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COPYRIGHT
“The author and the promoters give the authorization to consult and to copy parts of this thesis for personal
use only. Any other use is limited by the laws of copyright, especially concerning the obligation to refer to the
source whenever results from this thesis are cited."
Promotor Co-promotor
Prof. Dr. Kevin Braeckmans Raul Machado
Assistant researcher (CBMA)
Author
Julie Pijpers
Abstract
Essential oils are complex mixtures of volatile, hydrophobic components derived of plant material.
Problems with EO’s may arise due to their lack of physical stability. They are highly volatile, unstable and
sensitive to heat, oxygen and light. One of the possible solutions is nanoencapsulation which will lead to an
enhanced stability. Nanoencapsulation is mainly used in the development of therapeutic applications, but
essential oils are involved in numerous other applications. During the last decades, they already have been
reported being useful as antibacterial agents, antiviral agents, skin cleansers, food additives and so on.
The aim of this study is to develop a delivery platform for essential oils, more specific for Mentha piperita and
Eucalyptus globulus. The intention is to investigate the feasibility of encapsulating essential oils in chitosan
formulations by assessing their encapsulation and release profile. To achieve this, several techniques were
applied. The essential oils are characterized by UV-VIS spectrophotometry and infrared radiation. The
properties of chitosan nanoparticles are determined using dynamic light scattering. The essential oil loaded
chitosan particles are examined by calculating the encapsulation efficiency, the loading capacity with the help
of UV-VIS measurements. Lastly, a release profile for both oils is created with anew help of UV-VIS
spectrophotometry.
From the experimental data, it can be deduced that the formation of chitosan nanoparticles has been
successful. Depending on the type of application for which the particles will be used, parameters such as
chitosan concentration can be adjusted. The encapsulation efficiency values show that the encapsulation of
both oils into chitosan has been successful as well. However, there is still room for improvement. One of the
goals in the future could be an optimization of the release profile. For example, by experimenting with other
solvents or by applying other conditions.
Samenvatting
Etherische oliën zijn complexe mengsels van vluchtige, hydrofobe componenten afgeleid van plantaardig
materiaal. Problemen met etherische oliën kunnen zich voordoen als gevolg van hun gebrek aan fysieke
stabiliteit. Ze zijn zeer vluchtig, onstabiel en gevoelig voor warmte, zuurstof en licht. Eén van de mogelijke
oplossingen is nano-encapsulatie die tot een verbeterde stabiliteit zal leiden. Nano-encapsulatie wordt
voornamelijk gebruikt in de ontwikkeling van therapeutische toepassingen, maar essentiële oliën zijn ook
betrokken bij tal van andere toepassingen. In de afgelopen decennia zijn ze al gerapporteerd als antibacteriële
middelen, antivirale middelen, huidreinigers, voedingssupplementen enzovoort.
Het doel van deze studie is om een platform voor essentiële oliën te ontwikkelen, meer specifiek voor Mentha
piperita en Eucalyptus globulus. De bedoeling is om de haalbaarheid te onderzoeken van het inkapselen van
essentiële oliën in chitosan formuleringen door hun inkapselings- en vrijgaveprofiel te beoordelen. Om dit te
bekomen, werden verschillende technieken toegepast. De essentiële oliën worden gekarakteriseerd door UV-
VIS spectrofotometrie en infraroodstraling. De eigenschappen van de chitosan nanodeeltjes worden bepaald
met behulp van dynamische lichtverstrooiing. De olie-beladen chitosan deeltjes worden onderzocht door de
encapsulatie-efficiëntie en de ladingscapaciteit te berekenen met behulp van UV-VIS metingen. Tenlotte wordt
een release profiel voor beide oliën gecreëerd met behulp van UV-VIS spectrofotometrie.
Uit de experimentele gegevens kan afgeleid worden dat de vorming van chitosan nanodeeltjes succesvol is.
Afhankelijk van het type applicatie waarvoor de deeltjes zullen worden gebruikt, kunnen parameters zoals de
concentratie van chitosan worden aangepast. De encapsulatie-efficiëntie waarden tonen aan dat de
encapsulatie van beide oliën in chitosan ook geslaagd is. Er is echter nog ruimte voor verbetering. Eén van de
doelen in de toekomst kan een optimalisatie van het releaseprofiel zijn, bijvoorbeeld door te experimenteren
met andere oplosmiddelen of door andere condities toe te passen.
Acknowledgements
First of all, I would like to thank my promotor Prof. Kevin Braeckmans for giving me the opportunity to write this thesis .
I want to thank my supervisor Raul Machado for introducing me into the wonderful world of
nanoparticles, for his guidance through this project, for his expertise.
A big thank you to the people working in the laboratory of molecular biology to be there for me with tips and tricks whenever needed.
I would like to thank my parents and my brothers for their support through the many Skype
calls, for their advice when I needed it and most of all for the incredible opportunity they gave me to study abroad.
Last but not least, I would like to thank my international Erasmus friends for making these 4 months an adventure of a lifetime!
TABLE OF CONTENTS
1. INTRODUCTION.................................................................................................................................................................................................................... 1
1.1 ESSENTIAL OILS ........................................................................................................................................................................................................... 1
1.1.1 Mentha piperita (MP) ................................................................................................................................................................................... 3
1.1.2 Eucalyptus globulus (EG) .......................................................................................................................................................................... 4
1.2 CHITOSAN ..................................................................................................................................................................................................................... 5
1.3 PRODUCTION OF CHITOSAN NANOPARTICLES ..........................................................................................................................................6
1.3.1 By Ionic gelation ............................................................................................................................................................................................6
1.3.2 By emulsification and cross-linking .................................................................................................................................................. 7
1.3.3 By emulsification solvent diffusion method ............................................................................................................................... 8
1.3.4 By reverse micellar technique ............................................................................................................................................................. 8
1.4 ENCAPSULATION WITH CHITOSAN ...................................................................................................................................................................9
2. OBJECTIVE ..........................................................................................................................................................................................................................10
3. MATERIALS & METHODS ............................................................................................................................................................................................. 11
3.1 IDENTIFICATION AND CHARACTERIZATION OF THE ESSENTIAL OILS ........................................................................................... 11
3.1.1 UV-VIS .................................................................................................................................................................................................................. 11
3.1.2 ATR-FTIR ........................................................................................................................................................................................................... 12
3.2 PREPARATION OF CHITOSAN NANOPARTICLES ..................................................................................................................................... 13
3.2.1 Protocol ............................................................................................................................................................................................................ 14
3.2.2 Dynamic light scattering (DLS) .......................................................................................................................................................... 14
3.2.3 Yield of the particles ............................................................................................................................................................................... 15
3.3 PREPARATION OF ESSENTIAL OIL -LOADED CHITOSAN PARTICLES ............................................................................................ 16
3.3.1 Encapsulation efficiency ........................................................................................................................................................................ 16
3.3.2 Loading capacity ........................................................................................................................................................................................ 16
3.4 RELEASE PROFILE ................................................................................................................................................................................................. 17
3.4.1 Static conditions .......................................................................................................................................................................................... 18
3.4.2 Dynamic conditions .................................................................................................................................................................................. 18
3.5 TRACEABILITY .......................................................................................................................................................................................................... 18
4. RESULTS ............................................................................................................................................................................................................................. 20
4.1 UV-VIS ......................................................................................................................................................................................................................... 20
4.1.1 Calibration curve of Mentha piperita.............................................................................................................................................. 20
4.1.2 Calibration curve of Eucalyptus globulus .................................................................................................................................... 21
4.2 FT-IR ........................................................................................................................................................................................................................... 22
4.2.1 FTIR: Spectra of Mentha piperita ...................................................................................................................................................... 22
