FIELD OF INVENTION
[001] The present disclosure broadly relates to composition having anti-bacterial activity and particularly discloses a composition comprising thymol and berberine for inhibiting the growth of Staphylococcus epidermidis.
BACKGROUND OF INVENTION
[002] Miliaria or prickly heat or heat rash is a characteristic skin ailment of eccrine sweat gland, which is highly predominant in hot and humid climatic conditions. It has great affinity towards neonates, as they have immature sweat glands (Bukhari et al., 2016, Our Dermatology Online/Nasza Dermatologia Online, 7). However, it generally affects people of all ages. It is characterized by excessive sweating, with itchiness and discomfort.
[003] Depending on the severity of obstruction of sweat ducts, it is divided into three types, namely: (i) Miliaria crystalliana - superficial obstruction in stratum corneum; (ii) Miliaria rubra - obstruction in stratum malpighii with the development of primary lesions; Miliaria pustulosa - extension of primary lesions with characteristic formation of pustules; (iii) Miliaria profunda – obstruction at or within dermoepidermal junction (Wenzel and Horn 1998. Journal of the American Academy of Dermatology, 38, 1-20).
[004] The mismanagement of miliaria may sometimes culminate in serious implications such as anhidrosis wherein, the body fails to perspire, eventually leading to overheating of body and heat stroke (Bukhari et al., 2016, Our Dermatology Online/Nasza Dermatologia Online, 7). Thus, proper diagnosis and appropriate treatment of miliaria is an indispensable need. Moreover, the pathogenicity of miliaria is manifested primarily to be the result of plugging of sweat duct by periodic acid schiff’s (PAS) positive - extracellular polysaccharide substance (EPS) produced by Staphylococcus epidermidis (Mowad et al., 1995. Journal of the American Academy of Dermatology, 33, 729-733). S. epidermidis is a Gram positive and commensal microorganism, which turns out to be an opportunistic pathogen on favoring conditions.
[005] Formerly, the management of miliaria was accomplished using aminoglycosides class of antibiotics such as kanamycin and neomycin, which are

bactericidal against Staphylococcus species (Lyons et al., 1962 Archives of Dermatology, 86, 282-286). Numerous recent reports suggest the development of antibiotic resistance by microbes through various mechanisms, which devitalize the management of infectious diseases in clinical settings. Also, the excessive exposure of chemicals used in anti-miliaria products such as salicylic acid, boric acid and chlorphenesin are reported to possess certain degree of toxic effects on skin (Nitikhunkaesem et al., 2000, U.S. Patent 6,048,549).
[006] US 6613728 discloses a liquid disinfecting solution comprising peroxygen bleach and an antimicrobial active selected from the group consisting of thymol, eugenol, menthol, geraniol, vertenone, eucalyptol, pinocarvone, cedrol, anethol, carvacrol, hinokitiol, berberine, ferulic acid, cinnamic acid, methyl salicylic acid, methyl salicylate, terpineol, limonene and mixtures thereof. [007] CN 105663366 discloses a Chinese medical formulation for treating miliaria.
SUMMARY OF THE INVENTION
[008] In an aspect of the present invention, there is provided a composition
comprising: (a) thymol; and (b) berberine, wherein thymol to berberine weight
ratio is in the range of 1:0.2 – 1:2.
[009] In an aspect of the present invention, there is provided a process for
preparing a composition comprising: (a) thymol; and (b) berberine, wherein thymol
to berberine weight ratio is in the range of 1:0.2 – 1:2, said process comprising the
steps of: (i) obtaining thymol; (ii) obtaining berberine; and (iii) contacting thymol
with berberine to obtain the composition.
[0010] In an aspect of the present invention, there is provided a formulation
comprising: (i) an antibacterial composition comprising: (a) thymol; and (b)
berberine, wherein thymol to berberine weight ratio is in the range of 1:0.2 – 1:2;
and (ii) at least one cosmetically suitable carrier.
[0011] These and other features, aspects, and advantages of the present subject
matter will be better understood with reference to the following description and
appended claims. This summary is provided to introduce a selection of concepts in
a simplified form. This summary is not intended to identify key features or

essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
[0012] The following drawings form a part of the present specification and are
included to further illustrate aspects of the present disclosure. The disclosure may
be better understood by reference to the drawings in combination with the detailed
description of the specific embodiments presented herein.
[0013] Figure 1 depicts the MIC (minimum inhibitory concentration) of thymol
and berberine against S. epidermidis, in accordance with an embodiment of the
present disclosure.
[0014] Figure 2 depicts the MIC of zinc oxide and salicylic acid against S.
epidermidis, in accordance with an embodiment of the present disclosure.
[0015] Figure 3 depicts the degree of synergism between zinc oxide and salicylic
acid using checkerboard agar dilution assay against S. epidermidis growth
inhibition, in accordance with an embodiment of the present disclosure.
[0016] Figure 4 depicts the representative images of petriplates of CFU assay for
the assessment of anti-bacterial activity of thymol and berberine, and their
synergistic combinations against S. epidermidis growth inhibition, in accordance
with an embodiment of the present disclosure.
[0017] Figure 5 depicts the log reduction of S. epidermidis growth upon treatment
with thymol and berberine, in accordance with an embodiment of the present
disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0018] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features. Definitions

