Indolicidine-containing compositions for personal care / hygiene applications

Abstract

The present invention relates to antimicrobial combinations for topical application. Particularly, it relates to antimicrobial combinations comprising an Indolicidin peptide analogue (C2), one or more non-ionic surfactants and at least an organic acid and / or metal salt thereof based chelating agent.

Description

[0001] ANTIMICROBIAL COMBINATIONS WITH PEPTIDE FOR PERSONAL CARE / HYGIENE APPLICATIONS

[0002] FIELD OF INVENTION

[0003] The present invention relates to antimicrobial combinations for topical application. Particularly, it relates to antimicrobial combinations comprising an antimicrobial peptide and one or more non-ionic surfactants, with one or more organic acid based chelators.

[0004] BACKGROUND OF INVENTION

[0005] The well-known and currently used antimicrobial actives such as Triclosan mediate bacterial killing by specific mechanism that leads to selective pressure on the microorganism to develop resistance. In organisms such as Pseudomonas aeruginosa the resistance is plasmid mediated leading to faster spread of resistance. The efficacious antimicrobial actives such as Triclosan are under global regulatory agency’s scanner because of safety concern. Triclosan is associated with endocrine disruption and antimicrobial resistance.

[0006] World Health Organization (WHO) classifies the appearance of antibiotic resistance as one of the biggest threat to human health. Indiscriminate use of certain antimicrobial actives would exacerbate the problem. There are few active combinations that are efficacious as well as safe. The long-term systemic exposure to actives like Triclosan is an area of concern. Hence, there is a need to develop efficacious and safer antimicrobial active combinations.

[0007] Antimicrobial Peptides (AMPs) are part of innate defense for all living organisms. Antimicrobial properties are inherent to AMPs. There are few reports of determination of log reduction for AMPs. However, the first time point for measuring the log reduction is 30 minutes. For hand hygiene applications, the relevant time points are 1 minute and 5 minutes. For AMPs, such data is not cited in the prior art. Further, AMPs cited in prior art are tested in conditions that do not mimic real-life like scenario with respect to hand hygiene standard requirements. For example, mimicking soil conditions by testing in the presence of protein, Bovine Serum Albumin (BSA), and testing at temperature such as 20°C and for 1 minute / 5 minute as contact time for testing depending on the applications. The British Standard EN 1276 has laid out stringent protocol for antibacterial efficacy testing of the active system / formulation for hand hygiene applications.

[0008] Most of the personal care and hygiene linked products are used at a scale at which even a tiny amount of any environmentally non-friendly ingredient has the potential to cause substantial prejudice to the ecosystem. In view of this, the present inventors have come up with combinations and compositions that comprise environment friendly ingredients without any compromise on the effective log kill or rapid kill feature.

[0009] WO9958141A1 relates to indolicidin analogs and methods of using the analogs to reduce or inhibit microbial growth or survival.

[0010] US20020106802 relates to three single tryptophan analogs ILPWKLPLLPLRR-amide (IL4), ILPLKLPWLPLRR- amide (IL8) and ILPLKLPLLPWRR- amide (IL11), of indolicidin, a cationic tridecapeptide amide found in the granules of bovine neutrophils, said analogs having said amino acid only at 4th, 8thor 11thposition from N-terminal, with leucine at its all other conventional positions, said analogs selectively having only anti-microbial activity and no hemolytic activity of Indolicidin, and thereby providing therapeutic options and a method thereof.

[0011] IL19950112641 relates to indolicidin analogs, which are tryptophan-rich peptides that have substantially the same amino acid sequence as naturally occurring indolicidin, exhibit broad spectrum antimicrobial activity and have antimicrobial selectivity as compared to naturally occurring indolicidin.

[0012] W098 / 40401 Al relates to the combination of specific indolicidin analogues with antibiotic agent for treating infections. US201313745359 relates to antimicrobial metallodrugs comprising an antimicrobial peptide (“AMP”) and / or an antibiotic covalently bound to a metal binding moiety for the treatment of broad spectrum of pathogenic organisms.

[0013] US 19990318195 relates to an ophthalmic composition for storing, cleaning, or disinfecting a contact lens, said composition comprising: an indolicidin in an antimicrobially effective amount; and a buffer compatible with application to a mammalian eye.

[0014] JP5894457B2 discloses an antimicrobial composition containing an antimicrobial peptide as component (A), one or more cellulose derivatives selected from alkyl celluloses and hydroxyalkyl celluloses as component (B), and a chelating agent as component (C).

[0015] Guo, Hui, et al. "Synergistic antimicrobial system based on nisin and a- hydroxy acid." (2023) discloses synergistic effect between nisin and a-hydroxy organic acids on E. coli and S. aureus.

[0016] US6197315B2 relates to an antimicrobial wipe comprising an antimicrobial cleansing composition that comprises conventionally known non-peptide actives such as Triclosan®, Triclocarban®, Octopirox®, PCMX, ZPT, natural essential oils and their key ingredients along with surfactant / s and organic acid / s. However, it is silent on 5 log kill in one minute. Further, many of these actives can no longer be used in many products on account of the regulatory restrictions.

[0017] In IN202031033995, the co-owned application, the combination of antimicrobial peptide namely C2 peptide, chelator namely EDTA and / or non-ionic surfactant namely decyl glucoside was covered as showing synergistic bactericidal activity against four test bacteria E. coli, S. aureus, P. aeruginosa and E. hirae.

[0018] However, IN202031033995 does not disclose environment friendly organic acid based chelating agents.

[0019] There is a long felt need to develop new, potent peptide based antimicrobial combinations which are active against antibiotic resistant entities and provide rapid and effective log kill. The present inventors have surprisingly developed novel effective antimicrobial combinations and compositions which ameliorate the aforesaid shortcomings of the prior art.

[0020] OBJECTS OF THE INVENTION

[0021] It is an object of the present invention to provide effective antimicrobial combinations and compositions which provide rapid and effective kill with improved spectrum and improved potency.

[0022] It is another object of the present invention to provide cost-effective, environment friendly antimicrobial combinations and compositions.

[0023] It is yet another object of the present invention to provide effective antimicrobial combinations and compositions which can be used topically for various purposes.

[0024] It is a further object of the present invention to provide antimicrobial combinations and compositions that comprise environment friendly and biodegradable chelating agents.

[0025] SUMMARY OF THE INVENTION

[0026] According to one aspect there is provided an antimicrobial combination comprising:

[0027] (i) Indolicidin peptide analogue (C2) as set forth in SEQ ID 1, or salts and derivatives thereof at a concentration ranging from 0.01 wt. % to 0.15 wt. %;

[0028] (ii) non-ionic surfactant ranging from 0.5 wt.% to 5 wt.%

[0029] (iii) at least an organic acid and / or a metal salt thereof chelating agent ranging from 0.05 wt.% to 0.15 wt.%.

[0030] According to another aspect there is provided an anti-microbial composition comprising the combination as mentioned above. According to another aspect there is provided an anti -acne composition comprising:

[0031] (i) Indolicidin peptide analogue (C2) as set forth in SEQ ID 1, or salts and derivatives thereof;

[0032] (ii) at least an organic acid and / or a metal salt thereof chelating agent selected from the group consisting of malic acid, PESA and GLDA; and

[0033] (iii) non-ionic surfactant selected from the group consisting of Decyl glucoside and caprylyl / capryl glucoside; along with cosmetically acceptable excipients.

