Preparation and evaluation of clove oil in emu oil self-emulsion for hair conditioning and hair loss prevention

Abstract

Introduction: From a consumer perspective, developing a hair care formulation that offers multi-purpose products to enhance routine hair care such as conditioning, cleaning and grooming hair and stimulating hair follicles is important. Eugenol comprising about 70% of clove essential oil shows an androgenic activity and stimulates hair root to feed and hence could be a good candidate for developing an anti-hair loss formulation. Thus in hair research, hair follicle is of great interest. The aim of this study was to develop a self-emulsifying product containing eugenol in emu oil as a carrier. Methods: Eugenol was identified in clove oil extraction by UV spectrophotometer. Emu oil was characterized according to national oil standards. All formulations were prepared and best one was selected for further pharmaceutical examinations such as pH, particle size, content uniformity and drug release. The optimum formulation was clinically evaluated on rats back compared with minoxidil standard lotion as a positive control and distilled water as a negative control. Results: The selected formulation was demonstrated to condition hair with grooming and enhanced hair growth with longer lag time compared with minoxidil but after one week the hair growth accelerated. Conclusion: The formulation containing clove oil in emu oil self-emulsion shows a conditioning and grooming property with hair shaft repair and hair growth.

Full text

Journal of HerbMed Pharmacology
Journal homepage: http://www.herbmedpharmacol.com
J HerbMed Pharmacol. 2016; 5(2): 72-77.
Preparation and evaluation of clove oil in emu oil self-emulsion
for hair conditioning and hair loss prevention
*Corresponding author: Atefeh Sadat-Hosseini, Student Research
Committee, School of Pharmacy and Pharmaceutical Sciences, Isfahan
University of Medical Sciences, Isfahan, Iran. Email: atefesadat.hoseini@
yahoo.com
Introduction
Hair is one of the vital fragments of the body derived
from ectoderm of skin and contains a protein called
keratin that is produced in hair follicles in the outer layer
of skin (1-3). Hair loss or alopecia is one of the most
common problems of many communities causing many
economical and physiological problems (4). Alopecia is
a dermatological disorder that has been known for more
than 2000 years and is considered a common problem in
cosmetics as well as primary health care practice (1,2).
Normally, 50-100 hairs are lost per day and an increase
in lost hairs up to more than 100 is considered hair loss
(3-5). There are many types of hair loss and the most
common type is referred to as male-pattern baldness
(when it occurs in women it is called female-pattern
baldness) which affects over 95% of people with hair loss
(6,7). Androgenetic alopecia is hereditary thinning of the
hair induced by androgens in genetically predisposed men
and women (1,2,8). Alopecia areata is an autoimmune
illness affecting nearly 2% of the US population. Alopecia
areata affects both sexes similarly and occurs at all ages,
although children and young adults are affected most
often (6-8). The treatment of hair loss is sometimes
difficult because of deficient efficacy and limited options
(4). Two drugs have been approved by the Food and Drug
Administration (FDA) for the treatment of androgenetic
alopecia, minoxidil and finasteride. Minoxidil is a special
lotion applied twice a day to enhance blood supply to the
follicles and papillae and encourage hair growth (6,7).
Minoxidil is a powerful vasodilator and appears safe for
Mohammad Ali Shahtalebi1, Atefeh Sadat-Hosseini2*, Leila Safaeian3
1Department of Pharmaceutics, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
2Student Research Committee, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
3Department of Pharmaceutical Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
Implication for health policy/practice/research/medical education:
Eugenol comprising about 70% of clove essential oil exerts an androgenic and antibacterial activities and stimulates hair root to
feed and hence could be a used by humans for these purposes.
Please cite this paper as: Shahtalebi MA, Sadat Hosseini A, Safaeian L. Preparation and evaluation of clove oil in emu oil self-
emulsion for hair conditioning and hair loss prevention. J HerbMed Pharmacol. 2016;5(2):72-77.
Introduction: From a consumer perspective, developing a hair care formulation that offers multi-
purpose products to enhance routine hair care such as conditioning, cleaning and grooming hair
and stimulating hair follicles is important. Eugenol comprising about 70% of clove essential oil
shows an androgenic activity and stimulates hair root to feed and hence could be a good candidate
for developing an anti-hair loss formulation. Thus in hair research, hair follicle is of great interest.
