Pharmaceutical

Communication

Biosci. Biotech. Res. Comm. 10(1): 103-108 (2017)

Preparation of nanoliposomes containing

Rosmarinus

of cinalis

L essential oil: A comparative study

Mohammad Hossein Arabi

, Hora Chabok

, Azam Mirzapour

, Mehdi Sha ee

Ardestani

and Mostafa Saffari

3,4

Biochemistry Department, School of Medicine, Kashan University of Medical Sciences, Kashan, Iran

Radiopharmacy Department, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

Department of Pharmaceutics & Medical Nanotechnology, School of Pharmacy, Pharmaceutical Branch of

Azad University, Tehran, Iran

Herbal medicine research center (HMRC) and Department of Pharmaceutics. Branch of Pharmaceutical

Sciences. Islamic Azad University, Tehran, Iran

ABSTRACT

According to progressive trend of herbal application in modern medicine, various studies has been shown several

properties for Rosemary essential oil (EO) such as antioxidant, anti-bacterial, anti-cancer and anti-in ammatory

effect. Owing to liposomal bene ts such as increased solubility, enhanced performance and increased stability of its

content, the main objective of this study was to design nanoliposomes containing rosemary essential oils to achieve

more ef cacy. Nano-liposomes containing EO was prepared by three different methods including thin  lm hydration,

sonication and extrusion methods. The physical properties of nanoliposomes such as particle size, poly dispersity

index, zeta potential, encapsulation ef ciency and release pro le of EO were studied. The mean size of liposomes

containing essential oils of rosemary prepared by sonication method was 162 nm which, is greater than extruder

method (470 nm) and liposomes were prepared by thin  lm hydration were about one µm. Encapsulation ef ciency

was higher in sonication method rather that extrusion method. Both methods lead to spherical particles. Release of EO

from liposomes in aqueous media was negligible (P value > 0.05). It was found that the method of liposomes prepa-

ration, cholesterol concentration and essential oil concentration is effective on the size and encapsulation ef ciency

of nanoliposome.

KEY WORDS: NANOLIPOSOME, ROSEMARY ESSENTIAL OIL, THIN FILM HYDRATION, EXTRUSION, SONICATION

103

ARTICLE INFORMATION:

*Corresponding Author: mostafa.saffary@gmail.com

Received 10

Jan, 2017

Accepted after revision 21

March, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

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Online Contents Available at: http//www.bbrc.in/

104 PREPARATION OF NANOLIPOSOMES CONTAINING

ESSENTIAL OIL OF ROSMARY

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Mohammad Hossein Arabi et al.

INTRODUCTION

Plants have been the basis for medical treatments through

much of human history, and such traditional medicine is

still widely practiced today. Modern medicine make use

of many plant-derived compounds as the basis for evi-

dence-tested pharmaceutical drugs, and phytotherapy

works to apply modern standards of effectiveness test-

ing to herbs and medicines that are derived from natural

sources. There are many forms in which herbs can be

administered, in many cases essential oils are used as

medicine. An essential oil is a concentrated hydropho-

bic liquid containing volatile aroma compounds from

plants. An oil is “essential” in the sense that it contains

the characteristic fragrance of the plant that it is taken

from (Oxford dictionary, 2014). Essential oils are com-

plex blends of a variety of volatile molecules such as

terpenoids, phenol-derived aromatic components, and

aliphatic components have been great interest in vari-

ous industries such as food and medicine (Bilia et al.,

2014).

Rosmarinus of cinalis L that belongs to the family

Lamiaceae (Lamiaceae) and have at least 1% (volume

/ weight) volatile oil Rosemary commonly used in the

food industry as spice and  avors. Rosemary of cinalis’s

essential oil possess a verity of properties include anti-

oxidant, antimicrobial, antitumor anti-HIV, anti-in a-

mation, analgestic effect and etc Peng et al., 2007). The

most important constituents of the Iranian Rosemary

are 1,8-cineole (23.47%), -pinene (21.74%), berbonone

(7.57%), camphor (7.21%) and eucalyptol (4.49%) (Alti-

nier et al., 2007, Honorio et al., 2015).

Rosemary essential oil used in various commercial

products on the market, Including bath additive for sup-

port the function of the skin and auxiliary treatment in

conditions of exhaustion, ointment for the symptomatic

treatment of muscle and joint pain, solutions for stimu-

lation of circulation (Jalali-Heravi et al., 2011).

Studies have shown that a new drug delivery meth-

ods such as encapsulation can reduce volatility, increase

solubility in water, increase dilution for use in the end

product and the ef cacy of essential oils (Sherry et al.,

2013). Nanocarriers can be structured by a great vari-

ety of material and designs (Saraf, 2010).