[0019] For convenience, before further description of the present disclosure,
certain terms employed in the specification, and examples are delineated here.
These definitions should be read in the light of the remainder of the disclosure and
understood as by a person of skill in the art. The terms used herein have the
meanings recognized and known to those of skill in the art, however, for
convenience and completeness, particular terms and their meanings are set forth
below.
[0020] The articles “a”, “an” and “the” are used to refer to one or to more than one
(i.e., to at least one) of the grammatical object of the article.
[0021] The terms “comprise” and “comprising” are used in the inclusive, open
sense, meaning that additional elements may be included. It is not intended to be
construed as “consists of only”.
[0022] Throughout this specification, unless the context requires otherwise the
word “comprise”, and variations such as “comprises” and “comprising”, will be
understood to imply the inclusion of a stated element or step or group of element or
steps but not the exclusion of any other element or step or group of element or
steps.
[0023] The term “including” is used to mean “including but not limited to”.
“Including” and “including but not limited to” are used interchangeably.
[0024] The term “at least one” is used to mean one or more and thus includes
individual components as well as mixtures/combinations.
[0025] Minimum inhibitory concentration (MIC) is the lowest concentration of a
antimicrobial (antibiotic antifungal or bacteriostatic) drug that will visibly inhibit
the growth of microorganism after incubation.
[0026] CFU refers to colony forming units, it refers to a unit used to estimate the
number of viable bacteria or fungal cells in a sample.
[0027] The term “liquid cleansing product” include shower gel, face wash, body
wash, hand wash, and paste. It is apparent that numerous other forms and
modifications of the liquid cleansing product will be obvious to those skilled in the
art and generally should be construed to cover all such obvious forms and
modifications which are within the true spirit and scope of the present disclosure.

[0028] The term “solid cleansing products” include cleansing bars, and films. It is apparent that numerous other forms and modifications of the solid cleansing product will be obvious to those skilled in the art and generally should be construed to cover all such obvious forms and modifications which are within the true spirit and scope of the present disclosure.
[0029] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a weight ratio range of about 1:0.2 to about 1:2 should be interpreted to include not only the explicitly recited limits of about 1:0.2 to about 1:2, but also to include sub-ranges, such as 1:0.2 to 1:0.5, 1:0.5 to 1:1, and so forth, as well as individual amounts, including fractional amounts, within the specified ranges, such as 1:0.25, 1:0.5, and 1:1, for example.
[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference. [0031] Natural products are used for centuries as a remedy for curing several human sicknesses (Saklani and Kutty 2008. Drug Discovery Today, 13, 161-171). As natural products are less toxic and possess vast structural diversity, they can be considered as a dependable alternative. Understanding the importance of miliaria disease and owing to the problems associated with it (as discussed previously), the current invention has been developed using phytochemicals. Combination drug therapy has gained more importance in recent past owing to the benefits of reduced toxicity, magnified antimicrobial spectrum and reduced chance of antibiotic

resistance, thereby exceeding the antimicrobial activity of individual drug candidate (Chanda and Rakholiya 2011. Microbiol Book Series, 520-529). [0032] The present invention focuses on the development of non-toxic, economical and effective anti-miliaria formulation with the ability to avoid multi-drug resistance development. Moreover, the current invention is also aimed to substitute the prevalently used chemical antibacterial agents in anti-miliaria products with plant derived actives and or reducing the toxic effects and enhancing the antibacterial activity of chemical antibacterial agents by phytochemicals through combinatorial approach.
[0033] The present invention relates to a composition comprising: thymol and berberine, wherein thymol to berberine weight ratio is in the range of 1:0.2 – 1:2. The composition is shown to be effective in synergistically inhibiting the growth of S. epidermidis. Thus, providing a better treatment against miliaria disease. As phytochemicals such as thymol and berberine have been used in the present composition, it is less likely to cause any side effects, therefore can be developed into an excellent alternative for antibiotics or other synthetic drugs. [0034] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein. [0035] In an embodiment of the present disclosure, there is provided a composition comprising: (a) thymol; and (b) berberine, wherein thymol to berberine weight ratio is in the range of 1:0.2 – 1:2. In another embodiment of the present disclosure, thymol to berberine weight ratio is in the range of 1:0.2 – 1:0.4. In yet another embodiment of the present disclosure, thymol to berberine weight ratio is in the range of 1:0.4 – 1:0.7. In alternate embodiment of the present disclosure, thymol to berberine weight ratio is in the range of 1:0.9 – 1:1.2. In another embodiment of the present disclosure, thymol to berberine weight ratio is in the range of 1:0.22 – 1:1.8.
[0036] In an embodiment of the present disclosure, there is provided a composition comprising: (a) thymol; and (b) berberine, wherein thymol to berberine weight

ratio is in the range of 1:0.2 – 1:2, wherein thymol to berberine weight ratio is
1:0.25.
[0037] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) thymol; and (b) berberine, wherein thymol to berberine weight
ratio is in the range of 1:0.2 – 1:2, wherein thymol to berberine weight ratio is
1:0.5.
[0038] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) thymol; and (b) berberine, wherein thymol to berberine weight
ratio is in the range of 1:0.2 – 1:2, wherein thymol to berberine weight ratio is 1:1.
[0039] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) thymol; and (b) berberine, wherein thymol to berberine weight
ratio is in the range of 1:0.2 – 1:2, and thymol has a weight percentage in the range
of 0.00256% to 0.0256% with respect to the composition. In another embodiment
of the present disclosure, thymol has a weight percentage in the range of 0.005% to
0.02% with respect to the composition. In yet another embodiment of the present
disclosure, thymol has a weight percentage in the range of 0.01% to 0.02% with
respect to the composition.
[0040] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) thymol; and (b) berberine, wherein thymol to berberine weight
ratio is in the range of 1:0.2 – 1:2, and berberine has a weight percentage in the
range of 0.00128% to 0.0128% with respect to the composition. In another
embodiment of the present disclosure, berberine has a weight percentage in the
range of 0.002%-0.01% with respect to the composition. In yet another
embodiment of the present disclosure, berberine has a weight percentage in the
range of 0.005%-0.01% with respect to the composition.
[0041] In an embodiment of the present disclosure, there is provided a formulation
comprising: (i) an antibacterial composition comprising: (a) thymol; and (b)
berberine, wherein thymol to berberine weight ratio is in the range of 1:0.2 – 1:2;
and (ii) at least one cosmetically suitable carrier.
[0042] In an embodiment of the present disclosure, there is provided a formulation
comprising: (i) an antibacterial composition comprising: (a) thymol having a

