International Journal of Cosmetic Science 19, 29 1-298 (1997)

Biological screening of 100 plant extracts for

cosmetic use (I): inhibitory activities of
tyrosinase and DOPA auto-oxidation
K . T . L E E , B . J . K I M and J . H . K I M *
R& D Center, Coreana Cosmetics Co. Ltd, Cheonan, 333-830, Korea

M . Y . H E 0 and H . P . K I M
College of Pharmacy. Kangwon National University, Chuncheon, 200-701, Korea

Presented at the 19th IFSCC Congress, 22-25 October 1996, Sydney, Australia
Received I 1 July 1997
Accepted 14 August 1997

Keywords: tyrosinase inhibition; DOPA auto-oxidation; plant extracts

Synopsis
The aim of this study was to evaluate several plant extracts with a view to developing melanogenesis inhibitors.
In this study, 100 plant extracts were screened to elucidate their whitening effects using in vitro inhibition of
tyrosinase and DOPA auto-oxidation activity. Several plant extracts such as Chaenomeles speciosa, Dryopteris
crassirhizoma, Gastrodia ellata, Glycyrrhiza glabra, Morus alba, Myristica fragrans. Rheum palmaturn and
Sophora japonica showed inhibition of mushroom tyrosinase activity. Plant extracts including Bupleurum
falcatum, Caragana sinica, Morus alba and Tussilago farfara showed inhibition of DOPA auto-oxidation
activity.

Resume
L’objet de cette Ctude Ctait d’tvaluer plusieurs extraits v6gCtaux dans le but de dtvelopper des inhibiteurs de la
mClanogCnkse. Dans cette Ctude, on a crib16 100 extraits vCgCtaux pour connaitre leurs effets blanchissants en
utilisant in vitro I’inhibition de la tyrosinase et de I’activitt auto-oxydante de la DOPA. Plusieurs extraits
vCgttaux tels que Chaenomeles speciosa, Dryopteris crassirhizoma, Gastrodia ellata, Glycyrrhiza glabra,
Morus a h a , Myristica fragrans, Rheum palmaturn et Sophora japonica prCsentent une activit6 d’inhibition de
la tyrosinase du champignon. Des extraits vtgCtaux comprenant Bupleurum falcatum, Caragana sinica, Morus
alba et Tussilago fatfara montrent une inhibition de I’activitC d’auto-oxydation de la DOPA.

Introduction
In East Asia, most women want to have whiter skin. To satisfy this desire many cosmetic
companies have been developing melanogenesis inhibitors, in order to find promising
active agents for use in cosmetic preparations for skin whitening. In cosmetic preparations,
many plant extracts such as Morus alba and Glycyrrhiza glabra have been used as
whitening agents. Melanin formation is believed to be induced mainly by UV-light and
other stimuli such as toxic chemical agents [l].
Melanin is believed to be formed by a tyrosine pathway involving auto-oxidation,
serving as a light absorption compound protecting the skin against harmful UV-light.

*To whom correspondence should be addressed.