Liposomes are

bilayer vesicles that obtained from association of amphi-

philic lipids that can carry up hydrophilic, hydrophobic

and amphiphilic compounds. Nanoliposomes accord-

ing to the method and ingredients can have different

shapes and sizes (Moghimipour et al., 2012; Mozafari

et al., 2010). The aim of this study was encapsulation of

Rosemary EO oil in nanoliposome that done by three dif-

ferent methods including thin  lm hydration, extruder

and sonication. Then prepared liposomes, will compared

for their physicochemical properties.

MATERIAL AND METHODS

Soybean lecithin, cholesterol and DOTAP taken from

Sigma–Aldrich (Steinheim, Germany), Merck KGaA

(Darmstadt, Germany) and Lipoid GmbH (Ludwigshafen,

Germany), Rosemary essential oil (taken from Barij

Essence Kashan, Iran), PBS were purchased from Sigma–

Aldrich (Steinheim, Germany).

Cholesterol, phosphatidyl choline and rosemary

essential oil in several ratios of essential oil to total

lipids, and several molar ratio of cholesterol: lecithin

and lipid composition(DOTAP), were solved in chloro-

form/ ethanol (2: 1, v/v) inside the 500 ml round-bottom

 ask then by using rotary evaporator in constant tem-

perature above the lipid phase transition temperature

(Tc), under negative pressure and high vacuum for 2

hour organic solvent was removed and a thin layer was

formed in bottom  ask. Then the obtained lipid  lm was

hydrated by Phosphate buffered saline (PBS pH: 7.4) for

1.5 hour at temperatures above Tc and in 120 rpm of

speed.

Initial ratio of essential oils/total lipid was 1/3, 1/4

and 1/5. Molar ratio of Cholesterol: Phosphatidyl Cho-

line was 1: 1, 1: 2 and 1: 3 in 100 mM total lipid con-

centration.

In the preparation of Cationic liposomes containing

essential oil, phosphatidyl choline (E80), 1,2-dioleoyl-

3-trimethylammonium-propane (DOTAP), and choles-

terol (Chol) at a molar ratio of E80:DOTAP:Chol = 1:1:1

Formulation was used (Table1).

Liposomes obtained from thin  lm hydration method

was passed through Nucleopore polycarbonate  lters

(0.2 and 0.1 m in series, Whatman, UK) under strong

pressure at above the lipid transition temperature in

three stages by using the extruder device.

Table 1: Composition of cationic liposomes

formulation

Formulation

name

Chol: PC:

DOTAP

Rosemary essential

oil/total lipid

1:1:0 1/3

H2 1:1:0 1/4

H3 1:1:0 1/5

H4 1:2:0 1/3

H5 1:3:0 1/3

H6 1:1:1 1/3

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS PREPARATION OF NANOLIPOSOMES CONTAINING

ESSENTIAL OIL OF ROSMARY

105

Mohammad Hossein Arabi et al.

Table 2: Characterization of Rosemary EO containing nanoliposomes

Formulation

Extrusion method Sonication method

Thin  lm hydration

method

MD(nm) PDI) EE (%) MD(nm) PDI EE (%) MD(nm) PDI EE (%)

358.6±

1.3

0.46 83 ± 6.3 122 ±

3.2

0.35 98.9 1833.5 ±

926.5

0.51 100

346.9 ±

9.3

0.3 67.4 ± 6.9 104.33 ± 3.4 0.22 99.03 2747.5 ±

1632.5

0.50 100

230.9±

0.26 41.9 ± 4.3 92.36 ± 4.5 0.24 99.3 2453.9 ±

2186.1

0.65 100

366.9 ± 9 0.19 64.7 ± 3.3 154 ± 3.2 0.15 95.3 1757.5 ±

332.5

0.77 100

469.7 ±

2.5

0.19 47.7 ± 6.3 162 ± 4.3 0.12 96.3 1053

0.64 100

871.3 ±

0.29 97.5 ± 2.5 133 ±

1.0

0.23 95.3 2720 ±

0.71 100

MD: Mean diameter; PDI: Poly dispersity index; EE: encapsulation ef cacy;

Mean ± standard error of mean (SEM)

Liposomes obtained from thin  lm hydration method

were sonicated by probe sonicator (Hielscher UP400S,

Germany) at 70 Watt amplitude for 30 minutes.

The size and polydispersity (PDI) of formed liposome

was measured by Dynamic light scattering in a Zeta-

sizer Nano ZS (Malvern ZEN 3600). The samples were

diluted with phosphate buffer saline and measurement

was triplicate.

Charge on loaded vesicles surface and average zeta

potential is measured by zeta potential analyzers Zeta-

sizer instrument (Malvern ZEN 3600) (Kraft et al., 2014).