[0034] According to a further aspect there is provided an anti-dandruff composition comprising:

[0035] (ii) Indolicidin peptide analogue (C2) as set forth in SEQ ID 1, or salts and derivatives thereof;

[0036] (ii) at least an organic acid and / or a metal salt thereof chelating agent selected from the group consisting of malic acid, PESA and GLDA; and

[0037] (iii) non-ionic surfactant selected from the group consisting of Decyl glucoside and caprylyl / capryl glucoside; along with cosmetically acceptable excipients.

[0038] According to a further aspect there is provided wet wipe impregnated with an antimicrobial composition comprising:

[0039] (i) C2 peptide as set forth in SEQ ID No.l;

[0040] (ii) at least one non-ionic surfactant selected from the group consisting of Decyl glucoside and caprylyl / capryl glucoside; and

[0041] (iii) at least one organic acid based chelator selected from the group consisting of malic acid, PESA and GLDA; along with cosmetically acceptable excipients.

[0042] BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

[0043] Figure 1 illustrates HPLC chromatogram of C2 peptide

[0044] DETAILED DESCRIPTION OF THE INVENTION

[0045] The following description with reference to the accompanying drawings is provided to assist in a comprehensive understanding of exemplary embodiments of the invention. It includes various specific details to assist in that understanding but these are to be regarded as merely exemplary.

[0046] Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope of the invention. In addition, descriptions of well-known functions and constructions are omitted for clarity and conciseness.

[0047] The terms and words used in the following description and claims are not limited to the bibliographical meanings, but, are merely used by the inventor to enable a clear and consistent understanding of the invention. Accordingly, it should be apparent to those skilled in the art that the following description of exemplary embodiments of the present invention are provided for illustration purpose only and not for the purpose of limiting the scope of the invention as defined by the appended claims and their equivalents.

[0048] It is to be understood that the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0049] Features that are described and / or illustrated with respect to one embodiment may be used in the same way or in a similar way in one or more other embodiments and / or in combination with or instead of the features of the other embodiments.

[0050] It should be emphasized that the term “comprises / comprising” when used in this specification is taken to specify the presence of stated features, steps or components but does not preclude the presence or addition of one or more other features, steps, components or groups thereof. The term "peptide" as used herein refers to short polypeptides consisting from 2 to 50 amino acid residues, “Polypeptides” and “proteins” comprise more than 50 amino acid residues. A peptide may have a specific function. A peptide can be a naturally occurring peptide or a synthetically designed and produced peptide. The peptide can be, for example, derived or removed from a native protein by enzymatic or chemical cleavage, or can be prepared using conventional peptide synthesis techniques (e.g., solid phase synthesis) or molecular biology techniques (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)).

[0051] The term, "antimicrobial peptide" (AMP) as used herein refers to any peptide that has microbiocidal and / or microbio static activity. Thus, the term "antimicrobial peptide" as used herein refers in particular to any peptide having anti-bacterial, anti-fungal, anti-mycotic, antiparasitic, anti-protozoal, anti-viral, anti-infectious, anti-infective and / or germicidal, algicidal, amoebicidal, microbiocidal, bacteriocidal, fungicidal, parasitioidal, protozoacidal properties.

[0052] The term “derivatives” as used herein includes peptide analogues, peptide fragments and modified peptides. The derivatives may be a sequence that has at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1 of present invention.

[0053] The terms “environment friendly and biodegradable ingredient” as used herein means an ingredient that is relatively more environment friendly and more biodegradable as compared to EDTA based chelating agents such as disodium EDTA, Tetra-sodium EDTA etc.

[0054] The term “log kill / log reduction” as used herein denotes the number of cells killed in a test. If a log reduction is a whole integer, then its numerical value equals the number of nines in the percent reduction figure. For e.g. 1 log reduction = 90% reduction, 2 log reduction = 99% reduction, 3 log reduction = 99.9% reduction, 4 log reduction = 99.99% reduction, 5 log reduction = 99.999% reduction, 6 log reduction = 99.9999% reduction.

[0055] The term “minimum inhibitory concentration (MIC)” of an antimicrobial peptide is the lowest concentration of peptide that completely inhibits growth of the organism. In general, lower MIC values are preferred.

[0056] The term “Hygiene” as used herein means a series of practices performed to preserve health. According to the World Health Organization (WHO), "Hygiene refers to conditions and practices that help to maintain health and prevent the spread of diseases."

[0057] The term "skin" as used herein means the entire epidermis of the human body.

[0058] The term “peptide analogue” as used herein means a peptide designed to mimic a native peptide (in the present invention indolicidin).

[0059] The term “peptide fragment” as used herein means functional motifs / shorter fragments which retain the biological activity of the full-length peptide.

[0060] The term "skin" as used herein means the entire epidermis of any mammals including the humans.

[0061] The AMP active domains have potential to be used in personal hygiene products due to their ability to kill pathogenic microorganisms. In the present invention, inventors have disclosed analogues based on the natural AMPs.

[0062] Indolicidin (IL) is a 13-residue cationic anti-microbial peptide rich in tryptophan and proline having the sequence ILPWKWPWWPWRR-amide. The Indolicidins belong to family of cationic AMPs that adopt random coil confirmation. It is present in the cytoplasmic granules of bovine neutrophils. [Selsted et al., J. Biol. Chem. 267:4292, 1992] . The presence of five tryptophans out of 13 -residues renders its amino acid composition unique among host-defense endogenous anti-microbial peptides. It is cationic due to the presence of two arginines and one lysine and exhibits broad-spectrum anti-microbial activity. The bovine indolicidin per se is known to be cytotoxic. The bovine indolicidin peptide analogue C2 as set forth in SEQ ID No. 1 \Subbalakshmi et al., Requirements for antibacterial and hemolytic activities in the bovine neutrophil derived 13-residue peptide indolicidin. FEBS Lett. 1996 Oct 14;395(l):48-52.] has 62% sequence similarity with the native bovine indolicidin.

[0063] Typically, synthetic peptides are produced as salts of Trifluroacetate (TFA), Acetate, chloride, lactate. However, the peptide sequence is the active principle that is linked to its efficacy, that is, antimicrobial activity. Hence, all salt forms of the peptide (TFA, acetate etc) will have similar activities.

[0064] In one aspect, the present invention provides synergistic antimicrobial combinations comprising an effective concentration of antimicrobial peptide (indolicidin peptide analogue or derivatives thereof) and an effective concentration of at least one non-ionic surfactant along with an organic acid based chelating agent.

[0065] Further, the present invention also provides compositions that comprise these combinations. The compositions of the present invention are directed to specific personal care and hygiene end use cases such as wet wipes, anti-acne and anti-dandruff.

[0066] Organic acid as Chelators

[0067] It is established that for effective antimicrobial activity against bacteria, chelators are generally used. In the skin hygiene products such as for example wet wipes formulation, EDTA are typically used to sequester di and trivalent metal ions preventing precipitation of the salts responsible for water hardness and protecting against color changes caused by heavy metal ions.

