Evaluation of antioxidant activity linked to antimicrobial essential oils by

photochemiluminescence (PCL)

By ALEX AGUALEMA V.
Department of Energy and Mechanics. Petrochemical Engineer.
University of the Armed Forces ESPE
Student
E-mail: edmundoagualema21@gmail.com

ABSTRACT
In this article we study the antioxidant activities by the photochemiluminescence
(PCL) method in which the photochemical generation of free radicals is combined
with the sensitive detection using chemiluminescence by linking it with the
antimicrobial activity applying the method of diffusion in paper disc based on The
cultures of a gram-positive bacterium (Staphylococcus aureus) and a gram-
negative (Escherichia coli) and a yeast (Saccharomyces cerevisiae), concluding that
the most pronounced antimicrobial activity against yeasts and Gram-positive
organisms against Gram- Negative, environmentally friendly, reduced cost and
energy consumption; Cleaner features (such as no waste generation and no water
or solvent used)
KEY WORDS: antimicrobial activity; antioxidant activity; Essential oi
photochemiluminescence (PCL)

1. INTRODUCTION.
important antimicrobial activity
Essential oils (EOs) are liquid against bacteria, yeasts,
mixtures of volatile compounds dermatophyte and Aspergillus
obtained from aromatic plants, most strains, and have therapeutic
commonly by steam distillation. They potential, mainly in diseases
constitute what is called the essence involving mucosal, cutaneous and
of a plant and usually have pleasantly respiratory tract infections. The
scented fragrances. Aromatic plants major constituents of many of these
and EOs have been used for millennia oils are phenolic compounds
for their health benefits, well (terpenoids and phenylpropanoids)
documented in ancient literature.[1] like thymol, carvacrol or eugenol, of
Until recently, essential oils have which antimicrobial and antioxidant
been studied most from the activities are well documented [3].
viewpoint of their flavour and The literature outlines different
fragrance chemistry only for approaches within this trend and
flavouring foods, drinks and other both the biological screening of new
goods. Actually, however, essential essential oils and the evaluation of
oils and their components are gaining new properties of already marketed
increasing interest because of their oils have been done. In both cases,
relatively safe status, their wide different methodological approaches
acceptance by consumers, and their lead to scattered results, which are
exploitation for potential multi- hardly comparable and often
purpose functional use [2]. conflicting. A plethora of different
Previous works have suggested that antioxidant assays is available and,
several essential oils showed because results rely on different
mechanisms, they strictly depend on
the oxidant/antioxidant models
employed and on
lipophilic/hydrophilic. A single-
substance/single assay produces
relative results and it is perceived as
a reductive approach whenever a
phytocomplex is involved. Therefore,
a multiple-test and a simultaneous
chemical characterization must be
taken into account whenever assays
of essential oils are performed to
allow a balance between the sensory
acceptability and functional
properties.
1.1 Antioxidant Activity.
By definition, antioxidants are
compounds capable of slowing or
retarding the oxidation of an
oxidizable material, even when used
in very modest amount (<1%,
commonly 11000 mg/L) as
compared to the amount of material
they have to protect. Focusing on
processes of relevance in biological
systems or in food science, the
materials to protect are most
commonly lipids, proteins,
carbohydrates, and, to a minor extent,
other organic molecules that
compose animal or vegetal tissues.
Their oxidation occurs by a radical
chain reaction mediated by peroxyl
radicals (ROO) that parallels the
autoxidation of hydrocarbons [4].
Antioxidants retard oxidation and are
sometimes added to meat and poultry
products to prevent or slow oxidative
degradation of fats. Antioxidant
agents are effective due to different
mechanisms such as free radical
scavenging, chelating of pro-oxidant
metal ions or quenching singlet-
oxygen formation[3] .
In light of the differences among the
wide number of test systems
available, the results of a single-assay
can give only a reductive suggestion
of the antioxidant properties of
essential oils toward food matrices
and must be interpreted with some
caution. Moreover, the chemical
complexity of essential oils, often a
mixture of dozens of compounds with
different functional groups, polarity
and chemical behaviour, could lead to
scattered results, depending on the
test employed. Therefore, an
approach with multiple assays in
screening work is highly advisable.
Among the plethora of methods that
can be used for the evaluation of the
antioxidant activity (TEAC, TRAP,
LDL, DMPD, FRAP, ORAC, DPPH, PCL,
and b-carotene bleaching), very few
of them (TEAC, DPPH, PCL) are useful
for determining the activity of both
hydrophilic and lipophilic species,
thus ensuring a better comparison of
the results and covering a wider
range of possible applications [2].
1.2 Screening for antimicrobial
activity
Screening for antimicrobial activity
The paper disc diffusion method was
employed to determine the
antimicrobial activity of the essential
oils. For these assays the cultures of
the following micro-organisms were
used: one gram-positive
(Staphylococcus aureus) and one
gram-negative (Escherichia coli)
bacteria and one yeast
(Saccharomyces cerevisiae). All
micro-organisms were supplied from
the Algerian pharmaceutical industry
SAIDAL. Cultures of the micro-
