Uniwersytet Medyczny w Białymstoku - Repozytorium
Repository of Medical University of Bialystok
https://ppm.umb.edu.pl
Publikacja / Publication
DOI wersji wydawcy / Published version DOI
Adres publikacji w Repozytorium URL /
Publication address in Repository
Data opublikowania w Repozytorium /
Deposited in Repository on
Rodzaj licencji / Type of licence
Cytuj tę wersję / Cite this version
Polska Platforma Medyczna
Polish Platform of Medical Research
https://ppm.edu.pl
Composition development and in vitro evaluation of O/W emulsions based
on natural emulsifier Olivem 1000 as tea tree oil carriers,
Wróblewska Magdalena, Winnicka Katarzyna
http://dx.doi.org/10.32383/appdr/158784
https://ppm.edu.pl/info/article/UMB1401560e41454486b2e00e5b0b8a5d66/
Jan 26, 2023
Attribution - NonCommercial 4.0 (CC BY-NC 4.0)
Wróblewska Magdalena, Winnicka Katarzyna: Composition development
and in vitro evaluation of O/W emulsions based on natural emulsifier
Olivem 1000 as tea tree oil carriers, Acta Poloniae Pharmaceutica, vol. 79,
no. 5, 2022, pp. 687-705, DOI:10.32383/appdr/158784
 Acta Poloniae Pharmaceutica – Drug Research, Vol. 79 No. 5 pp. 687-705, 2022 ISSN 2353-5288
DOI: 10.32383/appdr/158784 Polish Pharmaceutical Society
Received 18 November 2022, Received in revised form 19 December 2022, Accepted 28 December 2022

PHARMACEUTICAL TECHNOLOGY

COMPOSITION DEVELOPMENT AND IN VITRO EVALUATION

OF O/W EMULSIONS BASED ON NATURAL EMULSIFIER
OLIVEM 1000 AS TEA TREE OIL CARRIERS
MAGDALENA WRÓBLEWSKA* and KATARZYNA WINNICKA

Department of Pharmaceutical Technology, Faculty of Pharmacy,

Medical University of Białystok, J. Kilińskiego 1, 15-089 Białystok, Poland

Abstract: The frequent occurrence of bacterial skin infections makes it necessary to search for new solu-
tions. Due to the limited effectiveness of traditional antibacterial agents, novel treatment alternatives are
investigated. The use of plant-origin compounds seems to be beneficial because they are usually well-
tolerated and do not cause as many side effects as synthetic substances. Tea tree oil (TTO) is noted for
its well-established antimicrobial activity, so it can be used in the treatment of various skin diseases. It is
effective against Gram-negative as well as Gram-positive bacteria. The aim of this work was to develop
the composition and preparation technology of emulsion with TTO using the natural emulsifier Olivem
1000 - a combination of cetearyl olivate and sorbitan olivate. The designed formulations were evaluated
by testing the pH, stability, and droplet size of the dispersed phase in the prepared emulsions. The type of
Pobrano z https://ppm.edu.pl / Downloaded from Repository of Polish Platform of Medical Research 2023-03-09

emulsions was determined by the selective phase staining method. In addition, rheological, mechanical,
and ex vivo bioadhesive properties of the prepared emulsions were studied and their antibacterial activ-
ity against selected strains was estimated. Obtained formulations were non-Newtonian systems, showing
a shear-thinning behavior and thixotropic properties, with proper textural features and beneficial bioad-
hesive properties. It was also demonstrated that formulations with lower viscosity showed higher antimi-
crobial efficacy against tested microorganisms and larger zones of inhibition were observed in the case of
Escherichia coli strains. The results of the study showed that the prepared emulsions appear to be a suit-
able form of topical administration of TTO.

Keywords: tea tree oil, O/W emulsion, antibacterial activity, bioadhesiveness, textural properties.

Semisolid formulations, such as creams, gels,
and lotions, are the preferred pharmaceutical ve-
hicles for topical therapy as they remain longer at
the site of application and usually provide an ex-
tended drug release. Over the years, the interest in
emulsions has increased, once they possess a sub-
stantial and extensive number of applications (1,
2). Emulsions are systems formed by two immis-
cible liquids, one of which is dispersed in the oth-
er as droplets. These have multiple applications in
pharmaceutical and cosmetic products. When de-
signing emulsions, some important factors must
be taken into consideration, as they should provide
a pleasant feeling during the application, ensure
drug activity, suitable appearance, and good stabil-
ity (1-5). The stabilization of droplets found in the
emulsion interface is achieved through the addition
of an emulsifier which increases the electrostatic
repulsion between droplets, steric hindrance, and
decreases the phase interfacial tension. Currently,
there is a great interest in natural emulsifiers ob-
tained from olive oil such as Olivem 1000. Olivem
1000 is composed of cetearyl olivate and sorbitan
olivate. Suitably matched emulsifiers with the to-
tal of HLB (hydrophilic - lipophilic balance) 12 in
Olivem 1000 ensure appropriate stabilization of oil-
water O/W emulsions. In addition to the emulsify-
ing properties, Olivem is characterized by many
other features that are crucial in skin care products:
it protects the skin from excessive loss of moisture,
helps soften the skin, and provides perfect spread-
ability. Olivem 1000  is an emulsifier that creates
liquid crystal emulsions, the type of emulsion imi-
tating the arrangement of lipids in the surface layer
of skin which makes emulsions formed with it bio-
compatible (6). There are some publications describ-
ing carriers with TTO (7-10). The intention of our
work was to prepare topical formulations contain-
ing TTO based on natural origin ingredients such
as emulsifier Olivem 1000  and thickening agents

