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Current Cosmetic Science, XXXX, X, X-XX 1
RESEARCH ARTICLE
2666-7797/XX $65.00+.00 ©XXXX Bentham Science Publishers
Formulation and In Vitro Characterization of Tea Tree Oil Anti-Dandruff
Shampoo
Humra Umar1, Tariq Mahmood1, Talib Hussain2,3, Rabia Aslam1, Yasser Shahzad2 and Abid
Mehmood Yousaf2,*
1Faculty of Pharmacy, University of Central Punjab, Lahore, Pakistan; 2Department of Pharmacy, COMSATS Univer-
sity Islamabad, Lahore Campus, Lahore, Pakistan; 3Provincial Pharmacovigilance Centre, Provincial Drug Control
Unit, Lahore, Pakistan
Abstract: Background: Dandruff is a common scalp condition affecting half of the population of the
world.
Objective: The current study aimed at developing anti-dandruff shampoos containing tea tree oil, which
is believed to be effective against Malassezia furfur, a fungus involved in dandruff production.
Methodology: Various shampoos containing tea tree oil in 0.5 to 3% concentration were prepared after
careful selection of various shampoo ingredients. The formulated shampoos were subjected to various
quality tests such as pH, viscosity, foam production, dirt dispersion, wetting time, surface tension, solid
contents, and antimicrobial activity against a model fungal strain, namely Candida albicans. The formu-
lated shampoos were also compared with the marketed shampoos for quality attributes.
Results: The results revealed that tea tree oil shampoos had pH values in the range of 5 – 6, which is
close to the slightly acidic skin’s pH and considered as good for hair. All other quality attributes were
comparable to the marketed products. The marketed shampoos had superior antifungal activity due to the
presence of zinc pyrithione or a higher concentration of salicylic acid or selenium sulfide. Notwithstand-
ing, the tea tree oil shampoos demonstrated an appreciable antifungal activity due to synergistic effects
of tea tree oil, sodium lauryl sulphate, and salicylic acid. Furthermore, the tea tree oil shampoos were
stable during two months-long stability testing.
Conclusion: Thus, tea tree oil anti-dandruff shampoos have the potential to address the dandruff problem.
A R T I C L E H I S T O R Y
Received: November 09, 2020
Revised: January 04, 2021
Accepted: January 10, 2021
DOI:
10.2174/2666779701666210426085302
Keywords: Anti-dandruff, antifungal, formulation, shampoo, tea tree oil, marketed shampoos.
1. INTRODUCTION
Dandruff is a non-inflammatory chronic dermal disorder
that affects almost half of the world’s population [1]. Dan-
druff is characterized as an unpleasant post-puberty problem
of hair and scalp, especially in males, which is often associ-
ated with itching due to flaky hair and scalp [2, 3]. A lipo-
philic yeast, namely Malassezia furfur, is associated with
dandruff production in sebum-rich areas such as the scalp
[4]. Dandruff is generally treated with shampoos containing
synthetic or natural ingredients. Synthetic ingredients such
as selenium sulfide or antifungals containing azole ring are
often used in chemical-based shampoos; however complete
eradication of dandruff is difficult with this regime [5].
Shampoos containing major components of synthetic origin
are also responsible for adverse reactions such as skin
*Address correspondence to this author at the Department of Pharmacy,
COMSATS University Islamabad, Lahore Campus, Lahore, Pakistan;
E-mail: abid.ucp@hotmail.com
irritation, allergy, hair breakage, skin and hair discoloration
[6]. On the other hand, herbal shampoos have shown prom-
ising results in addressing the issue of dandruff. Herbal
shampoos containing natural plant extracts are usually com-
patible with the skin and may not elicit side effects that are
often associated with synthetic ingredients [7].
Many natural extracts from plants have beneficial ef-
fects on skin and hair due to the presence of vitamins,
amino acids, sugars, glycosides, phytohormones, bioflavo-
noids, fruit acids, and essential oils and are commonly
used in shampoos [8]. Various plants such as Azadirachta
indica (Neem), Ocimum sanctum (Tulsi), Aloe vera (aloe),
Terminalia chebula (harda, haritaki), Terminalia bellirica
(bahera), Sapindus mukorossi (Reetha), Phyllanthus embli-
ca (Amla), and Acacia concinna (Sheekakai) have been
traditionally used for hair cleansing, conditioning, and
removing dandruff in sub-continent including Pakistan and
India [9]. Although these plants have been used for centu-
ries for hair care, yet commercial herbal shampoos mainly
2 Current Cosmetic Science, XXXX, Vol. X, No. X Umar et al.
consist of synthetic ingredients boosted with natural prod-
ucts [10].
Essential oil from the plant Melaleuca alternifolia, (tea
tree oil) has recently shown promising results against skin
infections because of its strong antibacterial, anti-fungal,
and anti-viral activities[11]. Tea tree oil contains a number
of hydrocarbons and terpenes; however, terpene–4–ol is the
major component.Terpene–4–ol is believed to have anti-
microbial activity and could possibly be effective against
Malassezia furfur[12]. Satchell and co-workers have
demonstrated that 5% tea tree oil shampoo was effective
against dandruff without any skin irritation [13]. However,
they did not disclose the complete composition of the sham-
poo they used in their study.
Here, we have described a new shampoo formulation
containing tea tree oil as a dandruff remedy. The prepared
shampoos were extensively characterized for various quality
attributes. More importantly, the prepared shampoos were
compared with five commercially available shampoos in
Pakistan, namely Vatika, Clear for Men, Head & Shoulder,
Selsun blue, and Garnier. The anti-dandruff activity of tea
tree oil shampoo and marketed shampoos was determined
by anti-microbial activity against Candida albicans as a
model fungal strain [14].
