Research article

Received: 5 October 2015, Revised: 14 January 2016, Accepted: 22 January 2016 Published online in Wiley Online Library: 25 February 2016

(wileyonlinelibrary.com) DOI 10.1002/ffj.3315

Chemical composition and the

anti-melanogenic potential of different
essential oils
Daniela Fiocco,a Marcella Arciuli,b Mattia Pia Arena,a,c Stefania Benvenutid
and Anna Galloneb*
Abstract: Tyrosinase is the key enzyme of melanogenesis, the process that determines skin and hair pigmentation in mammals.
In humans, various pigmentary disorders depend on excessive melanin production and accumulation. Moreover, tyrosinase
activity can cause the undesirable browning of plant-derived food. For such reasons, ongoing researches aim at developing
effective melanogenesis inhibitors for medical, cosmetic and food applications. Diverse biological activities have been observed
for certain plant essential oils (EOs) and their components. In this study, the EOs from nine different plant species were investi-
gated for their anti-melanogenic potential. The EOs chemical composition was assessed by gas chromatography-mass spectrom-
etry (GC-MS) revealing the presence of some constituents with known tyrosinase inhibitory ability. Most EOs inhibited
significantly and dose-dependently mushroom tyrosinase. Two samples also inhibited the activity of tyrosinase from murine
B16 melanoma cells. Our findings indicate that some of the analysed EOs may be promising natural sources for developing novel
anti-melanogenic agents, thus having a good potential in medical, cosmetic and food-processing industries. Copyright © 2016
John Wiley & Sons, Ltd.

Keywords: essential oil; tyrosinase; melanogenesis; tyrosinase inhibitors

Introduction medicinal, cosmetic, cleaning and food applications.[6] Compared

with drugs of a synthetic origin, natural products are generally less
Tyrosinase (monophenol, o-diphenol:oxygen oxidoreductase, EC toxic, more eco-friendly and better accepted by consumers, hence,
1.14.18.1) is a widespread, multifunctional, copper-enzyme, which they are good resources for developing anti-melanogenic agents.
catalyses the hydroxylation of monophenols to o-diphenols Indeed, some of the currently used depigmenting agents, includ-
(monophenolase or cresolase activity) and the oxidation of ing gallic acid, aloesin, arbutin and kojic acid, are compounds of
o-diphenols into o-quinones (diphenolase or catechol oxidase a natural origin.[7] Such substances can prevent the synthesis of
activity).[1] In animals, plants and lower organisms, tyrosinase melanin (i.e. exert anti-melanogenic effects) mainly by acting as
is involved in the biosynthesis of melanin pigments. As it is tyrosinase inhibitors. Indeed, as tyrosinase catalyses the key steps
responsible for the first and rate-limiting steps of melanogenesis of melanin biosynthesis, most of the strategies for developing
[i.e. the hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine antimelanogenic agents are based on the inhibition of such an
(L-DOPA) and the subsequent oxidation of L-DOPA to DOPA- enzyme.[4] Because concerns related to efficacy, toxicity, stability
quinone] (Figure 1), tyrosinase is the chief enzyme that underlies and adverse side effects do exist for some of the currently
the process of human skin pigmentation.[1] Moreover, tyrosinase commercialized tyrosinase inhibitors, the screening of substances
activity accounts for the enzymatic browning of plant tissues, which that may be developed into novel, more effective and safer
may impair the sensory and nutritive properties of plant-derived hypopigmenting agents continues to catalyse research efforts.[5,7]
food products.[2]
Over the past years, the search for safe and effective melano-
genesis inhibitors has gained attention for applications both in
* Correspondence to: Professor Anna Gallone, Department of Basic Medical
food quality management and in health issues related to the Sciences, Neuroscience and Sense Organs, University of Bari Aldo Moro,
clinical treatment and/or prevention of pigmentary disorders in Policlinico - Piazza G. Cesare, 70124, Bari, Italy. E-mail: [email protected]
humans.[3,4] Furthermore, as a lighter complexion is a desirable
a
aesthetic trait, especially in the Eastern culture, dermatological sci- Department of Clinical and Experimental Medicine, University of Foggia,
Foggia, Italy
ence has been interested in developing skin-lightening agents.[5]
Modern industries are constantly looking for alternative, more b
Department of Basic Medical Sciences, Neuroscience and Sense Organs,
natural drugs to be used as food preservatives, antimicrobial, University of Bari, Bari, Italy
antioxidant, pharmacological and crop protection agents. Plant
c
extracts represent a precious and yet partially unexplored source Department of Agriculture, Food and Environmental Science, University of
Foggia, Foggia, Italy
of such compounds. Since ancient times, herbal preparations have
been playing a relevant role in assuring human health and improv- d
Department of Life Sciences, University of Modena and Reggio Emilia,
255

