Chemical Engineering Communications

ISSN: 0098-6445 (Print) 1563-5201 (Online) Journal homepage: https://www.tandfonline.com/loi/gcec20

Encapsulation of antioxidant compounds in

biopolymer micelles

Jolanta Pulit-Prociak, Małgorzata Kabat, Ewelina Węgrzyn, Michał Zielina &

Marcin Banach

To cite this article: Jolanta Pulit-Prociak, Małgorzata Kabat, Ewelina Węgrzyn, Michał Zielina &
Marcin Banach (2019): Encapsulation of antioxidant compounds in biopolymer micelles, Chemical
Engineering Communications, DOI: 10.1080/00986445.2019.1602526

To link to this article: https://doi.org/10.1080/00986445.2019.1602526

Published online: 10 Apr 2019.

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CHEMICAL ENGINEERING COMMUNICATIONS
https://doi.org/10.1080/00986445.2019.1602526

Encapsulation of antioxidant compounds in biopolymer micelles

Jolanta Pulit-Prociaka, Małgorzata Kabata, Ewelina WeRgrzyna, Michał Zielinab, and Marcin Banacha
a
Department of Chemical Engineering and Technology, Cracow University of Technology, Cracow, Poland; bDepartment of
Environmental Engineering, Cracow University of Technology, Cracow, Poland

ABSTRACT KEYWORDS
This manuscript presents the method for encapsulation of antioxidant agents in micelles of Antioxidant compounds;
biopolymeric origin. The role of antioxidant agents is played by tannic acid or birch leaf Birch leaf extract; Drug
extract, which is a natural source of polyphenolic compounds. The products were obtained delivery; Micelles;
Nanoemulsion; Tannic acid
in the form of o/w nanoemulsions. The obtained nanoemulsions were characterized by
studying their physicochemical and utility properties. The size and electro-kinetic potential
of the micelles were measured by dynamic light scattering (DLS). The creaming index and
encapsulation efficiency are also provided. The use of statistical tools enabled analysis of
the influence of independent parameters on the limits describing the emulsions’ properties.
Nanoemulsions with birch leaf extract reached an encapsulation efficiency equal to 80–90%,
which remained quite constant over time. It was possible to obtain nanoemulsions with
birch extract that were homogeneous over the whole observation period. The values of
electro-kinetic potential ranged up to
40 mV, which proves the high stability of these
nanoemulsions. More desirable results were achieved in the case of obtaining nanoemul-
sions with tannic acid. In the beginning, the encapsulation efficiency was very effective
(approximately 70%), but after a few days it decreased to values of around 30%. That meant
that the tannic acid was released from the micelles with time. After 14 days of observation,
the creaming index values were very low, which proves their high homogeneity. These
nanoemulsions were stable in time, which was reflected by their electro-kinetic potential
values (>20 mV).

Introduction intestinal membrane, as well as mucosal perme-

Approximately 40% of newly discovered drugs or
agents for transdermal, parenteral, intravenous or
oral administration are highly hydrophobic sub-
stances with low water solubility. Recently, inter-
est in the use of nanoemulsions as a carrier
formulation for active substances delivery has
increased. Nanoemulsions, thanks in large to an
internal phase surface area and their nanometer
size, have become important biocarriers in the
process of the encapsulation and transport of
active substances. The essence of encapsulation is
the immobilization of the active substance with
therapeutic properties via micelle structures.
Encapsulation of the active substance, in a for-
mulation such as a nanoemulsion, provides pro-
tection against degradation, increases solubility,
and primarily facilitates diffusion through the
ability. Moreover, cosmetic nanoemulsions are
effective for the penetration of active substances
by a dermal route. However, the active substance
must not have a high affinity for the continuous
phase, or else it will not be released. Compared
with traditional drug delivery methods, nanoe-
mulsions are more biocompatible and enhance
favorable reactions to the medical formulations,
produced with their participation. In addition,
they are characterized by a high encapsulation
efficiency and better bioavailability.
Nanoemulsion classification criteria are not
strictly defined. The literature indicates that
micelle diameters range from 200 nm to 1000 nm
(Sivakumar et al. 2014; Zeeb et al. 2014). The
main considerations for the preparation of nano-
emulsions are related to their stability and the

