Field of the invention
The present invention relates to a process for identifying the anti dandruff actives which
will target persistent dandruff by using a drug resistant strains of Malassezia such as M
glohosa ITC5-1 and screen for potent anti-dandruff active(s) (such as hinokitiol and
ZPTO combinations) using the identified drug resistant strain(s) of Malassezia species.
The present invention further provides a composition comprising anti-dandruff effective
actives.
Background of the invention and prior arts
Nearly 50% of the global population at any given time suffers from dandruff. Although
it is not life-threatening, dandruff is socially associated with poor hygiene and can lead
to low self-esteem. Anti-dandruff formulations typically target the scalp fungus
Malassezia. Although many anti-fungal shampoos- medicated and cosmetic -have been
developed over the last 3-4 decades, there is room for more satisfactory solutions for the
consumer. It has been seen that in certain types of dandruff, anti-dandruff shampoos do
not completely clear the dandruff and frequent recurrence is seen.
This may be due to several reasons, including susceptibility of Malassezia to active
ingredients in anti-dandruff formulations. In particular, most of the commercial anti-
dandruff shampoos contain similar active ingredients. Thus, Malassezia have been
repeatedly exposed to these agents for over 2-3 decades and may have acquired
resistance to the same.
Conventionally, M. furfur and other standard laboratory strains have been used to screen
for actives and determine their effective concentrations. While M. globosa and M.
restricta are the species most commonly implicated in dandruff, the prevalence of these
Malassezia species in dandruff varies geographically and different sub-species/strains
vary in their susceptibility. Therefore, use of laboratory strains for screening of actives
that fight persistent dandruff may be misleading.

Hence there is a need in the market to identify potential active(s) to combat against
Malassezia species that is especially prevalent in Indian population which is resistant to
conventionally known cosmetic anti dandruff actives.
US 5658584 (herein referred to as '584) provides an antimicrobial composition
comprising hinokitiol, which is combined with other safe ingredients, in a specific ratio
in order to increase the antimicrobial activity of hinokitiol, in order to provide a broad
spectrum of activity against microorganisms including mold and Pseudomonas which
are not sufficiently inhibited by hinokitiol only, and in order to use hinokitiol for
toiletries and household articles for antiblastic and antifungal purposes. In order to
attain the objects of the present invention, carboxylic acids which are safe for humans
are used, and the antimicrobial activity of compositions comprising hinokitiol and the
carboxylic acids was investigated. Citronellic acid among several carboxylic acids was
selected, which has been conventionally utilized as a material for medicines and
fragrances and has been confirmed to be safe for human beings. As a result, it has been
found that a combination of citronellic acid in specific ratios increased the antimicrobial
activity of hinokitiol and enlarged the spectrum of antimicrobial effectiveness.
Specifically, the invention provides an antimicrobial composition comprising hinokitiol
and citronellic acid in a ratio ranging from about 1:1 to 3:1 by weight.
US 6310255 (herein referred to as '255) provides a process for producing hinokitiol by
synthesizing 1-isopropylcyclopentadiene from easily available cyclopentadiene, adding
a dichloroketene to this 1-isopropylcyclopentadiene, and subjecting the adduct to
solvolysis, wherein 1-isopropylcyclopentadiene is produced easily with high selectivity
at low cost without extreme nonaqueous conditions. Hinokitiol produced in '255 posses
not only antimicrobial and antifungal effect on a wide spectrum of bacteria and fungi
but also cell-activating effect, inhibitory effect on tyrosinase activity, inhibitory effect
on ethylene production in plants, etc. It is effective as an antimicrobial and antifungal
agent when incorporated into medicines, cosmetics, hair tonics, shampoos or soaps, and
it is incorporated also into freshness-maintaining films, antimicrobial coating materials,
etc.

