DESC:
FIELD OF INVENTION
The present invention relates to antimicrobial compositions for topical application. Particularly, it relates to antimicrobial compositions comprising an antimicrobial peptide with one or more actives. .

BACKGROUND OF INVENTION
In the recent past there has been increasing consumer awareness and sensitivity towards environmental and health impact of synthetic antimicrobial molecules that were widely used as active ingredients in many costumer products (e.g., oral care (toothpaste etc.) and hygiene products (hand wash, soap, body wash, deodorant in different formats such as liquid, gel, bar soap, rub, cream, lotion etc).
High impacting widely used synthetic antimicrobial active ingredients such as Triclosan, Triclocarban and other synthetic antimicrobials suffer from several short comings in terms of environmental impact, health impact as well as growing regulatory concerns in these areas.
The increasing consumer awareness and sensitivity regarding environmental and health impact of synthetic and widely used molecules has led to demand for safer, efficacious and nature friendly antimicrobials.
Hence it is imperative to develop sustainable antimicrobials that can maintain antibacterial properties with minimum side effects on environment and humans.
AMPs address the problems in following ways: a. it provides anti-resistant antimicrobial formulation (unlike the synthetic chemical ingredients); and b. it provides rapid killing (like the synthetic chemical ingredients); and c. it provides a rapid kill while ensuring an effective log kill.
Antimicrobial peptides (AMPs) are naturally occurring host defense molecules with potent antimicrobial activity. The native AMPs are known to undergo proteolytic degradation in vivo and the fragments generated carry out diverse functions.
The AMPs are considered as next generation antimicrobial molecules after the microorganisms have developed resistance against antibiotics due to their indiscriminate and rampant use. However, with current peptide synthesis technology, it is difficult to synthesize larger AMPs. Hence, their truncated versions are being investigated for potent antimicrobial activity.
US19990318195 is directed 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.
WO9522338 (A1) is directed to broad spectrum tryptophan-rich antimicrobial indolicidin analogs and their use as a therapeutic agent, a food preservative, a disinfectant or a medicament.
WO1999058141A1 is directed to indolicidin analogs which are truncated at the amino terminus by one and two amino acids and their use as a therapeutic agent, a food preservative, a disinfectant or a medicament.
US2004176300 A1 is directed to three single tryptophan analogs ILPWKLPLLPLRR-amide (IL4), ILPLKLPWLPLRR-amide (IL8) and ILPLKLPLLPWRR-amide (IL11), of Indolicidin and their compositions.
2795/MUMNP/2011 is directed to antimicrobial agents against Gram-negative bacteria, in particular to fusion proteins composed of an enzyme having the activity of degrading the cell wall of Gram-negative bacteria and an additional peptide stretch fused to the enzyme on the N- or C-terminus and pharmaceutical or cosmetic compositions comprising said fusion protein. The peptide is an antimicrobial peptide including indolicidin and cathelicidin.
Synergistic combinations of AMPs with antibiotics are also known. Mohammadi et al., In vitro synergistic effects of a short cationic peptide and clinically used antibiotics against drug-resistant isolates of Brucella melitensis. J Med Microbiol. 2017 Jul;66(7):919-926; Sabine et al., Synergistic Effects of Antimicrobial Peptides and Antibiotics against Clostridium difficile Antimicrobial Agents and Chemotherapy, October 2014, 58 (10) 5719–5725
WO98/40401 A1 are directed to the combination of specific indolicidin analogues with antibiotic agent for treating infections.
US201313745359 is directed 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.
Nagaoka et al. is directed to synergistic combination of antibacterial neutrophil defensins and cathelicidins in the presence of NaCl. [Reference: Nagaoka et al., Synergistic actions of antibacterial neutrophil defensins and cathelicidins. Inflamm Res. 2000 Feb;49(2):73-9.]
Conventionally, in sanitizers, alcohols (Ethanol and isopropyl alcohol) are used at 70% or higher concentration, which causes dehydration of skin. Ethanol is known to irritate the skin upon repeated applications. Hence it is important to develop compositions that provide a high bactericidal efficacy with lesser percentage of alcohol.
GASTRODIA ELATA PEPTIDE WASH DISINFECTANT https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=a43b4d40-d577-bff8-e053-2995a90ab7d1&audience=consumeris a liquid hand sanitizer comprising Gastrodia elata Blume peptide 5.5% v/v and alcohol 75% v/v. It has killing activity against Candida albicans, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli.
WO2014/207181 A1 is directed to cosmetic use of dermicidin, or analogues or fragments thereof, for preventing and/or treating body odour. Dermicidin is a natural AMP expressed in the sweat glands.
There is a long felt need to develop new and effective antimicrobial compositions which are anti-resistant, provide the rapid kill while ensuring an effective log kill, can be used in lower amounts, cost-effective, have minimum side effects on environment and humans and can be utilized in skin hygiene and sanitization.
The present inventors have surprisingly developed novel effective antimicrobial compositions which ameliorates the aforesaid shortcomings of the prior art.

OBJECTS OF THE INVENTION
It is one of the objects of the present invention to provide effective antimicrobial compositions which are anti-resistant, provide rapid kill while ensuring an effective log kill, can be used in lower amounts, cost-effective, have minimum side effects on environment and humans.
It is another object of the present invention to provide effective antimicrobial compositions which can be used topically for various purposes.

SUMMARY OF THE INVENTION
According to an aspect of the present invention there is provided an antimicrobial composition comprising an effective concentration of antimicrobial peptide (AMP) and an effective concentration of at least one antimicrobial active.
According to another aspect of the present invention there is provided an antimicrobial composition comprising an effective concentration of indolicidin peptide analogue and an effective concentration of chelating agent as active ingredients.
According to another aspect of the present invention there is provided an antimicrobial composition comprising an effective concentration of indolicidin peptide analogue and an effective concentration of at least one surfactant as active ingredients.
According to another aspect of the present invention there is provided an antimicrobial composition comprising an effective concentration of indolicidin peptide analogue, an effective concentration of chelating agent and an effective concentration of surfactant.
According to yet another aspect of the present invention there is provided an antimicrobial composition comprising an effective concentration of cathelicidin LL-37 fragment and an effective concentration of alcohol as active ingredients.
According to yet another aspect of the present invention there is provided an antimicrobial composition comprising antimicrobial peptides and antimicrobial actives alongwith selective cosmetically acceptable excipients.

BRIEF DESCRIPTION OF THE DRAWINGS
The above and other aspects, features and advantages of certain exemplary embodiments of the present invention will be more apparent from the following description taken in conjunction with the accompanying drawings wherein:
Figure 1. Antimicrobial efficacy: log reduction achieved after 1-minute contact time at 200C vs P. aeruginosa. log kill results of AMP C2, EDTA and combination of AMP C2 + EDTA versus P. aeruginosa. And log kill results of AMP D2, EDTA and combination of AMP D2 + EDTA versus P. aeruginosa
Figure 2 illustrates Antimicrobial efficacy: log reduction achieved after 1 minute contact time at 20?C vs E. coli. log kill results of AMP C2, EDTA and combination of AMP C2 + EDTA versus E. coli and log kill results of AMP D2, EDTA and combination of AMP D2+EDTA versus E. coli.
Figure 3 illustrates Antimicrobial efficacy: log reduction achieved after 1 minute contact time at 20?C vs S. aureus. log kill results of AMP C2, EDTA & combination of C2 + EDTA versus S. aureus and log kill results of AMP D2, EDTA & combination of D2 + EDTA versus S.aureus.
Figure 4. Antimicrobial efficacy: log reduction achieved after 1 minute contact time at 37?C vs E. hirae. log kill results of AMP C2, EDTA & combination of C2 + EDTA versus E. hirae and log kill results of AMP D2, EDTA & combination of D2 + EDTA versus E. hirae.
Figure 5A and 5B effect of lower concentration of EDTA and C2 combinations
Figure-6: Evaluation of activity of C2 in presence of citric acid
Figure 7A: Killing activity of surfactant alone against bacteria, E. coli.
Figure 7B: Killing activity of combination of C2 with different surfactants
Figure 8: Evaluation of activity of C2 with anionic (SLS) and amphoteric surfactant (CAPB)
Figures 9A-9D: Killing effect of 3 component composition of C2, EDTA and Decyl glucoside (DG) against P. aeruginosa, E. coli, S. aureus and E.hirae