2. MATERIALS AND METHODS
2.1. Materials
Tea tree oil was procured from Xpel marketing Ltd
(England). Sodium lauryl sulphate, salicylic acid, sodium
EDTA, guar gum, Tween 80, sodium hydroxide, urea, and
xanthan gum were purchased from Sigma-Aldrich (Germa-
ny). Lanolin was sourced from Suru Chemicals & Pharma-
ceuticals Private Ltd. (India). Triethanolamine was procured
from Merk (Germany). The purified water that was prepared
at an in-house facility was used throughout the studies.
2.2. Formulation of Anti-dandruff Shampoo
Initially, a total of eight formulation trials were conduct-
ed in order to find an optimal shampoo formulation. In all
eight trials, we fixed the quantities of SLS, lanolin, sodium
EDTA, Tween 80, urea, and salicylic acid, whilst we sys-
tematically studied the effect of xanthan gum, guar gum,
and gelatin, as given in Table 1. The shampoo formulations
were prepared by mixing the ingredients in distilled water
using a magnetic stirrer operating at 500 RPM. The final pH
of the shampoo was adjusted between 5 – 6 either by 0.4N
NaOH solution or 2% triethanolamine solution. The pre-
pared shampoo trials were evaluated for physical appear-
ance, and the most stable formulation was selected for mak-
ing the anti-dandruff shampoo by adding tea tree oil at vari-
ous concentrations, namely 0.5%, 1%, 1.5%, 2%, 2.5%, and
3% designated by S1, S2, S3, S4, S5, and S6, respectively.
2.3. Characterization of Shampoos
2.3.1. Organoleptic Properties
The prepared anti-dandruff shampoos were compared
with the marketed anti-dandruff shampoos based on sensory
evaluation. The shampoos were inspected for color, clarity,
odor, andtexture [15].
2.3.2. Determination of pH
The pH values of prepared shampoos and marketed se-
lected shampoos were determined using a digital pH meter
at ambient temperature 25 ± 0.2°C.
Table 1. Various trials of shampoo formulations.
Ingredients (Grams)
F1
F2
F3
F4
F5
F6
F7
F8
SLS
15
15
15
15
15
15
15
15
Lanolin
1
1
1
1
1
1
1
1
Sodium EDTA
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
Xanthan gum
3
-
2
-
3
-
2
-
Guar gum
-
1
-
-
-
1
-
-
Gelatin
-
-
-
2
-
-
-
2
Tween 80
1
1
1
1
1
1
1
1
Urea
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
Sodium hydroxide
q.s
q.s
q.s
q.s
-
-
-
-
Triethanolamine
-
-
-
-
q.s
q.s
q.s
q.s
Salicylic acid
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Distilled water
q.s. to make 100 mL
Formulation and In Vitro Characterization of Tea Tree Oil Current Cosmetic Science, XXXX, Vol. X, No. X 3
2.3.3. Viscosity Measurements
The viscosity of shampoos was determined using a
Brookfield viscometer (BDV-8S) equipped with an L3 spin-
dle, and the spindle rotation speed was set at 1.5, 3, 6, 12,
30, and 60 RPM. Viscosity measurements were performed
in triplicate at ambient temperature (25 ± 0.2°C).
2.3.4. Solid Contents Determination
The percentage of solid contents was measured by the
loss-on-drying method. Briefly, 5 g of each shampoo formu-
lation was poured in clean, dry, and pre-weighed Petri dish-
es, and the final weight was recorded. This was followed by
placing Petri dishes in a convection oven set at 50°C for 1 h
or until shampoos were completely dried. The driedPetri
dishes were weighed again, and the solid content after dry-
ing was estimated using the following formula [9]:
!"#$%!!"#$%#$&!%=
!
°−!
!
!
°
Where,Wo is the initial weight of the sample while W1 is the
weight of solid contents.
2.3.5. Surface Tension Determination
Surface tension was measured using a stalagmometer by
making a 10% dilution of each shampoo in distilled water at
ambient temperature (25 ± 0.2°C). The surface tension of
shampoos was determined in triplicate using the formula
given below:
!!!=!
!!×!!
!!×!!
×!!!
Where,γ1 is the surface tension of shampoo, n1is the number
of drops of shampoo, n2 is the number of drops of water, ρ1
is the density of shampoo, ρ2 is the density of water, and γ2 is
the surface tension of water.
2.3.6. Foam Volume
The foam volume of the prepared shampoos and the
marketed shampoos was determined by the shake cylinder
method, as described previously in a study[9]. Briefly, 50
mL of 10% shampoo solution was added to a 250mL gradu-
ated cylinder and shook 10 times. The total volume of foam
after 1, 2, 3, and 4 min of shaking was recorded immediate-
ly using the graduates of the cylinder. The process was re-
peated three times.
2.3.7. Dirt Dispersion
Two drops of shampoo were added to 10 mL of distilled
water in a test tube, followed by the addition of one drop of
Indian ink. The test tube was stoppered and gently shaken10
times. The amount of Indian ink was visually estimated in
the foam as none, light, moderate and heavy. Shampoos that
caused the color to stay in the foam were considered low
quality[16].
2.3.8. Wetting Time
The wetting time of shampoos was determined by drop-
ping 50 mL of 1% aqueous shampoo solution on 1g of wool
yarn in a 100 mL beaker [17]. The time when wool yarn
started to float at the surface of the shampoo solution and
when it started to sink was recorded carefully by a stop-
watch. The mean values and standard error of at least three
replicates were reported.
