCH 2 CH(OH)-], n- propyl (CH 3 CH 2 CH 2 -), 3-hydroxypropyl (HOCH 2 CH 2 CH 2 -), isopropyl [(CH 3 ) 2 CH—], 2-hydroxyisopropyl [HOCH(CH 3 )CH 2 -], 1,3-dihydroxypropan-2-yl [(HOCH 2 ) 2 CH-], 2-methylpropyl [CH 3 CH(OKs)CH 2 -], l-hydroxy-2-methylpropyl
[CH 3 CH(CH 3 )CH(OH)-], 2-butyl [CH 3 CH 2 CH(CH 3 )-], l-methyl-2- hydroxypropyl [HOCH(CH 3 )CH(CH 3 )-], mercaptomethyl (HSCH 2 - ), selenomethyl (HSeCH 2 -), 2-methylthioethyl (CH 3 SCH 2 CH 2 - ), carboxymethyl (HOOCCH 2 -), carboxy-hydroxymethyl [HOOCCH(OH)-], 2-carboxyethyl (HOOCCH 2 CH 2 -), 2-carboxy-2- hydroxyethyl [HOOCCH(OH)CH 2 -], carboxamidomethyl
(H 2 NCOCH 2 -), carboxamido-hydroxymethyl [H 2 NCOCH(OH)-], 2- carboxamidoethyl (H 2 NCOCH 2 CH 2 -), 2-carboxamido-2- hydroxyethyl [H 2 NCOCH(OH)CH 2 ], 3-aminopropyl
(H 2 NCH 2 CH 2 CH 2 -), 1-hydroxy-3-aminopropyl [H 2 NCH 2 CH 2 CH(OH)-
], 2-hydroxy-3-aminopropyl [H 2 NCH 2 CH(OH)CH 2 -], 4- aminobutyl (H 2 NCH 2 CH 2 CH 2 CH 2 -), 3-hydroxy-4-aminobutyl [H 2 NCH 2 CH(OH)CH 2 CH 2 -], 4-acetamidobutyl
(CH 3 CONHCH 2 CH 2 CH 2 CH 2 -), 3-guanidino propyl
[H 2 NC(=NH)NHCH 2 CH 2 CH 2 -], 2-hydroxy-3-guanidino propyl [H 2 NC(=NH)NHCH 2 CH(OH)CH 2 -], phenyl (C 6 H 5 -), 4- hydroxyphenyl methyl (4-HOC 6 H 4 CH 2 -), benzyl (C 6 H 5 CH 2 -), lH-imidazole-4-yl-methyl (C 3 H 3 N 2 -4-CH 2 -), lH-imidazole-4- yl-hydroxymethyl [C 3 H 3 N 2 -4-CH(OH)-], lH-imidazole-3- hydroxy-4-yl-methyl (3-HOC 3 H 2 N 2 -4-CH 2 -), lH-indol-3-yl- methyl (C 8 H 6 N-3-CH 2 -), 5-hydroxy-lH-indol-3-yl-methyl (5- HOC 8 H 5 N-3-CH 2 -); unsymmetrical compounds of general formula Ilia which are derived from amino acid L-proline ( 4a; Z = H) or 4- hydroxy-L-proline ( 4b; Z = OH) and amino acid derivatives IV with the group X defined above:
I a 4b : Z OH IV symmetrical L-proline-derived compound 3b and 4-hydroxy- L-proline 3c :
3b : Z = H
3c : Z = OH and, optionally,
B. one or more excipients selected from the groups comprising:
(iv) liquid diluents;
(v) suspending agents;
(vi) wetting agents;
(vii) pH adjusting agents;
(viii) solid diluents; and,
(ix) anti-caking agents; or mixtures thereof; from 0-90% w/w.
The excipients B. are selected from the following groups:
(iv) liquid diluents: water, glycerol, mixtures of water and glycerol;
(v) suspending agents: methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, xanthan gum;
(vi) wetting agents: polysorbates, poloxamers, lecithins, sorbitan esters;
(vii) pH adjusting agents: citric acid, malic acid, sodium hydroxide, trisodium citrate or its dihydrate;
(viii) solid diluents: sorbitol, sodium chloride; and,
(ix) anti-caking agents: colloidal silicon dioxide, sodium aluminium silicate.