[0068] In tune with growing environmental concern, the present invention also provides environment friendly and biodegradable organic acid based chelating agents.

[0069] The present invention is directed to find a relatively safer and acceptable chelating agent without adversely impacting the efficacy of the cleaning composition. Apart from the regulatory perspective, peptides being extremely sensitive moieties, the carrier formulations for such active combinations also requires agents that do not in any manner affect stability and efficacy of the peptide. After several experiments, the inventors have arrived at set of chelating agents that fulfills these broader criterions. Over and above this key aspect, the inventors also surprisingly found that the organic acid based chelating agents, complement the synergistic effect with the non-ionic surfactant

[0070] COMBINATIONS:

[0071] The present invention provides an antimicrobial combination comprising an effective concentration of Indolicidin peptide analogue (C2) as set forth in SEQ ID 1, or salts and derivatives thereof; at least one non-ionic surfactant; and at least an organic acid and / or a metal salt thereof as the chelating agent.

[0072] The antimicrobial combination of the present invention, comprises an effective concentration of Indolicidin peptide analogue (C2) as set forth in SEQ ID 1, or salts and derivatives thereof; at least one non-ionic surfactant; and at least an organic acid and / or a metal salt thereof as the chelating agent, such that it achieves a log-kill of at least 5, after a contact time of about 1 min against Gram positive and Gram-negative bacteria

[0073] In an embodiment the organic acid based chelating agent / chelator and / or a metal salt thereof is environment friendly and biodegradable.

[0074] In an embodiment, the organic acid based chelating agent / chelator and / or a metal salt thereof is selected from malic acid, Polyepoxy Succinic Acid (PESA), Glutamic Acid Diacetic Acid (GLDA), Malic acid.

[0075] Particularly preferred and exemplified organic acid-based chelators and their molecular formulas are as under: *This indicates a repeating unit of four carbon atoms, four hydrogen atoms, and five oxygen atoms, with "n" representing the number of repeating units in the polymer chain.

[0076] The present invention provides an antimicrobial combination comprising an effective concentration of indolicidin peptide analogue (C2) as set forth in SEQ ID 1, or salts and derivatives thereof, an effective concentration of at least a non-ionic surfactant and an effective concentration of at least an organic acid based chelating agent.

[0077] In an embodiment, the indolicidin peptide analogue (C2) as set forth in SEQ ID 1, or salts and derivatives thereof is at a concentration ranging from 0.01 wt.% to 0.15 wt. %.

[0078] In an embodiment, the organic acid based chelating agent has a concentration ranging from 0.05 wt.% to 0.15 wt.%.

[0079] In an embodiment, non-ionic surfactant ranging from 0.5 wt.% to 5 wt.%.

[0080] In an embodiment, the non-ionic surfactant is selected from the group of Alkyl-Poly Glucoside (APG).

[0081] In an embodiment, the Alkyl-Poly Glucoside (APG) is selected from Decyl- Glucoside, Lauryl Glucoside, Capryl glucoside, Caprylyl / Capryl Glucoside, Arachidyl glucoside or combinations thereof.

[0082] In an embodiment, the Alkyl-Poly Glucoside (APG) is Decyl-Glucoside.

[0083] In another embodiment the Alkyl-Poly Glucoside (APG) is Caprylyl / Capryl Glucoside.

[0084] The antimicrobial combination achieves a log-kill of at least 5, after a contact time of about 1 min against Gram positive and Gram negative bacteria.

[0085] The Gram positive and Gram negative bacteria include methicillin resistant S. aureus (MRSA), penicillin and carbapenem resistant E. coli.

[0086] Further the antimicrobial combination achieves a log-kill of at least 5, after a contact time of about 30 min against Cutibacterium acnes.

[0087] The antimicrobial combination achieves a log-kill of at least 4, after a contact time of about 1 min against Candida albicans. COMPOSITIONS:

[0088] The antimicrobial compositions of the present invention comprises the combination as mentioned above along with cosmetically acceptable excipients.

[0089] In an embodiment, the antimicrobial compositions of the present invention comprise a synergistic combination along with cosmetically acceptable excipients.

[0090] The composition according to the invention may be provided as an anti -acne composition comprising:

[0091] (i) Indolicidin peptide analogue (C2) as set forth in SEQ ID 1, or salts and derivatives thereof ;

[0092] (ii) at least an organic acid and / or a metal salt thereof chelating agent selected from the group consisting of malic acid, PESA and GLDA; and

[0093] (iii) non-ionic surfactant selected from the group consisting of Decyl glucoside and caprylyl / capryl glucoside; along with cosmetically acceptable excipients.

[0094] The amount of the C2 peptide in such anti-acne composition is from 0.01 to 0.15% The chelating agent may be present in such anti acne composition in amount of 0.05 to 0.15%

[0095] The anti-acne composition comprises non-ionic surfactant in amount of 0.25% to 2.5%

[0096] The composition according to the invention may be provided an anti -dandruff composition comprising:

[0097] (i) Indolicidin peptide analogue (C2) as set forth in SEQ ID 1, or salts and derivatives thereof;

[0098] (ii) at least an organic acid and / or a metal salt thereof chelating agent selected from the group consisting of malic acid, PESA and GLDA; and (iii) non-ionic surfactant selected from the group consisting of Decyl glucoside and caprylyl / capryl glucoside; along with cosmetically acceptable excipients.

[0099] The amount of the C2 peptide in such anti-dandruff composition is from 0.01 to 0.15%

[0100] The chelating agent may be present in such anti dandruff composition in amount of 0.05 to 0.15%

[0101] The anti-dandruff composition comprises non-ionic surfactant in amount of 0.25 to 5%

[0102] In an embodiment there is provided a wet wipe impregnated with an antimicrobial composition comprising:

[0103] (i) C2 peptide as set forth in SEQ ID No.l;

[0104] (ii) at least one non-ionic surfactant selected from the group consisting of Decyl glucoside and caprylyl / capryl glucoside; and

[0105] (iii) at least one organic acid based chelator selected from the group consisting of malic acid, PESA and GLDA; along with cosmetically acceptable excipients.

[0106] The wet wipe impregnated with an antimicrobial composition comprises C2 peptide in amount ranging from 0.0125wt% to 0.075wt%,

[0107] The wet wipe impregnated with an antimicrobial composition comprises non-ionic surfactant in ranges from 0.025wt% to 2wt%.

[0108] The wet wipe impregnated with an antimicrobial composition comprises organic acid based chelator and / or a metal salt in an amount that ranges from 0.025 wt% to 0.15wt%.

[0109] The wet wipe impregnated with an antimicrobial composition has a pH ranging between 5.0 to 7.0. In an embodiment, there is provided a wet wipe impregnated with an antimicrobial composition comprising:

[0110] (i) 0.025 wt.% of C2 peptide as set forth in SEQ ID No.l;

[0111] (ii) 1.65 wt.% of Decyl glucoside; and

[0112] (iii) 0.05 to 0.15 wt.% of malic acid; along with cosmetically acceptable excipients.