* Corresponding author: e-mail: magdalena.wroblewska@umb.edu.pl

687
 688 MAGDALENA WRÓBLEWSKA and KATARZYNA WINNICKA
sodium alginate or xanthan gum. Dermal formu-
lations containing natural products are highly val-
ued worldwide nowadays because they are consid-
ered safer, better tolerated, and less harmful to the
environment. The use of synthetic ingredients in
dermal products might cause adverse effects, e.g.
allergies. Olivem 1000 is a natural emulsifier with
unique properties creating stable emulsions with
the use of simple technics and relatively low cost.
Therefore, the preparation of stable emulsion sys-
tems using ingredients of natural origin seems to be
an interesting and important topic.
The natural products of plants such as essential
oils are highly valued worldwide. One such prod-
uct is TTO, the volatile essential oil derived mainly
from the Australian native plant Melaleuca alterni-
folia. TTO is transparent to slightly yellow in color,
with a cooling effect similar to that of menthol and
a strong odor just like camphor (11, 12). It can be
dissolved in organic solvents like alcohol and ace-
tone but is poorly soluble in water. The importance
Pobrano z https://ppm.edu.pl / Downloaded from Repository of Polish Platform of Medical Research 2023-03-09
of TTO is connected with its complex composition
which is characterized by high levels of antimicro-
bial and antioxidant ingredients. TTO is composed
of approximately 100 various chemical substances,
predominantly monoterpenes and sesquiterpenes,
with their respective alcohols, and the major com-
ponents of TTO are terpinen-4-ol and 1,8-cineole. It
is widely known because of its recognized biologi-
cal activities and this natural essential oil is consid-
ered safe and effective. Contemporary data reveal
the broad spectrum activity of TTO which includes
antibacterial, antifungal, antiviral, and antiprotozo-
al activities. It has been examined for its effects on
various superficial infections such as tinea, acne,
seborrheic dermatitis, scabies, dandruff, oral can-
didiasis, and cold sores (11-14).
The aim of this study was to design and evalu-
ate the physicochemical and application characteris-
tics of emulsions based on Olivem 1000 containing
TTO as an active compound. Moreover, the ex vivo
bioadhesive properties of the developed formula-
tions were examined and their antibacterial activ-
ity against Staphylococcus aureus ATCC 29213 and
Escherichia coli ATCC 25922 strains with the use
of diffusion method was estimated.
EXPERIMENTAL
Materials
Tea tree oil (TTO; ≤ 10% cineole, ≥ 34% ter-
pinen-4-ol), xanthan gum, and DMSO (dimethyl
sulfoxide) were obtained from Sigma Aldrich (St.
Louis, MO, USA). Glycerol 85%, tocopheryl acetate
(vitamin E), linseed oil, and liquid paraffin were
purchased from Fagron, Kraków, Poland. Tween
80, Span 80, and sodium alginate medium viscos-
ity - 2415  mPa·s of 1% solution at 25°C (Sigma
Aldrich, Steinheim, Germany), potassium sor-
bate and methylene blue (POCH, Gliwice, Poland),
Sudan III (Panreac, Barcelona, Spain), cetyl alco-
hol and glycerol stearate (Paulika, Kaczki, Poland),
Olivem 1000 - cetearyl olivate/sorbitan olivate (Zrób
Sobie Krem, Prochowice, Poland) were used as re-
ceived. All chemicals and solvents used for the ex-
periments were of analytical grade. Control strains:
S. aureus ATCC 29213, E. coli ATCC 25922 were
from TCS Biosciences Ltd, Buckingham, United
Kingdom. Mueller-Hinton agar was provid-
ed by Biomaxima (Lublin, Poland). Tribiotic
(5 mg + 0,833 mg + 0,01 g)/g ointment of the fol-
lowing composition: neomycin sulfate, polymyxin B
sulfate, zinc bacitracin, white petroleum jelly, used
as a control was a product of KETO LABS (series:
22311, expiration date: 04.2023; Warsaw, Poland).
Hairless mice skin from Cby.Cg-Foxn1nu/J was ob-
tained from the Experimental Medicine Center of the
Medical University of Białystok (the skin has been
achieved from hairless mice intended for the collec-
tion of organs and this procedure did not require the
approval of the Local Ethical Committee on Animal
Testing). Samples of the skin were stored at - 20°C.
On the day of the experiment, they were defrosted
and cut into 5 mm diameter pieces.
Composition development and preparation
of designed emulsions
Emulsions were prepared using paraffin or
linseed oil and various types of emulsifiers: Olivem
1000, combinations of Olivem 1000 with cetyl al-
cohol or glycerol stearate, and a mixture of Tween
80 and Span 80. Moreover, sodium alginate or xan-
than gum was added as thickeners and stabilizers.
The emulsions were obtained with the use of a me-
chanical stirrer RZR 2020 (Heidolph Instruments,
Schwabach, Germany). Placebo emulsions, with-
out an active substance, were initially prepared.
TTO in a  concentration of 10% w/w  was added
in the final stage of the emulsion production. The
concentration of the active substance was deter-
mined on the base of the available literature data
(11). Tables 1 and 2 show the compositions of the
prepared emulsions.
The ingredients of the aqueous phase of the
emulsion, i.e. water with glycerol, were weighed
into one vessel, then potassium sorbate was add-
ed and mixed until the substance was dissolved. In
the second vessel, ingredients of the oil phase were
Pobrano z https://ppm.edu.pl / Downloaded from Repository of Polish Platform of Medical Research 2023-03-09

Table 1. Compositions of the designed placebo emulsions with the addition of sodium alginate (F1 - F8) or xanthan gum (F1 G - F8 G).

Formulation code of placebo emulsions

Ingredient (g)
F1 F2 F3 F4 F5 F6 F7 F8 F1 G F2 G F3 G F4 G F5 G F6 G F7 G F8 G

Olivem 1000 8.0 8.0 8.0 8.0 8.0 8.0 - - 8.0 8.0 8.0 8.0 8.0 8.0 - -

Cetyl alcohol - 1.0 - - 1.0 - - - - 1.0 - - 1.0 - - -

Glycerol stearate - - 1.0 - - 1.0 - - - - 1.0 - - 1.0 - -

Tween 80 - - - - - - 4.0 4.5 - - - - - - 4.0 4.5

Span 80 - - - - - - 1.5 0.5 - - - - - - 1.5 0.5

Tocopheryl acetate 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05

Paraffin oil 20.0 20.0 20.0 - - - 20.0 - 20.0 20.0 20.0 - - - 20.0 -

Linseed oil - - - 20.0 20.0 20.0 - 20.0 - - - 20.0 20.0 20.0 - 20.0

Sodium alginate 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 - - - - - - - -

Xanthan gum - - - - - - - - 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Composition development and in vitro evaluation…

Potassium sorbate 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2

Glycerol 85% 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0
up to up to up to up to up to up to up to up to up to up to up to up to up to up to up to up to
Purified water
100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0
689
 690 MAGDALENA WRÓBLEWSKA and KATARZYNA WINNICKA

weighed: paraffin or linseed oil, emulsifi-

up to 100.0 up to 100.0 up to 100.0 up to 100.0 up to 100.0 up to 100.0 up to 100.0 up to 100.0 up to 100.0 up to 100.0 up to 100.0 up to 100.0
F6 G TTO ers (Olivem 1000, combination of Olivem

20.0

10.0
0.05
10.0

0.2
0.5
8.0

1.0
1000 with cetyl alcohol or glycerol stearate

-
-
-
and a mix of Tween 80 and Span 80), and
tocopheryl acetate. The oil phase was heated
F5 G TTO

to 70°C with constant mechanical stirring at

20.0

10.0
0.05
10.0

0.2
0.5
8.0
1.0

-
-
- 300 rpm until all the ingredients were melt-
ed and mixed. Next, the vessel containing
the water phase, heated to 70°C, was placed
F4 G TTO

on the mechanical stirrer and the oil phase

20.0

10.0
0.05
10.0

0.2
0.5
8.0

-
-
-
-

was gradually added with a stirring speed

of 800 rpm. After combining and mixing
both phases, thickener ingredients, i.e. so-
F3 G TTO

dium alginate or xanthan gum were added.

20.0

10.0
0.05
10.0

0.2
0.5
8.0

1.0

-
-
-

The formulation was mixed at 400 rpm un-

til it was cooled down to 35‑40°C and a sta-
ble emulsion was obtained. Active ingredi-
F2 G TTO

20.0

10.0
0.05
10.0

ent – TTO at 10.0% (w/w) was uniformly

0.2
0.5
8.0
1.0
Formulation code of emulsions with TTO

-
-
-

dispersed in previously prepared emulsion

vehicles and mixing was continued until
F1 G TTO

the ingredients were completely combined.

Pobrano z https://ppm.edu.pl / Downloaded from Repository of Polish Platform of Medical Research 2023-03-09

Table 2. Compositions of the designed emulsions with the addition of sodium alginate or xanthan gum containing tea tree oil (TTO).

20.0

10.0
0.05
10.0

0.2
0.5
8.0

Figure 1 presents a schematic diagram of

-
-
-
-

emulsion preparation.

Physicochemical and application
F6 TTO

10,0
20.0
0.05
10.0

0.2
0.5
8.0

1.0

properties of emulsions
-
-
-

Investigation of emulsion stability

The stability of the prepared emulsions
F5 TTO

was determined by the centrifuge test with

20.0

10.0
0.05
10.0

0.2
0.5
8.0
1.0

the use of the MPW-260 laboratory centri-

-
-

fuge (MPW Med. Instruments, Warsaw,

Poland). To indicate stability, the emulsion
F4 TTO

samples were centrifuged for 15  minutes

20.0

10.0
0.05
10.0

0.2
0.5
8.0

-
-
-
-

and the speed was set at 4000 rpm (5353 rcf)

(15).
F3 TTO

Determining the type of emulsion

20.0

10.0
0.05
10.0

0.2
0.5
8.0

1.0

-
-
-

by the dye method
The type of emulsion was determined
by the method of selective dyeing one of
F2 TTO

the phases by adding a dye dissolving only

20.0

10.0
0.05
10.0

0.2
0.5
8.0
1.0

-
-

in the water or the oil phase. Water-soluble

methylene blue and oil-soluble Sudan III
were used in this study. The stained sam-
F1 TTO

20.0

10.0
0.05
10.0

ples of emulsions were observed under the

0.2
0.5
8.0

-
-
-
-

optical microscope Motic BA 400 equipped

with a camera (Moticon, Wetzlar, Germany)
under 100x magnification (16).
Tocopheryl acetate

Potassium sorbate
Tea tree oil (TTO)

Glycerol stearate

Sodium alginate

Purified water
Ingredient (g)