US 2012/0149055 (herein referred to as '055) teaches a paper strip impregnated with
antibiotics according to a graded scale that has the characteristic of gradually releasing
the antibiotics once positioned in the microbial culture medium, so as to facilitate its
repositioning on the microbial culture medium. '055 concerns the determination of the
Minimum Inhibitory Concentration (MIC) using a paper strip impregnated with a
predetermined concentration gradient of the antimicrobial agent according to the known
technique, with a view to simplifying the execution of the relative test due to the fact
that the said strip of paper is thicker and permeable to air and possesses the
characteristic of releasing the antibiotics very slowly once positioned on the microbial
culture medium.
Surprisingly, the present invention details a screening tool for identifying novel anti-
Malassezia actives for resistant dandruff. This tool employs a strain of Malassezia
globosa isolated from the Indian geographical context (strain no. ITC5-1), which shows
resistance to common anti-dandruff actives such as ZPTO, azoles. By comparing the
efficacy of an active against this strain versus the standard laboratory strain, more
effective anti-fungal molecules for persistent dandruff can be identified. Figures 8 and 9
compare the efficacy of ZPTO and ZPTO/hinokitiol combinations against resistant
ITC5-1 strain and standard laboratory M. globosa strain. The combination of 0.5% each
of ZPTO and hinokitiol is as effective or more against ITC5-1 as is 0.5% of ZPTO alone
against standard M. globosa. This indicates that strains in the Indian population, such as
ITC5-1 are different in their susceptibility to anti-fungal agents from standard strains. A
more potent active combination is identified when using such resistant strains for
screening.
The present invention relates to a process to detect resistant strains of Malassezia
species and using the same to develop more effective anti-fungal actives for persistent
dandruff.
Object of the invention

It is an object of the present invention to overcome the drawbacks of the prior arts.
It is an object of the present invention to provide a process for identifying drug resistant
anti dandruff (AD) strain, e.g. M. globosa (ITC5-1).
It is yet another object of the present invention to provide a method of screening
potential anti-dandruff actives (e.g. Hinokitiol, Voriconazole) using drug resistant
Malassezia strains e.g. M. globosa (ITC5-1).
It is another object of the present invention to provide an anti dandruff personal care
composition comprising Hinokitiol (also known as beta Thujaplicin) in combination
with conventional anti dandruff actives such as ZPTO at the range of 0.1 wt% to 2 wt%
along with other conventional carriers.
Summary of the invention
The present invention relates to a screening process for identifying effective anti-
dandruff actives using drug resistant Malassezia strain comprising steps of
a. collecting the microbial samples from scalp with appropriate means;
b. isolating and purifying the species;
c. determining minimum inhibitory concentration (MIC) of drug for said
isolated species using active gradient strip;
d. identifying the drug resistance by comparing MIC to that of the standard
laboratory strain; and
e. using the identified drug resistant strain as a screening tool to identify
effective anti-dandruff actives.
The present invention further relates to a personal care composition comprising
a. anti dandruff actives;

b. ZPTO; and
c. other excipients
wherein said personal care composition helps combat Malassezia strains.
Brief Description of the Accompanying Figures
Figure-1 A and B: The E-test result obtained with commercially available strips
Figure-2 A and B: Illustrates the paper gradient method of the present invention.
Figure-3 A and B: Illustrates the MIC of ZPTO for M. globosa CBS 7966 and M. globosa
ITC5-1.
Figure-4 A and B: Illustrates the MIC of Ketoconazole for M. globosa CBS 7966 and M.
globosa ITC5-1.
Figure-5: Various concentrations of Hinokitiol tested against M. globosa CBS 7966 by
Zone of Inhibition method.
Figure-6: Various concentrations of Hinokitiol tested Against M. globosa ITC5-1 by
Zone of Inhibition method.
Figure-7 A and B: Illustrates the effect of hinokitiol on M. globosa CBS 7966 and M.
globosa ITC5-1 by the paper gradient method.
Figure 8: Hinokitiol and Zinc pyrithione combinations tested against M. globosa (CBS
7966) by Zone of Inhibition method.
Figure 9: Hinokitiol and Zinc pyrithione combinations tested against M. globosa (ITC5-
1) by Zone of Inhibition method.