[0113] In an embodiment, there is provided a wet wipe impregnated with an antimicrobial composition comprising:

[0114] (i) 0.025 wt.% of C2 peptide as set forth in SEQ ID No.l;

[0115] (ii) 1.65 wt.% of Decyl glucoside; and

[0116] (iii) 0.05 to 0.15 wt.% of Polyepoxy Succinic Acid and Glutamic Acid Diacetic Acid; along with cosmetically acceptable excipients.

[0117] In an embodiment, the wet wipe has a pH of 5.5 or 7.0.

[0118] The present invention also provides a cosmetic method of disinfecting a surface, the method comprising the steps of: providing a wet wipe impregnated with the antimicrobial composition as above; and wiping the surface with the wet wipe.

[0119] In an embodiment, the cosmetically acceptable excipients are selected from emulsifiers, rheology modifying agents, fillers / thickening agents, structurants, emollients, humectants, preservatives, fragrances and combinations thereof.

[0120] Thickening agents include but are not limited to hydrogels, PEG (polyethyelene glycol), polyacrylic acid, hydroxypropyl methylcellulose (HPMC), Bentone, Natural or synthetic gum. Rheology modifying agents include but are not limited to hydrophilic polymers. Structurants include but are not limited to hydrogenated vegetable oil, hydrogenated castor oil, fatty acids, beeswax, paraffin wax. Humectants include but are not limited to glycerin and derivatives. Emollients include but are not limited to fatty acids, fatty alcohol esters, hydrocarbons, mineral oils, polyorganosiloxane. Fragrances include but are not limited to give woody notes, floral notes, oriental notes and fresh notes. Preservatives include but are not limited to essential oils, organic acids and synthetic. The surfactants are preferably chosen from nonionic, amphoteric and anionic surfactants.

[0121] In an embodiment, the antimicrobial compositions of the present invention may be in the form of wet wipes, sanitizer, hand wash, body wash, soap, shampoo, wound healing cream / lotion, surface cleaner, gels, spray, foam, cream and the like.

[0122] The cosmetic composition according to the invention may thus be in the form of a lotion, a cream or a fluid gel dispensed as an aerosol spray, in a pumpdispenser bottle or as a roll- on, in the form of a thick cream dispensed in a tube and in the form of a stick or a powder, and, in this regard, may contain ingredients and propellants generally used in products of this type that are well known to those skilled in the art.

[0123] METHOD OF TREATMENT

[0124] The present invention also provides method of treatment of subjects comprising applying the composition of the present invention to the skin area to stop spread of infections caused by antibiotic resistant bacteria including methicillin resistant S. aureus (MRS A), Penicillin and Carbapenem resistant E. coli, Cutibacterium acnes, Candida albica.

[0125] The Gram positive and Gram negative bacteria include methicillin resistant S. aureus (MRSA), penicillin and carbapenem resistant E. coli.

[0126] The antimicrobial combination achieves a log-kill of at least 4, after a contact time of about 1 min against Candida albicans.

[0127] Further the antimicrobial combination achieves a log-kill of at least 5, after a contact time of about 30 min against Cutibacterium acnes. Drawbacks of prior art:

[0128] • To the best of inventor’s knowledge, there are no reports of demonstration of 5 log kill in 1 minute with AMP with specific surfactant composition against both Gram positive and Gram negative bacteria in presence of rigorous conditions that simulate hand hygiene conditions.

[0129] ■ In majority of prior art patents, AMP and antibiotic synergy is demonstrated.

[0130] ■ In other patents covered in prior art, AMPs are listed as anti-infective agents.

[0131] ■ In prior art, synergy is not demonstrated in terms of significant log kill i.e.

[0132] 5 log reduction, 99.999% reduction of microorganisms in 1 minute.

[0133] ■ In prior art, synergy is not demonstrated by active system against broad spectrum of bacteria including pathogenic Gram positive and Gram negative bacteria.

[0134] ■ The experimental conditions used in prior art do not mimic conditions required for hand hygiene applications. For example, 1 minute contact time, using bovine serum albumin (BSA) as interfering agent as per BS EN 1276.

[0135] Advantageous features of present invention:

[0136] ■ The present invention provides a unique and synergistic combination of AMP and chelating agent / surfactant that increases bacterial reduction. The specific ratios at which synergy in terms of bacterial reduction is demonstrated for the above-mentioned combination.

[0137] ■ The present invention has identified a synergistic combination of Indolicidin analog AMP and chelating agent / non-ionic surfactant that synergistically enhances bactericidal efficacy. The combination of AMP (peptide C2) and organic acid based chelator demonstrated synergy in antimicrobial efficacy than at tested individual concentrations.

[0138] ■ In 1 minute contact kill experiment (in presence of 3% BSA), the composition of peptide (C2) & chelating agent / non-ionic surfactant lead to higher log reduction against Gram negative bacteria Pseudomonas aeruginosa, Escherichia coli and Gram positive bacteria Staphylococcus aureus, Enterococcus hirae. The AMP+ chelating agent composition is working synergistically at lower concentrations providing 99.999% reduction against Gram positive and Gram negative under rigorous, challenging testing conditions.

[0139] ■ The typical costs of chemical synthesis of peptide are on higher side. There is significant reduction of peptide concentration achieved in presence of synergistic active.

[0140] The synergistic combinations of present invention can be used in the following products in any of the formats such as gel, cream, ointment, spray and wipes:

[0141] • Hand wash, body wash, bar soap, liquid soap, wipes, hand cream, feet cream, shave cream, feminine intimate hygiene, Aftershave lotion, After shave cream, face cleanser, shampoo, hair conditioner, surface cleanser and

[0142] • Hand sanitizer liquid, Hand sanitizer gel, deodorant, foot deodorant, and

[0143] • Vegetable & fruit wash, Vegetable & fruit spray, surface disinfectant, surface cleaner, floor cleaner and

[0144] • Other cosmetic products as preservatives / preservative system

[0145] • AMP compositions can be formulated in ointment base or as an antiseptic liquid for treating external skin wound such as skin abrasion, cuts / pricks, nicks, bums.

[0146] • Compositions for treating acne, dandruff, E. coli, Candida, infections

[0147] EXAMPLES:

[0148] The following examples are meant to illustrate the present invention. The examples are presented to exemplify the invention and are not to be considered as limiting the scope of the invention

[0149] Tests and Validation: Materials and methods

[0150] Description of the components of the invention

[0151] 1. Peptide C2 (Indolicidin analog): The 13 amino acid peptide with sequence ILPFKFPFFPFRR [SEQ ID NO.l] was custom synthesized from USV Pvt. Ltd, India at >98% purity based on HPLC profile. The HPLC chromatogram is enclosed (Figure 1). The molecular weight of the peptide is 1711 Daltons.