In one preferred embodiment of this invention, the principal and the secondary active ingredients in the preservative composition are selected to be:
(i) zinc bis-glycinate (1) or its monohydrate; from 0.5-20% w/w;
(ii) polyol: 5-hydroxymethyl-l,5,6,7,11-undecane-pentol ( 2 ) ; from 20-50% w/w; and,
(iii) 3,6-di(2-hydroxyethyl)piperazine-2,5-dione ( 3a) ; from 20-50% w/w
3a In another preferred option of this invention, the secondary active ingredient is selected to be 5-hydroxymethyl-l,5,6,7,11-undecane- pentol (2 ) , from 0.5-90% w/w.
Furthermore, in another preferred embodiment of this invention, the secondary active ingredient is selected to be 3,6-di(2-hydroxyethyl) piperazine-2,5-dione (3a) , from 0.5-90% w/w.
Additionally, in one preferred variant of this invention the secondary active ingredients are selected to be 5-hydroxymethyl- 1,5,6,7,11-undecane-pentol (2 ) , from 0.5-90% w/w; and 3,6-di(2- hydroxyethyl) piperazine-2,5-dione (3a) , from 0.5-90% w/w.
In addition, in the preferred embodiment of this invention, the preservative composition consists of the principal and the secondary active ingredients only; up to 100% w/w, without excipients. Said composition is selected to be:
(i) zinc bis-glycinate (1) or its monohydrate; from 0.5-20% w/w;
(ii) 5-hydroxymethyl-l,5,6,7,11-undecane-pentol (2 ) ; from 20-50% w/w; and,
(iii) 3,6-di(2-hydroxyethyl)piperazine-2,5-dione (3a) ; from 20-50% w/w.
Polyols of general formula II with linear- or branched-chain aliphatic hydrocarbon skeleton R with 5-18 carbon atoms and 4-8 hydroxyl (OH) groups in the molecule, can be used in all commercially available forms. Some of them are liquids, and others are solids. In the final form of the preservative composition, they are mixed with other active ingredients (i) and possibly (iii) and optionally with some of the auxiliary ingredients (excipients) (iv)- (ix) to form either a solid or a liquid. As a typical example of the polyol, the said 5-hydroxymethyl-l,5,6,7,11-undecane-pentol (2 ) can be used. But in addition to compound 2 , other described polyols with 4-8 hydroxyl groups can be employed. 3,6-Di(substituted)piperazine-2,5-diones III or diketopiperazines (DKP) are basically cyclic dipeptides, the derivatives of two molecules of the same (symmetrical DKP) or different (unsymmetrical DKP) a-amino acids IV, where X, X' is the residual moiety X (X') of the parent a-amino acid(s).
Ill IV
The preservative composition from the present invention can be formulated either as a homogeneous mixture of the principal active ingredient, compound 1, with one or more secondary active ingredients from the group (ii) and/or (iii), or, as a complex formulation with one or more above-defined auxiliary ingredients (excipients).
Wetting agents are selected from the groups consisting of polysorbates such as polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80; poloxamers, such as various symmetrical triblock copolymers of polyethylene oxide (PEO) and polypropylene oxide (PPO) of general formula (PEO) a ( PPO) b ( PEO) a , wherein the value for a and b can be different; e.g. in poloxamer 407, a = 104, b = 56, whilst in pluronic 123 a = 40, b = 70; lecithins such as soybean [Glycine max (L.) Merr.] lecithin, sunflower (Helianthus annuus L.) lecithin, hydrolyzed soybean lecithin, hydrolized sunflower lecithin; sorbitan esters such as sorbitan laurate, sorbitan palmitate, sorbitan stearate, and sorbitan oleate.
In the final physicochemical form, the preservative composition from the present invention can be a flowable powder or a liquid (either as a solution or suspension).