0 142-5463 0 1997 International Journal of Cosmetic Science
292 Lee et al.
Table I. Inhibitory activities of tyrosinase and DOPA auto-oxidation by 100 plant extracts
Inhibition of tyrosinase Inhibition of DOPA
f%) auto-oxidation (%)
3.3 333 100 500
Plant Part of plant pg mt’ pg mt’ pg mt’ pg mt’
Acorus gramineus Rhizoma -
Agastache rugosa Leaves 51
Akebia quinata Stem -
Alisma orientale Rhizoma -
Alpinia oficinarum Rhizoma -
Amomum cardamomum Fruit 40
Amomum xanthioides Seed 16
Anemarrhena asphodeloides Rhizoma -
Angelica dahurica Root 36
Angelica koreana Root 16
Angelica tenuissima Root -
Anthriscus sylvestris Root 47
Aralia cordata Root 33
Areca catechu Seed 34
Areca catechu Peel -
Arisaema heterophyllum Tuber -
Asiasarum sieboldii Root 5
Astragalus membraneaceus Root -
Atractylodes japonica Rhizoma 10
Belamcanda chinensis Rhizoma -
Brassica alba Seed -
Bupleurum falcatum Root 50
Cannabis sativa Seed 3
Caragana sinica Root 37
Chaenomeles speciosa Fruit 62
Chrysanthemum indicum Flower 10
Cinnamomum cassia Bark 5
Citrus aurantium Fruit 13
Citrus unshiu Peel -
Codonopsis pilosula Root 16
Commiphora molmol Resin -
Cornus officinalis Fruit -
Coptis japonica Rhizoma -
Corydalis ternata Tuber 20
Curcuma longa Rhizoma -
Curcuma zedoaria Rhizoma -
Cuscuta chinensis Seed -
Cyperus rotundus Rhizoma -
Dendrobium moniliforme Leaves 25
Dioscorea batatas Leaves -
Dryopteris crassirrhizoma Rhizoma 72
Equisetum hyemale Leaves -
Eucommia ulmoides Bark 49
Eugenia caryophyllata Flower 10
Evodia officinalis Fruit -
Foeniculum vulgare Fruit -
Gardenia jasminoides Fruit 40
Gastrodia ellata Rhizoma 85
Gleditsia sinensis Spina 8
Screening of plant extracts for cosmetic use (I) 293
Table I. Continued
Ihibition of tyrosinase Inhibition of DOPA
(%) auto-oxidation (%)
3.3 333 100 500
Plant Part of plant pg mt' pg mt' pg mt' pg rnt'
Glycyrrhiza glabra Root 68
Hordeum vulgare Fruit 34
Kochia scoparia Fruit 15
Leonurus sibiricus Leaves 48
Liriope platyphylla Tuber 15
Lindera strychnifolia Root -
Lycium chinensis Fruit 41
Morinda officinalis Root 24
Morus alba Stem 85
Myristica fragrans Seed 69
Nepeta japonica Leaves -
Paeonia suffruticosa Bark -
Perilla frutescens Leaves -
Perilla frutescens Bark -
Phellodendron arnurense Root 52
Phragmites communis Stem 12
Phylostachys nigra Tuber -
Pinellia ternata Seed 62
Plantago asiatica Root -
Platicodon grandiflorum Root -
Polygala tenuifolia Fruit -
Poncirus trifoliata Fruit -
Poria cocos (red) Hoelen -
Poria cocos (alba) Hoelen -
Prunus armeniaca Seed 26
Prunus rnume Fruit -
Prunus persica Seed 28
Pueraria thunbergiana Root -
Pueraria thunbergiana Flower -
Rehmannia glutinosa Root 48
Rhaphanus sativus Seed -
Rheum palmatum Rhizoma 68
Rhus verniciflua Resin -
Rubus coreanus Fruit -
Sanguisorba officinalis Root 19
Saussurea lappa Root 28
Schizandra chinensis Fruit 30
Sophora falvescens Root 26
Sophora japonica Flower 80
Sparganium stoloniferum Rhizoma 20
Taraxacum platycarpum Root -
Trapa bispinosa Fruit -
Terminalia chebula Fruit 38
Torilis japonica Fruit -
Trichosanthes kirilowii Root 18
Trichosanthes kirilowii Seed -
Tussilago farfara Flower -
Valeriana fauriei Root -
Zanthoxylum piperitum Peel -
Zingiber officinale Rhizoma 41
294 Lee et al.
Arbut in Kojic acid
100 100

4
c
0
.d
75
3 75
.d
4
.,-4
.I

2
H 50 2
H 50
25 * 25
0
0 50 100 150 200 250 300 0 50 100 150 200 250 300
Concentration (m/ml) Concentration (&ml)

fiaenaweles speciosa
100 100

75 15
..-I
c
0
U .-I
.w U

2 50 .

2
d

-
-3
-
.fi
50

25
* 25

0
0
0 250 500 750 1000
0 250 500 750 1000
Concentrat ion (pg/ml)
Concentration (pg/ml)

Figure 1. Concentration-dependent inhibition of tyrosinase.

However, whitening of facial skin and/or protection against skin darkening (melanin
formation) is considered desirable by some for cosmetic purposes, especially in Asia. Plant
extracts having an inhibitory effect on melanin formation may be a good choice for this
purpose because of their relatively lower side effects. Therefore, in this investigation 100
plant extracts were evaluated for their inhibitory activity on tyrosinase and DOPA auto-
oxidation.

Methods
Preparation of plant extracts
One hundred plants were obtained from the oriental medicinal market in Chuncheon,
South Korea. Each powdered plant (100 g) was soaked in 300 ml of 80% methanol
Screening of plant extracts for cosmetic use ( I ) 295
Rheum palmatum &rus alba
100

c 75
0
.H
4
.M

rs: 50
-dE
c(

* 25

0
0
1

250
I

500
I

750
I

1000
* 25t
- 0
0 250 500 750 1000
Concentrat ion (B/ml) Concentrat ion (m/ml)

Sophora japonica
100 r

0 250 500 750 1000

Concentration (m/ml)
Figure 1. continued.

solution and after filtration the filtrates were evaporated to dryness under vacuum. These
extracts were used for further biological study including inhibition of tyrosinase and
DOPA auto-oxidation activity.

