Formulation and evaluation of hair growth enhancing effects of oleogels made from Rosemary and Cedar wood oils

Abstract

Essential oils are one of the most popular natural products, with broad applications in dermatology. Hair loss is a disorder in which the hair falls out from skin areas where they are usually present, such as the scalp and the body.
The aim of the work was to formulate oleogels containing two essential oils (Cedarwood and Rosemary) singly and in combination and evaluate their hair growth enhancing effect on an animal model. Oleogels were formed using beeswax as the organogelator with concentrations of 10 % for the oils when used singly or 5 % each for the oils when formulated in combination. Characterization of oleogels were done using spreadability, oil binding capacity (OBC), gas chromatography (GC) and differential scanning calorimetry (DSC). Hair growth evaluation was carried out in 18 albino rats in six groups of threes. A hair removal cream was applied on the experimental animals and the Oleogel applied for six-weeks.
GC analysis of the oils revealed the presence of thujopsene, alpha-pinene, beta-pinene, pentane, alpha cedrenea, beta cedrenea, alpha cedrol, gamma-terpene, acetonitrite, atlantone, terpinolene in cedarwood oil while rosemary oil contained 1-8 cineole, camphor, alpha-pinene, beta-pinene, camphene, p-cymene, alpha-terpinene, gamma terpinene, gamma-humulene, beat-bisabolene, genaniol and terpinolene. DSC thermogram of the oleogel formulations showed varying degrees of amorphicity.
Spreadability results showed that the oleogels containing cedarwood oil were more spreadable. Conversely, oil binding capacity values were higher with Rosemary oil than Cedar wood oil with values ranging from 91 % (for the oleogel containing 10 % rosemary oil) to 81 % for the bland oleogel (no essential oil). Hair growth evaluation revealed that the oleogel containing rosemary oil (10 %) had similar effects as the positive control (Minoxidil, 2 %) at the end of the six-week period.
Oleogels made from cedar wood and rosemary oils have hair growth enhancing effects.

Full text

Editor: DR B Gyampoh
Journal Pre-proof
Formulation and evaluation of hair growth enhancing effects of
oleogels made from Rosemary and Cedar wood oils
Emmanuel Uronnachi , Chidiogo Atuegwu ,
Chukwuebuka Umeyor , Calistus Nwakile , Josephat Obasi ,
Chidalu Ikeotuonye , Anthony Attama
PII: S2468-2276(22)00130-2
DOI: https://doi.org/10.1016/j.sciaf.2022.e01223
Reference: SCIAF 1223
To appear in: Scientific African
Received date: 2 November 2020
Revised date: 21 February 2022
Accepted date: 16 May 2022
Please cite this article as: Emmanuel Uronnachi , Chidiogo Atuegwu , Chukwuebuka Umeyor ,
Calistus Nwakile , Josephat Obasi , Chidalu Ikeotuonye , Anthony Attama , Formulation and evalu-
ation of hair growth enhancing effects of oleogels made from Rosemary and Cedar wood oils, Scientific
African (2022), doi: https://doi.org/10.1016/j.sciaf.2022.e01223
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1
Formulation and evaluation of hair growth enhancing effects of oleogels made from
Rosemary and Cedar wood oils
Emmanuel Uronnachi1*, Chidiogo Atuegwu1, Chukwuebuka Umeyor1, Calistus Nwakile1,
Josephat Obasi1, Chidalu Ikeotuonye1, Anthony Attama2
1Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmaceutical
Sciences, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria.
2Department of Pharmaceutics, University of Nigeria, Nsukka, Enugu State, Nigeria.
ABSTRACT
Essential oils are one of the most popular natural products, with broad applications in
dermatology. Hair loss is a disorder in which the hair falls out from skin areas where they are
usually present, such as the scalp and the body.
The aim of the work was to formulate oleogels containing two essential oils (Cedarwood and
Rosemary) singly and in combination and evaluate their hair growth enhancing effect on an
animal model. Oleogels were formed using beeswax as the organogelator with concentrations of
10 % for the oils when used singly or 5 % each for the oils when formulated in combination.
Characterization of oleogels were done using spreadability, oil binding capacity (OBC), gas
chromatography (GC) and differential scanning calorimetry (DSC). Hair growth evaluation was
carried out in 18 albino rats in six groups of threes. A hair removal cream was applied on the
experimental animals and the Oleogel applied for six-weeks.
GC analysis of the oils revealed the presence of thujopsene, alpha-pinene, beta-pinene, pentane,
alpha cedrenea, beta cedrenea, alpha cedrol, gamma-terpene, acetonitrite, atlantone, terpinolene
in cedarwood oil while rosemary oil contained 1-8 cineole, camphor, alpha-pinene, beta-pinene,
camphene, p-cymene, alpha-terpinene, gamma terpinene, gamma-humulene, beat-bisabolene,
genaniol and terpinolene. DSC thermogram of the oleogel formulations showed varying degrees
of amorphicity.
Spreadability results showed that the oleogels containing cedarwood oil were more spreadable.
Conversely, oil binding capacity values were higher with Rosemary oil than Cedar wood oil with
values ranging from 91 % (for the oleogel containing 10 % rosemary oil) to 81 % for the bland
oleogel (no essential oil). Hair growth evaluation revealed that the oleogel containing rosemary
oil (10 %) had similar effects as the positive control (Minoxidil, 2 %) at the end of the six-week
period.
Oleogels made from cedar wood and rosemary oils have hair growth enhancing effects.
Key words: Oleogel, organogelator, Rosemary oil, Cedarwood oil, beeswax, hair growth.
*Corresponding author: Em.uronnachi@unizik.edu.ng