4.2.2 FTIR: Spectra of Eucalyptus globulus ............................................................................................................................................ 23
4.3 DLS – PARTICLE SIZE .......................................................................................................................................................................................... 23
4.4 DLS – CORRELOGRAMS ..................................................................................................................................................................................... 25
4.5 ENCAPSULATION EFFICIENCY ........................................................................................................................................................................ 26
4.6 LOADING CAPACITY ............................................................................................................................................................................................. 27
4.7 YIELD OF THE PARTICLES ................................................................................................................................................................................. 28
4.8 RELEASE PROFILE ................................................................................................................................................................................................ 29
4.9.1 Static conditions ........................................................................................................................................................................................30
4.9.2 Dynamic conditions ................................................................................................................................................................................. 33
5. DISCUSSION ...................................................................................................................................................................................................................... 36
5.1 IDENTIFICATION OF THE ESSENTIAL OILS ................................................................................................................................................. 36
5.1.1 UV-VIS spectroscopy ................................................................................................................................................................................. 36
5.1.2 FT-IR spectroscopy .................................................................................................................................................................................... 37
5.2 CHARACTERISATION OF CHITOSAN NANOPARTICLES with DLS.................................................................................................... 37
5.3 CHARACTERISATION OF ESSENTIAL OIL LOADED NANOPARTICLES ............................................................................................ 37
5.3.1 Encapsulation efficiency ....................................................................................................................................................................... 37
5.3.2 Loading capacity........................................................................................................................................................................................ 38
5.4 Yield of the particles ........................................................................................................................................................................................ 38
5.5 RELEASE PROFILE ................................................................................................................................................................................................ 38
5.5.1 Static conditions ......................................................................................................................................................................................... 38
5.5.2 Dynamic conditions ................................................................................................................................................................................. 39
6.CONCLUSION .................................................................................................................................................................................................................... 40
7. REFERENCES ..................................................................................................................................................................................................................... 41
Abbreviations
ATR-FTIR: Attenuated Total Reflectance Fourier Transform
DLS: Dynamic Light Scattering
EE: Encapsulation Efficiency
EG: Eucalyptus globulus
EO: Essential oil
LC: Loading Capacity
MP: Mentha piperita
PBS: Phosphate buffered saline
ST. DEV.: Standard Deviation
UV-VIS: Ultraviolet visible light
1
1. INTRODUCTION
1.1 ESSENTIAL OILS
According to the European Pharmacopoeia, essential oils are described as followed: “Odorant products,
generally of a complex composition, obtained from a botanically defined plant raw material, either by
driving by steam of water, either by dry distillation or by a suitable mechanical method without heating. An
essential oil is usually separated from the aqueous phase by a physical method that does not lead to
significant change in its chemical composition”. [1]
Essential oils (EO’s) are complex and contain highly volatile compounds. The main contributors to the volatility
are hydrocarbons (f.ex. limonene, pinene), alcohols (f.ex. menthol), aldehydes (f.ex. citral) and phenols (f.ex.
eugenol). These substances are commonly classified in two categories: terpenoids and phenylpropanoids. [2]
Besides volatile compounds, additional aromatic and aliphatic components can also be present. Usually, the
bioactivity of an essential oil is determined by one or two main components. [3] Applied to this project,
menthol and 1,8-cineole are the major components of respectively Mentha piperita and Eucalyptus globulus
essential oil.
Figure 1.1: Examples of hydrocarbons found in essential oils – Limonene (left) and P-cymene (right) (site 1)
Besides highly volatile, essential oils are also hydrophobic resulting in limited solubility in water. Their density
is slightly lower than water. The most commonly used solvent for EO’s is ethanol. [2] Essential oils have a
colorless to pale yellow color.
2
Going further on the physical properties of EO’s, it is known that these oils are unstable and sensitive to heat,
oxygen and light during processing, analysis and storage. [4] Oxidation can easily occur due to the presence of
double bounds and functional groups (f.ex. aldehydes, hydroxyl) in the structure of essential oils. This physical
instability can be overcome by, for example, nanoencapsulation. The latter will result in enhanced stability,
protection against oxidation, retention of volatile substances and so on… [5] In addition, the encapsulation of
essential oils also leads to improved efficacy and sustained release. At the moment, there is an increasing
interest in developing colloidal particles as a new application on the field of dermatology and local skin
therapy. [6] Dermatology and local skin therapy are by far not the only territories in which applications of
essential oils have been established. In the following paragraphs, some other applications are explained more
in detail.
The human body is exposed to essential oils through nutrition. EO’s are worldwide used as food additives. Think
of examples like lemongrass, spearmint and citrus peel. Thanks to their lipophilic character, essential oils can
easily be absorbed in the blood stream. It has not been thoroughly investigated yet what the average intake is
of essential oils through nutrition. [2]
Another feature of EO’s is their ability to act as an antibacterial agent. Since the disturbing rise in bacterial
resistance, there is a need to develop alternative therapies to assist antibiotics and the available drugs in its
limitations. These alternative therapies include the use of essential oils. These oils display a wide spectrum of
inhibitory activities against Gram positive as well as against Gram negative bacteria. The antibacterial potency
can vary between different essential oils. Overall, there are already great results published on the use of EO’s
in the treatment of bacterial infections. Essential oils can be used in low concentrations, for example 1%, and
still show enough antibacterial activity which is remarkable. Besides bacteria, essential oils are also active
against viruses. One of their established antiviral activities is the inhibition of viral replication which can be
contributed to the presence of phenylpropanoid components in EO’s. [3]
One of the challenges in the medical field nowadays is still trying to cure cancer, essential oils are also
involved in the research on this territory. It has been reported that several plant essential oils are able to
lower the number of malignant cells. Terpenoids for example – one of the major components of essential oils –
3
has been reported successful in the prevention of tumor cell proliferation by inducing apoptosis or necrosis.
These results assume that EO’s have anticancer potency and further research on this territory could possibly
lead to evolutionary results. [3]
1.1.1 Mentha piperita (MP)
Mentha piperita (or peppermint) oil is derived from the leaves of the plant Mentha arvensis. The most common
process to obtain the oil from the plant material is extraction by steam distillation. Subsequently a
modification and purification step is performed to make the oil ready for use. A pale-yellow liquid with a
watery viscosity is obtained. [7] The major chemical compounds of Mentha piperita essential oil are: limonene,
cineole, menthone, menthofuran, isomenthone, menthyl acetate, isopulegol, menthol, pulegone, carvone...
These compounds contribute to the hydrophobic character of Mentha piperita. This essential oil is soluble in
ethanol, ether and methylene chloride.[8] The chemical constituents can vary between different peppermint
oils, but overall menthol is present in the largest quantity. Menthol is responsible for the characteristic odor
and taste of peppermint. It triggers the nerves and sends signals to the brain which induces the cold sensation
and fresh breath. [9]
Figure 1.2: Structure of Menthol (site 2) Figure 1.3: Peppermint oil (site 3)
Mentha piperita essential oil has a lot of therapeutic properties such as antiseptic, analgesic, antispasmodic,
expectorant and so on. These characteristics are correlated with the uses of peppermint oil. It has been
assessed that Mentha piperita can help in the treatment of irritable bowel syndrome, hot flushes, depression,
nasal congestion, bronchitis and so on. In this project, the focus lies on external applications. Regarding the use
of peppermint essential oil in this context, it is known that Mentha piperita reduces itchiness, relieves pain and
cools down the skin. [site 3]
4
1.1.2 Eucalyptus globulus (EG)
Eucalyptus belongs to the Myrtaceae family. Overall, there are already more than 700 species of Eucalyptus
discovered around the world. Eucalyptus originates in Australia. The species from which essential oil is
obtained are: Eucalyptus globulus (Tasmanian blue gum), Eucalyptus camaldulensis (River red gum),
Eucalyptus citriodora (lemon-scented eucalyptus) and Eucalyptus polybractea (blue mallee). [10] Eucalyptus
globulus will be the species of interest throughout this work.
As well as for Mentha piperita, the most common production process of Eucalyptus globulus essential oil is by
steam distillation. Anew, a pale-yellow oil with a watery viscosity is obtained. However, one can easily
distinguish this essential oil from the previously mentioned peppermint oil based on their fragrance.