Encapsulation Ef ciency was determined by centrifu-

gation techniques (Bhatia et al., 2014).

In brief, liposomes

containing essential oil was isolated from unencap-

sulated materials by using centrifuge at 10000rpm for

10 minutes (laboratory centrifuge Hettich Universal

320 R). The liposomes were destroyed in ethanol 90%

(Merck,Germany) and encapsulated essential oil content

was measured by using UV/VIS spectrophotometer at

270 nm of wavelength.

The morphology of liposomal formulation were Stud-

ied by Scanning electron microscopy (SEM) using an

AIS2100 (Seron Technology, South Korea).The release

study was performed by using dialysis membranes

method. In summary, the 1000 L of the 14.20 mg/mL

essential oil encapsulated liposomal samples were inside

the dialysis bag (MWCO 12kDa, Thermo Fisher Scienti c).

The dialysis system was suspended in a release volume

of 100 mL PBS at 37°C and rotated at 100 rpm (1:100

dilution between donor and acceptor compartments). At

scheduled intervals, 1 ml of the release medium was col-

lected for the UV spectrophotometric assay. The same

volume of fresh PBS buffer at the same temperature was

added immediately to maintain constant release volume.

The length of the dialysis tubing was kept consistent

for all methods to ensure that the surface area available

for dialysis remained constant. To ensure that dilution

between the donor and acceptor compartments provided

sink conditions, a 1:100 dilution study was conducted

and release volume was set at 100 mL PBS.

Results are presented as mean ± SEM. Statistical

analysis was performed using SPSS Software (version

22). The mean values were compared by one-way analy-

sis of variance (ANOVA) followed by the post-hoc test.

The differences of P < 0.05 were considered as statisti-

cally signi cant.

RESULTS AND DISCUSSION

The results showed that nanoliposomes contain-

ing essential oils obtained from sonicator method are

smaller than extruder method. The average size of

nanoliposomes containing essential oil of three methods

were as follows: Thin Film> Extruder>sonicator. It was

also found that the concentration of essential oils affect

the size of the liposomes and nanoliposomes with low-

est essential oils have smaller size. On the other hand, it

was found that the ratio of cholesterol to total lipid in

the same concentration of Essential oil also affects the

particle size and in higher ratio of cholesterol to total

lipid, smaller nanoliposome can be achieved (Table 2).

The results showed that there was not signi cant

change on the PDI concentrations by changing the

Mohammad Hossein Arabi et al.

106 PREPARATION OF NANOLIPOSOMES CONTAINING

ESSENTIAL OIL OF ROSMARY

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

effective in loading of essential oil and increasing the

percentage of cholesterol lead to increase the loading of

the essential oil (Table 2).

THE MORPHOLOGY OF NANOLIPOSOMES

SEM Images of nanoliposome shown in Figure1. As can

be seen all containing particles are spherical shape and

a coherent. They are homogeneous in size and shape.

As showed in the Figure 2, Essential oil concentra-

tions within solvent is relatively stable. It indicates lack

of EO leakage from liposomes over time. After initial

release of less than 1 % of total EO no more release

could be seen. This behavior is very desirable in stor-

age stability of nanoliposomes. It is important that

liposomes can protect and keep inside their cargo before

reach target site.

DISCUSSION

Essential oils and their components are volatile and sen-

sitive to environmental factors such as light, heat, pH

and oxidation. Encapsulation of essential oils reduced

degradation and increased its stability before arriving

target site.

Compare liposomes with different ratio of essential

oil/ total lipid showed that the essential oils in uence

the size of nanoliposomes and increasing in Rosemary

essential oil concentrations, increased the size of the

liposomes. Probably due to the arrangement of essential

oil into the lipid bilayer of liposomes fusion of bilayer

fragment increase and larger liposomes would formed.

Also, results revealed that by increasing the concen-

tration of essential oils, encapsulation ef ciency is

increased (Stimac et al., 2017).

The results of this study

as well as previous studies conclude that the method

FIGURE 1. The SEM image of nanoliposomes contain-

ingrosemary essential oils; prepared by sonication

method (A) and extrusion method(B).

concentration of essential oil. But as the concentration

of cholesterol increased in both sonication and extrusion

methods, PDI was increased. EO containing liposomes

prepared by thin  lms have the highest PDI (0.9), but in

comparison the sonication and extrusion showed no sig-

ni cant difference (0.2; P value > 0.05). Add DOTAP to

the lipid composition of liposomes in comparison with

the same concentration of essential oils and cholesterol

showed not-signi cant difference, as shown in Table 2.

ZETA POTENTIAL

Nano-liposomes containing DOTAP have a zeta poten-

tial of +17 mV. Other liposomes due to the lack of charg-

ing ingredients, showed neutral charge.