Figures 10-13: Log kill results obtained with AMP FK16, Alcohol (Ethanol and IPA 19:1) and combination of AMP FK16 + Alcohol against Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Enterococcus hirae at 20°C in 1-minute contact time
Figures 14A and 14B: Stability profile of FK16 (C1) in alcohol and PVP (Polyvinylpyrrolidone)

DETAILED DESCRIPTION OF THE INVENTION
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.
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.
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.
It is to be understood that the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
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.
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)).
The term, "antimicrobial peptide" (AMP) as used herein refers to any peptide that has microbiocidal and/or microbiostatic 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.
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.
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.
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."
The term "skin" as used herein means the entire epidermis of the human body.
The term “peptide analogue” as used herein means a peptide designed to mimic a native peptide (in the present invention indolicidin).
The term “peptide fragment” as used herein means functional motifs/ shorter fragments which retain the biological activity of the full-length peptide (in the present invention LL-37)
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 and SEQ ID NO:8 of present invention.
The present invention relates to an antimicrobial composition comprising an antimicrobial peptide (AMP) and an antimicrobial active.
Anti-resistance nature friendly antimicrobials: Since their discovery in the 1980s, antimicrobial peptides (AMPs) have been viewed as one of the important solutions to the impending crisis of antimicrobial resistance. Antimicrobial peptides (AMPs), evolutionary ancient and conservative tools of the innate immune system providing immediate response to the large set of various pathogens. And Antimicrobial peptides (AMPs) produced by humans, animals, plant and bacteria. AMPs differ in amino acid sequence and structure, but predominantly are cationic and they can adopt an amphipathic conformation, thus, they are able to easily interact with the negatively charged components on the surface of bacterial cells and integrate into the lipid bilayer. The principle mechanism of antibacterial action of AMPs is related with their ability to alter membrane permeability and damage its structure. This leads to leakage of vital components, ions, and metabolites. Membrane destabilization additionally affects functioning of membrane-associated protein complexes. Some AMPs also have intracellular targets and interfere metabolic process such as DNA and Protein synthesis. Hence, Wide-scale multi targeted action is believed to be one of the reasons for the effectiveness of AMPs toward multidrug-resistant bacterial strains and an obstacle for the development of a high resistance level to such compounds
The AMP active domains have potential to be used in personal hygiene products such as deodorant and hand sanitizers due to their ability to kill pathogenic microorganisms. In the present invention, inventors have disclosed fragment/analogues based on the natural AMPs
Furthermore, the AMPs act on the bacterial membrane instead of single metabolic event. There will be less pressure on developing antimicrobial resistance to the two AMP actives in the present application. The AMPs act by disrupting the cell membrane, not on single step. Studies have demonstrated that the resistance level provided by point mutations and gene amplifications is very low in case of AMPs.
The present invention provides synergistic antimicrobial combinations comprising an effective concentration of antimicrobial peptide (AMP) and an effective concentration of antimicrobial active. The invention provides benefits of reducing pathogenic microbial load on skin and hence, helps in skin hygiene and sanitization.

Indolicidin peptide analogue compositions
According to an aspect of the present invention there is provided an antimicrobial composition comprising an effective concentration of an indolicidin peptide analogue or derivatives thereof and an effective concentration of additives. Preferably, the additive is selected from the group of chelating agent, surfactant, or a combination thereof.
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 present invention provides a synergistic combination of an indolicidin peptide analogue AMP and select chelating agents such as EDTA which synergistically enhances bactericidal efficacy.
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(1):48-52.] has 62% sequence similarity with the native bovine indolicidin.
The chelating agents such as EDTA are not known to have any bactericidal activity by itself. EDTA is used in hand wash and soaps to reduce the hardness of water by chelation of metal ions. The chelating activity of EDTA in formulation has benefit stripping of metal ions from the cell membrane of bacteria, making them susceptible to antimicrobial active. Chelating agents such as EDTA serve the dual purpose in hash wash/soap formulations. The AMP and EDTA combination has suitable applications in wash-off systems e.g. hand wash, soap etc. And hence the effective concentration required for efficacy of the active in the final formulation decreases, when EDTA is used as the additive. The present inventors have found that EDTA at specific concentrations potentiates the activity of the indolicidin peptide analogue, thereby reducing the effective concentration of antimicrobial actives required.
The present invention provides an antimicrobial composition comprising an effective concentration of indolicidin peptide analogue as set forth in SEQ ID 1 or derivatives thereof and an effective concentration of a chelating agent.
In an embodiment, the chelating agent is selected from the group of aminopolycarboxylic acids, polyphosphate, phosphonates or combination thereof.
In an embodiment, the chelating agent is Ethylenediaminetetraacetic acid (EDTA).
Non-limiting examples of other chelator that could be used in synergistic combination with C2 are STPP (Sodium tripolyphosphate), EHDP/Dequest® (Ethylene hydroxydiphosphonate).
In an embodiment, the indolicidin peptide analogue is present at a concentration ranging from 100 to 1800 µM and the chelating agent is present at a concentration ranging from 7.5 mM to 11.25 mM. In an embodiment, the indolicidin peptide analogue to chelating agent ratio present in the composition ranges from 1:7.36.to 1:3.27.
The present invention also provides an antimicrobial composition comprising an effective concentration of indolicidin peptide analogue as set forth in SEQ ID 1 or derivatives thereof and an effective concentration of a surfactant.
In an embodiment, the surfactant is a non-ionic surfactant selected from the group of Alkyl-Poly Glucoside (APG). Preferably, the Alkyl-Poly Glucoside (APG) is selected from Decyl-Glucoside, Lauryl Glucoside, Capryl glucoside, Arachidyl glucoside or combinations thereof.
In an embodiment, the non-ionic surfactant is present at a concentration ranging from 0.2 to 10% w/v.
The present invention also provides an antimicrobial composition comprising an effective concentration of indolicidin peptide analogue as set forth in SEQ ID 1 or derivatives thereof, an effective concentration of a surfactant and an effective concentration of chelating agent.

Cathelicidin LL-37 peptide fragment compositions
According to another aspect of the present invention there is provided an antimicrobial composition comprising an effective concentration of a cathelicidin LL-37 peptide fragment or derivatives thereof and an effective concentration of alcohol as active ingredients.
Cathelicidins are a family of evolutionarily conserved AMPs. hCAP-18 is the only cathelicidin in humans. This 18-kDa preproprotein consists of an N-terminal signal sequence, a cathelin-like domain, and a C-terminal AMP domain. Proteolytic cleavage of hCAP-18 releases a 37-residue, amphipathic, helical peptide known as LL-37. LL-37 exhibits a broad spectrum of antimicrobial activities (e.g. bacteria, fungi, and viruses), and also has the ability to neutralize bacterial lipopolysaccharides. [Reference: Ren SX, Shen J, Cheng AS, et al. FK-16 derived from the anticancer peptide LL-37 induces caspase-independent apoptosis and autophagic cell death in colon cancer cells [published correction appears in PLoS One. 2015;10(6):e0131750]. PLoS One. 2013;8(5):e63641. Published 2013 May 20. doi:10.1371/journal.pone.0063641] Human cathelicidin LL-37 has multiple roles in human defense and is expressed by epithelial cells and different immune cells.
The cost associated with chemical peptide synthesis is high. Therefore shorter /truncated fragments of LL-37 have been identified which retain the biological activity of the full-length peptide and can be produced with lower cost.
The present invention provides a synergistic combination of LL-37 peptide fragment AMP and select alcohols which synergistically enhances bactericidal efficacy.
LL-37 peptide fragment FK-16 as set forth in SEQ ID No. 8 corresponding to residues 17–32 retains antibacterial and antitumor effects. [Reference: Ren SX, Shen J, Cheng AS, et al. FK-16 derived from the anticancer peptide LL-37 induces caspase-independent apoptosis and autophagic cell death in colon cancer cells [published correction appears in PLoS One. 2015;10(6):e0131750]. PLoS One. 2013;8(5):e63641. Published 2013 May 20. doi:10.1371/journal.pone.0063641] FK16 kills a range of pathogens in both in vitro and in vivo systems. FK16 is capable of killing a variety of pathogens and do not elicit caustic immunologic responses and host tissue toxicity. FK16 covers 42% of full length parent, native human Cathelicidin LL-37 peptide.
Ethanol is known to irritate the skin upon repeated applications. Hence it may become important to develop the compositions that provide high bactericidal efficacy with lesser percentage of alcohol. The inventors noted that much less concentrations of respective individual ingredients are required to provide sanitizing effects. Whereas it is a well-known fact that alcohols tend to leave the surface dried up without moisturized feel, the inventors noted that when small amounts of select AMPs are added, much lesser quantities of alcohols are required to provide equivalent sanitizing end results. It helps reduce concentrations of alcohols (ethanol and isopropanol) required for skin sanitization applications. Thus, it helps prevent dehydration caused by high concentration of alcohols in sanitizers. Additionally, the inventors noted that the combinations of the present invention help in controlling body odour by killing microorganisms that would have otherwise grown on skin and produces odour causing metabolites. The invention thus finds strong applications in the deodorant industry.
The present invention provides an antimicrobial composition comprising an effective concentration of cathelicidin peptide fragment (FK16) as set forth in SEQ ID 8, or derivatives thereof and an effective concentration of alcohol.
Non-limiting examples of alcohols that could be used in synergistic combination with FK16 are Ethanol, Isopropyl alcohol or a combination thereof.
In an embodiment, the cathelicidin peptide fragment (FK16) is present at a concentration ranging from 40µM to 900 µM and the alcohol is present at a concentration ranging from 8% to 50%. The antimicrobial compositions of present invention are effective at lower alcohol percentages.
In an embodiment, the cathelicidin peptide fragment (FK16) to alcohol ratio present in the composition ranges from 1: 3130 to 1:196.
In an embodiment, the antimicrobial compositions of the present invention demonstrate a log kill in excess of 3, preferably in excess of 4 and more preferably in excess of 5.
The antimicrobial compositions of the present invention may further comprise cosmetically acceptable excipients selected from, emulsifiers, solvents, rheology modifying agents, fillers/thickening agents, structurants, emollients, diluents, humectants, feel enhancers, preservatives and fragrances.
Thickening agents include but are not limited to hydrogels, PEG (polyethyelene glycol), polyacrylic acid, hydroxypropyl methylcellulose (HPMC), Bentone, Natural or synthetic gum. Emulsifying agents include but are not limited to derivatives of PEG (Polyethylene Glycol), Polysorbate. Diluents include but are not limited to water or ethyl alcohol. 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 glycerine 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.
In an embodiment, the antimicrobial compositions of the present invention comprise a synergistic combination of antimicrobial peptides and antimicrobial actives alongwith cosmetically acceptable excipients.
In an embodiment, the antimicrobial compositions of the present invention may be in the form of hand wash, body wash, soap, shampoo and gels. Other forms include but not limited to wipes, feet sanitizer, hand cream.