2.3.9. Detergency Power
Detergency power (DP) of each shampoo was estimated
by soaking 5 grams of wool yarn in grease, weighing it, and
then placing it in 100 mL of 1% aqueous solution of sham-
poo in a 250 mL capacity flask. The flask was then fixed in
a shaking water bath that was set at 35°C and was shaken
for 4 min at the rate of 50 agitations per min. Afterward, the
solution was removed from the flask, and the wool yarn was
allowed to dry. After drying, the wool yarn was weighed,
and the detergency power was calculated by using the fol-
lowing equation [18]:
!"!%=1−
!
!
×100
Where, C is the initial weight of grease and T is the weight
of grease after removing it from the shampoo solution.
2.3.10. Antimicrobial Activity of Shampoos
Anti-microbial activity of formulated shampoos and the
marketed shampoos was determined using the agar well
diffusion method against Candida albicans culture[19, 20].
Briefly, Sabouraud Dextrose Agar (SDA) media was pre-
pared and poured into sterilized Petri dishes. After solidifi-
cation of media in the Petri dishes, 4 mm diameter hole was
made using a sterilized cork borer. This was followed by
adding 3 mL of Candida albicans culture and was spread
using a sterilized glass spreader. The holes were filled with
150 µL of shampoo, and Petri dishes were covered immedi-
ately with a lid and sealed with parafilm. The sealed Petri
dishes were placed in an incubator operating at 37°C for 24
h. After 24 h, zones of inhibition were measured and report-
ed as the mean and standard deviation of at least three repli-
cates. Tea tree oil solutions of respective concentrations (as
used in formulated shampoos) were used to compare zones
of inhibition of each formulated tea tree oil shampoo. This
was further compared with the antifungal activity of the
marketed shampoos.
2.3.11. Stability Studies
Stability studies on shampoos were conducted by plac-
ing them in an incubator at 45°C and 75% relative humidity
for 2 months and were regularly evaluated for a physical
appearance at 1, 4, 7, 15, 30, and 60 days.
2.3.12. Statistical Analysis
All the experiments were conducted in triplicate, and
their mean ± standard deviation was reported. One-way
ANOVA was applied to check the significance of the differ-
ence, and the p values<0.05 were considered significant.
The data was statistically analyzed using Origin software
(version 8.5, OriginLab, USA).
3. RESULT AND DISCUSSION
Here we have described a new anti-dandruff shampoo
formulation containing tea tree oil at various concentrations.
Initially, we have performed various trials to find a suitable
shampoo base to incorporate tea tree oil. Trial shampoos
4 Current Cosmetic Science, XXXX, Vol. X, No. X Umar et al.
were formulated using different natural polymers such as
xanthan gum, guar gum, and gelatin and were evaluated for
their physical appearance, pH, and day-long stability.
Among eight trial formulations, F2 appeared to be more
stable than any other formulation after keeping it at 45°C for
24 h, and it had a pH value of 5.8, which is very close to the
skin pH. Additionally, the physical appearance of shampoo
with a formulation containing guar gum (F2) was more ap-
pealing in terms of texture and clarity. Furthermore, sham-
poo formulation with triethanolamine as pH adjuster was
found to have a pungent smell as compared to the formula-
tion containing sodium hydroxide as a pH adjuster; thus
sodium hydroxide was deemed suitable. Finally, based on
initial observations, F2 formulation was chosen for incorpo-
ration of tea tree oil at 6 concentrations, namely 0.5%,1%,
1.5%, 2%, 2.5%, and 3%. The prepared tea tree oil sham-
poos were also compared with marketed shampoos, namely
Vatika, Clear for Men, Head & Shoulder, Selsun blue, and
Garnier Fructis.
3.1. Organoleptic Properties
All the formulated shampoos and marketed shampoos
were evaluated for color, clarity, odor, and texture. The re-
sults of organoleptic evaluations are summarized in Table 2.
In the formulated shampoos, we did not add any color or
fragrance; however, the presence of lanolin resulted in pale
yellow colored shampoo formulations. On the other hand,
all the marketed shampoos were perfumed and colored for
their aesthetic appeal. The tea tree oil shampoos appeared
cloudy with gel-type texture whilst the marketed shampoos
appeared shiny with gel-type texture. While the formulated
shampoos were acceptable, the marketed shampoos had a
clear edge over the tea tree oil shampoos in terms of appear-
ance and odor.
3.2. pH and Viscosity Measurements
Our prepared shampoos had pH in the range of 5.4 – 5.7,
which is close to the pH of the skin, as given in Table 2.
However, the marketed shampoos showed a markedly vari-
able pH value from 3.9 to 7.4. Hair consists of tiny scales
known as cuticles, which are sensitive to pH, especially al-
kaline pH, which opens up these scales, ultimately leading
to hair damage. Thus a slightly acidic pH is favourable [21].
Furthermore, the slightly acidic pH of shampoos increases
the hair’s quality, decreases eye irritation, and maintains the
ecological balance of the scalp [22].
The viscosity of tea tree oil shampoos and marketed
shampoos was determined at various spindle speeds. As can
be seen from Fig. (1A), the viscosity of formulated sham-
poos decreased from 4170.5cP to 1114.8cP with increasing
tea tree oil concentrations from 0.5% to 3%at a spindle
speed of 1.5 rpm. It was not clear why the viscosity of
shampoos decreased with increasing tea tree oil concentra-
tion; one possibility could be the modification in the gelling
ability of natural hydrophilic polymer by adding the essen-
tial oil. This would, in turn, had reduced the overall viscosi-
ty of the shampoo formulation. Previous research has re-
vealed an alteration in pH by increasing the tea tree oil con-
tents, thereby reducing the viscosity [23], however, this was
not true in our study, and the pH values remained fairly sta-
ble with increasing tea tree oil contents (Table 2). Neverthe-
less, the important factor is pseudo-plastic behaviour which
is a desirable attribute in shampoo formulations [24]. Re-
sults revealed that the viscosity of each of the tea tree oil
shampoo was decreased with increasing spindle speed from
Table 2. Organoleptic properties and pH of anti-dandruff shampoos.
