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OLEIC ACID
PRODUCTION, USES AND POTENTIAL
HEALTH EFFECTS
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BIOCHEMISTRY RESEARCH TRENDS
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BIOCHEMISTRY RESEARCH TRENDS
OLEIC ACID
PRODUCTION, USES AND POTENTIAL
HEALTH EFFECTS
LYNETTE WHELAN
EDITOR
New York
Copyright 2014 by Nova Science Publishers, Inc.
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CONTENTS
Preface vii
Chapter 1 Optimization of the Media Volume, Aeration Rate
and Inoculum Size for Sophorolipid Production
from Candida bombicola ATCC 22214 1
Stephanie Grieb, Fred J. Rispoli and Vishal Shah
Chapter 2 Influence of Oleic Acid on Self-Assembled Liquid
Crystalline Nanostructures 9
Intan Diana Mat Azmi and Anan Yaghmur
Chapter 3 Oleic Acid and Its Potential Health Effects 35
Igor Pravst
Chapter 4 Oleic Acid and Microbial Lipases:
An Efficient Combination 55
Fabiano Jares Contesini, Danielle Branta Lopes,
Elaine Berger Ceresino, Jose Valdo Madeira Junior,
Paula Speranza, Francisco Fbio Cavalcante Barros
and Ricardo Rodrigues de Melo
Chapter 5 Synthesis of Oleic Acid Alkil Esters
via Homogeneous Catalysis 83
Mrcio Jos da Silva and Abiney Lemos Cardoso
Chapter 6 Effects of Temperature on Oleic Acid Percentage
During Grain-Filling in Sunflowers and Other Oil
Crops 99
Rouxlne van der Merwe and Maryke Labuschagne
Index 129
PREFACE
Oleic acid is a monounsaturated fatty acid and natural constituent of a
number of foods, particularly vegetable oils. On the basis of proven beneficial
health effects it is also a possible ingredient in processed functional foods.
However, due to its high energy content it is not recommended to increase the
consumption of any particular fat, but to substitute other lipids with oleic acid.
While there is a well-established consensus that replacing saturated fats in the
diet with oleic acid or other unsaturated fats contributes to the maintenance of
normal blood cholesterol levels, a series of other effects has also been studied,
including the modulation of inflammatory markers, blood pressure, insulin
sensitivity, gastrointestinal functions and even various cancers. This book
discusses oleic acid's health effects, as well as its production, and how it is
used.
Chapter 1 In the current study the influence of aeration rate, inoculum
size and fermentation medium volume on the sophorolipids production from
the yeast Candida bombicola have been studied. Using the data obtained from
a two-level Placket-Burman experimental design, linear and cubic models
were obtained to understand the interaction amongst the ingredients. The cubic
model was used to find the optimal aeration rate, inoculum size and the
fermentation medium volume. The maximum production of SLs is predicted to
be obtained when the medium volume is 10 mL (in 125 mL Erlenmeyer flask),
is inoculated with 5% of the inoculum and incubated at 350 rpm.
Chapter 2 Various studies in the literature suggested a link between the
consumption of olive oil and different food products enriched with oleic acid
(OA) and various positive health effects. The central focus of this research
field is on learning and predicting how OA intake induces these health
benefits. In recent years, there is a growing interest in understanding the
biological role of this monounsaturated cis fatty acid in regulating cell
Lynette Whelan viii
membranes and its effect on biological processes. In this context, it is
interesting to explore the effect of its incorporation on the model membrane
characteristics and properties. These studies are considered as first steps
towards a deeper understanding of the molecular mechanisms underlying OA
beneficial health effects and their association with the biological membrane
properties.
This chapter summarizes recent studies conducted on the influence of OA
and its counterparts (saturated and trans fatty acids) on model lipid
membranes. In particular, the main focus is to present recent investigations on
the structural characterization and also the potential applications of lipidic
non-lamellar self-assembled nanostructures loaded with OA. These lyotropic
liquid crystalline (LLC) phases and microemulsions are attractive as drug
delivery systems. The most investigated LLC phases are the inverted-type
hexagonal (H
2
) and the inverted-type bicontinuous cubic (V
2
) nanostructures.
These unique inverted type self-assembled systems are compatible, digestible,
and bioadhesive matrices that are able to co-exist under equilibrium conditions
with excess water. They display nanostructures closely related to those
observed in biological membranes and posess interesting characteristics such
as the high interfacial area (specific interfacial area up to 400 m
2
/g), the high
solubilization capacities of drugs with different physicochemical properties
(hydrophilic, amphiphilic, and hydrophobic molecules), and the potential of
controlling drug release. In particular, there is an enormous interest in testing
the possibility of utilizing these LLC phases for enhancing the solubilization
of poorly water-soluble drugs, obtaining sustained drug release, and improving
the in vivo performance of various drug substances.
