A synergistic composition of an antimicrobial peptide (AMP) and natural active

Abstract

Provided herein is a composition comprising antimicrobial peptide having sequence of SEQ ID 1 and a natural active such that the composition provides 5 log reduction of bacteria in 1 minute. In the said composition the natural active is selected from medium chain fatty acid (6 to 12 carbon atoms) and selective essential oil like eugenol and citronellal from clove oil and citronella oil respectively.

Description

[0001] A SYNERGISTIC COMPOSITION OF AN ANTIMICROBIAL PEPTIDE (AMP) AND NATURAL ACTIVE

[0002] Field of Invention

[0003] The present invention relates to composition comprising antimicrobial peptide C2 (Indolicidin analog having 13 amino acid peptide sequence of ILPFKFPFFPFRR as provided in SEQ ID1) and natural(s) selected from medium chain fatty acid (6 to 12 carbon atoms) and essential oil. The said composition is synergistic and provide significant log kill i.e. 5 log (99.999%) reduction of microorganisms in 1 minute.

[0004] Background and prior art

[0005] Currently used and well known 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. World Health Organization (WHO) classifies the appearance of antibiotic resistance as one of the biggest threats 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.

[0006] 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. The long-term systemic exposure to actives like Triclosan is an area of concern

[0007] Hence, there is a need to develop efficacious and safer antimicrobial active combinations. Antimicrobial Peptides are part of innate defense for all living organisms. C2 is an antimicrobial peptide (AMP) which is an analogue of Indolicidin having peptide sequence of ILPFKFPFFPFRR. US2013 / 13745359 teaches use of AMP including Indolicidin in antimicrobial metallodrugs comprising AMP (that binds with target pathogen) covalently bound to a metal binding moiety. By incorporating a metal binding moiety with such antimicrobial agents (AMP) these antibiotics are rendered more potent. The first moiety i.e. the AMP are covalently bound to the metal binding moiety to form a complex. This complex is taken up by the pathogen or the cell of the subject hosting the pathogen and the uptake is promoted by the AMP moiety of the metallodrug. The antimicrobial composition may also contain antibiotic or conjugate comprising AMP and antibiotic in the first moiety.

[0008] US 19990318195 teaches ophthalmic solutions comprising an antimicrobial peptide that is an indolicidin and a buffer compatible with application to a mammalian eye, wherein the buffer is a Good's buffer or the buffer has a halide ion concentration less than 0.85 wt %. The compositions are useful for storing, cleaning, or disinfecting a contact lens. Having high tryptophan content has been used as antimicrobial and also can act as a solution preservative because they are selfsterilizing.

[0009] PCT / US 1999 / 009942 teaches indolicidin analogues those including at least 1 Tryptophan replaced by Phenylalanine and their use in antimicrobial activity. The peptide of SEQ ID 10 in Table 2 is the AMP referred herein as C2.

[0010] US2004 / 0176300 teaches 3 analogues of Indolicidin wherein the tryptophan is substituted by leucine at 3 positions. In an embodiment it also teaches a method of producing three single tryptophan analogs of Indolicidin namely, ILPWKLPLLPLRR-amide (IL4), ILPLKLPWLPLRR- amide (IL8) and ILPLKLPLLPWRR- amide (IL11), a cationic tridecapeptide amide found in the granules of bovine neutrophils, said analogs having said amino acid only at position 4, 8 or 11 from N-terminal, with leucine at its all other conventional positions, said analogs having selective anti-microbial activity and no haemolytic activity of Indolicidin, providing therapeutic options thereof. In the background this document teaches that presence of five tryptophan residues out of 13 -residues in Indolicidin renders its amino acid composition unique among host-defense endogenous antimicrobial peptides. It is cationic due to the presence of two arginine residues and one lysine residue and exhibits broad- spectrum anti-microbial activity. It causes lysis of erythrocytes and is cytotoxic to human and rat T lymphocytes. It further acknowledged that analogues of Indolicidin where proline was replaced by alanine and tryptophan by phenylalanine has anti-bacterial activity comparable to Indolicidin. Further, the presence of tryptophan appears to be essential for hemolytic activity as the phenylalanine analogs does not exhibit any haemolytic activity.

[0011] US5547939 teaches Indolicidin analogues, 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. The invention also provides methods of using an indolicidin analogue to inhibit or prevent the survival or growth of a microorganism in an environment capable of sustaining the microorganism.