ing the quality of life, by providing valuable components for Modena, Italy

Flavour Fragr. J. 2016, 31, 255–261 Copyright © 2016 John Wiley & Sons, Ltd.
 D. Fiocco et al.

Figure 1. Scheme of the first two reactions catalysed by tyrosinase during melanogenesis.

Essential oils (EOs), obtained from plants by steam distillation, Determination of EOs composition by gas
hydrodistillation or solvent extraction, are complex mixtures of chromatography-mass spectrometry (GC-MS) and gas
low-molecular-weight chemicals. EOs and/or their single constitu- chromatography-flame ionization detector (GC-FID)
ents have been ascribed various biomedical properties, such as
EOs were diluted 1:3 (v/v) with GC grade n-hexane and GC-MS
antimicrobial, anti-inflammatory and cancer-preventive abilities.[8]
analyses were carried out on a 6890 N gas chromatograph
Moreover, some recent papers have dealt with the effects of plant
(Agilent Technologies, Waldbronn, Germany), coupled to a 5973
EOs on tyrosinase and melanogenesis.[9–16] In a previous work, we
Network mass spectrometer (Agilent Technologies), as previously
observed that the hydrodistilled EOs from lavender and mint could
described.[17] Compounds were identified by comparing the reten-
dose-dependently inhibit mushroom tyrosinase.[17]
tion times with those of authentic samples, comparing their
In this paper, we investigated in vitro the anti-melanogenic
retention indices relative to the series of n-hydrocarbons and on
potential of EOs from nine different plants. For this purpose, we
computer matching against commercial mass spectral libraries NIST
studied the effect of EOs on the activity of mushroom and murine
98 and ADAMS,[18] as well as a homemade library, built up from pure
B16 melanoma cell tyrosinases. Our findings suggest that provided
substances or known oils.
there are further studies, some of the investigated EOs might
Semi-quantitative analysis was performed on a DANI 86-10 GC
provide helpful ingredients for food, cosmetic and clinical
with a flame ionization detector (FID) (DANI Instruments, Cologno
applications.
Monzese, Milan, Italy). Analyses were carried out on an HP-5 cross-
linked 5% phenyl – 95% methylpolysiloxane (25 m x 0.2 mm I.D.,
Materials and methods film thickness 0.5 μm) capillary (Agilent Technologies), as previ-
ously described.[17] The relative amounts of individual components
Reagents were expressed as % peak areas relative to the total peak area.
EOs, obtained by steam distillation from plant species of a different
origin (Table 1), were purchased from Alchima Natura (Modena,
Mushroom tyrosinase assay
Italy). EOs were stored at 4 °C, in airtight vials, shielded from light.
Immediately before the enzymatic assays, EOs were serially diluted The DOPA oxidase (i.e. diphenolase) activity of mushroom
in dimethyl sulfoxide (DMSO) 60% (v/v). tyrosinase was spectrophotometrically probed using L-DOPA as a
Kojic acid, 3,4-dihydroxy-L-phenylalanine (L-DOPA), 3-methyl-2- substrate and recording the increase in absorbance at 475 nm,
benzothiazolinonehydrazone hydrochloride (MBTH), Igepal Ca-630, associated with the formation of dopachrome (ε = 3700/M/cm).[19]
phenylmethylsulfonyl fluoride (PMSF), lyophilized mushroom The assay was conducted as previously described.[17] Dopachrome
tyrosinase from Agaricus bisporus (EC.1.14.18.1; 4187 U/mg) and formation was monitored by measuring the linear increase in
solvents were purchased from Sigma-Aldrich (St. Louis, MO, optical density at 475 nm (A475nm), in an EON Microplate
USA). All other reagents were of a high chemical grade. Aqueous Spectrophotometer (Biotek, VT, USA). The effects of test
solutions were prepared with water obtained by a Milli-Q Gradi- samples on tyrosinase activity were represented as %
ent A-10 system (18.2 MΩ/cm, organic carbon content ≤ 4 g/l; inhibition = (1-B/A) x 100, where A = ΔA475nm/min of the reaction
Merck-Millipore, Darmstadt, Germany). Mushroom tyrosinase mixture containing DMSO at 3% (v/v) the final concentration (con-
and L-DOPA were dissolved in 20 mM sodium-phosphate buffer trol reaction) and B = ΔA475nm/min of the reaction in the presence
(Na-phosphate buffer), pH 6.8. of the test sample. Concentrations of test samples that inhibited