CONTACT Jolanta Pulit-Prociak jolantapulit@chemia.pk.edu.pl Department of Chemical Engineering and Technology, Institute of Chemistry and
Inorganic Technology, Cracow University of Technology, Warszawska 24, Cracow 31-155, Poland.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/gcec.
ß 2019 Taylor & Francis Group, LLC
2 J. PULIT-PROCIAK ET AL.
smallest diameter of the internal phase droplets.
It has been shown that an increase in the hom-
ogenization pressure results in a lower average
diameter of droplets. The emulsification time is
also an important parameter, with its extension
favoring smaller micelles. The use of low molecu-
lar weight emulsifiers is also crucial in reducing
their diameter (Kaltsa et al. 2016).
Reactive oxygen species (ROS) (superoxide
radical O2
· , singlet oxygen 1O2, hydroxyl radical
OH· , hydroperoxyl radical HO2· , and hydrogen
peroxide H2O2) have unpaired electrons. These
radicals can exist independently, and due to their
high reactivity tend to pair free electrons, by
donating them or attaching them to other chem-
ical entities. Radicals are the product of cellular
metabolism and an effect of exogenous com-
pounds, as well as the influence of physical fac-
tors affecting the cell, such as ultrasounds,
ultraviolet, ionizing radiation or elevated tem-
perature. In the body, about 1–5% of oxygen
turns into reactive forms under physiological
conditions. The presence of reactive oxygen spe-
cies at physiological concentrations is necessary
for the proper functioning of organisms, but
exceeding a safe concentration generates various
types of diseases and disorders (neurodegenera-
tive diseases, cardiological diseases, nephrological
diseases, rheumatic diseases, tumors, and dia-
betes) (Stolarzewicz et al. 2013).
In the course of evolution, organisms have
developed a defense system that protects cells
from free radical damage. There are two main
mechanisms in this system: enzymatic and non-
enzymatic. The enzymatic mechanism consists of
antioxidant enzymes such as glutathione peroxid-
ase and cooperating catalase and superoxide dis-
mutase. The latter approach consists of numerous
low molecular antioxidants, which are able to
prevent or delay free radical processes even at
their very low concentrations, compared with
oxidized substances (Lobo et al. 2010).
Polyphenol compounds are common in plants,
mainly in the leaves and in ligated tissues and in
smaller amounts in the seeds and fruit. They are
also present in the mycobiota. The presence of
hydroxyl substituents causes polyphenol com-
pounds to have antioxidant properties. By trap-
ping free radicals, they themselves become free
radicals, but due to the chemical resonance phe-
nomenon, the free electrons are shifted so that
they become relatively stable (Pandey and
Rizvi 2009).
Birch leaves are one of the natural sources of
polyphenol compounds. Birch extract contains,
among other compounds: catechin, coumaric acid,
quercetin, kaempferol, apigenin, isoramnetin, and
triterpenoids, whose main constituents are betulin,
lupeol, betulinic acid, and oleanolic acid (German
o
et al. 2012; Malinowska et al. 2014). These com-
pounds are considered valuable active substances.
Thus, extracts from birch leaves may be an
important source of effective compounds in the
treatment of numerous diseases (Dehelean et al.
2007, 2012). Gallic acid (3,4,5-trihydroxybenzoic
acid) is a secondary metabolite present in birch
leaf extract. This metabolite has antioxidant, anti-
microbial, anti-inflammatory, and antitumor prop-
erties. It has been shown to have prophylactic and
therapeutic effects in many diseases caused by oxi-
dative stress, including cardiovascular diseases,
cancer, neurodegenerative disorders, and aging.
Gallic acid can also be found in birch leaf extract
(Nayeem et al. 2009; Piazzon et al. 2012; Hugo
et al. 2016). The solubility values of active com-
pounds that are contained in birch leaf extract are
provided in Table 1.
Tannic acid is an ester of gallic and digalic
acids combined with glucose (Aguilar-Zarate
et al. 2015). It occurs in bark, seeds, cones, and
fruits of trees such as oak, mahogany, beech, and
redwood. Tannic acid may also be found in galls
that are formed on the leaves of beech and oak
as a result of insect infiltration. Studies have
shown that a solution of tannic acid at a concen-
tration of 15 lg/cm3 is able to inhibit the lipid
peroxidation of linoleic acid by 97.9%. It is also
very effective to sweep away the DPPH radicals
and chelate peroxidized metals, mainly in the
form of Fe ions. Due to its very good antioxidant
properties, it can be used as a preservative of
both food and nutraceutics. It is also defined by
its antibiotic, antibacterial, and antiviral charac-
teristics (G€ulçin et al. 2010). The solubility of
tannic acid in various solvents is as follows:
2.86 g dissolves in 1 cm3 of water, 1 g dissolves in
1 cm3 of warm glycerol, and 0.1 g dissolves in
1 cm3 of ethyl alcohol. It is practically insoluble
 CHEMICAL ENGINEERING COMMUNICATIONS 3

Table 1. Solubility values of compounds contained in birch extract (PubChem Open Chemistry Database; Sigma Aldrich Product
Information).
Compound Solubility
Catechin 100 mg/1 cm3 of ethanol 50 mg/1 cm3 of DMSO 100 mg/1 cm3 of DMS
Quercetin 50 mg/1 cm3 of methanol 30 mg/1 cm3 of DMF 2 mg/1 cm3 of ethanol 60 mg/1 cm3 of water
Kaempferol 20 mg/1 cm3 of ethanol insoluble in water 25 mg/1 cm3 of DMSO
Apigenin 27 mg/1 cm3 of DMSO 50 mg/1 cm3 of KOH (1M) Practically insoluble in water
Isoramnetin Sparingly soluble in water Soluble in organic solvents (acetone, methanol, DMSO, etyhylacetate)
Betulin 2.5 mg/1 cm3 of DMF Sparingly soluble in water
Lupeol Practically insoluble in water 1 mg cm3 of ethanol 1 mg cm3 of DMF
Betulinic acid Partially miscible with water
Oleanolic acid Insoluble in water Soluble in organic solvents (acetone, chloroform, ethanol)
Gallic acid 11.5 mg/cm3 of water Insoluble in chloroform