The scope of this chapter also covers recent studies that have attempted to
shed light on the possible fragmentation of these inverted type self-assembled
nanostructures for forming nanoparticlulate formulations attractive for food
and pharmaceutical applications. These nanostructured aqueous dispersions
(mainly cubosomes, hexosomes, and micellar cubosomes) in which the
submicron-sized dispersed particles envelope distinctive well-defined self-
assembled nanostructures can be utilized in different applications owing to
their low viscosity as compared to the corresponding non-dispersed bulk liquid
crystalline phases and their biological relevance.
Chapter 3 Oleic acid is a monounsaturated fatty acid and natural
constituent of a number of foods, particularly vegetable oils. On the basis of
proven beneficial health effects it is also a possible ingredient in processed
functional foods. However, due to its high energy content it is not
recommended to increase the consumption of any particular fat, but to
Preface ix
substitute other lipids with oleic acid. While there is a well-established
consensus that replacing saturated fats in the diet with oleic acid or other
unsaturated fats contributes to the maintenance of normal blood cholesterol
levels, a series of other effects has also been studied, including the modulation
of inflammatory markers, blood pressure, insulin sensitivity, gastrointestinal
functions and even various cancers. Commercial communication of such
effects is only ethical where such effects are relevant to human health and
proven using the highest possible standards, preferably with well-performed,
double-blind, randomised, placebo-controlled human intervention trials. Most
intervention studies investigating the health effects of oleic acid are performed
using vegetable oils which also contain other fatty acids and minor
constituents. This represents a possible confounding factor and makes
interpretations difficult. In this chapter, the health effects of oleic acid are
discussed together with the possibilities of using oleic-acid-related health
claims on foods in commercial communications in the European Union.
Chapter 4 Oleic acid is a monounsaturated fatty acid found in high
concentrations in vegetable oils, presenting a broad number of applications in
many industrial areas, such as food, pharmaceutical, cosmetic, oleochemical
and biodiesel industries. Due to the lipophilicity, unsaturation and acidic
characteristics that this compound presents, oleic acid can be effectively used
in esterification and acidolysis, among other reactions. Recent studies have
used oleic acid as an efficient substrate for synthesis of trimethylolpropane
esters by esterification using lipase from Candida Antarctica, since this polyol
ester is widely applied in hydraulic fluids with several applications. Other
studies used C. antarctica lipase for improving the lipophilicity of bioactive
molecules, such as ferulic acid and L-ascorbic acid by esterification with oleic
acid, which is very interesting, taking into account that it increases the
solubility of these molecules in hydrophobic environments, resulting in higher
biological activities. On the other hand, some studies showed that lipases can
be used to convert oleic acid into epoxies, which are useful intermediates in
organic synthesis due to the high reactivity they present. They are used to
produce plasticizers that increase flexibility, workability or distensibility of
plastics, hence rendering them suitable for several applications. One study
reported biodiesel production by esterification of oleic acid with aliphatic
alcohols using immobilized Candida antarctica lipase, showing high yields of
biodiesel (above 90%) in less than 24 h with ethanol, n-propanol and n-
butanol; whereas with methanol, the enzyme was inactive after ten cycles of
reaction. In addition to the various reactions involving oleic acid as a
promising substrate for various reactions, oleic acid can also be used to induce
Lynette Whelan x
microbial lipase production, as seen in a study using the fungal strain Rhizopus
arrhizus. Therefore, different high-added-value compounds can be obtained
using oleic acid as a cheap and efficient substrate for microbial lipases, which
can be considered as environmentally friendly alternatives for chemical
catalysts. Within this context, this chapter reviews some studies and trends on
the use of oleic acid as an efficient substrate for microbial lipases.
Chapter 5 Recently, due to inevitable exhaustion of the fossil petroleum
reserves, and the environmental impact generated by the green-house effect
gas emission, to develop efficient processes for the production of fuels and
chemicals from the renewable feedstock has been pursued researchers in
worldwide. In this sense, since the oleic acid is a common component of
vegetal oils and animal fatty, it raise as a highly attractive raw material, due to
its high availability and affordability. In general, the oleic acid is present in
different feedstock as a free fatty acid or as glyceryl ester. Several chemicals
of interest for plentiful industries can be obtained via different catalytic
reactions starting from the oleic acid as source, such as alkyl esters or ethers
and epoxide-derivatives. Particularly, alkyl oleate esters are useful as
lubricant, surfactant, emulsifying agent, emollient, fuels additive and
biodiesel. Actually, the main component of biodiesel is in general the methyl
or ethyl oleate, which is manufactured by the alkaline transesterification of
edible or non-edible vegetable oils via a well-established industrial process.