Table 1. List of the essential oils analysed in the present study

Sample number Plant source: Latin name, common name, parts used for extraction Origin Country
1 Citrus x aurantium L., bitter orange, fruit peel Italy
2 Salvia officinalis L., sage, whole flowering plant Italy
3 Origanum vulgare L., oregano, whole flowering plant Italy
4 Cupressus sempervirens L., mediterranean cypress, buds France
5 Salvia sclarea L., clary sage, flowers and herbs Italy
6 Thymus vulgaris L., thyme, whole plant Italy
7 Cinnamomum zeylanicum Blume, cinnamon, leaves Ceylon
8 Rosmarinus officinalis L., rosemary, leaves Italy
9 Syzygium aromaticum L., clove, flowers Madagascar
256

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Essential oils inhibit mushroom and melanoma cell tyrosinases

50% the tyrosinase activity (IC50) were obtained by non-linear re- differences, as well as corresponding findings arise, depending
gression analysis of the inhibition plots (Origin software, OriginLab, on the sample. For instance, myrcene and citral could not be de-
Northampton, MA, USA). tected as components of C. aurantium EO (sample 1), yet they
were previously detected as main volatile constituents of EOs from
several Citrus species, and also demonstrated to account for the
Cell culture tyrosinase inhibitory activity exhibited by such EOs.[9] In contrast,
Murine B16 melanoma cells were cultured in DMEM (Gibco, the predominance of limonene in sample 1 is in accordance with
Carlsbad, CA, USA) supplemented with 100 U/ml penicillin, other previous analyses on EOs from citrus peel.[26] The chemical
100 μg/ml streptomycin and 10% (v/v) fetal bovine serum, at composition of the volatile oil obtained by supercritical CO2 fluid
37 °C in a humidified atmosphere with 5% CO2. Cells were sub- extraction from Cinnamomum zeylanicum bark, in a previous
cultured every 3–4 days to maintain the exponential growth. study, also differs from our findings, as it was mostly characterized
by (E)-cinnamaldehyde (77.1%) and β-caryophyllene (6.0%), with
lower percentages of eugenol (3.0 %).[27] In contrast, eugenol
Preparation of B16 cell crude extract and assay on cellular resulted in the chief component of the C. zeylanicum EO
tyrosinase analysed in the present study (EO 7), which also contained lower
Post-confluent B16 cells were solubilized in a lysis buffer (1% Igepal caryophyllene amounts (2.4%) and no (or undetectable levels of)
Ca-630, 0.1 mM PMSF, in PBS) and lysates were clarified by centri- cinnamaldehyde. Interestingly, both (E)-cinnamaldehyde and
fugation, according to Zanna et al.[20] The protein concentration eugenol were previously found to be mainly responsible for the
was determined using the Bio-Rad protein assay Kit (Bio-Rad, anti-tyrosinase effect exhibited by Cinnamomum extracts.[11,27]
Hercules, CA, USA).The DOPA oxidase activity of B16 tyrosinase The chemistry of C. sempervirens EO (sample 8) is in good accor-
was assayed using the Winder and Harris spectrophotometric dance with recent studies.[28] Even the chemical composition of
method,[21] with minor modifications. Briefly, a solution of clove EO (S. aromaticum, sample 9) agrees with previous works
0.1 mM Na-phosphate buffer (pH 6.8) containing 4 mM MBTH, indicating eugenol, β-caryophyllene and eugenyl acetate as the
600 μM of L-DOPA and 5 μl of the test sample was incubated at main constituents of the EOs obtained by hydrodistillation from
37 °C for 10 min in a 96-well plate. Subsequently, 20 μg of protein buds and leaves.[29,30] Interestingly, eugenol and eugenyl acetate
extract was added and the production of the dopaquinone-MBTH isolated from clove extracts were shown to inhibit melanin produc-
adduct was immediately monitored by measuring the increase in tion in B16 melanoma cells.[31]
optical density at 505 nm (A505nm). Control reactions were per- Overall, our findings confirm the great variability which usually