The aim of this study was to develop a self-emulsifying product containing eugenol in emu oil as
a ca rrier.
Methods: Eugenol was identified in clove oil extraction by UV spectrophotometer. Emu oil was
characterized according to national oil standards. All formulations were prepared and best one
was selected for further pha rmaceutical examinations such as pH, particle size, content uniformity
and drug release. The optimum formulation was clinically evaluated on rats back compared with
minoxidil standard lotion as a positive control and distilled water as a negative control.
Results: The selected formulation was demonstrated to condition hair with grooming and
enhanced hair growth with longer lag time compared with minoxidil but after one week the hair
growth accelerated.
Conclusion: The formulation containing clove oil in emu oil self-emulsion shows a conditioning
and grooming property with hair shaft repair and hair growth.
A R T I C L E I N F O
Keywords:
Hair loss
Eugenol
Emu oil
Conditioner
Article History:
Received: 14 November 2015
Accepted: 12 February 2016
Article Type:
Original Article
A B S T R A C T

Clove oil in emu oil self-emulsion and hair fall prevention
Journal of HerbMed Pharmacology, Volume 5, Number 2, April 2016
http://www.herbmedpharmacol.com 73
long-term treatment and anagen phase of hair growth,
and enlarges miniaturized and suboptimal follicles (8-11).
The main adverse reactions are itching, contact dermatitis
and dryness (10,12). Finasteride (Propecia)®, taken once
a day, is a an oral medication that specifically decreases
the production of dihydrotestosterone by blocking the
enzyme vital to its formation. Treatments of alopecia areata
include steroids (injection or topical ointment), minoxidil
and anthralin cream. Corticosteroid may be injected into
the bald patches or applied directly to the skin as a lotion
to stimulate hair growth. Usually, hair begins to grow
again within some weeks, and the injections are repeated
within a month. Anthralin cream may also be applied to
the hairless area; this irritant is used every day and rinsed
off an hour later. The treatment usually encourages hair
growth within two to three months (6,7).
The purpose of this study is to prepare self-emulsion
using appropriate amounts of eugenol and emu oil as
an emollient agent to reduce skin irritation and increase
penetration.
The emu oil can simply penetrate into skin because of
containing large amounts of oleic acid and similarity
to human sebum. It is an excellent transdermal carrier
which penetrates into the skin, increases the potency of
topical medications such as eugenol and provides long-
lasting effectiveness. Emu oil helps to repair scar tissue.
It also accelerates the development of fresh skin cells
by delivering the required bio-nutrients deep into skin
where fresh cells form and decrease the buildup of wound
tissue (13,14).
Materials and methods
Clove essential oil (eugenol) was supplied from Golchai
Co. (Iran), cetrimonium chloride and coconut fatty acid
diethanolamin were supplied from Merck Co. (Germany)
and polysorbate 80 from (Croda Chemicals Ltd, UK),
Emu oil was obtained from Abyaneh Cosmetic Company
(Isfahan, Iran). All other ingredients used in this study
were of analytical grade.
Authentication of eugenol
Eugenol was authenticated according to USP and BP
pharmacopeia by UV spectroscopy.
Preparation of standard curve
In order to generate standard curve, 19 mg of eugenol was
accurately weighed and solubilized in 19 ml ethanol 96%.
This solution was diluted to obtain standard solutions
at the concentrations of of 5-50 µg/mL. The absorbance
of standard solutions was measured by UV/Visible
spectrophotometer (Shimadzu, UV mini-1240CE) at 282
nm and the standard curve was generated (Figure 1).
Preparation of self-emulsion
To develop a stable emulsion, the formulations were
developed with different amounts of emulsifiers with
experimental design. Nine experiments were suggested
by software according to input variable and expected
output specification. The compositions of prepared
formulations are shown in Tab le 1. The oil phase of
emulsion was prepared by mixing emu oil and eugenol
1%. The oil phase was separately heated to 40-50ºC to
achieve homogeneity. Then the aqueous phase was added
to the oily phase with continuous stirring at 800 rpm to
obtain a clear, transparent micro emulsion. Based on the
primary evaluation of organoleptic and centrifuge test of
the prepared emulsions, formulation 6 (F6) was selected
to finish evaluation.
Evaluation of the selected formulation
The following physicochemical parameters were used for
the evaluation of formulation:
Determination of particle size
The particle size of emulsion was determined by zeta
analyzer (Malvern Instruments Ltd.).