Glycerol 85%
Xanthan gum
Cetyl alcohol
Olivem 1000

Paraffin oil
Linseed oil

Study of the droplets size of the dispersed

phase
The prepared emulsion formulations
were tested for the droplets’ size of the dis-
persed phase. The analysis was performed
 Composition development and in vitro evaluation…
aqueous phase of emulsion 70°C
sodium
stirring
alginate/xanthan
at 800 rpm
gum
mixing at 400 rpm
35-40°C
O/W emulsion
oil phase of emulsion
with emulsifiers 70°C
Pobrano z https://ppm.edu.pl / Downloaded from Repository of Polish Platform of Medical Research 2023-03-09
Figure 1. Schematic diagram of emulsion preparation.
using Motic BA 400 optical microscope equipped
with a  camera (Moticon, Wetzlar, Germany).
Samples of emulsions were observed under 100x
magnification, microscopic images were recorded
and the droplets’ size of the dispersed phase was
measured. The emulsions were dropped on a glass
slide and covered afterward with a coverslip. Fifty
emulsion droplets were captured by a  calibrated
Motic Image Plus 3.0 program and the diameter of
the droplets was measured, then the median size was
calculated (17).
pH determination
The pH measurement of prepared emulsions
was undertaken by employing the pH meter mod-
el Orion 3 Star (Thermo Scientific, Waltham, MA,
USA). The test involved immersing the electrode of
the apparatus six times in a sample of the prepared
emulsion, recording the determined pH value, and
calculating the average from the measurements. The
pH meter was calibrated before each use with stan-
dard buffer solutions.
Viscosity measurement and determination of
rheological properties
The rheological properties of the prepared
emulsions were tested using a Brookfield plate/cone
viscometer (RVDV-III Ultra, Brookfield Engineering
Laboratories, Middleboro, MA, USA), equipped
with the CPA52Z cone (plate diameter 24 mm, cone
angle 3°, the gap between the cone and the plate
691
tea tree oil
(TTO)
mixing at 400 rpm
25°C
O/W emulsion
with TTO
0.013 mm). 0.5 mL of the emulsion was applied to
the vessel of the apparatus, the number of rotations
was determined, and then the viscosity [mPa∙s] and
the shear stress [N/m 2] values were measured at the
specified shear rate [s-1]. During the measurement,
16 points were determined, 8 with increasing and
8 with decreasing speed of rotation of the cone. The
viscosity of emulsions at a shear rate of 8.0 s-1 was re-
corded and the rheograms were evaluated by plotting
the obtained values of shear stress versus shear rate
Test was carried out at a temperature of 25°C ± 5°C
(the apparatus was thermostated to maintain a con-
stant temperature).
Texture analysis
The evaluation of texture properties was car-
ried out using a TA.XT Plus texture analyzer (Stable
Micro System, Godalming, UK) for backward ex-
trusion measurements. The samples of emulsions
(30 g) were subjected to a stress of 10.0 g, and the
probe was immersed to a depth of 5 mm at the speed
of 2 mm·s-1. The measurements were performed at
25°C ± 5°C. Using the Texture Exponent software,
the obtained data were collected, analyzed, and the
hardness [g], compressibility [g·s], and adhesive-
ness [-g·s] were determined on the basis of force-
time plots (18).
Ex vivo bioadhesive properties of emulsions
Bioadhesive properties of the prepared pla-
cebo emulsions as well as formulations containing
 692 MAGDALENA WRÓBLEWSKA and KATARZYNA WINNICKA
TTO were performed using TA.XT.Plus Texture
Analyser (Stable Micro Systems, Godalming, UK)
on the skin of hairless mice as a model of the ad-
hesive layer. The skin samples, previously frozen
at -20°C, were prepared and separated into frag-
ments approximately 5 mm in diameter on the day
of the experiment. They were stored at 25°C ± 5°C
no longer than 30 minutes in physiological saline
(0.9% NaCl). Then, the pieces of mice skin were
attached to a cylindrical probe with cyanoacrylate
glue, which was placed over the samples of tested
preparations (0.5 g of emulsion). Measurements were
performed at 32°C ± 1°C, to mimic in vivo condi-
tions. The test parameters included: pretest speed
0.5 mm∙s-1, test speed 0.1 m∙s-1, contact time 150 s,
post-test 0.1  mm∙s-1, applied force 0.5 N. The ad-
hesive properties of the prepared emulsion prepa-
rations were defined as the maximum detachment
force Fad and the work of adhesion Wad (calculated
from the area under the force versus distance curve,
expressed in µJ) (18, 19).
Pobrano z https://ppm.edu.pl / Downloaded from Repository of Polish Platform of Medical Research 2023-03-09
The work of adhesion was calculated using
the formula:
Wad = A · 0.1 · 1000
where: A – area under the force – distance curve;
multiplication by 0.1 – conversion time measurement
to distance (the sampler was raised at 0.1 mm·s-1),
then multiply by 1000 to express the result in µJ.
This study did not require approval from the
Local Ethics Committee for Animal Experiments.
Antibacterial activity of prepared emulsions
with TTO against S. aureus and E. coli strains
The antibacterial activity of the prepared
emulsion systems was determined by the diffusion
method according to European Pharmacopoeia 10th
Edition, using the Mueller-Hinton medium (20).
Tested microorganisms included Gram(+) bacte-
ria: S. aureus ATCC 29213 and Gram(-) bacteria:
E. coli ATCC 25922. 20  mL of the medium was
poured into Petri dishes with a diameter of 90 mm.
The Mueller-Hinton agar plates were then allowed to
solidify and dry for 24 h at 37°C, then 100 μL of the
suspension of testing microorganisms were inocu-
lated (the density of the suspension was equivalent
to 0.5 McFarland scale: 1.5 x 108/mL, which corre-
sponds to the optical density of 0.125 at 550 nm) and
left to dry at room temperature for 15 minutes. In
the next stage, wells with a diameter of 5 mm were
cut out in agar plates, into which prepared formula-
tions (100 mg) were placed. The plates were incu-
bated at 37°C for 24 h. The results were recorded by
measuring the zones of growth inhibition surround-
ing the wells using a caliper (Mitutoyo, Kawasaki,
Japan) with an accuracy of 0.1 mm (21). Emulsions
placebo, TTO 100%, TTO at concentrations of 10%,
5%, 2.5% (solutions in DMSO), and commercially
available product (Tribiotic ointment) were used as
controls.
Statistical analysis
The results of the experiments are presented
as the mean ± standard deviation (SD) based on six
independent experiments. Statistical analysis was
done by one-way analysis of variance (ANOVA)
using Statistica 12.0  software (StatSoft, Kraków,
Poland). A probability level of P < 0.05 was consid-
ered significant.
RESULTS AND DISCUSSION
Composition development and preparation of
emulsions with tea tree oil
Preparations intended for use on the skin
surface should be properly designed to maintain
the structure and functions of the skin. There is
a  hydro-lipid coat on the surface of the skin and
its primary role is protection against external fac-
tors and microorganisms and reducing water loss.
Accurately selected moisturizing and nourishing
ingredients strengthen the protective hydro-lip-
id coat of the skin. When choosing dermatologi-
cal products, the type of dosage form, as well as
its composition, are of great importance, as they
might affect the effectiveness and safety (22, 23).
Pharmaceutical products commonly used topically
in various skin conditions include simple O/W (oil
in water emulsions - the oily phase is dispersed in
water) or W/O (water in oil - the phase dispersed
in oil is water). Emulsions are promising formu-
lations enabling allow both hydrophilic and lipo-
philic active substances to be delivered locally to
the skin surface. O/W emulsions usually are light,
skin-soothing, non-greasy, easily washable, and
quickly absorbing preparations. The content of oil
combined with the water phase usually does not
exceed 20-30%. This type of emulsion is charac-
terized by a reduced content of lipid components
therefore, they slightly protect the skin against dry-
ness, but additionally provide a feeling of cooling
and refreshment (24, 25). To obtain a stable disper-
sion of two immiscible liquid phases, it is neces-
sary to use an emulsifier that acts as a stabilizer.
Compounds facilitating emulsification increase the
stability of the emulsion by reducing the interfa-
cial tension and preventing the aggregation of the
 Composition development and in vitro evaluation…
dispersed phase droplets (26, 27). In this work, var-
ious emulsifiers or their mixtures were employed
in order to increase the stability of designed for-
mulations. Olivem 1000 – a biodegradable, natu-
ral emulsifying compound and the combinations of
Olivem 1000 with cetyl alcohol or glycerol stearate
were applied. Olivem 1000  is an emulsifier that
creates liquid crystal emulsions. The lowest effec-
tive concentration of Olivem 1000 usually used is
2-3%, and at this concentration, a strong lamellar
network is formed. The lamellar liquid crystals
are similar to the stratum corneum lipidic struc-
ture and they have a great skin hydration potential.
Emulsion creation with an emulsifier that forms
a liquid crystal phase reduces transepidermal wa-
ter loss (TEWL) without solubilizing proteins and
lipids naturally contained in the skin. The ability
of Olivem 1000 emulsions to prevent TEWL may
last for several hours after application thus provid-
ing long-term moisturizing and hydrating benefits.
This type of emulsion mimics the arrangement of
Pobrano z https://ppm.edu.pl / Downloaded from Repository of Polish Platform of Medical Research 2023-03-09
lipids in the surface layer of the skin and this aspect
makes emulsions formed with Olivem 1000  bio-
compatible (6). In addition, a mixture of non-ionic
emulsifiers often used in pharmaceutical technol-
ogy, i.e. Tween 80 and Span 80, was used to obtain
emulsions. In order to increase the viscosity and
stability of the designed emulsions, it was neces-
sary to use thickeners such as sodium alginate or
xanthan gum. Polysaccharides are widely used as
excellent emulsion stabilizers during the prepara-
tion of emulsions. Various polysaccharides have
different mechanisms for improving emulsion sta-
bility. Some polysaccharides, such as pectin, aca-
cia gum, or chitosan can form a complex with the
emulsifier, which prevents the coalescence of the
droplets by electrostatic interaction and steric hin-
drance. Other polysaccharides increase the viscosi-
ty of the emulsion by thickening or forming a three-
dimensional gel-like network in the aqueous phase
to prevent droplets from aggregation e.g. xanthan
gum, guar gum. Sodium alginate is a linear anionic
polysaccharide, it consists of mannuronic and gu-
luronic acid residues. It is obtained from brown sea
algae by extraction, is biodegradable and biocom-
patible, as well as non-toxic, therefore it is widely
used for pharmaceutical and biomedical applica-
tions (28, 29). It stabilized emulsion droplets due
to its charging, thickening, and gelling properties.
Hence, the addition of sodium alginate influences
the microstructure, textural attributes, and gela-
tion characteristics of the resultant emulsion sys-
tem, thereby improving its stability and function-
al properties. Another thickening agent frequently
693
used in emulsion systems is xanthan gum, which is
a negatively charged high molecular weight poly-
saccharide. It is obtained by fermentation from
Xanthomonas campestris, and its skeleton resem-
bles the structure of cellulose. Xanthan gum shows
bioadhesive properties, biocompatibility, and bio-
degradability. It significantly increases the viscos-
ity even at low concentrations, therefore it has been
often used in the production of emulsions and sus-
pensions as a stabilizer and thickening agent (30,
31). It is usually added to the aqueous phase to im-
prove the stability of O/W emulsions since it forms
a three-dimensional gel-like network in the aque-
ous phase and this provokes a delay in the creaming
process. The thickening ingredients used in formu-
lations additionally stabilized the emulsion systems
by increasing the viscosity of the dispersing phase
and preventing the colloidal particles from joining
together into larger aggregates, which could nega-
tively affect the emulsion stability (29-31). Two oils
were used as components of the lipid phase of the
prepared dermatological emulsions: liquid paraf-
fin and linseed oil. Liquid paraffin is a mineral oil
with good chemical and physical stability. After
application to the skin, it forms an occlusive layer
on its surface, thus reducing the loss of water. It
also acts as an emollient and makes the skin more
smooth and soft, and indirectly moisturizes the skin
(32). Linseed oil contains vitamin E and fatty ac-
ids, including omega-3, omega-6, and omega-9. It
is characterized by an anti-inflammatory activity
and it normalizes the function of sebaceous glands,
therefore it is recommended for oily, acne-prone
skin and skin susceptible to inflammation. In addi-
tion, linseed oil reduces irritations, accelerates the
regeneration of the epidermis, and effectively im-
proves the condition of the skin, and the vitamins
contained in it make the skin smooth and properly
taut. These properties are used in dermatological
preparations and linseed oil can be helpful in the
treatment of eczema (20, 33).
The prepared formulations were visually in-
spected for color, homogeneity, consistency, and
phase separation. Depending on the oil used, the
emulsions were white (paraffin oil) or light yellow
(as a result addition of linseed oil). The emulsions
which included the mixture of Span 80 and Tween
80 as emulsifiers (formulations F7, F8, F7 G, F8 G)
were unstable, as shown in Figure 2. The coales-
cence and the separation of the aqueous phase 24 h
after preparation were observed.
Stable emulsions were obtained with the use of
Olivem 1000 at a concentration of 8% and the combi-
nation of Olivem 1000 (8%) with cetyl alcohol (1%)
 694 MAGDALENA WRÓBLEWSKA and KATARZYNA WINNICKA