[0012] W098 / 040401 discloses cationic peptides, including analogues of indolicidin and cecropin / melittin fusion peptides, in combination with antibiotics such that the combination is either synergistic, able to overcome microorganismal tolerance, able to overcome resistance to antibiotic treatment, or further provides other related advantages.

[0013] Giovagnoni et.al teaches the role of Medium chain fatty acids(MCFA) as well as some antibiotics against the bacteria Brachyspira hyodysenteriae. (Dual Antimicrobial Effect of Medium-Chain Fatty Acids against an Italian Multidrug Resistant Brachyspira hyodysenteriae Strain Giulia Giovagnoni 1 , Benedetta Tugnoli 2, Andrea Piva 1,2 and Ester Grilli : Microorganisms 2022, 10, 301 ). It acknowledges that organic acids are widely used as feed additives in swine farming due to their dual use as antimicrobials and growth promoters in the dose range of 0.2-3%. Depending on their acid dissociation constant, undissociated organic acids can pass through the bacterial membrane and, in response to the release of H+ and RCOO- ions, are able to exert their antimicrobial action: on one hand, bacteria waste energy to restore intracellular pH and, furthermore, the anion has a toxic effect by targeting replication and metabolic functions. In particular, antibacterial properties of MCFA are recognized as the most effective ones against Grampositive bacteria and, to a lesser extent, against Gram-negative bacteria . MCFA antibacterial activity against B. hyodysenteriae varied depending on the pH conditions and the length of the carbon chain. The longer is the carbon chain and the lower is the pH, the stronger is the antimicrobial activity, with dodecanoic acid being the most powerful antimicrobial against three strains of B. hyodysenteriae, including the ATCC 27164. According to the authors of this article Decanoic and dodecanoic acid showed the lowest MIC, also confirming the longer the chain, the more effective the antibacterial properties of MCFA are, the only exception being hexanoic acid with a lower MIC compared to octanoic acid, as already shown for other Gram-negative bacterial species, like Escherichia coli. Despite the difference in relation to pH and the carbon chain length, in our study, MIC values of MCFA were more consistent between the two strains compared to the tested antibiotics. In fact, resistance development to organic acids is much less frequent and problematic than for antibiotics, because the bacterial susceptibility to MCFA is an intrinsic property, meaning that the bioactive power of these molecules meets a broad spectrum of microorganisms with lower probability of failure.

[0014] Man et al (Antimicrobial Activity of Six Essential Oils Against a Group of Human Pathogens: A Comparative Study: Pathogens 2019, 8, 15) investigated the role of 6 essential oils on common human pathogens. They tested frankincense, myrtle, thyme, lemon, oregano and lavender essential oils against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Both micellar and aqueous extracts were used for determination of their minimal inhibitory (MIC) and bactericidal concentrations (MBC). The most active oils were oregano, thyme, lemon and lavender, while the least active were frankincense and myrtle. Prabuseenivasan, et al ( In vitro antibacterial activity of some plant essential oils : BMC Complementary and Alternative Medicine 2006, 6:39) investigated the role of 21 essential oils including aniseed oil, calamus oil, camphor oil, lemon grass oil, cinnamon oil, citronella oil, lavender oil, rosemary oil, eucalyptus oil, clove oil and others. It was found that out of 21 essential oils tested, 19 oils showed antibacterial activity against one or more strains out of which Cinnamon, clove, geranium, lemon, lime, orange and rosemary oils exhibited significant inhibitory effect. Cinnamon oil showed promising inhibitory activity even at low concentration, whereas aniseed, eucalyptus and camphor oils were least active against the tested bacteria. Cinnamon, clove and lime oils were found to be inhibiting both grampositive and gram-negative bacteria. In general, B. subtilis was the most susceptible, while K. pneumoniae exhibited low degree of sensitivity.

[0015] Scotti et al (Fighting Microbial Infections from Escherichia coli O157:H7: The Combined Use of Three Essential Oils of the Cymbopogon Genus and a Derivative of Esculentin-la Peptide : Antibiotics 2024, 13, 86. 2024) discloses effect of antimicrobial peptide esculentin- la in combination with essential oils from 3 species of lemon grass namely Cymbopogon citratus, C. flexuosus and C. martini. The fractional inhibitory concentration (FIC) was analysed by analyzing the combination of the peptide derived from esculentin- la, Esc(l-21), with the three essential oils. Also the expression of different genes implicated in motility, biofilm formation and stress responses were analysed. In conclusion, it was found that the three essential oils used in combination with the peptide showed synergy against the E. coli O157:H7, proving attractive as an alternative strategy against E. coli pathogen infections.