organisms were maintained on
nutrient agar (NA) medium. Briefly, a
suspension of the tested micro-
organism (107 108 CFU/ml) was
spread on the solid media plates.
Filter paper discs of 6 mm diameter
(Whatman no. 1) were individually
impregnated with 50 ml of essential
oil, then laid on to the surface of the
inoculated plates. At the end of
incubation time (24 h at 37 C for
bacteria, 48 h at 25 C for yeasts),
positive antibacterial and antifungal
activities were established by the
presence of measurable zones of
inhibition. The antimicrobial activity
was recorded as the width (in
millimetres, diameter of the disc
included) of the zone
of inhibition after incubation. Each
test was performed in three
replicates and repeated twice.
1.2.1 Screening for antioxidant
activity.
Antioxidant activity of essential oils
was determined using the
photochemiluminescence (PCL) in
which the photochemical generation
of free radicals is combined with the
sensitive detection by using
chemiluminescence. This reaction is
induced by optical excitation of a
photosensitizer S which results in the
generation of the superoxide radical
O2-.
S+hv+O_2[S*O_2 ]S^++ O2 calibration curve [2].
The free radicals are visualised with
the chemiluminescent detection
reagent luminol. It works as
photosensitizer as well as oxygen
radical detection reagent. The
essential oils were measured in the
Photochem with the ACL kit
(AnalytikJena, Jena, Germany). A 2.2
ml portion of reagent 1 (solvent and
dilution reagent), 200 ll of reagent 2
(buffer solution), 25 ll of reagent 3
(photosensitizer), and 100 ll of
standard (trolox reagent in reagent 1)
or sample (essential oil in methanol)
solution were mixed and measured. A
light emission curve was recorded
over 180 s, using inhibition as the
parameter to evaluate antioxidant
potential. The antioxidant capacity
was then determined by using the
integral under the curve and was
expressed as mmol/l of trolox used as
standard to obtain calibration curve
[2].
1.2.1 Photochemiluminescence
The luminol-
photochemiluminescence assay was
carried out with the procedure
described by Popov and Lewin
(1999) and adapting the [2] standard
protocol. The essential oils were
measured in the Photochem with the
ACL kit. A 2.30 ml portion of reagent
1 (solvent and dilution reagent), 200 l
of reagent 2 (buffer solution), 25 ll of
reagent 3 (photosensitizer), and 10 ll
of standard (trolox solution in
reagent 1) or simple (essential oil in
methanol) solution were mixed and
measured. A light emission curve was
recorded over 130 s, using inhibition
as the parameter to evaluate
antioxidant potential. The antioxidant
capacity was then determined by
using the integral under the curve
and was expressed as mmol/l of
trolox used as standard to obtain a
2. RESULTS AND
DISCUSSION.
2.1 Antimicrobial activity.
Antimicrobial activity The
antimicrobial activity of the essential
oils from rosemary leaves, isolated by
two isolation methods, against three
species of micro-organisms by the
disc diffusion method was recorded
in Table 2. The essential oils showed
inhibition zones against all micro-
organisms tested. The data obtained
from disc diffusion method using
rosemary essential oil, indicated that
S. cereveciae was the most sensitive
micro-organism tested with the
largest inhibition zone (2420 mm)
and S. aureus exhibited the smallest
inhibition zone (1712.5 mm).
Overall, the essential oils displayed a
broad antimicrobial spectrum and
exerted a stronger antimicrobial
effect against gram-positive bacteria
than gram-negative bacteria. The
antimicrobial activity of the rosemary
essential oils obtained by MHG is
slightly higher than that obtained
with HD. The antimicrobial activity of
MHG essential.
2.2 Antioxidant activity.
Photochemiluminescence is a modern
technique for the estimation of the
total antioxidant capacity. It is based
on an antioxidantsensitive inhibition
of a photo-induced,
chemiluminescence accompanied
auto oxidation of luminol. The
luminol is a photosensibilitiser,
generating superoxide, radicals, and
also a chemiluminogenic probe for
free radicals. Because superoxide
radical is a deleterious by-product of
oxygen metabolism, responsible for
the most important diseases, the
values obtained by the PCL method
give an evaluation of the protective
capacity of a given ingredient against
ROS which are the most dangerous
species of free radicals for leaving
beings. Data obtained from PCL
testing show very small differences
between the antioxidant activities of
the oils obtained with each extraction
technique. In this assay, rosemary
essential oil obtained by MHG showed
a slightly better antioxidant capacity
value, which is equivalent to 4.53
0.02 mmol of trolox per litre of
sample, than the essential oil
obtained by HD with 3.68 0.02
mmol of trolox per litre of sample
(Table 2). These small differences
could be due to the higher proportion
of the oxygenated compounds
contained in MHG essential oil.
REFERENCIAS BIBLIGRAFICAS:
1. Baratta, M.T., et al., Antimicrobial and antioxidant properties of some
commercial essential oils. Flavour and fragrance journal, 1998. 13(4): p. 235-244.
2. Sacchetti, G., et al., Comparative evaluation of 11 essential oils of different
origin as functional antioxidants, antiradicals and antimicrobials in foods. Food
chemistry, 2005. 91(4): p. 621-632.
3. Lopes-Lutz, D., et al., Screening of chemical composition, antimicrobial and
antioxidant activities of Artemisia essential oils. Phytochemistry, 2008. 69(8): p.
1732-1738.
4. Amorati, R., M.C. Foti, and L. Valgimigli, Antioxidant activity of essential oils.
Journal of Agricultural and Food Chemistry, 2013. 61(46): p. 10835-10847.