Eucalyptus globulus has a very clear characteristic smell. It is mainly derived from Eucalyptol or 1,8-cineole
which is also the major compound in this essential oil. Besides the aroma, 1.8-cineole also contributes the most
to the therapeutic properties and chemical behavior of Eucalyptus globulus. Other chemical compounds that
can be present are: α-pinene, o-cymene, limolene, isopulegol… [11] This oil is soluble in ethanol, ether and
slightly soluble in carbon tetrachloride. (site 11)
Figure 1.4: Structure of 1,8-cineole (site 4) Figure 1.5: Eucalyptus globulus (site 3)
Eucalyptus globulus has a broad range of biological properties such as anti-microbial, antiseptical,
antifungicidal... In the field of therapeutics, numerous applications are also known. This essential oil helps in
the treatment of respiratory infections like asthma, throat infections and sinusitis. It has been discovered that
Eucalyptol leads to a decrease in inflammation and mucus production which in turn leads to a relief of
headache when fighting a cold or an allergic reaction for example. In the framework of this project, the
5
spotlight is on the external use. Eucalyptus globulus essential oil has an invigorating and sanitizing effect on
the body. It is used inter alia to sooth wounds, as a skin cleanser and moisturizer. (site 3)
1.2 CHITOSAN
Chitosan is a polysaccharide consisting of N-acetyl glucosamine and D-glucosamine units. It is a derivative of
chitin, which is found in the skeletons of crustacea. Animals belonging to the crustacea include shrimps, crabs
and lobsters. [12] Chitin can also be found in the cell wall of bacteria or fungi. There are 4 steps in the
production of chitosan. Firstly, chitin is deacetylated by the enzyme chitinase or by NaOH as illustrated in
figure. The degree of deacetylation varies between 50-95%, however it is never fully deacetylated which
results in an alteration of acetylated and deacetylated monomers in the final structure. The following steps are
deproteinization, demineralization and decolorization. There are different types of chitosan, depending on the
way it was produced. Regarding the structure of the obtained chitosan, each monomer contains one primary
amine group and two free hydroxyl groups. Chitosan is a weak base with a pKa value in the range of 6.2-7.0.
The primary amine group in its protonated form plays a critical role in different processes such as the
solubilization of chitosan and the interaction with crosslinking agents in order to form chitosan nanoparticles.
The solubility of chitosan in organic acids is restricted. However, it is soluble in lactic acid, formic acid and
acetic acid. These acids are most used in their 1% concentration when dissolving chitosan. Inorganic acids like
phosphoric acids and sulphuric acids cannot be used, since chitosan is insoluble in it. [13]
Figure 1.6: Structure of Chitosan (site 5)
6
Commercially available chitosan occurs in the form of white to yellow colored flakes. During the years, there
has been a growing interest in chitosan because of its appealing properties. This polymer is biocompatible,
non-toxic, biodegradable and anti-immunogenic. Chitosan has applications in pharmaceutical and cosmetic
industries as well as in food industries.
Regarding the use of chitosan in pharmaceutical industries, the amount of publications on this subject has
been increasing since the last decade. Chitosan plays a role in several applications in drug delivery. Some
examples of these applications are: hydrogels for controlled and localized drug delivery, targeted delivery for
low molecular drugs and nanostructures for the delivery of essential oils which will be further discussed in this
work. [13]
1.3 PRODUCTION OF CHITOSAN NANOPARTICLES
Chitosan nanoparticles can be produced by several different methods. Particularly the physical properties of
the particles are most influenced by the mode of production. Some common methods are discussed in the
following paragraphs.
1.3.1 By Ionic gelation
Ionic gelation is the applied method in this project. It is the most simplistic protocol, yet very efficient. The
mechanism responsible for the formation of the nanoparticles is the electrostatic interaction between the
positively charged amino groups of chitosan and the negatively charged groups of a chemical crosslinker as
illustrated below. Examples of crosslinkers used in the process of ionic gelation are: tripolyphosphate (TPP),
sulphuric acid or inorganic ions like Fe(CN)64 and calcium ions. In this work, TPP is used as a crosslinker.
Besides chitosan and the crosslinker, the addition of a stabilizing agent such as Tween or polyethylene glycol is
very common. This agent will prevent fusion of the nanoparticles. The size of the obtained particles can be
influenced by altering the ratio of chitosan and crosslinker/stabilizing agent. [13]
7
Figure 1.7: Mechanism of Ionotropic gelation (site 6)
Green lines: chitosan polymer with positively charged amino groups
Grey lines: crosslinking agent with negatively charged anions
1.3.1.1 Tripolyphosphate as a crosslinker
Tripolyphosphate (TPP) occurs in the form of sodium tripolyphosphate. It is a white very hygroscopic powder
which implies its good solubility in water. This chemical is used as an emulsifier, detergent, stabilizer, chelating
agent… As figure illustrates, TPP is highly charged due to the phosphate ion. These chemically reactive groups
make TPP greatly suitable for crosslinking. [14]
Figure 1.8: Structure of Sodium tripolyphosphate (site 7)
1.3.2 By emulsification and cross-linking
In this method, the cross-linking is not the main principle behind the formation of the nanoparticles. It is used
as an additive method to harden the obtained particles. In a first step, a W/O (water-in-oil) emulsion is formed,
consisting of chitosan solution (oil phase) and an aqueous phase with surfactant (Span 80 as an example
illustrated in figure 1.9). Secondly, a cross-linking agent – glutaraldehyde in the illustrated example – is added
and reacts with the amino groups of chitosan. It has been assessed that with this method, the particle size is
highly influenced by the stirring speed. Although this method is often used, it contains some disadvantages
8
such as the use of harsh cross-linking agents which is not recommended since they might react with the active
agent. [13]
Figure 1.9: Emulsification and cross-linking technique (site 8)
1.3.3 By emulsification solvent diffusion method
This method relies on the partial miscibility of an organic solvent and water. As a first step, an O/W (oil-in-
water) emulsion is made by adding an organic phase into the aqueous phase which contains a chitosan
solution and a stabilizing agent under mechanical stirring. This emulsion is homogenized by high pressure. In a
next step the emulsion is diluted with additional water to conquer organic solvent miscibility in water. As a
consequence of the diffusion of the organic solvent in the water, the polymer will start to precipitate what
ultimately will lead to the formation of chitosan nanoparticles. Comparing this technique with ionic gelation,
they are both simple to execute, but the solvent diffusion method has higher risk of unstable particles due to
the high shear forces used during the production process. [13]
1.3.4 By reverse micellar technique
This technique gives rise to ultrafine chitosan nanoparticles, nevertheless it is a time-consuming preparation
process. As a first step, a surfactant is dissolved in an organic solvent to obtain reverse micelles. Aqueous
solutions of chitosan are secondly added to the organic solvent under constant vortexing. In a next step, a
cross-linking agent is mixed with the solution and cross-linking occurs during stirring overnight. Afterwards a
9
suitable salt needs to be added to precipitate the surfactant. Then the mixture can be centrifuged and
nanoparticles are obtained. [13]
1.4 ENCAPSULATION WITH CHITOSAN
As mentioned before, chitosan has attractive features like biodegradable, biocompatible and non-toxic which
make this polymer an optimal candidate for the encapsulation of bioactive compounds. It is also capable to
form gels, beads and nanoparticles. There is a wide range of components that already have been encapsulated
with chitosan. Examples are: proteins, vitamins and phenolic compounds. It also has been established that
chitosan can be used as an envelope for active compounds because this cationic polymer can be crosslinked
with negatively charged substances. [12] However, there is no report yet on the encapsulation of Mentha
piperita and Eucalyptus globulus essential oil.