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1. Introduction
Complementary and alternative medicines are used by 60–80 % of developing countries because
they are one of the most prevalent medicines worldwide (Kasparaviciene et al., 2018). Essential
oils are among the most popular natural products, with significant applications in dermatology.
The essential oils are natural, nontoxic, non-pollutive, and biodegradable compounds with a wide
range of therapeutic benefits and a low risk of side effects after their use. Currently, semisolid
products have been of paramount importance in the pharmaceutical, cosmetics, nutraceuticals
and food industries. They have been used as gels, lotions, creams, ointments and jellies. In
general, gel-based products are classified depending on the polarity of the liquid component, as
hydrogels, emulgels, and organogels or oleogels. The use of oleogels has been exploited in
pharmaceutical, cosmetics, and nutraceutical industries for their desired rheological, physical,
and chemical stabilities in semisolid formulations (Balasubramanian et al., 2012). Oleogels are
characterized by many favourable features such as mucoadhesion, thixotropy or ease of
spreadability (Tomczykowa et al., 2018). Oleogels are composed of lipophilic fluids gelled with
suitable gelling agents. They can be designed to deliver both lipophilic (such as essential oils)
and hydrophilic drugs (Tomczykowa et al., 2018).
The process of hair growth occurs through different distinct phases: hair fiber production phase
(anagen); a transient regression phase (catagen) and a final resting phase (telogen) [Ma et al.,
2018]. Hair loss is a disorder in which the hair falls out from skin areas where they are usually
present, such as the scalp and the body (Blume-Peytavi et al., 2011). This loss interferes with the
many useful biologic functions of the hair like sun protection (mainly to the scalp) and dispersal
of sweat gland products (Blume-Peytavi et al., 2011).

3
Current United States Food and Drug Administration (FDA)-approved treatment options for hair
loss are restricted to topical Minoxidil (for men and women), oral Finasteride (men only), and
low-level light therapy (men and women) (FDA, 2010; Ashique et al., 2020). Unfortunately, all
therapies are limited by their incomplete efficacy and risk of recurrence after cessation, with
untoward side effects (Harries et al., 2010; Vesoulis et al., 2014; Mysore and Shashikumar,
2016). Corticosteroids can be administered orally, topically, or as intralesional injections;
however, they have only short-term benefits and systemic use is associated with multiple adverse
effects (Hosking et al., 2019).
Cedarwood, lavender, thyme, and rosemary oils have been used anecdotally for over 100 years to
treat hair loss (Hosking et al., 2019). Other medicinal herbs have also been explored for their
potential in enhancing hair growth (Lee et al., 2010; Dhanotia et al., 2011; Seo et al., 2013; Oh
et al., 2014; Jain and Dass, 2015; Jain et al., 2016; Shahtalebi et al., 2016; Ash et al., 2019).
Current literature reviews suggest that oleogel is a promising base for various drugs to design
topical formulations (O’Sullivan et al., 2016). The absence of an aqueous component offers a
better physical, microbiological and chemical stability when compared with conventional topical
bases, while the manufacturing process is simple. The work was therefore aimed at formulating
oleogels containing cedarwood and rosemary essential oils singly and in combination and
evaluating their hair growth enhancing effect on an animal model.
2. Materials and Methods
2.1 Materials
Weighing balance (HX-T, China), paraffin oil, beeswax (Qualikem, India), cedarwood oil and
rosemary oil (Vanity oil, Lagos Nigeria), olive oil (Goya, Spain).