Shampoo
Color
Clarity
Odor
Texture
pH
Tea Tree Oil Anti-Dandruff Shampoos
-
S1
PY
C
PL
GT
5.7 ± 0.0
S2
PY
C
PL
GT
5.6 ± 0.0
S3
PY
C
PL
GT
5.7 ± 0.1
S4
PY
C
PL
GT
5.6 ± 0.1
S5
PY
C
PL
GT
5.4 ± 0.0
S6
PY
C
PL
GT
5.5 ± 0.1
Marketed Shampoos
-
Head & Shoulder
W
S
PL
GT
7.4 ± 0.2
Selsun Blue
BG
S
PL
GT
3.9 ± 0.0
Garnier Fructis fortifying shampoo
SB
S
PL
GT
4.9 ± 0.6
Clear
PB
S
PL
GT
5.4 ± 0.1
Vatika
Y
S
PL
GT
6.3 ± 0.1
Abbreviations: PY=Pale Yellow, C=Cloudy, GT=Gel Type, W=White, BG=Blue-Green, SB=Sky Blue, PB=Pale Blue, Y=Yellow, S=Shiny, PL=Pleasant.
Formulation and In Vitro Characterization of Tea Tree Oil Current Cosmetic Science, XXXX, Vol. X, No. X 5
1.5 rpm to 60 rpm, which confirmed the pseudo-plastic be-
haviour of prepared shampoos [25]. On the other hand, the
marketed shampoos had significantly higher(p<0.05) viscos-
ity as compared with that of the formulated tea tree oil
shampoos (Fig. 1B), possibly because of compositional dif-
ference in formulated and marketed shampoos. Neverthe-
less, a similar trend of decreasing viscosity with increasing
spindle speed was observed for the marketed shampoos.
Generally, a decrease in viscosity with increasing shear rate
is a favorable activity, which helps in the easy spreading of
shampoo on hair [26].
Fig. (1). Viscosities of (A) Tea Tree Anti-Dandruff shampoos and
(B) marketed shampoos. (A higher resolution / colour version of
this figure is available in the electronic copy of the article).
3.3. Percentage Solid Contents
For good quality anti-dandruff shampoos, solid content
should ideally be between 20 – 30%. This allows for easy
rinsing and removal from the hair [7]. Fig. (2A & B) depicts
solid contents in the prepared tea tree oil anti-dandruff
shampoos and the marketed shampoos. The solid contents in
formulated shampoos ranged from 20.03 – 22.25%, whilst
the solid contents in marketed shampoos ranged from 21.96
– 32.20%. All the tea tree oil shampoos had a lower range of
solid contents, thus deemed excellent. On the other hand,
marketed shampoos, namely Garnier Fructis and Clear had
slightly higher solid content, whilst Head & Shoulder, Sel-
sun Blue, and Vatika shampoos had solid content within an
ideal range.
Fig. (2). Percentage of solid contents in (A) tea tree anti-dandruff
shampoos and (B) marketed shampoos. (A higher resolution / col-
our version of this figure is available in the electronic copy of the
article).
3.4. Measurement of Surface Tension
Surface tension is linked with the cleaning ability of
shampoos. Surfactants have the ability to reduce the surface
tension and the lesser the surface tension, the higher the
cleaning ability. Good shampoos generally reduce the sur-
face tension of water, which is approximately 72.28
dyn/cm[25]. The measured surface tensions of tea tree oil
and marketed shampoos are listed in Table 3. As can be
seen from Table 3, all the shampoos had surface tension in
the range of 24.02 – 36.27 dyn/cm, which is sufficiently
below the surface tension of pure water. Thus, all shampoos
were able to significantly reduce the surface tension of wa-
ter and were deemed good for their cleaning ability. How-
ever, the surface tension of marketed shampoos was signifi-
cantly less (p<0.05) than that of the tea tree oil shampoos.
Furthermore, all tea tree oil shampoos reduced the surface
tension significantly, yet no correlation existed between tea
tree oil concentration and reduction in surface tension.
3.5. Foam Volume
From the customer perspective, foaming has paramount
importance in the quality of shampoos, however, there is no
correlation existed between foaming and cleaning [25]. Fig.
6 Current Cosmetic Science, XXXX, Vol. X, No. X Umar et al.
(3A & B) depicts the volume of foam produced by tea tree
oil shampoos and marketed shampoos in distilled water at
different time points. All the shampoos (formulated and
marketed) resulted in foam in higher than 100 mL volume,
and the foam was stable throughout the testing time. The
foam volume produced by tea tree oil shampoos and mar-
keted shampoos was comparable and no significant difference
existed. The foaming ability of shampoos is correlated with
the surface tension reduction by the surfactants. Without the
presence of surfactants, there will be enough surface tension
that could pull the bubbles closed, resulting in limited or no
foam at all. However, the presence of surfactants at the
air/water interface reduces the surface tension to a point
where the pull caused by the tension becomes negligible and
air bubbles stabilize for a longer duration of time[27]. Since
our formulated shampoos and marketed shampoos had surfac-
tants, a reduced surface tension resulted in bubble formation
as revealed by the foaming of shampoos.
3.6. Dirt Dispersion
Dirt dispersion is an important quality attribute of sham-
poos and it is linked with the cleansing action of shampoos.