in chloroform, benzene and petroleum ether
(PubChem Open Chemistry Database; Sigma
Aldrich Product Information, St. Louis, MO).
Low stability and poor bioavailability of poly-
phenols mean that their potential health benefits
in inhibiting aging, inflammatory, and neurode-
generative diseases are limited. Encapsulation of
polyphenols significantly improves their mechan-
ical resistance and strengthens their bioavailabil-
ity. Encapsulation also allows for preservation of
their chemical activity and gives a slow and con-
trolled release from the formulation (Lu et al.
2015; Puligundla et al. 2017). The aim of this
work is to encapsulate antioxidant compounds in
the form of birch leaf extract and tannic acid in
biopolymer micelles suspended in emulsion
formulations.
The novelty of the studies performed was to use
statistical methods for process optimization.
Thanks to these, it was possible to determine the
values of input parameters that allowed the most
desirable products to be obtained. This approach is
of particular importance in technological processes.
Statistical methods are applied widely in the devel-
opment of stable nanoemulsions. For example,
Yang et al. used central composite design to define
the influence of six different factors on the encap-
sulation efficiency, particle size and drug loading
when obtaining hydrocortisone butyrate-loaded
PLGA nanoparticles (Yang et al. 2014).
Miastkowska et al. prepared Silybum marianum
seed oil-based-nanoemulsions based on statistical
design and were as a result able to assess the influ-
ence of the type of surfactant, emulsifier concentra-
tion, speed of stirring, pre-emulsification time,
premix time, and output power of ultrasound on
the physicochemical properties of the nanoemul-
sions obtained (Miastkowska et al. 2017). Alzorqi
et al. used central composite design to obtain
palm-olein based nanoemulsions by using an ultra-
sound technique. They defined the influence of
input variables such as water content, oil/surfactant
ratio, ultrasonic power, irradiation time, and com-
binations of these parameters on the output varia-
bles (droplets size, polydispersity index, and
viscosity) (Alzorqi et al. 2016).
Materials and methods
Materials
Dried birch leaves (Betula pendula) from
Mazurski Spichlerz were purchased in a local
herbal store, where they are sold as a commercial
herb infusion. The following compounds were
used in the study: gallic acid (99%), sodium
carbonate (p.a.), Folin–Ciocalteu reagent (p.a.),
2,2-diphenyl-1-picrylhydrazyl (DPPH) (p.a.),
ethyl alcohol (99.8%), cholesterol (99%), soy
lecithin (p.a.), tannic acid (p.a.), gelatin (p.a.),
castor oil containing 2% of palmitic acid,
2.5% of stearic acid, 2.5–6.0% of oleic acid,
85% of ricinoleic acid, 2.5–7% of linoleic acid,
1% of linolenic acid (p.a.), coconut oil contain-
ing 40–50% of lauric acid, 12–20% of myristic
acid, 7–12% of palmitic acid, 4–9% of decanoic
acid, 4–10% of olefinic acid (p.a.), methylene
blue (p.a.), and Sudan dye (p.a.). All compounds
were provided by Sigma Aldrich, Germany. All
solutions were prepared with deionized water.
Methods
Nanoemulsions with birch leaf extract as a source
of antioxidant compounds
Obtaining birch leaf extract: Extraction was per-
formed using a Soxhlet apparatus. Water
4 J. PULIT-PROCIAK ET AL.
Figure 1. Schematic diagram showing the process for obtaining nanoemulsions with birch leaf extract (A) and tannic acid (B).
(300 mL) which serves as extracting solvent was
placed in a round-bottomed flask. Dried and
ground birch leaves (7 g) were placed in a cellu-
lose filter cup which was inserted into the
Soxhlet chamber. A flask with solvent was heated
under reflux. The extraction process was con-
ducted for 5 h. The obtained extract was stored at
5  C. The content of total polyphenol compounds
in the obtained extract was determined using the
Folin–Ciocalteu method, according to a known
protocol (Blainski et al. 2013). The method is
based on colorimetric assessment when using
Folin–Ciocalteu reagent, which consists of phos-
phomolybdic and phosphotungstic acids
(Ballesteros et al. 2017). In an alkaline environ-
ment, phenolic compounds which are present in
the analyte are oxidized while the salts are
reduced. The reaction product is characterized by
a blue color. Briefly, an aqueous solution of
Folin–Ciocalteu reagent at a concentration of
100 mg/dm3, an aqueous solution of sodium car-
bonate at a concentration of 0.7076 mol/dm3, and
an aqueous stock solution of gallic acid at a con-
centration of 0.0065 mol/dm3 were prepared. By a

dilution method, a series of gallic acid solutions
with known concentrations were prepared. The
analyzed sample contained 0.5 cm3 of analyte
(gallic acid standard solution, extract or water in
the case of the blind test), 2.5 cm3 of
Folin–Ciocalteu reagent solution and 2 cm3 of
sodium carbonate solution. After 15 min of stor-
age under dark conditions, their absorbance
against water as reference material was measured
at k ¼ 765 nm. The results were provided as a gal-
lic acid equivalent.
The antioxidant activity of the extract against
the DPPH free radical was also assessed. An etha-
nolic solution of DPPH at a concentration of
0.08 mM was prepared. The blind test was
obtained by mixing 0.4 cm3 of deionized water
with 30 cm3 of DPPH solution. Samples with ana-
lyzed extract were prepared in the same way.
Absorbance against ethyl alcohol as reference
material was measured at k ¼ 515 nm. The anti-
oxidant activity (AA) was calculated based on
formula 1 (Brand-Williams et al. 1995):
A0
A
AA ¼  100% (1)
A0
A0 – absorbance of DPPH ethanolic solution
A – absorbance of analyzed extract etha-
nolic solution.
What is more, as the experiment proceeded, in
the absence of antioxidants, the absorbance val-
ues of the DPPH were constant in time.
However, when the analyte contains antioxidants,
consumption of free radicals occurs (Fuentes
et al. 2016). Considering this fact, the kinetic pro-
file for DPPH consumption has been studied
as well.
Obtaining nanoemulsions with birch leaf
extract: The process consisted of preparing oil
and water phases and their further homogeniza-
tion. First, cholesterol was mixed and stirred with
castor oil, and the resulting mixture was heated
to 60  C until the solid was dissolved completely.
At the same time, a mixture of soy lecithin and
water was stirred and heated to 60  C for 10 min.
The solution was then mixed with the previously
obtained birch leaf extract and the entire water
phase. After this, the sonication horn was intro-
duced to the oil phase, and under a continuous
homogenization process (30 kHz, 50 W), portions
CHEMICAL ENGINEERING COMMUNICATIONS 5
of water phase were dropped into the oil phase.
The homogenization process was conducted
using a Hielscher UP50H homogenizer. A sche-
matic diagram of the procedure is presented in
Figure 1(A).
The experiment was designed using Statistica
v.10 software (StataCorp, College Station, TX).
The purpose of using statistical data analysis
techniques in planning the experiments and ana-
lyzing the obtained results was to plan the
research such that a reliable interpretation of the
obtained results would be possible. This approach
contributes significantly to the efficiency of the
laboratory work and the effectiveness of formu-
lating conclusions. Design of experiments is an
interdisciplinary field of science, combining ele-
ments of metrology, applied mathematics, statis-
tics, and computer science. The purpose of this
stage is to enable planning of the research at the
lowest possible cost, but also to receive the max-
imum amount of useful information. The object
of research is – in the case of the presented work
– a technological process which, in the light of
experimental design techniques, is perceived as a
“black box.” It is characterized by constant
parameters, input parameters that remain under
the control of the researcher, and output parame-
ters that constitute a set of observed and meas-
ured quantities. In addition, the object may be
influenced by other factors that the researcher
often has no control over. The combination of
both groups of variables results in the creation of
a research object function, which, in the light of
statistical analysis, is the basic scientific informa-
tion, through which it is possible to select the
most favorable parameter values and numerical
control of the process. The process parameters
were divided into groups of independent (tested)
and dependent (resultant) parameters. On the
basis of preliminary research, input variables that
could influence the resultant (dependent) factors
were selected. These were the homogenization
time, the concentration of gelatin, and the vol-
ume ratio of oil phase to water phase.
Preliminary research allowed us to determine the
ranges of the independent parameters. A central
composite plan was used, which is a form of
multilevel plan. It allows linear analysis, as well
as square and mixed parameters in a relatively
6 J. PULIT-PROCIAK ET AL.