weight percentage in the range of 0.00256%-0.0256% with respect to the composition; and (b) berberine having a weight percentage in the range of 0.00128%-0.0128% with respect to the composition, wherein thymol to berberine weight ratio is in the range of 1:0.2 – 1:2; and (ii) at least one cosmetically suitable carrier.
[0043] In an embodiment of the present disclosure, there is provided a formulation comprising: (i) an antibacterial composition comprising: (a) thymol; and (b) berberine, wherein thymol to berberine weight ratio is 1:0.25; and (ii) at least one cosmetically suitable carrier.
[0044] In an embodiment of the present disclosure, there is provided a formulation comprising: (i) an antibacterial composition comprising: (a) thymol; and (b) berberine, wherein thymol to berberine weight ratio is 1:0.5; and (ii) at least one cosmetically suitable carrier.
[0045] In an embodiment of the present disclosure, there is provided a formulation comprising: (i) an antibacterial composition comprising: (a) thymol; and (b) berberine, wherein thymol to berberine weight ratio is 1:1; and (ii) at least one cosmetically suitable carrier.
[0046] In an embodiment of the present disclosure, there is provided a formulation comprising: (i) an antibacterial composition comprising: (a) thymol; and (b) berberine, wherein thymol to berberine weight ratio is in the range of 1:0.2 – 1:2; and (ii) at least one cosmetically suitable carrier, and the at least one cosmetically suitable carrier comprises additives selected from the group consisting of anti-caking agent, absorbent, skin protectant, viscosity modifier, opacifying agent, preservative, skin conditioning agent, cooling agent, odour enhancer, hydrophilic polymer, UV stabilizer, pH adjusting agent, chelating agent, deodorant, perfumes, antimicrobial, antioxidant, humectant, conditioning ingredients, propellants, salts, colorants, dyes, diluent, and combinations thereof. In another embodiment of the present disclosure, the adsorbent is selected from the group consisting of corn starch, rice bran starch, maize starch, and combinations thereof, the anti-caking agent is talc, the hydrophilic polymer is selected from the group consisting of polyethylene glycols (PEGs), polyvinylpyrrolidones (PVP), hydroxypropyl

methylcellulose (HPMC), and poloxamers; UV stabilizer is benzophenone-3 and other known UV stabilizers in the art; preservative is selected from the group consisting of phenoxyethanol, benzyl alcohol, methyl paraben, and propyl paraben; pH adjusting agent is selected from the group consisting of lactic acid, citric acid, sodium citrate, succinic acid, phosphoric acid, sodium hydroxide, and sodium carbonate; antimicrobials is selected from the group consisting of farnesol, zinc phenolsulphonate, and ethylhexylglycerin; humectants is selected from the group consisting of tribehenin, and glycerine; the cooling agent is selected from the group consisting of trimethyl isopropyl butanamide, peppermint and menthol; the skin conditioning ingredient is selected from the group consisting of panthenol, panthetine, pantotheine, panthenyl ethyl ether, allantoin, and combinations thereof; propellant is selected from the group consisting of propane, isopropane, butane, and isobutene; salts is selected from the group consisting of potassium acetate, sodium chloride and combinations thereof; the antioxidant is selected from the group consisting of tocopheryl acetate, propyl, octyl and dodecyl esters of gallic acid, butylated hydroxyanisole (BHA, usually purchased as a mixture of ortho and meta isomers), butylated hydroxytoluene (BHT) , nordihydroguaiaretic acid, Oxynex (Oxynex ST liquid is a mixture of diethylhexyl syringyliden-emalonate and caprylic/capric triglyceride), vitamin A, vitamin E and vitamin C; the diluent is selected from the group consisting of water, alcohol, silicone, oils, and combinations thereof; the chelating agent is selected from the group consisting of ethylene diaminetetraacetic acid (EDTA), EDTA disodium, calcium disodium edetate, EDTA trisodium, citric acid, EDTA tetrasodium and EDTA dipotassium; the viscosity modifier is selected from the group consisting of aluminium starch octenylsuccinate, crosslinked polyacrylate polymers, carboxylic acid polymers, polyacrylamide polymers, acrylic acid/ethyl acrylate copolymers and the carboxyvinyl polymer, and mixtures thereof. In yet another embodiment of the present disclosure, the viscosity modifier is selected from resins as are described in, e.g., U.S. Pat. Nos. 5,288,814 and 5,468,814, and in Amjad et al., Carbomer Resins: Past, Present and Future Cosmetics & Toiletries 107 (1992), pp 81-85. These resins consist essentially of a colloidally water-soluble polyalkenyl polyether