Figure 10: Various concentrations of Hinokitiol and Lemon Grass Oil (LGO) tested against
M. furfur by Zone of Inhibition method.
Figure 11: Combination of Ketoconazole + Hinokitiol against M. globosa CBS 7966
and M. globosa ITC5-1 by Zone of Inhibition method.
Figure 12: Combination of 0.07% ZPTO+1.25% Hinokitiol tested against M. globosa
(CBS 7966) and M. globosa (ITC5-1).
Detailed Description of the Invention
The present invention provides a method for the identification of the drug resistant
Malassezia strains comprising the steps of
- collecting the microbial samples from scalp with appropriate means
isolate and purify the Malassezia species
- determine minimum inhibitory concentration (MIC) of drug for isolated
Malassezia species using active gradient strip
identify the drug resistance by comparing the MIC of isolates with that of
standard laboratory strains for a given anti-dandruff active (MIC difference
of greater than 4-fold is defined as resistance).
Further, upon identifying the drug-resistant Malassezia strains, potent anti-dandruff
agents are identified using the said drug-resistant strains of Malassezia species. The
present invention provides a process for identifying the anti-Malassezia actives against
resistance strains comprising the steps of:
a) Filter paper strips are cut 51mm of height and 4 mm of width and internally
divided into 7 squares and each square corresponds to 7 mm of height and
4mm of width;
b) Using appropriate means approximately 2.5 micro liters of serial dilutions of
actives are added to form a gradient, higher to lower concentrations;

c) The prepared strip is placed onto a plate spread with M. globosa inoculum
adjusted to 1 OD at 600 nm and the plates are incubated at 30 deg C for
about 7 days;
d) The elliptical zone formed by lack of organism growth is observed and MIC
determinate by the concentration at the point at which the zone meets the
filter strip.
The present invention also relates to an anti-dandruff composition comprising hinokitiol
in an amount of 0.001 wt% to 1 wt%. in combination with ZPTO in an amount of 0.001
wt% to 2 wt% along with other excipients.
The shampoo composition further comprises supplementary agent selected from a group
comprising a surfactant, emulsifiers, a thickening agent, a preservative, cationic
conditioning agents, a humectants, fragrance, moisturizers and other ingredients in any
combinations thereof.
Suitable anionic surfactants of the present invention include, but are not limited to, the
alkyl sulphates, alkyl ether sulphates, alkarylsulphonates, alkanoylisethionates, alkyl
succinates, alkyl sulphosuccinates, N-alkoylsarcosinates, alkyl phosphates, alkyl ether
phosphates, alkyl ether carboxylates, and alpha-olefin sulphonates, especially their
sodium, magnesium ammonium and mono-, di- and triethanolarnine salts. The alkyl and
acyl groups generally contain from 8 to 18 carbon atoms and may be unsaturated. The
alkyl ether sulphates, alkyl ether phosphates and alkyl ether carboxylates may contain
from 1 to 10 ethylene oxide or propylene oxide units per molecule, and preferably
contain 2 to 3 ethylene oxide units per molecule. Examples of suitable anionic surfactants
of the present invention include, but are not limited to, sodium oleyl succinate,
ammonium lauryl sulphosuccinate, ammonium lauryl sulphate, sodium
dodecylbenzenesulphonate, triethanolaminedodecylbenzenesulphonate, sodium
cocoylisethionate, sodium law oylisethionate and sodium N-lauryl sarcosinate. The most
preferred anionic surfactants are sodium lauryl sulphate, triethanolarnine lauryl sulphate,
triethanolaminemonolauryl phosphate, sodium lauryl ether sulphate 1E0, 2E0 and 3E0,
ammonium lauryl sulphate and ammonium lauryl ether sulphate 1E0, 2E0 and 3E0.

