Preparaon and Characterizaon of Liposome Containing Minoxidil and
Rosemary Essenal Oil
Gita Kiaee1*, Hamid Akbari Javar1, Bita Kiaee2 and Shadi Kiaei3
1Tehran University of Medical Sciences, Tehran, Iran
2Islamic Azad University of Medical Science, Tehran, Iran
3Portland State University, USA
*Corresponding author: Gita Kiaee, Doctor of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran, Tel: 98218889 6692; E-
Received date: July 2, 2016; Accepted date: Aug 3, 2016; Published date: Aug 10, 2016
Copyright: © 2016 Kiaee G, et al. This is an open-access arcle distributed under the terms of the Creave Commons Aribuon License,
which permits unrestricted use, distribuon, and reproducon in any medium, provided the original author and source are credited.
Citaon: Kiaee G, Javar HA, Kiaee B, et al. Preparaon and Characterizaon of Liposome Containing Minoxidil and Rosemary Essenal Oil. J In
Silico In Vitro Pharmacol. 2016, 2:3.
Minoxidil and Rosemary essenal oil have been used for
several years to smulate hair growth. Therefore, co-
administraon of both Minoxidil and Rosemary essenal
oil could enhance hair growth. The chemical/biological
characteriscs of liposomes, which encapsulate both
hydrophobic and hydrophilic drugs, can be ulized to
encapsulate the herbal and chemical drug concocon
concomitantly. A thin-lm hydraon method was used to
prepare the liposomes. The entrapment ecacy of the
liposomes was determined for Minoxidil and Rosemary
essenal oil using UV spectrophotometry and
hydrodisllaon; as dictated in the European
pharmacopeia. Furthermore, a dynamic light scaering
(DLS) analysis was conducted to determine the parcle
size and zeta potenal of the prepared liposome. In
addion, the storage stability of the liposome was
checked aer 60 days. The results showed that co-
encapsulaon of Minoxidil and Rosemary essenal oil
increased the encapsulaon ecacy of Minoxidil while
the entrapment ecacy of essenal oil was not
signicantly inuenced. In addion, according to the DLS
results, the parcle size and zeta potenal of prepared
liposomes didn’t change signicantly during 60 days of
Liposomes; Hydrodisllaon; Spectrophotometry; Co-
Minoxidil and Rosemary essenal oil have been known for
their hair growth smulatory properes for several years
(Arcle 1999) [1,2] and there are several explanaons that
aribute to the Minoxidil mechanism of acon . One of the
biochemical acons of Minoxidil for hair growth is its
smulatory eect on prostaglandin and Vascular endothelial
growth factor (VEGF) synthesis . However, a limitaon to the
applicaon of Minoxidil is its poor skin penetraon ability and
water solubility . In order to enhance Minoxidil’s
penetraon and solubility it has to be formulated in an ethanol
based soluon  which has been known to cause dermas
irritaon, pruritus, erythema, scaling and dryness of skin . In
addion, Terpene compounds such as cineol, limonene, and
Nerolidol have been observed to improve the penetraon of
Minoxidil in the skin . These Terpene compounds, especially
cineol constute the major fracon of Rosemary essenal oil
[8-10]. Therefore, co-administraon of Minoxidil and
Rosemary essenal oil can lead to the high potency for a hair
The advents of novel drug delivery system provide the
appropriate means to encapsulate both hydrophilic and
hydrophobic compounds . Liposomes have capability of
increasing the concentraon of topically applied drugs in the
dermis while reducing the unfavourable risk by restricng the
absorbance of systemic drug . Moreover, the similarity of
lipid composion of liposomes and membranes of intercellular
lamellae and keranocytes render the improvement in drug
release properes and skin compability . Therefore,
liposomes appear to be an appropriate vehicle to co-
encapsulate Minoxidil and the Rosemary essenal oil .