4
2.2 Methods
2.2.1 Oleogel formulation
The oleogel was formulated using the formula below:
Table 1: Batch composition of preparations
Batch 1
Batch 2
Batch 3
Batch 4
Batch 5
Batch 6
Ingredients
Percentage quantities (%)
Beeswax
20
20
20
20
20
20
Rosemary oil
_
_
10
10
5
_
Cedarwood oil
10
10
_
_
5
_
Propylene
glycol
5
_
5
_
5
5
Olive oil
65
70
65
70
65
75
Briefly, about 26 g of the olive oil and 8 g of beeswax were weighed and transferred to a beaker.
This was put in a paraffin bath and placed on a magnetic stirrer, and the temperature set to 70 oC
and a stirring speed of 200 rpm until the beeswax completely dissolved. Afterwards, 2 g of
propylene glycol was transferred into the beaker. After ensuring a proper mix, the temperature of
the bath was reduced to 40 oC before adding 4 g of essential oil (cedarwood oil) in aliquot into
the beaker while stirring at the same time. After completely adding the essential oil, stirring was
continued for another five (5) minutes to ensure complete homogenization. The homogenized

5
preparation was then brought down from the paraffin bath to allow it to cool after which it was
carefully transferred into the container.
2.2.2 Characterization of Oleogel
2.2.2.1 Oil binding capacity (OBC)
This property was measured according to a modified method of Yılmaz and Öğütcü (2014).
The empty eppendorf tubes were labelled appropriately and weighed using the weighing balance
(HX-T, China). Afterwards, 1 g of the preparation (oleogel) in each batch was transferred into
the eppendorf tube, reweighed and recorded. The eppendorf tubes were refrigerated at a
temperature of 4 oC for 1 h. After refrigeration, the eppendorf tubes were re-weighed and
centrifuged using a refrigerated centrifuge (TGL-20M, China) at a speed of 9167 x g for 15 min.
These were done in triplicate and the OBC was then calculated using the formula:
% Released oil = =(b−a) −(c−a)
(b−a) x 100 ………………. (1)
% OBC = 100- Released oil ……………… (2)
Where:
a = weight of empty Eppendorf tube
b = weight of Eppendorf tube after refrigeration
c = weight of Eppendorf tube after centrifugation
2.2.2.2 Spreadability
This was performed using the slide and weight method according to Mbah (2013) as reported by
Kenechukwu et al. (2017) with slight modification (100 g weight was used). About 0.5 g of
oleogel formulation was placed on a glass slide and a second glass slide was placed over it.
Subsequently, the diameter of the spread was measured. Then, a weight of 100 g was placed to
rest at the upper glass slide for 5 min. The increase in diameter (in cm) due to spreading of the

6
oleogel was recorded. This was done in triplicate and spreadability was calculated using the
formula below:
Spreadability (%) = 𝐼𝑛𝑐𝑟𝑒𝑎𝑠𝑒 𝑖𝑛 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟
Initial diameter X 100 ………………………… (3)
2.2.2.3 Thermal analysis (DSC)
This was carried out using a differential scanning calorimeter (DSC 204, NETZSCHE,
Germany). Samples were heated from 10 oC to 250 oC at a rate of 10 oC per minute and baseline
correction was observed.
2.2.2.4 Gas chromatography
Samples of Rosemary oil and Cedarwood oil were prepared according to standard procedure
(AOAC 1990) and analysed using Gas Chromatography (GC).
2.2.3 Hair growth evaluation in experimental animal
Eighteen albino rats were allocated into six groups of three rats and used for the experiment.
Before commencement of the experiment, the rats were acclimatized for a period of seven (7)
days under standard environmental conditions of temperature, relative humidity, and 12 hours
dark/ light cycle. All animal experiments were conducted in compliance with the ethical
guidelines of the animal ethics committee of the Faculty of Pharmaceutical Sciences, Nnamdi
Azikiwe University, Awka, Nigeria and NIH guide for care and use of laboratory animals (Pub
NO: 85-23 Revised 1985).
2.2.3 1 Induction of hair loss
Hair loss was induced in the experimental animals using a commercially available hair removal
cream (Veet ®). This was done for all the experimental animals allocated into the different
groups.
2.2.3.2 Treatment of hair loss
This was done according to the following sequence: Batches 1, 3, 5 and 6 of the oleogel
formulation were applied to the experimental animals in groups 1, 2, 3 and 4 respectively. While