Suitable cationic surfactants of the present invention include, but are not limited to,
quaternary ammonium compounds and esterquats, more particularly quaternized fatty acid
trialkanolamine ester salts, aliphatic mono, di and polyamines derived from fatty and rosin
acids, amine oxides, ethoxylated alkyl amines and imidazolines.
The cationic surfactants are present in an amount ranging from 1 to 30% w/w.
The nonionic surfactants of the present invention suitable for use in compositions of the
invention may include condensation products of aliphatic (C8-C18) primary or secondary
linear or branched chain alcohols or phenols with alkylene oxides, usually ethylene oxide
and generally having from 6 to 30 ethylene oxide groups. Other suitable nonionics
include mono- or di-alkyl alkanolamides. Example includes coco mono- or di-
ethanolamide and coco mono-isopropanolamide.
The amphoteric and zwitterionic surfactants suitable for use in compositions of the
invention may include alkyl amine oxides, alkyl betaines, alkyl amidopropylbetaines,
alkyl sulphobetaines (sultaines), alkyl glycinates, alkyl carboxyglycinates, alkyl
amphopropionates, alkylamphoglycinates alkyl amidopropylhydroxysultaines, acyl
taurates and acyl glutamates, wherein the alkyl and acyl groups have from 8 to 19 carbon
atoms. Examples include lauryl amine oxide, cocodimethylsulphopropylbetaine and
preferably lauryl betaine, cocamidopropylbetaine and sodium cocamphopropionate.
The amphoteric and zwitterionic surfactants are present in an amount ranging from 1 to
30% w/w.
Suitable emulsifiers are well known in the art and include anionic and nonionic
surfactants. Examples of anionic surfactants used as emulsifiers for the silicone particles
are alkylarylsulphonates, e.g., sodium dodecylbenzenesulphonate, alkyl sulphates e.g.,
sodium lauryl sulphate, alkyl ether sulphates, e.g., sodium lauryl ether sulphate nEO,
where n is from 1 to 20 alkylphenol ether sulphates, e.g., octylphenol ether sulphate nEO

where n is from 1 to 20, and sulphosuccinates, e.g., sodium dioctylsulphosuccinate.
Conditioners that may be added to the shampoo composition in the form of organic cationic
conditioning agents for the purpose of providing more hair grooming if deemed necessary
such as cationic conditioning agents that may include homopolymersof dimethyldiallyl
ammonium chloride; copolymers of acrylamide and dimethyldiallylammonium chloride;
homopolymers or copolymers derived from acrylic acid or methacrylic acid which
contain cationic nitrogen functional groups attached to the polymer by ester or amide
linkages and copolymers of vinylpyrrolidone and acrylic acid esters with quaternary
nitrogen functionality. Specific materials include Polyquatenium 11 other possible
candidates include Polyquatenium 8 and 23 provided that clarity can be maintained.
The conditioners are present in an amount ranging from 0.1 to 10% w/w.
Water is used as an aqueous carrier in the present invention. It helps in producing
optimum viscosity to the product. The hydrating shampoo compositions of the present
invention are aqueous systems which comprise from about 27% to about 94.5%),
preferably from about 55% to about 85%, more preferably from about 60% to about 75%, of
water by weight of the shampoo composition.
Such conventional optional ingredients are well known to those skilled in the art and may be
selected from the group comprising but not restricted to preservatives such as benzyl
alcohol, methyl paraben, propyl paraben, imidazolidinyl urea,
methylchloroisothiazolinone and methylisothiazolinone; thickeners and viscosity
modifiers such as coconut ethanolamide, sodium chloride, ammonium chloride, sodium
sulfate, carboxymethyl cellulose, methyl cellulose, polyvinylalcohol, and ethyl alcohol;
perfumes; dyes, sequestering agents such as disodium ethylenediaminetetraacetate; pH
adjusting agents such as citric acid, succinic acid, phosphoric acid, sodium hydroxide,
sodium carbonate, etc. and a foam booster such as lauricdiethanolamide.
The conventional ingredients like preservatives, thickeners, viscosity modifiers, perfumes,

dyes, sequestering agents, pH adjusting agents and foam booster are present in an amount
ranging from 0.001 to 10% w/w.
Other cationic conditioning agents useful in the shampoos of the present invention
include cationic polyamide polymers such as the low molecular weight
adipicacid/diethylene-triamine polyamide and the copolymers of vinylpyrrolidone
and dimethylaminoethyl methacrylate quaternised with dimethyl sulphate (Gafquat
755, GAF Corporation); the graft cationic copolymer containing N-
vinylpyrrolidone, dimethyaminoethyl methacrylate and polyethylene glycol; the
mineral acid salts of the amino alkyl esters of homo- and copolymers of unsaturated
carboxylic acids having from 3 to 5 carbon atoms.
The high molecular weight polymers sold under the trade mark Merquat by Merck
&Co. Inc. are cationic polymers which are also suitable for use in the present
shampoos. Representative ones are Merquat 100, a highly charged cationic
dimethyldiallylammonium chloride homopolymer, and Merquat 550, a highly charged
cationic copolymer prepared with dimethyldiallylammonium chloride and acrylamide.
These materials are designated in the CFTA dictionary as Quaternium-40 and
Quaternium-41, respectively.
The moisturizing system disclosed in the instant invention comprises at least one amino
acid, derivative thereof, at least one vitamin and derivative thereof. The at least one
amino acid is selected from the group comprising alanine, cysteine, aspartic acid,
glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine,
asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine,
valine, tryptophan, tyrosine and combinations thereof. In a preferred embodiment of the
invention the moisturizing system comprises glycine.
Such fillers and carriers may be selected from a group comprising but not limited to
starches such as corn starch, wheat starch, maize starch, fumed silica, talc, clays
,Kaolin, manganese containing clays, mica, silica, polyamide powder, poly-beta-alanine

