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Aims: The purpose of this study was to investigate the action of Citrus limonum essential oil to control free radical-induced lipid peroxidation and preventing tissue damage in skin. Place and Duration of Study: Department of Internal Medicine (University of Roma “Tor Vergata) and A.R.P.A (Aging Research, Prevention and Therapy Association, www.antiaging.it), between January 2010 and June 2011. Methodology: The essential oil was subjected to GC-MS analysis. The superoxide anion scavenging activity of essential oil was evaluated by the enzymatic hypoxanthine/xanthine oxidase system. The same oil diluted in DMSO or grape-seed oil was spread on the face of human volunteers after UV exposition. A sample of skin lipids was collected and the presence of peroxyl radicals was detected based on the measurement of light emitted (chemiluminescence) when the excited carbonyl and singlet oxygen decay to ground state. Results: Our data demonstrate that the lemon essential oil is more active than -tocopherol against *O2- and peroxide free radical inhibition at 1:100 dilution. A protocol for controlling free radical-induced lipid peroxidation in human skin was thus proposed. Conclusion: The scavenging action of lemon essential oil could have a practical application for treating human skin against oxidative damage.
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*Corresponding author: Email: paola.angelini@unipg.it;
European Journal of Medicinal Plants
3(1): 1-9, 2013
SCIENCEDOMAIN international
www.sciencedomain.org
Antioxidative Action of Citrus limonum
Essential Oil on Skin
G. Bertuzzi1, B. Tirillini2, P. Angelini3* and R. Venanzoni3
1Department of Internal Medicine, University of Roma “Tor Vergata”, Via Montpellier,
1 – 00133 Roma (RM), Italy.
2Department of Biomolecular Science, University of Urbino, Via Bramante, 28 - 61028
Urbino (PU), Italy.
3Department of Applied Biology, University of Perugia, Borgo XX giugno 74 - 06121 Perugia
(PG), Italy.
Authors’ contributions
The work presented here was carried out with the collaboration of all the authors. Authors
GB and BT defined the research theme and designed the methods and experiments,
analyzed the data, interpreted the results and wrote the paper. Author PA was involved in
the writing process of the manuscript, author RV co-designed the experiments, discussed
the analyses, interpretation, and presentation of data. All authors have contributed to, seen
and approved the manuscript.
Received 7th August 2012
Accepted 8th October 2012
Published 4th December 2012
ABSTRACT
Aims: The purpose of this study was to investigate the action of Citrus limonum essential
oil to control free radical-induced lipid peroxidation and preventing tissue damage in skin.
Place and Duration of Study: Department of Internal Medicine (University of Roma “Tor
Vergata) and A.R.P.A (Aging Research, Prevention and Therapy Association, www.anti-
aging.it), between January 2010 and June 2011.
Methodology: The essential oil was subjected to GC-MS analysis. The superoxide anion
scavenging activity of essential oil was evaluated by the enzymatic hypoxanthine/xanthine
oxidase system. The same oil diluted in DMSO or grape-seed oil was spread on the face
of human volunteers after UV exposition. A sample of skin lipids was collected and the
presence of peroxyl radicals was detected based on the measurement of light emitted
(chemiluminescence) when the excited carbonyl and singlet oxygen decay to ground
state.
Research Article
European Journal of Medicinal Plants, 3(1): 1-9, 2013
2
Results: Our data demonstrate that the lemon essential oil is more active than -
tocopherol against *O2-and peroxide free radical inhibition at 1:100 dilution. A protocol for
controlling free radical-induced lipid peroxidation in human skin was thus proposed.
Conclusion: The scavenging action of lemon essential oil could have a practical
application for treating human skin against oxidative damage.
Keywords: Anti-aging; GC-MS; grape seed oil; superoxide anion scavenging.
1. INTRODUCTION
The inhibition of lipid oxidation by essential oils such as Origanum spp., Thymus spp.,
Satureja spp. and Rosmarinus officinalis, have already been reported in literature [1, 2, 3].