containing TTO demonstrated smooth and homoge-

neous consistency and possessed a specific menthol-
camphor spicy smell.
In order to determine the type of prepared
emulsions, the method of selective staining of the
emulsion phases with two dyes was used. In the
case of emulsions stained with methylene blue, the
microscopic images showed colorless drops of the
oil phase in the blue-colored aqueous phase. On the
other hand, in the case of emulsion samples stained
with Sudan III, the microscopic image showed pink-
stained drops of the oil phase dispersed in the aque-
ous phase (data not shown). Thus, the obtained ob-
servations indicated that the prepared preparations
F7 F8
belong to the O/W emulsions.

Physicochemical, rheological, and textural

Instability of prepared emulsions characteristics of prepared emulsions
– separation of the aqueous phase
In emulsions intended for application to the
Figure 2. Phase separation in emulsions containing Tween 80 and skin, the droplet size of the dispersed phase is im-
Span 80. portant because it firmly affects the stability of the
Pobrano z https://ppm.edu.pl / Downloaded from Repository of Polish Platform of Medical Research 2023-03-09

formulation. Both the degree of dispersion of the

or glycerol stearate (1%). They were characterized
by a uniform creamy appearance and good spread-
ability. Furthermore, obtained emulsion bases were
not greasy in touch, with no sign of phenomena as-
sociated with the physical instability of emulsions
such as creaming or sedimentation, flocculation, co-
alescence, or phase inversion. They were selected as
vehicles and TTO at a concentration of 10% was add-
ed in the final stage of emulsions preparation. The
addition of the essential oil did not adversely affect
the stability of the designed emulsions. Formulations
internal phase and the homogeneity of the droplets
are important for maintaining the stability of the
preparation. It was proven that smaller droplet sizes
have a positive effect on the stability of the emul-
sion, preventing phenomena leading to its destabi-
lization, such as creaming, flocculation, or coales-
cence (34). Based on the data presented in Table 3
and Figures 3 and 4, it was found that the size of the
droplets in the emulsions with the addition of so-
dium alginate was in the range of 5-262 μm, while
in the formulations with xanthan gum it was in the

Table 3. Droplets size of the dispersed phase in prepared emulsions containing TTO with the addition of sodium alginate
(F1 TTO - F6 TTO) or xanthan gum (F1 G TTO - F6 G TTO).

