232 JOURNAL OF COSMETIC SCIENCE
growing interest today in cosmetic products that come from naturally derived components.
Studies have screened abundant components from natural products, represented by arbutin,
tetrahydrocurcumin, and pterostilbene, all of which show favorable effects on pigmentation
reduction.5–7
Naturally whitening active ingredients can inhibit the formation and transfer of melanin
through multiple pathways.8 Many of these actives are reported to inhibit the transferring
of melanin from melanocytes to keratinocytes.9 For example, glabridin, a prenylated
isoflavonoid of G. glabra L. roots, has been associated with a wide range of biological
properties, including regulation of estrogenic, energy metabolism, neuroprotective,
and skin whitening in previous studies.10 The inhibition activity against tyrosinase and
melanin synthesis of glabridin may be the main whitening mechanisms.11–13 While the
present studies have mainly focused on the whitening effects of glabridin at the cell
level (melanocytes), few clinical reports on the effects of glabridin on melasma have been
published.
In this study, a skin 3D model was established and the effects of a whitening serum
containing glabridin on the apparent chromaticity, L* value, along with the content and
distribution of melanin were studied. Meanwhile, the whitening effect of the serum on the
melasma area and nonmelasma area was studied through clinical experiments.
MATERIALS AND METHODS
MATERIALS
Glabridin (98.5%) was bought from Push Bio-Technology (Push Bio-Technology, Ltd.,
Chengdu, China). Dimethyl sulfoxide and kojic acid were bought from Sigma-Aldrich
(Sigma, Massachusetts, USA). The 3D skin model provided was MelaKutis® (Guangdong
Boxi Biological Technology Co., Ltd., Guangdong, China). All other chemicals used were
of analytical grade.
PIGMENTATION IN 3D SKIN EQUIVALENT MODEL
The reconstructed human 3D epidermis (MelaKutis®) consisted of normal human-derived
epidermal keratinocytes and normal human epidermal melanocytes that had been cultured
to form a multilayered, highly differentiated model, similar to real skin structure. The blank
control (BC) group changed the culture medium every day without any other treatment
the negative control (NC) group, the positive control (PC) group with kojic acid (500 µg/
mL), and the whitening serum group were required to change the culture medium every
day and undergo ultraviolet-B (UVB) stimulation every day (50 mJ/cm2). The whitening
serum and PC groups used samples on the model surface on day 3 and day 5.The test
samples were applied topically in a volume of 10 µL. Controls were not treated. The change
in media and application of test material was repeated every 2 days for 7 days. The chemical
was added in the lower well of the reconstructed epidermis system then penetrated through
a membrane to reach the basal cells. The epidermis was then fixed with 4% formalin
in phosphate-buffered saline, followed by Fontana-Masson staining to visualize melanin
pigments. All tissues were photographed using a digital camera for a visual assessment of
depigmentation. It was then processed further for determining skin lightening as a measure

233 IMPROVEMENT OF MELASMA WITH GLABRIDIN
of the tissue luminance values (L* unit) using a CM-2600d Spectrophotometer® (Konica
Minolta, Tokyo, Japan), and the melanin reduction was quantified spectrophotometrically
by solubilizing the tissue using solvable.
MELANIN CONTENT MEASUREMENT
Briefly, the cell pellets were solubilized in 1 mol/L NaOH (80°C) for 40 minutes. The
melanin was evaluated at 405 nm using a spectrophotometer. Melanin was determined by
the protein’s absorbance from the cell extract.
MELANIN DISTRIBUTION
All samples were fixed with 4% formaldehyde solution for 24 hours after 8 days of cell
culture. The sliced samples were stained according to the silver staining kit directions,
and then photographed under the microscope. Intel® Integrated Performance Primitives
(Intel, California, USA) software was used to determine the relative integral optical
density (IOD).
CLINICAL STUDY
Subjects. 33 Chinese urban women between the ages of 33 and 51 were enrolled in
the study. These selected volunteers showed the following characteristics: lackluster
complexion, color spot density on the cheek of ≥2, at least one 2 mm independent
color spot on the face, no obvious redness, and skin lesions or scars on the face. During
the study period, strong sun exposure was to be avoided. All subjects participating in
this study signed an informed consent form. Patients were excluded from the study
if they were pregnant, breastfeeding, in hormone or corticosteroid therapy, or had
a history of endocrine disorders or allergies. Additionally, potential volunteers who
used depigmenting or whitening products (oral or topical) in the past 6 weeks were
excluded.
Test materials and procedure. The test formula contained glabridin was in a vehicle base. A
neutral cream without glabridin was used as a placebo. Additionally, the whitening essence
passed the safety test. Both study formulas were identical in appearance and supplied with
two identical pump dispensers. One test formula was applied twice a day on the subjects’
right face and the other one on the left face for 56 days, and the subjects were also asked to
not use any cosmetics containing ingredients that could potentially interfere with their skin
color status during the study period. The skin color (Colorimeter® CL400, EnviroDerm
Services, Longhope, UK), melanin index value (Mexameter® MX18, EnviroDerm Services,
Longhope, UK), the gloss of skin (Glossymeter® GL200, EnviroDerm Services, Longhope,
UK) and photographic documentation (VISIA-CR, Canfield Scientific Inc., New Jersey,
USA) were evaluated at baseline, then at 14 days, 28 days, and 56 days after treatment.
During evaluation, the entire study was conducted under controlled environmental
conditions of temperature and relative humidity. Participants were asked to rest for at least