Furthermore, previous studies of liposomal encapsulaon of
Minoxidil has led to the higher concentraon of drugs in the
pilosebace units in comparison to convenonal Minoxidil
formulaons . In addion, the organic solvent deleon of
convenonal formulaons reduces the adverse side eects of
long term applicaon. Moreover, liposomal encapsulaon of
Rosemary essenal oil could protect the essenal oil against
degradaon factors such as pH and light, and increase its
http://www.imedpub.com/ Journal of In Silico & In Vitro Pharmacology
© Copyright iMedPub | This article is available from: http://pharmacology.imedpub.com/archive.php 1
Therefore, the aim of this study is to prepare liposomes
containing Minoxidil and rosemary essenal oil that presented
acceptable physicochemical properes.
Materials and Methods
The Rosemary essenal oil was purchased from Barij
essence company and the other consumed substances were
purchased from Merck Company.
Liposomes were prepared by a thin-lm hydraon method
as reported in the literature accurately weighed quanes of
the Egg Phosphadyl choline (EPHC) and Cholesterol (CHOL)
with the molar rao of 7:3 were dissolved in methanol:
Chloroform (1:3) mixture. The soluon was placed in a rotary
evaporator (Rotavapor R 200/205, Buchi) at 55°C unl a thin
lipid lm on the wall of a round-boomed ask was obtained.
The resulng lipid lm was kept under a vacuum overnight in
order to eliminate traces of organic solvents. The lipid lm was
then hydrated with 10 mL of the aqueous soluon described in
(Table 1) for one hour. Aerwards the homogenous
suspension of the liposome was spun in the centrifuge for 45
minute at 15000 rpm. Following the separaon of the
supernatant, the sediment was rehydrated with dislled water
and it was sonicated for 10 min by using an ultrasound bath
(Transonic 460 H, Singen), and nally the liposome mixture
was extruded with a 400 nm lter.
Table 1: Content of aqueous soluon for hydrang a thin-lm
- 1 mg/ml - 1 mg/ml
- - 300 µl 300 µl
Determinaon of Minoxidil-entrapment
The supernatant of centrifuged suspension containing
liposome were analyzed at 285 nm spectrophotometrically.
The percent drug entrapment for the prepared liposome was
calculated by using equaon (1).
(Total drug added – non entrapped drug/Total drug added) ×
Determinaon of Rosemary essenal oil-
The quanty of entrapped essenal oil in the liposome was
determined via a method introduced in the European
pharmacopeia . The method was carried out by means of
steam disllaon by placing the prepared liposomal soluon in
the ask described below and heat it for 30 min (Figure 1).
Aer 30 min, heang was stopped, and following 10 min of
cooling a room temperature the volume of the essenal oil
was read o. The drug entrapment percentage was calculated
using equaon (2).
(The volume of essenal oil in the tubed aer 30 minutes of
heang/Total volume of added essenal oil) × 100 (2)
Figure 1: Hydro disllaon method: Clevenger type
Size and zeta potenal
The vesicle size and zeta potenal analysis of the liposomes
were carried out by using a Malvern Zetasizer 2000 HS
(Malvern instrument limited, Malvern, UK, NIPER, SAS Nagar,
Storage stability studies
In order to determine the physical stability of the liposomes,
size of the parcle and polydispersity index (PDI) were
measured by Malvern Zetasizer. The vesicles were stored at
4°C for up to 2 months under light protecon . In
predetermined me intervals, vesicles were characterized for
their vesicle size and PDI.
Results and Discussion
The inuence of rosemary essenal oil on
Minoxidil encapsulaon ecacy
The Minoxidil encapsulaon ecacy (EE) with and without
the essenal oil was 73% and 64% respecvely. Based on our
results, the liposome formulaon containing essenal oil
represented larger EE% of Minoxidil which was accompanied
by increase in the parcle size and zeta potenal of the
vesicles. The high zeta potenal frequently led to an increase
in the repulsion forces of the bilayer structure of the vesicles
Journal of In Silico & In Vitro Pharmacology
2This article is available from: http://pharmacology.imedpub.com/archive.php
which consequently increased the size of the liposomes.