Droplets size of the dispersed phase in Droplets size of the dispersed phase in
Formulation code
formulations - average (µm) formulations - size range (µm)
F1 TTO 64.5 ± 61.5 6-262 μm
F2 TTO 58.5 ± 63.1 7-240 μm
F3 TTO 64.7 ± 80.3 5-239 μm
F4 TTO 53.6 ± 32.3 6-115 μm
F5 TTO 53.3 ± 30.6 6-115 μm
F6 TTO 53.4 ± 40.8 6-140 μm
F1 G TTO 59.7 ± 52.6 5-145 μm
F2 G TTO 63.0 ± 42.3 6-220 μm
F3 G TTO 48.3 ± 39.7 5-182 μm
F4 G TTO 51.4 ± 28.9 5-115 μm
F5 G TTO 60.3 ± 55.8 5-169 μm
F6 G TTO 50.0 ± 52.2 6-119 μm
 Composition development and in vitro evaluation…
F1 TTO
90 μm
F2 TTO
Pobrano z https://ppm.edu.pl / Downloaded from Repository of Polish Platform of Medical Research 2023-03-09
90 μm
F3 TTO
90 μm
Figure 3. Microscopic images of prepared emulsions containing TTO with the addition of sodium alginate (F1 TTO-F6 TTO) under
magnification 100×.
range of 5-220 μm. The mean of the droplets’ size
of the dispersed phase was greater in the emulsions
prepared with the addition of sodium alginate com-
pared to the systems with xanthan gum. In formu-
lations with xanthan gum based on paraffin oil and
linseed oil as components of the lipid phase, larger
droplets size was observed in emulsions containing
mixture of Olivem 1000 and cetyl alcohol used as
an emulsifier. Based on the tests performed, it can
also be concluded that formulations containing par-
affin oil were characterized by larger droplets size
695
F4 TTO
90 μm
F5 TTO
90 μm
F6 TTO
90 μm
of the dispersed oil phase. The particle size depends
on numerous factors such as the type and concen-
tration of emulsifiers, phase volume fractions, inter-
facial properties, viscosity ratios, and composition
of the phases. It was also demonstrated that higher
droplet size might be caused by the higher viscosity
of the oil used in the formulation. A more viscous
oil phase might be difficult to disperse into smaller
droplets. Upon increasing a difference in the vis-
cosity of the non-miscible phases, the size of the oil
droplets might grow (35, 36).
 696 MAGDALENA WRÓBLEWSKA and KATARZYNA WINNICKA
F1 G TTO
90 μm
F2 G TTO
Pobrano z https://ppm.edu.pl / Downloaded from Repository of Polish Platform of Medical Research 2023-03-09
90 μm
F3 G TTO
90 μm
Figure 4. Microscopic images of prepared emulsions containing TTO with the addition of xanthan gum (F1 G TTO-F6 G TTO) under
magnification 100×.
Preparations intended for application to the
skin should be characterized by an appropriate pH,
which is important for the safety of the product. The
studies showed that desired pH of the dermal formu-
lations is regarded as ≤ 7. Obtaining the proper pH
of the topical products enable avoiding irritation at
the site of application, and also maintains the proper
functioning of the skin (37, 38). The pH of all for-
mulated placebo emulsions was in the range of 5.9-
6.6 (Figure 5). Therefore, it was in accordance with
the pH expected for preparations applied to the skin.
F4 G TTO
90 μm
F5 G TTO
90 μm
F6 G TTO
90 μm
Higher pH values (6.2-6.6) were found for systems
containing sodium alginate as a viscosity-enhanc-
ing agent compared to formulations with the addi-
tion of xanthan gum (5.9-6.4). In the case of formu-
lations containing TTO, a slight increase in pH was
noted (6.3-6.6 - preparations with sodium alginate,
6.2‑6.4 – emulsions with xanthan gum), however,
these pH values can be considered appropriate for
dermal preparations.
Rheological features have a  significant im-
pact on the quality and application properties of
 Composition development and in vitro evaluation… 697

A Placebo emulsions Emulsions with TTO

6.7

6.6

6.5

6.4

6.3
pH

6.2

6.1

6.0

5.9
F1 F1 F2 F2 F3 F3 F4 F4 F5 F5 F6 F6
TTO TTO TTO TTO TTO TTO

B
Placebo emulsions Emulsions with TTO
Pobrano z https://ppm.edu.pl / Downloaded from Repository of Polish Platform of Medical Research 2023-03-09

6.5

6.4

6.3

6.2

6.1

6.0
pH

5.9

5.8

5.7

5.6
F1 G F1 G F2 G F2 G F3 G F3 G F4 G F4 G F5 G F5 G F6 G F6 G
TTO TTO TTO TTO TTO TTO

Figure 5. The pH of prepared placebo emulsions with the addition of sodium alginate F1-F6, emulsions with the addition of sodium
alginate containing TTO F1 TTO-F6 TTO (A), and placebo emulsion with the addition of xanthan gum F1 G-F6 G, emulsions with the
addition of xanthan gum containing TTO F1 G TTO-F6 G TTO (B).

dermatological formulations. Due to the fact that
pharmaceutical emulsions are subjected to high
shear forces during preparation, the quality of the
product may deteriorate. The drug forms intended
for application to the skin should be of suitable vis-
cosity. Higher viscosity means that the preparation
stays longer at the application site (skin surface),
while the active substance is released slowly from
the carrier, which may be beneficial in the treat-
ment of superficial dermatological infections (39).
This parameter also affects the spreadability of
emulsions and the conditions to be applied during
production processes (mixing and filling contain-
ers). The use of substances increasing the viscosity
extends the shelf life and ensures a homogeneous
consistency of the products (26, 36). The conducted
research shows that emulsions containing xanthan
gum were characterized by higher viscosity values
compared to preparations with sodium alginate used
as a thickening agent (Figure 6). Moreover, it can
be concluded that in all prepared emulsions the ad-
dition of TTO caused a noticeable increase in vis-
cosity. The highest (1.4 times) growth in viscosity
after the addition of TTO was observed in the case
of the F6 TTO formulation, which contained sodium
alginate, linseed oil, and a mixture of the Olivem
1000 with glycerol stearate. In contrast, the small-
est change of the tested parameter (1.1-fold increase
 698 MAGDALENA WRÓBLEWSKA and KATARZYNA WINNICKA

in viscosity) was observed in the F2 TTO emulsion
with sodium alginate, paraffin oil, Olivem 1000,
and cetyl alcohol. The effect of the addition of es-
sential oils on the viscosity of preparations was also
described in other research studies. Mut with co-
workers showed that cinnamon oil (3%) increased
the viscosity of the gel formulation by 1.3 times (40).
Moreover, the addition of TTO also caused growth
in the viscosity of Pluronic® F-127 gels with keto-
conazole. In this research work, it was found that
the addition of 5% TTO to the designed formula-
tions resulted in an 11‑fold increase in the viscos-
ity of the preparation (19). Differences in the vis-
cosity of the formulations after adding TTO may
result from the varied and complex chemical com-
position of this essential oil and its polarity, which
might contribute to the interaction between the oil
components and the emulsion ingredients, i.e. the
polymers used (sodium alginate, xanthan gum) or
emulsifiers (19, 40, 41).
On the basis of the obtained rheograms, it can
be concluded that all prepared emulsions belong to
the non-Newtonian shear thinning systems. The
hysteresis loops illustrate the behavior of emulsion
when it is subjected to shear stress. As it was dem-
onstrated, with the increase of the shear rate, the sys-
tem is thinned, and thus its viscosity decreases. The
resulting loops, presented in Figures 7 and 8, allow
the tested preparations to be classified as thixotropic
systems. They are characterized by the ability to re-
structure, and thus restore the consistency lost under
shear stress (18, 36).

A Placebo emulsions Emulsions with TTO

16000

14000
Viscosity [mPa·s]
Pobrano z https://ppm.edu.pl / Downloaded from Repository of Polish Platform of Medical Research 2023-03-09

12000

10000

8000

6000

4000

2000

0
F1 F1 F2 F2 F3 F3 F4 F4 F5 F5 F6 F6
TTO TTO TTO TTO TTO TTO

B Placebo emulsions Emulsions with TTO

16000

14000

12000
Viscosity [mPa·s]

10000

8000

6000

4000

2000

0
F1 G F1 G F2 G F2 G F3 G F3 G F4 G F4 G F5 G F5 G F6 G F6 G
TTO TTO TTO TTO TTO TTO
Figure 6. The viscosity of prepared placebo emulsions with the addition of sodium alginate F1-F6, emulsions with the addition of sodium
alginate containing TTO F1 TTO-F6 TTO (A), and placebo emulsions with the addition of xanthan gum F1 G-F6 G, emulsions with the
addition of xanthan gum containing TTO F1 G TTO-F6 G TTO (B).
 Composition development and in vitro evaluation… 699

A F1 F2 F3 F4 F5 F6
180

160

140
Shear stress [N/m2]

120

100

80

60

40

20

0
0 2 4 6 8 10 12 14 16 18
Shear rate [s-1]

B F1 TTO F2 TTO F3 TTO

F4 TTO F5 TTO F6 TTO
180
Pobrano z https://ppm.edu.pl / Downloaded from Repository of Polish Platform of Medical Research 2023-03-09

160
Shear stress [N/m2]

140

120

100

80

60

40

20

0
0 2 4 6 8 10 12 14 16 18
Shear rate [s-1]

Figure 7. Rheograms of prepared placebo emulsions with the addition of sodium alginate F1-F6 (A) and emulsions with the addition
of sodium alginate containing TTO F1 TTO-F6 TTO (B).