Organoleptic evaluation
The prepared self-emulsion was examined visually for
color and homogeneity.
Centrifuge test
The prepared formulation was centrifuged at 3000 rpm
for 30 minutes (HETTIC D-7200, Germany) 24 hours
after preparation and then once a week for 28 days (15).
Determination of pH
pH of the prepared formulation was measured by a
digital pH meter (Metrohm, Switzerland) (15). The
determinations were carried out in triplicate and the
average value of three readings was recorded.
Freeze-thaw cycle
Freeze-thaw treatment of the emulsion was performed
closely after preparation. Samples (20 mL) were stored at
-20°C for 48 hours. The frozen samples were subsequently
thawed at room temperature for 48 hours (16). This test
carried out in triplicate for each sample.
Drug content
To determine drug content, a certain amount of emulsion
(10 puffs equal to 1 g of emulsion) was collected and
introduced into a screw-capped tube. Then phosphate
buffer (pH 7.4) was added to the emulsion up to the
volume of 50 mL. After shaking for 2 hours in orbital
water bath shaker (Gallen KAMP, Germany) at 37°C,
the diluted emulsion was filtered through a 0.45 µm
Whatman filter. The amount of eugenol was determined
spectrophotometrically (Shimadzu, UV mini-1240CE) by
measuring the absorbance of the filtrate at 282 nm (17).
y = 0.005x + 0.032
R² = 0.9973
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
010 20 30 40 50 60
Absorbance
Concentration(μg/ml)
Figure 1. Ultraviolet spectrum of standard eugenol.

Shahtalebi MA et al
Journal of HerbMed Pharmacology, Volume 5, Number 2, April 2016 http://www.herbmedpharmacol.com
74
This procedure was performed for an emulsion system
containing no drug as the blank.
Stability test
The stability was examined at 8ºC (in refrigerator), 25ºC
(in room) and 40ºC (in oven) (15,18). At 1 week intervals
for 1 month, drug content and physical appearance
(organoleptic characteristics) were examined.
In-vitro drug release study
Franz diffusion cell (25 mL volume) was used to study the
drug release. The receiver compartment was filled with
ethanol 96% obtained after examination of sink condition
and 10 puffs (equal to 1 g emulsion) of self-emulsion were
applied on the surface of cellulose acetate membrane.
The membrane was clamped between the donor and the
receiver compartments. The donor compartment was
exposed to the receiver compartment at 37°C temperature.
The solution on the receiver compartment was stirred by
magnetic stirrer. At predetermined time intervals, 0.5 mL
of solution from receiver compartment was pipetted out
and closely replaced with fresh 0.5 mL of receiver medium.
After appropriate dilution the drug concentration on the
receiver medium was determined spectrophotometerically
at 282 nm. The experiment was carried out in triplicate.
To study drug release kinetics, data obtained from in vitro
investigation of release were fitted to the zero, first and
Higuchi kinetic models (16,19,20).
Kinetic analysis of drug release
To study drug release kinetics, data obtained from in
vitro investigation of release were fitted in Zero, First and
Higuchi kinetic models (20-22).
Animal study
Male Wistar albino rats weighing 200-250 g were used for
in vivo studies. They were kept in standard environmental
conditions with free access to special diet and drinking
water. All animal experiments were carried out in
accordance with the guidelines of Institutional Animal
Ethics Committee of Isfahan University of Medical
Sciences.
Skin irritation test
Three healthy male rats were selected for this study. Each
rat was separately kept in a cage during the study period.
The hair from the back of each rat (test sites of 1 cm2)
was shaved on the side of the spine to expose sufficiently
large test areas. The test sites were cleaned with surgical
spirit and 1 g of F6 was applied over the respective test
sites of one side of the spine. The test sites were observed
for erythema and edema for 48 hours after application
(23,24).
Application of test formulations for hair growth evaluation
The rats were divided into four groups of five each and a
4 cm² area of dorsal section of all rats was shaved. Group
I (control) received no treatment. Group II (positive
control) was treated with standard formulation including
1 mL of 5% minoxidil ethanolic solution applied on the
shaved area once a day. Group ΙΙΙ (blank) was treated with
the prepared formulation (1 g) without eugenol applied
on the shaved area once a day, and Group IV animals
were treated with 1 g of prepared formulation under study
once a day. All treatments lasted for 28 days. During the
treatments, hair growth initiation and completion time
was observed. Hair was randomly plucked from the test
area of each rat on days 7, 14, and 21 of the experiment.