[0016] Thus there are disclosure of AMPs including indolicidin analogue as antimicrobial, essential oils and medium chain fatty acids (6 to 12 carbon atoms) each is also known to have antibacterial activity and even combination of AMP namely Esculentin- la along with lemon grass oils has been shown to have antimicrobial effect. There are also disclosures of synergistic activity of AMP with antibiotics. However, the antimicrobial property cited in the prior art is mainly related to determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC).

[0017] 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 the time for testing depending on the applications. The British Standard EN1276 has laid out stringent protocol for antibacterial efficacy testing of the active system / formulation for hand hygiene applications.

[0018] There are no prior art reports of AMP alone or in combination of naturals like medium chain fatty acid (6 to 12 carbon atoms) and selective essential oil in a composition achieving 5 log kill in 1 minute versus both Gram positive and Gram negative test organisms as per the British Standard BS EN 1276:2009.

[0019] Further even the medium chain fatty acids (6 to 12 carbon atoms) or the natural oils are also not known to achieving 5 log kill in 1 minute versus both Gram positive and Gram negative test organisms as per the British Standard BS EN 1276:2009

[0020] Surprisingly, the present inventors have found that naturals like caprylic acid as a medium chain fatty acid, selective essential oil exhibit synergistic antimicrobial effect with AMP in achieving the 5 log kill in 1 minute against both Gram positive and Gram negative bacteria in presence of rigorous conditions that simulate hand hygiene conditions.

[0021] Objects of the Invention

[0022] According to one object of the invention is to overcome the drawback of the prior art According to one aspect of present invention there is provided a synergistic composition comprising antimicrobial peptide (C2) (having sequence ILPFKFPFFPFRR as provided in SEQ ID1) and a natural selected from medium chain amino acid and essential oil.

[0023] According to another object of the invention is to provide a synergistic composition comprising peptide C2 and caprylic acid is medium chain fatty acid (6 to 12 carbon atoms)

[0024] According to another object of the invention is to provide a synergistic composition comprising peptide C2 and essential oils selected from clove oil and citronella oil

[0025] Another object is to provide such composition so as to 5 log kill in 1 minute against both Gram positive and Gram negative bacteria in presence of rigorous conditions that simulate hand hygiene conditions

[0026] Summary of the invention

[0027] According to one aspect of present invention there is provided a synergistic composition comprising antimicrobial peptide (having sequence as in SEQ ID 1) and a natural selected from medium chain fatty acid (6 to 12 carbon atoms) and essential oil such that the composition is capable of achieving 5 log reduction of bacteria in 1 minute.

[0028] Brief Description of the accompanying Drawing

[0029] Figure 1 : The HPLC chromatogram of C2 peptide

[0030] Detailed description of the invention

[0031] The antimicrobial property cited in the prior art is mainly related to determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). 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.

[0032] Surprisingly, the present inventors have now found a composition comprising specifically the indolicidin analogue peptide (C2) having amino acid sequence of SEQ ID 1 to act synergistically with naturals selected from specific medium chain fatty acid namely caprylic acid and selective essential oils like clove oil and citronella oil so as to achieve 5 log reduction of both gram positive and gram negative bacteria in 1 minute in presence of rigorous conditions that simulate hand hygiene conditions.

[0033] The combination of AMP (peptide C2) and natural demonstrated synergy in antimicrobial efficacy than at tested individual concentrations.

[0034] The AMP and natural work synergistically at lower concentrations providing 99.999% reduction against Gram positive and Gram negative under rigorous, challenging testing conditions.

[0035] In 1 minute contact kill experiment (in presence of 3% BSA), the composition of peptide (C2) and natural lead to higher log reduction against Gram negative bacteria Pseudomonas aeruginosa, Escherichia coli and Gram positive bacteria Staphylococcus aureus, Enterococcus hirae.

[0036] Thus the composition provides quick reduction of bacteria and to a high level which is required for instant kill of the bacteria as in handwash, sanitizer, wipes etc.

[0037] The present invention is described with reference to specific embodiments and the figures / tables including the best mode contemplated by the inventors for carrying out the invention. This description is not meant to be construed in a limiting sense, as various alternate embodiments of the invention will become apparent to persons skilled in the art, upon reference to the description of the invention. It is therefore contemplated that such alternative embodiments form part of the present invention.