Results of preservative efficacy of the composition from the present invention The preservative efficacy of the composition from the present invention was analyzed using the standard method in cosmetics microbial challenge test - against the following strains:
(i) Pseudomonas aeruginosa (P. aeruginosa) ATCC 9027;
(ii) Staphylococcus aureus (S. aureus ATCC 6538;
(iii) Escherichia coli (E. coli) ATCC 8739;
(iv) Candida albicans (C. albicans) ATCC 10231; and,
(v) Aspergillus brasiliensis (A. brasiliensis) ATCC 16404; following the ISO 11930:2012 method.
The cosmetic base for the microbial challenge tests was an oil-in water (O/W) emulsion (like a body cream). The formulation of the base O/W emulsion is shown in Table 1, and the preparation is described in Example 1 in the experimental section.
Table 1. The formulation of the base oil-in-water (O/W) emulsion for microbial challenge testing of the preservative composition from the present invention; also see Example 1, a a The pH value of the resulting model O/W emulsion was 6.0-6.5.
The unpreserved sample of the O/W emulsion was used as a control. Additional controls were the active substances, compounds 1, 2, and 3a, individually added to the base O/W emulsion and analyzed; see Table 2. The active testing substances from the present invention shown in the Examples 2-4, were mixed in the laboratory batches of the base O/W emulsion before the microbial challenge testing. The composition of the control and testing samples are shown in Table 2.
Table 2. Percentage values of the compounds in the preservative composition from the present invention in the control and test samples (products from Examples 2-4, in the base oil- in-water (O/W) emulsion. a ) a The preparation of the control samples and the testing samples with the formulations from the present invention is described in Example 6. b Active substances tested: zinc bis-glycinate (1), 5-hydroxymethyl- 1,5,6,7,11-undecane-pentol (2) as the selected polyol II, and 3,6- di(2-hydroxyethyl)piperazine-2,5-dione (3a) as the selected DKP derivative III.
Experimental procedure for the microbial challenge test of the control and the preserved emulsion from the present invention (Examples 2-4) is described in Example 6. The average results are shown in Tables 3-6.
The results show that zinc bis-glycinate (1) acts as an effective antimicrobial agent at the concentration of 0.25% w/w in the formulation of the base O/W emulsion against all tested microorganisms except P. aeruginosa where it showed no effect and S. aureus where its effect weakens throughout the duration of the test; see Table 3.
The selected polyol, 5-hydroxymethyl-l,5,6,7,11-undecane-pentol (2 ) , in the concentration 0.35% w/w, in the base O/W emulsion shows weak antimicrobial effect against three microorganisms, but does not affect P. aeruginosa and A. brasiliensis; see Table 3.
Table 3 . Results of microbial challenge testing for individual active substances from the preservative composition of the present invention. The antimicrobial effects are shown for zinc bis-glycinate (1), 5-hydroxymethyl-l,5,6,7,11- undecane-pentol (2 ) and 3,6-di(2-hydroxyethyl)piperazine- 2,5-dione ( 3a) and compared to unpreserved base emulsion. a
a The experimental procedure for performing microbial challenge test is described in Example 6.
CFU = colony forming units; TO = the initial inoculum number; 7d = MB count of CFU/g after 7 days; 14d = MB count of CFU/g after 14 days; 28d = MB count of CFU/g after 28 days; log c = log change relative to the initial inoculum number value; Control-1 = unpreserved base O/W emulsion,; 1 = base O/W emulsion with 0.25% w/w zinc bis-glycinate (1); 2 = base O/W emulsion with 0.35% w/w 5- hydroxymethyl-1,5,6,7,11-undecane-pentol (2); 3a = base O/W emulsion with 0.80% w/w 3,6-di(2-hydroxyethyl)piperazine-2,5-dione (3a); n.d. = not determined.
Selected DKP derivative 3,6-di(2-hydroxyethyl)piperazine-2,5-dione (3a) showed an antimicrobial effect on all of the tested microorganisms similarly, albeit weak; see Table 3.