10
2. OBJECTIVE
Essential oils are complex mixtures of volatile, aromatic components. They are becoming more and more
important in the therapeutic field and have been used already for several different applications. The biggest
challenge for developing essential oil-based applications is their physical stability. These oils are volatile,
unstable and sensitive to oxidation by light, heat and oxygen. It already has been reported that
nanoencapsulation is a possible solution to enhance the stability. Nanoencapsulation is a promising technique
in context of drug delivery. Several small molecules and/or drugs have yet been successfully encapsulated
leading to improved stability and delivery. There are various techniques available for nanoencapsulation, but
it’s an art to develop a simple, yet effective method. Up till now, the encapsulation of Mentha piperita and
Eucalyptus globulus essential oil with chitosan, by the method of ionic gelation, has not been assessed.
The main objective of this project is to develop a delivery platform for essential oils, more precisely for Mentha
piperita and Eucalyptus globulus. The intention is to investigate the feasibility of encapsulating essential oils
into chitosan by assessing their encapsulation and release profile.
As a first step, both essential oils are characterized by UV-VIS spectrophotometry and FT-IR spectroscopy. The
purpose of subjecting these oils to UV-VIS is a quantitative, namely the development of a calibration curve. FT-
IR is used for identification, to assess characteristic functional groups of the oils. In a second step, chitosan
nanoparticles are produced. Different concentrations of chitosan, stabilizing agent and crosslinker are used to
determine the conditions that will lead to the best results. The nanoparticles are characterized by dynamic
light scattering. After selecting two favorable conditions, the essential oils are encapsulated into chitosan.
Subsequently, the encapsulation is criticized by calculating the encapsulation efficiency and loading capacity.
These calculations are assisted by UV-VIS spectrophotometry. In a final step, the release of the essential oils is
assessed in order to evaluate the feasibility of encapsulation as delivery platform for essential oils. This is
accomplished by performing UV-VIS measurements at specific time points.
11
3. MATERIALS & METHODS
3.1 IDENTIFICATION AND CHARACTERIZATION OF THE ESSENTIAL OILS
3.1.1 UV-VIS
UV-VIS spectroscopy is a very widely used method for the quantification of substances in a sample. This
technique is based on the quantum theory. This theory correlates energy of a photon with its frequency.
Molecules or atoms appear in defined energy states and can change in between energy levels by absorbing a
unit of energy, a photon. [15]
The main equation of the quantum theory is as followed:
Equation 3.1
E = energy of a photon absorbed or emitted
h = Planck’s constant
ν = frequency of the photon
A UV-VIS spectrophotometer records the absorbance of the sample. From this, the concentration can be derived
using the law of Lambert-Beer:
Equation 3.2
A = absorbance
ε = extinction coefficient
c = concentration
l = path length of the cuvette
As a first step of identification, the essential oils are subjected to UV-VIS (ultraviolet-visible light)
spectrophotometry. The aim of this experiment is to develop a calibration curve for every essential oil. To
accomplish this, the following dilutions are made: 0.01% - 0.1% - 0.5% - 1.0% - 1.5% - 2.0%- 2.5% - 3.0% - 3.5% -
4.0% - 4.5% - 5.0% V/V. The essential oil is dissolved in absolute ethanol. The exact volumes of essential oil and
ethanol are described in table 4.1. The absorbance is measured using a 3-ml quartz cuvette with a path length
of 10 mm. The spectra are recorded within a wavelength range of 1100-190 nm.
12
The baseline of the spectra is formed by measuring the empty cuvette. Secondly, a sample with pure ethanol is
measured to see if ethanol is a feasible solvent for the oils. After measuring the ethanol, 2 samples with 100 %
essential oil are measured to see if the ethanol interferes with the spectra. Finally, the absorbance of each
dilution is recorded, starting with the lowest concentration.
3.1.2 ATR-FTIR
Besides UV-VIS, ATR-FTIR (Attenuated Total Reflectance – Fourier Transform Infrared) spectrophotometry is
used to identify typical functional groups in the oils.
Infrared spectroscopy is based on the interaction of infrared radiation with matter. The electromagnetic
spectrum of infrared can be divided into three regions: near-, mid- and far IR region. Each region corresponds
with transitions in molecules. Electronic transitions are situated in the UV/VIS range, while vibrational
transitions are near- and mid the IR-range. Besides these two types of transitions, there are also rotational
transitions and they are situated in the far IR region. [16]
The infrared spectrum of a sample is obtained by sending a beam of light through the sample. When the
frequency of IR is identical as the vibrational frequency of a bond or collection of bonds, absorption occurs.
Observation of the transmitted light shows how much energy was absorbed at each frequency (or wavelength).
A monochromator can be used for scanning the wavelength range, but a Fourier transform instrument will
probably be more appropriate. With a Fourier transform instrument the entire wavelength range is measured
and an absorbance or transmittance spectrum is obtained.
Fourier transform infrared spectrophotometer (FT-IR) uses an interferometer to collect a spectrum. The
interferometer contains a source, a beam splitter, two mirrors, a laser and a detector. The beam splitter splits
the energy coming from the source into two beams. One part is reflected to a fixed mirror and the other part is
led to the moving mirror. When the beams are recombined at the beam splitter, an interference pattern is
constructed. This pattern then goes from the beam splitter back to the sample where some energy is
transmitted and some is absorbed. The transmitted part reaches the detector. The signal goes from the
detector to a computer where an algorithm called a Fourier transform is performed and a single beam
spectrum is obtained.
13
Figure 3.1: Schematic representation of FT-IR (site 10)
A reference single beam was also collected and rated as a background spectrum to achieve a transmittance
spectrum. By taking the negative log10, this spectrum can be converted into an absorbance spectrum. There are
multiple advantages of FT-IR spectroscopy. It’s a rapid, cheap and nondestructive technique. Also, due to the
laser in the instrument, accuracy and precision in infrared spectra are very high.
Infrared spectroscopy can be used for qualitative as well as for quantitative purpose.
The quantitative aspect is based on the law of Lambert-Beer while the qualitative aspect includes
identification and characterization of the chemical structure, which is applied in this experiment. [17]
3.2 PREPARATION OF CHITOSAN NANOPARTICLES
Chitosan nanoparticles are produced by ionic gelation. The formation of the particles is based on the
electrostatic interaction between the positively charged amino groups of chitosan and negatively charged
groups of a crosslinking agent, in this case TPP (tripolyphosphate).
14
3.2.1 Protocol
Chitosan (Sigma,417963) is dissolved in 1% V/V acetic acid. The following polymer concentrations are used: 0.5
mg/ml, 0.75 mg/ml, 1.0 mg/ml and 2.5 mg/ml. Tween 80 (0.5-1.5% v/v) is added as a stabilizing agent to prevent
particle aggregation. The solutions are then stirred for 2 hours at 50 degrees to acquire homogenous mixtures.
In the next step, the pH is raised to a value of 4.6-4.8 with 1 M NaOH. Subsequently, the solutions are filtered
through a 0.45 µm micron filter (Millipore).
Figure 3.2 Dissolving chitosan in acetic acid Figure 3.3: Obtained particles after centrifugation
TPP (penta-sodiumtriphosphate, MERCK) is dissolved in distilled water to obtain the following concentrations:
0.5 mg/ml, 0.75 mg/ml and 1.0 mg/ml. These solutions are also filtered, but through a 0.22 µm filter due to a
lower viscosity.