Minoxidil, being parally hydrophobic, was expected to be
localized in the membrane compartment of lipid vesicles .
In Baranl et al.  study it was shown that Terpen compounds
such as cineol increased the entrapment ecacy of
hydrophobic drugs , thus the increase in EE could be
related to Terpen compound of essenal oil.
The inuence of Minoxidil on Rosemary
essenal oil encapsulaon ecacy
The essenal oil encapsulaon ecacy with and without
Minoxidil was 50% and 55% respecvely. There was no
signicant change to the encapsulaon ecacy of Rosemary
essenal oil accompanied with Minoxidil.
Size and Zeta potenal of liposomes
Zeta potenal and parcle size of the formulated vesicles
aer probe sonicaon is presented in Table 2. The results
showed that the average size of liposomes containing
Minoxidil was 169 nm with a PDI of 0.261, while the average
size of the liposomes containing Minoxidil and essenal oil was
183 nm with a PDI of 0.174. In cases of vesicles containing
solely essenal oil, the vesicle size was 187 nm with a PDI of
0.105. The parcle size of free-drug liposomes was 118 nm.
The increasing of parcle size of liposomes containing Terpene
was observed. These ndings are in agreement with previous
study . The PDI of the invesgated formulaons was below
0.3, which indicates the homogeneity of the prepared
liposomes . Regarding the zeta potenal measurements,
all liposomal dispersions had a negave surface charge
indicang that the formulaons were more stable and
homogeneous in distribuon. Moreover, liposomes containing
Terpene are more negave than convenonal liposomes.
These negave charge values of the obtained liposomes are
aributed to the presence of ethanol .
Table 2: Size and Zeta potenal of Formulaon (1): Liposome
without drugs; Formulaon (2): Minoxidil loaded liposome;
Formulaon (3): Rosemary essenal oil loaded liposome;
Formulaon (4): Minoxidil and Rosemary essenal oil loaded
Particle size Zeta potential
Formulation 1 118 -18.8
Formulation 2 169 -34
Formulation 3 187 -37.5
Formulation 4 183 -37
The physical stability of the four liposome formulaons
which were stored at 4°C for 60 days presented in Figures 2
and 3. The stability results showed minimal changes of parcle
size and PDI of the invesgated liposomes. The parcle size
and the PDI of liposomal dispersions had slightly increased
aer 60 days of storage. These results showed that the co-
existence of the Minoxidil and Rosemary essenal oil in the
vesicular formulaons did not aect the vesicle’s stability
Figure 2: Size of liposome parcle in 60 days of storage for
Formulaon (1): Liposome without drugs; Formulaon (2):
Minoxidil loaded liposome; Formulaon (3): Rosemary
essenal oil loaded liposome; Formulaon (4): Minoxidil
and Rosemary essenal oil loaded liposome formulaon.
Figure 3: Zeta potenal of liposome parcle in 60 days of
storage for Formulaon (1): Liposome without drugs;
Formulaon (2): Minoxidil loaded liposome; Formulaon
(3): Rosemary essenal oil loaded liposome; Formulaon
(4): Minoxidil and Rosemary essenal oil loaded liposome
Minoxidil and Rosemary essenal oil successfully entrapped
in the liposome with appropriate size and entrapment ecacy
which possible its consumpon as the future hair growth
smulator formulaon. The stability of formulaon in terms of
size and zeta potenal remained appropriate during 60 days of
Journal of In Silico & In Vitro Pharmacology
© Copyright iMedPub 3
1. Al-Serei MR, Abu-Amer KM, Sena P (1999) Pharmacology of
rosemary (Rosmarinus oicinalis) and its therapeuc potenals.
IJEB 37: 124-130.