However, the conventional alkaline homogeneous process results in large
generation of effluents and residues of neutralization, in addition the laborious
steps to remove the non-reusable catalyst, being because of these reasons a
non-friendly environment process. In this work, the authors wish the recent
advances achieved in the development of catalytic processes for the production
of alkyl esters of oleic acid via acid catalysis, however, using recyclable
catalysts. They will pay special attention to development of homogeneous
catalysts that can be recovery and reusable without loss of activity in the oleic
acid esterification reactions. These catalysts are solid when pure and soluble in
the reaction being thus recovered after solvent distillation and extraction of
products. Numerous industries in all parts of world have crescent demand by
developing of environmentally friendly technologies for the production of
biodiesel and chemicals, which are especially attractive when are based on
reusable catalysts. Herein, the authors focus the use of two different sorts of
catalysts: the former, Lewis acid such as tin compounds, and the second one,
Brnsted acid catalysts, which are based on Keggin-type heteropolyacids. The
catalysts performance it was assessed in the esterification reactions with short
chain alkyl alcohols (i.e., methyl, ethyl, propyl, isopropyl and butyl alcohols).
Preface xi
A comparison with the traditional catalysts used in these reactions also was
performed. The development of new, efficient, and environmentally benign
catalytic processes that may lead to high value added products, starting of
renewable raw material such as oleic acid, is still an challenge to be overcome.
The authors hope that this work can significantly contribute to improvement of
this important research field.
Chapter 6 Most vegetable oils are obtained from beans or seeds, which
furnish valuable and high quality oil commodities in the world oil market.
Seed oil quality is related to oil percentage and fatty acid composition and
defines the oils value for industry. With emerging new markets and increased
concerns about the health risks of foods, changes in the oil quality of various
crops have been demanded. Plant breeders have been successful in developing
novel oil types in sunflower, soybean, peanut and others with increased
percentages of oleic acid. Genotype is the most important factor that defines
the oil fatty acid composition, but environmental factors, particularly during
the grain-filling period, can widely affect both oil content and oleic acid
percentage. Various environmental factors including temperature (heat and
cold, day/night differences), solar radiation, humidity, day length and moisture
availability (rainfall distribution and intensity, drought or flooding) affect seed
oil percentage and composition. When environmental factors deviate from the
optimal quantity or intensity for the crop plant, stress is caused. Changes in
both oil percentage and fatty acid composition caused by environmental stress
could have a dynamic effect on the quantity and quality of oil that is
extractable by seed processors. Temperature is a major environmental factor
that determines the rate of oil accumulation. Generally warm temperatures
during the entire growing season or a period of heat stress during grain-filling
favors the production of oleic acid, while cooler temperatures favor the
production of linoleic acid in traditional oil crops. However, not all genotypes
are similarly affected by temperature and show strong genotype by
environment interaction. Generally the novel sunflower genotypes with
increased oleic acid contents display more stable oleic to linoleic acid ratios
across different environments than standard types with high linoleic acid
percentages. In novel soybean varieties, the high oleic acid content fluctuates
with temperature differences. In order to improve oil quality in traditional oil
crops, it is necessary to understand the temperature effects on oleic acid
content. In addition, since agricultural and management practices can alter
temperature and other important environmental factors that plants are exposed
to during grain-filling, altered production practices could contribute to
modified oleic acid contents in vegetable oil crops.
In: Oleic Acid ISBN: 978-1-63117-576-3
Editor: Lynette Whelan 2014 Nova Science Publishers, Inc.
Chapter 1
OPTIMIZATION OF THE MEDIA VOLUME,
AERATION RATE AND INOCULUM SIZE
FOR SOPHOROLIPID PRODUCTION FROM
CANDI DA BOMBI COLA ATCC 22214
Stephanie Grieb
1
, Fred J . Rispoli
2
and Vishal Shah
*
1
1
Department of Biology, Dowling College, Oakdale, NY, US
2
Department of Mathematics, Dowling College, Oakdale, NY, US
ABSTRACT
In the current study the influence of aeration rate, inoculum size and
fermentation medium volume on the sophorolipids production from the
yeast Candida bombicola have been studied. Using the data obtained
from a two-level Placket-Burman experimental design, linear and cubic
models were obtained to understand the interaction amongst the
ingredients. The cubic model was used to find the optimal aeration rate,
inoculum size and the fermentation medium volume. The maximum
production of SLs is predicted to be obtained when the medium volume is
10 mL (in 125 mL Erlenmeyer flask), is inoculated with 5% of the
inoculum and incubated at 350 rpm.
*
Corresponding author: Phone: 631-244-3339; Fax: 631-244-1033; Email: ShahV@dowling.
edu.