Table 2. Values of the process parameters for obtaining nanoemulsion with birch leaf extract.
Sample Voil phase : Vwater phase Voil phase (cm3) Vwater phase (cm3) Vwater (cm3) Ccholesterol (%) Homogenization time (min)
E1 0.5 12 24 22 1 2
E2 0.5 12 24 22 3 2
E3 0.5 12 24 22 1 22
E4 0.5 12 24 22 3 22
E5 2.0 24 12 10 1 2
E6 2.0 24 12 10 3 2
E7 2.0 24 12 10 1 22
E8 2.0 24 12 10 3 22
E9 0.5 12 24 22 2 11
E10 2.0 24 12 10 2 11
E11 1.25 20 16 14 2 2
E12 1.25 20 16 14 2 22
E13 1.25 20 16 14 1 11
E14 1.25 20 16 14 3 11
E15 1.25 20 16 14 2 11
E16 1.25 20 16 14 2 11

quick way. This kind of plan consists of is also performed by a tabular presentation of
imbedded factorial design, star points for estima- changes in the performance value at the extreme
tion of curvature and center points for evaluating change of the given input parameter, but
the experimental reproducibility. By using it, unchanged values of other independent factors.
researchers can significantly reduce the number Statistically significant effects are characterized by
of experiments that are needed to determine a lower value of significance in relation to its
mathematical trends without losing the good assumed value.
quality of the final product. The obtained model The highlighted parameters are the input inde-
provides information on the influence of several pendent parameters whose influence on the phys-
variables on the response. Based on it, one can icochemical properties of the obtained emulsions
predict the optimum level of input variables that is being studied. A series of studies were con-
are necessary to obtain the desired response ducted immediately after obtaining the nanoe-
(Yang et al. 2014). The group of constant param- mulsions and after fixed time intervals:
eters included the volume of added extract, which t0 – day of obtaining nanoemulsion
was equal to 2 cm3 (0.055 cm3/1 cm3 of final t3 – third day after obtaining nanoemulsion
product), the mass of soy lecithin, which was t5 – fifth day after obtaining nanoemulsion
equal to 1 g (28 mg/1 cm3 of final product), and t10 – 10th day after obtaining nanoemulsion
the power of the ultrasounds, which was equal to t17 – seventeenth day after obtaining
50 W. The group of independent (input) parame- nanoemulsion
ters consisted of: the homogenization time, the t21 – 21st day after obtaining nanoemulsion.
concentration of cholesterol, and the volume
ratio of oil phase to water phase. Table 2 presents Nanoemulsions with tannic acid as an antioxi-
the values of the process parameters. In order to dant compound
interpret the obtained results, analysis of variance The first stage of the process was to prepare oil
(ANOVA) was used, and the standardized effects and water phases which were further homogen-
(impact of input parameters on the output ized. The oil phase consisted of coconut oil which
parameters) were graphically presented in a was heated in order to make it liquid. At the
Pareto chart. On the Pareto chart, statistically sig- same time, a mixture of soy lecithin and water
nificant effects are separated from the irrelevant was stirred and heated to 30  C for 10 min. The
lines corresponding to the assumed significance solution was then mixed with the previously
level a (with the assumed significance level obtained tannic acid and gelatin solutions.
a ¼ 0.05). In this way, it is possible to determine Gelatin was not present in all experiments and its
how large the impact of the input factors on the presence was dependent on the experimental
output factors should be so that the effect is stat- design. These two or three solutions were water
istically significant. The evaluation of the effects phase. After that, the sonication horn was
 CHEMICAL ENGINEERING COMMUNICATIONS 7

Table 3. Values of the process parameters for obtaining nanoemulsion with tannic acid.
Voil phase : Vsolution of Vsolution of tannic Vsolution of Homogenization
Sample Vwater phase Voil phase (cm3) Iwater phase (cm3) lecithin(cm3) acid (cm )
3
gelatin(cm3) Cgelatin (mg/cm3) time (min)
T1 0.25 8 32 16 16 0 0 2
T2 0.25 8 32 16 8 8 2.5 2
T3 1.00 20 20 10 10 0 0 2
T4 1.00 20 20 10 5 5 2.5 2
T5 0.25 8 32 16 16 0 0 20
T6 0.25 8 32 16 8 8 2.5 20
T7 1.00 20 20 10 10 0 0 20
T8 1.00 20 20 10 5 5 2.5 20
T9 0.50 13.4 26.6 13.3 6.65 6.65 1.25 2
T10 0.50 13.4 26.6 13.3 6.65 6.65 1.25 20
T11 0.25 8 32 16 8 8 1.25 11
T12 1.00 20 20 10 5 5 1.25 11
T13 0.50 13.4 26.6 13.3 13.3 0 0 11
T14 0.50 13.4 26.6 13.3 6.65 6.65 2.5 11
T15 0.50 13.4 26.6 13.3 6.65 6.65 1.25 11
T16 0.50 13.4 26.6 13.3 6.65 6.65 1.25 11