2. Aburjai T, Natsheh FM (2003) Plants Used in Cosmecs.
Phytother Res 17: 987-1000.
3. Meisheri KD, Cipkus LA, Taylor CJ (1988) Mechanism of Acon of
Minoxidil Sulfate-Induced Role for Increased K Permeability
Vasodilaon: a role for increased K+ permeability. JPST 245:
4. Messenger AG, Rundegren J (2004) Minoxidil : mechanisms of
acon on hair growth. BJD 150: 186-194.
5. Moser K, Kriwet K, Naik A, Kalia YN, Guy RH (2001) Passive skin
penetraon enhancement and its quancaon in vitro. EJD p:
6. Balakrishnan P, Shanmugam S, Seok W, Mo W, Oh J, et al. (2009)
Formulaon and in vitro assessment of minoxidil niosomes for
enhanced skin delivery. Int J Pharm 377: 1-8.
7. Mura S, Manconi M, Sinico C, Valen D, Maria A (2009)
Penetraon enhancer-containing vesicles ( PEVs ) as carriers for
cutaneous delivery of minoxidil. Int J Pharm 380: 72-79.
8. Ahad A, Aqil M, Kohli K, Sultana Y, Mujeeb M, et al. (2011) Role
of novel terpenes in transcutaneous permeaon of valsartan:
eecveness and mechanism of acon. Drug Dev Ind Pharm 37:
9. Pres MLO, Ragusa S, Trozzi A, Dugo P, Mondello L, et al. (2005)
A comparison between dierent techniques for the isolaon of
rosemary essenal oil. J Sep Sci 28: 273-280
10. Badran M, Shazly G, El-badry M (2012) Eect of terpene
liposomes on the transdermal delivery of hydrophobic model
drug, nimesulide: Characterizaon, stability and in vitro skin
permeaon. Afr J Pharm Pharmacol 6: 3018-3026.
11. Kiaee G, Etaat M, Kiaee B, Kiaei S, Akbari H (2016) Mullayered
Controlled Released Topical Patch Containing Tetracycline for
Wound Dressing. J In Silico In Vitro Pharmacol 2.
12. Schreief H (1994) Liposomes and niosomes as topical drug
carriers: dermal and transdermal drug delivery. J control Release
13. Maghraby GM El, Barry BW, Williams AC (2008) Liposomes and
skin: From drug delivery to model membranes. Eur J Pharm Sci
14. Egbaria K, Weiner N (1990) Liposomes as a topical drug delivery
system. Adv Drug Deliv Rev 5: 287-300.
15. Mura S, Pirot F, Manconi M, Fadda AM, Falson F, et al. (2007)
Liposomes and niosomes as potenal carriers for dermal
delivery of minoxidil. J Drug Target 15: 101-108.
16. Saraf S, Ajazuddin (2010) Applicaons of novel drug delivery
system for herbal formulaons. Fitoterapia 81: 680-689.
17. Hossein M, Sourmaghi S, Kiaee G, Golfakhrabadi F, Khanavi M
(2015) Comparison of essenal oil composion and
anmicrobial acvity of Coriandrum savum L. extracted by
hydrodisllaon and microwave-assisted hydrodisllaon. JFST
18. Nasr M, Mansour S, Mortada ND, Elshamy AA (2008) Vesicular
aceclofenac systems: A comparave study between liposomes
and niosomes. J Microencapsul 25: 499-512.
19. Biruss B, Dietl R, Valenta C (2007) The inuence of selected
steroid hormones on the physicochemical behaviour of DPPC
liposomes. Pharmacological Reports 148: 84-90.
20. Touitou E, Dayan N, Bergelson L, Godin B, Eliaz M (2000) novel
vesicular carriers for enhanced delivery: characterizaon and
skin penetraon properes. J Control Release 65: 403-418.
Journal of In Silico & In Vitro Pharmacology
4This article is available from: http://pharmacology.imedpub.com/archive.php