Stephanie Grieb, Fred J. Rispoli and Vishal Shah 2
Biosurfactants have become increasingly popular in the recent times
owing to their environmental friendly properties. One of the biosurfactants that
is gaining attraction for its biological properties are Sophorolipids (SLs). SLs
are low-molecular weight biosurfactants produced by yeasts such as Candida
bombicola, Yarrowia lipolytica, Candida apicola, and Candida bogoriensis
when grown on carbohydrates and lipophilic substrates. [1] The biological
properties of the compounds include anticancer [2], antibacterial [3],
antifungal [4], antiviral [5] and spermicidal activity. [6] In addition, SLs have
also shown to be an effective septic shock antagonist [7,8] and have been
proposed to have applications in food thickening, herbicide and pesticide
formulations, consumer product manufacturing (e.g. detergents and
cosmetics), and lubricant formulations. [9]
Not many studies have been published to optimize the fermentation
conditions for obtaining maximum SL yields. In our recent study, we
optimized the fermentation medium for the maximum production of SLs using
the yeast Candida bombicola ATCC 22214. [8] Sixteen different media
ingredients were screened and the fermentation medium composed of sucrose,
malt extract, oleic acid, K
2
HPO
4
and CaCl
2
was shown to provide the highest
yield of the glycolipids. However, no physical parameters were optimized in
the earlier study. Using a two-level Placket-Burman design, three physical
process parameters are optimized in the current study to obtain high yields of
SLs under batch fermentation. The process parameters are aeration rate,
medium volume and the age of the inoculum. Aeration rate and medium
volume are critical in determining the amount of oxygen transferred into the
fermentation medium. Oxygen supply is important in the SL fermentation
because the yeast is very sensitive to the oxygen limitation during their
exponential growth phase Guilmanov et al. have carried out a detailed
investigation on the influence of oxygenation on the SL production under fed-
batch conditions using shake-flask method [9]. They reported that higher
levels of oxygenation resulted in increased SL formation and that the oxygen
transfer rate has to be between 50 and 80 mM O
2
/L h
-1
for obtaining high
yields. The study however was carried out using an un-optimized media of
glucose, yeast extract and urea, and also included a step of centrifuging the
cells from the inoculum media before introducing them into the fermentation
media. In our preliminary study, we found that centrifugation of cells before
introducing them to the fermentation media decreases the yield of SL (data not
shown). Thus, the process parameters of media volume and agitation rate were
selected in the current study. As the culture flasks will be of identical size,
cultures of higher medium volumes represent lower oxygenation rate and those
Optimization of the Media Volume, Aeration Rate 3
with smaller volumes represent higher oxygenation. Higher aeration rate
results in higher oxygenation rate, and smaller aeration rates results in lower
oxygenation rate. Inoculum volume was selected as the third parameter
because it is known that the production of SL begins only when the nitrogen in
the fermentation media is depleted. [10] The inoculum size would determine
how many yeast cells are introduced in the fermentation medium and hence
the rate at which the nutrients are utilized.
Candida bombicola ATCC 22214 was used for SL production. The
protocol described in Rispoli et al. [8] was used for Sophorolipid production.
The fermentation was carried out in 125 mL Erlenmeyer flasks and the
fermentation media was composed of sucrose, 125 g/L; oleic acid, 166.67 g/L;
CaCl2, 2.5 g/L; K2HPO4, 1.5 g/L and malt extract 25 g/L. The amount of
fermentation medium in the flask and the volume of inoculum added to the
media were varied as per the experimental design described in Table 1. The
flasks were incubated for 8 days at 30 1.5 C in a rotary shaker. The
extraction and estimation of SLs was carried out following the protocol
described earlier [8] A Plackett-Burman two-level experimental design was
obtained with one block for three independent variables. Fusion Pro version
7.3.20 (S-Matrix Corp., USA) software was used to obtain the design. The
obtained design is shown in Table 1. The statistical analysis of data was
carried out using Statistica release 8 (StatSoft Inc., USA).
Table 1. Experimental design matrix and the obtained yields of
Sophorolipids under each condition
Experiment
Number
Aeration
(rpm)
Media
volume
a
(mL)
Inoculum
(%)
SL Yield
(g/L)
1 50 10 5 26.14
2 50 10 15 23.33
3 50 40 5 9.67
4 50 40 15 7.85
5 200 25 10 15.49
6 350 10 5 87.84
7 350 10 15 74.2
8 350 40 5 15.29
9 350 40 15 15.2
a
The media volume is the final volume in the flask after addition of the inoculum.
As can be seen in Table 1, the media volume in the flask was varied from
1/10 (10 mL) of the total flask volume to 1/3 (40 mL). Similarly, the aeration
Stephanie Grieb, Fred J. Rispoli and Vishal Shah 4
was varied from 50 rpm to 350 rpm. Thus, experiment number 6 and 7 which
have a volume of 10 mL and were incubated at 350 rpm receive highest
oxygenation. Whereas experiment number 3 and 4 have the lowest
oxygenation. SL yield indicates that the highest yield was obtained when the
yeast received high amount of oxygen. When one compares the SL yield
obtained in experiment 1 and 3, 6 and 8 it can be concluded that increasing the
media volume decreases the production of SLs. These comparisons were
carried out because between the experiments, the other two variables have
same value. Comparison between experiments 1 and 6, 2 and 7, indicates that
increasing aeration has a positive influence on the yield.