The present invention provides an antimicrobial hand wash comprising:
a) An effective concentration of Indolicidin peptide analogue as set forth in SEQ ID No. 1 or derivatives thereof and
b) An effective concentration of a chelating agent or a surfactant or combinations thereof.

In another embodiment, the antimicrobial compositions of the present invention may be in the form of hand sanitizer or body deodorant.

The present invention provides an antimicrobial hand sanitizer comprising:
a an effective concentration of cathelicidin peptide fragment (FK16/C1) as set forth in SEQ ID 8, or derivatives thereof and;
b. an effective concentration of an alcohol.
The hand sanitizer may be in the form of gel, foam, cream, spray or wipes.
The present invention also provides an antimicrobial body deodorant comprising:
a an effective concentration of cathelicidin peptide fragment (FK16) as set forth in SEQ ID 8, or derivatives thereof and;
b. an effective concentration of an alcohol.

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 pump-dispenser 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.
The synergistic combinations of C2+ EDTA/ C2+surfactant/ C2+EDTA+surfactant and FK16+alcohol can be used in the following products in any of the formats such as gel, cream, ointment, spray and wipes:
• Hand wash, body wash, bar soap, liquid soap, wipes, hand cream, feet cream, shave cream, Aftershave lotion, After shave cream, face cleanser, shampoo, hair conditioner, surface cleanser and
• Hand sanitizer liquid, Hand sanitizer gel, deodorant, foot deodorant, and
• Vegetable & fruit wash, Vegetable & fruit spray, surface disinfectant, surface cleaner, floor cleaner and
• wound healing cream/ointment/gel/spray and
• other cosmetic products as preservatives/ preservative system
• 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, burns.
The present invention also identifies compatible product formulation such as:
• The C2+EDTA compatibility in different surfactant category combinations and
• FK16 compatibility in different alcohols and combinations
The present invention also provides a method of screening the peptide for rapid bactericidal applications, based on the bioinformatics analysis of physicochemical descriptors of the peptides (example, charge and hydrophobicity) and sequence similarity.
The present invention provides synergistic antimicrobial compositions demonstrating anti-bacterial and anti-viral efficacy, requires a lower effective concentration of actives, is cost-effective, has minimum side effects on environment and humans, and can be utilized in various skin hygiene, sanitization applications and for treating skin infections.

EXAMPLES:
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

EXAMPLE -1:
Materials:
Antimicrobial Peptides (AMPs)
AMPs used in the present study were custom synthesized from China Peptides, China & USV Pvt. Ltd., Mumbai. The peptides were > 90% purity based on HPLC profile. The peptide purity was confirmed before testing. All AMPs are cationic in nature. The details of peptides are provided in Table 1. The stock solution of the peptide was prepared by weighing specific quantity of lyophilized peptide & dissolving in required amount of Milli-Q® water. The peptide solution was further aliquoted & stored at -200C till further use.
The details (length, sequence and properties) are listed in the table 1.
1. Peptide C2: The 13 amino acid peptide was custom synthesized from China Peptides, China
2. Peptide D2: The 12 amino acid peptide was custom synthesized from USV Pvt. Ltd, India.
3. Peptide E2: The 13 amino acid peptide was custom synthesized from GenScript, USA.

The below peptides of different length were custom synthesized from USV Pvt. Ltd. India:
4. Antimicrobial peptide LL37, FK16. It is peptide containing 16 amino acids. It is C-terminal fragment of human cathelicidin LL-37. FK16 is also referred to as C1 in the present invention.
5. Antimicrobial peptide LL37- Full length LL37 containing 37 amino acids
6. Antimicrobial peptide LL23
7. Antimicrobial peptide KR20
8. Antimicrobial peptide FK13 &
9. Antimicrobial peptide Dermicidin
Table 1. Sequence & Physicochemical parameters of peptides used.
Peptide Sequence Length SEQ ID No:
Peptide C2 ILPFKFPFFPFRR*
13 1
Peptide D2 VRLIVAVRIWRR 12 2
Peptide E2 ILPLKLPLLPWRR*
13 3
Dermicidin SSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDSVL 48 4
LL37 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES 37 5
LL23 LLGDFFRKSKEKIGKEFKRIVQR 23 6
KR20 KRIVQRIKDFLRNLVPRTES 20 7
FK16 (C1) FKRIVQRIKDFLRNLV 16 8
FK13 FKRIVQRIKDFLR 13 9
*C-terminal was amidated.

Peptide C2 & E2 is modified analogue of the AMP, bovine indolicidin.
Peptide D2 belongs to Innate Defense Regulator (IDR) peptide which is derived from host defense peptides (HDP).

Chelating Agent:
The chelating agent, Tetra sodium EDTA was procured from Sigma- Aldrich (cat #03701). 0.25 M EDTA solution was prepared, sterilized by membrane filtration & was used

Alcohols:
1. Ethanol: Antimicrobial activity
2. Isopropyl alcohol: Antimicrobial activity

Testing of bactericidal efficacy as per EN1276 protocol
Test Organisms:
1. Pseudomonas aeruginosa ATCC 15442
2. Escherichia coli ATCC 10536
3. Staphylococcus aureus ATCC 6538
4. Enterococcus hirae ATCC 10541

Microorganism: The glycerol stock was stored at -800C. The culture was maintained on Tryptone soya agar (TSA) and sub-cultured every two weeks.

Antimicrobial properties of AMPs and their fragments described in the prior art are mainly related to determination of Minimum Inhibitory Concentration (MIC) & Minimum Bactericidal Concentration (MBC). There are few reports of determination of log reduction for AMPs; their first time point for measuring the log reduction is 30 minutes. However, for hand hygiene applications, the relevant time points are shorter at 1 - 5 minutes. In prior art, synergy is not demonstrated in terms of significant log kill i.e. >5 log kill i.e. 99.999% reduction of microorganisms in 1 minute.
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. The British Standard EN1276 has laid out stringent protocol for antibacterial efficacy testing of the active system/formulation for hand hygiene applications. To the best of inventor knowledge, there are no prior art report of AMP containing active tested under such stringent conditions. For example, mimicking soil conditions by testing in the presence of Bovine Serum Albumin (BSA), and testing at temperature such as 200C for short duration of 1 /5 minute, achieving >5 log kill in 1 minute versus both Gram positive and Gram negative test organisms.
The AMP compositions of present invention have been tested under stringent conditions as per EN1276 protocol.