[0038] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, pH, temperature described herein are those well-known and commonly used in the art. Some of the terms are defined briefly here below; the definitions should not be construed in a limiting sense.

[0039] It is to be understood that the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0040] 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

[0041] The use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and this detailed description are exemplary and explanatory only and are not restrictive.

[0042] The term “plurality” as used herein is defined as “one, or more than one”. Accordingly, the terms “one”, “at least one” would all fall under the definition of “plurality”.

[0043] Whether or not a certain feature or element was limited to being used only once, either way, it may still be referred to as “one or more features” or “one or more elements” or “at least one feature” or “at least one element” 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)).

[0044] In one embodiment the present invention provides a composition of caprylic acid as natural active along with the C2 peptide, wherein the caprylic acid is also selective in terms of its spectrum while other medium chain fatty acids (6 to 12 carbon atoms) do not provide such effect, which is surprising. Specially, when it is known that the antimicrobial effect of such fatty acids increase with increasing carbon chain length, the synergistic activity of caprylic acid (C8) with C2 peptide is indeed surprising when the decanoic acid (CIO) or dodecanoic acid (C12) do not provide such effect. The combination of caprylic acid and C2 peptide in given ratios exhibited synergy selectively against gram positive bacteria, namely E. hirae and S. aureus.

[0045] In another embodiment the present invention provides a composition of selective essential oil namely citronella oil or eugenol (clove oil) as a natural active along with the C2 peptide, wherein the citronella oil or eugenol is also selective in terms of its spectrum while other essential oils do not provide such effect, which is surprising. Specially, when the antimicrobial effect of such essential oils are known, the synergistic activity of only citronella oil or eugenol with C2 peptide is indeed surprising when the other essential oils like lemongrass oil, eucalyptus oil, rosemary oil do not provide such effect. The typical costs of chemical synthesis of peptide is on higher side. The present invention for the composition having such peptide and natural achieves significant reduction of peptide concentration which in turn leads to economic advantage.

[0046] In the composition the ratio of C2 peptide and caprylic acid is from 0.111 : 1 to 0.4: 1. The ratio of C2 peptide and eugenol is from 0.04: 1 to 0.08:1 and the ratio of C2 peptide and citronellal is from 0.04: 1 to 0.08: 1.

[0047] The composition of the present invention may be applied used as Wipes, sanitizers, body wash and hand wash, deodorant, disinfectant, acne control, wound healing cream / lotion, surface cleaner and the like.

[0048] The excipients present in the composition are selected from humectants, pH modifier, preservative, surfactants, fragrance solubilizer and the like.

[0049] The humectant is glycerol while the pH modifier is selected from Triethanolamine and citric acid.

[0050] The preservative is selected from phenoxyethanol, DMDM hydantoin, benzoic acid.

[0051] The surfactant is selected from non-ionic surfactants, Decyl Glucoside, alcohol ethoxylates. The fragrance solubilizer is selected from polysorbate 20, PEG 40 hydrogenated castor oil.

[0052] Though specific formulation examples of wet wipes with caprylic acid, eugenol and citronellal as illustrative examples are provided here, similar formulations with other natural are also covered within the scope of the present invention.

[0053] The present invention is described with reference to the following nonlimiting illustrative examples. Examples

[0054] Provided below are the actives used in the composition of the present invention and the assays used

[0055] Materials and methods:

[0056] Description of the components of the invention

[0057] • Peptide C2 (Indolicidin analog): The 13 amino acid peptide with sequence ILPFKFPFFPFRR (SEQ ID1) was custom synthesized. The molecular weight of the peptide is 1711 Daltons at >98% purity based on HPLC profile. The HPLC chromatogram is shown in Figure 1.

[0058] • Caprylic acid (Ocatanoic acid): Sigma-Aldrich C2875, USA

[0059] • Caproic acid (hexanoic acid): Sigma -Aldrich 21530, USA

[0060] • Capric acid (decanoic acid) : Sigma- Aldrich C1875, USA

[0061] • Eugenol: Sigma- Aldrich W246719, USA

[0062] • Citronellal: Sigma- Aldrich W230715, USA

[0063] Microorganism :

[0064] The microorganisms used in the study to demonstrate working of the present invention are:

[0065] 1. Pseudomonas aeruginosa, ATCC 15442

[0066] 2. Escherichia coli ATCC 10536

[0067] 3. Staphylococcus aureus ATCC 6538

[0068] 4. Enterococcus hirae ATCC 10541

[0069] 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.