crosslinked polymer of acrylic acid crosslinked with 0.75% to 2.00% of a crosslinking agent such as for example polyallyl sucrose or polyallyl pentaerythritol.
[0047] In an embodiment of the present disclosure, there is provided a formulation comprising: (i) an antibacterial composition comprising: (a) thymol; and (b) berberine, wherein thymol to berberine weight ratio is in the range of 1:0.2 – 1:2; (ii) anti-caking agent; (iii) absorbent; (iv) skin protectant; (v) viscosity modifier; (vi) opacifying agent; (vii) preservative; (viii) skin conditioning agent; (ix) cooling agent; (x) odour enhancer; (xi) hydrophilic polymer; (xii) UV stabilizer; (xiii) pH adjusting agent; (xiv) chelating agent; (xv) deodorant; (xvi) perfumes; (xvii) antimicrobial; (xviii); (xix) antioxidant; (xx) humectant; (xxi) conditioning ingredients; (xxii) propellants; (xxiii) salts; (xxiv) colorants; (xxv) dyes; and (xxvi) diluent.
[0048] In an embodiment of the present disclosure, there is provided a formulation comprising (a) thymol having a weight percentage in the range of 0.00256 – 0.0256% with respect to the formulation;(b) berberine having a weight percentage in the range of 0.00128 – 0.0128% with respect to the formulation; (c) the anti-caking agent having a weight percentage in the range of 60 – 70% with respect to the formulation; (d) the absorbent having a weight percentage in the range of 10 – 15% with respect to the formulation; (e) the skin protectant having a weight percentage in the range of 10 – 15% with respect to the formulation; (f) the viscosity modifier having a weight percentage in the range of 0 – 20% with respect to the formulation; (g) the opacifying agent having a weight percentage in the range of 0 – 10% with respect to the formulation; (h) the preservative having a weight percentage in the range of 0 – 5% with respect to the formulation; (i) the skin conditioning agent having a weight percentage in the range of 0 – 1.5% with respect to the formulation; (j) the cooling agent having a weight percentage in the range of 0 – 2% with respect to the formulation; (k) the odour enhancer having a weight percentage in the range of 0.5 – 3% with respect to the formulation; (l) the perfume having a weight percentage in the range of 0.5 – 30% with respect to the formulation; (m) the antioxidant having a weight percentage in the range of 0.001 –

5% with respect to the formulation; (n) chelating agent having a weight percentage
in the range of 0.001-0.2% with respect to the composition, wherein thymol to
berberine acid weight ratio is in the range of 1:0.2 – 1:2.
[0049] In an embodiment of the present disclosure, there is provided a process for
preparing a composition comprising: (a) thymol; and (b) berberine, wherein thymol
to berberine weight ratio is in the range of 1:0.2 – 1:2, said process comprising the
steps of: (i) obtaining thymol; (ii) obtaining berberine; and (iii) contacting thymol
with berberine to obtain the composition.
[0050] In an embodiment of the present disclosure, there is provided a process for
preparing a composition comprising: (a) thymol; and (b) berberine, wherein thymol
to berberine weight ratio is in the range of 1:0.2 – 1:0.4, said process comprising
the steps of: (i) obtaining thymol; (ii) obtaining berberine; and (iii) contacting
thymol with berberine to obtain the composition.
[0051] In an embodiment of the present disclosure, there is provided a process for
preparing a composition comprising: (a) thymol; and (b) berberine, wherein thymol
to berberine weight ratio is in the range of 1:0.4 – 1:0.7, said process comprising
the steps of: (i) obtaining thymol; (ii) obtaining berberine; and (iii) contacting
thymol with berberine to obtain the composition.
[0052] In an embodiment of the present disclosure, there is provided a process for
preparing a composition comprising: (a) thymol; and (b) berberine, wherein thymol
to berberine weight ratio is in the range of 1:0.9 – 1:1.2, said process comprising
the steps of: (i) obtaining thymol; (ii) obtaining berberine; and (iii) contacting
thymol with berberine to obtain the composition.
[0053] In an embodiment of the present disclosure, there is provided a process for
preparing a composition comprising: (a) thymol; and (b) berberine, wherein thymol
to berberine weight ratio is 1:0.25, said process comprising the steps of: (i)
obtaining thymol; (ii) obtaining berberine; and (iii) contacting thymol with
berberine to obtain the composition.
[0054] In an embodiment of the present disclosure, there is provided a process for
preparing a composition comprising: (a) thymol; and (b) berberine, wherein thymol
to berberine weight ratio is 1:0.5, said process comprising the steps of: (i) obtaining

thymol; (ii) obtaining berberine; and (iii) contacting thymol with berberine to obtain the composition.
[0055] In an embodiment of the present disclosure, there is provided a process for preparing a composition comprising: (a) thymol; and (b) berberine, wherein thymol to berberine weight ratio is 1:1, said process comprising the steps of: (i) obtaining thymol; (ii) obtaining berberine; and (iii) contacting thymol with berberine to obtain the composition.
[0056] In an embodiment of the present disclosure, there is provided a process for preparing a formulation comprising: i) the antibacterial composition comprising: (a) thymol; and (b) berberine, wherein thymol to berberine weight ratio is in the range of 1:0.2 - 1:2; and (ii) at least one cosmetically suitable carrier, said process comprising the steps of: a) contacting the anti caking agent, the absorbent and the skin protectant to obtain a first mixture; b) contacting thymol, berberine, the odour enhancer, the viscosity modifier, the opacifying agent, the preservative, the skin conditioning agent, the cooling agent with the first mixture to obtain a second mixture; and c) processing the second mixture to obtain the formulation. [0057] In an embodiment of the present disclosure, there is provided a process for preparing a formulation as described herein, wherein contacting the anti-caking agent, the absorbent and the skin protectant is carried out at a temperature in the range of 22 - 28°C at a stirring speed in the range of 20 - 40 rpm for a period in the range of 10 - 120 minutes to obtain a first mixture. In another embodiment of the present disclosure, contacting the anti-caking agent, the absorbent and the skin protectant is carried out at a temperature in the range of 24 - 26°C at a stirring speed in the range of 25 - 35 rpm for a period in the range of 30 - 100 minutes to obtain a first mixture.
[0058] In an embodiment of the present disclosure, there is provided a process for preparing a formulation as described herein, wherein contacting thymol, berberine, the odour enhancer, the viscosity modifier, the opacifying agent, the preservative, the skin conditioning agent, the cooling agent with the first mixture is carried out at a temperature in the range of 22 - 28°C at a stirring speed in the range of 20 - 40 rpm for a period in the range of 10 - 120 minutes to obtain the second mixture. In