The length of 10 hairs per animal was estimated and the
average length was recorded (Tabl e 4). At the completion of
the treatments, two rats from each group were euthanized
and skin biopsies were taken from shaved areas. Specimen
was preserved in 10% formalin. Tissues were embedded
in paraffin wax, sectioned into uniform thickness of 10
µm and stained with hematoxylin and eosin. The sections
were evaluated microscopically for the number of hair
follicles (24,25).
Ethical issues
All the animals were handled in accordance with the
internationally accepted principles and guidelines for
the care and use of laboratory animals in 2010 (26) and
the Ethics Committee of Isfahan University of Medical
Sciences.
Results
Authentication of eugenol
UV spectrum eugenol
As Figure 2 shows, the maximum absorption of eugenol
at 5-50 μg/mL concentrations in ethanol 96% at 200-400
nm was seen at 282 nm. The UV spectrum obtained from
eugenol sample in ethanol 96% solution was similar to the
standard eugenol.
Self-emulsion preparation
As Table 1 shows, the best formulation was F6 containing
eugenol (1%), emu oil (5%), cetrimonium chloride (0.5%),
Table 1. Self-emulsion formulation (oil/water%)
Formulaon
Ingredient (g) 1 2 3 4 5 6 7 8 9
Eugenol 1 1 1 1 1 1 1 1 1
Emu oil 5 5 5 5 5 5 5 5 5
Cetrimonium chloride 0.5 2 2 1 1 0.5 2 1 0.5
Cremophor 6 2 6 2 6 2 4 4 4
Cetyl Alcohol 4 4 3 3 2 2 2 4 3
Tween 80 5 3 1 5 3 1 5 1 3
Coconut fay acid diethanolamin 2 2 2 2 2 2 2 2 2
Canola oil up to 100 ml 76.5 81 80 81 80 86.5 79 82 81.5

Clove oil in emu oil self-emulsion and hair fall prevention
Journal of HerbMed Pharmacology, Volume 5, Number 2, April 2016
http://www.herbmedpharmacol.com 75
cremophor (2%), cetyl alcohol (2%), tween 80 (1%),
coconut fatty acid diethanolamin (2%) and canola oil (up
to 100%). The oil phase was separately heated to 50-60°C.
Then the aqueous phase (95 mL water) was added to 5 mL
of oil phase with continuous stirring. When the emulsion
cooled to room temperature, control tests were done.
Quality control tests of selected formulation
The F6 had light white color and a specific odor (Table
2). Microscopic examination of the prepared formulation
revealed homogeneity of globule and internal phases.
Determination of particle size of the prepared emulsion
In order to reach to a particle size less than 10 μm able to
penetrate into follicular structure, the average particle size
of F6 in the present study was approximately 7 μm.
In-vitro release profile of eugenol from the prepared
formulation
The in vitro investigation of the drug release of the
formulation presented a controlled drug release for a
period of 4 hours (Figure 3).
Kinetic analysis of drug release
The in vitro investigation of drug release of the formulation
exhibited release for a period of 4 hours. According to the
release based on the correlation coefficient, first-order
kinetic is dominant.
Stability studies
According to Table 3, F6 did not show changes in color
and phase separation at 8°C, 25°C and 40°C within one
month. The drug content of the formulation was found
to be in the range of 96.5%-98% at 8°C, 25°C and 40°C,
which represents a permitted range (100 ± 5) percentage
of variation (27).
Skin irritation test
Evaluation of the studied formulation for possible
irritation on intact skin of rats showed no erythema or
edema on outer layer of skin, indicating the safety of the
prepared F6 containing 1% eugenol for topical application.
Hair growth activity evaluation
Hair growth initiation significantly increased by treatment
with standard drug and the studied formulation. The hair
growth was initiated in denuded area on day 15 in control
rats, while it was initiated after the first week in positive
control group and in the F6-treated group. However, the
completion time was not affected by different treatments
in this study. Table 4 shows the average hair length of each
group at different time intervals during the experimental
period. The average hair length significantly increased
with different treatments compared to the control group.
Discussion
Self-emulsifying drug delivery systems (SEDDSs) are
mixtures of oils, surfactants and co-surfactants. Sometimes
co-solvents are also used to increase the solubility.