[0070] Antimicrobial activity Assay to determine Microbicidal Efficacy:

[0071] 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.

[0072] • BSA: 3% BSA [3 g I’1bovine serum 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 (Catalogue no A8022).

[0073] • 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.

[0074] • The media chemicals Tryptone Soya Agar (TSA), Tryptone soya broth (TSB) and other chemicals were procured from HiMedia Laboratories, Mumbai, India.

[0075] • Diluent: Diluent consisted of Tryptone 1.0 g / 1 Sodium chloride (NaCl) 8.5 g / 1 in water.

[0076] Briefly, the bacterial 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 predetermined time after contact time duration was completed by diluting with neutralizer, and finally, counting the number of surviving bacteria to calculate the level of killing achieved by the test samples.

[0077] 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, lOOpl of BSA was added. Within 5 minutes, the bacterial suspension was added to 800pl of test samples and 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 log io reduction factor that was calculated as the difference between logarithms of CFU / ml before and after exposure to test samples.

[0078] Example 1

[0079] Comparative study of medium chain fatty acids to evaluate antimicrobial activity

[0080] Medium-chain fatty acids (MCFAs) are saturated or unsaturated fatty acids with 6 to 12 carbon atoms. The four commonly known medium chain fatty acids are evaluated for antimicrobial activity in combination with C2 peptide to assess synergistic activity. Antimicrobial efficacy against test bacteria as per EN1276 Standard with contact time of 1 minute, dirty condition (BSA as interfering / soiling substance at 20°C was first carried out.

[0081] Table 1: Properties of Medium chain Fatty acids

[0082] Lauric acid is solid at room temperature and was partially soluble in water, hence was not included for further screening. Other fatty acids, Caproic, Caprylic, Capric acids were evaluated alone and in combination with C2 peptide.

[0083] Medium -chain fatty acids caproic acid, caprylic acid and capric acid combinations were tested against the most challenging organism E. hirae. EN1276 protocol was carried out. Log reduction achieved after contact time of 1 minute under dirty conditions is reported and provided below.

[0084] Table 2. Antimicrobial Efficacy in terms of log reduction achieved against in E. hirae after 1 minute contact time (EN1276, dirty condition) by C2 peptide, MCFA and their combinations. Among the three tested medium chain fatty acids, caprylic acid demonstrated synergistic bactericidal activity against E. hirae in 1 minute. Caprylic acid is an eight-carbon chain fatty acid and is found naturally in coconuts. The ratio of C2 peptide and caprylic acid tested was 0.111: l.and 0.2: 1.

[0085] Example 2: Alcohol free Antimicrobial Wipes

[0086] Base formulation for wet wipe (Wipe base) was prepared with the following composition:

[0087] 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.

[0088] Formulation example comprising C2 peptide and caprylic acid.

[0089] The antimicrobial wipe formulation was tested for rapid bactericidal activity as per EN 1276 standard protocol using 4 bacteria prescribed by the standard. Bacteria tested were Staphylococcus aureus ATCC 6538, Enterococcus hirae ATCC 10541, Escherichia coli ATCC 10536 & Pseudomonas aeruginosa ATCC 15442. The conditions used for the assay was dirty (0.3% final BSA concentration) and contact time was 1 minute. The antimicrobial efficacy of this formulation base supplemented with C2, Caprylic acid and their combination are described in Table 3 A, 3B, 3C.1

[0090] Table 3A. Antimicrobial Efficacy (log reduction) achieved by C2 in wet wipes base, in 1 and 5 minute contact time at 20°C under dirt condition as per EN 1276 Standard against E. coli, S. aureus, E. hirae, P. aeruginosa

