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*Correspondence: N. R. B. Raposo. Universidade Federal de Juiz de
Fora, Campus Universitário. Rua José Lourenço Kelmer, s/n, São Pedro -
36036-900 - Juiz de Fora - MG, Brazil. Tel/fax: 55 32 3229 3809. E-mail:
nadiafox@gmail.com
Article
Brazilian Journal of
Pharmaceutical Sciences
vol. 48, n. 3, jul./sep., 2012
Mushroom tyrosinase inhibitory activity and major fatty acid
constituents of Amazonian native flora oils
Raquel da Silva Teixeira1, Paula Rafaela Rocha1, Hudson Caetano Polonini1,2, Marcos Antônio
Fernandes Brandão1,2, Maria das Graças Afonso Miranda Chaves2, Nádia Rezende Barbosa Raposo1, 2,*
1NIQUA, Federal University of Juiz de Fora, 2NUPICS, Federal University of Juiz de Fora
In order to treat hyperpigmentation-related problems, there has been a global trend in developing cosmetics
claiming to have skin-whitening properties, which act by inhibiting melanin biosynthesis. The objective
of this work was to evaluate the in vitro mushroom tyrosinase inhibitory activity of ve Amazonian
native ora oils, and so to verify the possibility of their incorporation into cosmetic products. In addition,
the fatty acid composition of the essential oils was determined by gas chromatography-ame ionisation
detection in order to determine the main components of these oils. The tyrosinase inhibitory activity of
the tested oils was found to be in the following order: açaí (IA50 = 66.08 µg mL-1) > tucumã > patauá
> pracaxi > castanha do Brasil. This study suggests that açaí oil has great potential in the treatment of
hyperpigmentation and other related disorders, due to its considerable tyrosinase inhibitory activity.
Uniterms: Amazonian plants/evaluation/dermatological use. Skin lightening agents. Tyrosinase/
inhibition/in vitro evaluation. Melanin. Hyperpigmentation/treatment.
Com o intuito de se tratar problemas dermatológicos de hiperpigmentação, há uma tendência mundial no
desenvolvimento de cosméticos que possuam propriedades despigmentantes, os quais agem inibindo a
biossíntese de melanina. O objetivo deste trabalho foi avaliar in vitro a atividade de inibição da tirosinase
de cogumelo de cinco óleos de plantas nativas da Amazônia e, desta forma, vericar a possibilidade de sua
incorporação em produtos cosméticos. Ainda, a composição de ácidos graxos dos óleos foi determinada
por cromatograa gasosa com detecção por ionização de chama, no intuito de determinar os principais
componentes destes óleos. A atividade de inibição da tirosinase dos óleos testados foi encontrada na
seguinte ordem: açaí (IA50 = 66,08 µg mL-1) > tucumã > patauá > pracaxi > castanha do Brasil. Este
estudo sugere que o óleo de açaí possui grande potencial para o tratamento da hiperpigmentação cutânea
e doenças correlatas, devido à sua considerável atividade de inibição da tirosinase.
Unitermos: Plantas amazônicas/avaliação/uso dermatológico. Despigmentantes. Tirosinase/inibição/
avaliação in vitro. Melanina. Hiperpigmentação cutânea/tratamento.
INTRODUCTION
The major determinant of the colour of mammalian
skin and hair is the content and distribution in keratino-
cytes of the melanins, coloured polymorphous biopoly-
mers produced from complex and multi-step oxidative
reactions involving the amino acid L-tyrosine (Adhikari
et al., 2008; Lin et al., 2011). This pigment acts primarily
by defending animals from ultraviolet (UV) radiation.
However, its abnormal production is related to an increase
in the number of melanocytes and/or increased activity
of melanogenic enzymes. This can result in considerable
aesthetic problems due to hyperpigmentation, namely
freckles, melasma, liver spots, age spots and others (Ad-
hikari et al., 2008).