All the essential oils studied have shown a strong phenolic profile characterized by the
presence of phenylpropanoids which are believed to be the active component of the
essential oils [4, 5, 6, 7, 8, 9]. Citrus essential oil has also been reported to have
antioxidative activities against linoleic acid oxidation [10] and to both Cu2+-induced and 2, 2’-
azobis (2-aminopropane) hydrochloride-induced oxidation of human low-density lipoprotein
in vitro [11]. Among the compounds tested in Citrus essential oil, γ-terpinene had the
strongest antioxidant effect [11], but no clear relationship could be shown between the
antioxidant activity and the essential oil composition of the extracts [12]. When skin is
exposed to air that is irradiated by ultraviolet (UV) light consisting of UVA (320-400 nm) and
UVB (290-320 nm), reactive oxygen species (ROS) including superoxide anion radical (*O2-),
hydrogen peroxide (H2O2), hydroxyl radical (*OH), singlet oxygen (*O2), lipid peroxides
(LOOH), and their radicals (LOO*) are formed. These in turn induce skin aging, phototoxicity,
inflammation and malignant tumors [13, 14, 15, 16, 17].
Recently, consumer interest and the media have focused specifically on products that use
natural ingredients, such as plant extract. There is some evidence that these ingredients
could have possible in vitro anti-aging activity, but the question remains whether it is
possible to deliver adequate doses to the skin in vivo. Lemon oil, traditionally used for its
aromatic properties, has recently been investigated for its effects on skin [18]. The purpose
of this study was to investigate the effectiveness of Citrus limonum Risso essential oil in
controlling free radical-induced lipid peroxidation and preventing tissue damage in skin.
2. MATERIALS AND METHODS
2.1 Plant Material
The lemon (Citrus limonum, Rutaceae) essential oil used in this study was obtained from
cold pressed oil extracted from the peel of the fruit (collected in the north of Sicily Island)
according to the methods of Sawamura and Kuriyama [19]. The cold pressed oil was then
hydrodistilled for 1h in an all-glass Clevenger apparatus. Voucher specimens of C. limonum
plants, identified following the Italian botanical standard treatise [20] were deposited in the
Herbarium of the Dept. of Applied Biology (University of Perugia, Italy).
European Journal of Medicinal Plants, 3(1): 1-9, 2013
3
2.2 GC and GC-MS Analysis
The GC analyses were carried out using a Varian 3300 instrument equipped with an FID and
an HP-InnoWax capillary column (30 m x 0.25 mm, film thickness 0.17 µm), working from
60ºC (3 min) to 210ºC (15 min) at 4ºC/min or an HP-5 capillary column (30 m x 0.25 mm,
film thickness 0.25 µm) working from 60ºC (3 min) to 300ºC (15 min) at 4ºC/min; the injector
and detector temperature was 250ºC. Helium was used as the carrier gas, with a flow rate of
1 ml/min, and the split ratio was 1:10.
GC-MS analyses were carried out with a Hewlett Packard 5890 GC-MS system operating in
the EI mode at 70 eV, using the two above-mentioned columns. The operating conditions
were analogous to those reported in the GC analyses section. The injector and transfer line
temperatures were 220ºC and 280ºC, respectively. Helium was used as the carrier gas, with
a flow rate of 1 ml/min and the split ratio was 1:10.
2.3 Identification of the Components
The components were identified by matching the spectra with those from mass spectral
libraries; the identity of each component was confirmed by comparing the retention indices,
from both columns, relative to the C6-C22 n-alkanes, with those from the literature [21, 22,
23, 24, 25]. When reported,co-elution gas chromatography with reference compounds was
also used for an additional confirmation of the compound identity. The percentage
composition of the essential oil was obtained by the normalization method from the GC peak
areas, without using correction factors.