[0152] 2. DL Malic acid - Aldrich #240176

[0153] 3. Lactic Acid- Sigma- Aldrich #27714, Merck

[0154] 4. Citric acid - BDH GPR™ 279844E, VWR International Limited

[0155] 5. Caprylyl / capryl Glucoside- Seppic Oramix CG 110

[0156] Microorganism :

[0157] The microorganisms used in the study are:

[0158] 1. Pseudomonas aeruginosa, ATCC 15442

[0159] 2. Escherichia coli ATCC 10536

[0160] 3. Staphylococcus aureus ATCC 6538

[0161] 4. Enterococcus hirae ATCC 10541

[0162] The glycerol stock of these bacteria was stored at -80°C. The culture was maintained on Tryptone soya agar (TSA) and sub-cultured every two weeks.

[0163] Antimicrobial activity Assay to determine Microbicidal Efficacy:

[0164] Antimicrobial activity was determined using quantitative suspension test (contact kill) using Dilution-neutralization method with contact time of 1 minute and 5 minutes. The contact kill experiment was conducted at 20°C.

[0165] • BSA: 3% BSA [3 g I’1bovine albumin (final concentration)] was used as interfering substance in the assay. Bovine serum albumin (heat shock fraction, pH 5.2, >96%) was procured from Sigma Chemical Company (Catalog no A8022).

[0166] • Neutralizer solution: The neutralizer was used to stop the activity of antimicrobials at determined time points. The composition of neutralizer was polysorbate 80, 30g / l + saponin, 30 g / 1 + lecithin, 3 g / 1 & pH 7.2.

[0167] • The media chemicals Tryptone Soya Agar (TSA), Tryptone soya broth (TSB) and other chemicals were procured from HiMedia Laboratories, Mumbai, India. • Diluent: Diluent consisted of Tryptone 1.0 g / 1 Sodium chloride (NaCl) 8.5 g / 1 in water.

[0168] Briefly, the cells were added to test samples for 1 minute ±5 seconds at 20°C in a dry bath set at 20°C (Eppendorf Thermomixer comfort), the antimicrobial activities of samples were stopped at the end of predetermined contact time by diluting with neutralizer, and finally, counting the number of surviving bacteria to calculate the level of killing achieved by the test samples.

[0169] Colonies of bacteria from the TSA plate were suspended in diluent to prepare bacterial cell suspension of about 108CFU / ml. The absorbance of bacterial suspension was measured in Thermo Multiskan at 600nm using cuvette (BRAND, Polystyrene 2.5 mL volume cuvette). Different concentrations of peptide and combination with naturals were prepared in Milli-Q water. Bacterial suspension, test samples and BSA solutions were equilibrated at 20°C for 10 min. To lOOul of bacterial suspension, lOOul of BSA was added. Within 5 minutes, the bacterial suspension was added to 800ul of test samples & immediately vortexed. To stop the action of antimicrobial, an aliquot of assay content was transferred to neutralizer solution and left for 5 minute. The neutralized samples were serially diluted and the surviving cell counts in the dilutions were determined on TSA plates after incubation at 37°C for 24 hours. Activity was evaluated by logio reduction factor that was calculated as the difference between logarithms of CFU / ml before and after exposure to test samples.

[0170] MIC determination and Checkerboard assay:

[0171] Minimum inhibitory concentration (MIC) of actives was determined by CLSI method. The Muller Hilton (MH) broth was used to grow bacteria at 37C. The MIC determination was done in 96 well plate.

[0172] A] Minimum Inhibitory Concentration (MIC) of C2 and malic acid alone:

[0173] Initially, the MIC of C2 and malic acid was evaluated using broth microdilution assay in 96 well microtiter plate. MIC of C2 and malic acid was determined as 0.4% and 0.2%, respectively. These values were important in establishing the individual contribution of the combination components towards inhibition of bacterial growth.

[0174] B] Checkerboard assay to determine the synergism between C2 and malic acid:

[0175] The synergistic effect of combination of C2 peptide + malic acid was established by two-dimensional checkerboard assay using 96 well microtiter plate. 11 different concentrations of C2 and 7 different concentrations of malic acid were tested. C2 at 2X MIC (0.8%) was added to the last column of 96 well microtiter plate, which was followed by the addition of two-fold diluted concentration of C2 for every consecutive column. Thus, the test concentrations of C2 were added in the range of 0.8% to 0 %. Similarly, malic acid at MIC (0.2%) was added to the first row, which was diluted two-fold lesser concentration for every successive row. The last test concentration of each active was set at 0%, to examine the activity of individual active at various test concentrations in combination. Appropriate control, negative control and blank were taken into account.

[0176] EXAMPLE 1:

[0177] Comparative Example Formulation of C2 peptide+ EDTA chelator+ decyl glucoside

[0178] In the previously filed co-owned patent application (IN202031033995), the below combination was covered as showing synergistic bactericidal activity against four test bacteria E. coli, S. aureus, P. aeruginosa and E. hirae.

[0179] • C2 peptide (50 pM to 900 pM i.e. 0.01% to 0.15%) + EDTA (7.5 mM i.e.0.25%) + decyl glucoside / DG (0.5% to 5%).

[0180] With the above, wet wipes formulation containing C2 peptide, Decyl glucoside (DG) and EDTA was developed. The wet wipes composition was as below.

[0181] Glycerin (1%), PEG-40 Hydrogenated Castor Oil1(0.25%), Decyl Glucoside (1.65%), Phenoxyethanol (0.3%), Propylene Glycol (0.2%), Disodium EDTA (0.2%), C2 peptide (0.025%), Fragrance (0.2%) and water q.s. pH was adjusted with Citric acid / Triethanolamine to pH 5.

[0182] The antimicrobial efficacy of C2 + Decyl Glucoside + EDTA combination in wet wipes base against test bacteria E. coli, P. aeruginosa, E. hirae and .S'. aureus was tested. The efficacy was measured in terms of log reduction and is presented in table 1.

[0183] Table 1. Antimicrobial efficacy of C2 + surfactant and / or chelator combinations (EN1276, contact time: 1 min, dirt conditions) against test bacteria, E. coli, P. aeruginosa, S. aureus, E. hirae

[0184] The wipes formulation initially contained 0.25% disodium EDTA. Further iterations demonstrated that 0.05% disodium EDTA is sufficient for bactericidal activity (>5 log reduction) against four test bacteria as demonstrated in the above examples (table 1). Example 2: Inventive antimicrobial combinations of the present invention with Organic acid based Chelators :

[0185] The inventors of the present invention come up with compositions comprising environment friendly and biodegradable, chelating agents. The experiments linked with use of various plausible environment friendly and biodegradable chelating agents are provided herein below:

[0186] Antimicrobial combination C2 peptide + Caprylyl / Capryl Glucoside + Malic acid

[0187] Antibacterial efficacy of AMP C2 + Capryryl / capryl glucoside (CG) + Organic Acid against bacteria as per EN1276.

[0188] Antimicrobially effective amount of C2 peptide was admixed with at least one non-ionic surfactant [Capryryl / capryl glucoside (CG)] to obtain an admixture. The environment friendly chelating agents were then introduced to obtain the inventive combinations. The specific amounts of the constitutents as well as the organisms against which the antimicrobial effect was tested are provided in Table 2 below.

[0189] More particularly the anti microbial activity was tested agaisnt E.coli, P. aeruginosa, S. aureus, E.hirae using malic acid as chelting agent. The results are provided in the Table 2 below.