formed in the presence of the EO solvent alone, DMSO 1.5% (v/v) characterizes the EOs composition, as observed earlier by other
final concentration, or 4 mM Kojic acid. The effects of the EOs on authors.[32] Plant EOs are complex mixtures of several lipophilic
B16 extracts tyrosinase activity were represented as % inhibi- compounds. Monoterpenes, sesquiterpines and phenolics are
tion = (1-B/A) x 100, where A = ΔA505nm min-1 of the reaction often the predominating constituents. However, the chemistry of
mixture containing DMSO at 1.5% of (v/v) final concentration EOs, along with their biological properties, can considerably
(control reaction), and B = ΔA505nm min-1 of the reaction in the change, as it may be greatly affected by several factors, including
presence of a test sample. the geographical and climate differences, seasonal changes,
agronomic practices, soil type, as well as different extraction
procedures.[8,33]
Results and discussion
Composition of the essential oils (EOs) Inhibitory effect of the EOs on mushroom tyrosinase activity
In the present work, we sought to study nine EOs from different Nine different essential oils of a plant origin were tested on mush-
plants, with a diverse geographical distribution and comprising room tyrosinase. As shown in Figure 2, the tyrosinase DOPA oxidase
mostly species which have been traditionally and widely used in activity was inhibited when the different test samples were added
food preparation for their flavouring properties (Table 1). The to the reaction mixtures. Conversely, the oil solvent alone (DMSO
GC-MS and GC-FID analyses resulted in the identification and 3%) poorly affected the enzymatic activity. It was apparent that,
quantification of the EO constituents, as reported in Table 2. Cyclic although used at the same concentration (218 μg/ml), the various
monoterpenes, terpenoids, sesquiterpenes and monophenols EOs inhibited the enzymatic activity to a different extent, suggest-
were among the main components identified. EOs from Salvia ing a diverse anti-tyrosinase potential, specific for each oil and
officinalis (sample 2) and from Cupressus sempervirens (sample 4) possibly reflecting their characteristic composition, the presence
exhibited the most diversified composition, including up to 12 of active specific constituent and/or the synergistic effects.
different constituents identified and quantified by the applied A clearer differentiation of the inhibitory effect of the EOs
method. In contrast, only two main constituents could be became more evident when they were used in the enzymatic
detected and quantified in the EOs from Citrus aurantium (EO 1) assays at different concentrations and the initial rate of the
and Syzygium aromaticum (EO 9). dopachrome formation was evaluated (kinetic analysis) and
As indicated in Table 2, tyrosinase inhibition and/or anti- compared with that of the control reaction (Figure 3). All the EOs
melanogenic effects were previously reported for some of exhibited a dose-dependent inhibitory effect on mushroom
the observed EO constituents, including linalyl acetate,[14] tyrosinase. EO 1 resulted in the best inhibiting sample, reaching
bornyl acetate,[13] thymol,[22] carvacrol,[23] eugenol[24] and a maximal inhibitory effect of 96% (at 870 μg/ml, i.e. the highest
β-caryophyllene.[25] concentration at which EOs could be tested, as a result of solubility
By comparing the observed composition with analogous data limit) and with an estimated IC50 of 156 μg/ml on tyrosinase
available in the scientific literature and obtained for EOs activity. Even EOs 8, 7, 3 and 9 performed well, exerting maximal
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extracted from the same and/or related species, some inhibitory effects of 78%, 73%, 62% and 66%, with an estimated

Flavour Fragr. J. 2016, 31, 255–261 Copyright © 2016 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/ffj
 258