7
minoxidil (2 %) was applied to the experimental animals in group 5 (positive control) and
nothing was applied on the experimental animals in group 6 (negative control).
2.2.3.3 Evaluation of hair growth in experimental animal
Three hair samples from each of the grouped experimental animals were collected and the length
was measured in centimetre using a flexible meter rule. The hair density was also taken using a
scoring approach of 0 for no hair growth; 1 for less than 25 % hair growth; 2 for 25 to < 50 %
hair growth; 3 for 50 to < 75 % hair growth; 4 for 75 to < 100 % hair growth (Ma et al., 2018;
Oh et al., 2014). These were done for basal and also for a six-week period at regular intervals.
3. Results and Discussion
3.1 Characterization of Oleogel
The spreadability profile was assessed using weight method. Results as shown in fig. 1 revealed
that the spreadability of the oleogel formulation (Batch 1) containing cedarwood with propylene
glycol was higher than other batches. The presence of propylene glycol in the batches did not
improve the spreadability as was evidenced in the lower spreadability of batch 3 containing
rosemary oil when compared with batch 4 that contained the same rosemary oil but had no
propylene glycol. However, batch 1 had a higher spreadability than batch 2 that contained similar
ingredients but no propylene glycol. The difference in the spreadability values may have been
caused by the rheological properties of the individual oils (cedar wood and rosemary oil) used in
the study. Spreadability of semi solid formulations, that is the ability of a gel to evenly spread on
the skin, plays an important role in the administration of a standard dose of a medicated
formulation to the skin. The spreadability of dermal applications is a guide to the ease of
application of such samples to a thinly layered surface (Ogutcu and Yilmaz, 2014). This will
affect the effective surface area of the application, as more spreadable substances will require

8
less friction to spread thereby occupying a greater surface area for both cosmetic and therapeutic
effect.
Fig 1: Spreadability Chart
The oil binding capacity shown in Fig. 2 revealed that all the batches had good oil binding
capacity (greater than 80 %) with batch 3 having the highest capacity of 91 %. The results also
revealed that the oleogel had a better oil binding capacity for Rosemary oil than Cedarwood oil.
This effect was noticed even in the batch incorporating equal ratios of both oils (Batch 5) as its
oil binding capacity was greater than that of Cedarwood oil alone.
0
50
100
150
200
250
Batch 1 Batch 2 Batch3 Batch 4 Batch 5 Batch 6
spreadability (%)
Batches

9
Fig 2: Oil Binding Capacity Chart
Oil binding capacity is also another measure of the stability and spreadability of oleogels
(Ogutcu and Yilmaz, 2014). Lower oil binding capacities could give rise to softer oleogels,
which would affect the firmness and texture of the preparations (Da Reve et al., 2010; Co and
Marangoni, 2012). Also, the nature and concentration of organogelator used has an effect on the
oil binding capacity of the oleogel. Lower concentrations of organogelator may give rise to very
soft oleogels that may not sufficiently hold the oil; conversely, a high organogelator
concentration could give rise to a very viscous oleogel that would be poorly spreadable. A
suitable concentration has to be determined experimentally. Preliminary investigations carried
out using lower concentrations of oleogelator (5, 10 and 15 %) showed different degrees of
firmness with higher concentrations of oleogelator being less fluid. For our study, the beeswax
74
76
78
80
82
84
86
88
90
92
94
Batch 1 Batch 2 Batch 3 Batch 4 Batch 5 Batch 6
OBC (%)
Batches

10
(organogelator) concentration of 20 % yielded oleogels with the desired firmness, oil binding
capacity and spreadability. Our results are in concordance with that of some researchers who
have demonstrated the effect of increasing oleogelator concentrations on firmness and oil
binding capacity of oils (Yang et al., 2017; Ogutcu and Yilmaz, 2015). In addition, the ability of
an oleogelator to gel a solvent is a function of the solubility of the gelator in a given solvent (oil)
[Co and Marangoni, 2012]. This solubility is influenced by the nature and polarity of the solvent
(Wright and Marangoni, 2006). These may explain the differing oil binding capacity results
obtained with the batches incorporating the different oils (Cedarwood and Rosemary). The
presence of propylene glycol had a positive effect on the oil binding capacities of the
formulations containing the oils- Cedarwood and Rosemary oil as was evidenced in the greater
oil binding capacities of batch 1 over batch 2 and batch 3 over batch 4. In addition, the nature of
the oils may have played a role as oleogels containing rosemary oil (batches 3, 4 and 5) had
higher oil binding capacities than those containing cedarwood, in the presence or absence of
propylene glycol.
Thermal analysis of cedarwood oil, rosemary oil, olive oil, beeswax, and various batches of
oleogel formulation shown in Supplementary materials 1 and 2 revealed that beeswax is
crystalline while the oils are amorphous. The thermogram of crystalline substances gives sharp
peaks while the thermogram of amorphous substances gives broad peaks.
Gas chromatogram of cedarwood oil and rosemary oil was utilized to evaluate the content of the
Rosemary oil and Cedarwood oil shown in Supplementary materials 3 and 4.
Supplementary material three (3) shows GC chromatogram of Cedarwood oil. Compounds
observed include Thujopsene, Alpha-pinene, Beta-pinene, pentane, Alpha cedrenea, Beta
cedrenea, Alpha cedrol, Gamma-terpene, Acetonitrite, Atlantone, Terpinolene.