another embodiment of the present disclosure, wherein contacting thymol,
berberine, the odour enhancer, the viscosity modifier, the opacifying agent, the
preservative, the skin conditioning agent, the cooling agent with the first mixture is
carried out at a temperature in the range of 24 - 26°C at a stirring speed in the range
of 25 - 35 rpm for a period in the range of 30 - 100 minutes to obtain the second
mixture.
[0059] In an embodiment of the present disclosure, there is provided a process for
preparing a formulation as described herein, wherein the processing the second
mixture comprises filtration to obtain the antibacterial formulation. The filtration
can be carried out using a 60-mesh filter.
[0060] In an embodiment of the present disclosure, there is provided a formulation
as described herein, wherein the formulation is dispensed in a form selected from
powders, sprays, emulsion, solution, antiseptics, gels, patches, face washes, body
washes, hand washes, pastes, solid cleansing products such as cleansing bars and
films.
[0061] Although the subject matter has been described in considerable detail with
reference to certain examples and implementations thereof, other implementations
are possible.
EXAMPLES
[0062] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may apply. [0063] The present section will be elaborated by depicting the working examples of the composition comprising thymol and berberine. The examples cover the

synergistic effect of the composition on the MIC, CFU (colony forming units) of S. epidermidis strain ATCC 35984. The checker board assay has been depicted to establish the synergism. The synergism of the present composition has been compared with known substances available for the treatment of miliaria.
Example 1
Determination of minimal inhibitory concentration (MIC) of thymol and berberine against reference strain S. epidermidis (ATCC 35984)
[0064] Method used – Thymol, a phytochemical was procured from Alfa Aesar, UK and berberine, another phytochemical was procured from Alfa Aesar, UK. The MIC of both the above-mentioned phytochemicals was evaluated using broth micro dilution assay in 96 well microtitre plate. One percent inoculum (v/v) of overnight culture of S. epidermidis was added to 200 μL of tryptic soy broth (TSB) with increasing concentrations (1 μg/mL – 512 μg/mL) of thymol and berberine separately. Wells devoid of treatment and wells containing only TSB was deliberated as control and blank, respectively. The assay was performed in triplicate. The plate was incubated at 37°C for 24 h. Then, the optical density (OD) of the test samples was measured spectrophotometrically at 600 nm. MIC is generally determined as minimum concentration at which complete visible growth inhibition is observed. The percentage of growth inhibition was calculated using following formula.
% of growth inhibition = (Control OD - Treated OD / Control OD) * 100 [0065] Results obtained: Figure 1 depicts the MIC of thymol and berberine against S. epidermidis as per the method detailed above. Referring to Figure 1, thymol at 512 μg/mL and berberine at 256 μg/mL showed significant inhibition of growth (> 95%). Hence, MIC of thymol and berberine was determined as 512 μg/mL and 256 μg/mL, respectively.
Example 2
Determination of minimal inhibitory concentration (MIC) of salicylic acid and
zinc oxide against reference strain S. epidermidis (ATCC 35984)

[0066] The Example 1 depicted the MIC of thymol and berberine. In order to compare the effect of the composition of the present disclosure on S. epidermidis with the current anti-miliaria products, the present example has been illustrated with determining the MIC of salicylic acid and zinc oxide. Thus, in the present disclosure, zinc oxide and salicylic acid were used as positive controls. Since, the present invention is focused to substitute the predominantly used aforementioned antibacterial agents and or enhancing their activity in combination with phytochemicals, determination of MIC of zinc oxide and salicylic acid is mandatory.
[0067] Method used: Owing to the insoluble nature of zinc oxide, MIC of zinc oxide (provided by ITC, LSTC) and Salicylic acid (Hi-Media, India) was determined using agar dilution method. Briefly, TSB agar plates supplemented with varying concentrations (1 to 2048 μg/mL) of Zinc oxide and Salicylic acid separately were prepared. Two microliter of overnight culture of S. epidermidis was used to inoculate a spot at center of the agar plates. Plain TSB agar plate and TSB agar plate devoid of treatment was deliberated as blank and control, respectively. Then, the plates were incubated at 37 °C for 24 h. After incubation, the plates were documented using high resolution charge-coupled device (CCD) camera (GelDoc XR+, Bio-Rad).
[0068] Result obtained: Figure 2 depicts the petriplates in which the experiment was performed. Referring to Figure 2, it can be appreciated that zinc oxide and salicylic acid exhibited significant growth inhibition (> 90%) of S. epidermidis at the concentration of 2048 μg/mL. Hence, MIC of zinc oxide and salicylic acid was determined as 2048 μg/mL.
Example 3
Determination of synergism between thymol and berberine, and zinc oxide
and salicylic acid
[0069] As the invention is directed towards the substitution of prevalently used
chemical based antibacterial agents, appraising the synergistic effect of
combination of phytochemicals (thymol + berberine) and combination of chemical