Further, the antimicrobial effects of the preservative composition from the present invention were tested. The mixture of zinc bis- glycinate (1; 0.25% w/w) and 5-hydroxymethyl-l,5,6,7,11-undecane- pentol (2; 0.5% w/w) as the selected polyol II (product described in Example 2) showed significant synergic antimicrobial effects, evident as an effect on P. aeruginosa and full suppression of S. aureus, wherein individual compound 1 was not active. The difference between the determined CFU/g for P. aeruginosa in the preservative composition from the present invention (Example 2) was in the range of 10 2 lower (see Table 4) compared to either individual compound 1 or unpreserved base O/W emulsion (see Table 3). The result suggests a synergic effect of polyol 2 on antimicrobial activity of compound 1 against tested microorganisms. The preservative composition from the present invention, the product described in Example 2, eliminated all other tested microorganisms, and no viable CFU could be isolated.
Similarly, the mixture of zinc bis-glycinate (1; 0.25% w/w) and 3,6- di(2-hydroxyethyl)piperazine-2,5-dione (3a; 0.50% w/w) as the selected DKP derivative also exhibited significant synergic antimicrobial effects against P. aeruginosa and S. aureus. The product of Example 3 also showed about 10 2 reduction of CFU value for P. aeruginosa and complete supression of S. aureus compared to either compound 1 (see Table 3) or the unpreserved O/W emulsion; see Table 4. The efficacy of the preservative composition from the present invention, the product described in Example 3, was also effective against all other tested microorganisms, wherein no viable CFU could be isolated.
Table 4. Results of microbial challenge testing of the preservative composition from the present invention, the product described in Example 2. The result shows synergic antimicrobial effects of zinc bis-glycinate (1) and 5- hydroxymethyl-1,5,6,7,11-undecane-pentol (2) as the selected polyol. a a The experimental procedure for the microbial challenge test is described in Example 6.
CFU = colony forming units; TO = initial inoculum number; 7d = MB count of CFU/g after 7 days; 14d = MB count of CFU/g after 14 days; 28d = MB count of CFU/g after 28 days; log c = log change relative to initial inoculum number value; Control-1 = unpreserved base O/W emulsion, without any active substance; Example-2 = testing sample of base O/W emulsion with mixed in 0.25% w/w zinc bis-glycinate (1) and 0.50% w/w 5-hydroxymethyl-l,5,6,7,11-undecane-pentol (2), product described in Example 2; n.d. = not determined.
Table 5. Results of microbial challenge testing of the composition from the present invention, the product described in Example 3. The synergic antimicrobial effects of zinc bis- glycinate (1) and 3,6-di(2-hydroxyethyl)piperazine-2,5- dione (3a). a a The experimental procedure for the microbial challenge testing is described in Example 6. CFU = colony forming units; TO = initial inoculum number; 7d = MB count of CFU/g after 7 days; 14d = MB count of CFU/g after 14 days; 28d = MB count of CFU/g after 28 days; log c = log change relative to initial inoculum number value; Control-1 = unpreserved base O/W emulsion; Example-3 = testing sample of base O/W emulsion with mixed in 0.25% w/w zinc bis-glycinate (1), 0.5% w/w 3,6-di(2-hydroxyethyl) piperazine-2,5-dione (3a), product from Example 3; n.d. = not determined.
Finally, the ternary composition of zinc bis-glycinate (1; 0.25% w/w), 5-hydroxymethyl-l,5,6,7,11-undecane-pentol (2; 0.50% w/w) as selected model polyol, and 3,6-di(2-hydroxyethyl)piperazine-2,5- dione (3a; 0.5% w/w) as selected model DKP derivative, exhibited significant synergic antimicrobial effects against all tested microorganisms, especially against P. aeruginosa. Herein, the similar decrease of CFU value at the range of 10 2 was observed after 28 days of the challenge test, compared to the CFU value determined for either compound 1 or the unpreserved O/W emulsion; see Table 3 and Table 5. The preservative composition from the present invention, product of Example 4, was also effective against all other tested microorganisms, wherein no viable CFU could be isolated, except for A. brasiliensis that showed some viability at day 28.
Table 6. Results of microbial challenge testing of the preservative composition from the present invention, product described in Example 4. The synergic antimicrobial effects of zinc bis-glycinate (1), 5-hydroxymethyl-l,5,6,7,11-undecane- pentol (2) as selected polyol and 3,6-di(2- hydroxyethyl)piperazine-2,5-dione (3a) as selected DKP derivative. a
a The experimental procedure for performing microbial challenge test is described in Example 6.