EXAMPLE -2: Compositions of Indolicidin peptide analogue
A] Identification of peptide analogue of Indolicidin:
Two indolicidin analogues with amino acid substitution have been created out of original indolicidin parent sequence. Two such indolicidin analogs (C2, E2) and one AMP [D2- Innate Defense Regulator (IDR) peptide which are derived from host defense peptides (HDP)] unrelated to indolicidin family were used in the present study. Out of 3 AMPs tested, the C2 with 62% sequence similarity showed better efficacy than E2, D2 and was chosen for the further synergy experiments with chelator.

B] Bactericidal Efficacy Evaluation of indolicidin peptide analogue (C2) + EDTA
Reagent Details:
o BSA: 3% BSA.3 g l-1 bovine albumin (final concentration) was used as interfering substance [mimicking soil conditions by testing in the presence of BSA]
o Neutralizer solution: The neutralizer was prepared in 250mM potassium phosphate buffer pH 7.2. The composition was polysorbate 80, 30g/l + saponin, 30 g/l + lecithin, 3 g/l.
o The media chemicals Tryptone Soya Agar (TSA), Tryptone soya broth (TSB) and other chemicals were procured from HiMedia Laboratories, Mumbai, India.
o Diluent: Tryptone sodium chloride solution
Tryptone sodium chloride solution, Consisting of: Tryptone, pancreatic digest of casein 1,0 g Sodium chloride (NaCl) 8,5 g Water (5.2.2.2) to 1 000,0 ml
o Hard Water Preparation:

The hard water was freshly prepared under aseptic conditions and used within 12 h.
For the preparation of 1000 ml of hard water, the following procedure was used:
? solution A: dissolve 19,84 g magnesium chloride (MgCl2) and 46,24 g calcium chloride (CaCl2) in water and dilute to 1 000 ml. The same was sterilized by membrane filtration.
? solution B: dissolve 35,02 g sodium bicarbonate (NaHCO3) in water and dilute to 1000 ml. The same was sterilized by membrane filtration.
? To 700 ml of water in a 1000 ml volumetric flask and add 6,0 ml of solution A, then 8,0 ml of solution B. Mix and dilute to 1000 ml with water.

Bactericidal Efficacy Evaluation:
Contact Kill Experiment:
Antimicrobial/bactericidal efficacy of antimicrobial peptides and combinations was tested by contact kill experiment. Dilution-neutralization method was followed. The time of contact was 1 minute. One challenge microorganism was used in each experiment.
Briefly, the cells were exposed to different concentrations of peptide and peptide + EDTA combinations for 1 minute ±5 seconds at 200C. The control contained no peptide but was treated in identical conditions.
The experiment was conducted at temperature bath set at required temperature, generally 20?C (Eppendorf Thermomixer comfort). The contact kill was experiment was conducted at 20?C against all 3 microorganisms (P. aeruginosa, E. coli & S. aureus). The temperature for experiment against E. hirae was 37?C. Single colony of bacterium was picked from the TSA plate & grown in TS broth at 37?C. The absorbance of bacterial suspension was measured in Thermo Scientific™ Multiskan™ GO at 600nm using cuvette (BRAND, Polystyrene 2.5 mL volume cuvette).
Bacterial suspension of about 108 CFU/ml was prepared. Different concentrations of peptide and combinations were prepared in Milli-Q water. Bacterial suspension and peptides were equilibrated at 20?C for 10 min. To 50 µl of bacterial suspension, 450 µl of peptide with defined concentrations was added & immediately vortexed. To stop the action of AMP, reaction aliquot containing cells was transferred to neutralizer solution. After the contact time of 1 minute, 20ul aliquot of sample reaction mixture was immediately pipetted into 96-well plate containing 180 µl of neutralizer solution and left for 5 minutes. The suitably serially diluted aliquots were spotted on TSA plates. The plates were incubated at 37?C for 24 hours. The colonies in the plate were counted and fraction of surviving bacteria was calculated. Bacterial killing activity of AMPs was evaluated by log10 reduction factor that was calculated as the difference between logarithms of CFU/ml before and after exposure to tested concentrations of AMP, EDTA, surfactant and AMP+EDTA, AMP + surfactant and AMP + EDTA + surfactant compositions.

Results & Discussion:
Figure 1 illustrates Antimicrobial efficacy: log reduction achieved after 1-minute contact time at 20?C vs P. aeruginosa - log kill results of AMP C2, EDTA and combination of AMP C2 + EDTA versus P. aeruginosa. And log kill results of AMP D2, EDTA and combination of AMP D2 + EDTA versus P. aeruginosa.
AMP C2 and EDTA when tested individually were not active. However, AMP C2 + EDTA combination exhibited synergistic effect. > 5 log kill i.e. 99.999% reduction was achieved at 4 of 6 tested combinations. Only when EDTA concentration in composition was 3.75 mM, synergy was not observed. EDTA concentration above 11.25 mM are not used because of regulatory concerns.
AMP D2 and EDTA when tested individually were not active. Even, when AMP D2 + EDTA combination was tested synergistic effect was not seen at any of the tested concentration. This suggests the specific sequence features of AMP C2 are responsible for significant log kill observed. The simple apparent additive feature observed with AMP D2 and EDTA is not significant. =3 log kill is considered as bactericidal. The minor increase in log kill is due to increased susceptibility of Gram negative organisms such as Pseudomonas aeruginosa, Escherichia coli towards EDTA because of their membrane composition.
Figure 2 illustrates Antimicrobial efficacy: log reduction achieved after 1 minute contact time at 200C vs E. coli. log kill results of AMP C2, EDTA and combination of AMP C2+ EDTA versus E. coli and log kill results of AMP D2, EDTA and combination of AMP D2+EDTA versus E. coli.
The AMP C2 and EDTA when tested individually were not active. However, when AMP C2 + EDTA combination demonstrated synergistic effect. > 5 log kill ie. 99.999% reduction was achieved at 4 out of 6 tested combinations against E. coli. Only when EDTA concentration in the composition was 3.75 mM, synergy was not observed.
The AMP D2 and EDTA when tested individually were not active. Even, when AMP D2 + EDTA combination was tested synergistic effect was not seen at any of the tested concentration. This suggests the specific sequence features of AMP C2 are responsible for significant log kill observed with AMP C2 against E. coli. The simple apparent additive feature observed with AMP D2 and EDTA is not significant. =3 log kill is considered as bactericidal. The insignificant increase in log kill is due to increased susceptibility of Gram-negative organisms such as Pseudomonas aeruginosa, Escherichia coli towards EDTA because of their membrane composition.
Figure 3 illustrates Antimicrobial efficacy: log reduction achieved after 1 minute contact time at 200C vs S. aureus. log kill results of AMP C2, EDTA & combination of C2 + EDTA versus S. aureus and log kill results of AMP D2, EDTA & combination of D2 + EDTA versus S.aureus.
AMP C2 and EDTA when tested individually were not active. However, when AMP C2 + EDTA were tested synergistic effect was evident. > 5 log kill i.e. 99.999% reduction was achieved at 4 of 6 tested combinations against S.aureus. Only when EDTA concentration in composition was 3.75 mM, synergy was not observed suggesting the threshold.
AMP D2 and EDTA when tested individually were not active. Even, when AMP D2 + EDTA combination was tested synergistic effect was not seen at any of the tested concentration. This suggests the specific sequence features of AMP C2 are responsible for significant log kill observed against S.aureus. There was no additive effect of D2 + EDTA combination vs Gram positive S.aureus. This could be due to different membrane composition and decreased membrane susceptibility of Gram positive compared to Gram negative bacteria.
Figure 4 illustrates Antimicrobial efficacy: log reduction achieved after 1 minute contact time at 370C vs E.hirae. log kill results of AMP C2, EDTA & combination of C2 + EDTA versus E.hirae and log kill results of AMP D2, EDTA & combination of D2 + EDTA versus E.hirae.
AMP C2 and EDTA when tested individually were not active. However, when AMP C2 + EDTA were tested synergistic effect was evident. > 5 log kill i.e. 99.999% reduction was achieved at 2 of 3 tested combinations against E. hirae. Only when EDTA concentration in the combination was 3.75 mM, synergy was not observed suggesting the threshold.
AMP D2 and EDTA when tested individually were not active. Further, when AMP D2 + EDTA combination was tested synergistic effect was not seen at any of the 3 tested concentrations. This suggests the specific sequence features of AMP C2, that are responsible for significant log kill observed against E. hirae.
And no additive effect of D2 + EDTA combination vs Gram positive E.hirae was seen. This could be due to different membrane composition and decreased membrane susceptibility of Gram positive (E.hirae, S.aureus) compared to Gram negative bacteria (P. aeruginosa, E. coli)