powder and polyethylene powder, tetrafluoroethylenepolymer, lauroyllysine, starch,
boron nitride, elastomeric polyorganosiloxane particles, precipitated calcium carbonate,
magnesium carbonate and magnesium hydrocarbonate, hydroxyapatite, zinc,
magnesium or lithium stearate, zinc laurate or magnesium myristate. The fillers may be
present in a proportion of from 0 to 15% by weight, typically 0.01% to 10% by weight
relative to the total weight of the composition.
Emollients that may be used in the present invention include but are not limited to
silicone oils and modifications thereof such as linear and cyclic polydimethylsiloxanes;
amino, alkyl, alkylaryl, and aryl silicone oils; fats and oils including natural fats and oils
such as jojoba, soybean, sunflower, rice bran, avocado, almond, olive, sesame, persic,
castor, coconut, mink oils; cacao fat; beef tallow, lard; hardened oils obtained by
hydrogenating the aforementioned oils; and synthetic mono, di and triglycerides such as
myristic acid glyceride and 2-ethylhexanoic acid glyceride; waxes such as carnauba,
spermaceti, beeswax, lanolin, and derivatives thereof; hydrophobic plant extracts;
hydrocarbons such as liquid paraffin, petrolatum, microcrystalline wax, ceresin,
squalene, pristan and mineral oil; higher fatty acids such as lauric, myristic, palmitic,
stearic, behenic, oleic, linoleic, linolenic, lanolic, isostearic, arachidonic and poly
unsaturated fatty acids (PUFA); higher alcohols such as lauryl, cetyl, stearyl, oleyl,
behenyl, cholesterol and 2-hexydecanol alcohol; esters such as cetyloctanoate, myristyl
lactate, cetyl lactate, isopropyl myristate, myristylmyristate, isopropyl palmitate,
isopropyl adipate, butyl stearate, decyloleate, cholesterol isostearate, glycerol
monostearate, glycerol distearate, glycerol tristearate, alkyl lactate, alkyl citrate and
alkyl tartrate; essential oils and extracts thereof such as mentha, jasmine, camphor,
white cedar, bitter orange peel, ryu, turpentine, cinnamon, bergamot, citrus unshiu,
calamus, pine, lavender, bay, clove, hiba, eucalyptus, lemon, starflower, thyme,
peppermint, rose, sage, sesame, ginger, basil, juniper, lemon grass, rosemary, rosewood,
avocado, grape, grapeseed, myrrh, cucumber, watercress, calendula, elder flower,
geranium, linden blossom, amaranth, seaweed, ginko, ginseng, carrot, guarana, tea tree,
jojoba, comfrey, oatmeal, cocoa, neroli, vanilla, green tea, penny royal, aloe vera,
menthol, cineole, eugenol, citral, citronelle, borneol, linalool, geraniol, evening