introduced to the oil phase and, under a continu-
ous homogenization process (30 kHz, 50 W), por-
tions of water phase were dropped into the oil
phase. The schematic diagram of this mechanism
is presented in Figure 1(B).
The experiment was designed using Statistica
v.10 software (StataCorp, College Station, TX).
The central composite plan was used. The group
of constant parameters included the concentra-
tion of tannic acid in a reaction mixture (10 mg/
cm3), the concentration of lecithin in the final
product (25 mg/cm3), and the power of ultra-
sounds which was equal to 50 W. The group of
independent (input) parameters consisted of the
homogenization time, the concentration of gel-
atin, and the volume ratio of oil phase to water
phase. Table 3 presents the values of the pro-
cess parameters.
The highlighted parameters are the input inde-
pendent parameters whose influence on the phys-
icochemical properties of the obtained emulsions
is being studied. A series of studies were con-
ducted immediately after obtaining the nanoe-
mulsions and after fixed time intervals:
t0 – day of obtaining nanoemulsion
t1 – first day after obtaining nanoemulsion
t2 – second day after obtaining nanoemulsion
t6 – sixth day after obtaining nanoemulsion
t9 – ninth day after obtaining nanoemulsion
t14 – 14th day after obtaining nanoemulsion.
Characteristics of obtained nanoemulsions
Determination of emulsion type
 Optical microscopy
For effective visualization of the phases, a solu-
tion of water-soluble dye (methylene blue) and a
solution of oil-soluble dye (Sudan) were intro-
duced into the tested emulsion. After mixing,
the emulsion was observed using an optical
microscope (Nicon Eclipse LV 100, Shimadzu,
Kyoto, Japan).
 The “blending of droplets” method
A drop of the emulsion was taken with a
Pasteur pipette and placed on a watch glass.
Next, a drop of water was added to it. It was
then observed whether the two drops would
mix together.
Assessment of polyphenolic compounds’ encap-
sulation efficiency: The idea was that polyphenolic
compounds, which are not trapped, could be ana-
lyzed using the Folin–Ciocalteu method (Isailovic
et al. 2012; Tumbas Saponjac et al. 2016). The
protocol for the determination of the total poly-
phenol content was completed as described
above. In order to investigate the time depend-
ence, the analyses were conducted at established
time intervals. The encapsulation efficiency (EE)
was calculated based on the formula given in the
following equation:
C0
C
EE ¼  100% (2)
C0

The following analysis of the nanoemulsions C – concentration of oxidant agent in the ana-
was performed. lyzed sample [mg/cm3]
8 J. PULIT-PROCIAK ET AL.
C0 – initial concentration of oxidant agent in
the analyzed sample [mg/cm3].
Dynamic light scattering (DLS) analysis: The
DLS technique serves to assay the average
micelles size and to investigate the electro-kinetic
potential values (f), which reflect the stability of
nanoemulsions. The size of micelles was meas-
ured after established time intervals, and the elec-
tro-kinetic potential was determined only once, at
i0. The values of electro-kinetic potential are
given as the average from three independent
measurements. Analysis was conducted using a
Zetasizer Nano, from Malvern. The refractive
index was set to that of gallic acid (RI ¼ 1.73).
Creaming stability measurement: To determine
the progress of phase separation, and thus the
instantaneous height of the oil and water phases,
around 20 cm3 of the obtained emulsions were
transferred into transparent containers. Next, visual
observations were completed and the appearance
of the emulsions was photographed. Pictures were
taken after the previously established time intervals.
The values of the creaming index (CI) were calcu-
lated based on the formula given in the following
equation (Varka et al. 2012):
Haq
CI ¼  100% (3)
Htotal
Haq – height of water phase
Htotal – height of total emulsion in container.
The measurement of the height of the emulsion
phases was determined by means of an applied
background, in which one grate corresponded to a
length of 0.5 cm.pH analysis: The pH values of the
obtained nanoemulsions were investigated at the
established time intervals. Analysis was conducted
using an Elmetron EPS-1 electrode.
Statistical analysis
Statistical analysis was performed based on one-
way analysis of variance (ANOVA). In order to
evaluate the significance of the differences, a test-
F was used. A value of p < 0.05 was established
as significant in all cases. The profiles of the util-
ity function with respect to certain independent
parameters, thanks to which it was possible to
determine the changes in the dependent
parameters when changing the values of the inde-
pendent parameters, were prepared.
The resulting approximation profiles allow us
to provide specific values of input factors that
ensure the most desirable (useful) estimated val-
ues of the output factors. Approximated values
are converted into a utility scale which includes
the values of 0 (undesired effects) to 1 (desired
effects). From the approximation profiles
obtained, it was possible to optimize the usability
of this product (Jiju 2014; Fox 2016).
Results
Nanoemulsions with birch leaf extract as a source
of antioxidant compounds
Birch leaf extract
Based on the calibration curve (R2 ¼ 0.9996)
(Figure 2(A)), the calculated total content of poly-
phenolic compounds is equal to 7.24 ± 0.09 mg/
cm3. The antioxidant activity in relation to the
DPPH is equal to 89.48 ± 0.54%. The calculated val-
ues indicate that the birch leaf extract is a very
strong antioxidant (Da Porto et al. 2000).
Figure 2(B) presents the disappearance pattern
of DPPH free radicals in the presence of tannic
acid aqueous solution and aqueous birch extract
within a time period of 600 s. The DPPH con-
sumption profile has a characteristic biphasic
course. In the initial phase, a very fast degrad-
ation occurs which is followed by slower decay.
The first fast degradation is attributed to the fol-
lowing reaction (Nyau et al. 2015):
i. DPPH þ (Y-H) ! DPPH-H þ Y
(purple) (yellow)
ii. Y þ DPPH ! products
The subsequent slower step may be explained
by degradation of by-products. The consumption
rate is much faster in the case of using tannic
acid as antioxidant agent. After 100 s, the con-
sumption rate is two times greater for tannic acid
than for birch extract.
Nanoemulsions with birch leaf extract
Sixteen emulsions with pale yellow colors
were obtained.