Finally, all combinations with the different Chitosan-, TPP- and Tween 80 concentrations are tested using the
next protocol: A ratio of Chitosan: TPP 2.5:1 is used, therefore 12 ml of TPP solution is dropwise added to 30 ml
of Chitosan solution under magnetic stirring. The formation of particles starts spontaneously and the solutions
are left on the stirrer for half an hour. The particles are collected by centrifugation at 12.000 G’s for 30 min at
4 degrees. The supernatants are discarded and the pellets are resuspended in half of their original volume,
using distilled water. Storage of the pellets occurs at 4 degrees. [18], [19], [20]
3.2.2 Dynamic light scattering (DLS)
Dynamic light scattering (DLS) is an ideal technique to determine the characteristics of nanoparticles. It
determines the particle size and the polydispersity of the sample. DLS measures the Brownian motion and
correlates this motion with the particle size. The Brownian motion is about the movement of the particles
when they randomly encounter the molecules of the solvent that surrounds them. The connection between the
Brownian motion and the particle size is described by Stokes-Einstein equation:
15
Equation 3.1
DH = hydrodynamic diameter
k = Boltzmann constant
T = Temperature
η = viscosity
D = diffusion coefficient
An important principle in order to understand dynamic light scattering is that small particles move quicker
than larger particles. The DLS device contains a laser which sends a light through the sample. The particles in
the sample will scatter the light leading to fluctuations in light intensity. The equipment measures these
fluctuations and derives the particle size from this. [21]
Figure 3.4: Dynamic light scattering – setup (site 11)
3.2.3 Yield of the particles
The yield of the particles is calculated based on the following formula:
Equation 3.2
The experimental weight of the particles is determined after 8 days of freeze-drying. [22] Firstly, the particles
are stored at -80 degrees for 2 days. Subsequently they are put in the lyophilizer for 8 days. The theoretical
weight of the particles is the sum of the amount of chitosan and the amount of TPP. NaOH and Tween 80 are
disregarded, they will not contribute to the weight after freeze-drying.
16
3.3 PREPARATION OF ESSENTIAL OIL -LOADED CHITOSAN PARTICLES
After analyzing the results from the DLS, two conditions are chosen for the encapsulation of the essential oils.
The first one is the following: 0.5 mg/ml chitosan – 1.5% V/V Tween 80 – 0.5 mg/ml TPP. These conditions give
rise to the smallest particles according to the data obtained with DLS.
The second condition which is used is: 2.5 mg/ml chitosan – 1.5% V/V Tween 80 – 0.75 mg/ml TPP.
The protocol is very similar as for the production of chitosan nanoparticles. Chitosan is again dissolved in 1%
(V/V) acetic acid. 1.5% V/V Tween 80 is added and the solution is left on a magnetic stirrer for 2 hours at 50
degrees. Afterwards, the pH is raised to a value of 4.6-4.8 with 1N NaOH. Subsequently the solutions are
filtered through a 0.45 microfilter. At this point, the essential oil is added dropwise to the mixture in a weight
ratio of chitosan: essential oil 1:1, which correlates with 16.5 µl and 82.5 µl respectively for 0.5 mg/ml chitosan
and the 2.5 mg/ml chitosan solutions. After adding the oil, the solution is left on the stirrer for 15 min to give
time for the encapsulation before mixing with TPP. [23]
3.3.1 Encapsulation efficiency
To determine the amount of oil which is encapsulated, the encapsulation efficiency is assessed by UV-VIS
spectrophotometry. Each sample is made in triplicate. After centrifuging the samples at 4 degrees at 12.000 G’s
for 30 min, the supernatants are collected and the absorbances are measured in the spectrophotometer. From
the absorbances, the amount of loaded essential oil is derived using the aforementioned calibration curves.
[23]
The encapsulation efficiency (EE%) is calculated using the following formula:
Equation 3.3
3.3.2 Loading capacity
17
The loading capacity displays the contribution of the essential oil to the weight of the nanoparticles. In an
analogous manner as the encapsulation efficiency, the amount of loaded essential oil is measured. The weight
of the particles has been determined after 8 days of freeze-drying. [23] The loading capacity (LC%) is
calculated using the following formula:
Equation 3.4
3.4 RELEASE PROFILE
The release of the essential oil has been assessed using different conditions, namely static and dynamic.
Measurements were performed at the following specific time points: 24 hours, 48 hours, 72 hours, 8 days, 15
days. On these time points, the samples are centrifuged at 4 degrees at 12.000 G’s for 30 minutes.
Subsequently 2.5 mL supernatant of each sample is measured using UV-VIS spectrophotometry. After
measuring, the 2.5 ml of the sample is replaced by fresh medium. Two different media are used to determine
their impact on the release. Initially, the particles are resuspended after centrifugation in water. In a
subsequent experiment, the particles are resuspended in a mixture of PBS (phosphate buffered saline) and
ethanol. [5]
The ratio of PBS and ethanol is 60:40 and is made as followed: 300 ml of PBS and 200 ml of absolute ethanol
are mixed in a volumetric flask of 500 ml. PBS (10x) itself was present in the lab. It was produced according to
the following procedure: 80 g of NaCl, 2.0 g of KCl, 14.4 g of Na2HPO4 and 2.4 g of KH2PO4 are dissolved in 800
ml distilled water. After mixing well, the pH is adjusted to 7.4. Finally, the volume is adjusted to 1 liter with
additional distilled water. This obtained PBS was diluted by a factor 10 before using it in the mixture with
ethanol.
18
3.4.1 Static conditions
As a first step in assessing a release profile, static conditions are applied. The samples are stored at 4 degrees
in between measurements. At the specific time points, 2.5 ml of supernatant is measured by UV-VIS
spectrophotometry and the volume is replaced in the sample by fresh medium.
3.4.2 Dynamic conditions
After determining the release of both essential oils in static conditions, dynamic conditions are applied. In this
experiment, the samples are placed under continuous agitation, more specific at 140 rpms at 16 degrees. At the
specific time points, 2.5 ml of supernatant is measured by UV-VIS spectrophotometry and the volume is
replaced in the sample by fresh medium.
3.5 TRACEABILITY
Table 3.1: Traceability chemicals
Chemical Company Lot number
Absolute ethanol SIGMA-ALDRICH SZBF1900V
Acetic acid (99.7%) PANREAC 0000-449766
Chitosan SIGMA 417963 – 100G
Deionized water Lab /
Eucalyptus globulus essential oil PLENA NATURA 8000-48-4/84625-32-1
Mentha piperita essential oil PLENA NATURA 8806-90-4/84082-70-2
NaOH MERCK B988-298
PBS (phosphate buffered saline) Lab /
TPP (penta-sodiumtriphosphate) MERCK 207-F469799
Tween 80
(polyoxyethylene sorbitan
monooleate)
SIGMA 87H0648
19
Table 3.2: Traceability equipment
Apparatus Company Lot number
Balance METTLER TOLEDO AG245
Centrifuge 5804 R EPPENDORF 0031568
FT-IR instrument PERKIN ELMER /
DLS instrument MALVERN Zetasizer nano series
Filter MERCK MILLIPORE 51260103
Lyophilisizer BIOBLOCK SCIENTIFIC CHRIST Alpha 2-4 LO
20
4. RESULTS
4.1 UV-VIS
Table 4.1: Dilution series
Concentration (% V/V) Amount of EO (µl) Amount of ethanol (µl)
0.1 2.5 2497.5
0.5 12.5 2487.5
1.0 25.0 2475.0
1.5 37.5 2462.5
2.0 50 2450.0
2.5 62.5 2437.5
3.0 75.0 2425.0
3.5 87.5 2412.5
4.0 100.0 2400.0
4.5 112.5 2387.5
5.0 125.0 2375.0
4.1.1 Calibration curve of Mentha piperita
y = 0,0129x - 0,007 R² = 0,9962
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
0 20 40 60 80 100 120 140
Ab
sorp
tio
n
Amount of essential oil (µl)
Calibration curve MP at 314 nm
21
Equation derived from the calibration curve, for Mentha piperita:
R² = 0.9962
Equation 4.1
4.1.2 Calibration curve of Eucalyptus globulus
Equation derived from the calibration curve, for Eucalyptus globulus:
R² = 0.9989 Equation 4.2
y = 0,017x - 0,0078 R² = 0,9989
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
2
2,2
2,4
0 20 40 60 80 100 120 140
Ab
sorp
tio
n
Amount of essential oil (µl)
Calibration curve EG - 294 nm
22
4.2 FT-IR
The FT-IR spectra are recorded over a wavelength range 6500-450 nm.