Table 2. Relative percentage of the main constituents of essential oils detected by gas chromatography (GC) analysis. Components with known anti-melanogenic properties are in bold
and references are reported

LRI(a) (1) Citrus (2) Salvia (3) Origanum (4) Cupressus (5) Salvia (6) Thymus (7) Cinnamomum (8) Rosmarinus (9) Syzygium

wileyonlinelibrary.com/journal/ffj
aurantium officinalis vulgare sempervirens sclarea vulgaris zeylanicum officinalis aromaticum
α-Pinene 931 2.8 1.9 45.1 1.2 28.5
Camphene 946 2.2 0.4 0,3 7.2
β-Pinene 975 1.2 2.3 0.7 8.1 3.8
δ3-Carene 1007 15.6
p-Cymene 1024 4.4 7.2
Limonene 1027 74.1 3.8 2.4
Eucalyptol 1029 7.3 5.9 7.2 25.8
γ-Terpinene 1058 2.4 0.9 8.0
Terpinolene 1087 2.8
Linalool 1101 18.4 1.9 1.8
α-Thujone 1104 31.9
β-Thujone 1116 7.0
Camphor 1143 9.5 0.2 0.4 5.6
Borneol 1165 2.7 0.7 1.8
Terpinen-4-ol 1177 2.0
α-Terpineol 1191 1.0
Linalyl acetate[14] 1263 45.1

Copyright © 2016 John Wiley & Sons, Ltd.

Bornyl acetate[13] 1290 1.8 0.8 1.1
Thymol[22,24] 1295 6.6 42.9
Carvacrol[23,24] 1306 61.0 6.5
α-Terpinyl acetate 1347 4.0
Eugenol[24,27,31] 1356 70.0 61.0
β-Caryophyllene[25] 1424 5.3 3.2 1.2 4.1 2.4 1.5 5.7
β-Cedrene 1430 1.3
α-Humulene 1459 8.4 1.2
Caryophyllene- 1592 3.4
oxide
a
LRI: linear retention indices calculated on HP-5 column.