Figure 2. Calibration curve for determination of total content of polyphenolic compounds (A), kinetic profile of DPPH consumption
by both tannic acid and antioxidants present in birch extract (B), microphotograph of emulsion with colored oil and water phases
(sample E12) (C), photo of delaminated sample (E1) taken in t21 (D).
Determination of the emulsion type
Both optical microscopy and the “blending of
droplets” method indicated that all products are
o/w emulsions. When staining with dyes, it was
observed that the external phase was dyed blue
and the inner phase dyed orange, which means
that the oil phase is dispersed within the water
phase. This result was confirmed in the “blending
of droplets” method in which the droplets of the
emulsion and water were mixed easily. Figure
2(c) presents the microphotograph taken with an
optical microscope.
The results are in line with other researchers,
who confirmed that soy lecithin is an effective o/
w emulsifier (Yamamoto and Araki 1997; Pichot
et al. 2013).
Assessment of polyphenolic compounds’ encapsula-
tion efficiency
The changes of the encapsulation efficiency along
time are presented in Figure 3(A). In all prod-
ucts, the encapsulation efficiency reached a high
level of approximately 80–90%. Within the ana-
lysis period (21 d), it remains quite constant. This
means that the release of antioxidant substances
is limited over this time. Thus, the availability of
active agents does not change. The direction fac-
tor, which was determined in the trend line equa-
tion, was the subject of statistical analysis which
is presented below.
CHEMICAL ENGINEERING COMMUNICATIONS 9
DLS analysis
Results of electro-kinetic potential (f) are pre-
sented in Figure 4(A). The achieved values of the
electro-kinetic potential, ranging from
10 to

40 mV, are enough to form an efficient energy
barrier made by anionic molecules on the emul-
sion micelles’ surface (Shanmugam and
Ashokkumar 2014). One may observe that the
clear majority of emulsions have a good colloidal
stability, and thus possess the ability to hold
micelles in the colloidal dispersion. This is indi-
cated by the values of the electro-kinetic poten-
tial, whose absolute values are greater than
20 mV. The results of the average size measure-
ment confirm the presence of nanomicelles in the
products obtained. The dependence of the aver-
age size upon time is presented in Figure 3(B).
It may be concluded that, in general, the aver-
age size of the nanomicelles increased with time.
This dependence was observed especially in the
case of the Voil phase:Vwater phase, the values of
which were equal to 0.5 and 1.25. However, in
some cases, the size of micelles decreased on the
last day of analysis. When Voil phase:Vwater phase¼
2.0, the particle sizes were distributed randomly
in time.
Creaming stability measurement
The creaming index (CI) values were calculated
based on photos taken on the 21st day of
10 J. PULIT-PROCIAK ET AL.

Figure 3. Dependence of EE upon time (A), dependence of micelles average size upon time (B), dependence of CI upon time (C),
and dependence of pH upon time (D).

observation. Figure 2(D) presents an example of
the method for the CI calculation.
The dependence of CI on time is presented in
Figure 3(C). The best CI values (0%) were
obtained for samples E3, E4, and E12. These
products were homogeneous over the whole
observation period. A satisfactory CI (17%) was
obtained for sample E7 (Juttulapa et al. 2017).
pH analysis
Changes in the pH values with time are pre-
sented in Figure 3(D). In all samples, the pH

Figure 4. Results of electro-kinetic potential for nanoemulsions with birch leaf extract measured in t0 (A) and with tannic acid
measured at t0 (B).
value is expected to be acidic. The pH value may
be correlated with the EE. The assumption is that
as the efficiency of the extract encapsulation
increases, the concentration of the acidic com-
pounds that birch extract consists of should
decrease. The nature of the birch extract’s com-
position is the reason for this. The more acidic
the compounds encapsulated in micelles are, the
lower their content is outside the micelle and,
hence, the higher the pH value. This analogy is
presented in Figure 5(A–F).
Nanoemulsions with tannic acid as a source of
antioxidant compounds
It was possible to obtain sixteen emulsions with a
pale yellow color.
Assessment of polyphenolic compounds’ encapsula-
tion efficiency
The dependence of the EE upon time is pre-
sented in Figure 6(A).
CHEMICAL ENGINEERING COMMUNICATIONS 11
It is observed that the initial encapsulation effi-
ciency rises slightly. This means that, for the vast
majority of samples, tannic acid was encapsulated
for up to 2 d, indicating that the encapsulation of
the active ingredient did not occur only during
emulsification of the product but also during its
storage. When Voil phase:Vwater phase is equal to
0.5, the encapsulation of tannic acid at the begin-
ning is very effective (approximately 70%), but
after a few days it decreases to values of around
30%. This means that the tannic acid was
released from the micelles over time, which is
highly desirable. A similar situation is observed
when Voil phase:Vwater phase is equal to 1.0. The
direction factor, which was determined in the
trend line equation, was the subject of statistical
analysis, which is presented below.
DLS analysis
Figure 4(B) provides the results of the electro-
kinetic potential (f) for the nanoemulsions with
12 J. PULIT-PROCIAK ET AL.
Figure 5. Juxtaposition of the pH and EE for the nanoemulsions with birch leaf extract (A – E1, B – E2, C – E7, D – E8, E – E11, F
– E12) and for nanoemulsions with tannic acid (G – T4, H – T7, I – T8, J – T12).
tannic acid. In almost all cases, the emulsions
were characterized by high absolute values of the
electro-kinetic potential, which were greater than
20 mV. Therefore, micelles were held in an effi-
cient colloidal dispersion (Ostolska and
Wisniewska 2014). The average size of micelles
indicates that they were on a nanometric scale.
The dependence of the average size upon time is
presented in Figure 6(B).
Apart from some samples (T3, T4, T8), one
may conclude that the average size of the nano-
micelles was quite constant over time. Moreover,
with a greater ratio of oil phase to water phase,
the average size of the nanomicelles increased.
Creaming stability measurement
Since all the emulsions were visually stable, it was
not possible to assess the delamination of the
products. The values for the creaming index
measured in t14 are provided in Figure 7.
pH analysis
The measured pH values for the obtained prod-
ucts are provided in Figure 6(C). It was con-
firmed that all the samples were acidic. A general
interaction of pH and EE was expected. One may
assume that when the encapsulation efficiency
increases, the pH should increase as well. This is
due to the fact that tannic acid is strongly acidic
(its pH is equal to 3) and. when it is trapped in
micelles, the pH value should be greater (Hem
1960). This hypothesis was mostly observed when
Voil phase:Vwater phase was equal to 1.0 and it is
presented in Figure 5(G–J).
Statistical analysis
Nanoemulsions with birch leaf extract
Figure 8(A) presents the Pareto charts for our
parameters. Parameters that are statistically sig-
nificant for the established p value (p < 0.05) are
denoted with a red line.
The homogenization time in linear and square
functions, as well as the product of the choles-
terol concentration and homogenization time, are
the parameters that affect the encapsulation effi-
ciency with a statistical significance. The electro-
kinetic potential is dependent only on the choles-
terol concentration. No input parameter influen-
ces the average size of the micelles with the
established p values. The homogenization time in
linear and quadratic functions, as well as the
product of the volume of the oil phase to the vol-
ume of water phase ratio and the homogenization
time, influence the values of the creaming index.
The approximation profiles are presented in
Figure 9(A).
Based on an assessment of the approximation
profiles, the influence of independent parameters
on dependent variables and determination of the
specific values of input parameters ensure that
reaching the desired values of the output parame-
ters is possible.
Higher encapsulation efficiency is more desir-
able. Taking into consideration the ratio of the
volume of oil phase to the volume of water