4.2.1 FTIR: Spectra of Mentha piperita
Figure 4.1: IR spectrum of Mentha piperita
Table 4.2: Characteristic wavenumbers of Mentha piperita
Wavenumber Assignment
2955 Hydroxyl group, contributed to menthol
2850 Methyl group, contributed to menthol
1615 C-C multiple bond
1296 C-H bonding, contributed to limonene
1031 C-O bonding
960 C-H bonding, contributed to limonene
23
4.2.2 FTIR: Spectra of Eucalyptus globulus
Figure 4.2: IR spectrum of Eucalyptus globulus
Table 4.3: Characteristic wavenumbers of Eucalyptus globulus
Wavenumber Assignment
2965 C-H stretching, contributed to 1.8-cineole
1450 C-H bonding, contributed to 1.8-cineole
1060 C-O stretching, contributed to 1.8-cineole
965 C-H bonding, contributed to 1.8-cineole
840 C-bonding, contributed to 1.8-cineole
4.3 DLS – PARTICLE SIZE
The following data are obtained from dynamic light scattering.
Table 4.4: Overview particle size
Concentration Chitosan
(mg/ml)
Concentration Tween 80
(V/V %)
Concentration TPP
(mg/ml)
Mean particle size (nm)
Polydispersity
0,5 mg/ml 0,5 0,5 141,9 0,343
1,5 0,5 106,2 0,265
0,5 0,75 AGGREGATES 0,53
1,5 0,75 AGGREGATES 0,748
0,5 1 AGGREGATES AGGREGATES
1,5 1 AGGREGATES AGGREGATES
24
Concentration Chitosan
(mg/ml)
Concentration Tween 80
(V/V %)
Concentration TPP
(mg/ml)
Mean particle size (nm)
Polydispersity
0,75 mg/ml 0,5 0,5 239,9 0,404
1,5 0,5 202,7 0,357
0,5 0,75 308,5 0,384
1,5 0,75 591,5 0,772
0,5 1 AGGREGATES 0,47
1,5 1 AGGREGATES 0,566
1 mg/ml 0,5 0,5 198,1 0,511
1,5 0,5 123,3 0,675
0,5 0,75 485,3 0,706
1,5 0,75 457,8 0,596
0,5 1 252,6 0,44
1,5 1 154,6 0,442
2,5 mg/ml 0,5 0,5 2158,2 1
1,5 0,5 1455 0,959
0,5 0,75 1556 1
1,5 0,75 1508,6 0,9728
0,5 1 1708 1
1,5 1 1469 0,941
25
4.4 DLS – CORRELOGRAMS
Besides the exact values of the particle sizes, DLS also displays correlograms. The particle size and
polydispersity can be derived of these graphs. More precisely, the time when the decay starts gives an
indication about the particle size. As the particle size increases, from the first to the second correlogram, the
decay moves to longer times. The polydispersity of the sample can be derived from the gradient of the curve.
Figure 4.3: Correlogram 1
Parameters: 0.5 mg/ml chitosan, 1.5% V/V Tween 80, 0.5 mg/ml TPP.
->This leads to an average particle size of 106.2 nm and a PDI of 0.265
26
Figure 4.4: Correlogram 2
Parameters: 2.5 mg/ml chitosan, 0.5% V/V Tween 80, 0.75 mg/ml TPP.
->This leads to an average particle size of 1556 nm and a PDI of 1.00.
4.5 ENCAPSULATION EFFICIENCY
Equation 4.3
27
Table 4.5: Overview Encapsulation Efficiency
EO
Chitosan
(mg/ml) T80 (% V/V) TPP (mg/ml) EE % Absorbance λ
MP1
MP2
MP3
0,5
0,5
0,5
1,5
1,5
1,5
0,5
0,5
0,5
71,99
87,70
87,59
0,05261
0,01917
0,01941
314
314
314
82,43 Mean
7,38 St. Dev.
MP1
MP2
MP3
2,5
2,5
2,5
1,5
1,5
1,5
0,75
0,75
0,75
77,11
87,45
90,19
0,23657
0,12659
0,09741
314
314
314
84,92 Mean
5,63 St. Dev.
EG1
EG2
EG3
0,5
0,5
0,5
1,5
1,5
1,5
0,5
0,5
0,5
64,62
77,72
73,46
0,09143
0,05469
0,06665
294
294
294
71,93 Mean
5,46 St. Dev.
EG1
EG2
EG3
2,5
2,5
2,5
1,5
1,5
1,5
0,75
0,75
0,75
92,32
88,17
92,09
0,09985
0,15808
0,10315
294
294
294
90,86 Mean
1,90 St. Dev.
4.6 LOADING CAPACITY
Table 4.6: Loading capacity
Condition Amount EO encapsulated (mg) Weight freeze-dried particles (mg) Loading Capacity (%)
0,5 MP 12.35 47.52 25.99
0,5 EG 10.78 51.66 20.86
2,5 MP 63.61 85.24 74.63
28
Condition Amount EO encapsulated (mg) Weight freeze-dried particles (mg) Loading Capacity (%)
2,5 EG 68.06 88.37 77.02
4.7 YIELD OF THE PARTICLES
The yield of the particles is calculated by the following formula:
Equation 4.4
Table 4.7: Yield of the particles
Chitosan
concentration
(mg/ml)
TPP concentration
(mg/ml)
Theoretical
weight (mg)
(Chitosan + TPP)
Experimental
weight (mg)
Yield (%)
0.5 0.5 21 13.0 61.90
0.5 0.5 21 12.5 59.52
0.5 0.5 21 14.2 67.62
63.02 ±
3,40
Average
yield
0.75 1.0 34.5 27.0 78.26
0.75 1.0 34.5 27.5 79.71
0.75 1.0 34.5 27.3 79.13
79.03 ±
0,60
Average
yield
1.0 0.75 39 10.3 26.41
1.0 0.75 39 7.0 17.95
1.0 0.75 39 8.6 22.05
29
Chitosan
concentration
(mg/ml)
TPP concentration
(mg/ml)
Theoretical
weight (mg)
(Chitosan + TPP)
Experimental
weight (mg)
Yield (%)
22.14 ±
3,45
Average
yield
2.5 0.75 84 76.8 91.43
2.5 0.75 84 74.3 88.45
2.5 0.75 84 77.5 92.26
90.71 ±
1,64
Average
yield
4.8 RELEASE PROFILE
The release profile is assessed by calculating the cumulative release percentage with the following formula:
Equation 4.5
Mt = cumulative amount of essential oil released at the specific time point M0 = initial weight of the essential oil loaded in the sample
At the specific time points, the supernatants are measured by the spectrophotometer. In order to determine
the released amount of essential oil, the equations from the calibration curves are re-used.
For Mentha piperita, the equation is again:
For Eucalyptus globulus, the equation is again:
30
4.9.1 Static conditions
4.9.1.1 With water as a medium
There has been no release using static conditions and with water as a medium. The absorbance values are very
similar with the ones in the following tables (4.8-4.15) but there is no signal of release, hence there could not
be a release profile determined.