Generally, shampoos that cause the ink to stay in the foam
instead of the liquid portion are considered poor quality prod-
ucts because of difficulty in washing away the ink, conse-
quently, the dirt would be difficult to remove from the hair
[16]. Among the prepared tea tree oil shampoos, S2, S3, S5,
and S6 had light ink in their foam section while no ink was
detected in the S1 and S4 shampoo formulation (Table 3). On
the other hand, all marketed shampoos except Selsun Blue
shampoo showed light ink distribution in the foam. Thus, tea
tree oil shampoos were similar to marketed shampoos, while
S1 and S4 shampoo formulations were found to be superior in
terms of cleansing action on the basis of dirt dispersion.
3.7. Wetting Time
The wetting ability of shampoos describes their efficacy
as cleansing products and it depends on the surfactant used.
The marketed shampoos had wetting time in the range of
4.24 – 6.03s (Table 3). In comparison, the formulated tea
tree oil shampoos showed wetting time in the range of 4.54
– 7.55s (Table 3). The results revealed that there was no
significant difference (p>0.05) in the wetting time of formu-
lated tea tree oil shampoos and the marketed shampoos.
Fig. (3). Foam Volume of (A) tea tree oil anti-dandruff shampoos
and (B) marketed shampoos. (A higher resolution / colour version
of this figure is available in the electronic copy of the article).
Table 3. A comparison of surface tension, dirt dispersion, and wetting time of tea tree oil and marketed shampoos.
Shampoo
Surface Tension (dyn/cm)
Dirt Dispersion
Wetting Time (Seconds)
S1
35.61 ± 1.12
None
5.46± 0.09
S2
36.27 ± 0.98
Light
4.65± 0.11
S3
34.73 ± 0.66
Light
4.54± 0.32
S4
36.09 ± 0.32
None
7.30± 0.15
S5
35.15 ± 0.11
Light
7.55± 0.12
S6
34.54 ± 1.09
Light
6.98± 0.50
Head & Shoulder
27.24 ± 0.87
Light
4.40± 0.08
Selsun Blue
32.96 ± 0.54
None
4.24± 0.57
Garnier Fructis fortifying shampoo
24.02 ± 0.21
Light
5.29± 0.03
Clear
27.03 ± 0.33
Light
6.03± 0.12
Vatika
29.28 ± 1.99
Light
5.04± 0.85
Formulation and In Vitro Characterization of Tea Tree Oil Current Cosmetic Science, XXXX, Vol. X, No. X 7
3.8. Cleaning Action
The prepared tea tree oil shampoos and the marketed
shampoos were subjected to evaluation of detergency power
or cleaning action. Although experimental assessment of
detergency power is difficult to standardize, yet it is a gen-
eral agreement that good quality shampoos should effective-
ly remove the grease or oil from the hair [18]. Our results
showed that the cleaning action of tea tree oil shampoos was
in the range of 23.0 – 30.7% as compared to 28.6 – 30.5%
for the marketed shampoos, as depicted in Fig. (4A & B).
With little or no difference in cleaning action, we can say
that our formulated shampoos were as good as the marketed
shampoos.
Fig. (4). Cleaning action of (A) tea tree oil anti-dandruff shampoos
and (B) marketed shampoos. (A higher resolution / colour version
of this figure is available in the electronic copy of the article).
3.9. Antimicrobial Activity of Tea Tree Oil Anti-
Dandruff Shampoos
Antifungal activity of tea tree oil shampoos and the mar-
keted shampoos was studied against a model fungal strain,
namelyCandida albicans. Moreover, pure tea tree oil solu-
tions were also studied to compare the shampoo formula-
tion. In this case, pure tea tree oil solutions were considered
as a control to compare the effect of other ingredients of
formulations for their antifungal activity, and it was found
that the solutions had the same concentration as used in the
shampoo formulations. The antifungal activity in terms of
zones of inhibitions of pure tea tree oil, tea tree oil sham-
poos, and the marketed shampoos is listed in Table 4.
As can be seen from Table 4, pure tea tree oil solutions
had antifungal activity against Candida albicans, as demon-
strated by zones of inhibition. The inhibition of fungal
growth was concentration-dependent for pure tea tree oil
solutions. The fungal growth inhibition was more pro-
nounced in the formulated tea tree oil shampoos as demon-
strated by the values. Although no concentration-dependent
inhibition was observed, yet the inhibition zones were larger
than those produced with pure tea tree oil solution. The en-
hanced antifungal activity of tea tree oil shampoos was at-
tributed to the presence of SLS and salicylic acid, which
have some antifungal activity and contributed synergistical-
ly against the fungal strain [28, 29]. On the other hand, the
marketed shampoos had significantly higher (p<0.05) zones
of inhibition, thus had the higher antifungal activity against
Candida albicans. However, Selsun Blue had the lowest
antifungal activity as compared to other marketed shampoos
and its antifungal activity was insignificantly different
(p>0.05) from our formulated shampoos (Table 4). Overall,
the higher antifungal activity of the marketed shampoos was
attributed to a combined effect of zinc pyrithione, SLS, and
salicylic acid. Conclusively, the tea tree oil shampoos had
demonstrated an appreciable antifungal activity and could
serve as an excellent alternative to the marketed shampoos.
3.10. Stability Studies
Finally, the tea tree oil shampoos and the marketed
shampoos were compared in terms of their stability at vari-
ous temperatures, such as 4°C, 25°C, and 40°C (75% RH).
The shampoos were stored at each of the temperatures for 2
Table 4. Zone of inhibition of tea tree oil shampoos and their Standards.