Figure 6. Dependence of EE upon time (A), dependence of micelles average size upon time (B), and dependence of pH upon
time (C).
phase, there is an inflection point. Initially, when
this ratio increases, the EE value increases as
well. After Voil phase:Vwater phase reaches a value of
1.25, the EE begins to decay. When the choles-
terol concentration increases, the encapsulation
efficiency also increases. Neither short nor long
homogenization time favors the efficient encapsu-
lation of the birch extract. The best time value is
equal to 11.5 min. A higher absolute value of the
electro-kinetic potential is desired, so the lower
its value the better. Micelles are more stable if
the ratio of the volume of oil phase to the vol-
ume of water phase decreases, and if the choles-
terol content is lower. This is due to the fact that
cholesterol favors the formation of w/o emulsion
and it exerts an antagonistic effect (Moustafa
CHEMICAL ENGINEERING COMMUNICATIONS 13
1995). Extension of the homogenization time
does not favor the production of stable micelles.
Lower values of the creaming index are desirable.
They are favored by decreasing the ratio of the
volume of the oil phase to the volume of the
water phase and by extension of the homogeniza-
tion time.
Analysis of direction factor
The influence of the input parameters on the dir-
ection factor, which was determined in the trend
line equation in plots of the dependence of EE
upon time, was analyzed. Negative values are
desirable as the negative value of the direction
factor means that the active encapsulation effi-
ciency decreases with time and thus the active
14 J. PULIT-PROCIAK ET AL.
Figure 7. CI values for emulsions with tannic acid at t14.
substance is released. The impact of the inde-
pendent variables on the direction factor values is
presented in Figure 10(A).
Based on the results obtained, it is obvious
that the direction factor, which reflects the releas-
ing of the birch extract components, is mostly
dependent on the homogenization time. There is
an inflection point in the values over time that
gives the most desirable value of the direction
factor. This occurs at 17 min. Lower and higher
values of the homogenization time mean that the
release rate is weaker. However, one must be
aware that the EE values themselves in general
do not differ much. Thus, this result has a theor-
etical meaning.
Nanoemulsions with tannic acid
Figure 8(B) presents the Pareto charts for our
parameters. The parameters which are statistically
significant for the established p value (p < 0.05)
are marked with a red line.
The encapsulation efficiency is mostly depend-
ent on the ratio of oil phase to water phase over
square and linear functions. The product of the
gelatin concentration and the ratio of the oil
phase to the water phase also have a significant
effect. The remaining parameters are statistically
not significant. No input parameter influences
the electro-kinetic potential. The average size of
micelles is affected only by the product of the
ratio of the oil phase to the water phase and the
time of homogenization. The gelatin concentra-
tion is the only parameter that influences the
creaming index.
The approximation profiles are presented in
Figure 9(B). Based on the approximation profiles,
it is possible to assay the influence of the inde-
pendent parameters on the dependent variables
and to determine the specific values of the input
parameters that ensure the desired values of the
output parameters are reached.
The encapsulation efficiency increases slightly
when the concentration of gelatin also increases.
In order to obtain a higher EE, the value of the
Voil phase:Vwater phase should be adjusted to 0.625.
The average size of micelles increases when the
gelatin concentration increases. The smallest
micelles are obtained when no gelatin is used.
The lowest value of the Voil phase:Vwater phase ratio
gives the smallest micelles. The homogenization
favors reduction of the size of the micelles; the
longer the homogenization time, the smaller the
micelles obtained. Again, the absence of gelatin
gives the most stable emulsions, since the Cgel is
equal to 0, and the corresponding CI values are
the lowest. This may be due to the presence of
gelatin disintegration products, which are formed
under sonication frequencies, and which destabil-
ize the emulsion. Increasing the oil phase content
favors a reduction in the CI values.
Analysis of the direction factor
The influence of the input parameters on the dir-
ection factor, which was determined in the trend
line equation in the plots of the dependence of
EE upon time, was analyzed. Negative values of
the direction factor are most desirable. The
impact of the independent variables on the direc-
tion factor values is presented in Figure 10(B).
 CHEMICAL ENGINEERING COMMUNICATIONS 15

Figure 8. Pareto charts for nanoemulsions with birch leaf extract (A) and nanoemulsions with tannic acid (B).