primrose, camphor, thymol, spirantol, penene, limonene and terpenoid oils; and
mixtures of any of the foregoing components, and the like. Advantageously emollients
may be used from about 5, 10 or 15% by wt. to about 20, 25, 30, 35, 40, 45% by wt.
Essential oils and extracts that may be used in the present invention include but are not
limited to mentha, jasmine, camphor, white cedar, bitter orange peel, ryu, turpentine,
cinnamon, bergamot, citrus unshiu, calamus, pine, lavender, bay, clove, hiba,
eucalyptus, lemon, starflower, thyme, peppermint, rose, sage, sesame, ginger, basil,
juniper, lemon grass, rosemary, rosewood, avocado, grape, grapeseed, myrrh, cucumber,
watercress, calendula, elder flower, geranium, linden blossom, amaranth, seaweed,
ginko, ginseng, carrot, guarana, tea tree, jojoba, comfrey, oatmeal, cocoa, neroli, vanilla,
green tea, penny royal, aloe vera, menthol, cineole, eugenol, citral, citronelle, borneol,
linalool, geraniol, evening primrose, camphor, thymol, spirantol, penene, limonene,
rose, lemon grass, peach, honey, almond oil, olive oil, shea butter, olive butter,
bearberry, black currant, rosemary, blue lotus, white lily, and terpenoid oils.
The composition of the present invention may utilize a fragrance composition
comprising a blend of essential oils and synthetic aroma compounds. The blend is often
diluted with a carrier like propylene glycol, vegetable oil, or mineral oil. Some
examples of synthetic aroma compound that are suitable for temporary dye
compositions of the present invention include, but are not limited to benzaldehyde,
citral, vanillin, ethyl acetate, fructone, octyl acetate, pentylbutanoate, pentylpentanoate,
methyl salicylate, isoamyl acetate, limonene, citronellol, and mixtures thereof.
Preferably, the fragrance containing the essential oil is present in the composition of the
invention in an amount between approximately 0.1% to approximately 2% by weight.
The hinokitiol and ZPTO in a ratio of about Hinokitiol preferably at 0.001 to 1% by wt,
more preferably at 0.01 to 0.5% and most preferably 0.1 to 0.25% by wt, ZPTO
preferably at 0.001 to 2% by wt, more preferably at 0.01 to 1% and most preferably 0.1
to 0.5%) by wt. is found to be most effective in the antimicrobial activity.

According to Figure 3A and 3B of the present invention, it is demonstrated that ZPTO
inhibits growth of M globosa standard laboratory strain (CBS7966) more effectively (as
observed by area of the clear zone) than M. globosa (ITC 5-1) thereby indicating that M
globosa (ITC 5-1) is resistant to ZPTO. Further according to Figure 4A and 4B of the
present invention it is demonstrated that ketoconazole inhibits growth of M. globosa
standard laboratory strain (CBS7966) more effectively (as observed by area of the
clear zone) than M. globosa (ITC 5-1) thereby indicating that M. globosa (ITC 5-1) is
resistant to ketoconazole. The strain of M. globosa (ITC 5-1) which is resistant to
ketoconazole is deposited at --, dated with an
accession number of .
Further, according to the present invention, there is provided a technique for detecting
the effective active using the above identified resistant strain by paper gradient
technique. It is stated that the commercial methods available for estimating the
minimum inhibitory concentration (MIC) are prone to false-positive results (Figure 1A
and IB). Thus the present invention provides a simple, reliable method to determine
actives effective against drug resistant Malassezia species without any false-negative
positives (Figure 2A and 2B).
According to Figure 5 and 6, Hinokitiol shows equal anti fungal effect against M.
globosa (CBS 7966) and M. globosa (ITC5-1) resistant strain in initial screening by
ZOI. According to Figure 7 A, B, using the above paper gradient method of determining
MIC, it is confirmed that the Hinokitiol is an effective active against the resistant ITC5-
1 strain, compared to the standard laboratory strain (CBS 7966) Figure 7A and 7B of the
present invention demonstrates that hinokitiol inhibits growth of M. globosa standard
strain and M. globosa ITC 5-1 at the same active concentration indicating that there is
no resistance towards hinokitiol. Thus the strain such as M. globosa (ITC5-1), resistant
to common antidandruff actives such as ZPTO or ketoconazole, demonstrates no
resistance to hinokitiol. Further, M. globosa (ITC 5-1) strain is effective as a screening
tool for identifying the actives for prevention of dandruff conditions.