[0190] Table 2 Antimicrobial combination containing AMP C2, Caprylyl / Capryl Glucoside and Malic acid It is evident from above that various combinations with different concentrations of Capryryl / capryl glucoside (CG) and malic acid as chelating agent provide log reduction of >5 in 1 minute.

[0191] 5 Comparative evaluation of different organic acid chelators:

[0192] The below listed naturally occurring organic acids were compared for their potential to mediate bactericidal activity.

[0193] Table 3. Organic acids.

[0194] 10 Evaluating compatibility of different organic acids with C2 peptide + Decyl glucoside combinations

[0195] Antimicrobial efficacy of 0.025% C2+ 1.65% decyl glucoside with different organic acids i.e. citric acid, lactic acid and malic acid.

[0196] Log reduction achieved in antimicrobial efficacy assay against test bacteria

[0197] 15 Pseudomonas aeruginosa and Staphylococcus aureus in 1 minute contact time is reported (EN1276, dirty condition).

[0198] Table 4. Antimicrobial efficacy of C2 + surfactant- DG + citric acid (EN1276, contact time: 1 min, dirt conditions) against test bacteria P. aeruginosa, S. aureus

[0199] 20 as comparative example.

[0200] Table 5. Antimicrobial efficacy of C2 + surfactant- DG + lactic acid (EN1276, contact time: 1 min, dirt conditions) against test bacteria P. aeruginosa, S. aureus as comparative example.

[0201] 5

[0202] Table 6. Antimicrobial efficacy of C2 + surfactant- DG + malic acid (EN1276, contact time: 1 min, dirt conditions) against test bacteria P. aeruginosa, S. aureus (Present invention)

[0203] The results from Table 6 suggest that malic acid at 0.05%, 0.10% and 0.15% in combination with 0.025% C2 peptide and 1.65% DG was able to achieve the efficacy 3 to >5 log reduction against two tested bacteria P. aeruginosa and S. aureus. Malic acid alone (0.01% and 0.15%) or malic acid (0.15%) in combination with 1.65% DG had no activity (< 1 log kill)

[0204] It was observed that comparative examples of either citric acid or lactic acid in combination with C2 peptide + DG were not able to demonstrate similar compatibility in maintaining rapid bactericidal efficacy (Table 4 & 5).

[0205] EXAMPLE 3:

[0206] Malic acid as organic chelator.

[0207] In order to further understand in vitro interaction of malic acid and C2 peptide, MIC based synergy experiments were carried out. Initially minimum inhibitory concentration (MIC) was calculated for C2 peptide and malic acid. The checkerboard assay method was used to evaluate synergistic potential of the combination of malic acid with C2 peptide.

[0208] The results of experiment demonstrating synergistic activity of C2 peptide and malic acid against bacteria S. aureus is presented below.

[0209] Checkerboard assay to determine the synergism between C2 and malic acid:

[0210] The synergistic effect of combination of C2 peptide + malic acid was established by two-dimensional checkerboard assay using 96 well microtiter plate. The results of the experiment are provided below in Table 7 & 8.

[0211] Al Demonstration of synergy between C2 peptide and malic acid against S. aureus (SA) based on FIC calculated from MIC’s of C2 and Malic acid alone. Table 7. Demonstration of synergy between C2 peptide and malic acid against S. aureus based on FIC

[0212] (+), (-) indicate the presence and absence of antibacterial activity, respectively.

[0213] 5 Values indicate the FIC index.

[0214] Synergy: was based the Fraction Inhibitory Concentration (FIC).

[0215] Synergy was defined as a FIC <0.5, indifference as a FIC between >0.5 and 4 and antagonism as a FIC > 4

[0216] 10

[0217] Based on the results of table 5, FIC index of combination of malic acid (MA) and C2 peptide is 0.27, clearly suggesting the synergy between peptide C2 (FIC: 0.25) and malic acid (FIC: 0.015) against S. aureus

[0218] Bl Demonstration of synergy between C2 peptide and malic acid against E. coli 15 based on FIC: The results of experiment demonstrating synergistic activity of C2 peptide and malic acid against bacteria E. coli presented below in table 6.

[0219] Table 8. Demonstration of synergy between C2 peptide and malic acid

[0220] 5 against E. coli (EC):

[0221] (+), (-) indicate the presence and absence of antibacterial activity, respectively.

[0222] Values indicate the FIC index.

[0223] Based on the results of table 7, FIC index of combination of malic acid (MA) and C2 peptide is 0.08, clearly suggesting the synergy between peptide C2 (FIC: 0.0625) and malic acid (0.015) against E. coli. EXAMPLE 4:

[0224] Antimicrobial combinations employing Polyepoxy Succinic Acid and 1GLDA1 Glutamic Acid Diacetic Acid as organic acid based chelator

[0225] Antimicrobial combinations were prepared with C2 + DG Active system with natural chelators [PESA (Polyepoxy Succinic Acid) and GLDA Glutamic Acid Diacetic Acid] in presence 0.005% Calcium and the antimicrobial activity of the same are tested.

[0226] The combinations are prepared by process as provided in Example 2.

[0227] More particularly the combinations contained 0.03% C2 + 1.65% DG + 0.005 %CaC12 + 0.05% PESA + 0.05% GLDA.

[0228] PESA and GLDA are used to chelate 0.005% CaCh. The active system 0.03% C2 + 1.65% DG + 0.005 %CaCl2+ 0.05% PESA + 0.05% GLDA was tested against four bacteria as per EN1276 using dirty soiling conditions at 20 °C with a contact time of 1 minute

[0229] The efficacy of this Antimicrobial combinations was tested against four bacteria as per EN1276. The result is provided in the following table 9.

[0230] Table 9: Efficacy evaluation of 0.03% C2 + 1.65% DG + 0.005 %CaCl2+ 0.05%PESA + 0.05% GLDA against four bacteria. It is found that the Antimicrobial combinations with natural chelators led to significant, 5 log reduction. The compatibility of biodegradable natural chelators with C2 + DG active system as demonstrated above is useful for various personal hygiene products including baby care such as wet wipes, sanitizers.

[0231] Example 5

[0232] Testing and evaluation of Antimicrobial combinations comprising Caprylyl / Capryl Glucoside as non ionic surfactant a) Inventive Antimicrobial combinations were prepared with Caprylyl / Capryl Glucoside as non ionic surfactant and the efficacy of the combination was measured against antibiotic resistant bacteria.

[0233] The combinations are prepared by process as provided in Example 2.

[0234] The Antimicrobial combination was prepared with 0.06% C and 0.5% Caprylyl / Capryl Glucoside. The same was tested against antibiotic resistant E. coli and S. aureus as per EN1276 using dirty soiling conditions at 20°C with a contact time of 1 minute.

[0235] Table 10. Efficacy evaluation of 0.06% C2 + 0.5% CG (Caprylyl / Capryl

[0236] Glucoside) against antibiotic resistant bacteria As evident from above, the Antimicrobial combination demonstrated >5 log reduction against both antibiotic resistant bacteria. b) Inventive Antimicrobial combinations were prepared with Caprylyl / Capryl Glucoside as non ionic surfactant and the efficacy of the combination was measured against acne causing bacteria were tested.