CFU = colony forming units; TO = initial inoculum number; 7d = MB count of CFU/g after 7 days; 14d = MB count of CFU/g after 14 days; 28d = MB count of CFU/g after 28 days; log c = log change relative to initial inoculum number value; Control-1 = unpreserved base O/W emulsion; Example-3 = testing sample of base O/W emulsion with mixed in 0.25% w/w zinc bis-glycinate (1), 0.5% w/w 5-hydroxymethyl- 1,5,6,7,11-undecane-pentol (2), and 0.5% w/w 3,6-di(2-hydroxyethyl) piperazine-2,5-dione (3a), product described in Example 4; n.d. = not determined.
Preparation of the preservative composition from the present invention
The process of preparation of the preservative composition according to the present invention includes the homogenization of:
A. active ingredients (i)-(iii); with or without,
B. one or more excipients selected from (iv)-(ix); at 10-100°C for 1-60 minutes.
For the preparation of the preservative composition from the present invention in the liquid form, such as solution or suspension, the following auxiliary ingredients (excipients) can be used: (iv) liquid diluents: water, glycerol, mixtures of water and glycerol;
(v) suspending agents: methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methycellulose, xanthan gum;
(vi) wetting agents: polysorbates, poloxamers, lecithins, sorbitan esters; and if needed,
(vii) pH adjusting agents: citric acid, malic acid, sodium hydroxide, trisodium citrate, or its dihydrate.
For the preparation of the preservative composition in the flowable powder form, the following excipients can be used:
(viii) solid diluents: sorbitol, sodium chloride; and,
(ix) anti-caking agents: colloidal silicon dioxide, sodium aluminium silicate.
All standard devices can be used for the mixing of liquids, either as solutions or suspensions. In the preparation of the solid forms like powders, various blenders for raw powder materials can be employed, e.g., V-blender, bin-blender, etc.
Illustrative examples of the process for the preparation of the preservative composition from the present invention are described in experimental Examples 2-5.
The use of the preservative composition from the present invention
The preservative composition from the present invention is used in the production of water-based cosmetic, pharmaceutical, household, or chemical formulations to protect against their microbial spoilage.
The preservative composition from the present invention acts as an antimicrobial agent. It shows a biocidal effect at the upper end of the proposed concentrations and biostatic at the lower end of the recommended levels. It is active against bacteria, yeasts, and molds in various water-based products that are due to high water content, prone to microbial spoilage.
The preservative composition from the present invention is typically used in concentrations from 0.1-20% w/w within the formulation of the final product.
Examples
General remarks
All percentage (%) values of materials are expressed as weight portions (% w/w). The term „room temperature" relates to a temperature range of 20-25 °C. The mixing speed is expressed as the number of revolutions of the bin per minute (rpm). The term CFU means colony-forming units, which is the number of viable cells in a given sample.
The starting raw materials were purchased from the following suppliers: zinc bis-glycinate (1) from Ambinter (FR); 5- (hydroxymethyl ) -1,5,6, 7,11-undecanepentol (2 ) and 3,6-di(2- hydroxyethyl ) piperazine-2,5-dione (3a) from Merck AG (DE) ; purified water from Adrialab Ltd (HR); carbomer and colloidal silicon dioxide from Evonik Industries AG (DE); xanthan gum from Fagron (HR); glycerol from Emery Oleochemicals GmbH (DE); cetostearyl alcohol from BTC Europe GmbH; Myritol caprylic/capric triglyceride from BASF AG (DE); sunflower seed oil from Henry Lamotte Oils GmbH (DE); dimethicone from Dow Corning GmbH (DE); hydrogenated polyisobutene (and) sucrose polystearate from BASF AG (DE); glyceryl stearate from BTC Europe GmbH; sodium hydroxide solution (20% w/w) from Kemika (HR). All other starting raw materials, microbiological broths, and laboratory consumables were purchased from a local supplier.