In order to treat hyperpigmentation-related prob-
lems, there has been a global trend in developing cosmetics
claiming to have skin-whitening properties, which act by
inhibiting melanin biosynthesis (Lin et al., 2011). This can
be achieved by protecting the skin from solar light expo-
sure, removing melanin by corneal ablation, blocking or
diminishing the proliferation of melanocytes or inhibiting
R. S. Teixeira, P. R. Rocha, H. C. Polonini, M. A. F. Brandão, M. G. A. M. Chaves, N. R. B. Raposo
400
tyrosinase activity, since melanin is derived from tyrosine
and its biosynthesis is to a large extent regulated by this
copper-containing enzyme (Wang et al., 2006; Ha et al.,
2005).
Current research is focused on both synthetic and
natural sources for reducing melanin formation (Arung
et al., 2011; Park et al.., 2011). With respect to natural
substances, the Amazon biome is a major source of new
species and therefore molecules that may have pharma-
cological activity, skin-whitening included. In this light,
the objective of this work was to evaluate the mushroom
tyrosinase inhibitory activity of ve Amazonian native
ora oils, and so to verify the possibility of their incorpo-
ration into cosmetic products. In addition, the fatty acid
composition of the essential oils was determined by gas
chromatography in order to determine the main compo-
nents of these oils.
MATERIAL AND METHODS
Plant material
Vegetable oils of Astrocaryum vulgare Mart. (Are-
caceae, popular name tucumã), Bertholletia excelsa Bonpl.
(Lecythidaceae, popular name castanha do Brasil). Euter-
pe oleraceae M. (Areaceae, popular name açaí) Oenocar-
pus bataua Mart. (Arecaceae, popular name patauá) and
Pentaclethra macroloba (Willd.) Kuntze (Mimosaceae,
popular name pracaxi) were acquired from the Amazonian
oil industry (Pará, Brazil).
Tyrosinase inhibition: qualitative enzymatic
reaction screening
The assay was performed according to the protocol
described by Macrini et al. (2009), with some modifi-
cations. Aliquots of 10 µL of a solution composed of
125 U mL-1 of mushroom tyrosinase (Sigma-Aldrich,
USA) were added to 96-well microplates, and then 70 µL
of pH 6.8 phosphate buffer solution and 60 µL of the
Amazonian native ora oils (350 µg mL-1, in 25% dimethyl
sulphoxide, DMSO) were also added. For the positive
control, 60 µL of kojic acid (17.5 µg mL-1 in 25% DMSO)
was used instead of the Amazonian oil, and for the negative
control, 60 µL of 2.5% DMSO was added. To the resultant
mixture, 70 µL of L-tyrosine (Sigma-Aldrich, USA) were
added at a concentration of 0.3 mg mL-1 in distilled water
(the nal volume in the wells was 210 µL).
The absorbance of the microplate wells was read
using a microplate spectrophotometer (SpectraCount,
Packard, USA) at 510 nm (T0). Then, the microplates were
incubated at 30 ± 1ºC for 60 min and the absorbance was
measured again (T1). An additional incubation period of
60 min at 30 ± 1ºC was done and, after this period, a new
spectrophotometric reading was taken (T2).
The inhibitory percentage at the two time points (T1
and T2) was obtained according to the formula:
where IA% = inhibitory activity; C = negative control ab-
sorbance; S = sample or positive control absorbance (ab-
sorbance at time T1 or T2 minus the absorbance at time T0).
Tyrosinase inhibition: quantitative enzymatic
reaction assay
For the samples that reached an IA% greater than
35%, a quantitative assay was conducted. For this pur-
pose, the above experimental protocol was followed,
with modications (a 500 U mL-1 tyrosinase solution was
used instead of 125 U mL-1 and the optical densities were
measured every 10 min for 1 h).
The quantitative determination was obtained
through an analytical curve and the respective equation
of the line. For this, the oils were diluted in the microplate
wells to concentrations of 100, 80, 40, 20 and 10 µg mL-1
with 25% DMSO, and the kojic acid was diluted to con-
centrations of 10, 5, 2.5, 1.25 and 0.625 µg mL-1. The
analytical curve was plotted between tyrosinase inhibition
activity percentages at each time point and the concentra-
tions of the oils/ positive control. Using the equation of
the line, the inhibitory activity at 50% (IA50, in µg mL-1)
was calculated.