based antibacterial agents (zinc oxide + salicylic acid) is essential. Thus, two-dimensional checkerboard assay was employed to assess the in vitro interactions between thymol + berberine and zinc oxide + salicylic acid. [0070] Method used:
[0071] For thymol and berberine: Initially, the interactions between Thymol and Berberine were evaluated using 96 well microtitre plate. Six different concentrations of each drug candidate were tested. Thymol at MIC (512 μg/mL) was added to the first column of 96 well microtitre plate, which was followed by the addition of two-fold lesser concentration of Thymol to every consecutive column. Thus, the test concentrations of Thymol were added in the range of 512 μg/mL to 0 μg/mL. Similarly, Berberine at MIC (256 μg/mL) was added to the first row, which was diluted two-fold lesser concentration for every successive row. The last test concentration of each drug candidate was set at 0 μg/mL, so as to examine the activity of individual drug candidate at various test concentrations in combination. Thus, the combination of two phytochemicals was accomplished in checkerboard broth micro dilution assay. Appropriate control, negative control and blank were considered. Each combination and independent dilutions were made in triplicate in 96 well microtitre plate as two separate experiments. [0072] For zinc oxide and salicylic acid: Similarly, the in vitro interactions between positive controls Zinc oxide and Salicylic acid were tested using agar dilution method. Totally six different concentrations were considered for each drug candidate. Initially, 36 petriplates were aligned in 6 X 6 arrays (i.e. 6 rows & 6 columns). Similar to broth dilution assay, TSB agar supplemented with Zinc oxide at MIC (2048 μg/mL) was added to the first column, which was followed by addition of two-fold lesser concentration of Zinc oxide to every consecutive column. Thus, the test concentrations of Zinc oxide were added in the range of 2048 μg/mL to 0 μg/mL. In a similar way, Salicylic acid at MIC (2048 μg/mL) was added to the first row, which was followed by the addition of two-fold lesser concentrations to every consecutive row. The last concentration in agar dilution method was also set at 0 μg/mL, so as to examine the activity of individual drug candidate at various test concentrations in combination. Thus, the combination of

Zinc oxide and Salicylic acid was made using agar dilution method. Two microliter
of overnight culture was used to inoculate a spot at the center of the agar plates and
incubated at 37 °C for 24 h. After incubation, the plates were documented using
high resolution CCD camera (GelDoc XR+, Bio-Rad).
[0073] Synergism between Thymol and Berberine was evaluated using the
following formula: (Meletiadis et al., 2010. Antimicrobial Agents and
Chemotherapy, 54, 602-609.)
[0074] FIC index = FICA + FICB
[0075] FICA =MIC of A in combination with B / MIC of A alone
[0076] FICB=MIC of B in combination with A / MIC of B alone
[0077] FIC index ≤ 0.5 ==synergistic
[0078] 0.5 > FIC index < 2 == indifferent
[0079] FIC index > 2 == Antagonistic
[0080] FIC Thymol =0.25
[0081] FIC Berberine = 0.25
[0082] FICIndex = FIC Thymol +FIC Berberine (0.25 + 0.25)
Results obtained for thymol and berberine: Table 1 depicts the synergistic activity
of thymol and berberine.
[0083] Table 1: Checker boar assay for depicting the synergistic activity of
thymol and berberine.


64 μg/mL +
1.25 +
0.75 + 0.5 + 0.375 - -

32 μg/mL +
1.125 +
0.625 + 0.375 +
0.25 - -

16 μg/mL +
1.0625 +
0.5625 - - - -

0 μg/mL + - - - - -
(+), (-) indicate the presence and absence of antibacterial activity, respectively. IC - Inoculum control. IC was found to be (-) for all the combinations. Values indicate the FIC index.
[0084] Referring to table 1, it can be appreciated that synergistic antibacterial activity with more than 90% growth inhibition was found in four different combinations namely: 128 μg/mL (thymol)+64 μg/mL (berberine); 128 μg/mL (thymol)+32 μg/mL (berberine); 64 μg/mL (thymol)+64 μg/mL (berberine); 64 μg/mL (thymol)+32 μg/mL (berberine), and the corresponding FIC indices of aforesaid active combinations are 0.5, 0.375 0.375 and 0.25 respectively. Thus, the combination of thymol and berberine is able to synergistically inhibit the growth of S. epidermidis at three different w/w ratios of thymol and berberine of 1:0.25, 1:0.5, and 1:1.
Results obtained for zinc oxide and salicylic acid:
[0085] Figure 3 depicts the degree of synergism between zinc oxide and salicylic acid using the checkerboard assay. Referring to Figure 3, it can be appreciated that the synergistic antibacterial activity with more than 85% of growth inhibition of S.

epidermidis was observed in 512 μg/mL + 512 μg/mL of Zinc oxide and Salicylic acid combination. The corresponding FIC index is 0.5.
[0086] Moreover, the synergistic concentration of Zinc oxide and Salicylic acid combination was found to be approximately four times more than that of working synergistic concentrations of Thymol and Berberine. Thus, the proficient synergistic antibacterial activity of phytochemical combination than chemical based antibacterial agents is verified. Henceforth, the synergistic phytochemical combination of Thymol and Berberine could be used as an effective substitute for prevalently used antibacterial agents, which are reported to pose certain toxic effects at their effectual concentration.
Example 4
Non-working example pertaining to either of thymol and berberine [0087] Initially, the minimum inhibitory concentration (MIC) of phytochemicals namely Eugenol, Thymol, Berberine and Undecanoic acid was determined at increasing concentrations (1 to 1024 μg/mL) against Staphylococcus epidermidis (ATCC 35984) using microbroth dilution method as described in Example 1. Eugenol at 1024 μg/mL, Thymol at 512 μg/mL, Berberine at 256 μg/mL and Undecanoic acid at 256 μg/mL showed significant inhibition of growth (> 95%). Hence, MIC of Eugenol, Thymol, Berberine and Undecanoic acid was determined as 1024 μg/mL, 512 μg/mL, 256 μg/mL and 256 μg/mL, respectively. The non-working examples as depicted here exemplifies the experimental effort put in to arrive at a working composition comprising thymol and berberine. [0088] Method used: Further, two-dimensional checkerboard assay was employed to assess the in vitro interactions between Eugenol + Thymol, and Berberine + Undecanoic acid. Initially, the interactions between Eugenol and Thymol were evaluated using 96 well microtitre plate. Six different concentrations of each drug candidate were tested. Eugenol at MIC (1024 μg/mL) was added to the first column of 96 well microtitre plate, which was followed by the addition of two-fold lesser concentration of Eugenol for every consecutive column. Thus, the test concentrations of Eugenol were added in the range of 1024 μg/mL to 0 μg/mL. Similarly, Thymol at MIC (512 μg/mL) was added to the first row, which was