The analysis of mechanical properties, which
include hardness, compressibility, and adhesive-
ness, provides the assessment of the behavior of the
preparations during their application to the skin.
It is important that the product adheres well to the
skin surface and that it can be easily removed from
the packaging container. Hardness is defined as the
maximum force required to induce deformation.
Compressibility is described as the work required
to deform the preparation under applied pressure.
Adhesiveness, on the other hand, is described as the
work required to overcome the pull forces between
the tested product and the surface of the measur-
ing device (36, 42). High adhesiveness prolongs the
contact time of the preparation with the skin (24, 42)
During the study, it was shown that the emulsions
prepared with the use of the mixture of emulsifi-
ers are characterized by higher values ​​of hardness,
compressibility, and adhesiveness compared to for-
mulations containing single Olivem 1000 (Table
4). Moreover, it was noticed that the addition of tea
tree oil contributed to an increase in the values of
the examined mechanical parameters in all ana-
lyzed emulsions. The lowest values of hardness,
compressibility, and adhesiveness in cease of emul-
sions containing TTO were recorded for the formu-
lation with sodium alginate, Olivem 1000, and par-
affin oil (F1 TTO). On the other hand, high values
of the analyzed parameters were observed in the
emulsion with xanthan gum, the combination of the
Olivem 1000 with glycerol stearate, and linseed oil
(F6 G TTO). It can be concluded that the prepared
 700 MAGDALENA WRÓBLEWSKA and KATARZYNA WINNICKA

A F1 G F2 G F3 G F4 G F5 G F6 G
180

160

140
Shear stress [N/m2]

120

100

80

60

40

20

0
0 2 4 6 8 10 12 14 16 18
Shear rate [s-1]

F1 G TTO F2 G TTO F3 G TTO

B F4 G TTO F5 G TTO F6 G TTO

180
Pobrano z https://ppm.edu.pl / Downloaded from Repository of Polish Platform of Medical Research 2023-03-09

160

140
Shear stress [N/m2]

120

100

80

60

40

20

0
0 2 4 6 8 10 12 14 16 18
Shear rate [s-1]

Figure 8. Rheograms of prepared placebo emulsion with the addition of xanthan gum F1 G-F6 G (A) and emulsions with the addition of
xanthan gum containing TTO F1 G TTO-F6 G TTO (B).

emulsions showed adequate compressibility, essen-
tial for the formulation to retain at the application
site. Formulation characterized by good compress-
ibility is easier to spread as a continuous thin layer
on the surface of the skin and enables better retain-
ing at the application site. These parameters might
also indicate the appreciable mechanical strength of
the preparation, easy applicability, and ease of extru-
sion from the tube, which are valuable for an ideal
topical formulation.
Ex vivo bioadhesive properties of prepared
emulsions
The effectiveness of dermatological prepara-
tions largely depends on their contact time with
the skin surface. Bioadhesion is defined as the
phenomenon of adhesion of two natural materials
or synthetic materials to biological surfaces. This
kind of connection is possible due to the formation
of specific bonds, mostly hydrogen, and hydropho-
bic, as well as the van der Waals interactions. The
analysis of bioadhesive properties makes it possible
to determine the ability of the preparation to remain
in the place of its application, as well as to deter-
mine the adhesion strength to the skin surface. The
bioadhesion properties can be enhanced by adding
polymers to the designed drug forms that rise vis-
cosity, ensure better adhesion, and extend the resi-
dence time at the application site, which in turn
leads to the increase in the effectiveness of the for-
mulation when it is applied topically (43, 44). Based
on the data obtained using the skin of the hairless
 Composition development and in vitro evaluation… 701

Table 4. Textural properties of prepared placebo emulsions with the addition of sodium alginate (F1-F6) or xanthan gum (F1 G-F6 G) and
emulsions containing tea tree oil made with the addition of sodium alginate (F1 TTO-F6 TTO) or xanthan gum (F1 G TTO-F6 G TTO).

Formulation code Firmness (g) Compressibility (g·s) Adhesiveness (-g·s)

F1 34.4 ± 0.7 73.2 ± 0.8 -81.9 ± 0.9
F2 58.8 ± 2.3 121.9 ± 5.9 -127.4 ± 1.6
F3 43.1 ± 0.4 88.3 ± 1.2 -100.7 ± 2.6
F4 63.6 ± 0.9 131.2 ± 0.8 -134.7 ± 2.4
F5 157.0 ± 1.4 265.6 ± 50.0 -276.9 ± 4.6
F6 84.7 ± 4.6 170.8 ± 9.5 -173.4 ± 5.2
F1 G 116.6 ± 0.5 201.4 ± 20.6 -153.9 ± 6.6
F2 G 138.0 ± 5.0 255.2 ± 35.4 -215.2 ± 2.0
F3 G 132.7 ± 3.8 237.0 ± 10.1 -181.2 ± 5.9
F4 G 122.3 ± 1.8 205.5 ± 23.3 -136.6 ± 6.0
F5 G 155.7 ± 9.9 262.3 ± 7.5 -171.1 ± 264.2
F6 G 156.8 ± 2.5 247.6 ± 18.1 -156.7 ± 18.7
F1 TTO 52.7 ± 4.5 113.1 ± 12.7 -121.0 ± 5.2
F2 TTO 62.9 ± 5.5 134.1 ± 16.9 -135.4 ± 11.8
Pobrano z https://ppm.edu.pl / Downloaded from Repository of Polish Platform of Medical Research 2023-03-09

F3 TTO 85.9 ± 4.8 190.5 ± 15.6 -203.4 ± 9.4

F4 TTO 67.2 ± 1.3 139.2 ± 16.9 -135.5 ± 4.0
F5 TTO 158.4 ± 4.1 268.0 ± 28.3 -280.8 ± 2.0
F6 TTO 143.4 ± 3.6 248.2 ± 16.9 -243.8 ± 10.4
F1 G TTO 138.5 ± 6.6 220.0 ± 24.7 -169.9 ± 18.0
F2 G TTO 189.4 ± 7.5 256.9 ± 10.3 -217.9 ± 9.9
F3 G TTO 172.2 ± 4.3 259.1 ± 23.5 -203.3 ± 11.6
F4 G TTO 138.5 ± 5.2 250.7 ± 2.7 -193.4 ± 11.7
F5 G TTO 202.1 ± 7.9 305.7 ± 18.3 -191.6 ± 224
F6 G TTO 235.9 ± 2.5 351.2 ± 14.6 -215.8 ± 10.1

mice as a model of the adhesive layer (Figure 9), it
can be concluded that all prepared emulsions dem-
onstrated bioadhesive properties. The preparations
with the addition of xanthan gum were character-
ized by slightly higher values of Wad and Fmax com-
pared to emulsions containing sodium alginate. It
can also be noticed that in all formulations, the ad-
dition of TTO increased the strength and work of
adhesion.
Antibacterial activity of designed emulsions
with TTO
The increase in bacterial skin infections and
drug resistance is a crucial challenge in contem-
porary medicine. Due to the limited effectiveness
of commonly used antibacterial agents and the
widespread occurrence of drug-resistant strains,
new treatment alternatives are investigated. They
should be highly effective and safe. The use of
compounds of plant origin seems to be a promising
solution because they are usually better tolerated
and do not cause as many side effects as synthet-
ic compounds (45). TTO, due to its proven an-
timicrobial activity, is used in the treatment of
many dermatological diseases (46-48). It is effec-
tive against both Gram-negative and Gram-positive
bacteria. The main component responsible for the
antimicrobial activity of TTO is terpinen-4-ol (12).
TTO has proven bactericidal effects against vari-
ous bacterial species such as Bacillus cereus, B.
subtilis, E. coli, Pseudomonas putida, and S. au-
reus (11, 12). The antibacterial properties are re-
lated to disturbing the structural and functional
integrity of the bacterial membrane. The mecha-
nism of action appears to be an alteration of the
permeability and integrity of bacterial cell mem-
branes leading to leakage of intracellular mate-
rials, inhibition of respiration, and as the conse-
quence - a decrease in the viability of bacterial
cells (11, 14).
 702 MAGDALENA WRÓBLEWSKA and KATARZYNA WINNICKA