Discussion:
The bactericidal efficacy was tested for 1-minute contact time with 4 challenge microorganisms in suspension, followed by dilution-neutralization-method as per the EN1276 standard.
The results in Figures 1-4, clearly demonstrate synergistic efficacy of the AMP C2 + EDTA versus four different microorganisms that include 2 Gram negative -P. aeruginosa, E. coli & 2 Gram positive bacteria S.aureus, and E. hirae. There was no synergy when another AMP D2 & EDTA was used at same concentrations in the combination.
The C2 peptide demonstrated higher efficacy when tested in combination with EDTA. The 62% sequence similarity and unique amino acid composition leads to achieve achievement in activity when combined with EDTA.
The C2 peptide with EDTA also showed equivalent efficacy as the antimicrobial standard, Benzalkonium chloride (BKC) in 1-minute log kill studies. The BKC (at 0.5% w/v) is demonstrated to achieve >5 log kill in 1 minute under similar test conditions.
From the aforesaid, it is observed that the active system, C2 and EDTA combination achieves 99.999% reduction (>5 log kill) against a broad spectrum of bacteria (Gram positive and Gram negative) in one minute. The test conditions mimicked real life hand wash conditions. The 3% Bovine Serum Albumin, a protein present in the test condition mimics soil/dirt on the hand. In prior art, the bactericidal efficacy evaluation is not carried out under the conditions that mimic the real life conditions.

C] Experimental data to show that C2+EDTA combination below and beyond the given concentration value 300 to 450 uM and 7.5 mM to 11.25 mM does not show synergism
Figure 5A and 5B illustrates effect of lower concentration of EDTA and C2 combinations against a Gram negative, E. coli, and a Gram positive bacteria, S. aureus. The results demonstrate that at tested concentrations (0.9 mM or 1.8 mM EDTA with 25 µM or 50 µM C2 peptide), insignificant activity of around 1 log kill was observed.
C] Other Chelators
Several other chelators were tested.
Figure 6 represents citric acid compatibility with C2 in presence and absence of EDTA. Citric acid an organic acid based chelator was evaluated for compatibility. Addition of citric acid to C2 and C2 + EDTA decreased its bacterial killing activity. Thus, citric acid is incompatible with C2 based active system under the tested conditions.

D]Peptide-Peptide combination:
AMP may show synergy in terms of antimicrobial activity with other class of AMPs. However, the same was tested. The C2 peptide was tested in combination with FK16. There was no bactericidal activity, hinting there was no compatibility.

E] Compatibility of C2 with Surfactants:
Surfactant compatibility of C2 was evaluated using various non-ionic surfactants such as Lauramine Oxide (LO),Lauryl Glucoside (LG),IGEPAL CO-520, Polyoxyethylene cholesteryl ether (ChEO30) and amphoteric surfactant, Cocoamidopropyl hydroxysultaine (CAHS), Cocamidopropyl betaine (CAPB) and Sodium Laureth Sulfate (SLS) and Decyl glucoside (DG) were procured from Galaxy surfactants Limited, Mumbai. Other surfactants used in the study were procured from Sigma-Aldrich. Citric acid was from Sigma, Merck. All other reagents used were from Sigma, Merck
The bacterium E.coli was grown in LB media till mid-log phase (3 X108 cfu/ml). The culture was diluted to get a final OD of 3 X 106 cfu/ml. The cells were collected by centrifugation and the cell pellet was washed with 10mM HEPES buffer (pH 7.4). The cell suspension was mixed with different test solutions of surfactants with C2 (100 µM) and without C2. The contact between cells and test solution was allowed for 10 minutes, after which, the sample was diluted and surviving bacterial counts were measured on LB agar plates. The log reduction of bacterial cells was calculated compared to control sample to obtain antimicrobial activity of test solutions.
In order to find appropriate partners for C2, surfactants were evaluated for their compatibility with the antimicrobial active. Non-ionic surfactants were preferred, as the cationic nature of the peptide is compatible and gives impetus to peptide activity. Figure 7A represents killing activity of surfactant alone against bacteria, E. coli. The testing was done to assess the baseline i.e. effect of surfactant alone. The killing activity observed with combinations of C2 with each of these surfactant is represented in Figure 7B. C2 alone at 100 µM showed 1.22 log reduction of E. coli cells. Presence of surfactants, CAHS and CHEO3 decreased the activity of C2 indicating that these are not compatible with C2. Among the tested surfactants, Lauryl Glucoside(LG) at 0.75% surprisingly showed enhanced killing activity with100 µM of C2 achieving log kill value of 4.79. The Lauryl glucoside(LG) belongs to Alkyl-Glucoside group of surfactants, and has carbon chains that are 12-14 units long. Decyl glucoside (DG) has C8-16 carbon chain.

Evaluation of activity of C2 with anionic (SLS) and amphoteric surfactant (CAPB)
It was of interest to check the compatibility of C2 in commonly used surfactant systems consisting of CAPB and SLS. Both amphoteric (CAPB) and anionic (SLS) surfactants significantly affected the activity of C2. It’s possible that cationic nature of peptide might interact in a non-favourable way with SLS, anionic surfactant (Figure 8)

F] Examples for the 3 component system of C2+EDTA+Decyl glucoside
The effect of Decyl glucoside and C2 peptide concentration was assessed against four test organisms (P. aeruginosa, E. coli, S. aureus and E.hirae). Furthermore, additional testing with various concentrations of C2 were performed against E.coli and S .aureus.
It can be observed from Figures 9A-9D that the enhanced bacterial killing effect was observed at higher peptide concentration (300 µM, 600 µM and 900 µM) with decyl glucoside and EDTA. The enhanced activity effect was not observed at lower peptide concentrations of 50 µM.

EXAMPLE -3:
Compositions of LL-37 peptide fragment
A] Identification of shorter versions/functional motifs of LL37.
Full length human Cathelicidin peptide LL37, known for its antimicrobial activity was chosen. Different truncated versions varying in length were synthesized and tested for activity. A series of four different shorter versions of LL37 were synthesized. They were 35% (FK13), 42% (FK16), 54% (KR20), 62% (LL23) & 100% (LL37). Some of these fragments were tested in the screening assay, MIC determination against bacteria. The promising leads were tested in contact kill assay which measures the rapid killing efficacy of the active. LL37 fragment with at least 35% similarity to C-terminal portion of LL37 provides enhancement in antimicrobial activity in combination with conventional additives. Another different AMP, dermicidin which doesn’t belong to this LL37 family was used as control. Total of six AMPs were tested.
It was observed that FK16 showed remarkable synergistic activity with alcohol.