Table 2. Linear and cubic model obtained by analyzing the data described
in Table 1
Variable Linear model Cubic model
R
2
= 0.71 R
2
= 0.94
x
1
50.72 86.30
x
2
-21.53 8.13
x
3
14.74 21.79
x
1
.x
2
- -80.68
x
1
.x
3
- -35.43
x
2
.x
3
- -23.61
x
1
.x
2
.x
3
- 37.16
Both a linear and a cubic model were obtained using regression analysis
(Table 2). The primary effect of each of the variables can be evaluated based
on a liner regression model. Based on the coefficients, aeration has the highest
positive influence on the yield, whereas media volume has a strong negative
influence. The amount of inoculum added also has a positive influence on the
production of SLs. The low fit of the linear model with the experimental data
is an indication that apart from the primary effect for each independent
variable, there is a high degree of interaction that is undetected by the linear
model. The quadratic model result has R
2
value of 0.94. The improvement of
the R
2
value from 0.70 to 0.94 is due to the two-way and three-way interaction
terms incorporated into the cubic model. Interestingly, the cubic model shows
that the primary effects of all the variables (including media volume) are
positive and the observed overall effect for each variable is due to the
interactions with other variables. The model shows that all the two-way
interactions are negative. Confirmation of the interaction can be obtained from
Optimization of the Media Volume, Aeration Rate 5
the ternary plot illustrated in Figure 1. Maximum yield is predicted near the
vertex of the aeration and along the inoculum aeration axis. Very low yield
is predicted when the aeration has a lower value (along the inoculum media
volume axis).
Figure 1. Ternary plot of the quadratic model predicting the production of
Sophorolipids under various conditions.
The optimization of the process variables was carried out using Frontline
Solver, optimization software built into Microsoft Excel. The cubic model
described in Table 2 was selected as the objective function. The optimal
solution obtained was aeration of 350 rpm, inoculum volume of 5% and media
volume of 10 mL and the maximum yield predicted is 86.29 g/L under optimal
conditions. The optimal conditions predicted by Solver are similar to those in
experiment 6, and the yield obtained experimentally was 87.84 g/L.
In conclusion, the influence of the aeration, inoculum volume and media
volume have been studied in the current study and the optimal values of the
three obtained to achieve highest SL yield. During the course of study we have
also identified several confounding variables including the amount of cells in
the inoculum and the physiology of the organisms (data not shown). Studies
Stephanie Grieb, Fred J. Rispoli and Vishal Shah 6
are now being carried out in our laboratory to investigate how these variables
influence the SL production by Candida bombicola. In addition, it has been
recently shown that the structural composition of SL is highly dependent on
the aeration rate. [12] A regression model that is able to predict the
composition of the SL based on the fermentation conditions is also being
developed.
ACKNOWLEDGMENT
The study was funded by National Science Foundation (Grant # CBET
0828292).
REFERENCES
[1] Gobbert, U., Lang, S. and Wagner, F. (1984) Biotechnol Lett. 6, 225-
230.
[2] Chen, J., Song, X., Zhang, H. and Qu, Y. (2006) Enzyme Microbial
Technol. 39, 501-506.
[3] Shah, V., Badia, D. and Ratsep, P. (2007) Antimicrobial Agents and
Chemotheraphy. 51, 397-400.
[4] Gross, R. and Shah, V. (2004) Antifungal properties of various forms of
sophorolipids. US Patent application No. 20050164955.
[5] Shah, V., Doncel, G. F., Seyoum, T., Eaton, K. M., Zalenskaya, I,
Hagver, R., Azim, A. and Gross, R. (2005) Antimicrobial Agents and
Chemotherapy. 49, 4093-4100.
[6] Bluth, M.H., Kandil, E., Mueller, C. M., Shah, V., Lin, Y. Y., Zhang, H.,
Dresner, L., Lempert, L., Nowakowski, M., Gross, R., Schulze, R. and
Zenilman, M. E. (2006) Crit. Care Med. 34, 188-195.
[7] Solaiman, D. K. Y. (2005) Inform. 16, 408-410.
[8] Rispoli, F. J., Badia, D. and Shah, V. (2010) Biotechnol. Progress, 26,
938-944
[9] Guilmanov, V., Ballistreri, A., Impallomeni, G.. and Gross, R. A. (2002)
Biotechnol. Bioeng, 77, 489-494.
[10] Lien, C-C. (2007) Ph. D. Thesis. Polytechnic University of New York.
2007.
Optimization of the Media Volume, Aeration Rate 7
[11] Shah, V., Jurjevic, M. and Badia, D. (2007) Biotechnol. Prog. 23, 512-
515.
[12] Ratsep, P. and Shah, V. (2009) J. Microbiol. Methods. 78, 354-356.