[0091] 1Cremophor CO -40

[0092] The wet wipes base alone (without added C2) showed significant activity against Gram negative bacteria E. coli and P. aeruginosa and no activity against S. aureus and E. hirae (both Gram positive bacteria). The peptide C2 was titrated in the range 0.005 to 0.015% versus four test bacteria in base. Significant bactericidal activity versus noted against E. coli and P. aeruginosa (Gram negative bacteria) >0.005%. Against Gram positive S. aureus and E. hirae no significant activity was noticed with C2 at three tested concentrations i.e. 0.005, 0.01 and 0.015%. Table 3B. Antimicrobial Efficacy (log reduction) achieved by Caprylic acid in wet wipes base, in 1 and 5 minute contact time at 20°C under dirt condition as per EN 1276 Standard against E. coli, S. aureus, E. hirae, P. aeruginosa The caprylic acid was tested in the range 0.04 to 0.14% against four test bacteria in wipe formulation base. Significant bactericidal activity noted against E. coli and P. aeruginosa (Gram negative bacteria) starting from 0.04% onwards. Against Gram positive S. aureus significant activity was noticed with caprylic acid at >0.07% and against E. hirae >0.11%. Only at 0.14% caprylic acid, >5 log kill activity was achieved against all four tested bacteria.

[0093] Table 3C. Antimicrobial Efficacy (log reduction) achieved by C2 + caprylic acid in wet wipes base, in 1 and 5 minute contact time at 20°C under dirt condition as per EN 1276 Standard against E. coli, S. aureus, E. hirae, P. aeruginosa

[0094] Synergy:

[0095] The synergy was defined as enhancement in killing of bacteria by at least 1 log (i.e. 10-fold in percentage value) by a combination compared to additive effect of individual actives that are part of the combination. Further, the combination needs to exhibit at least 3 log reduction of viable counts of bacteria to be considered as strong antimicrobial for its utility.

[0096] The wipes base alone led to significant log reduction of >3 against S. aureus, E. coli and P. aeruginosa. Hence the synergy was applicable with respect to only Gram positive bacteria E. hirae.

[0097] Among four test bacteria, E. hirae is considered as challenging owing its membrane architecture. Antimicrobials including naturals tend to demonstrate limited bactericidal activity against E. hirae specifically under EN 1276 dirt conditions with 1 minute contact time. Hence demonstrating synergy against E. hirae is important for developing appropriate formulation.

[0098] The proportion of the C2 and caprylic acid which resulted in synergy are provided below.

[0099] Bactericidal activity against E. hirae in 1 minute:

[0100] The C2 + caprylic combinations of below ratios demonstrated 3 to >5 log kill: 0.067: 1, 0.1: 1, 0.111:1, 0.143: 1, 0.15: 1, 0.2: 1, 0.214: 1, 0.25: 1, 0.3: 1, 0.375: 1

[0101] Synergy against Gram positive E. hirae in 1 minute

[0102] The below ratios demonstrated synergy

[0103] • 0.111: 1, 0.143: 1, 0.15: 1, 0.2: 1, 0.214: 1, 0.25: 1, 0.3: 1, 0.375: 1

[0104] The below ratio did not demonstrate synergy

[0105] 0.067: 1, 0.1: 1 The test sample containing mixture of caprylic acid + C2 at various ratios in a wet wipes base were tested against four test bacteria. Significant bactericidal activity versus noted against E. coli and P. aeruginosa followed by .S'. aureus, E. hirae. Example 3: Comparative study of naturals for synergistic activity with C2 peptide.

[0106] Natural actives including essential oils in combinations with peptide were studied against Gram positive E. hirae by quantitative suspension test (EN 1276 - 1 minute, dirt conditions). The purpose of study is to demonstrate synergistic actives that could reduce the effective concentration of C2 peptide. Gram positive E. hirae is relatively challenging test bacteria among four test bacteria (other three are Gram positive S. aureus and Gram negative E. coli and P. aeruginosa) used in EN 1276.

[0107] Table 4. study of naturals for synergistic activity with C2 peptide. Log reduction achieved with different naturals in combination with 0.01% C2 peptide after 1 minute, contact time at 20°C against E. hirae in dirt conditions as per EN1276.

[0108] Among the tested naturals two actives, 0.25% citronellal and 0.25% eugenol, in combination with 0.01% peptide C2 demonstrated synergistic effect in terms of demonstrating significant log reduction (>5 log reduction) against Gram positive E. hirae.

[0109] Hence, citronellal and eugenol were further tested for evaluating synergy.

[0110] Example 4: Citronellal + C2 peptide combination

[0111] Citronellal is a monoterpenoid, the main component of citronella oil which gives it its distinctive lemon aroma. It was evaluated in combination with C2 peptide for synergy.

[0112] The peptide C2 (0.01%) and citronellal (0.25 and 0.125%) combination is tested against three test bacteria.

[0113] C2 peptide alone is not active at 0.01%.