diluted two-fold lesser concentration for every successive row. The last test concentration of each drug candidate was set at 0 μg/mL, so as to examine the activity of individual drug candidate at various test concentrations in combination. Thus, the combination of two phytochemicals was accomplished in checkerboard broth micro dilution assay. Appropriate control, negative control and blank were considered. Each combination and independent dilutions were made in triplicate in 96 well microtitre plate as two separate experiments. The in vitro interactions between Berberine + Undecanoic acid were also evaluated in a similar fashion. [0089] The In vitro interactions between phytochemicals were evaluated using the following formula mentioned in Example 3
[0090] Results obtained for the combination of eugenol and thymol: Table 2 depicts the checkerboard assay for said combination. Table 2: Checkerboard assay

Concentration of Eugenol
1024 μg/mL 512 μg/mL 256 μg/mL 128 μg/mL 64 μg/mL 0 μg/mL

512 μg/mL +
2 .0 +
1.5 +
1.25 +
1.125 +
1.0625 +

256 μg/mL +
1.5 +
1.0 +
0.75 -
0.625 -
0.5625 -

128 μg/mL +
1.25 +
0.75 -
0.5 -
0.375 - -

64 μg/mL +
1.125 +
0.625 -
0.375 - - -

32 μg/mL +
1.0625 -
0.5625 - - - -

0 μg/mL + - - - - -
(+), (-) indicate the presence and absence of antibacterial activity, respectively. IC -
Inoculum control. IC was (-) for all the combinations. Values indicate the fractional
inhibitory concentration (FIC) index.
Results obtained for the combination of berberine and undecanoic acid: Table 3
depicts the checkerboard assay for said combination.
Table 3: Checkerboard assay

Concentration of Berberine
256 μg/mL 128 μg/mL 64 μg/mL 32 μg/mL 16 μg/mL 0 μg/mL

256 μg/mL + + + + + +
2 .0 1.5 1.25 1.125 1.0625

128 μg/mL + + + - - -
1.5 1.0 0.75 0.625 0.5625
64 μg/mL + + - - - -
1.25 0.75 0.5 0.375

32 μg/mL + - - - - -
1.125 0.625 0.375

16 μg/mL + - - - - -

1.0625 0.5625

0 μg/mL + - - - - -
(+), (-) indicate the presence and absence of antibacterial activity, respectively. IC -Inoculum control. IC was observed (-) for all the combinations. Values indicate the FIC index.
[0091] Referring to Table 2 and Table 3, it can be appreciated that neither the combination of eugenol and thymol nor the combination of berberine and undecanoic acid is showing a synergistic inhibition of S. epidermidis at any concentration. Thus, it is proved that a mere combination of any antibacterial active or any phytochemical active fails to exhibit a synergistic inhibition of the pathogen.
Example 5
Determination of synergistic inhibition effect of a combination of thymol and berberine by studying colony forming units (CFU) of S. epidermidis [0092] The antibacterial activity of synergistic combinations of Thymol and Berberine was further confirmed using colony forming unit (CFU) assay. [0093] Method used: Working synergistic combinations of Thymol and Berberine (128 μg/mL+64 μg/mL; 128 μg/mL+32 μg/mL; 64 μg/mL+64 μg/mL; 64 μg/mL+32 μg/mL) and individual drug candidates at synergistic combinations (128 μg/mL, 64 μg/mL, 32 μg/mL) were added separately to the tubes containing 1 mL of TSB. Tubes without any treatment and tubes containing TSB alone were deliberated as control and blank, respectively. All tubes were inoculated with 1% v/v of overnight culture of S. epidermidis and incubated at 37°C for 24 h in shaker incubator (160 rpm). After incubation, the cells were harvested by centrifugation (8000 rpm for 10 min) and subsequently suspended in 1 mL of phosphate buffered saline (PBS). The cell suspensions were serially diluted and 100 μL of 10-6 dilution of each sample was spread over the TSB agar plates. TSB agar plate spread with 100 μL of PBS was considered as blank. The plates were incubated

overnight at 37°C for 24 h. Then, the number of colonies in each plate was counted manually and CFU/mL was calculated. The log reduction was calculated using the following formula
Log reduction = Log (CFU/mLControl) – Log (CFU/mLTreated) [0094] Results obtained: Figure 4 depicts the representative images of the petriplates of CFU assay. Figure 5 depicts the log reduction of S. epidermidis growth upon treatment with thymol and berberine in isolation and in different combinations. The CFU assay further affirmed that the synergistic combinations of T (thymol)+B (berberine) potentially inhibited the growth of S. epidermidis cells than that of the individual T and B (Figure 1 and 4). In addition, the log reduction of S. epidermidis growth upon treatment with individual T and B and their combinations was calculated and depicted as a bar chart (Figure 5). The result reveals that the synergistic combinations of T+B exhibited more than one log reduction of S. epidermidis growth (~ 99 % of growth inhibition) when compared to that of individual T and B, which showed less than one log reduction. Altogether, the obtained results verified the anti-miliaria potential of synergistic T+B combinations than that of their individual effect.
Example 6
Formulation comprising thymol and berberine
[0095] The present example depicts the formulation comprising thymol and berberine, wherein the formulation would comprise carriers as disclosed previously in the present disclosure.
[0096] Table 4 depicts the ingredients used in preparing the formulation (anti-miliaria or prickly heat powder preparation) as disclosed in the present disclosure. The present invention consists of anti-caking agent such as talc in the range of 60%-70% or Q.S. to 100%. However, the said anti-caking agent or fell enhancer is not limited to talc and also includes other known anti-caking agents. [0097] The hydrophobic benefit agent of the present invention may also include one or more perfumes. The one or more perfumes may be selected from any perfume or perfume chemical suitable for topical application to the skin and/or hair and suitable for use in personal care compositions. The concentration of the

perfume in the personal care composition should be effective to provide the desired
aroma including, but not limited to, unscented. Generally, the concentration of the
scented primary perfume is from about 0.5% to about 30% with respect to the
formulation.
Table 4: Anti-miliaria or prickly heat powder formulation of the present disclosure