In: Oleic Acid ISBN: 978-1-63117-576-3
Editor: Lynette Whelan 2014 Nova Science Publishers, Inc.
Chapter 2
INFLUENCE OF OLEIC ACID
ON SELF-ASSEMBLED LIQUID
CRYSTALLINE NANOSTRUCTURES
I ntan Diana Mat Azmi and Anan Yaghmur
*
Department of Pharmacy, Faculty of Health and Medical Sciences,
University of Copenhagen, Denmark
ABSTRACT
Various studies in the literature suggested a link between the
consumption of olive oil and different food products enriched with oleic
acid (OA) and various positive health effects. The central focus of this
research field is on learning and predicting how OA intake induces these
health benefits. In recent years, there is a growing interest in
understanding the biological role of this monounsaturated cis fatty acid in
regulating cell membranes and its effect on biological processes. In this
context, it is interesting to explore the effect of its incorporation on the
model membrane characteristics and properties. These studies are
considered as first steps towards a deeper understanding of the molecular
mechanisms underlying OA beneficial health effects and their association
with the biological membrane properties.
This chapter summarizes recent studies conducted on the influence of
OA and its counterparts (saturated and trans fatty acids) on model lipid
*
Corresponding author: Tel.: +45 35 33 65 41, Fax: +45 35336030, e-mail: anan.yaghmur
@sund.ku.dk.
Intan Diana Mat Azmi and Anan Yaghmur 10
membranes. In particular, the main focus is to present recent
investigations on the structural characterization and also the potential
applications of lipidic non-lamellar self-assembled nanostructures loaded
with OA. These lyotropic liquid crystalline (LLC) phases and
microemulsions are attractive as drug delivery systems. The most
investigated LLC phases are the inverted-type hexagonal (H
2
) and the
inverted-type bicontinuous cubic (V
2
) nanostructures. These unique
inverted type self-assembled systems are compatible, digestible, and
bioadhesive matrices that are able to co-exist under equilibrium
conditions with excess water. They display nanostructures closely related
to those observed in biological membranes and posess interesting
characteristics such as the high interfacial area (specific interfacial area
up to 400 m
2
/g), the high solubilization capacities of drugs with
different physicochemical properties (hydrophilic, amphiphilic, and
hydrophobic molecules), and the potential of controlling drug release. In
particular, there is an enormous interest in testing the possibility of
utilizing these LLC phases for enhancing the solubilization of poorly
water-soluble drugs, obtaining sustained drug release, and improving the
in vivo performance of various drug substances.
The scope of this chapter also covers recent studies that have
attempted to shed light on the possible fragmentation of these inverted
type self-assembled nanostructures for forming nanoparticlulate
formulations attractive for food and pharmaceutical applications. These
nanostructured aqueous dispersions (mainly cubosomes, hexosomes, and
micellar cubosomes) in which the submicron-sized dispersed particles
envelope distinctive well-defined self-assembled nanostructures can be
utilized in different applications owing to their low viscosity as compared
to the corresponding non-dispersed bulk liquid crystalline phases and
their biological relevance.
INTRODUCTION
The negative health effects associated with the consumption of food
products containing trans-fatty acids (TFAs) remain a major concern for the
consumers [1]. The overall awareness about the significant role of these fatty
acids in human nutrition has been raised since 1980s [2-4]. These unsaturated
fatty acids contain at least one double bond of trans configuration and are
mainly generated by the process of partial hydrogenation of vegetable oils,
which is used in food manufacturing industry to commercially produce edible
solid fats with an increased shelf life that can substitute animal fats in diet [2,
5-7]. The major concern is that the trans configuration affects not only the
Self-Assembled Liquid Crystalline Nanostructures 11
physicochemical properties of the fatty acids [2, 7] but also it attributes to
multiple negative effects [6-14]. Various epidemiologic and clinical studies
reported on the influence of the TFA intake on increasing the risk of coronary
heart disease [5-8, 15] and cancer [6, 7, 16], increasing the blood low density
to high density lipoprotein (LDL/HDL) ratio [6, 17, 18]. More than one third
of cancer incidence and other chronic diseases such as cardiovascular risk
factors were claimed to be associated with the nutrition-related attitudes [19-
22]. In addition, different studies suggested an important link between the
TFA intake and insulin sensitivity [6], systemic inflammation [6, 23], and
impairing the endothelial function [14]. Diabetes was also reported to be
associated with the TFA dietary that stimulated a greater adipogenic effect [11,
24]. A growing body of evidence on the adverse negative health effects
associated with TFA consumption suggests introducing TFA-free food
products to the market [25-27].