According to Figures 8 and 9, out of various Zinc pyrithione and Hinokitiol
combinations tested, combination of 1% ZPTO + 1% Hinokitiol, 0.5% ZPTO + 0.5%
Hinokitiol and 0.25% ZPTO + 0.25% Hinokitiol combination provided better activity
against resistant M. globosa ( ITC5-1) than ZPTO alone. According to figure 12,
combination of 0.07% ZPTO + 1.25% Hinokitiol combination does not demonstrate any
additive/synergism compared to individual ZPTO, against STD M. globosa (CBS 7966).
According to figures 10 and 11, although Lemon grass oil and Hinokitiol are known
anti-fungal actives, when this combination was tested by Zone of inhibition method
against M. furfur, no additive or synergistic effect was seen. Hence, it is inferred that the
combination of ZPTO and Hinokitiol is not obvious even for a skilled individual.
Similarly, the combinations of Hinokitiol and ketoconazole were tested by Zone of
inhibition method against M. globosa standard (CBS 7966) and M. globosa resistant
(ITC5-1) strains. Though both the molecules were known for anti fungal activity, they
failed to show any additive or synergistic effects. Hence it is concluded that a
combination of ZPTO and Hinokitiol is not-obvious for better efficacy or efficacy
against resistant strains.
According to Table 1, in addition to hinokitiol, another effective AD molecule
Voriconazole was identified for persistent dandruff by using M. globosa (ITC5-1) as a
screening tool. Compared to other azoles, which showed resistance when tested against
M. globosa (ITC5-1), Voriconazole provided us the equal or higher ZOI value as
compared to the standard M. globosa (CBS 7966) strain. It is concluded that M. globosa
(ITC5-1) can be used as a screening tool to identify the potent anti-dandruff actives
against persistent/resistant dandruff.
Table 1: AD molecule identified for persistent dandruff by using M. globosa ITC5-1 as



The present invention is now illustrated by way of non limiting examples:
Example 1: Anti dandruff composition

Example 2: Process of preparation of the anti dandruff composition
A homogenous solution of surfactant is made in water under continuous stirring.
When the solution becomes homogenous emulsifiers, cationic guar gum derivatives,
thickening agent, cationic conditioning agents, humectants, moisturizers and other
ingredients are added one at a time under continuous stirring. Thereafter preservative
and fragrance are added under continuous stirring at temperatures below 40°C. Anti-
dandruff actives are added at room temperature. Viscosity and pH are adjusted using

Sodium Chloride and NaOH respectively to obtain the final composition.
Example 3: A screening process for identifying drug resistant anti dandruff (AD)
strain
• The scalp was divided into four parts and subjected for visual observation. The
scalp was classified as normal or dandruff affected based on presence of flakes
• From the dandruff afflicted scalp, samples were collected by rubbing sterile
cotton swap previously immersed in the sterile distilled water into the affected
portion front and back and immediately spread over the pre-poured agar plates
containing Leeming Notman agar (LN agar) with CC supplement.
• The plates were allowed to incubate for 15 days at 30°C
• After incubation, Malassezia colonies were subcultured into fresh LN agar
plates followed by inoculated into LN broth for glycerol stocks, DNA extraction
to identify the species and further culturing.
• Genomic DNA was extracted by the following protocol:
o Malassezia cell pellet was resuspend with 200 .1 of lysis buffer (2%
Triton X 100, 1% SDS, 100 mM NaCl, 10 mM Trsi-HCl, pH 8.0, 1 mM
EDTA ) and incubated at -80°C for five mins, 99oC in dry bath for five
minutes, this cycle being repeated once again.
o Samples were vortexed for 30 Seconds and subject to phenolxhloroform
extraction to isolate nucleic acids.
o Nucleic acids were precipitated with ice cold 100% ethanol. And the
nucleic acid pellet was resuspended in 50 l of TE buffer (10 mM Tris
(pH 8.0)1 mMEDTA)
• DNA samples were subject to sequencing to identify the Malassezia species.
• To determine whether the isolated species was resistant to standard anti-fungal
agents, the e-test method was employed.

o The standard laboratory Malassezia strain chosen for comparison is to be
the same species as the suspected resistant scalp isolate strain, as
determined by DNA sequencing.
o The E test strip (Commercial or In-house strip) containing anti-fungal
agents were placed onto a plate spread with 100 l of Malassezia sp
culture (Inoculum was prepared by dispersing culture in sterile PBS and
adjust to required OD: M. restricta & M. globosa - 2 OD @ 600 nm) and
the plates were incubated at 30 deg C for 7 days.
o After incubation period, the elliptical zone formed by lack of organism
growth was observed and MIC determined by the concentration at the
point at which the zones meets the filter strip.
o When the MIC value of the anti-fungal agent for the scalp isolate strain
is 4 or more times greater than the MIC value of the same agent for the
standard laboratory strain of the same species, the isolate is defined as
resistant.
Example 4: A screening process for identifying effective actives against drug
resistant anti dandruff (AD) strain
The following method was adopted to identify effective anti-fungal agents against
resistant Malassezia strains:
1) In-house E test strip dimensions were designed in silico and prepared by taking a
printout on Whatmann filter paper (with pre-defined dimensions as mention
below).



