Free Radical Scavenging and Tyrosinase Inhibition Activity of Aromatic

Volatile Oil from Thai Medicinal Plants for Cosmetic Uses

A. Manosroi and J. Manosroi
Pharmaceutical-Cosmetic Raw Materials and Natural Products Research and
Development Center (PCRNC)
Institute for Science and Technology Research and Development
Faculty of Pharmacy, Chiang Mai University Thailand

Keywords: antiwrinkle, betel vine, holy basil, kaffir lime, kojic acid, vitamin C, vitamin
E, whitening
Abstract
Free radical scavenging and tyrosinase inhibition activity of thirteen aromatic
oils from Thai medicinal plants were investigated. A stable free radical DPPH (1, 1-
diphenyl-2-picryhydrazyl) was used in the free radical scavenging assay and
mushroom tyrosinase enzyme was used in the tyrosinase inhibition test. The two
assays were performed in 96-well plates and the absorbance measured by a well
reader. Betel vine and holy basil oils gave the highest free radical scavenging activity
with percent residual rate of absorbance (%RRA) of ≈ 15%, which is comparable to
vitamin E and vitamin C, widely used in antiwrinkle cosmetics. Kaffir lime leaf and
fruit peel oils showed the highest tyrosinase inhibition activity of ≈ 86.8 and 100%,
respectively, which is similar in potency to kojic acid, a popular ingredient in
whitening cosmetics. This study demonstrated the potential use of aromatic volatile
oils extracted from Thai medicinal plants in commercial cosmetics.
INTRODUCTION
Recently natural plant extracts are being studied for use in cosmetics, since use of
synthetic chemicals in cosmetics usually have inappropriate physical properties causing
irritation and allergy to skin. Two main acclaimed activities of these extracts are anti-
aging and whitening effects. The mechanisms of these two activities are free radical
scavenging and tyrosinase enzyme inhibition. Many natural compounds (vitamin E,
vitamin C, carotenoids and plant phenolic compounds) extracted from plants have been
successfully used in cosmetics (Haraguchi et al., 1995; Yoshino et al., 1994; Teguno et al.,
1998; Yen et al., 2000; Pietta, 2000).
Thailand has high plant biodiversity, and many Thai plants have been traditionally
used in folk medicines, in foods as spices, in massage and in aromatherapy. Thailand
imports many active chemicals including aromatic oils for use in cosmetics each year. In
order to make use of its own natural resources, especially aromatic oils for cosmetic
application, the activities of free radical scavenging and tyrosinase inhibition activity of
various aromatic volatile oils extracted from Thai medicinal plants were investigated.
MATERIALS AND METHODS
A total of 13 aromatic volatile oils from Thai medicinal plants were obtained from
Thai-China Flavors and Fragrances Industry Co., Ltd. (TCFF, Ayudthaya, Thailand).
These oils were from holy basil (Ocimum sanctum, Linn), sweet basil (Ocimum basilicum,
Linn), tumeric (Curcuma longa, L.), lesser galanga (Alpinia officinarum), betel vine
(Piper betel, Linn), lemon grass (Cymbopogon citratus, Stapf), citronella (Cymbopogon
nardus, Rendle), plai (Zingiber montanum), vetiver (Vetiveria zizanioides), lavender
(Lavandula angustifola, P.), Kaffir lime leaf (Citrus hystrix, DC), Kaffir lime fruit peel
(Citrus hystrix, DC) and black pepper (Piper nigrum, L.). They were tested for free
radical scavenging activity using an electrochemical method (Yamasaki et al., 1994), and
tyrosinase inhibition assay (Matsuda et al., 1996).
In the free radical scavenging assay system, acetate buffer (pH 5.5, 80 µl),

Proc. WOCMAP III, Vol.6: Traditional Medicine & Nutraceuticals 97

Eds. U.R. Palaniswamy, L.E. Craker and Z.E. Gardner
Acta Hort. 680, ISHS 2005
absolute alcohol (50 µl), a stable free radical DPPH (1,1-diphenyl-2-picrylhydrazyl, 100
-3
µL 2.53 x 10 M, Sigma Co., St. Louis, Missouri) and 40-80 µL 0.5% oil samples or
standards (vitamin E or vitamin C, Sigma Co.) were mixed in 96 -well plates and the
absorbance at 570nm were kinetically determined by a well reader (Seikagaku
Corporation, Tokyo, Japan) at 30 seconds, 1, 2, 3, 5, 10, 17 and 21 min. The time that
gave maximum absorbance in all samples was selected. The % RRA (residual rate of
absorbance) of each oil sample was calculated from the maximum O.D. compared to
control according to the equation:

% RRA = O.D. of sample or standard with DPPH - O.D without DPPH X 100
O.D. control (with DPPH but no sample) X amount (mg) of sample
or standard

For tyrosinase inhibition assay, the system contained L-tyrosinase (80 mg, T7755
Sigma Co.), phosphate buffer (0.1 M, pH 6.8), 18µg mushroom tyrosinase enzyme (Fluka,
Madrid, Spain) and 40-80 µL of 0.5% oil sample or the standards (kojic acid, Wako
Chemical Co., Japan; vitamin C, Carlo Erba, Spain). The mixture in a 96-well plate was
incubated at 37±0.5°C and the absorbance (450 nm) at 5, 15, 30, 50 and 60 min was
measured by a well reader. The time that gave minimum absorbance in all samples was
selected. The % tyrosinase inhibition activity of each oil sample was calculated from
minimum absorbance of sample system compared to control using the equation:

% melanin formation = (O.D. sample t=t-O.D. sample t=o) X 100

(O.D. control t=t – O.D. control t=0)
where, % tyrosinase inhibition = 100 - % melanin formation; t = t, time at each time
interval; t = 0, initial time. For colored oil samples, the absorbances were subtracted from
that which contained oil sample without DPPH or tyrosinase enzyme in free radical
scavenging and tyrosinase inhibition assay, respectively.
RESULTS AND DISCUSSION
Betel vine and holy basil oils showed the highest free radical scavenging activity
(Table 1). These two oils gave ≈ 15% RRA of DPPH, which is the same as vitamin C and
Vitamin E. The main chemical constituents were hydroxychavicol and eugenol in betel
vine oil and linalol (40-45%) and methyl chavicol (23.8%) in holy basil oil (Lawless,
1995; Ohloff, 1994). Generally, polyphenolic compounds are strong free radical
scavengers. The scavenging activity of these monophenolic compounds appear to be due
to the double bonds or hydroxyl groups attached to the aromatic ring. Only moderate to
less free radical scavenging activity was found in other oil samples. No free radical
scavenging activity was seen in sweet basil and vetiver oils.
The tyrosinase enzyme, known as polyphenol oxidase, a copper (Cu) containing
monooxygenase, which catalyzes both hydroxylation of monophenols and oxidation of
diphenols. Tyrosinase, a tetramer (molecular weight = 113,000 daltons) is a key enzyme
for pigmentation in plants, animals and humans. Kojic acid, used as standard in this study,
is an effective tyrosinase inhibitor. The Cu in tyrosinase enzyme is chelated by the ketone
group at position 4 and hydroxyl group at position 5 of kojic acid. Kojic acid is one of the
popular chemicals used in whitening cosmetics. Although betel vine and holy basil oil
showed no tyrosinase inhibition activity (Table 2), their strong free radical scavenging
activities may be through an antioxidative mechanism, which can contribute to the
inhibition of melanogenesis pathway. Aromatic oils from leaves and fruit peels of Kaffir
lime gave the highest tyrosinase inhibition activity of 86.8% and 100%, similar to that of
kojic acid. The main chemical constituents in these oils were limonene, pinene, camphene

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and coumarin (Lawless, 1995; Ohloff, 1994). The chemical structures indicate that they
may have chelating activity similar to kojic acid. However, these two oils showed no free
radical scavenging activity since their main constituents were not phenolic compounds.
Moderate to low tyrosinase inhibition activities were demonstrated in other oils.
Citronella oil showed no tyrosinase inhibition activity, and contained mainly citronello
and geraniol, which may not facilitate chelating of Cu in tyrosinase enzyme. This study
showed that the mechanism of tyrosinase enzyme inhibition of oils were not free radical
scavenging pathway, and can possibly be used in anti-wrinkle and whitening products.
ACKNOWLEDGEMENTS
We thank Ms. Bang-on Kiethanakorn at Thai-China Flavors and Fragrances
Industry Co., Ltd. for supplying the Thai aromatic oil samples and Mr. Chalerm Jansom
for assisting in the experiments in this study.
Literature Cited
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Ohloff, G. 1994. Scents and Fragrances, Springer-Verlag, NewYork.
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Tables

Table 1. Percentages of RRA and percentages of free radical scavenging of Thai aromatic
oil samples compared to vitamin C.

Sample (5 mg) % RRA % Free radical scavenging

compared to vitamin C
Vitamin C 2.70 100
Vitamin E 18.00 84.3
Betel Vine Oil 15.40 86.9
Holy Basil Oil 17.40 84.9
Citronella Oil 57.10 44.1
Lesser Galanga Oil 76.70 23.9
Lavender Oil 86.50 13.8
Lemon Grass Oil 87.56 12.8
Black Pepper Oil 87.90 12.4
Plai Oil 89.90 10.4
Tumeric Oil 90.00 10.3
Kaffir Lime Fruit Oil 90.60 9.66
Kaffir Lime Leaf Oil 92.70 7.50
Sweet Basil Oil 100 0
Vetiver Oil 100 0

Table 2. Percentages of melanin formation compared to control and percentages of

tyrosinase inhibition of Thai aromatic oil samples.

Samples % Melanin formation % Tyrosinase inhibition

compared to control
Control 100.0 -
Kojic Acid -0.631 100
Vitamin C 20.80 72.9
Kaffir Lime Fruit Oil -5.6 100
Kaffir Lime Leaf Oil 13.2 86.8
Tumeric Oil 36.2 63.8
Lavender Oil 40.3 59.7
Sweet Basil Oil 43.5 56.5
Lesser Galanga Oil 55.2 44.8
Vetiver Oil 61.8 38.2
Plai Oil 67.5 32.5
Black Pepper Oil 80.1 19.9
Lemon Grass Oil 88.3 11.7
Citronella Oil 103.5 0
Holy Basil Oil 103.8 0
Betel Vine Oil 108.5 0

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