lib.ugent.be · een release profiel voor beide oliën gecreëerd met behulp van uv-vis...

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“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


4.9.1.1 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


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




2.5 MP1 -0.04297 0.00 -0.03357 0.00 -0.0166 0.00

31


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




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



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




0.5 EG1 -0.06592 0.00 -0.03784 0.00

32


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




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




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


4.9.2.1 With water as a medium

Table 4.16: Dynamic release profile with water as a medium – part 1


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


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



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



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




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




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



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




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




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


St. Dev. 0,00345

0.27 0,00080

0.58




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.

37

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.

38

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


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


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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3. http://plena-natura.pt/index.php?route=product/category&path=12 (13-04-17)

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7. https://nl.wikipedia.org/wiki/Pentanatriumtrifosfaat (27-03-17)

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http://www.sigmaaldrich.com/catalog/product/aldrich/c121452?lang=en&region=BE

http://www.sigmaaldrich.com/catalog/product/aldrich/w504505?lang=en&region=BE

http://www.sigmaaldrich.com/catalog/product/sial/m2772?lang=en&region=BE

http://plena-natura.pt/index.php?route=product/category&path=12

http://www.sigmaaldrich.com/catalog/product/aldrich/c80601?lang=en&region=BE

https://www.researchgate.net/figure/227609076_fig2_Figure-1-Deacetylation-of-chitin-to-chitosan-and-acid-base-equilibrium-of-chitosan-The

https://www.researchgate.net/figure/227609076_fig2_Figure-1-Deacetylation-of-chitin-to-chitosan-and-acid-base-equilibrium-of-chitosan-The

https://www.researchgate.net/figure/281541246_fig7_Figure-4-Schematic-synthesis-of-chitosan-calcium-ion-hybrid-nanoflower-Chitosan-binds

https://www.researchgate.net/figure/281541246_fig7_Figure-4-Schematic-synthesis-of-chitosan-calcium-ion-hybrid-nanoflower-Chitosan-binds

https://nl.wikipedia.org/wiki/Pentanatriumtrifosfaat

https://www.researchgate.net/figure/221797419_fig1_Figure-1-Schematic-representation-of-the-method-of-emulsification-and-cross-linking

https://www.researchgate.net/figure/221797419_fig1_Figure-1-Schematic-representation-of-the-method-of-emulsification-and-cross-linking

https://en.wikipedia.org/wiki/Fourier_transform_infrared_spectroscopy

https://chem.libretexts.org/@api/deki/files/277/revisions

https://pubchem.ncbi.nlm.nih.gov/compound/Eucalyptol#section=Solubility

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