B] Bactericidal Efficacy Evaluation:
Materials and Methods:
• Bacterial cells: Pseudomonas aeruginosa, Staphylococcus aureus,
• Media: MH broth, TSA,
• Diluent: Phosphate buffered Saline,
• Alcohols: Ethanol, Isopropyl alcohol

Testing of antimicrobial activity of chemicals - Determination of Minimum Inhibitory Concentration:
Antimicrobial activities of AMPs, ethanol and isopropyl alcohol were studied by determining their Minimum Inhibitory Concentration (MIC) against two bacteria, Pseudomonas aeruginosa ATCC 15442 and Staphylococcus aureus ATCC 6538, representing Gram negative and Gram positive bacteria, respectively. MIC is the minimum concentration required for growth inhibitory activity of antimicrobial substances. It was determined using capped 2 ml Eppendorf tubes to prevent evaporative loss of alcohol. Stock solutions of Antimicrobial peptides (512, 256, 128, 64, 32, 16, 8, 4, 2 and 1 µM) and alcohols (32%, 16%, 8%, 4%, 2%, 1%) were prepared in MH broth using 2-fold serial dilution procedure in MH broth to obtain various concentrations of the test substances.
Log phase cultures of Pseudomonas aeruginosa ATCC 15442 and Staphylococcus aureus ATCC 6538 adjusted to O.D.600 nm of 0.1 were diluted 1:100 in MH broth and 250 µl aliquots were added to the tubes containing 250 µl volume of various concentration of antibacterial actives to obtain cell density of 5 × 105 cells/ml. Addition of bacteria lead to the dilution of 1:2 of all the concentrations prepared. The tubes were incubated at 37°C for 21 hours. The growth or absence of growth of bacterial cells was measured by visual observations for turbidity in the growth medium. The lowest concentration that showed no visible growth indicates the MIC.
Table 2: MIC of Antimicrobials
No. Antimicrobial MIC against
Pseudomonas aeruginosa ATCC 15442 Staphylococcus aureus ATCC 6538
AMPs:
1 FK16 128 ?M (0.026%) 32 ?M
2 LL37 512 ?M (0.0512%) 512 ?M
3 Dermicidin >512 ?M (0.0512%) >512 ?M
4 LL23 >512 ?M >512 ?M
Alcohols:
5 Ethanol 4% 8%
6 Isopropanol 4% 8%

Table 2 reports the MIC values obtained. AMP FK16 exhibits antimicrobial activity with MIC of 128 and 32. ?M against Pseudomonas aeruginosa ATCC 15442 and Staphylococcus aureus ATCC 6538, respectively. However, other AMPs tested did not show antimicrobial activity at concentrations tested. Both, ethanol and isopropanol exhibited MIC of 4% and 8% against Pseudomonas aeruginosa ATCC 15442 and Staphylococcus aureus ATCC 6538, respectively.

C] Determination of synergy by checkerboard assay and rapid log kill assay
Checkerboard assay is 24 hour contact time and log kill assay has 1 minute contact time. Hence they are operating at different concentrations; higher concentration is required for log kill assay as raid killing is exhibited. It is closer equivalent of product formulation setting than the checkerboard assay. For checkerboard assay generally, 100 times higher concentration can be used. For e.g. Triclosan will show killing at 0.0001% in checkerboard but will need 0.1% for log kill and product formulation.

I] Checkerboard assay to evaluate interaction of combination of FK-16 AMP and alcohol:
The combined activity and interactions (synergism, sum of activities, or antagonism) between peptides and ethanol against bacteria was determined using checkerboard assay method (7 × 7 matrix format) in matrix of 2 ml Eppendorf tube with total volume of 0.5 ml. Six concentrations of 2-fold serial dilutions of Test peptides and ethanol in MH broth were created to obtain 4X concentrations of the required concentrations. The seventh tube for each series (test peptide and ethanol) consisted of 0 concentration, i.e. MH broth only. 125 µL of test peptide dilution and 125 µL of ethanol dilution were added in each tube of the matrix. To this, 250 µL of the bacterial suspension (prepared as described in above sections) was added to each tube to obtain final cell density of 5 × 105 cells/mL. After 21 h of incubation at 37°C, the tubes were visually examined for growth.
The Fractional Inhibitory Concentration (FIC) index for combination of FK-16 AMP and ethanol against bacterial strain was calculated as [(MIC of FK-16 AMP in combination with ethanol)/(MIC of FK-16 AMP alone)] + [(MIC of ethanol in combination with peptide)/(MIC of ethanol alone)]. The results were interpreted as FIC = 0.5, synergistic; 0.5 < FIC = 1, additive; 1 < FIC = 4, indifferent; FIC > 4, antagonistic (Ng et al., 2018).

Table 3A: Growth of P. aeruginosa ATCC 15442 at various FK-16 AMP and Ethanol concentration in checkerboard assay
Ethanol conc. FK-16 AMP conc (µM)
% mM 128 64 32 16 8 4 0
8 1390 n n n n n n n
4 695 n n n n n n n
2 347.5 n n n n n y y
1 173.8 n n n y y y y
0.5 86.9 n y y y y y y
0.25 43.4 n y y y y y y
0 0 n y y y y y y
Key: n = No growth (Inhibition of growth observed), y: Growth (No inhibition of growth)

Table 3B: Fractional inhibitory concentration (FIC) index of combination against Pseudomonas aeruginosa ATCC 15442:
MIC of active (when used alone) Conc. of active in combination at MIC FIC
FK-16 AMP (?M) 128 ?M 32 ?M FIC(FK-16 AMP) = 0.25
Ethanol (%) 4% 1% FIC(ethanol) = 0.25
FIC of combination - - 0.5

The combination of 32?M FK16 AMP and 1% ethanol exhibits antimicrobial activity against P. aeruginosa (Table 3A). The concentrations of individual actives within the combination are lower than individual MICs. The FIC index was calculated to be 0.5 indicating that the combination shows synergistic activity against P. aeruginosa (Table 3B).

Table 4A: Growth of S. aureus at various FK16 AMP and Ethanol concentration in checkerboard assay
Ethanol conc. FK16 AMP conc (µM)
% mM 64 32 16 8 4 2 0
8 1390 n n n n n n n
4 695 n n n n n n y
2 347.5 n n n n y y y
1 173.8 n n y y y y y
0.5 86.9 n n y y y y y
0.25 43.4 n n y y y y y
0 0 n n y y y y y
Key: n = No growth (Inhibition of growth observed), y: Growth (No inhibition of growth)

Table 4B: Fractional inhibitory concentration (FIC) index of combination against S. aureus
MIC of active (when used alone) Conc. of active in combination at MIC FIC
FK16 AMP (?M) 32 ?M 8 ?M FIC(FK-16 AMP) = 0.25
Ethanol (%) 8% 2% FIC(ethanol) = 0.25
FIC of combination - - 0.5

The combination of 8 ?M FK16 AMP and 2% ethanol exhibits antimicrobial activity against S. aureus (Table 4A). The concentrations of individual actives within the combination are lower than individual MICs. The FIC index was calculated to be 0.5 (Table 4B) indicating that this combination has synergistic activity similar to the activity observed against P. aeruginosa.

II] Rapid Kill activity (1-minute contact time);
The antimicrobial active is required to be shown to have a rapid killing activity in order to be suitable for application. Hence, killing of bacterial strains at 1 minute of contact time was evaluated. The bacteria included in the analysis were Pseudomonas aeruginosa ATCC 15442, Escherichia coli ATCC 10536, Staphylococcus aureus ATCC 6538 and Enterococcus hirae ATCC 10541. The temperature for the contact kill assay was maintained at 20°C. The initial bacterial count was set to 1.5-5 X 107 cells / ml. The 10X bacterial cells were treated 1:1 with 0.3% Bovine serum albumin to stimulate dirty condition on surface for 2 minute and the test substance (alone or combination) was added. After 1 minute, 0.1 ml of the mixture was collected, and added to 0.9 ml of neutralizer to inactivate antimicrobial active. The surviving bacterial counts were determined by plating on TSA plates to determine the extent of killing. The log kill was calculated to evaluate the efficacy of active systems to kill bacterial cells.
As sanitizers containing ethanol also contain small amount of isopropanol, the log kill study was conducted using ethanol spiked with isopropanol in 19:1 ratio to match with the commercial products.
Table 5: Log kill obtained with Antimicrobial active system against bacteria at 20°C in 1-minute contact time

Active System Concentration Log kill obtained against
S. aureus E. coli P aeruginosa E. hirae
FK16 10 µM 0.2 0.3 0 0
20 µM 0.3 0.4 0.12 0
40 µM 1.15 1.23 1.75 0.05
80 µM 2.04 2.23 2.2 0.1
160 µM 3.12 2.8 3.36 0.23
Alcohol -Ethanol and IPA (19:1) 2% 0 0 0 0
4% 0.1 0.2 0 0
8% 0.1 0.3 0.1 0.01
16% 0.5 0.8 0.12 0.01
32% 1.8 5.6 2.03 0.06
48% 5.4 5.5 5.5 5.5
FK16+Alcohol
10 µM + 2% 0.1 0.2 0 0
10 µM + 8% 0.2 0.3 0 0
10 µM + 32% 1.7 5.3 2.1 0.1
40 µM +8% 2.05 1.97 3.98 0.01
40 µM +32% 5.5 5.5 5.5 5.4
80 µM +16% 4.7 4.23 5.06 2.62
80 µM + 32% 5.5 5.5 5.5 5.5