The preparation of a base O/W emulsion was done as an outsourcing service by Adrialab Ltd (HR). Example 1. Preparation of the base oil-in-water (O/W) emulsion for microbial challenge testing of the preservative composition from the present invention
Formulation (for 1,000 g of base O/W emulsion):
(1) 70.90% w/w (709.00 g) deionized water
(2) 12.00% w/w (120.00 g) carbomer (2% aqueous solution)
(3) 0.10% w/w (1.00 g) xanthan gum
(4) 1.00% w/w (10.00 g) glycerol
(5) 2.00% w/w (20.00 g) cetearyl alcohol
(6) 5.00% w/w (50.00 g) caprylic/capric triglycerides
(7) 2.00% w/w (20.00 g) Helianthus annuus (sunflower) seed oil
(8) 2.00% w/w (20.00 g) dimethicone
(9) 3.00% w/w (30.00 g) sucrose polystearate (and) hydrogenated polyisobutene
(10) 1.75% w/w (17.50 g) glyceryl stearate
(11) 0.25% w/w (2.50 g) sodium hydroxide (20% aqueous solution)
Total: 100% w/w (1,000.00 g)
Preparation: carbomer and xanthan gum are dispersed in water and heated to 70-75 °C, when glycerine is added (this is Phase-1). Cetearyl alcohol, caprylic/capric triglyceride, Helianthus annuus (sunflower) seed oil, dimethicone, sucrose polystearate (and) hydrogenated polyisobutene and glyceryl stearate are mixed separately and also heated to 70-75 °C until everything is melted and mixed (Phase-2).
Then the Phase-2 is slowly added to Phase-1 at 70-75 °C temperature, and all is mixed until uniform. The mixture is cooled down to 60 °C, and the pH is adjusted with an aqueous solution (20% w/w) of sodium hydroxide (NaOH) to pH 6.0-6.5. The mixture is further cooled down with mixing to room temperature, and the pH is again adjusted to pH 6.0-6.5, if necessary. The product is white, almost odorless O/W emulsion. The formulation of the base O/W emulsion is given in Table 1.
Example 2: Preparation of the preservative composition from the present invention based on 10% w/w zinc bis-glycinate (1) and 20% w/w 5-hydroxymethyl-l,5,6,7,11-undecane-pentol (2)
Composition (for 100 g solution):
(1) 10.00% w/w (10.00 g) zinc bis-glycinate (1)
(2) 20.00% w/w (20.00 g) 5-hydroxymethyl-l,5,6,7,11-undecane- pentol (2)
(3) 70.00% w/w (70.00 g) purified water
(4) q.s. citric acid anhydrous, to pH 6.0-6.5
Total: 100% w/w (100.00 g)
Preparation: Purified water is added to the compounds 1 and 2 and dissolved by rocking at room temperature for half to one hour. The pH of the dissolved product is adjusted to pH = 6.0-6.5 with small amounts of anhydrous citric acid. The final product is in the form of a slightly yellow opaque solution with a faint oily odor.
Example 3: Preparation of the preservative composition from the present invention based on 10% w/w zinc bis-glycinate (1) and 20% w/w 3,6-di(2-hydroxyethyl)piperazine-2,5-dione (3a)
Composition (for 100 g solution):
(1) 10.00% w/w (10.00 g) zinc bis-glycinate (1)
(2) 20.00% w/w (20.00 g) 3,6-di(2-hydroxyethyl)piperazine-2,5- dione (3a)
(3) 70.00% w/w (70.00 g) purified water
Total: 100% w/w (100.00 g) Preparation: Purified water is added to compounds 1 and 3a and dissolved by stirring at room temperature for 15 minutes. The product is in the form of an opaque white solution without a distinctive odor.
Example 4: Preparation of the composition from the present invention based on 10% w/w zinc bis-glycinate (1), 20% w/w 5-hydroxymethyl-
1,5,6,7,11-undecane-pentol (2), and 20% w/w 3,6-di(2-hydroxyethyl) piperazine-2,5-dione (3a)
Composition (for 100 g solution):
(1) 10.00% w/w (10.00 g) zinc bis-glycinate (1)
(2) 20.00% w/w (20.00 g) 5-hydroxymethyl-l,5,6,7,11-undecane- pentol (2)
(3) 20.00% w/w (20.00 g) 3,6-di(2-hydroxyethyl)piperazine-2,5- dione (3a)
(4) 50.00% w/w (50.00 g) purified water
Total: 100% w/w (100.00 g)
Preparation: Purified water is added to compounds 1, 2, and 3a and dissolved by rocking at room temperature for half to one hour. The product is in the form of an opaque white-to-yellow solution with a faint oily odor.