2) Filter paper strips were cut 51 mm of height and 4 mm of width and internally
divided into 7 squares, each square corresponding to 7 mm of height and 4 mm
of width. Then the required amount of strips were kept in a Petri dish and
autoclaved at 121°C for 15 minutes.
3) Using appropriate means 2.5 ul of serial dilutions of actives was added to form a
gradient, higher to lower concentrations [ZPTO (ppm) - 2.5, 1.25, 0.63, 0.31,
0.16 and 0.08. Hinokitiol (ppm) - 75, 37.5, 18.75, 9.4, 4.3 and 0.8 and
Climbazole (ppm) - 2.5, 1.25, 0.63, 0.31, 0.16 and 0.08]. The concentration
range was designed keeping in mind the MIC values for each active, obtained by
broth dilution method.
4) The strip was placed onto a plate spread with 100 ul of standard Malassezia sp
or resistant Malassezia strain of the same species (inoculum was prepared by
dispersing culture in sterile PBS and adjust to required OD: M. restricta and M
globosa - 2 OD @ 600 nm) and the plates were incubated at 30 deg C for 7 days.

5) After incubation period, the elliptical zone formed by lack of organism growth
was observed and MIC determined by the concentration at the point at which the
zones meets the filter strip.
6) Experiments are repeated a minimum of three times. Effective actives are
defined as those which show similar MIC values for the standard laboratory
strain and the resistant strain.
According to Figure 3A and 3B of the present invention, it is demonstrated that ZPTO
inhibits growth of M. globosa standard laboratory strain (CBS7966) more effectively (as
observed by area of the clear zone) than M. globosa (ITC 5-1) thereby indicating that M.
globosa (ITC 5-1) is resistant to ZPTO. Further according to Figure 4A and 4B of the
present invention it is demonstrated that ketoconazole inhibits growth of M. globosa
standard laboratory strain (CBS7966) more effectively (as observed by area of the
clear zone) than M. globosa (ITC 5-1) thereby indicating that M. globosa (ITC 5-1) is
resistant to ketoconazole.
Figure 7A and 7B of the present invention demonstrates the ability of the screening
process employing ITC5-1 strain to identify effective actives against resistant dandruff.
Hinokitiol inhibits growth of M globosa standard strain and M globosa ITC 5-1 at the
same active concentration indicating that there is no resistance towards hinokitiol. Thus
the strain such as M. globosa (ITC5-1), resistant to common antidandruff actives such
as ZPTO or ketoconazole, demonstrates no resistance to hinokitiol.

WE CLAIM:
1. A screening process for identifying effective anti-dandruff actives using drug
resistant Malassezia strain comprising steps of
a) collecting the microbial samples from scalp with appropriate means;
b) isolating and purifying the species;
c) determining minimum inhibitory concentration (MIC) of drug for said
isolated species using active gradient strip;
d) identifying the drug resistance by comparing MIC to that of the standard
laboratory strain; and
e) using the identified drug resistant strain as a screening tool to identify
effective anti-dandruff actives.

2. The screening process as claimed in claim 1, wherein said Malassezia strain is
ITC 5-1.
3. The screening process as claimed in claim 1, wherein said drug resistance is
MIC difference of greater than 4-fold.
4. A personal care composition comprising

a) anti dandruff actives;
b) ZPTO;and
c) other excipients
wherein said personal care composition helps combat Malassezia strains.
5. The composition as claimed in claim 4, wherein said anti-dandruff actives are
selected from a group consisting of Hinokitiol, Voriconazole.

6. The composition as claimed in claim 4, wherein amount of anti dandruff active
ranges from 0.001 to 1% by wt.
7. The composition as claimed in claim 4, wherein amount of ZPTO ranges from
0.001 to 2% by wt.
8. The composition as claimed in claim 4, wherein said Malassezia strain is ITC 5-
1.