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J HerbMed Pharmacol. 2016; 5(2): 72-77.

Journal of HerbMed Pharmacology

Journal homepage: http://www.herbmedpharmacol.com

Preparation and evaluation of clove oil in emu oil self-emulsion

for hair conditioning and hair loss prevention
Mohammad Ali Shahtalebi1, Atefeh Sadat-Hosseini2*, Leila Safaeian3
1
Department of Pharmaceutics, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
2
Student Research Committee, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
3
Department of Pharmaceutical Pharmacology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

ARTICLE INFO ABSTRACT

Article Type: Introduction: From a consumer perspective, developing a hair care formulation that offers multi-
Original Article 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
Article History: shows an androgenic activity and stimulates hair root to feed and hence could be a good candidate
Received: 14 November 2015 for developing an anti-hair loss formulation. Thus in hair research, hair follicle is of great interest.
Accepted: 12 February 2016 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
Keywords: characterized according to national oil standards. All formulations were prepared and best one
Hair loss was selected for further pharmaceutical examinations such as pH, particle size, content uniformity
Eugenol and drug release. The optimum formulation was clinically evaluated on rats back compared with
Emu oil minoxidil standard lotion as a positive control and distilled water as a negative control.
Conditioner 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.

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 baldness) which affects over 95% of people with hair loss
Hair is one of the vital fragments of the body derived (6,7). Androgenetic alopecia is hereditary thinning of the
from ectoderm of skin and contains a protein called hair induced by androgens in genetically predisposed men
keratin that is produced in hair follicles in the outer layer and women (1,2,8). Alopecia areata is an autoimmune
of skin (1-3). Hair loss or alopecia is one of the most illness affecting nearly 2% of the US population. Alopecia
common problems of many communities causing many areata affects both sexes similarly and occurs at all ages,
economical and physiological problems (4). Alopecia is although children and young adults are affected most
a dermatological disorder that has been known for more often (6-8). The treatment of hair loss is sometimes
than 2000 years and is considered a common problem in difficult because of deficient efficacy and limited options
cosmetics as well as primary health care practice (1,2). (4). Two drugs have been approved by the Food and Drug
Normally, 50-100 hairs are lost per day and an increase Administration (FDA) for the treatment of androgenetic
in lost hairs up to more than 100 is considered hair loss alopecia, minoxidil and finasteride. Minoxidil is a special
(3-5). There are many types of hair loss and the most lotion applied twice a day to enhance blood supply to the
common type is referred to as male-pattern baldness follicles and papillae and encourage hair growth (6,7).
(when it occurs in women it is called female-pattern Minoxidil is a powerful vasodilator and appears safe for

*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
 Clove oil in emu oil self-emulsion and hair fall prevention

long-term treatment and anagen phase of hair growth, 0.35

and enlarges miniaturized and suboptimal follicles (8-11). 0.3

The main adverse reactions are itching, contact dermatitis 0.25 y = 0.005x + 0.032
R² = 0.9973
and dryness (10,12). Finasteride (Propecia)®, taken once 0.2
a day, is a an oral medication that specifically decreases

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

http://www.herbmedpharmacol.com Journal of HerbMed Pharmacology, Volume 5, Number 2, April 2016 73

Shahtalebi MA et al

Table 1. Self-emulsion formulation (oil/water%)

Formulation
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 fatty 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

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

74 Journal of HerbMed Pharmacology, Volume 5, Number 2, April 2016 http://www.herbmedpharmacol.com

 Clove oil in emu oil self-emulsion and hair fall prevention

cremophor (2%), cetyl alcohol (2%), tween 80 (1%), and phase separation at 8°C, 25°C and 40°C within one
coconut fatty acid diethanolamin (2%) and canola oil (up month. The drug content of the formulation was found
to 100%). The oil phase was separately heated to 50-60°C. to be in the range of 96.5%-98% at 8°C, 25°C and 40°C,
Then the aqueous phase (95 mL water) was added to 5 mL which represents a permitted range (100 ± 5) percentage
of oil phase with continuous stirring. When the emulsion of variation (27).
cooled to room temperature, control tests were done.
Skin irritation test
Quality control tests of selected formulation Evaluation of the studied formulation for possible
The F6 had light white color and a specific odor (Table irritation on intact skin of rats showed no erythema or
2). Microscopic examination of the prepared formulation edema on outer layer of skin, indicating the safety of the
revealed homogeneity of globule and internal phases. prepared F6 containing 1% eugenol for topical application.

Determination of particle size of the prepared emulsion Hair growth activity evaluation
In order to reach to a particle size less than 10 μm able to Hair growth initiation significantly increased by treatment
penetrate into follicular structure, the average particle size with standard drug and the studied formulation. The hair
of F6 in the present study was approximately 7 μm. growth was initiated in denuded area on day 15 in control
rats, while it was initiated after the first week in positive
In-vitro release profile of eugenol from the prepared control group and in the F6-treated group. However, the
formulation completion time was not affected by different treatments
The in vitro investigation of the drug release of the in this study. Table 4 shows the average hair length of each
formulation presented a controlled drug release for a group at different time intervals during the experimental
period of 4 hours (Figure 3). period. The average hair length significantly increased
with different treatments compared to the control group.
Kinetic analysis of drug release
The in vitro investigation of drug release of the formulation Discussion
exhibited release for a period of 4 hours. According to the Self-emulsifying drug delivery systems (SEDDSs) are
release based on the correlation coefficient, first-order mixtures of oils, surfactants and co-surfactants. Sometimes
kinetic is dominant. co-solvents are also used to increase the solubility.
Therefore, SEDDSs have also become an important tool
Stability studies in novel drug delivery in recent years. SEDDSs emulsify
According to Table 3, F6 did not show changes in color 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

0.8 First order

log amount of drug remained to release

Figure 2. Eugenol standard curve in ethanol 96%. 0.7

y = -0.1499x + 0.7036
0.6 R² = 0.9948
0.5
Table 2. Physicochemical evaluation of formulation
0.4
Parameters Results
0.3
Physical appearance Light white color and an specific odor,
0.2
complete homogeny
Centrifuge +++ 0.1
Freeze-thaw +++ 0
pH 5.873 ± 0.146 0 1 Time (h) 2 3 4
Drug content 98.09 ± 0.36 Figure 3. In vitro release of eugenol through cellulose acetate
+: poor after test, ++: good after test, +++: excellent after test. membrane.

http://www.herbmedpharmacol.com Journal of HerbMed Pharmacology, Volume 5, Number 2, April 2016 75

Shahtalebi MA et al

Table 3. Stability of the studied parameters of selected formulation

Result
Parameters
Condition Initial 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 Ethical considerations
at various intervals Ethical issues (including plagiarism, misconduct, data
Group Day 7 Day 14 Day 21 Day 28 fabrication, falsification, double publication or submission
Control - - 0.445±0.089 0.57±0.115 and redundancy) were completely observed by authors.
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 Funding/Support
Treatment 0.53±0.1 1.07±0.082 2.2±0.188 2.85±0.164 This work was funded by School of Pharmacy and
Pharmaceutical Sciences of Isfahan University of Medical
day 3 after application, but the hair growth accelerated Sciences (grant no. 393543).
after one week. The animals which were treated with
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