2.4 Superoxide Anion Scavenging (*O2-)
Superoxide anion was generated by a hypoxanthine-xanthine oxidase system [26]. Reaction
mixtures with 100 µl EDTA (30 mmol/l), 10 µl hypoxanthine (30 mmol/l), 100 µl cytochrome c
(3 mmol/l) or nitroblue tetrazolium (3 mmol/l) were added to 150 µl of lemon essential oil
(solubilized in DMSO 10%) at various concentrations in a final volume of 3 ml buffered in
KH2PO4(50 mmol/l), pH 7.4 [27]. The reaction was started by adding 200 µl xanthine
oxidase (1U/ml) and the rate of reduced cytochrome c or nitroblue tetrazolium was
measured at 550 nm, and 560 nm, respectively, against a reference. The amount of *O2-
generated was calculated using the extinction coefficient ε550 = 2.1 x 10-2 µmol–1 per cm and
the *O2-inhibition was expressed as percentage values. The sample tested did not interfere
with the xanthine oxidase activity (measured at 290 nm). The positive response was tested
using α-tocopherol. Ten repetitions were carried out.
2.5 Randomized Controlled Trial
2.5.1 Subjects
Subjects were selected from among men aged 18 to 52 (mean 33±11) years who were
found to have no serious illness on physical checkup at A.R.P.A. (Aging Research,
Prevention and Therapy Association), www.anti-aging.it (Civita Castellana, VT, Italy). Eighty
volunteers (average age: 33±11 years) who gave their written consent to participate in the
test were selected as subjects from January 2010 to June 2011.
European Journal of Medicinal Plants, 3(1): 1-9, 2013
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2.5.2 Extraction of skin lipids from healthy volunteers
Skin lipids were collected with acetone-wetted cotton swabs from the forehead over a 9 cm2
area from healthy volunteers (80 men, 18–52 years old–mean 33±11) in the morning for 7
days. The sampling procedure was identical for all the subjects. The volunteers were
randomly divided into four groups (A, B, C, D). In group A the forehead was treated daily for
a week with α-tocopherol in ethanol (20%), group B with lemon essential oil solubilized in
DMSO (1:100), group C with lemon essential oil solubilized in grape-seed oil (1:100), and
group D was left untreated. In accordance with the European norm EN 60335-2-27 and
under medical supervision, volunteers were irradiated daily with UVA and UVB of 0.3
W/m2/mm from sunlamps for 7 min at each session. The participants were asked not to
expose themselves to direct sunlight and to avoid the use of face creams or hair lotions for
the entire duration of the experiment. Twenty-four hours after the last treatment, the skin
lipids were collected.
Extracts were taken twice from the wet cotton swabs using 3 ml of chloroform/methanol
(1:2.5) for two hours (10 µg heneicosanoic acid was used for the recovery test). The raw
extracts were partitioned between 1% NaCl in 0.01 M HCl and chloroform. The chloroform
extracts were washed with methanol/water (1:1) and dried under N2stream. The samples
were stored at –20Cº in 3 ml of chloroform/ethanol (2:1).
2.6 Lipid Peroxidation Analyzed by Chemiluminescence
Chemiluminescence is an index of oxidative stress that quantifies lipid peroxidation and was
measured according to the method of Gonzalez-Flecha et al. [28]. This method is based on
the measurement of light emitted (chemiluminescence) when the excited carbonyl and
singlet oxygen produced by peroxyl radicals decay to ground state. This light is due to the
generation of reactive oxygen species in whole lipids. Skin lipids were incubated with 3 mM
t-BHP for 60 min at 37ºC. Lipid peroxidation was initiated by adding a small amount of stock
solution of t-butyl hydroperoxide (80 mM) to each vial which was then maintained at 37ºC,
and measured by monitoring light emission [29] with a liquid scintillation analyzer Packard
1900 TR. Chemiluminescence was measured over a 60 min period and recorded as counts
per minute (cpm) every 12 min. Each reaction was terminated by adding 5 ml
chloroform/methanol (2:1, v/v) containing 0.01% butylated hydroxytoluene (BHT). This also
inhibited any further oxidation during the lipid extraction. The DMSO had no antioxidative
action and gave a chemiluminescence curve that could be superimposed on to that of the
control.
2.7 Statistical Analysis
Analysis of variance, significances, correlations and other statistical analysis were performed
using Graph Pad Prism version 5.00, (GraphPad Software, San Diego, California, USA).