Bioadhesive properties of emulsions (ALG)

0.25 450

400
0.2 350

300

Wad [μJ]
0.15
Fmax [N]

250

200
0.1
150

0.05 100

50

0 0
F1 F2 F3 F4 F5 F6 F1 F2 F3 F4 F5 F6 Cp
TTO TTO TTO TTO TTO TTO

Fmax Wad
Pobrano z https://ppm.edu.pl / Downloaded from Repository of Polish Platform of Medical Research 2023-03-09

Bioadhesive properties of emulsions (XG)

0.25 450

400
0.2 350

300
0.15

Wad [μJ]
250
Fmax [N]

200
0.1
150

0.05 100

50

0 0
F1 G F2 G F3 G F4 G F5 G F6 G F1 G F2 G F3 G F4 G F5 G F6 G Cp
TTO TTO TTO TTO TTO TTO

Fmax Wad
Figure 9. Ex vivo bioadhesive properties of prepared placebo emulsions with the addition of sodium alginate F1 - F6, emulsions with the
addition of sodium alginate containing TTO F1 TTO-F6 TTO (ALG), placebo emulsion with the addition of xanthan gum F1 G-F6 G,
emulsions with the addition of xanthan gum containing TTO F1 G TTO-F6 G TTO (XG) and control – cellulose paper (Cp) determined as
the maximum detachment force Fmax and the work of adhesion Wad.

In order to determine the effectiveness of the
prepared TTO emulsions, their activity both against
the bacteria Gram (+) (S. aureus) and Gram (-)
(E. coli) was tested. On the basis of the obtained re-
sults (Tables 5 and 6), the concentration-dependent
antibacterial activity of TTO against tested strains
was found. 100% TTO had greater antimicrobial
activity than the tested commercial preparation,
however, its application to the skin is not recom-
mended due to the possibility of causing irritation.
According to the literature data, the use of TTO in
concentrations of 1-10% is regarded as safe (12, 14).
In the case of the prepared emulsions containing
10% of TTO, zones of growth inhibition with a larg-
er area were observed in E. coli compared to S. au-
reus. Lower antibacterial activity against the tested
strains was found for emulsion systems character-
ized by a higher viscosity (formulations F6 TTO
and F6 G TTO). This may be due to the weaker dif-
fusion of the active substance from emulsion bases
containing a lipophilic component and character-
ized by high viscosity (19,49). The antibacterial ac-
tivity of TTO against S. aureus and E. coli was also
demonstrated in the work characterizing nanogels
 Composition development and in vitro evaluation… 703

Table 5. Antibacterial activity of prepared emulsions with the addition of sodium alginate containing TTO (F1 TTO-F6 TTO)
and controls: placebo emulsions (F1-F6), TTO 2.5%, 5%, 10% in DMSO, TTO 100% and commercially available product (C).

Name of the strain

S. aureus E. coli
Formulation code
ATCC 29213 ATCC 25922
Zone of inhibition (mm)
Control (C) 20.3 ± 1.0 18.0 ± 0.6
DMSO 0.0 ± 0.0 0.0 ± 0.0
TTO 100% 32.7 ± 0.8 37.7 ± 4.0
TTO 10%/DMSO 19.8 ± 1.2 22.5 ± 1.4
TTO 5%/DMSO 13.3 ± 0.5 16.5 ± 0.5
TTO 2.5%/DMSO 10.7 ± 0.5 12.3 ± 0.5
F1 0.0 ± 0.0 0.0 ± 0.0
F2 0.0 ± 0.0 0.0 ± 0.0
F3 0.0 ± 0.0 0.0 ± 0.0
F4 0.0 ± 0.0 0.0 ± 0.0
F5 0.0 ± 0.0 0.0 ± 0.0
F6 0.0 ± 0.0 0.0 ± 0.0
F1 TTO 14.5 ± 0.5 17.8 ± 0.8
Pobrano z https://ppm.edu.pl / Downloaded from Repository of Polish Platform of Medical Research 2023-03-09

F2 TTO 14.5 ± 0.5 17.8 ± 0.8

F3 TTO 11.3 ± 0.5 16.0 ± 0.6
F4 TTO 12.2 ± 0.4 17.5 ± 0.5
F5 TTO 11.5 ± 0.5 14.5 ± 0.5
F6 TTO 11.5 ± 0.5 13.2 ± 0.8

Table 6. Antibacterial activity of prepared emulsions with the addition of xanthan gum containing TTO (F1 G TTO-F6 G TTO)
and controls: placebo emulsions (F1 G-F6 G), TTO 2.5%, 5%, 10% in DMSO, TTO 100% and commercially available product (C).