[0114] Citronellal alone (0.125 to 0.5%) is effective against only E. coli leading to log reduction of 4.9 to 5.1.

[0115] Table 5. Antimicrobial efficacy: log reduction achieved with various combinations of peptide C2 and citronellal after 1 minute contact time at 20 C vs E. coli, E. hirae and S. aureus (EN1276, dirt condition).

[0116] The synergy was defined as enhancement in killing of bacteria by at least 1 log (i.e. 10-fold in percentage value) by a combination compared to additive effect of individual actives that are part of the combination. Further, the combination needs to exhibit at least 3 log reduction of viable counts of bacteria to be considered as strong antimicrobial for its utility.

[0117] The synergy is demonstrated:

[0118] • against E. hirae & S. aureus by 0.01% C2 + 0.25% Citronellal (C2 and citronellal ratios 0.04: 1) and 0.01% C2 + 0.125% Citronellal (ratios 0.08: 1)

[0119] • against E. coli by 0.01% C2 + 0.125% Citronellal (ratios 0.08: 1)

[0120] Example 4. Synergistic antimicrobial efficacy of C2+eugenol combinations

[0121] Eugenol is an allyl chain-substituted guaiacol, a member of the allylbenzene class of chemical compounds from clove oil Eugenol (0.0625 to 0.5%) was evaluated for antibacterial activity against 3 test bacteria viz., E. coli, E. hirae and S. aureus. At 0.125%, eugenol showed significant activity against E. coli, at 0.25% against S. aureus and at only at 0.5% concentration demonstrated significant activity against E. hirae. C2 peptide at the tested concentrations (0.005 to 0.02%) had no activity on its own. Table 6. Antimicrobial efficacy: log reduction achieved with various combinations of C2 + eugenol after 1 minute contact time at 20°C vs E. coli, E. hirae & S. aureus (EN1276, dirt conditions)

[0122] The synergy is demonstrated against E. hirae-.

[0123] • in the combinations of 0.005% C2 + 0.25% Eugenol, 0.01% C2 + 0.25% Eugenol & 0.02% C2 + 0.25% Eugenol (C2: Eugenol ratios of 0.02: 1, 0.04: 1 and 0.08: 1, respectively) • in the combinations of 0.01% C2 + 0.125% Eugenol and 0.02% C2 +

[0124] 0.125% Eugenol (C2 and eugenol ratios 0.08: 1 & 0.16: 1) Example 5: Formulation example comprising C2 peptide and eugenol

[0125] The efficacy of C2 and eugenol combination was evaluated in wet wipe formulation base.

[0126] Formulation for Wet wipes:

[0127] Formulation for Wet wipes was prepared with composition: Glycerin (1%), PEG- 40 Hydrogenated Castor Oil (0.25%), Decyl Glucoside (0.2%), Phenoxyethanol (0.3%), Propylene Glycol (0.2%), Disodium EDTA (0.2%), Fragrance (0.2%) and water q.s. pH adjusted with citric acid / triethanolamine to pH 6.

[0128] This formulation base was supplemented with C2 alone, Eugenol alone or combinations of C2 and eugenol in 0.04: 1 and 0.08: 1 ratios. The antimicrobial efficacy of these formulations was tested and the results are described below in table 7.

[0129] Table 7. Antimicrobial efficacy: log reduction achieved with various combinations of C2 + eugenol in wipe formulation base after 1 minute contact time at 20°C vs E. coli, E. hirae, S. aureus, P. aeruginosa (EN1276, dirt condition) The formulation base alone (no added C2 and Eugenol) exhibited log reduction of 4.4 against E. coli but lacked ability to kill E. hirae and S. aureus. Addition of combinations of C2 peptide and eugenol exhibited enhanced killing activity against all 3 tested bacteria E. coli, E. hirae and S. aureus

[0130] Two combinations of C2 peptide and eugenol showed synergy against test bacteria E. coli, E. hirae and S'. aureus.

[0131] • 0.01% C2 + 0.25% Eugenol (C2 peptide to eugenol ratio of 0.04: 1) was the most synergistic leading to >5 against tested 3 bacteria.

[0132] • Similarly, 0.01% C2 + 0.125% Eugenol (C2 peptide to eugenol ratio of 0.08: 1) demonstrated synergy (3 to >5 log reduction).

[0133] Example 6: Formulation example comprising C2 peptide and Citronellal

[0134] The efficacy of C2 and citronellal combination is evaluated in wet wipe formulation base.