[0237] The combinations are prepared by process as provided in Example 2.

[0238] Antimicrobial combination comprising 0.06% C2 + 0.5% CG (Caprylyl / Capryl Glucoside) along with Malic acid chelator was tested against Cutibacterium acne as per EN 1276 using dirty soiling conditions at 20°C with a contact time of 30 minute.

[0239] The results are provided in the following table

[0240] Table 11. Efficacy evaluation of 0.06% C2 + 0.5% CG (Caprylyl / Capryl Glucoside) along with Malic acid chelator active system against acne causing bacteria

[0241] As evident from above table 11, the Antimicrobial combinations led to significant log kill of 5, demonstrating potential for use in anti-acne formulations.

[0242] The Antimicrobial combinations comprising 0.06% C2 + 0.5% CG (Caprylyl / Capryl Glucoside) along with Malic acid chelator was tested against antibiotic resistant Malassezia globosa which causes dandruff as per EN 1650 using dirty soiling conditions at 20°C with a contact time of 30 minute.

[0243] The results are provided in the Table 12 Table 12. Efficacy evaluation of 0.06% C2 + 0.5% CG (CaprylyU Capryl Glucoside) along with Malic acid chelator, pH 5.5 against dandruff causing bacteria

[0244] As evident from the above Table 12, the Antimicrobial combination led to log reduction of 5, demonstrating use in anti-acne formulations.

[0245] Example 6

[0246] Further wipe composition containing AMP C2, Caprylyl / Capryl Glucoside and Malic acid were prepared.

[0247] Method as used in Example 2 above was used to prepare the combination of AMP C2, Caprylyl / Capryl Glucoside and Malic acid. This was then added to wipe base formulation having components a mentioned below the following table, to prepare the wipe compositions.

[0248] Various compositions having different amounts of malic acid as chelator were tested for their role against bacteria and the results are provided below.

[0249] Table 13 : Wipe composition containing AMP C2, Caprylyl / Capryl Glucoside and Malic acid

[0250] *Glycerin (1%), PEG-40 Hydrogenated Castor Oil (0.25%), Phenoxyethanol (0.3%), Propylene Glycol (0.2%), Fragrance (0.2%) and water q.s. pH was adjusted with Citric acid / Triethanolamine, pH 5.5.

[0251] As evident form above the compositions without the malic acid chelating agent has low log reduction value after 1 minute and so is the composition having only malic acid and no C2 and CG. The composition having C2 +CG + malic aid provides >5 log reduction value after 1 minute demonstrating synergistic effect of the composition of the present invention.

[0252] EXAMPLE 7:

[0253] Examples of Composition employing malic acid as organic acid chelator

[0254] Composition for wet wipes was prepared with composition: C2 peptide (0.025%) Glycerin (1%), PEG-40 Hydrogenated Castor Oil [Cremophor CO-40] (0.25%), Decyl Glucoside (1.65%), Phenoxyethanol (0.3%), Propylene Glycol (0.2%), Fragrance (0.2%). pH adjusted with Citric acid / Triethanolamine, pH 5.5. 0.25% disodium EDTA was replaced with different concentrations of malic acid as mentioned in the composition example (Table 14).

[0255] This was to evaluate if malic acid could replace EDTA, synthetic chelator. Different concentrations of malic acid were evaluated in composition base set to pH 5.5 and 7.0. Composition base without C2 peptide and Decyl glucoside was used as control to assess efficacy of malic acid alone.

[0256] Al Wet wipes Composition at pH 5.5:

[0257] Compatibility of malic acid in combination with C2 peptide in wet wipes composition base pH 5.5 was evaluated based on log reduction observed at different concentrations of malic acid versus four test bacteria (EN1276, 1 min, dirt condition). Log reduction is presented in Table 14. Table 14. Log reduction observed at different concentrations of malic acid versus four test bacteria (EN1276, 1 min, dirt condition).

[0258] Composition base composition* Glycerin (1%), PEG-40 Hydrogenated Castor Oil(0.25%), Phenoxyethanol (0.3%), Propylene Glycol (0.2%), Fragrance (0.2%) and water q.s. pH was adjusted with Citric acid / Triethanolamine, pH 5.5.

[0259] The malic acid was dosed in the wipes composition (pH 5.5) from 0.01% to 0.15%. The malic acid (0.05%, 0.1% and 0.15%) dosed composition demonstrated >3 to > 5 log kill activity against all four test bacteria. It was observed that 0.05% to 0.15% of malic acid in composition base was superior in efficacy and could replace 0.05% EDTA in wipe composition.

[0260] When malic acid concentration was 0%, there was reduction in activity against all four test bacteria suggesting the requirement of organic chelator in the base for achieving significant bactericidal efficacy. EXAMPLE 8

[0261] Wet wipes composition at pH 7:

[0262] Previous example (table 14) demonstrates efficacy of malic acid (replacement of EDTA) and C2 +DG containing wipe composition at skin friendly pH 5.5.

[0263] Taking cue from pH 7 data, the compatibility of the same composition was evaluated at neutral pH 7. Log reduction observed at different concentrations of malic acid versus four test bacteria (EN1276, 1 min, dirt condition) is presented in Table 15.

[0264] Table 15. Compatibility of malic acid in wet wipes composition base pH 7.

[0265] Composition base composition*: Glycerin (1%), PEG-40 Hydrogenated Castor Oil (0.25%), Phenoxyethanol (0.3%), Propylene Glycol (0.2%), Fragrance (0.2%) and water to q.s. The pH was adjusted with Citric acid / Triethanolamine to pH 7. Disodium EDTA was replaced with different concentrations of malic acid as shown above.

[0266] The malic acid was dosed in the wipes composition (pH 7) from 0.01% to 0.15%. The malic acid (0.10 to 0.15%) in composition base containing 0.025% + 1.65% DG was superior in efficacy achieving >5 against tested bacteria and could potentially replace 0.05% EDTA. Thus, it was found that malic acid 0.10% and 0.15% is effective in wet wipes base composition cited above replacing 0.05% disodium EDTA showed significant bactericidal efficacy versus all test bacteria at pH 5.5 and 7.

[0267] EXAMPLE 9 a) Wet wipe composition with Caprylyl / Capryl Glucoside as non ionic surfactant

[0268] Three wet wipe compositions A, B, C were prepared with Caprylyl / Capryl Glucoside as non ionic surfactant & more biodegradable chelators, Polyepoxy

[0269] Succinic acid (PESA) and Glutamic acid diacetic acid (GLDA), in lieu of EDTA were prepared as provided in following table 16:

[0270] Table 16: Wet Wipes compositions containing environmentally friendly chelator.

[0271] By replacing the EDTA with organic acid based chelator and / or salt thereof, not only improved the environment friendliness and biodegradability profile but it also enhanced the spectrum while retaining the same microbial kill and contact time constraints for all the microbes and fungi. b) Activity against Candida albicans

[0272] Wet wipe compositions using 0.06% C2 and 1.65% Caprylyl / Capryl Glucoside in Wipes composition base was prepared. The effect on the Candida is provided in Table 17.