Example 5: Preparation of the composition from the present invention based on 10% w/w zinc bis-glycinate (1), 20% w/w 5-hydroxymethyl- 1,5,6,7,11-undecane-pentol (2), and 50% w/w 3,6-di(2-hydroxyethyl) piperazine-2,5-dione (3a)
Composition (for 100 g free-flowing powder):
(1) 10.00% w/w (10.00 g) zinc bis-glycinate (1)
(2) 20.00% w/w (20.00 g) 5-hydroxymethyl-l,5,6,7,11-undecane- pentol (2) (3) 50.00% w/w (50.00 g) 3,6-di(2-hydroxyethyl)piperazine-2,5- dione (3a)
(4) 20.00% w/w (20.00 g) colloidal silicon dioxide, type Aerosil 200F
Total: 100% w/w (100.00 g)
Preparation: Previously weighted components (1-4) are combined in the overhead stirrer and homogenized by mixing for 15 minutes at 200 rpm. Powder product obtained in this way is further de-agglomerated by passing through a conical mill equipped with 1 mm sieve. The product is in the form of white-to-yellow free-flowing powder with a faint oily odor.
Example 6: Microbial challenge test of the preservative composition from the present invention
The testing of preservative efficacy of the preservative composition from the present invention was performed following the ISO11930:2012 standard on the below-listed microbial strains:
(i) Pseudomonas aeruginosa (P. aeruginosa) ATCC 9027;
(ii) Staphylococcus aureus (S. aureus) ATCC 6538;
(iii) Escherichia coli (E. coli) ATCC 8739;
(iv) Candida albicans (C. albicans) ATCC 10231; and,
(v) Aspergillus brasiliensis (A. brasiliensis) ATCC 16404.
The testing was performed using the base oil-in-water (O/W) cosmetic cream-like emulsion that is a suitable medium for microbial growth. The formulation of the base O/W emulsion is shown in Table 1, and its preparation is described in experimental Example 1.
For the control, the unpreserved base O/W emulsion, as well as the formulations obtained by mixing in of individual active substances, compounds 1, 2, and 3a, in the base O/W emulsion were used; see
Table 2. Each preservative composition in the present invention described in Examples 2-4, was individually mixed in the testing batches of base O/W emulsion and used in microbial challenge tests. The preservative composition of the control and testing samples is shown in Table 2.
Bacteria (P. aeruginosa, S. aureus, E. coli) were grown on tryptic soy agar (TSA) at 32.512.5 °C, yeast C. albicans on Sabouraud dextrose agar (SDA) at 22.512.5 °C, and mold A. brasiliensis at Potato dextrose agar (PDA) at 22.512.5 °C. For the inoculation, the microorganisms are harvested and washed with sterile saline solution. The number of microorganisms for inoculation was adjusted to Ixl0 5 -lxl0 6 CFU/ml for bacteria and Ixl0 4 -lxl0 5 CFU/ml for fungi.
Before their addition to the base O/W emulsion (the product described in Example 1), each active substance was first dissolved in purified water. Specifically, 5-(hydroxymethyl)-1,5,6,7,11- undecanepentol (2) was dissolved in purified water as the 50% solution, whilst 3,6-di(2-hydroxyethyl) piperazine-2,5-dione (3a) , and zinc bis-glycinate (1) each as 10-20% w/w solutions, all at room temperature. The pH value of each solution was tested and adjusted with citric acid to pH 6-6.5 if needed. The dissolved active components are added at the end of formulation preparation, at a temperature of 40 °C and thoroughly mixed into the formulation while cooling. The pH is corrected with citric acid to pH = 6-6.5 when cooled to room temperature, if necessary. Zinc bis-glycinate (1 ) was added to the final formulation at 0.25% w/w, while 5- (hydroxymethyl)-1,5,6,7,11-undecanepentol (2) and 3,6-di(2- hydroxyethyl) piperazine-2,5-dione (3a) were added at at 0.35% w/w and 0.8% w/w, respectively.