3. RESULTS AND DISCUSSION
3.1 Chemical Composition of the Essential Oil
Citrus oils are a mixture of volatile compounds and consist mainly of monoterpene
hydrocarbons [30]. Citrus essential oils, on the other hand, generally contain some amount
of coumarins or furanocoumarins [31], flavonoids [32] and tocopherols [33] in the non-volatile
European Journal of Medicinal Plants, 3(1): 1-9, 2013
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fractions of citrus oils. Coumarins and furanocoumarins may have an important role in skin
photosensitization. Hydrodistillation of the cold pressed oil prevents this hazard. Nineteen
compounds were identified in the GC and GC/MS analyses. The percentage composition of
Citrus limonum essential oil is shown in Table 1. The components are listed in the order of
elution from the HP-5 column. The main component was limonene (54.6%) followed by γ-
terpinene (19.1%) and β-pinene (14.5%). The monoterpene hydrocarbons (87.7%)
constituted the main fraction of lemon oil. This oil composition, as reported in the literature, is
similar to other volatile fractions characterized by the high content of limonene [34].
Table 1. Percentage composition of the essential oil from C. limonum
Compound
%
α-pinene
3,9
β-pinene
14,5
myrcene
1,5
α-terpinene
0,3
p-cymene
0,1
limonene
54,6
γ-terpinene
19,1
terpinolene
0,8
linalool
0,1
citronellal
0,1
terpinen-4-ol
0,1
α-terpineol
0,3
citronellol
0,1
nerol
0,1
neral
1,1
geraniol
0,1
linalyl acetate
0,1
geranial
2,3
geranyl acetate
0,8
aRetention index, relative to C9-C22 n-alkanes on the HP-5 column.
3.2 In Vitro and In Vivo Free Radical Scavenging Activity of Essential Oil
The superoxide anion scavenging activity of Citrus limonum essential oils was evaluated
using the enzymatic hypoxanthine/xanthine oxidase system. Among the concentrations
tested (Fig.1), the 1:100 dilution of lemon essential oil in DMSO had an *O2-inhibition that
was comparable to that of α-tocopherol. The 1:1000 dilution inhibited *O2-less than α-
tocopherol but the level of inhibition was about 76% and 65% of the α-tocopherol activity on
cytochrome c and tetrazolium nitroblue, respectively.
European Journal of Medicinal Plants, 3(1): 1-9, 2013
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Fig. 1. Percentage cytochrome c inhibition and percentage nitroblue tetrazolium
reduction
Test significant from normal control (P < 0.05). Mean ±S.E.M of ten experiments
The peroxidation data as evidenced by the light emission are shown in Fig 2.
Fig. 2. Chemiluminescence over time in four groups of volunteers
-tocopherol = group A; EO-DMSO = group B ; EO-grape-seed = group C;control = group D.
Test significant from normal control (P < 0.05). Mean ± S.E.M of twenty experiments
The lipids from untreated volunteers showed the highest chemiluminescence and are
considered to be the normal response to the peroxyl radical action. Lower emissions were
recorded for the lipids from volunteers treated with antioxidative substances and the lemon
essential oil was more effective than α-tocopherol as an antioxidant. The grape-seed oil
showed a slightly higher antioxidative action that was added to the action of lemon essential
European Journal of Medicinal Plants, 3(1): 1-9, 2013
6
Fig. 1. Percentage cytochrome c inhibition and percentage nitroblue tetrazolium
reduction
Test significant from normal control (P < 0.05). Mean ±S.E.M of ten experiments
The peroxidation data as evidenced by the light emission are shown in Fig 2.
Fig. 2. Chemiluminescence over time in four groups of volunteers
-tocopherol = group A; EO-DMSO = group B ; EO-grape-seed = group C;control = group D.
Test significant from normal control (P < 0.05). Mean ± S.E.M of twenty experiments
The lipids from untreated volunteers showed the highest chemiluminescence and are
considered to be the normal response to the peroxyl radical action. Lower emissions were
recorded for the lipids from volunteers treated with antioxidative substances and the lemon
essential oil was more effective than α-tocopherol as an antioxidant. The grape-seed oil
showed a slightly higher antioxidative action that was added to the action of lemon essential
European Journal of Medicinal Plants, 3(1): 1-9, 2013
6
Fig. 1. Percentage cytochrome c inhibition and percentage nitroblue tetrazolium
reduction
Test significant from normal control (P < 0.05). Mean ±S.E.M of ten experiments
The peroxidation data as evidenced by the light emission are shown in Fig 2.