In contrast to trans-fat dietary, the consumption of olive oil, which is rich
in oleic acid (a monounsaturated fatty acid with the natural cis configuration),
is associated with positive health effects [28-30]. In European countries such
as Greece and Italy and in the Middle East the intake of olive oil is high and is
linked in different regions to a relatively reduced blood pressure and a reduced
risk of developing coronary heart disease, a reduced breast cancer, and a low
level of plasma cholesterol [30-32]. The past decade has witnessed a
tremendous interest in understanding why the consumption of oleic acid-rich
diet is important to our health and wellness. It was reported that oleic acid
(OA) reduces a cluster of prevalence metabolic syndrome (MetS) including
obesity, hypertension, impaired fasting glucose (insulin resistance at pre-
diabetic state), blood pressure, high-density lipoprotein cholesterol [HDL-C]
levels, and the risk of coronary heart disease [20, 33-39]. It was also found that
this monounsaturated fatty acid (MUFA) is an active component that
influences the proliferation of immune cells in comparison with other fatty
acids [36, 40] as well as it reduces the risk of ulcerative colitis (UC) disease
[41]. Not only that, OA is used as a penetration enhancer to increase the
permeability of active molecules to the skin [42-44]. Most interestingly, the
role of OA in inhibiting cell proliferation and inducing apoptosis in carcinoma
cells has received great attention [16, 45, 46]. It was suggested therefore to use
OA as an antitumoral agent [29, 40, 47-50]. In an interesting report, it was
found that the combination of OA with the drug trastuzumab leads to the
occurrence of a synergistic cytotoxic effect towards breast cancer [51].
There is a growing research interest on exploring the effect of OA on
biological membrane structures due to the implications of its daily
Intan Diana Mat Azmi and Anan Yaghmur 12
consumption in vital biological processes related to health and disease, and its
possible use as one of the main components in the formation of soft lipidic
nanoparticlulate formulations attractive for delivering drugs or functional
foods [25, 52-55]. Understanding the effect of this free cis-fatty acid on
regulating cell membranes is considered as first step towards a deeper
understanding of the biological membrane properties and the molecular
mechanisms underlying OA beneficial health effects. In this contribution, the
main attention is to focus on the influence of OA and its counterparts
(saturated and trans fatty acids) on the structural characterization and the
potential pharmaceutical applications of lipidic non-lamellar lyotropic liquid
crystalline (LLC) phases and their corresponding aqueous dispersions
(cubosomes and hexosomes).
I. OLEIC ACID: BIOLOGICAL ACTIVITY
AND PHARMACEUTICAL USES
OA-rich diets are associated with increasing the level of this fatty acid in
human plasma membrane [56, 57]. The health benefits of OA intake has been
subjected to a large number of reports [16, 46, 58], but its specific mechanism
of action remains poorly understood. It was suggested that OA intake
modulates the structure of cell membranes [59-61]. For instance, a recent
interesting study suggested an important role of this monounsaturated cis-fatty
acid in modulating the adrenoreceptor signaling pathway that induces a
reduction in the blood pressure (BP) [62]. This G protein-associated signaling
activity was found in both in vivo (in human) and in cell culture studies, but
apparently not detected in the membrane-free system [62, 63]. In contrast, the
counterparts elaidic (EA, trans C18:1t9) or stearic (SA, C18:0) acids, which
are structurally different than OA at the molecular level, do not induce
significant activity on the adrenoreceptor signaling pathway. This structural
difference between trans- (a rod-like structure) and cis-FA (a boomerang-
shaped structure with prominent kink in the molecular backbone) leads to
important biophysical and biological consequences [64]. It was reported that
the conformational flexibility of OA molecule induces a major structural
alteration of the hydrophobic core of the lipid bilayer and perturb the
membrane structure as compared to the rod-like molecular structure of trans-
FA that leads only to a little disorganization [62].
Self-Assembled Liquid Crystalline Nanostructures 13
It was reported that the molecular mechanisms by which OA affects the
biological membrane involve a very specific link between the membrane lipid
structure and the BP regulation [39]. In this context, it was demonstrated that
the penetration of OA molecules into the lipid membrane structure leads to a
marked reduction in the lamellar (L
)-to-hexagonal (H
2
) phase transition as
compared to the trans or saturated FA counterparts [59, 60]. The presence of
this non-lamellar prone lipid in the cell membrane significantly alters the
membrane curvature strain to be more negative [60]. It was assumed that the
transition to hexagonal (H
2
) phase favors the docking of certain peripheral
signaling G protein, which in turn affects the BP [39, 65]. It is also interesting
that the structural analogue of synthetic OA, 2-hyroxyoleic acid (2OHOA) acts
as a potent antitumor drug for glioma by inducing important signaling changes
that end up with cell death [66, 67]. Martnez et al. reported on the propensity
of 2OHOA to organize the lipid membrane into a non-lamellar phase, which
promotes the recruitment of protein kinase C (PKC) to the cell [68]. It was
suggested that the transition to the H
2
phase leads to impair of cell progression
and simultaneously inhibits the growth of the tumor cells [68]. In another
report, the apoptotic activity of OA/protein complexes, known as HAMLET
(Human Alpha-lactalbumin Made LEthal to Tumor cells) was attributed also
to the role of OA in membrane perturbation. As an initial step of killing the
tumor cells, OA alters the membrane and compromises its integrity [64, 69,
70].