Figures 10-13 illustrates Log kill obtained with Antimicrobial active system against four bacteria at 20°C in 1-minute contact.
Log kill data shows that combinations of AMP FK16 and Alcohol have higher killing efficacy compared to individual active (Table 5, Figures 10-13).For example: Figure 13 which illustrates the log kill activity against E. hirae demonstrates that AMP FK16 and alcohol when tested individually were not active. Only alcohol alone when used at a higher concentration of 48% was effective. However, when combinations of AMP FK16 + alcohol were tested using low alcohol percentage (16%, 32%), a synergistic effect was observed. Also, > 5 log kill i.e. 99.999% reduction was achieved.
In the present active system FK16 and alcohol achieve significant bacterial reduction 99.999% (>5 log kill) against a broad spectrum of bacteria (Gram positive and Gram negative) in 1 minute under tested conditions.
To the best of inventor’s knowledge, there are no reports of demonstration of > 5 log kill in 1 minute with AMP based composition against both Gram positive and Gram negative in presence of rigorous conditions including BSA to simulate dirty condition for the action area.
It is also observed from Table-5 that at the tested concentration ranges below 40 µM to 160 µM FK-16 and 8% to 32% alcohol, activity was evidently significantly diminished or absent (<1 log kill).
While ethanol is known to be antimicrobial and is widely used, it is used at very high concentration (generally 70% or more) to ensure efficacy. As it evaporates rapidly, it needs to act very fast. Such rapid killing activity is not obvious at alcohol concentration as low as 32%.
Also, it was observed that the log kill activity obtained with FK16 was equivalent to the standard antimicrobial, Triclosan when tested under similar conditions.
It is observed from the aforesaid that both active combinations [(C2 + EDTA) ,(C2+DG+EDTA) & (FK16 + Alcohol)] independently achieve 99.999% reduction (>5 log kill) against four test microorganisms in 1 minute under the tested conditions.

EXAMPLE-4
Formulations comprising the synergistic combinations of present invention.
A] Hand wash Formulation:
C2 peptide 600µM
Tetra sodium EDTA 9mM
Decyl glucoside (DG) 2.5%
Lauryl glucoside (LG) 1%
Capryl glucoside 1%
Arachidyl glucoside 1%
PEG 400 1.5%
Propylene Glycol 3.5%
Hydroxymethyl Cellulose (HPMC) 0.5%
Glycerin 3%
Phenoxyethanol / preservative 0.6%
Fragrance 0.1%
Water -QS

B] Sanitizer formulation
Ethanol 38%
FK-16 (C1) 100µM
Isopropanol 2%
HPMC 0.2%
Glycerine 0.5%
Fragrance 0.2%
QS- water

C] Body deodorant formulation
Ethanol: 47.5%
FK16 (C1) 100µM
Isopropanol 2.5%
Fragrance 1.5%
Phenoxyethanol 0.4%

D] Antiseptic formulation to control skin infection
C2 peptide 600µM
Tetra sodium EDTA 9mM
Propylene Glycol 2%
PEG 2000 2%
Glycerol 2%
Water QS

EXAMPLE-5
I] Demonstration of anti-viral activities of FK-16 (C1) and C2 based active systems
The antiviral activity of FK-16 (C1) and C2 against Human SARS-CoV-2 virus was tested in BSL-3 facility at CSIR-CCMB, Hyderabad using Real-Time PCR based protocol standardized by the institute. The test samples were combinations of FK-16 (C1) with alcohol, alcohol control and C2 with DG and EDTA. Briefly, 10 µl of virus suspension containing ~8000 viral particles treated with 10 µl of test sample. After 15 minutes contact time, 80 µl of serum free cell media was added to stop killing activity. 50 µl volume was added to monolayer of host mammalian cells (Vero cells), and incubated for 2 hours to allow entry of surviving (infectious) viral particles into the host cells. The solution was removed and replaced with fresh cell medium supplemented with 10% serum and test sample at 10%. Host Vero cells were incubated further for 72 hours. The media was collected, viral RNA was extracted, reverse transcribed to cDNA and SARS coronavirus specific RNA segment was quantified using Real-Time PCR. Antiviral activity of test sample was determined based on Log reduction values calculated compared to of virus counts obtained in control samples.
Table 6. Activity of FL-16 (C1) and C2 based active system against novel human SARS CoV-2 virus
Active System Concentration Log viral titre Log Reduction Percent Reduction
FK16 (µM) +alcohol (%) Control 6.5 0 0
400 µM+40% 4.7 1.8 98.42
100 µM+10% 2 4.5 99.997
50 µM+5% 2.9 3.6 99.975
25 µM+2.5% 6.5 0 0
Control (alcohol] Control 6.6 0 0
40% 6.5 0.06 12
20% 6.5 0.12 24
10% 6.5 0.12 24
C2 (µM)
with 0.5% DG+7.5 mM EDTA Control 6.5 0 0
600 µM 2.8 3.7 99.98
300 µM 6.5 0 0

Table 6 depicts the anti-SARS-CoV-2 efficacy of FK-16 (C1) and C2 active systems. The results suggested that FK-16 (C1) and alcohol was very effective, achieving >4 log kill at 15 minute time point. The C1 and alcohol system showed 3.6 log kill at 50uM of C1 and 5% alcohol. The alcohol used alone at 10%, 20% and 40% had no effect on levels of Human SARS CoV-2 virus in test conditions. The C2 in presence of DG (0.5%) and EDTA (7.5 mM) reduced viral levels at 600 µM achieving significant log kill of 3.7. It did not show any activity when tested at 300 µM in the same system. Preferably, a higher concentration of C2 peptide =600 µM is required for antiviral activity against SARS-CoV-2. There is possibility that C2 active system may show anti-viral activity against other viruses at < 600uM i.e lower concentrations.

II] Demonstration of activity of FK-16 (C1) and C2 peptides against malodor causing bacteria and differential killing
Antimicrobial activity of C1 and C2 against axillary bacteria: Potential of C1 and C2 to control body malodour
Body malodour is primarily due to action of bacteria on secretions of apocrine sweat glands. It is most intense in the axillary (underarm) area where higher moisture allows bacteria to grow. The bacteria from axillary area of human volunteers were collected using a sterile cotton swab wetted in 100 mM Phosphate buffer (pH 7.4). The wetted swab was rubbed for 1 minute over 10 cm2 of auxiliary area. The swab was placed in phosphate buffer and mixed to suspend bacteria from swab in to the buffer. This cell suspension was used as total axillary bacterial suspension for testing antimicrobial activity of FK-16 and C2 peptides. The contact time for bacteria with FK-16 and C2 peptide was 1 hour. Surviving bacterial counts were evaluated and log kill values were calculated as done for quantitative suspension test experiments described above.
Table 7A.Log Kill of axillary bacteria by FK-16 (C1) and C2 in the suspension test after contact time of 60 minutes.

Conc (µM) Peptide FK-16 (C1) Peptide C2
360 3.74 3.74
180 3.74 3.28
90 2.79 2.57

Both C1 and C2 Peptides exhibits antimicrobial activity in 60 minutes against mixed bacteria collected from axillary skin. Thus demonstrating the potential of the C1 and C2 to control axillary malodor.

Antimicrobial activity of FK-16 and C2 against standard skin bacteria: Potential of FK-16 (C1) and C2 to control body malodour.
The antimicrobial activity of FK-16 and C2 peptides was evaluated against Corynebacterium striatum as a representative of Corynebacteria species responsible for axillary malodour. Additionally, killing activities of FK-16 was also checked against Staphylococcus epidermidis, which is considered as normal resident of human skin and may have beneficial role to human physiology. The killing of these two bacteria was measured after a contact time of 5 minutes using quantitative suspension test.
Table 7B.Log Kill of skin associated bacteria by FK-16 and C2 in the suspension test at 5 minutes

Peptide
concentration
Time (minutes) Peptide FK-16 Peptide C2
C. striatum S. epidermidis C. striatum S. epidermidis
At 180 µM 5 min 4.66 2.62 4.81 1.24
At 45 µM 5 min 4.21 1.43 3.24 0.95

The human skin secretions such as axilla secretions contain non-odoriferous precursors. These are transformed by bacteria to chemicals that are responsible for body malodour (Shelly et al., Arch. Dermatol. Syphilol. 68, 430-446, 1953). A strong correlation was found between a high population of Corynebacteria and a strong axillary odor formation [Levden et al., J Invest Dermatol. 77: 413–416, 1981; Natsch et al, J. Biol. Chem. 278(8), 5718-5727, 2003].
Both, FK-16 and C2 killed C. striatum after 5 minutes of contact time showing >4 log kill when used at 180µM . The S. epidermidis was less sensitive to peptide action, with less than 3 log kill achieved. The differential killing of Corynebacterium and S. epidermidis is favorable for killing malodour associated bacteria without disrupting skin microbiome. This could be perhaps due to human and mammalian origin of C1 and C2 peptides respectively. Most deodorant products controls axillary malodour by non-specifically killing all bacteria on the skin. This could be detrimental over long time as destruction of human skin microbiome may render the skin sensitive to attack by pathogenic bacteria.
It is observed from aforesaid, that both peptides have always demonstrated compatibility and enhanced activity in their respective active systems i.e. C2+chelator (EDTA)+surfactant(DG) and C1 +alcohol against range of Gram positive and Gram negative bacteria tested (S.aureus, E.hirae and P.aeruginosa, E.coli).
Hence activity of C2 and C1 peptide alone against Corynebacterium striatum, Staphylococcus epidermidis and other bacteria from auxiliary skin suggests that the active systems will provide impetus to enhanced activity.