Standard (Tea Tree
Oil Solution)
Zone of Inhibition
(mm)
Tea Tree Oil
Shampoos
Zone of Inhibition
(mm)
Marketed
Shampoos
Zone of Inhibition
(mm)
0.5%
14.2± 0.4
S1
23.8± 1.1
Head & Shoulder
33.35 ± 2.1
1%
16.2± 0.6
S2
22.3± 0.8
Selsun Blue
20.45 ± 0.9
1.5%
17.4± 0.2
S3
24.8± 0.3
Garnier Fructis
41.85 ± 2.8
2%
18.6± 0.8
S4
26.9± 1.4
Clear
35.5 ± 1.9
2.5%
22.9± 0.9
S5
20.1± 0.7
Vatika
35.8 ± 2.6
3%
21.7± 0.4
S6
25.7± 1.7
-
-
8 Current Cosmetic Science, XXXX, Vol. X, No. X Umar et al.
months, and the physical appearance and pH values were
determined at various time points, as shown in Table 5-7.
As can be seen from the stability data at various conditions,
no change in physical appearance was observed, however,
an insignificant change (p>0.05) in pH values was observed
at all storage conditions. Since the pH change was small, we
can say that all the formulations were sufficiently stable
over the 2 months stability testing period.
Table 5. Stability studies on tea tree oil shampoos and the marketed shampoos at 4°C.
Stability Studies at 4°C
Shampoo Formulations
At 1st Day
At 4th Day
At 7th Day
At 15th Day
At 1 Month
At 2 Months
S1
5.70± 0.01
5.73± 0.07
5.75± 0.21
5.78± 0.42
5.81± 0.00
5.88± 0.13
S2
5.60± 0.18
5.61± 0.04
5.64± 0.08
5.66± 0.00
5.70± 0.02
5.76± 0.09
S3
5.70± 0.14
5.70± 0.21
5.73± 0.07
5.75± 0.03
5.82± 0.08
5.88± 0.00
S4
5.60± 0.00
5.63± 0.04
5.65± 0.03
5.69± 0.07
5.73± 0.00
5.76± 0.00
S5
5.40± 0.04
5.42± 0.18
5.45±0.09
5.48± 0.22
5.52± 0.11
5.59± 0.10
S6
5.50± 0.03
5.50± 0.00
5.53± 0.17
5.56± 0.2
5.60± 0.15
5.67± 0.06
Marketed Shampoos
Head & Shoulder
7.41± 0.01
7.43± 0.01
7.46± 0.01
7.49± 0.01
7.54± 0.01
7.59± 0.01
Selsun Blue
3.90± 0.00
3.91± 0.11
3.95± 0.16
4.01± 0.03
4.08± 0.08
4.12± 0.10
Garnier fructis
4.92± 0.00
4.95± 0.02
4.97± 0.10
5.02± 0.20
5.09± 0.19
5.14± 0.12
Clear
5.43± 0.17
5.47± 0.05
5.49± 0.00
5.54± 0.02
5.60± 0.01
5.64± 0.09
Vatika
6.30± 0.04
6.33± 0.11
6.36± 0.03
6.41± 0.01
6.46± 0.08
6.52± 0.00
Table 6. Stability studies on tea tree oil shampoos and the marketed shampoos at 25°C.
Stability Studies at 25°C
Shampoo Formulations
At 1st Day
At 4th Day
At 7th Day
At 15th Day
At 1 Month
At 2 Months
S1
5.70± 0.05
5.75± 0.00
5.77± 0.13
5.81± 0.05
5.84± 0.03
5.91± 0.09
S2
5.60± 0.00
5.63± 0.07
5.67± 0.01
5.68± 0.21
5.73± 0.11
5.79± 0.06
S3
5.70± 0.02
5.73± 0.09
5.76± 0.10
5.77± 0.11
5.85± 0.16
5.92± 0.05
S4
5.60± 0.06
5.65± 0.03
5.68± 0.14
5.72± 0.12
5.76± 0.04
5.79± 0.07
S5
5.40± 0.02
5.44± 0.07
5.49± 0.03
5.51± 0.05
5.54± 0.11
5.62± 0.00
S6
5.50± 0.09
5.52± 0.14
5.55± 0.11
5.59± 0.12
5.63± 0.07
5.69± 0.03
Marketed shampoos
Head & Shoulder
7.41± 0.00
7.45± 0.04
7.48± 0.07
7.53± 0.03
7.57± 0.00
7.63± 0.15
Selsun Blue
3.90± 0.19
3.93± 0.11
3.97± 0.08
4.04± 0.05
4.11± 0.07
4.16± 0.10
Garnier fructis
4.92± 0.03
4.96± 0.00
4.99± 0.05
5.05± 0.02
5.09± 0.03
5.18± 0.08
Clear
5.43± 0.01
5.49± 0.05
5.52± 0.21
5.57± 0.11
5.63± 0.13
5.67± 0.09
Vatika
6.30± 0.22
6.36± 0.11
6.38± 0.06
6.43± 0.02
6.49± 0.03
6.56± 0.04
Formulation and In Vitro Characterization of Tea Tree Oil Current Cosmetic Science, XXXX, Vol. X, No. X 9
Table 7. Stability studies on tea tree oil shampoos and the marketed shampoos at 40°C.