Besides the widespread research interests in understanding the role of OA
in regulating biological functions, the use of OA as a main essential
constituent in various drug nanoparticulate formulations including liposomes,
microemulsions, and nanoemulsions has attracted a great attention in the last
two decades [1, 55, 71]. For instance, the utilization of OA-loaded liposomes
(LipoOA) as promising candidates in transdermal applications was suggested
in the literature due to the therapeutic efficacy of these soft drug nanocarriers
in eradicating drug resistance and enhancing its skin penetration [72, 73]. It
was also reported that the association of OA in lipidic nanoparticles (LNPs)
enhances the cellular uptake and hepatic delivery of siRNA and microRNA
[74]. In addition, self-assembled gelatin-OA nanoparticles and OA-loaded
microemulsion were found attractive candidates for improving the
solubilization of poorly water-soluble drugs and controlling their release [71,
75-77].
Intan Diana Mat Azmi and Anan Yaghmur 14
II. FORMATION OF SELF-ASSEMBLED NANOSTRUCTURES
Surfactant-like lipids adopt either normal (type 1) or inverted (type 2) self-
assembled phases, resulting in either oil-in-water (o/w) phases with convex
curvature lipid/water interface or water-in-oil (w/o) phases with a concave
interface, respectively. The formation of a normal or an inverted self-
assembled nanostructure in water mainly depends on the lipids molecular
shape, as discussed in the seventies by Israelachvili and co-workers [78]. In
this regard, the geometric shape of the lipid can be a useful tool for predicting
the water-lipid interface curvature and also can be helpful in understanding the
phase behavior of binary, ternary, and even multi-component systems [79].
For this purpose, the shape factor or more commonly known in the literature as
the critical packing parameter (CPP) was defined [78] as:
(1)
where v
s
is the effective hydrophobic chain volume, a
0
is the headgroup area,
and l is the hydrophobic chain length. The inverted type phases are favored
when CPP > 1 and therefore are generally formed when adding to water
wedge-shaped lipids with hydrophobic tails having a relatively large volume
(v
s
) as compared to the hydrophobic chain length (l) and the headgroup area
(a
0
). Balanced surfactants with CPP 1 tend to form planar bilayers (the
lamellar phase); whereas normal type liquid crystalline phases and micellar
solutions are displayed in the presence of surfactants having CPP < 1. It is
worth noting that the CPP is affected by different variables including lipid
composition, hydration level, electrostatic interactions, presence of
hydrophilic, hydrophobic and amphiphilic additives, and applied experimental
conditions [79-82].
From applicational point of view, there is a noteworthy difference in the
hydration behavior between the normal and inverted type self-assembled
phases. The normal type phases can be easily destabilized in the presence of
excess water, as the surfactant monomers are dissolved in the aqueous
environment when approaching a concentration lower than its critical micellar
concentration (cmc). In contrast, the inverted type phases are independent of
water content under full hydration conditions and therefore are stable against
water dilution [83]. Thus, these systems have recently gained considerable
CPP
v
s
a
0
l
Self-Assembled Liquid Crystalline Nanostructures 15
interest in designing drug and functional food delivery systems due to their
unique properties [84].
Owing to this attractiveness to potential pharmaceutical applications, the
focus in the next sections will exclusively be on describing the formation and
the characterization of inverted type dispersed and non-dispersed phases.
III. INVERTED TYPE LYOTROPIC LIQUID
CRYSTALLINE PHASES
Certain biologically relevant amphiphilic (surfactant-like) lipids including
monoglycerides, glycolipids, and phospholipids have the ability to self-
assemble upon hydration into inverted type lyotropic liquid crystalline (LLC)
phases or micellar systems [79, 85].
This process of self-assembly depends on various parameters including
the chemical structure of the lipid and its composition [86]. It results under
certain experimental conditions on the formation of highly ordered liquid
crystalline phases or micellar solutions consisting of discrete aqueous and
lipidic regions upon direct contact of the surfactant-like lipid with water [87].
These self-assembled systems include lamellar (L
V
2
H
2
I
2
L
2
with increasing solubilized oil content and/or
temperature, ranking the inverse phases by increasing values of their mean-
interfacial curvature or CPP value [83, 99]_ENREF_96. The CPP increases
with temperature due to the increased fluctuation of the hydrophobic chains of
the investigated surfactant-like lipid [83].
Figure 1. Left: Phase diagram of the binary MLO-water system. Right: Phase sequence in a binary or ternary lipid system that is
displayed upon increasing temperature and/or solubilizing oil. The phases are the following: (A) a fluid lamellar (L