Fatty acids assay
For essential oil lipid hydrolysis, 10 mg of the oils
were dissolved in 100 µL of a mixture of ethanol and
1 mol L-1 potassium hydroxide (95:5, v/v) in 2 mL cryo-
genic tubes. After 10 s of vigorous mechanical agitation,
the oils were hydrolysed in a microwave (Panasonic, Ja-
pan), using 80 W of power for 5 min; they were then cooled
and the following reagents were added to the tubes, in this
order: 400 µL of 20% hydrochloric acid, one spatula tip of
sodium chloride and 600 µL of ethyl acetate. Sample were
agitated for a further 10 s, followed by a 5 min rest period.
A 300 µL aliquot from the organic layer was transferred
to a microcentrifuge tube and dried in an oven in order to
obtain the free fatty acids. The residues were methylated
with 14% boron triuoride in methanol and heated in a
Mushroom tyrosinase inhibitory activity and major fatty acid constituents of Amazonian native ora oils 401
water bath for 10 min at 80 ºC. The resulting solution was
injected into an HP5890 gas chromatograph (HP, USA)
with a flame ionisation detector. The instrument was
equipped with a 15 m x 0.25 mm HP-INNOWax column,
1 µL injection (1/50 split) with hydrogen as the carrier
gas (2 mL min-1). The temperature of both the detector
and the injector was maintained at 250 ºC, and the column
was subjected to a temperature gradient (initial = 80 ºC,
then an increase of 7 ºC min-1 to 240 ºC). The identica-
tion of the peaks was made by comparison with Supelco
37 methylated fatty acids (Supelco, USA) using the same
conditions described above.
Statistical analysis
All data were reported as mean (n=3). The com-
parison between the values obtained with the different oils
and the reference drug (kojic acid) was achieved through
a Student’s t-test to calculate p-values (α = 0.05), using
the software Statistical Package for the Social Sciences®
(SPSS) v. 14.0.
RESULTS AND DISCUSSION
Oils from plants are increasingly gaining more at-
tention from pharmacists, physicians and chemists due to
their great scope of applications in different elds such
as cosmetics, perfumery, pharmaceuticals and the food
industry. Since they have a wide range of pharmacological
applications due to their highly complex chemical compo-
sition, currently, there is a global trend toward replacing
synthetic substances with natural extracts (Al-Mamary et
al., 2011).
One of the abilities of plant materials currently being
researched is the inhibition of the tyrosinase enzyme, but
these studies have mainly been focused on crude drugs or
plant extracts, and not on plant oils (Adhikari et al., 2008).
Since the Brazilian Amazon is renowned worldwide for
its great plant diversity and potential for new pharmaceu-
ticals, this study selected five well-known Amazonian
species for screening their capability in being used as raw
materials for skin-whitening agents, which are sought after
by the cosmetic market.
According to Momtaz et al. (2008), vegetable oils
are rich in compounds containing hydrophobic compo-
nents, which can act as competitive inhibitors for the en-
zyme tyrosinase, and so on melanin synthesis. According
to these authors, plant oils have great reducing power, this
being the principle for exploring the tyrosinase inhibitory
potential of plants, which may also have phytoconstitu-
ents that chelate the copper ions present in the active site
of the tyrosinase enzyme. It is also important to discover
new skin-whitening agents because some of the currently
used substances are characterised by high toxicity, as hy-
droquinone (Macrini et al., 2009).
To our knowledge, this is the first report of such
activity for these species, and all of the ve Amazonian
vegetable oils screened for their tyrosinase inhibition
activity showed some degree of activity, as can be seen
in Table I. The tyrosinase inhibitory activity of tested oils
was found to be in the following order: açaí > tucumã >
patauá > pracaxi > castanha do Brasil. Thus, the natural oil
obtained from the açaí was the most active in the present
study, as it had an IA50 close to kojic acid, the gold standard
for this activity.
Our methodology included two screenings and a
nal quantitative step. The tyrosinase concentration of
125 U mL-1 in the rst test was adopted to increase the
chances of discovering an oil with potential skin-whit-
ening activity. As all oils seemed to possess such activity
(p < 0.05), they were submitted to the second screening,
and as they continued to show some activity, they were all
subjected to the quantitative analysis in order to discover
which was the most effective and therefore has the greatest
potential for the cosmetic/pharmaceutical industry.