Name of the strain

S. aureus E. coli
Formulation code
ATCC 29213 ATCC 25922
Zone of inhibition (mm)
Control (C) 20.3 ± 1.0 18.0 ± 0.6
DMSO 0.0 ± 0.0 0.0 ± 0.0
TTO 100% 32.7 ± 0.8 37.7 ± 4.0
TTO 10%/DMSO 19.8 ± 1.2 22.5 ± 1.4
TTO 5%/DMSO 13.3 ± 0.5 16.5 ± 0.5
TTO 2.5%/DMSO 10.7 ± 0.5 12.3 ± 0.5
F1 G 0.0 ± 0.0 0.0 ± 0.0
F2 G 0.0 ± 0.0 0.0 ± 0.0
F3 G 0.0 ± 0.0 0.0 ± 0.0
F4 G 0.0 ± 0.0 0.0 ± 0.0
F5 G 0.0 ± 0.0 0.0 ± 0.0
F6 G 0.0 ± 0.0 0.0 ± 0.0
F1 G TTO 12.0 ± 0.6 16.0 ± 0.6
F2 G TTO 12.0 ± 0.5 16.3 ± 0.8
F3 G TTO 11.3 ± 0.5 15.3 ± 0.5
F4 G TTO 12.7 ± 0.5 17.8 ± 0.4
F5 G TTO 12.5 ± 0.5 17.7 ± 0.5
F6 G TTO 11.2 ± 0.4 12.3 ± 0.5
 704 MAGDALENA WRÓBLEWSKA and KATARZYNA WINNICKA
as a  new form of drug carrier that can be used
in dermatological infections. The reported zones
of growth inhibition were 15.33  mm  ±  0.88  mm
for S. aureus and 14.3 mm ± 1.20 mm for E. coli,
respectively (7).
CONCLUSIONS
Stable O/W emulsion systems containing TTO
were obtained with the use of Olivem 1000 at 8%
concentration and combinations of Olivem 1000
(8%) with cetyl alcohol (1%) or glycerol stearate
(1%). The prepared formulations were homogeneous,
smooth in texture, easily spreadable on the skin sur-
face, and showed adequate stability. Additionally,
it was also necessary to add viscosity-increasing
agents - sodium alginate or xanthan gum, to im-
prove the stability and application properties of de-
signed emulsions. Preparations containing xanthan
gum as a thickening agent and linseed oil as a com-
ponent of the lipid phase showed smaller droplet
Pobrano z https://ppm.edu.pl / Downloaded from Repository of Polish Platform of Medical Research 2023-03-09
sizes in the dispersed phase. The pH values of all
TTO emulsion systems were in the range of 6.2-6.6,
which is considered suitable for dermal preparations.
Formulations containing xanthan gum as a  thick-
ening agent were characterized by higher viscosity
values. In addition, TTO caused an increase in the
viscosity of designed emulsions. All prepared emul-
sions exhibited thixotropic properties and they were
classified as shear-thinning non-Newtonian systems.
Based on the analysis of mechanical properties,
it was concluded that the formulations containing
TTO possessed higher hardness, cohesiveness, and
adhesion compared to placebo formulations. All
prepared emulsion systems were characterized by
bioadhesive properties and higher Fmax and Wad val-
ues were recorded for emulsions with TTO. Based
on the investigation of the antimicrobial activity of
designed emulsions containing TTO, it was found
that the formulations with lower viscosity showed
higher antimicrobial efficacy against S. aureus and
E. coli, and larger zones of inhibition were observed
in the case of E. coli strains. The results of the study
showed that the prepared emulsions appear to be
a suitable form of topical administration of TTO to
the skin and they might be a promising alternative
to commonly used dermatological antimicrobial
formulations.
Acknowledgments
This research was supported by the Medical
University of Białystok grant (number SUB/2/
NN/21/001/2215).
Conflicts of interest
The authors declare no conflict of interest.
REFERENCES
1. Singh Malik D., Mital N., Kaur G.: Expert Opin.
Ther. Pat. 26, 213 (2016).
2. Esposito T., Mencherini T., Sansone F.,
Auriemma G., Gazzerro P., et al.: Pharmaceutics
13, 1634 (2021).
3. Otto A., du Plessis J., Wiechers J.W.: Int. J.
Cosmet. Sci. 1, 19 (2009).
4. Bordes C., Bolzinger M.A., El Achak M., Pirot
F., Arquier D., et al.: Int. J. Cosmet. Sci. 4, 445
(2021).
5. Antonijević D., Novac O., O’Hagan B.: Clin.
Cosmet. Investig. Dermatol. 11, 465 (2018).
6. Wojciechowska K., Walczak A., Rostowska E.,
Poleszak E.: Curr. Issues Pharm. Med. 34, 223
(2021).
7. Sinha P., Srivastava S., Mishra N., Singh D.K.,
Luqman S., et al.: Drug Dev. Ind. Pharm. 42,
1445 (2016).
8. Muta T., Parikh A., Kathawala K., Haidari
H., Song Y., et al.: Pharmaceuticals 12, 1091
(2020).
9. Biju S.S., Ahuja A., Khar R.K., Chaudhry R.:
Pharmazie 60, 208 (2005).
10. Erosh Y., Rekha R., Sunil K., Sheefali M.,
Prakriti V.: Nat. Prod. J. 10, 286 (2020).
11. Pazyar N., Yaghoobi R., Bagherani N.,
Kazerouni A.: Int. J. Dermatol. 52, 790 (2013).
12. Carson C.F., Hammer K.A., Riley T.V.: Clin.
Microbiol. Rev. 19, 62 (2006).
13. Chin K.B., Cordell B.: J Altern. Complement.
Med. 19, 945 (2013).
14. de Groot A.C., Schmidt E.: Contact Derm. 75,
143 (2016).
15. Kulawik-Pióro A., Drabczyk A.K., Kruk J.,
Wróblewska M., Winnicka K., Tchórzewska J.:
Materials (Basel) 14, 4723 (2021).
16. Hu Y.T., Ting Y., Hu J.Y., Hsieh S.C.: J. Food
Drug Anal. 1, 26 (2017).
17. Burapapadh K., Kumpugdee-Vollrath M.,
Chantasart D., Sriamornsak P.: Carbohydr.
Polym. 82, 384 (2010).
18. Carvalho F.C., Calixto G., Hatakeyama I.N.,
Luz G.M., Gremião M.P., et al.: Drug Dev. Ind.
Pharm. 39, 1750 (2013).
19. Wróblewska M., Szymańska E., Winnicka K.:
Int. J. Mol. Sci. 22, 11326 (2021).
20. The European Pharmacopoeia 10.0, Council of
Europe: Strasbourg, France, 2021.
 Composition development and in vitro evaluation…
21. Wróblewska M., Szymańska E., Szekalska M.,
Winnicka K.: Polymers (Basel) 12, 680 (2020).
22. Zeichner J.A., Del Rosso J.Q.: J. Clin. Aesthet.
Dermatol. 9, 32 (2016).
23. Bettoli V., Coutanceau C., Georgescu V.: Clin.
Cosmet. Investig. Dermatol. 10, 769 (2019).
24. Kowalska M., Turek P., Żbikowska A., Babut
M., Szakiel J.: Biomolecules 11, 213 (2021).
25. Mohsin S, Akhtar N. Acta Pol. Pharm. 74, 945
(2017).
26. Abdolmaleki K., Alizadeh L., Hosseini S.,
Nayebzadeh K.: Food Sci. Biotechnol. 29, 1693
(2020).
27. Liu Y., Wei Z.C., Deng Y.Y., Dong H., Zhang
Y., et al.: Molecules 25, 458 (2020).
28. Lin X., Ma Q., Su J., Wang C., Kankala R.K., et
al.: Molecules 24, 2089 (2019).
29. Bialik-Wąs K., Królicka E., Malina D.:
Molecules 26, 2381 (2021).
30. Patel J., Maji B., Moorthy N.S.H.N., Maiti S.:
RSC Advances 10, 27136 (2020).
Pobrano z https://ppm.edu.pl / Downloaded from Repository of Polish Platform of Medical Research 2023-03-09
31. Alves A., Miguel S.P., Araujo A.R.T.S., de Jesús
Valle M.J., Sánchez Navarro A., et al.: Polymers
(Basel) 12, 99 (2020).
32. Handbook of Pharmaceutical Excipients
6th ed. Pharmaceutical Press and American
Pharmacists Association 2009.
33. Petry T., Bury D., Fautz R., Hauser M., Huber
B., et al.: Toxicol. Lett. 5, 78 (2017).
34. Jiang Q., Sun N., Kumar P., Li Q., Liu B., et al.:
Molecules 25, 2904 (2020).
35. Weiss J., Muschiolik G.: J. Dispers. Sci. Tech.
28, 703 (2007).
© 2022 by Polish Pharmaceutical Society. This is an open-access article under the CC BY NC license
(https://creativecommons.org/licenses/by-nc/4.0/).
705
36. Huynh A., Garcia A.G., Young L.K., Szoboszlai
M., Liberatore M.W., et al.:. Int. J. Cosm. Sci.
43, 11 (2021).
37. Ali S.M., Yosipovitch G.: Acta Derm. Venereol.
93, 267 (2013).
38. Lambers H., Piessens S., Bloem A., Prank H.,
Finkel P.: Int. J. Cosmet. Sci. 5, 359 (2006).
39. Mohammed A.M., Faisal W., Saleh K.I., Osman
S.K.: Curr. Drug Deliv. 14, 926 (2017).
40. Mut A.M., Vlaia L., Coneac G., Olariu I., Vlaia
V., et al.: Farmacia 66, 256 (2018).
41. Fürtjes T., Weiss K.T., Filbry A., Rippke.F,
Schreml S.: Skin Pharmacol. Physiol. 30, 297
(2017).
42. Osmałek T., Milanowski B., Froelich A., Górska
S., Białas W., et al.: Pharm. Dev. Technol. 20, 1
(2016).
43. Palacio M.L., Bhushan B.: Philos. Trans.
A Math. Phys. Eng. Sci. 370, 2321 (2012).
44. Zhang Y., Zhang J., Chen M., Gong H.,
Thamphiwatana S., et al.: ACS Appl. Mater.
Interfaces 8, 18374 (2016).
45. Hammer K.A., Carson C.F., Riley T.V., Nielsen
J.B.: Food Chem. Toxicol. 44, 616 (2006).
46. Oliva A., Costantini S., De Angelis M., Garzoli
S., Božović M., et al.: Molecules 23, 2584 (2018).
47. Wińska K., Mączka W., Łyczko J., Grabarczyk
M., Czubaszek A., et al.: Molecules 24, 2130
(2019).
48. Low W.L., Kenward K., Britland S.T., Amin
M.C., Martin C.: Int. Wound J. 14, 369 (2017).
49. Abd-Allah F.I., Dawaba H.M., Ahmed A.M.:
Drug Discov. Ther. 4, 275 (2010).