11
A few of the compounds such as thujopsene and alpha cedrol have been noted to have
therapeutic activities, which include anti-inflammatory, antispasmodic, tonic, astringent, diuretic,
sedative, insecticidal and antifungal activities (Jeong et al., 2014). Terpinolene has some
anticancer effect (Okumura et al., 2012).
Supplementary material four (4) shows GC Chromatogram of rosemary oil. Compounds
observed include 1-8 cineole, camphor, alpha-pinene, beta-pinene, camphene, p-cymene,alpha-
terpinene, gamma terpinene, gamma-humulene, beat-bisabolene, genaniol and terpinolene.
A few have been noted to have activities such as 1-8 cineole known for its mucolytic and
spasmolytic action on the respiratory tract (Juergens, 2014). Camphor relieves pain and promotes
hair growth (Cronkleton, 2018).
3.2 Hair growth evaluation in experimental animals
Fig. 3 showed the hair length of the experimental animals during the six-week period interval. It
was observed that the oleogel formulation increased the hair length of the experimental animals.
In week 1, there was no pronounced growth in hair length. In week 2, there was pronounced
growth in batch 3 and very pronounced growth in positive control when compared with Negative
control. In week 3, 4 and 5, there were very pronounced growth in both batch 3 and positive
control. In week 6, there was a pronounced growth in batch 5 and very pronounced growth in
batch 3 and positive control when compared with the negative control. Batch 3 had almost the
same effect with the positive control which means that it has hair growth enhancing effect.

12
Fig. 3: Hair Length Chart
Fig. 4: Hair Density Chart
-0.5
0
0.5
1
1.5
2
2.5
Basal Week 1 Weeek 2 Week 3 Week 4 Week 5 Week 6
hair length ( cm)
Treatment groups
batch 1 batch 3 batch 5 batch 6 positive control Negative control
-1
0
1
2
3
4
5
6
7
8
Basal Week 1 Week 2 Week 3 Week 4 Week 5 Week 6
Mean score
Treatment groups
Batch 1 Batch 3 Batch 5 Batch 6 Positive control Negative control

13
Fig. 4 showed the hair density of the experimental animals during the six weeks period interval.
Hair density means “hair fullness” i.e. the number of strands. It was observed that the application
of the oleogel formulation on the experimental animals within the scheduled period of study
increased their hair density. The hair densities were found to be within the scale of 0-4. The
batch 3 oleogel had a hair density of 75-100 % (score of 4) while the negative control had a hair
density of only 25-50 % (score of 2) indicating that the oleogel formulation had hair density
enhancing effect.
The results of hair length and hair density evaluations illustrate the effect of the animal’s body to
grow hair on its own, as evidenced in the growth observed with the negative group. However, as
was also observed, the growth arising from the formulations and the positive control exceeded
that of the negative control alone. Subtractions of the growth of the negative control from other
formulations were made and are presented in supplementary material 5. These figures give the
growth that could be attributable to the formulations and positive control alone.
Similar works investigating the ability of essential oils to promote hair growth have been done
and these include studies by Hay et al. (1998), Oh et al. (2014), Shatalebi et al. (2016) and
Goren & Naccarato (2018). These studies illustrated the hair growth enhancing effects of
essential oils.
4. Conclusion
Oleogels containing rosemary oil and cedarwood oil were successfully formulated using
beeswax as the organogelator and their hair growth potential evaluated. The formulation
containing rosemary oil (10 %) and propylene glycol (Batch 3) had the best hair growth
enhancing effect similar to that obtained from commercial preparations of minoxidil (2 %). The

14
combination of both oils (Cedarwood and Rosemary oils) did not offer any synergistic effect.
Future investigations can target the effect of essential oil concentration on improving hair
growth. In addition, further research is necessary to ascertain the toxicity of this preparation in
comparison with commercially available preparations (e.g. minoxidil).
Declaration of Interests: None.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial,
or not-for-profit sectors.
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