S.No. Name of ingredient % weight Functionality
1. Ta l c 60 – 70 or Q.S. to 100 Anti-caking agent
2. Maize starch or corn starch 10 – 15 Absorbent
3. Zinc oxide 10 – 15 Skin protectant
4. Aluminium starch octenylsuccinate 0 – 20 Viscosity modifier
5. Titanium dioxide 0 – 10 Opacifying agent
6. Boric acid 0 – 5 Preservative
7. Salicylic acid 0 – 1.5 skin conditioning agent
8. Menthol 0 – 2 Cooling agent
9. Thymol 0.00256 – 0.0256 Active
10. Berberine 0.00128 – 0.0128 Active
11. Fragrance 0.5 – 3 Odour enhancer
Method for preparing the formulation
[0098] The disclosure has been illustrated with a working example, which is
intended to explain the working template for the commercial application of
synergistic combination of T and B used in the present disclosure. However, this
working example does not imply any limitations on the scope of the present
disclosure.

[0099] The ingredients of anti-miliaria or prickly heat powder comprising synergistic combination of T+B is mentioned in Table 4. The method of preparation contains the following steps:
(a) Talc, maize or corn starch and zinc oxide were continuously mixed in a blender at 20-40 rpm at a temperature in the range of 22 - 28°C for a time in the range of 10 - 120 minutes until a homogenous mixture - a first mixture is obtained; (b) synergistic combination of T+B, odour enhancer such as fragrances and other optional ingredients such as aluminium starch octenylsuccinate, titanium dioxide, boric acid, salicylic acid and menthol were added to the first mixture at a temperature in the range of 22 - 28°C at a stirring speed in the range of 20 - 40 rpm for a period in the range of 10 - 120 minutes to obtain a second mixture; (c) successively, the second mixture was passed through 60 mesh size.
Advantages of the present disclosure
[00100] The present disclosure discloses a composition comprising thymol and berberine for inhibiting the growth of S. epidermidis thereby providing a solution to the problem of miliaria. The composition comprises thymol and berberine in a w/w ratio in a range of 1:0.2 to 1:2. The present composition has also been formulated which can be made available commercially in the form of powders, sprays, emulsion, solution, antiseptics, gels, patches, face washes, body washes, hand washes, pastes, solid cleansing products such as cleansing bars and films. The present composition is a promising alternate to synthetic drugs and antibiotics without displaying any side effects associated with the synthetic drugs of antibiotics. As the components used in the present composition are naturally occurring phytochemicals, hence the possibility of attaining resistance simultaneously towards both the components is meager. Thus, the present composition also provides answer to the drug resistance problem that has engaged the entire research community towards finding alternative.

I/We Claim:
1. An antibacterial composition comprising:
a) thymol; and
b) berberine,
wherein thymol to berberine weight ratio is in the range of 1:0.2 – 1:2.
2. The antibacterial composition as claimed in claim 1, wherein thymol to berberine weight ratio is 1:0.25.
3. The antibacterial composition as claimed in claim 1, wherein thymol to berberine weight ratio is 1:0.5.
4. The antibacterial composition as claimed in claim 1, wherein thymol to berberine weight ratio is 1:1.
5. An antibacterial formulation comprising:

a) an antibacterial composition as claimed in any of the claims 1-4; and
b) at least one cosmetically suitable carrier.

6. The antibacterial formulation as claimed in claim 5, wherein the at least one cosmetically suitable carrier is selected from the group consisting of anti-caking agent, absorbent, skin protectant, viscosity modifier, opacifying agent, preservative, skin conditioning agent, cooling agent, odour enhancer, hydrophilic polymer, UV stabilizer, pH adjusting agent, chelating agent, deodorant, perfumes, antimicrobial, antioxidant, humectant, conditioning ingredients, propellants, salts, colorants, dyes, diluent, and combinations thereof.
7. A process for preparing the composition as claimed in claim 1, said process comprising the steps of: a) obtaining thymol; b) obtaining berberine; and c) contacting thymol with berberine to obtain the composition.
8. A process for preparing the formulation as claimed in any of the claims 5-6, said process comprising the steps of: a) contacting the anti caking agent, the absorbent and the skin protectant to obtain a first mixture; b) contacting thymol, berberine, the odour enhancer, the viscosity modifier, the opacifying agent, the preservative, the skin conditioning agent, the cooling

agent with the first mixture to obtain a second mixture; and c) processing the second mixture to obtain the formulation.
9. The process as claimed in claim 8, wherein contacting the anti caking agent, the absorbent and the skin protectant is carried out at a temperature in the range of 22 - 28°C at a stirring speed in the range of 20 - 40 rpm for a period in the range of 10 - 120 minutes to obtain a first mixture.
10. The process as claimed in claim 8, wherein contacting thymol, berberine, the odour enhancer, the viscosity modifier, the opacifying agent, the preservative, the skin conditioning agent, the cooling agent with the first mixture is carried out at a temperature in the range of 22 - 28°C at a stirring speed in the range of 20 - 40 rpm for a period in the range of 10 - 120 minutes to obtain the second mixture.
11. The formulation as claimed in any of the claims 5-6, wherein the formulation is dispensed in a form selected from powders, gels, sprays, emulsion, solution, antiseptics, patches, face washes, body washes, hand washes, pastes, solid cleansing products such as cleansing bars and films.