Stability Studies at 40°C and 75% RH
Shampoo Formulations
At 1st Day
At 4th Day
At 7th Day
At 15th Day
After 1 Month
After 2 Months
S1
5.70± 0.07
5.78± 0.02
5.80± 0.11
5.85± 0.21
5.86± 0.06
5.95± 0.00
S2
5.60± 0.02
5.66± 0.08
5.71± 0.04
5.73± 0.11
5.76± 0.04
5.84± 0.05
S3
5.70± 0.11
5.76± 0.05
5.79± 0.02
5.79± 0.08
5.88± 0.03
5.96± 0.13
S4
5.60± 0.06
5.68± 0.03
5.72± 0.11
5.75± 0.09
5.79± 0.00
5.83± 0.04
S5
5.40± 0.01
5.47± 0.04
5.52± 0.13
5.54± 0.07
5.57± 0.11
5.65± 0.05
S6
5.50± 0.01
5.55± 0.00
5.57± 0.01
5.63± 0.07
5.67± 0.02
5.73± 0.11
Marketed shampoos
Head & Shoulder
7.41± 0.14
7.47± 0.21
7.51± 0.08
7.56± 0.19
7.62± 0.07
7.66± 0.02
Selsun Blue
3.90± 0.06
3.95± 0.11
4.02± 0.09
4.09± 0.04
4.18± 0.01
4.23± 0.03
Garnier fructis
4.92± 0.03
4.98± 0.05
5.04± 0.18
5.09± 0.10
5.15± 0.02
5.26± 0.01
Clear
5.43± 0.08
5.52± 0.03
5.56± 0.00
5.62± 0.09
5.67± 0.11
5.71± 0.06
Vatika
6.30± 0.07
6.39± 0.11
6.42± 0.15
6.48± 0.09
6.54± 0.03
6.62± 0.00
CONCLUSION
The current study demonstrated successful preparation
of tea tree oil anti-dandruff shampoos at various concentra-
tions of tea tree oil. The formulated shampoos had excellent
properties in terms of pH, viscosity, wetting time, detergen-
cy and cleaning action, dirt dispersion, and antifungal activi-
ty. Furthermore, the formulated shampoos had comparable
properties with the marketed shampoos. In conclusion, tea
tree oil can be used to formulate anti-dandruff shampoos at
various concentrations, and different concentrations may be
advantageous to various skin and hair types. However, fur-
ther studies are warranted to establish the efficacy of formu-
lated shampoos in human volunteers with different skin and
hair types.
ETHICS APPROVAL AND CONSENT TO PARTICI-
PATE
Not applicable.
CONSENT FOR PUBLICATION
Not applicable
AVAILABILITY OF DATA AND MATERIAL
Not applicable.
HUMAN AND ANIMAL RIGHTS
Not applicable.
FUNDING
None.
CONFLICT OF INTEREST
The authors have no conflicts of interest to declare.
ACKNOWLEDGEMENTS
The authors would like to thank Professor Muhammad
Jamshid, Dean of Faculty of Pharmacy, the University of
Central Punjab, for providing laboratory facilities to conduct
this research.
REFERENCES
[1] Pal, R.S.; Saraswat, N.; Wal, P.; Wal, A.; Pal, Y. Preparation &
assessment of poly-herbal anti-dandruff formulation. Open Der-
matol. J., 2020, 14(1)
http ://dx.doi.org/10.2174/1874372202014010022
[2] Xu, Z.; Wang, Z.; Yuan, C.; Liu, X.; Yang, F.; Wang, T.; Wang,
J.; Manabe, K.; Qin, O.; Wang, X.; Zhang, Y.; Zhang, M.
Dandruff is associated with the conjoined interactions between
host and microorganisms. Sci. Rep., 2016, 6(1), 24877.
http ://dx.doi.org/10.1038/srep24877 PMID: 27172459
[3] Pramodani, M.; Wickramarachchi, W. A clinical study to evaluate
the efficacy of selected traditional hair pack for dandruff. Interna-
tional Journal of Research-Granthaalayah, 2017, 5(1), 12-21.
http ://dx.doi.org/10.29121/granthaalayah.v5.i1.2017.1685
[4] Rao, K.J.; Paria, S. Anti-Malassezia furfur activity of natural
surfactant mediated in situ silver nanoparticles for a better anti-
dandruff shampoo formulation. RSC Advances, 2016, 6(13),
11064-11069.
http ://dx.doi.org/10.1039/C5RA23174D
[5] Sanfilippo, A.; English, J. An overview of medicated shampoos
used in dandruff treatment. P&T, 2006, 31(7), 396.
[6] Sharma, R.M.; Shah, K.; Patel, J. Evaluation of prepared herbal
shampoo formulations and to compare formulated shampoo with
marketed shampoos. Int. J. Pharm. Pharm. Sci., 2011, 3(4), 402-
405.
[7] Punyoyai, C.; Sirilun, S.; Chantawannakul, P.; Chaiyana, W. De-
velopment of antidandruff shampoo from the fermented product of
ocimum sanctum linn. Cosmetics, 2018, 5(3), 43.
http ://dx.doi.org/10.3390/cosmetics5030043
[8] Gubitosa, J.; Rizzi, V.; Fini, P.; Cosma, P. Hair care cosmetics:
from traditional shampoo to solid clay and herbal shampoo, a re-
view. Cosmetics, 2019, 6(1), 13.
http ://dx.doi.org/10.3390/cosmetics6010013
[9] Al Badi, K.; Khan, S.A. Formulation, evaluation and comparison
of the herbal shampoo with the commercial shampoos. Beni-Suef
10 Current Cosmetic Science, XXXX, Vol. X, No. X Umar et al.
University Journal of Basic and Applied Sciences, 2014, 3(4), 301-
305.
http ://dx.doi.org/10.1016/j.bjbas.2014.11.005
[10] Arora, P.; Nanda, A.; Karan, M. Shampoos based on synthetic
ingredients vis-a-vis shampoos based on herbal ingredients: A re-
view. Int. J. Pharm. Sci. Rev. Res., 2011, 7(1), 42-46.