[0273] The efficacy evaluation against Candida albicans was carried out as per EN1650 using dirty soiling conditions at 20°C with a contact time of 1 minute. The wipe composition achieved >4 log kill demonstrating rapid yeasticidal activity.

[0274] Table 17: Efficacy evaluation of Wipes against Candida albicans.

[0275] Composition base composition* Glycerin (1%), PEG-40 Hydrogenated Castor Oil(0.25%), Phenoxyethanol (0.3%), Propylene Glycol (0.2%), Fragrance (0.2%) and water q.s. pH was adjusted with Citric acid / Triethanolamine, pH 5.5. Antimicrobial combination added to base: 0.06% C2 + 1.65% Caprylyl / Capryl Glucoside.

[0276] EXAMPLE 10

[0277] Anti-acne composition containing AMP C2 and Caprylyl / Capryl Glucoside along with Malic acid chelator is provided in Table 18. Table 18: Anti-acne composition

[0278] EXAMPLE 11 Anti-dandruff composition containing AMP C2 and Caprylyl / Capryl Glucoside along with Malic acid chelator is provided in the following table 19.

[0279] Table 19: Anti-dandruff composition It is to be understood that the present invention is susceptible to modifications, changes and adaptations by those skilled in the art. Such modifications, changes, adaptations are intended to be within the scope of the present invention.

Claims

CLAIMS:

1. An antimicrobial combination comprising:(i). Indolicidin peptide analogue (C2) as set forth in SEQ ID 1, or salts and derivatives thereof at a concentration ranging from 0.01 wt. % to 0.15 wt. %;(ii) non-ionic surfactant ranging from 0.5 wt.% to 5 wt.%. ; and(iii) at least an organic acid and / or a metal salt thereof chelating agent ranging from 0.05 wt.% to 0.15 wt.%.

2. The antimicrobial combination as claimed in claim 1, wherein said antimicrobial combination achieves a log-kill of at least 5, after a contact time of about 1 min against Gram positive and Gram-negative bacteria.

3. The antimicrobial combination as claimed in claim 1 , wherein the non-ionic surfactant is selected from the group of Alkyl-Poly Glucoside (APG).

4. The antimicrobial combination as claimed in claim 3, wherein the Alkyl- Poly Glucoside (APG) is selected from Decyl-Glucoside, Caprylyl / Capryl Glucoside, Lauryl Glucoside, Capryl glucoside, Arachidyl glucoside or combinations thereof.

5. The antimicrobial combination as claimed in claim 4, wherein the Alkyl- Poly Glucoside (APG) is Decyl-Glucoside.

6. The antimicrobial combination as claimed in claim 5, wherein the Alkyl- Poly Glucoside (APG) Caprylyl / Capryl Glucoside.

7. The antimicrobial combination as claimed in claim 1 to 6, wherein said Gram positive and Gram negative bacteria, include methicillin resistant S. aureus (MRSA), Penicillin and Carbapenem resistant E. coli.

8. The antimicrobial combination as claimed in claim 1 to 6, wherein said antimicrobial combination achieves a log-kill of at least 5, after a contact time of about 30 min against Cutibacterium acnes.

9. The antimicrobial combination as claimed in claim 1 to 6, wherein said antimicrobial combination achieves a log-kill of at least 4, after a contact time of about 1 min against Candida albicans.

10. The antimicrobial combination as claimed in claim 1 to 6, wherein said antimicrobial combination achieves a log-kill of at least 3, after a contact time of about 30 min against Azole resistant Malassezia globosa.

11. The antimicrobial combination as claimed in claim 1, wherein the organic acid based chelator and / or a metal salt thereof is environment friendly and biodegradable.

12. The antimicrobial combination as claimed in claim 1 and 11, wherein the organic acid based chelator and / or a metal salt thereof is selected from malic acid, Polyepoxy Succinic acid (PESA), Glutamic acid diacetic acid (GLDA).

13. The antimicrobial combination as claimed in claim 1 and 11, wherein the organic acid based chelator is Polyepoxy Succinic Acid (PESA) and Glutamic Acid Diacetic Acid (GLDA)14. An antimicrobial composition comprising the antimicrobial combination as claimed in any one of the claims 1 to 13 along with cosmetically acceptable excipients.

15. The antimicrobial composition as claimed in claim 14, wherein the cosmetically acceptable excipients are selected from emulsifiers, solvents, rheology modifying agents, fillers / thickening agents, structurants,emollients, diluents, humectants, feel enhancers, preservatives, fragrances, pH modifiers and combinations thereof.

16. The antimicrobial composition as claimed in claim 14, wherein the composition is in a form selected from wipes, gel, foam, cream or spray.

17. An anti -acne composition comprising:(i) Indolicidin peptide analogue (C2) as set forth in SEQ ID 1, or salts and derivatives thereof;(ii) at least an organic acid and / or a metal salt thereof chelating agent selected from the group consisting of malic acid, PESA and GLDA; and(iii) non-ionic surfactant selected from the group consisting of Decyl glucoside and caprylyl / capryl glucoside; along with cosmetically acceptable excipients.

18. An anti -dandruff composition comprising:(ii) Indolicidin peptide analogue (C2) as set forth in SEQ ID 1, or salts and derivatives thereof;(ii) at least an organic acid and / or a metal salt thereof chelating agent selected from the group consisting of malic acid, PESA and GLDA; and(iii) non-ionic surfactant selected from the group consisting of Decyl glucoside and caprylyl / capryl glucoside; along with cosmetically acceptable excipients.

19. A wet wipe impregnated with an antimicrobial composition comprising:(i) C2 peptide as set forth in SEQ ID No.1;(ii) at least one non-ionic surfactant selected from the group consisting of Decyl glucoside and caprylyl / capryl glucoside; and(iii) at least one organic acid based chelator selected from the group consisting of malic acid, PESA and GLDA; along with cosmetically acceptable excipients.

20. The wet wipe impregnated with an antimicrobial composition as claimed in claim 19 wherein the amount of C2 peptide ranges from 0.0125wt% to 0.075wt%,21. The wet wipe impregnated with an antimicrobial composition as claimed in claim 19 wherein the amount of non-ionic surfactant ranges from 0.025 wt% to 2wt%.

22. The wet wipe impregnated with an antimicrobial composition as claimed in claim 19 wherein the amount of organic acid based chelator and / or a metal salt ranges from 0.025 wt% to 0.15wt%.

23. The wet wipe as claimed in claim 19, wherein the wet wipe has a pH ranging between 5.0 to 7.0.

24. A cosmetic method of disinfecting a surface, the method comprising the steps of: providing a wet wipe as claimed in any one of claims 19 to 23; and wiping the surface with the wet wipe.

25. A method of treatment of a subject infected with bacteria selected from Gram positive bacteria, Gram negative bacteria, , methicillin resistant S. aureus (MRSA), Penicillin and Carbapenem resistant E. coli, Cutibacterium acnes, said treatment comprising applying the antimicrobial composition as claimed in claim 14.

26. A method of treatment of a subject infected with Candida albicans, said treatment comprising applying the antimicrobial composition as claimed in claim 14.

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