4.9.1.2 With PBS/EtOH as a medium
Table 4.8: Static release profile of Mentha piperita – part 1
Cumulative release Cumulative release Cumulative release
Condition Absorbance 16 h (%) Absorbance 24 h (%) Absorbance 48 h (%)
0.5 MP1 -0.03882 0.00 -0.03857 0.00 -0.04163 0.00
0.5 MP2 -0.03906 0.00 -0.04138 0.00 -0.04358 0.00
0.5 MP3 -0.04785 0.00 -0.04224 0.00 -0.04346 0.00
Mean value -0,04191 0.00 -0,04073 0.00 -0,04289 0.00
St. Dev. 0,00420 0.00 0,00157 0.00 0,00089 0.00
Table 4.9: Static release profile of Mentha piperita – part 2
Cumulative release Cumulative release
Condition Absorbance 8 d (%) Absorbance 15 d (%)
0.5 MP1 -0.05310 0.00 -0.02832 0.00
0.5 MP2 -0.05554 0.00 -0.02979 0.00
0.5 MP3 -0.05566 0.00 -0.03052 0.00
Mean value -0,05477 0.00 -0,02954 0.00
St. Dev. 0,00118 0.00 0,00092 0.00
Table 4.10: Static release profile of Mentha piperita – part 3
Cumulative release Cumulative release Cumulative release
Condition Absorbance 16 h (%) Absorbance 24 h (%) Absorbance 48 h (%)
2.5 MP1 -0.04297 0.00 -0.03357 0.00 -0.0166 0.00
31
Cumulative release Cumulative release Cumulative release
2.5 MP2 -0.05103 0.00 -0.03796 0.00 -0.01941 0.00
2.5 MP3 -0.05115 0.00 -0.03796 0.00 -0.01990 0.00
Mean value -0,04838
0.00 -0,03650
0.00 -0,01864
0.00
St. Dev. 0,00383 0.00 0,00207 0.00 0,00145
0.00
Table 4.11: Static release profile of Mentha piperita – part 4
Cumulative release Cumulative release
Condition Absorbance 8 d (%) Absorbance 15 d (%)
2.5 MP1 -0.0365 0.00 -0.02478 0.00
2.5 MP2 -0.03491 0.00 -0.02722 0.00
2.5 MP3 -0.03711 0.00 -0.02747 0.00
Mean value -0,03617
0.00 -0,02649
0.00
St. Dev. 0,00093 0.00 0,00121 0.00
Table 4.12: Static release profile of Eucalyptus globulus – part 1
Cumulative release Cumulative release Cumulative release
Condition Absorbance 16 h (%) Absorbance 24 h (%) Absorbance 48 h (%)
0.5 EG1 -0.0531 0.00 -0.04907 0.00 -0.05579 0.00
0.5 EG2 -0.05188 0.00 -0.04858 0.00 -0.05518 0.00
0.5 EG3 -0.04700 0.00 -0.04626 0.00 -0.05579 0.00
Mean value -0,05066 0.00 -0,04797 0.00 -0,05559
0.00
St. Dev. 0,00264 0.00 0,00123 0.00 0,00029 0.00
Table 4.13: Static release profile of Eucalyptus globulus – part 2
Cumulative release Cumulative release
Condition Absorbance 8 d (%) Absorbance 15 d (%)
0.5 EG1 -0.06592 0.00 -0.03784 0.00
32
Cumulative release Cumulative release
0.5 EG2 -0.06616 0.00 -0.03979 0.00
0.5 EG3 -0.06665 0.00 -0.03931 0.00
Mean value -0,06624 0.00 -0,03898 0.00
St. Dev. 0,00030 0.00 0,00083 0.00
Table 4.14: Static release profile of Eucalyptus globulus – part 3
Cumulative release Cumulative release Cumulative release
Condition Absorbance 16 h (%) Absorbance 24 h (%) Absorbance 48 h (%)
2.5 EG1 -0.06726 0.00 -0.04883 0.00 -0.02979 0.00
2.5 EG2 -0.06494 0.00 -0.04651 0.00 -0.0271 0.00
2.5 EG3 -0.06531 0.00 -0.04688 0.00 -0.02759 0.00
Mean value -0,06584
0.00 -0,04741
0.00 -0,02816 0.00
St. Dev. 0,00102 0.00 0,00102
0.00 0,00117 0.00
Table 4.15: Static release profile of Eucalyptus globulus – part 4
Cumulative release Cumulative release
Condition Absorbance 8 d (%) Absorbance 15 d (%)
2.5 EG1 -0.04749 0.00 -0.03711 0.00
2.5 EG2 -0.04578 0.00 -0.03552 0.00
2.5 EG3 -0.04504 0.00 -0.03491 0.00
Mean value -0,04610
0.00 -0,03585 0.00
St. Dev. 0,00103
0.00 0,00093
0.00
33
4.9.2 Dynamic conditions
4.9.2.1 With water as a medium
Table 4.16: Dynamic release profile with water as a medium – part 1
Cumulative release Cumulative release
Condition Absorbance 24 h (%) Absorbance 48 h (%)
0.5 MP -0.03333 0.00 -0.02759 0.00
0.5 EG -0.04199 0.00 -0.03455 0.00
2.5 MP 0.01929 2.91 0.00952 4.74
2.5 EG -0.02722 0.00 -0.01501 0.00
Table 4.17: Dynamic release profile with water as a medium – part 2
Cumulative release Cumulative release
Condition Absorbance 72 h (%) Absorbance 8 d (%)
0.5 MP -0.0321 0.00 -0.03552 0.00
0.5 EG -0.03992 0.00 -0.03333 0.00
2.5 MP 0.03918 9.85 0.00696 11.39
2.5 EG -0.00061 0.56 0.00073 1.23
4.9.2.2 With PBS/EtOH as a medium
Table 4.18: Dynamic release profile of Mentha piperita with PBS/EtOH – part 1
Cumulative release Cumulative release
Condition Absorbance 24 h (%) Absorbance 48 h (%)
0.5 MP1 -0.00073 3.57 -0.00549 4.43
0.5 MP2 0.01624 13.25 -0.00549 14.11
0.5 MP3 0.01489 12.48 -0.00696 12.50
Mean value 0,01013
9.77 -0,00598
10.35
St. Dev. 0,00770
4.39 0,00069
4.24
34
Table: 4.19: Dynamic release profile of Mentha piperita with PBS/EtOH – part 2
Cumulative release Cumulative release
Condition Absorbance 8 d (%) Absorbance 15 d (%)
0.5 MP1 -0.00305 6.69 0.02808 26.68
0.5 MP2 0.00012 18.19 0.00232 23.50
0.5 MP3 -0.00464 13.84 0.00073 18.25
Mean value -0,00252
12.91 0,01038
22.81
St. Dev. 0,00198
4.74 0,01253
3.48
Table: 4.20: Dynamic release profile of Mentha piperita with PBS/EtOH – part 3
Cumulative release Cumulative release
Condition Absorbance 24 h (%) Absorbance 48 h (%)
2.5 MP1 0.01587 2.53 0.00659 4.03
2.5 MP2 0.03491 4.64 0.00842 6.34
2.5 MP3 0.05542 6.91 0.007505 8.51
Mean value 0,03540
4.69 0,00751
6.29
St. Dev. 0,01615
1.79 0,00075
1.83
Table: 4.21: Dynamic release profile of Mentha piperita with PBS/EtOH – part 4
Cumulative release Cumulative release
Condition Absorbance 8 d (%) Absorbance 15 d (%)
2.5 MP1 0.00757 5.65 -0.00037 6.38
2.5 MP2 0.00867 8.08 0.00244 9.12
2.5 MP3 0.01111 10.52 -0.00525 10.71
Mean value 0,00912 8.08 -0,00106
8.74
St. Dev. 0,00148
1.99 0,00318
1.79
35
Table 4.22: Dynamic release profile of Eucalyptus globulus with PBS/EtOH – part 1
Cumulative release Cumulative release
Condition Absorbance 24 h (%) Absorbance 48 h (%)
0.5 EG1 0.01147 9.55 -0.00793 9.55
0.5 EG2 -0.01343 0.00 -0,00818 0.00
0.5 EG3 -0.01404 0.00 -0.00811 0.00
Mean value -0,00533
3.18 -0,00808
3.18
St. Dev. 0,01188
4.50 0,00011 4.50
Table 4.23: Dynamic release profile of Eucalyptus globulus with PBS/EtOH – part 2
Cumulative release Cumulative release
Condition Absorbance 8 d (%) Absorbance 15 d (%)
0.5 EG1 -0.00513 10.81 -0.01733 10.81
0.5 EG2 -0.00806 0.00 -0.02039 0.00
0.5 EG3 -0.00513 1.32 -0.02014 1.32
Mean value -0,00611
4.04 -0,01929 4.04
St. Dev. 0,00138
4.82 0,00139
4.82
Table 4.24: Dynamic release profile of Eucalyptus globulus with PBS/EtOH – part 3
Cumulative release Cumulative release
Condition Absorbance 24 h (%) Absorbance 48 h (%)
2.5 EG1 0.01599 1.87 0.01123 2.57
2.5 EG2 0.02393 2.49 0.01001 3.89
2.5 EG3 0.02246 2.37 0.00928 3.71
Mean value 0,02079 2.24 0,01017
3.39
36
Cumulative release Cumulative release
St. Dev. 0,00345
0.27 0,00080
0.58
Table 4.25: Dynamic release profile of Eucalyptus globulus with PBS/EtOH – part 4
Cumulative release Cumulative release
Condition Absorbance 8 d (%) Absorbance 15 d (%)
2.5 EG1 -0.00037 3.15 -0.00464 3.39
2.5 EG2 -0.00012 4.49 -0.00452 4.75
2.5 EG3 -0.00098 4.25 -0.00378 4.57
Mean value -0,00049
3.96 -0,00431 4.24
St. Dev. 0,00036
0.58 0,00038
0.60
5. DISCUSSION
5.1 IDENTIFICATION OF THE ESSENTIAL OILS
5.1.1 UV-VIS spectroscopy
The equations derived from the calibration curves look very similar. The difference in slope is only 0.0041. In
line with the equations, both curves look almost identical. The graphs are pretty linear which means that UV-
VIS spectroscopy is a valuable technique for the quantification of essential oils. The concentrations used in the
dilutions series are low, but must be seen in the right context, namely the purpose of encapsulation of the oils.