[11] Carson, C.F.; Hammer, K.A.; Riley, T.V. Melaleuca alternifolia
(Tea Tree) oil: A review of antimicrobial and other medicinal pro-
perties. Clin. Microbiol. Rev., 2006, 19(1), 50-62.
http ://dx.doi.org/10.1128/CMR.19.1.50-62.2006 PMID:
16418522
[12] Carson, C.F.; Riley, T.V. Antimicrobial activity of the major com-
ponents of the essential oil of Melaleuca alternifolia. J. Appl. Bac-
teriol., 1995, 78(3), 264-269.
http ://dx.doi.org/10.1111/j.1365-2672.1995.tb05025.x PMID:
7730203
[13] Satchell, A.C.; Saurajen, A.; Bell, C.; Barnetson, R.S. Treatment
of dandruff with 5% tea tree oil shampoo. J. Am. Acad. Dermatol.,
2002, 47(6), 852-855.
http ://dx.doi.org/10.1067/mjd.2002.122734 PMID: 12451368
[14] Umer, H. Anti-dandruff activity of different commercially availab-
le shampoo.. Academic Thesis, University of Central Punjab,
2019.
[15] Aghel, N.; Moghimipour, E.; Raies Dana, A. Formulation of a
herbal shampoo using total saponins of Acanthophyllum squarro-
sum. Iran. J. Pharm. Res., 2010, (3), 167-172.
[16] Saad, A.H.; Kadhim, R.B.; Rasool, B. Formulation and evaluation
of herbal shampoo from Ziziphus spina Christi leaves extract. Int.
J. Res. Ayurveda Pharm., 2011, 2, 1802-1806.
[17] Krunali, T.; Dhara, P.; Meshram, D.; Mitesh, P. Evaluation of
standards of some selected shampoo preparation. World J. Pharm.
Pharm. Sci., 2013, 2(5), 3622-3630.
[18] Mainkar, A.R.; Jolly, C.I. Evaluation of commercial herbal sham-
poos. Int. J. Cosmet. Sci., 2000, 22(5), 385-391.
http ://dx.doi.org/10.1046/j.1467-2494.2000.00047.x PMID:
18503425
[19] Bakr, R.O.; Amer, R.I.; Fayed, M.A.A.; Ragab, T.I.M. A comple-
tely polyherbal conditioning and antioxidant shampoo: a phyto-
chemical study and pharmaceutical evaluation. J. Pharm. Bioallied
Sci., 2019, 11(2), 105-115.
http ://dx.doi.org/10.4103/JPBS.JPBS_214_18 PMID: 31148886
[20] Lucia, A.; Guzmán, E. Emulsions containing essential oils, their
components or volatile semiochemicals as promising tools for in-
sect pest and pathogen management. Adv. Colloid Interface Sci.,
2021, 287, 102330.
http ://dx.doi.org/10.1016/j.cis.2020.102330 PMID: 33302055
[21] CORBETT, J.F. The chemistry of hair-care products. J. Soc.
Dyers Colour., 1976, 92(8), 285-303.
[22] Gavazzoni Dias, M.F.; de Almeida, A.M.; Cecato, P.M.; Adriano,
A.R.; Pichler, J. The shampoo pH can affect the hair: Myth or
Reality? Int. J. Trichology, 2014, 6(3), 95-99.
http ://dx.doi.org/10.4103/0974-7753.139078 PMID: 25210332
[23] Wulansari, A.; Jufri, M.; Budianti, A. Studies on the formulation,
physical stability, and in vitro antibacterial activity of tea tree oil
(Melaleuca alternifolia) nanoemulsion gel. International Journal
of Applied Pharmaceutics, 2017, 9(1), 135-139.
http ://dx.doi.org/10.22159/ijap.2017.v9s1.73_80
[24] Kinjuit, H.; Surugau, N. In Journal of Physics: Conference Series.
IOP Publishing, 2019, 1358, 012022.
[25] AlQuadeib, B.T.; Eltahir, E.K.D.; Banafa, R.A.; Al-Hadhairi, L.A.
Pharmaceutical evaluation of different shampoo brands in local
Saudi market. Saudi Pharm. J., 2018, 26(1), 98-106.
http ://dx.doi.org/10.1016/j.jsps.2017.10.006 PMID: 29379340
[26] Kumar, A.; Mali, R.R. Evaluation of prepared shampoo formulati-
ons and to compare formulated shampoo with marketed shampoos.
Evaluation, 2010, 3(1), 25-27.
[27] Cornwell, P.A. A review of shampoo surfactant technology: Con-
sumer benefits, raw materials and recent developments. Int. J.
Cosmet. Sci., 2018, 40(1), 16-30.
http ://dx.doi.org/10.1111/ics.12439 PMID: 29095493
[28] da Rocha Neto, A.C.; Maraschin, M.; Di Piero, R.M. Antifungal
activity of salicylic acid against Penicillium expansum and its pos-
sible mechanisms of action. Int. J. Food Microbiol., 2015, 215,
64-70.
http ://dx.doi.org/10.1016/j.ijfoodmicro.2015.08.018 PMID:
26340673
[29] Leong, C.; Schmid, B.; Buttafuoco, A.; Glatz, M.; Bosshard, P.P.
In vitro efficacy of antifungal agents alone and in shampoo formu-
lation against dandruff-associated Malassezia spp. and Staphy-
lococcus spp. Int. J. Cosmet. Sci., 2019, 41(3), 221-227.
http ://dx.doi.org/10.1111/ics.12525 PMID: 30851208
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