III] Stability studies of FK16 (C1) peptide at 4°C, 25°C and 40°C.
2 mg of the FK-16 peptide was dissolved in 1 ml ethanol (Haymen and Merck) in a polypropylene tube. Similarly, FK-16 peptide (2 mg) was mixed with 2mg of viscosity enhancer, polyvinylpyrrolidone (PVP) and the volume was made up to 1 ml. The samples were stored at 4°C, 25°C and 40°C for the stability studies. And end of designated time points i.e. 4 week, 8 week and 12 week, an aliquot was taken out and HPLC analysis was carried out to determine percent of peptide remaining in the test samples. The peptide amount detected in samples at each time point was normalized to the samples analyzed at the start of the stability study (at zero week).
Figures 14A and 14B pertains to stability profile of FK-16 (C1) in two conditions. The stability studies demonstrated that C1 peptide in ethanol was stable with 85% remaining intact compared to PVP (61% remaining) after 12 weeks at 40°C.

IV] Bioinformatics analyses of the peptides
For the listed 9 peptides, bioinformatics analysis was carried out. The calculation of the mean hydrophobicity() and calculation of the net charge (z) was carried out using the server https://heliquest.ipmc.cnrs.fr
Table 8. Bioinformatics analysis of the peptides.

Peptide Mean Hydrophobicity Average
Net Charge
C2 0.89 0.31
E2 0.90 0.31
D2 0.62 0.33
FK16 (C1) 0.40 0.25
FK13 0.32 0.31
Dermicidin
(DCD-1L) 0.21 -0.04
LL37 0.20 0.16
KR20 0.20 0.20
LL23 0.14 0.22

The physicochemical properties such as net charge and hydrophobicity play a key role antimicrobial nature, i.e. membrane insertion and rupture action of bacteria by the peptides. In addition, the spatial distribution and ratio hydrophobic and positive charged amino acids known to contribute to the membranolytic potential of the peptides
The higher net positive charge is required to attract and anchor to the bacterial membrane. The higher mean hydrophobicity is required for insertion depolarization and subsequent leakage of the bacterial membrane.
The peptide C2 possesses high mean hydrophobicity and average net charge, while the C1 displayed moderate hydrophobicity and net charge. For the reasons not clear, E2 and D2 do not demonstrate rapid and significant bactericidal activity. Interestingly, the HPLC retention time is higher for C1 and C2, (based on the CoA) than other peptides tested. The higher retention time on HPLC is correlated to hydrophobicity and is contributing to rapid bactericidal actions. Further, it is possible that other factors such as secondary structure and oligomerization/aggregation at the membrane play a role in rapid bactericidal mechanisms.
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:
1. An antimicrobial composition comprising:
(i). Indolicidin peptide analogue (C2) as set forth in SEQ ID 1 or derivatives thereof at a concentration ranging from 100 to 1800 uM; and
(ii.). an additive selected from:
(a) a chelating agent at a concentration ranging from 7.5 mM to 11.25 mM;
(b) a non-ionic surfactant at a concentration ranging from 0.2 to 10% w/v; or
(c) combinations thereof.

2. The antimicrobial composition as claimed in claim 1, wherein the Indolicidin peptide analogue (C2) is present at a concentration ranging from 300 to 900 uM.

3. The antimicrobial composition as claimed in claim 1, wherein the chelating agent is selected from the group of aminopolycarboxylic acids, polyphosphate, phosphonates or combination thereof.

4. The antimicrobial composition as claimed in claim 3, wherein the chelating agent is selected from Ethylenediaminetetraacetic acid (EDTA), Sodium tripolyphosphate (STPP), Ethylene hydroxydiphosphonate (EHDP) or combinations thereof.

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

6. The antimicrobial composition as claimed in claim 5, wherein the Alkyl-Poly Glucoside (APG) is selected from Decyl-Glucoside, Lauryl Glucoside, Capryl glucoside, Arachidyl glucoside or combinations thereof.

7. The antimicrobial composition as claimed in claim 6, wherein the Alkyl-Poly Glucoside (APG) is Decyl-Glucoside present at a concentration ranging from 0.5 to 5% w/v.

8. An antimicrobial composition comprising:
(a) Indolicidin peptide analogue (C2) as set forth in SEQ ID 1 or derivatives thereof at a concentration ranging from 100 to 1800 uM;
(b) a chelating agent at a concentration ranging from 7.5 mM to 11.25 mM; and
(c) a non-ionic surfactant at a concentration ranging from 0.2 to 10% w/v.

9. The antimicrobial composition as claimed in claim 8, wherein the chelating agent is selected from Ethylenediaminetetraacetic acid (EDTA) and the non-ionic surfactant is selected from Decyl glucoside.

10. An antimicrobial composition comprising:
(i). Cathelicidin peptide fragment (FK-16) as set forth in SEQ ID 8, or derivatives thereof at a concentration ranging from 40µM to 900 µM; and
(ii). An alcohol at a concentration ranging from 8% to 50%.

11. The antimicrobial composition as claimed in claim 10, wherein the Cathelicidin peptide fragment (FK-16) is present at a concentration ranging from 40µM to 160 µM.

12. The antimicrobial composition as claimed in claim 10, wherein the alcohol is selected from Ethanol, Isopropyl alcohol or a combination thereof.

13. The antimicrobial composition as claimed in claim 10, wherein the cathelicidin peptide fragment (FK-16) to alcohol ratio present in the composition ranges from 1: 3130 to 1:196.

14. The antimicrobial composition as claimed in claim 10, wherein the alcohol is a combination of ethanol and isopropanol present at a concentration ranging from 8 to 40%.

15. The antimicrobial composition as claimed in any one of the preceding claims, further comprising cosmetically acceptable excipients selected from polyethylene glycol (PEG), propylene glycol, hydroxymethyl cellulose (HPMC), glycerin, phenoxyethanol, fragrance, water and combinations thereof.

16. The antimicrobial composition as claimed in any one of the preceding claims, wherein the composition is in a form selected from gel, foam, cream, spray or wipes.

17. A synergistic hand wash formulation comprising:
300µM to 900µM of C2 peptide as set forth in SEQ ID 1;
7.5mM to 11.25mM of Tetra sodium EDTA;
0.5 to 5% of Decyl glucoside (DG);
0.25 to 2% of Lauryl glucoside (LG);
0.2 to 2% of Capryl glucoside;
0.2 to 2% of Arachidyl glucoside;
0.1 to 1% of Hydroxymethyl Cellulose (HPMC);
Water.

18. A synergistic sanitizer formulation comprising
38% Ethanol;
40 to 160µM of FK-16 (C1) as set forth in SEQ ID 8;
2% Isopropanol;
0.1 to 0.3% HPMC;
Water.

19. A synergistic body deodorant formulation comprising:
47.5% Ethanol;
40 to 160µM of FK16 (C1) as set forth in SEQ ID 8;
2.5% Isopropanol.

20. A synergistic antiseptic formulation comprising:
300µM to 900µM of C2 peptide as set forth in SEQ ID 1;
7.5mM to 11.25mM of Tetra sodium EDTA;
0.1 to 4% Propylene Glycol ;
0.1 to 4% PEG 2000;
0.1 to 4% Glycerol; and
Water.

21. A cosmetic method of reducing body odour by topically applying an antimicrobially effective amount of a body deodorant as claimed in claim 19 to a mammal.