Results showed that the preparation method of

nanoliposomes is effective on encapsulation of essential

oil. Nanoliposome prepared by sonicator and extrusion

have the maximum and Minimum of Essential oil load

respectively. Also increasing concentration of essential

oils is increasing its load into liposomes. On the other

hand, it was found that the percentage of cholesterol is

FIGURE 2. release curve for Rosemary Essential

Oil from H3 nanoliposomes containing Rose-

mary essential oils in hours

Mohammad Hossein Arabi et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS PREPARATION OF NANOLIPOSOMES CONTAINING

ESSENTIAL OIL OF ROSMARY

107

of preparation of liposomes is effective on the size and

encapsulation ef ciency (Stimac et al., 2017; Saffari

et al., 2013).

In comparison with the three methods of

nanoliposome preparation: thin  lm hydration, sonica-

tion and extrusion, the lowest and largest size of the

liposomes was related to sonication and thin  lm hydra-

tion method but the Essential loaded into the liposomes

is more in the sonication method compare to extrusion

method one.

Against studies that showed use of sonication method

reduces the encapsulation ef ciency in nanoliposomes

production and may also lead to damage liposomes

phospholipid and loaded compounds (Stimac et al., 2017;

Khatibi et al., 2015)

this study doesn’t show a signi cant

reduction in encapsulation ef ciency in preparation of

liposome by sonication method.

Poly dispersity index is an index of particle size dis-

tribution in a system, which low level of that, re ects

the uniformity of particle diameter distribution. PDI less

than 0.1, indicating homogeneous particle diameter and

the value of that greater than 0.3 indicates heterogene-

ous distribution of particle diameter (Sinico et al., 2005).

In previous studies it has been reported that essential oil

containing liposome have wider size distribution than

liposomes without essential oils (Ruozi et al., 2005).

Comparisons between different concentrations of essen-

tial oil in the preparation of liposomes showed that

change in oil concentration had not a signi cant effect

on the liposome size distribution. (P value: 0.178) in

preparation of rosemary containing liposomes.

Also in the comparison between three methods of lipo-

some preparation, Method of preparation of liposomes

did not have signi cant effect on PDI, However, thin

 lm hydration method was highest PDI.

Contrary to Ortan et al. reports, the results of this study

showed that cholesterol help to entrapment of essential

oil into nanoliposome structure, (Ortan et al., 2009) Unlike

previous studies that had been done on MLV liposome,

this study showed that in both methods of extrusion and

sonication, in the presence of essential oil, increasing the

concentration of cholesterol reduces the size of nanoli-

posomes (Arriaga et al., 2009; Detoni et al., 2009).

In analyze of SEM image of Nanoliposome contain-

ing Rosemary Essential Oil, particles had monodisper-

sity and spherical structure. This can reveal that critical

packing parameter of our composition is suitable for EO

containing liposomes.

The release study showed that essential oil release

from nanoliposome was very low during 24 hours

incubation time, which is probably due to lipophilic

properties of essential oil. Results showed impression

of multiple factor must be considered in this regard as

mentioned by different works (Fathi Moghaddam et al.,

2008; Rezaee et al., 2015)

EO can be trapped well inside

the liposomes. Adding DOTAP to the lipid composition

of liposomes enhances the encapsulation Ef ciency of

essential oil and on the other hand, increase the size of

the liposomes.

In brief, this study showed that the method of prepa-

ration of nanoliposomes containing Rosemary essential

oil has been effective on the particle size, dispersity and

encapsulation of essential oil. The study also found that

changes in formulation, percentage of cholesterol, addi-

tion of ionic lipid and using different ratio of essential oil

can cause changes in the physicochemical properties of EO

containing nanoliposomes. Present study also, revealed

that due to lipophilic and connection with nanoliposomes

rosemary EO have fewer tendency to release in physiolog-

ical environments. To determine the ef ciency and effec-

tiveness of drug delivery of liposomes, in-vivo studies on

animal models and MIC studies are useful.

ACKNOWLEDGEMENTS

The authors would like to thank Ms. Zahra Abbasian

& Ms. Astaraki for their valuable technical assistance.

This research was performed with support from research

Center of Kashan University of Medical Sciences.

Con ict of interest

There is no con ict of interest.

ABBREVIATIONS

EO: Essential Oil Tc: Transition temperature,PBS: Phos-

phate Buffered Saline, DOTAP: 1, 2-dioleoyl-3-trimeth-

ylammonium-propane, PC: Phosphatidyl Choline , Chol:

Cholesterol, PDI: Polydispersity Index, EE: Encapsula-

tion Ef ciency SEM: Scanning Electron Microscopy

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