Flavour Fragr. J. 2016, 31, 255–261

D. Fiocco et al.
Essential oils inhibit mushroom and melanoma cell tyrosinases

of cinnamaldehyde. Therefore, its capacity to inhibit mushroom

tyrosinase is probably ascribable to the predominant presence of
eugenol (70% of the extract), an ortho-substituted monophenol
with well-known anti-tyrosinase activity.[24] Likewise, eugenol,
which is also the main constituent of S. aromaticum EO (sample
9) could account for the good tyrosinase inhibitory effect observed
for such extract, confirming previous studies on the anti-
melanogenic properties of methanol extracts from clove buds.[31]
Interestingly, both EOs 7 and 9, which exhibited a good anti-
tyrosinase capacity, share a very similar chemical composition,
with eugenol and β-caryophyllene as the predominant constitu-
ents. In this regard, it is worth mentioning that β-caryophyllene
from lime mint essential oil was found to inhibit melanogenesis,
but not directly tyrosinase.[26]
EO from Origanum vulgare (sample 3), which was also a good
tyrosinase inhibitor, was particularly rich in carvacrol (61%) and
thymol (6.6%), two other ortho-substituted monophenols, with
documented anti-tyrosinase activity.[22,24] A significant tyrosinase
inhibitory activity was recently reported for the water-distilled EO
from an O. vulgare subspecies, which was similarly rich in thymol
and carvacrol (58% and 16%, respectively).[35]
Intriguingly, the predominance of known tyrosinase inhibitors
on the EO composition does not strictly relate to the observed
activity of the EOs. Indeed, although sample 6 (EO from Thymus
vulgaris) was rich in thymol (42.9%) and presented significant
amounts of carvacrol (6.5%), both chemicals being well-known
tyrosinase inhibitors,[24] such an extract resulted in the weakest
mushroom tyrosinase inhibitor among all tested EOs. This might
Figure 2. Time course of dopachrome formation catalysed by mushroom
depend on interactions between components of the EO. In fact,
tyrosinase in presence of the tested essential oils (EOs). Dopachrome forma-
tion was monitored by recording the increase in absorbance at 475 nm although the biological properties of a particular EO are usually
(A475nm) over a 15-min period. The DOPA oxidase activity of mushroom ty- determined by either one or two of its main components,
rosinase was evaluated in Na-phosphate buffer (tyr, open circle), in the pres- sometimes its overall bioactivity can be greatly influenced by the
ence of the oil solvent (3% [v/v] DMSO, grey diamond), in the presence of combined action of different molecules.[8]
1 mM kojic acid (KA, solid triangle) (reported as a positive inhibition control The major compound of Salvia sclarea EO (sample 5) was linalyl
in both A and B) and in the presence of the different EO samples (1–4 in acetate (45.1%), which was recently demonstrated to account for
panel A; 5–9 in B) at 218 μg/ml concentration. Values are mean of at least the anti-melanogenic action of Achillea millefolium EO, by down-
two independent experiments performed in triplicate. regulating tyrosinase activity in α-MSH-stimulated B16 cells.[14]
However, in our study, EO 5 resulted in a weak mushroom tyrosi-
nase inhibitor, thus suggesting that linalyl acetate may not directly
IC50 of 177, 257, 375 and 403 μg/ml, respectively. Conversely, EOs inhibit the enzymatic action and that its anti-melanogenic effect
2, 4 and 5 exhibited a lower tyrosinase inhibitory action, reaching depended mainly on its ability to interfere with the signalling path-
a maximal inhibition of 45%, 28% and 31%, respectively, and IC50 ways that trigger tyrosinase gene expression, as also suggested by
could not be determined in the range of the tested concentrations. Peng et al.[14]
Sample 6 was the poorest inhibiting EO (only 6% inhibition at the To our knowledge, no specific record exists on the anti-
maximal concentration used). melanogenic action of EO from Rosmarinus officinalis (sample 8),
The very good tyrosinase inhibition exhibited by EO 1 (from C. which resulted in a very good mushroom tyrosinase inhibitor in
aurantium) is in accordance with previous reports, which analysed the present study. In fact, anti-tyrosinase properties do not seem
the composition and anti-tyrosinase activity of EOs from different to be widespread in extracts from plants of the Lamiaceae
Citrus species, including a variety of C. aurantium.[9] As we could family,[36] whose R. officinalis is a member. Interestingly, EO 8 was
not detect those chemical constituents (i.e. myrcene and citral) particularly rich in eucalyptol (25.8 %), also known as 1,3 cineol,
to which the inhibitory effect had been previously ascribed, it is which was previously found to be the major ether component of
plausible that the anti-tyrosinase activity of our sample might Eucalyptus camaldulensis flowers EO, which exhibited anti-
depend on some other constituents. In this regard, it is worth tyrosinase and anti-melanogenic effects.[16] Lamiaceae also
mentioning that a recent study found a significant anti-tyrosinase include Salvia and Thymus species, and, indeed, based on our data,
activity in citrus EOs which contained very low levels of myrcene the EOs from S. officinalis (sample 2), S. sclarea (sample 5) and T.
and citral, but was extremely rich in limonene,[34] as was C. vulgaris (sample 6) exerted a poor anti-tyrosinase action compared
aurantium EO 1. with the other tested EOs. Accordingly, Dadashpour et al.[37] found
Even results obtained with EO 7, from C. zeylanicum, agree with that Thymus daenensis EO inhibited mushroom tyrosinase much
the well-documented tyrosinase inhibitory activity of both whole less than that extracted from Anethum. Interestingly, the ethanol
extracts and single constituents from cinnamon species.[11,12,27] extracts from other Salvia species have been reported to inhibit
Based on our data on its chemical composition, and in contrast mushroom tyrosinase[38] and, recently, a combination of Salvia
259

to previous works,[27] C. zeylanicum EO lacks substantial amounts hispanica seed and pomegranate fruit extracts were found to

Flavour Fragr. J. 2016, 31, 255–261 Copyright © 2016 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/ffj
 D. Fiocco et al.