[0135] This formulation base is supplemented with C2 alone, Citronellal alone or combinations of C2 and Citronellal in 0.04: 1 and 0.08: 1 ratios. The antimicrobial efficacy of these formulations was tested and the results are described below in table 8.

[0136] Table 8. Antimicrobial efficacy: log reduction achieved with various combinations of C2 + Citronellal in wipe formulation base after 1 minute contact time at 20°C vs E. coli, E. hirae, S. aureus, P. aeruginosa (EN1276, dirt condition)

[0137] The formulation base alone (no added C2 and Citronellal) exhibited log reduction of 4.4 against E. coli but lacked ability to kill E. hirae and S. aureus. Addition of combinations of C2 peptide and citronellal exhibited synergistic killing activity against all 3 tested bacteria E. coli, E. hirae and S. aureus.

[0138] • 0.01% C2 + 0.25% citronellal (C2 peptide to citronellal ratio of 0.04: 1) was the most synergistic leading to >5 against tested 3 bacteria.

[0139] • Similarly, 0.01% C2 + 0.125% citronellal (C2 peptide to citronellal ratio of 0.08: 1) demonstrated synergy (>4 log reduction).

[0140] Example 7: Formulation example comprising C2 peptide and Lemongrass oil

[0141] The efficacy of C2 and lemongrass oil combination was evaluated in wet wipe formulation base comprising Glycerin (1%), PEG-40 Hydrogenated Castor Oil1(0.25%), Decyl Glucoside (1.65%), Phenoxyethanol (0.3%), Propylene Glycol (0.2%), Disodium EDTA (0.2%), Fragrance (0.2%) and water q.s. pH was adjusted with Citric acid / Triethanolamine to pH 5.

[0142] This formulation base was supplemented with C2 alone, lemongrass oil alone or combinations of C2 and lemongrass oil in 0.04: 1 and 0.08: 1 ratios. The antimicrobial efficacy of these formulations was tested and the results are described below in table 9. Table 9. Antimicrobial efficacy: log reduction achieved with various combinations of C2 + lemongrass oil in wipe formulation base after 1 minute contact time at 20°C vs E. coli, E. hirae, S. aureus, P. aeruginosa (EN1276, dirt condition)

[0143] Two combinations of C2 peptide and lemongrass oil did not show synergy against test bacteria E. hirae and S. aureus. The formulation base alone (no added C2 and lemon grass oil) exhibited log reduction of 4.4 against E. coli but lacked ability to kill E. hirae and S. aureus. Addition of Lemongrass oil has not significant enhancement effect indicating lack of synergy in combination of C2 and an essential oil, Lemongrass oil.

[0144] The above data thus shows that not all medium chain fatty acids or essential oil act synergistically with C2 peptide to achieve log reduction of bacteria. Only caprylic acid (C8) and citronellal / eugenol act synergistically with peptide C2 to achieve log reduction of bacteria and are thus appropriate for hygiene product like wipes, hand wash, sanitizer.

Claims

Claims:

1. A synergistic composition comprising antimicrobial peptide having sequence of SEQ ID 1 and a natural active.

2. The synergistic composition as claimed in claim 1 , wherein the composition provides 5 log reduction of bacteria in 1 minute.

3. The synergistic composition as claimed in claim 1, wherein natural active is selected from medium chain fatty acid (6 to 12 carbon atoms) and selective essential oil.

4. The synergistic composition a claimed in claim 3, wherein the medium chain fatty acid is caprylic acid.

5. The synergistic composition a claimed in claim 4, wherein the ratio of C2 peptide and caprylic acid is from 0.111 : 1 to 0.4: 1.

6. The synergistic composition a claimed in claim 3, wherein the essential oil is selected from citronellal and eugenol.

7. The synergistic composition as claimed in claim 6, wherein the ratio of C2 peptide and eugenol is from 0.04: 1 to 0.08: 1.

8. The synergistic composition as claimed in claim 6, wherein the ratio of C2 peptide and citronellal is from 0.04: 1 to 0.08: 1.

9. The synergistic composition as claimed in any one of claims 1-6 comprising excipients selected from humectant, pH modifier, preservative, surfactants, fragrance solubilizer.

10. The synergistic composition as claimed in any one of claims 1-6 which is a hygiene product selected from wipe, hand wash, sanitizer, deodorant, disinfectant, acne control, wound healing cream / lotion, surface cleaner.

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