The results suggest that only one independent of the oil phase to the volume of the water phase.
variable has a significant impact on the direction An inflection point is observed. This means that
factor in general. This is the ratio of the volume the most desirable value of the volumetric ratio
16 J. PULIT-PROCIAK ET AL.
Figure 9. Approximation profiles for nanoemulsions with birch leaf extract (A) and nanoemulsions with tannic acid (B).
lies between 0.625 and 0.813. Any lower and
higher values of this ratio give products that
release tannic acid in a reduced time.
Discussion
Nanoemulsions are effective formulations for
active substance transfer, and thanks to their bio-
logical origin they do not pose a threat to human
tissues and the environment. Hussein et al.
(2017) conducted similar research, where they
studied the impact of nano-encapsulated and
nanoemulsion forms containing carvacrol on liver
fibrosis. Carvacrol is a phenolic compound which
is known for its anti-inflammatory, immunomo-
dulatory, and chemopreventive properties.
Carvacrol was incorporated into o/w nanoemul-
sions which were prepared with the use of sonic-
ation frequencies. The authors conclude that the
incorporation of carvacrol by nanoemulsions
enhances its bioavailability. Moreover, such a for-
mulation could be an effective product in pro-
tecting against liver fibrosis.
Pulicharla et al. (2016) have confirmed that
formulations containing encapsulated strawberry
extract improved the bioavailability and are char-
acterized by a sustained release of active
substances. Researchers used chitosan as the host
compound, with its negative amino groups that
attract positive groups of polyphenolic com-
pounds, and thus formed micelles that are more
stable. The achieved encapsulation efficiency was
approximately 60%. Also, the pH of the sur-
rounding medium was studied. It was confirmed
that this factor has a large impact on the release
of polyphenolic compounds.
Similar studies were conducted by Donsi et al.
(2011). They encapsulated resveratrol and curcu-
min, which are known for their antioxidant and/
or antimicrobial properties, in a nanoemulsion-
based delivery system. The aim of encapsulation
was to obtain the desired dispersion of active
substances, and to improve their bioavailability
and protect them against degradation factors.
Resveratrol was trapped in peanut oil-based
nanoemulsions, using soy lecithin and sugar
esters as emulsifiers. Curcumin was encapsulated
in solid lipid nanoemulsions, using stearic acid
and/or palm oil as the lipid phase. The homogen-
ization technique was employed in the studies. It
was confirmed that the highest concentration of
active substances that can be encapsulated with-
out delamination of the emulsions is 0.1% and
0.01% for curcumin and resveratrol, respectively.

Figure 10. Impact of independent variables on direction factor values for nanoemulsions with birch leaf extract (A) and nanoemul-
sions with tannic acid (B).
Arulmozhi et al. (2013) researched the encap-
sulation of ellagic acid into chitosan-based nano-
particles by an ionic gelation method. They also
studied the release of the antioxidant substance
and its activity against tumors in the KB cell line.
The results show that the release of the ellagic
acid in a phosphate buffer is rapid up to 3 h, and
over the subsequent 45 h the velocity is gradual
and sustained. According to the authors, the
rapid rate may be due to the desorption of ellagic
acid from polymer particles, while their release
from the inner space of nanoparticles is slower.
Thus, such a formulation may be considered as
an effective drug delivery system. Consequently,
trapped ellagic acid exhibits anticancer activity
which was confirmed against the human oral
cancer cell line.
Paini et al. (2015) conducted research on the
possibility of apigenin encapsulation in liposome
micelles. Apigenin is an active polyphenolic com-
pound that has a large activity against tumors
and cardiovascular disorders. However, it is
defined by its low stability and poor bioavailabil-
ity. The authors used lecithin as the liposome
shell that traps the active substance and the
whole system is thus more resistant to mechan-
ical treatment. The encapsulation efficiency
reached 98% and the average size of micelles was
around 160 nm. It was observed that the process
parameters, such as the sonication amplitude and
CHEMICAL ENGINEERING COMMUNICATIONS 17
time, are crucial in controlling the properties of
the products.
Our formulations also meet the requirements
for the stability, size of micelles, encapsulation
efficiency, and controlled release of active sub-
stances that are desirable for drug delivery sys-
tems, especially in the case of tannic acid.
Similarly, the bioavailability of curcumin may
be enhanced by loading silane–hydrogen nano-
particles with this lipophilic active substance.
Krausz et al. (2015) conducted studies on the
wound healing efficacy of such a formulation. A
high antimicrobial activity against Staphylococcus
aureus and Pseudomonas aeruginosa was con-
firmed. This effect is explained by the greater
topical administration of curcumin, which has a
nanometric size, such that its chemical activity
against some microorganisms occurring in
wounds is also accelerated.
Conclusion
It was possible to encapsulate active compounds
(birch leaf extract and tannic acid) with antioxi-
dant properties into the biopolymeric micelles
which are contained in o/w emulsions.
Considering the encapsulation of birch leaf
extract, we conclude that, despite the fact that its
encapsulation efficiency is high, it remains quite
constant for 21 d, which means that the studied
18 J. PULIT-PROCIAK ET AL.
conditions did not allow the formation of emul-
sions that can give controlled release of active
substances. Moreover, the average size of the
micelles increases with time and most of the
emulsions delaminate. However, kinetically stable
emulsions were obtained successfully, and these
had the lowest content of the oil phase and were
obtained when the homogenization time was
the longest.
The encapsulation of tannic acid gave satisfac-
tory results. It is possible to obtain an emulsion
with controlled release of the active substance.
The average size of the micelles remains constant
and their electro-kinetic potential absolute values
are high, which means that the micelles them-
selves are stable. All products were stable kinetic-
ally and this was confirmed by the low values of
the creaming index. We confirm that the pres-
ence of gelatin is not desirable since the products
of its decomposition cause the encapsulation effi-
ciency to decrease, the micelles size to increase,
and favor the delamination of the emulsions. The
action of lecithin as an emulsifier is sufficient to
obtain the desired formulation.
Future work will cover obtaining nanoemul-
sions containing antioxidant agents on a larger
laboratory scale. It will then be possible to con-
firm the so-far selected process parameters that
would allow the large-scale manufacturing of
desirable products.
Disclosure statement
The authors report no conflicts of interest.
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