Figure 3. Dose-dependent inhibitory effects exerted by the essential oils (EOs) on mushroom tyrosinase activity. The DOPA oxidase activity was evaluated
at different concentrations of the EOs, ranging from 36 to 870 μg/ml. (A) solid diamonds, EO 1; grey squares, EO 2; open triangles, EO 3; (B) crosses, EO 4; open
squares, EO 6; grey triangles, EO 7; (C) open diamond, EO 5; solid squares, EO 8; solid circles, EO 9. (D) Overview of the inhibition exerted by the different EOs
used at the concentration of 435 μg/ml, grey bars, and 218 μg/ml, white bars. The effect on tyrosinase is shown as a function of the EO concentration and is
represented as a percentage of inhibition. The inhibition percentage was calculated relative to the reaction mix containing tyrosinase and substrate in the
presence of the oil solvent only (3% DMSO), control reaction. Values are average (± SE) of at least two independent experiments run in triplicate. Theoretical
fitting curves are also shown. Significantly different from the control value (Student’s t-test): P < 0.05 (*); P < 0.01 (**).

reduce melanin synthesis through the downregulation of shown). EO 7 (C. zeylanicum) inhibited B16 tyrosinase by 37% and
melanogenesis-related genes.[39] 10% when used at 435 and 218 μg/mL, respectively. Likewise, EO
9 (from S. aromaticum) could inhibit the reaction by 25% and 12%.
EOs effects on tyrosinase activity in B16 cell extracts
Cinnamomum zeylanicum and S. aromaticum EOs (samples 7
When assayed in crude extracts from B16 cells, only EOs 7 and 9 and 9, respectively), which were good mushroom tyrosinase inhib-
exerted an appreciable and dose-dependent inhibitory action on itors (see above), also significantly inhibited B16 tyrosinase. Our
the DOPA-oxidase activity of tyrosinase (Figure 4), whereas the other finding partially confirms a recent paper reporting that the
tested EOs gave either no inhibition or inconsistent results (data not steam-distilled EO from a related Cinnamomum species (i.e. C.
cassia) possessed both mushroom tyrosinase inhibitory properties
and antimelanogenic activities on B16 cells.[12] Moreover, the
methanol extracts from clove (S. aromaticum) were formerly found
to reduce melanin formation in B16 melanoma cells.[31] Interest-
ingly, as highlighted above, both EO 7 and 9 contain high levels
of eugenol, which was demonstrated to inhibit tyrosinase by
acting as an alternative substrate to L-tyrosine and L-DOPA in
the monophenolase and diphenolase activities, respectively.[24]
Therefore, based on earlier and present data, eugenol-containing
extracts have a high potential as effective ingredients of both
anti-food-browning and skin-whitening drugs.
Much fewer EOs were able to inhibit tyrosinase-catalysed reaction
in crude cell extracts compared with the effect observed in mush-
room tyrosinase assays (see Figures 3D and 4). Moreover, the levels
Figure 4. Inhibitory effects of selected essential oils (EOs) on tyrosinase ac- of inhibition observed for EOs 7 and 9 were lower on cell extracts
tivity in B16 cell crude extracts. The DOPA oxidase activity was evaluated
rather than on purified mushroom tyrosinase. Such a difference
using EOs 7 (from C. zeylanicum) and 9 (from S. aromaticum) at 435 μg/ml,
might partly reflect the different spectrophotometric methods used
grey bars, and 218 μg/ml, white bars. The percentage of inhibition was
calculated relative to the reaction mix containing B16 cell extracts and to assay the DOPA oxidase activity of mushroom and B16 tyrosi-
substrate in presence of the oil solvent only (1.5% DMSO), control reaction. nases. In this study, the MBTH-based method was preferred to assay
Values are the average (± SD) of at least two independent experiments run the mammalian enzyme for its higher sensitivity[21] and because,
in triplicate. Significantly different from the control value (Student’s t-test): under our experimental conditions, it was more effective at detect-
260

P < 0.05 (*); P < 0.01 (**). ing the tyrosinase activity of cell crude extracts. However, the use of

wileyonlinelibrary.com/journal/ffj Copyright © 2016 John Wiley & Sons, Ltd. Flavour Fragr. J. 2016, 31, 255–261
Essential oils inhibit mushroom and melanoma cell tyrosinases

two different enzymatic assays may not facilitate the direct compar- 2. M. V. Martinez, J. R. Whitaker. Trends Food Sci. Technol. 1995, 6, 195.
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4. F. Solano, S. Briganti, M. Picardo, G. Ghanem. Pigment Cell Res. 2006,
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19, 550.
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