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Evaluation of antioxidant activity linked to antimicrobial essential oils by photochemiluminescence


Department of Energy and Mechanics. Petrochemical Engineer.
University of the Armed Forces ESPE

In this review 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)-

The literature outlines different approaches
Essential oils (EOs) are liquid mixtures of within this trend and both the biological
volatile compounds obtained from aromatic screening of new essential oils and the
plants, most commonly by steam distillation. evaluation of new properties of already
They constitute what is called the “essence” of marketed oils have been done. In both cases,
a plant and usually have pleasantly scented different methodological approaches lead to
fragrances. Aromatic plants and EOs have been scattered results, which are hardly comparable
used for millennia for their health benefits, and often conflicting. A plethora of different
well documented in ancient literatura [1]. antioxidant assays is available and, because
results rely on different mechanisms, they
Until recently, essential oils have been strictly depend on the oxidant/antioxidant
studied most from the viewpoint of their models employed and on lipophilic/hydrophilic.
flavour and fragrance chemistry only for A single-substance/single assay produces
flavouring foods, drinks and other goods. relative results and it is perceived as a
Actually, however, essential oils and their reductive approach whenever a phytocomplex
components are gaining increasing interest is involved. Therefore, a multiple-test and a
because of their relatively safe status, their simultaneous chemical characterization must
wide acceptance by consumers, and their be taken into account whenever assays of
exploitation for potential multi-purpose essential oils are performed to allow a balance
functional use [2]. between the sensory acceptability and
functional properties [4].
Previous works have suggested that several
essential oils showed important antimicrobial 1.1 Antioxidant Activity.
activity against bacteria, yeasts, dermatophyte
and Aspergillus strains, and have therapeutic By definition, antioxidants are compounds
potential, mainly in diseases involving mucosal, capable of slowing or retarding the oxidation of
cutaneous and respiratory tract infections. The an oxidizable material, even when used in very
major constituents of many of these oils are modest amount (<1%, commonly 1−1000 mg/L)
phenolic compounds (terpenoids and as compared to the amount of material they
phenylpropanoids) like thymol, carvacrol or have to protect. Focusing on processes of
eugenol, of which antimicrobial and antioxidant relevance in biological systems or in food
activities are well documented [3]. science, the materials to protect are most
commonly lipids, proteins, carbohydrates, and,
to a minor extent, other organic molecules that bacteria, 48 h at 25 C for yeasts), positive
compose animal or vegetal tissues. Their antibacterial and antifungal activities were
oxidation occurs by a radical chain reaction established by the presence of measurable
mediated by peroxyl radicals (ROO•) that zones of inhibition. The antimicrobial activity
parallels the autoxidation of hydrocarbons [4]. was recorded as the width (in millimetres,
diameter of the disc included) of the zone of
Antioxidants retard oxidation and are inhibition after incubation. Each test was
sometimes added to meat and poultry products performed in three replicates and repeated
to prevent or slow oxidative degradation of twice [1].
fats. Antioxidant agents are effective due to
different mechanisms such as free radical 1.2.1 Screening for antioxidant activity.
scavenging, chelating of pro-oxidant metal ions
or quenching singlet-oxygen formation[3]. Antioxidant activity of essential oils was
determined using the photochemiluminescence
In light of the differences among the wide (PCL) in which the photochemical generation of
number of test systems available, the results of free radicals is combined with the sensitive
a single-assay can give only a reductive detection by using chemiluminescence. This
suggestion of the antioxidant properties of reaction is induced by optical excitation of a
essential oils toward food matrices and must be photosensitizer S which results in the
interpreted with some caution. Moreover, the generation of the superoxide radical O2-.
chemical complexity of essential oils, often a
mixture of dozens of compounds with different S+hv+ O2- →[S*O2- ]→S+ + O2-
functional groups, polarity and chemical
behaviour, could lead to scattered results, The free radicals are visualised with the
depending on the test employed. Therefore, an chemiluminescent detection reagent luminol. It
approach with multiple assays in screening works as photosensitizer as well as oxygen
work is highly advisable. Among the plethora of radical detection reagent. The essential oils
methods that can be used for the evaluation of were measured in the Photochem with the ACL
the antioxidant activity (TEAC, TRAP, LDL, kit (AnalytikJena, Jena, Germany). A 2.2 ml
DMPD, FRAP, ORAC, DPPH, PCL, and b-carotene portion of reagent 1 (solvent and dilution
bleaching), very few of them (TEAC, DPPH, PCL) reagent), 200 ll of reagent 2 (buffer solution),
are useful for determining the activity of both 25 ll of reagent 3 (photosensitizer), and 100 ll
hydrophilic and lipophilic species, thus ensuring of standard (trolox reagent in reagent 1) or
a better comparison of the results and covering sample (essential oil in methanol) solution were
a wider range of possible applications [2]. mixed and measured. A light emission curve
was recorded over 180 s, using inhibition as the
1.2 Screening for antimicrobial activity. parameter to evaluate antioxidant potential.
The antioxidant capacity was then determined
Screening for antimicrobial activity The paper by using the integral under the curve and was
disc diffusion method was employed to expressed as mmol/l of trolox used as standard
determine the antimicrobial activity of the to obtain calibration curve [2].
essential oils. For these assays the cultures of
the following micro-organisms were used: one 1.2.1 Photochemiluminescence.
gram-positive (Staphylococcus aureus) and one
gram-negative (Escherichia coli) bacteria and The luminol-photochemiluminescence assay
one yeast (Saccharomyces cerevisiae). All was carried out with the procedure described
micro-organisms were supplied from the by Popov and Lewin (1999) and adapting the [2]
Algerian pharmaceutical industry SAIDAL. standard protocol. The essential oils were
Cultures of the micro-organisms were measured in the Photochem with the ACL kit. A
maintained on nutrient agar (NA) medium. 2.30 ml portion of reagent 1 (solvent and
Briefly, a suspension of the tested micro- dilution reagent), 200 l of reagent 2 (buffer
organism (107 –108 CFU/ml) was spread on the solution), 25 ll of reagent 3 (photosensitizer),
solid media plates. Filter paper discs of 6 mm and 10 ll of standard (trolox solution in reagent
diameter (Whatman no. 1) were individually 1) or simple (essential oil in methanol) solution
impregnated with 50 ml of essential oil, then were mixed and measured. A light emission
laid on to the surface of the inoculated plates. curve was recorded over 130 s, using inhibition
At the end of incubation time (24 h at 37 C for 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 calibration curve [2].