Preservative compositions from the present invention in the final forms described in Examples 2-4, were added in the same manner into the base O/W emulsion, at the concentrations corresponding to:
(i) 0.25% w/w of the compound 1 and 0.5% w/w of the compound 2 ;
Example 2; (ii) 0.25% w/w of the compound 1 and 0.5% of the compound 3a; Example 3; and,
(iii) 0.25% w/w of the compound 1, 0.5% w/w of the compound 2, and 0.5% w/w of the compound 3a; Example 4; respective to the amount of the final base O/W emulsion testing batches.
One hundred grams of the O-W emulsion with the mixed-in control individual active substances 1, 2, 3a, or the preservative compositions from the present invention described in Examples 2-4 was divided into five sterile containers, and each was inoculated with a different microorganism. The containers were stored at 22.5 °C ± 2.5 °C.
At time points 7 days (7d), 14 days (14d), and 28 days (28d), each test sample (except for A. brasiliensis that is omitted at 7d) is plated by spreading of a determined amount on agar plates in duplicates. The plates are incubated at 32.512.5 °C for 48-72 hours for bacteria and at 22.512.5 °C for fungi. The growth of yeast C. albicans is counted after 48-72 hours, and the mold A. brasiliensis after 3-5 days.
Before experimentation, neutralization and validation of the method are performed by dilution and growth in LT 100 broth.
The results of this study for the unpreserved base O/W emulsion (Control-1) and individual active substances 1, 2, and 3a (control experiments) are presented in Table 3.
The results for:
(i) the binary preservative compositions: a) zinc bis-glycinate (1) and 5-hydroxymethyl-l,5,6,7,11- undecane-pentol (2) are presented in Table 4; and, b) zinc bis-glycinate (1) and 3,6-di(2-hydroxyethyl) piperazine-2,5-dione (3a) are given in Table 5; whilst,
(ii) the ternary preservative composition: zinc bis-glycinate (1), 5-hydroxy methyl-1,5,6,7,11-undecane-pentol (2), and 3,6- di(2-hydroxy ethyl)piperazine-2,5-dione (3a) are shown in Table 6.
Conclusion
The preservative composition from the present invention based on the mixture of:
(i) zinc bis-glycinate (1) or its monohydrate as the principal active ingredient; from 0.5-20% w/w; and one or more secondary active ingredients selected from the following groups:
(ii) polyols of general formula II with linear- or branched-chain aliphatic hydrocarbon skeleton R with 5-18 carbon atoms and 4-8 hydroxyl (OH) groups in the molecule, e.g., 5-hydroxy methyl-1,5,6,7,11-undecane-pentol (2); from 0.5-90% w/w;
(iii) 3,6-di(substituted)piperazine-2,5-diones III wherein the substitution groups X, X' are the same or different, and defined as above, e.g., 3,6-di(2-hydroxyethyl)piperazine- 2,5-dione (3a); showed an effective antimicrobial activity against bacteria, yeasts, and molds in the model base O/W emulsion, as demonstrated with the microbial challenge tests for a standard set of microorganisms Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Candida albicans, and Aspergillus brasiliensis following the
ISO11930:2012 method. This antimicrobial activity was significantly stronger than in the case of the individual compound 1, which is considered the closest prior art solution (see literature references 4-6). Due to this fact, the inventive step of this invention is unambiguously demonstrated.
All active ingredients used in the composition from the present invention are of very low toxicity and high antimicrobial efficacy. Thus, the preservative composition from the present invention represents an effective and safe alternative to existing conventional preservatives that are of medium-to-high toxicological concern. The present invention also clearly surpasses the efficacy of zinc bis-glycinate (1), as the only antimicrobial agent (see literature references 4-6).
Industrial Applicability
The preservative composition from the present invention is used in the manufacturing of various water-based cosmetic, pharmaceutical, or chemical products as a preservative against their microbiological spoilage. In this manner, the industrial applicability of the present invention is obvious.