Therefore, SEDDSs have also become an important tool
in novel drug delivery in recent years. SEDDSs emulsify
spontaneously and produce fine oil-in-water emulsions if
introduced into an aqueous phase under gentle agitation
(28,29).
The prepared emulsion formulation, with good
characteristics based on pharmaceutical evaluation,
consists of eugenol, emu oil, setrumonium chloride,
cremophor, cetyl alcohol, tween 80, coconut fatty acid
diethanolamine, canola oil and water.
A salient feature of this formulation is use of emu oil. Emu
oil is compatible with human skin lipid and can be used
as an enhancer and drug carrier to help the penetration of
active ingredients through the skin.
The results showed that the effect of emulsion (F6) on
hair growth began on day 6 after the treatment and
commercial minoxidil 5% solution exerted its effect on
Figure 2. Eugenol standard curve in ethanol 96%.
Table 2. Physicochemical evaluation of formulation
Parameters Results
Physical appearance Light white color and an specic odor,
complete homogeny
Centrifuge +++
Freeze-thaw +++
pH 5.873 ± 0.146
Drug content 98.09 ± 0.36
+: poor aer test, ++: good aer test, +++: excellent aer test.
Figure 3. In vitro release of eugenol through cellulose acetate
membrane.
y = -0.1499x + 0.7036
R² = 0.9948
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 1 2 3 4
log amount of drug remained to release
Time (h)
First order

Shahtalebi MA et al
Journal of HerbMed Pharmacology, Volume 5, Number 2, April 2016 http://www.herbmedpharmacol.com
76
day 3 after application, but the hair growth accelerated
after one week. The animals which were treated with
blank formulation showed greater conversion of follicle
compared with the control group on day 28 after the
treatment. This may be due to the effect of emu oil on
hair growth and gentle rubbing of the shaved skin area
during treatment which may enhance blood flow to the
hair follicles. A clinical study on the activity of emu oil
on hair growth revealed that there was a 20% increase in
DNA synthesis, hair and skin re-growth stimulated and
hair follicles robustness increased (30). In a study on rats,
when treated similarly with water, the whole denuded area
was covered at the end of the treatment course because of
gentle rubbing of the site (31).
Conclusion
The prepared eugenol in self-emulsion formulation was
stable by different pharmaceutical evaluations. The emu
oil used in this formulation could increase penetration.
This formulation could be a good candidate for hair loss
treatment.
Acknowledgments
This article was derived from the PhD thesis of Ms. Atefeh
Sadat-Hosseini. Authors gratefully thank the School
of Pharmacy and Pharmaceutical Sciences of Isfahan
University of Medical Sciences for funding this study and
Dr. Baradaran Laboratory.
Authors’ contributions
All authors contributed to the design of the study. ASH
carried out the study. MAS, ASH and LS prepared and
confirmed the final manuscript.
Conflict of interests
The authors declared no competing interests.
Table 3. Stability of the studied parameters of selected formulation
Parameters Result
Condion Inial 7 days 14 days 28 days
Physical appearance
8 °C √ √ √ √
25 °C √ √ √ √
40 °C √ √ √ √
Drug content (mean ±SD)
8 °C 98.07% ± 0.41 97.7 ± 1 97.6 ± 1.02 97 ± 1.07
25 °C 98.07% ± 0.41 97.9 ± 1.02 97.5 ± 1.16 97.56 ± 1.16
40 °C 98.07% ± 0.41 97 ± 1 96.88 ± 1.023 96.5 ± 1.07
√: Unchanged
Table 4. Mean (standard deviation) hair length in different groups
at various intervals
Group Day 7 Day 14 Day 21 Day 28
Control - - 0.445±0.089 0.57±0.115
Standard 1.08±0.113 1.58±0.122 2.16±0.183 2.5±0.163
Blank - 0.51±0.061 0.93±0.105 1.11±0.11
Treatment 0.53±0.1 1.07±0.082 2.2±0.188 2.85±0.164
Ethical considerations
Ethical issues (including plagiarism, misconduct, data
fabrication, falsification, double publication or submission
and redundancy) were completely observed by authors.
Funding/Support
This work was funded by School of Pharmacy and
Pharmaceutical Sciences of Isfahan University of Medical
Sciences (grant no. 393543).
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