Fig. 2. Chemiluminescence over time in four groups of volunteers
-tocopherol = group A; EO-DMSO = group B ; EO-grape-seed = group C;control = group D.
Test significant from normal control (P < 0.05). Mean ± S.E.M of twenty experiments
The lipids from untreated volunteers showed the highest chemiluminescence and are
considered to be the normal response to the peroxyl radical action. Lower emissions were
recorded for the lipids from volunteers treated with antioxidative substances and the lemon
essential oil was more effective than α-tocopherol as an antioxidant. The grape-seed oil
showed a slightly higher antioxidative action that was added to the action of lemon essential
European Journal of Medicinal Plants, 3(1): 1-9, 2013
7
oil; the chemiluminescence curve is a little lower than that of the lemon essential oil
dissolved in DMSO, but the data belong to the same set according to the one-way ANOVA.
These results show that these two oils had a similar scavenging action against peroxide free
radicals in vitro and in vivo [35].
The exposure of human skin to UV radiation can generate ROS in both the epidermis and
dermis. The depth of penetration of UV radiation, as well as its damaging potential in deeper
skin cells have been demonstrated [36]. Among the scavenging substances, α-tocopherol
was chosen as a reference for comparing the scavenging action of lemon essential oil. The
anti-oxidant activity of oil-in-water emulsion containing α-tocopherol has been reported over
a wide range of conditions and test systems [37]. Our data demonstrate that the lemon
essential oil is more active than α-tocopherol against *O2-and peroxide free-radical inhibition
at 1:100 dilution. Lemon essential oil is used instead of other lemon extracts, to avoid the
toxic action that furanocoumarins have under UV exposure.
4. CONCLUSIONS
The results of this study suggest that lemon essential oil has properties that could benefit
human skin as it undergoes environmental and chronological ageing. The scavenging action
of lemon essential oil solubilized in grape-seed oil could have a practical application in
aesthetic medicine (a branch of medicine focused on satisfying the aesthetic desires and
goals of patients) for treating human skin against oxidative damage. Therefore, continuous
application of lemon essential oil solubilized in grape-seed oil might contribute to the
prevention of lifestyle-related skin diseases by regulating the balance of oxidative stress.
ETHICAL APPROVAL
All authors hereby declare that all experiments have been examined and approved by the
appropriate ethics committee and have therefore been performed in accordance with the
ethical standards laid down in the 1964 Declaration of Helsinki.
COMPETING INTERESTS
The authors declare that no competing interests exist.
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... Some valuable phenolic compounds such as naringin, hesperidin, neohesperidin which are found in the essential oil extracted from the peel of the citrus species has the potential to turn the food rancidity 16 . Essential oil extracted from citrus species is found to have antioxidative potential and in some traditional medicines lemon oil is used as they contain different aromatic properties 17 . ...
... Therefore, the compound limonene has wide range of applications most commonly as dietary supplement, fragrance ingredient for cosmetic products, in food manufacturing industries, in some medicines, bath products, personal care products, botanical insecticide, cleaning products, solvents, paint and in air fresheners also 34,35 . From pharmaceutical and medical point of view there were evidences that C. limon essential oil possess antimicrobial, antioxidant activities 12,17 . Therefore C. limon peel essential oil could also be implemented for the herbal formulation of cost effective, easily available antimicrobial and antioxidant drugs. ...