According to Table I, it can be noted that the oils
maintained their 60 min activity in the second screening,
and so one can infer that they did not lose their tyrosinase
inhibition activity. Conversely, kojic acid went from
73.13% inhibition in the rst hour to 42.77% in the second
hour. Açaí oil, with a good IA50, had the longest duration of
action, compared to kojic acid, which may be a desirable
characteristic for a skin-whitening product as it decreases
the need for constant reapplication of the product to the
skin.
TABLE I - Tyrosinase inhibitory activity of oils from the Amazon
Sample IA 60 min
(%)
IA 120 min
(%)
IA50
(µg mL-1)
Açaí 38.56* 38.98 66.08*
Castanha do
Brasil 34.69* 34.70 1605.27
Patauá 35.15* 34.86 668.03
Pracaxi 37.75* 35.43 781.52
Tucumã 38.38* 37.69 557.91
Kojic acid 73.13 42.77 5.86
IA (%) = percentage of inhibitory activity; IA50 = inhibitory
activity at 50%; * p < 0.05 versus control.
R. S. Teixeira, P. R. Rocha, H. C. Polonini, M. A. F. Brandão, M. G. A. M. Chaves, N. R. B. Raposo
402
In order to know the composition of the studied oils,
a gas chromatography analysis was also performed, as can
be seen in Table II and Figure 1. The results show that the
oils possessed both saturated and unsaturated fatty acids,
the latter being considered essential fatty acids. This,
coupled with the tyrosinase inhibition results, led to the
inference that they can be included in functional foods
and cosmetics.
Theoretically, one can imagine that the high skin-
whitening property found in açaí oil may be due to the
presence of oleic acid (C18:1) in its composition, since this
was the main component in the chromatographic analysis.
However, further studies must be conducted with its com-
ponents separately in order to determine which specic
compound is responsible for enzyme inhibition. However,
it has been noted that natural products generally exert their
activity by synergy between their various compounds,
and a single substance is generally not responsible for the
biological effects found in natural products.
FIGURE 1 - Gas chromatograms of the studied Amazonian oils. Acids: C12:0 – lauric; C14:0 – myristic; C16:0 – palmitic; C16:1 –
palmitoleic; C18:0 – stearic; C18:1 – oleic; C18:2 – linoleic; C18:3 – linolenic; C20:0 – arachidic; C22:1 – erucid; C24:0 – lignoceric.
Mushroom tyrosinase inhibitory activity and major fatty acid constituents of Amazonian native ora oils 403
TABLE II - Main constituents of Amazonian native oils
Fatty acid (%) Oil
Açaí Castanha do Brasil Patauá Pracaxi Tucumã
C12:0 nd 0.3 nd nd nd
C14:0 nd 0.4 nd nd nd
C16:0 22.8 15.3 11.8 2.7 21.2
C16:1 4.2 0.3 0.5 nd nd
C18:0 1.9 10.6 3.8 5.3 5.1
C18:1 60.7 42.6 79.1 53.3 66.6
C18:2 9.6 30.2 4.5 25.5 5.3
C18:3 0.3 nd nd 2.5 1.8
C20:0 nd nd nd 1.3 nd
C22:0 nd nd nd 5.0 nd
C22:1 nd nd nd 1.6 nd
C24:0 nd nd nd 1.8 nd
nd = not detectable. Acids: C12:0 – lauric; C14:0 – myristic; C16:0 – palmitic; C16:1 – palmitoleic; C18:0 – stearic; C18:1 – oleic;
C18:2 – linoleic; C18:3 – linolenic; C20:0 – arachidic; C22:0 – behenic; C22:1 – erucid; C24:0 – lignoceric.
CONCLUSIONS
This study suggests that açaí oil has great potential
for the treatment of hyperpigmentation and other related
disorders due to its considerable tyrosinase inhibitory
activity. Thus, this may be an interesting candidate for
evaluation in more complex biological assays such as me-
lanocyte cultures and eventually in animal/human assays.
However, for the raw material be effectively incorporated
into the cosmetic market, it is now required to develop an
adequate delivery system/formulation for its topical use
in humans and for this activity be assessed in animal cells.
ACKNOWLEDGEMENTS
This research was supported by FAPEMIG, CNPq,
CAPES and PROPESQ.
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Received for publication on 10th February 2012
Accepted for publication on 3rd May 2012
