36 JOURNAL OF COSMETIC SCIENCE
and mulberry. These agents are all tyrosinase inhibitors, which inactivate tyrosinase (the
enzyme responsible for skin pigmentation) by chelating with its vital copper ion and
suppressing tautomerization from dopachrome to DHICA. Normal skin color is formed
by melanin, a natural pigment that also determines hair and eye color. In the skin, the
enzyme tyrosinase biochemically converts the amino acid tyrosine into melanin. Hyper-
pigmentation occurs when too much melanin is produced and forms deposits in the skin.
Hyperpigmentation is not a medically harmful condition. However, it is always advis-
able to have new brown spots checked by a dermatologist to make sure they are not skin
cancers (2). Arbutin is a naturally occurring glycoside of hydroquinone (Figure 1).
Arbutin is found in the bark and leaves of various plants, usually occurring together with
methylarbutin. Naturally occurring arbutin was first characterized by Kawalier (3), who
obtained it from bearberry leaves. It is also found in the leaves of blueberry, cranberry,
and pear. Synthetic arbutin was first reported by Mannich (4), and later by others.
Commercial arbutin is almost always synthetic in origin. Because of its antibacterial
properties, arbutin is a constituent of the traditional medicine uva ursiJ and it is widely
used in a variety of formulations (5). The ability of arbutin to inhibit human melanin
synthesis has given rise to its wide use in many cosmetic formulations (6). Arbutin
protects the skin against damage caused by free radicals. It is a skin-whitening agent that
is very popular in Japan and Asian countries for skin depigmentation. Arbutin inhibits
the formation of melanin pigment by inhibiting tyrosinase activity (7). Back in the 18th
century arbutin was first used in medical areas as an anti-inflammatory and antibacterial
agent. It was used particularly for cystitis, urethritis, and pyelitis. These uses have been
applied until today, when natural medicine uses only natural ingredients to treat any
disease. It may be used to repress the virulence of bacterial pathogens and to repress
contaminating bacteria. It is also used for treating allergic inflammation of the skin. It
can be used to whiten the skin, to prevent liver spots and freckles, to treat sunburn
marks, and to regulate melanogenesis (8).
Arbutin is a very safe skin-whitening agent for external use, which does not have
toxicity, a stimulating effect, an unpleasant odor, or side effects such as hydroquinone
(Figure la). Hydrophilic arbutin can be incorporated in lipophilic media by encapsu-
lation. Arbutin has three main properties: a whitening effect, an anti-aging effect, and
a UVB/UVC filter (9). Arbutin is determined by many methods: the spectrophotometric
method (10-12), capillary zone electrophoresis (13 ),and thin-layer chromatography-
densitometry (14). The proposed HPLC method for determining arbutin in skin-
whitening cosmetic products and some medicinal plants is more sensitive, precise, and
less time-consuming than the previous HPLC methods described in the literature (15-
OH
Hydroquinone
(a)
Arbutin
(b)
Figure 1. Chemical structures of hydroquinone (a) and arbutin (b).
HPLC DETERMINATION OF ARBUTIN 37
18). Furthermore, there is no data in the literature about the isolation and quantitative
analysis of arbutin in Betula alnoides Buch. Ham., Clerodendrum petasites S. Moore, Cur-
culigo latifolia Dryand. Var. latifolia, and Hesperethusa crenulata (Roxb.) Roem. Therefore,
it is interesting to investigate the arbutin content in these medicinal plants, because
naturally occurring arbutin is very safe skin-whitening agent.
EXPERIMENT AL
APPARATUS
HPLC analyses were carried out with Hewlett Packard Model ll00 liquid chromato-
graph with autosampler, thermostatic column compartment, online degasser, and a
UV-visible detector model G 1313 A. The column used was ODS Hypersil® C
18
(125
mm x 4 mm, 5 .0 µm [Chromtech, Stockholm, Sweden}) with a Lichrosphere® 100
RP-18 (4 mm x 4 mm, 5.0 µm) guard column. The mobile phase was a mixture
containing varying ratios of methanol, water, and 0.1 M hydrochloric acid, vacuum-
filtered through 0.45-µm nylon membranes (Germany) before use. The following in-
struments were also used: a simultaneous spectrophotometer (Spekol 1200), a pH-meter
(Model pH 900, Precisa, Switzerland), a water bath and shaker (Model SB-200-10,
Thailand), an ultrasonicator (Model 889, Cole Parmer, USA), and a rotary evaporator
(EYELA N-N series).
REAGENTS
The following standard reagents were used: arbutin HPLC grade 98% (Sigma, St Louis,
MO) and resorcinol 98% (Fluka). The following reagents were used: hydrochloric acid
(AR) (Farmitalia Carlo Erba, Italy), glacial acetic acid (AR) (Farmitalia Carlo Erba),
acetonitrile (HPLC grade) (Lab-Scan Analytical Sciences, Ireland), ethyl acetate (AR)
(BDH laboratory supplies, England), methanol (HPLC grade) (Lab-Scan Analytical Sci-
ences), and ether (AR) (Lab-Scan Analytical Sciences). De-ionized distilled water was
used throughout.
SAMPLES
Skin whitening products. Arbuwhite® cream (Nature Best Health Product Co., Ltd.,
Thailand), Super Whitening® cream (Aunyamanee Herbs, Thailand), and Shiseido®
cream (Shiseido Co., Ltd., Tokyo, Japan) were used.
Plant material and location. The bark of Betula alnoides was collected at Bah Bae Village,
Mae Dtang District, Chiang Mai Province, Thailand, in October 2003 and was iden-
tified by W. Thongchai 1. Voucher specimens have been deposited at CMU Herbarium,
Chiang Mai University, Chiang Mai, Thailand. The roots of Clerodendrum petasites S.
Moore were collected from Chiang Dow, Cl1aing Mai, Thailand. The tubers of Curculigo
latifolia Dryand. Var. latifolia were collected from Papae, Maetang, Chiang Mai, Thai-
land. The trunk of HeJperethusa crenulata (Roxb.) Roem. was collected from Mae Sai,
Chiang Rai, Thailand.
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