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Citrus limon L. Burmf (commonly known as lemon) is a small thorn bearing tree which belongs to the family Rutaceae cultivated in the tropical and subtropical regions of the world and mostly cultivated in North Eastern region of India. It bears many medicinal properties and used in the cosmetics industries and pharmaceutical industries. Apart from the plant, extracted peel essential oil of C. limon has many medicinal properties so it can be used in the treatment of different diseases. Essential oil was extracted using hydrodistillation and analyzed by using Gas chromatography/mass spectroscopy (GC/MS). A total of forty three compounds were identified of which limonene (55.40 %) and neral (10.39 %) were found as major compound followed by trans-verbenol (6.43 %) and decanal (3.25 %). The other compounds present were ethyl cinnamate (2.21 %), ethyl p-methoxycinnamate (2.21 %), cis-α-bergamotene (1.60 %), geraniol (1.48 %), trans-carveol (1.33 %), nonanal (1.19 %), linalool (1.16 %), α-terpineol (1.07 %), p-mentha-2,8-dien-1-ol (0.90 %), estragole (0.73 %), α-fenchene (0.46 %), perillol (0.46 %), β-curcumene (0.45 %), trans-d-limonene oxide (0.43 %), 1-naphthalenamine (0.42 %), camphor (0.34 %), 3-carene (0.34 %), β-santalene (0.27 %), β-sesquiphellandrene (0.25 %), α-pinene (0.17 %), terpinen-4-ol (0.17 %), 3-terpinolenone (0.17 %), zizaene (0.16 %), germacrene -D (0.16 %), α-bisabolol (0.16 %), β-bisabolol (0.14 %), methyl n-hexadecanoate (0.14 %), citronellal (0.12 %), o-cymene (0.12 %), 1-undecanol (0.11 %) and sabinol (0.11 %) were identified as in minor quantities. Limonene was the major chemical compound in the C. limon peel essential oil which possess many medicinal properties could be used as a pharmaceutical industries.
... In fact, the combined EO was mainly rich in eugenol (32.35%), thymol (25.49%) and limonene (21.30%). There were many other reports regarding the chemical composition of lemon peel and all these researchers confirmed that limonene was the main component ranging from 29.52 to 98.40% (Ayendoun and Sossou, 1996;Mahalwal and Ali, 2003;Yoo et al., 2004;Monajemi et al., 2005;Kamal et al., 2011;Jomaa et al., 2012;Bertuzzi et al., 2013;Wu et al. 2014;Hong et al., 2017;Himed et al., 2019;Kakoos, 2019). The major component of clove essential oil is usually considered eugenol (34.10-88.58%) ...
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Essential oils are natural products composed of a mixture of volatile and aromatic compounds extracted from different organs of plants that have been widely studied for their antibacterial activities against pathogens. In this study, clove, lemon peel and thyme essential oils and their mixture were assessed for their antimicrobial activities using a panel of pathogenic Gram-positive, and Gram-negative, strains. Cytotoxicity and anti-inflammatory activity were also evaluated. Lemon peel essential oil was characterized by the predominance of limonene. Eugenol was the main component in clove essential oil and thymol in thyme essential oil. Clove, lemon peel and thyme essential oils and their combination had potent antibacterial, cytotoxic and anti-inflammatory activities. This study demonstrates that the use of essentialvoils is an effective alternative for pathogenic bacterial control, alone or in combination with antibiotic therapy.
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... Synergistic effects against pathogens from the complex mixture of chemically different components of the extract may be the explanation for the result obtained 26 . The composition of Citrus auranitifolia has been attributed to properties like free radical scavenging, anti-helminthic, antimicrobial, anti-inflammatory and analgesic activity 27 . The antimicrobial activity of the essential oil-based hand sanitizer was found highest against S. aureus while lowest activity was found against Escherichia coli which are facultative anaerobic, Gram-negative bacteria. ...
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We studied the fumigant toxicity and repellent properties of essential oil of Citrus limonum (Sapindales: Rutaceae L.) against Sitophilus granarius (L). adults. The chemical composition of this essential oil by gas chromatography-mass spectrometry (GC-MS) showed that it contains 50 compounds and the major compounds were: Limonene (19.81 %), α-Citral (17.16 %) and Neryl acetate (16.23 %). The essential oil showed fumigant toxicity, with dose-response relationship and repellent activity against S.granarius.
... c Essential oil composition not reported; essential oil composition of commercial (dōTERRA International, Pleasant Grove, Utah, USA). d Essential oil composition not reported; essential oil composition obtained from [82]. e Essential oil composition not reported; essential oil composition obtained from [83]. ...
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