Tyrosinase inhibition
Tyrosinase activity is generally determined by spectrophotometry. The procedure followed
that described by Vanni et al. [2]. The test reaction mixture was prepared by adding 0.5 ml
of each plant extract, to which 70 units of mushroom tyrosinase had been added, to 0.5 ml
of 0.1 mg ml-' L-tyrosine and 0.5 ml of 0.05 mM sodium phosphate buffer (pH 6.8). The
test mixture (1.5 ml) was incubated for 10 min at 37°C and the absorption at 475 nm was
measured. The same mixture but without the plant extract was used as the control. The
296 Lee et al.
IC,,, the concentration of plant extract at which half the original tyrosinase activity is
inhibited, was determined for each plant extract.
The percent inhibition of tyrosinase activity was calculated as follows:
% inhibition = ( A - B)/A X 100

where A = absorbance at 475 nm without test sample, and B = absorbance at 475 nm with
test sample.

Inhibition of DOPA auto-oxidation

In an assay tube, DOPA (500 p ~in) 0.05 M sodium phosphate buffer (pH 6.8) was
incubated at 37°C for 2 days. The total reaction volume per tube was 2 ml. Each plant
extract dissolved in the same buffer was added before starting incubation. The absorbance
was measured at 475 nm following the procedure of Joshi et al. [3].

Results and Discussion

We have screened 100 plant extracts for their inhibitory activities of tyrosinase and DOPA
auto-oxidation. Table I represents the result of the preliminary screening test. Twelve plant
extracts - Agastache rugosa, Bupleurum falcatum, Chaenomeles speciosa, Dryopteris
crassirrhizoma. Gastrodia ellata, Glycerrhiza glabra, Morus alba, Phellodendron amur-
ense, Pinellia ternata, Rheum palmatum, Sophora japonica - showed higher inhibitory
activity than 50% inhibition at the concentration of 333 pg ml-'. Among plant extracts M.
alba and Rh. palmatum showed strong inhibition of tyrosinase activity.
It was also found that nine plant extracts - Astragalus membranaceus, Bupleurum
falcatum, Caragana sinica, Foeniculum vulgare. Liriope platyphylla, M . alba, Phragmites
communis, Trichosanthes kirilowii, Tussilago f a ~ a r a- showed more than 50% inhibition
of DOPA auto-oxidation activity. Morus alba and Bupleurum falcatum showed good
inhibition of DOPA auto-oxidation activity. The potency of several plant extracts, was
compared using the 1C5,. Arbutin and kojic acid currently used as whitening agents were
also compared. Figure 1 shows the concentration effect on the inhibition of tyrosinase
activity of several selected plant extracts. The IC,, values for M . alba, G . glabra, Rh.
palmatum, S.japonica and C . speciosa were determined as 16.6, 71.0, 86.3, 93.4 and
328.8 pg ml-' respectively, compared to those of the reference compounds, arbutin
(65.2 pg ml-*) and kojic acid (5.8 pg ml-I), Figure 2 shows the concentration effect on the
inhibition of DOPA auto-oxidation activity.
The IC,, values of Trichosantheskirilowii (root), Lycium chinensis, M. alba, Bupleurum
falcatum and Liriope platyphylla were found to be 155.6, 297.6, 329.3, 335.7 and
362.4 pg ml-' respectively. These compared with the values for the reference compound,
kojic acid (197.2 pg ml-I). Among the extracts tested M . alba showed the highest
inhibition of tyrosinase and DOPA auto-oxidation activity.
From all of these results, it is clear that some plant extracts were able to inhibit melanin
formation, at least in vitro. This study suggests that several plant extracts have a potential
as whitening agents as single ingredients or in combination with other of the above
extracts.
Screening of plant extracts for cosmetic use ( I ) 297

100 -
Kojic acid
Rupleurum falca tvm

.-
4

25 ~

1 1 I J
0 0 L - L U

0 200 400 600 800 1000 0 200 400 600 80b 1000
Concentrat ion (n/ml) Concentrat ion (pg/ml)

Lycium chinensis
100 j-
a
0
'S 75 -
.3

f,
.

*50 -
1$ 1
'Z 75
d-

I I I I I

0 200 400 600 800 1000 0 200 400 600 800 1000
Concentrat ion (lrg/ml) Concentration (alml)

Trichosanthes kjrilorii
Uorus alba
100 r 100

* 25 1 * 25

0 0
0 200 400 600 800 1000 0 200 400 600 800 1000
Concentration ( ~ / m l ) Concentrat ion (lrg/ml)

Figure 2. Concentration-dependent inhibition of DOPA auto-oxidation activity.

298 Lee et al.
References
1. Kubo, M. and Matsuda, H. Development studies of cuticle and medicinal drugs from natural
sources on melanin biosynthesis. Fragrance J. 8, 48-55 (1995).
2. Vanni, A., Gastaldi, D. and Giunata, G. Kinetic investigations on the double enzymatic activity
of the tyrosinase mushroom. Annali di Chimica 80, 35-60 (1990).
3. Joshi, P.C.,Carraro, C. and Path&, M.A. Involvement of reactive oxygen species in the
oxidation of tyrosine and dopa to melanin and in skin tanning. Biochem. Biophys. Res. Comm.,
142, 265-74 (1987).