It is not intended to encapsulate huge amounts of oil.
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5.1.2 FT-IR spectroscopy
Essential oils contain several different components corresponding with a lot of characteristic absorption
bands. In general, the spectrum redirects to that of the main component, more precisely to the spectra of
menthol and 1.8-cineole, respectively for Mentha piperita and Eucalyptus globulus. The characteristic
wavenumbers of these main components have indeed been observed.
5.2 CHARACTERISATION OF CHITOSAN NANOPARTICLES with DLS
Much information is collected from dynamic light scattering. One of the first things that strike from table 4.4 is
the appearance of aggregates under certain conditions. There is a certain correlation found between the
concentrations used and the presence of aggregates. When the TPP concentration is higher than the chitosan
concentration the particles tend to coalescence and form aggregates. The concentration of Tween 80 does not
seem to play a role in this.
Pure looking at the influence of stabilizing agent, Tween 80, the obtained particle sizes and polydispersity
values do not differ a lot between the two used concentrations of Tween 80. However, this stabilizing agent is
indispensable in the formation of chitosan nanoparticles. Tween 80 ensures the homogenization and stability
by functioning as a surfactant.
5.3 CHARACTERISATION OF ESSENTIAL OIL LOADED NANOPARTICLES
5.3.1 Encapsulation efficiency
The encapsulation of both oils has been very successful. The values of the encapsulation efficiency are very
consistent between the different conditions as well as between the two essential oils. There is just a bit more
difference between the data of Eucalyptus globulus than with Mentha piperita, but overall one can decide that
the results regarding efficiency are very good. This indicates that a great part of the initially added amount of
oil was encapsulated.
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5.3.2 Loading capacity
The data of the loading capacity give a slightly different image than the ones from the encapsulation
efficiency. Here, the results of both oils are quite similar, the difference is shown between the two used
concentrations. The loading capacity is lower when lower concentrations of chitosan and TPP are used. The
explanation behind this can be found in the fact that the loading capacity takes into account the weight of the
particles. Two scenarios are possible. Either the ratio of the amount of oil to the weight of the particles stays
the same for the two conditions, or the higher concentration of chitosan leads to a higher loading capacity.
From these results, it turns out to be the last one, indicating that a higher volume of oil will make a larger
contribution to the weight of the particles.
5.4 Yield of the particles
Regarding the data from table 4.7, it is shown that the yield increases as the concentration of chitosan rises.
The correlation is almost linear, except for the yield of the particles made with the following concentrations:
1.0 mg/ml chitosan and 0.75 mg/ml TPP. These concentrations are not correlated with the appearance of
aggregates or something else irregular regarding the particle size. Possible statements could be: it is an
outlier or an error occurred during the practical execution. The chance of a specific correlation of the 1.0 mg/ml
chitosan concentration and the low yield is almost impossible since a straightforward relationship between
the concentration of chitosan and the yield of the particles is the most plausible statement.
5.5 RELEASE PROFILE
5.5.1 Static conditions
Overall, there has been no release established using static conditions. The explanation must be sought in the
hydrophobicity of the oils. These hydrophobic essential oils will not have the urge to move to a polar solvent,
water in this case. Also, the fact that the samples were stored at 4 degrees in a static way, with no movement
in between the measurements, is not conducive for the release.
39
5.5.2 Dynamic conditions
Since no significant release was noticeable using static conditions, dynamic conditions were applied in a next
experiment. Firstly, one sample of each essential oil in the two selected concentrations were subjected to
dynamic conditions. Water was used as a medium. In contrast with Eucalyptus globulus, Mentha piperita
encapsulated in chitosan with a concentration of 2.5 mg/ml shows release already after 24 hours. Eucalyptus
globulus also shows release, but starting after 72 hours and in much lower amounts. The amount released
after 8 days of Mentha piperita is almost ten times the amount of Eucalyptus globulus. This is quite rare since
these oils do not differ so much in chemical and physical properties. Therefore, this result could be an outlier or
there could be a specific component in Mentha piperita contributing to the release. The question is whether
this discrepancy will pass if the medium changes.
Since water is not the optimum solvent in any case to induce the release of hydrophobic compounds, no
additional experiments were performed with this medium and PBS/EtOH was used in the following
experiments.
From observing the release data using PBS/EtOH as a medium and with dynamic conditions, Mentha piperita
has again the best release. But there is way higher release with the 0.5 mg/ml concentration of chitosan than
with the 2.5 mg/ml concentration which is the opposite of what have been assessed with water. If the previous
release result of Mentha piperita (release with water) is in fact an outlier, it would mean that Mentha piperita
encapsulated in chitosan with a concentration of 0.5 mg/ml and with PBS/EtOH as a medium has the best
outcome.
Regarding the release of Eucalyptus globulus for dynamic conditions and with PBS/EtOH as a medium, there is
not so much difference in release between the two used chitosan concentrations. Also, the percentages are
clearly lower than these of Mentha piperita. This can indicate that Eucalyptus globulus essential oil is less
sensitive to PBS/EtOH, is probably also less hydrophobic than Mentha piperita which translates into a reduction
of release in the PBS/EtOH medium.
Taking into account all the release data obtained with dynamic conditions, it is clear that PBS/EtOH is a
possible medium for the release of these essential oils, although it is not the best.
40
6.CONCLUSION
Chitosan is a suitable polymer to encapsulate the following essential oils: Mentha piperita and Eucalyptus
globulus. The experimental data obtained from dynamic light scattering prove that ionic gelation is an efficient
method to produce chitosan nanoparticles. It is obvious that the concentration of chitosan as well as the
concentration of stabilizing agent Tween 80 and crosslinker TPP contribute to the achieved particle size.
Although the selected conditions for the encapsulation of the oils differ greatly in particle size and
polydispersity index, the encapsulation efficiency results display a reduction in this contrast. From the
encapsulation efficiency can also be decided that the encapsulation has been successful, with no lower value
than 70%.
From the release data, one can decide that static conditions are (almost) not able to induce release of the
essential oils. Comparing the 2 solvents used, PBS/EtOH is significantly better than water. The main reason
behind this is the hydrophobicity of the essential oils. The biggest release is obtained with Mentha piperita
with a chitosan concentration of 2.5 mg/ml and a TPP concentration of 0.75 mg/ml. However, these release
values are still relatively low. This may point to the fact that PBS/EtOH is probably not the optimum solvent.
The search for the optimal release parameters can be one of the goals in the future.
41
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