auto oxidation of luminol. The luminol is a

2.1 Antimicrobial activity. photosensibilitiser, generating superoxide,
radicals, and also a chemiluminogenic probe for
Antimicrobial activity The antimicrobial free radicals. Because superoxide radical is a
activity of the essential oils from rosemary deleterious by-product of oxygen metabolism,
leaves, isolated by two isolation methods, responsible for the most important diseases,
against three species of micro-organisms by the the values obtained by the PCL method give an
disc diffusion method was recorded in Table 2. evaluation of the protective capacity of a given
The essential oils showed inhibition zones ingredient against ROS which are the most
against all micro-organisms tested. The data dangerous species of free radicals for leaving
obtained from disc diffusion method using beings. Data obtained from PCL testing show
rosemary essential oil, indicated that S. very small differences between the antioxidant
cereveciae was the most sensitive micro- activities of the oils obtained with each
organism tested with the largest inhibition zone extraction technique. In this assay, rosemary
(24–20 mm) and S. aureus exhibited the essential oil obtained by MHG showed a slightly
smallest inhibition zone (17–12.5 mm). Overall, better antioxidant capacity value, which is
the essential oils displayed a broad equivalent to 4.53 ± 0.02 mmol of trolox per
antimicrobial spectrum and exerted a stronger litre of sample, than the essential oil obtained
antimicrobial effect against gram-positive by HD with 3.68 ± 0.02 mmol of trolox per litre
bacteria than gram-negative bacteria. The of sample (Table 2). These small differences
antimicrobial activity of the rosemary essential could be due to the higher proportion of the
oils obtained by MHG is slightly higher than oxygenated compounds contained in MHG
that obtained with HD. The antimicrobial essential oil [2].
activity of MHG essential [2].

1.3 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

1. Baratta, M.T., et al., Antimicrobial and antioxidant properties of some

commercial essential oils. Flavour and fragrance journal, 1998. 13(4): p.
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.