22 JOURNAL OF COSMETIC SCIENCE
N-Succinyl-Ala-Ala-Ala-p-nitroanilide (1.015 mM) was prepared in 0.1232 M Tris-Cl
buffer (pH 8.0) This solution (1300 µl) was added to the sample stock (100 µl). Each
sample stock solution was diluted to final concentrations of 100, 50, and 10 µg/ml. The
solutions were then vortexed and preincubated for 10 min at 25°C, and elastase (0.0375
unit/ml) stock solution (100 µl) was added. After vortexing, the solutions were placed
in a water bath for 10 min at 25°C. The absorbance was measured at 410 nm.
CYTOTOXICITY ASSAY ON HUMAN FIBROBLASTS
Human fibroblast cells (American Type Culture Collection, ATCC, CRL-2076) were
seeded in 24-well plates with DMEM +10% FBS at a density of 1 x 105 cells per well
and cultured at 37°C in 5% CO2 .After one day, the medium was exchanged with fresh
media containing 2% serum, and the cells were allowed to incubate in a CO2 incubator
at 37°C in the presence of samples (100 µ1/ml) for 24 h. The cells were then treated with
100 µl of 2.5 mg/ml MTT and incubated at 37°C for an additional 4 h. The medium
containing the MTT was discarded, the MTT formazan produced was extracted with 1
ml of DMSO, and the absorbance was read at a wavelength of 5 70 nm with a reference
wavelength of 650 nm. The level of cell viability was calculated as:
Cell viability(%)=(OD
57 o
(
sample/OD570
(comrol ))X 100
where OD 57 o(sample) is the absorbance of the treated cells at 570 nm and OD
57 o(control)
is the absorbance of the negative control (non-treated cells) at 5 70 nm.
INHIBITION ASSAY ON MMP-1 EXPRESSION BY RT-PCR
Human fibroblasts were cultured with DMEM +10% FBS, 50 U/mol penicillin, and 50
µg/ml of streptomycin the medium was changed every two or three days. Cells were
cultured at 3 7 ° C in 5 %CO2 • When the cells reached confluence, they were separated
by treatment with 0.25% trypsin-0.03% EDTA (ethylenediamine tetraacetic acid) so-
lution. Cells were seeded into a 100-mm dish at a density of 2 x 106 cells and cultured
at 37°C in 5% CO2. After one day, fresh medium containing 2% serum was added to
the cells, which were then treated with samples for 24 hours. Total RNA was isolated
from the cells with TRizol (Invitrogen) according to the instructions of the manufac-
turer. First-strand cDNA synthesis was performed using random hexamers. The se-
quences of primers are as follows: 5 '-TGGGAGCAAACACATCTGA-3' (sense) and
5'-ATCACTTCTCCCCGAATCGT-3' (anti-sense) for MMP-1 5'-GAGACCTTCA-
ACACCCCAGCC-3' (sense) and 5'-GGCCATCTCTTGCTCGAAGTC-3' (anti-sense)
for �-actin. For MMP-1 RT-PCR reactions, reverse transcription was performed at 50°C
for 30 min, and denaturing was performed at 96°C for 3 min, followed by 22 cycles at
94°C for 1 min, 48°C for 1 min, and 72°C for 1 min, followed by an extension step cycle
at 72°C for 10 min. For �-Actin RT-PCR reactions, reverse transcription was performed
at 50°C for 30 min, and denaturing was performed at 96°C for 3 min, followed by 29
cycles at 94°C for 1 min, 70°C for 1 min, and 72°C for 1 min, followed by an extension
step cycle at 72°C for 10 min. The final products were detected with 1.5% agarose gel.
The gels were photographed, and the intensity of the stained PCR fragments was
quantified from photographs by densitometric analysis using Gel Doc 2000 (Bio-Rad
Laboratories, Segrate, Milan, Italy). EGCG ((-)epigallocatechin-3-gallate) was used as a
positive control.

ANTI-AGING COMPOUNDS FROM JEJU ISLAND 23
RES UL TS AND DISCUSSION
PREPARATION OF PLANT EXTRACTS
About 60 plants obtained from Jeju Island were extracted with 70%(v/v) aqueous
ethanol at room temperature. The yields were calculated by comparison of the amount
of raw material used with that of the extracts obtained. The results of these calculations
are shown in Table I. The highest yield was 32. 70% for Hizikia fusiformis (whole plant)
extract, and the yield was 26.15% for Cimicifuga acerina (root), 22.14% for Daucus carota
L. (leaf), and 21.08% for Cassia nomame (whole plant).
FREE RADICAL SCA VEN GING ACTIVITY
It was reported that free radicals induced by ultraviolet radiation or oxidative stress
accelerate skin aging (16). Free radical scavenging capacity assays were carried out using
the DPPH method. DPPH is one of a limited number of stable, commercially available
organic nitrogen radicals, and has a UV-Vis absorption maximum at 515 nm. Upon
reduction, the color of the solution fades the progress of the reaction is conveniently
monitored by a spectrophotometer. The free radical scavenging capacity of native plant
extracts was measured at each concentration (1 -400 µg/ml), and the results are shown
in Table II. The free radical scavenging capacity was expressed as SC
50 ,the concentration
needed to reduce 50% of DPPH.
Twelve extracts, Erigeron annuus (leaves and twigs, root), Typha orientalis (seed), Torreya
nucifera (leaf), Cassia nomame (whole plant), Myriophyllum spicatum L. (whole plant),
Miscanthus sinensis (flower), Persicaria hydropiper (whole plant), Euphorbia jolkini (whole
plant), Elsholtzia splendens (whole plant), Nymphaea tetragona (rhizome), Cimicifuga acerina
(root), and Pyrrosia hastata (root) had efficient free radical scavenging capacity (SC50
30 µg/ml). Among these, Nymphaea tetragona (rhizome) extract had the highest free
radical scavenging activity (SC50 =4.7 µg/ml). The SC50 values were 6.1 µm/ml for
Euphorbia jolkini (whole plant), 9.4 µg/ml for Euphorbia jolkini (seed), and 9.5 µg/ml for
Persicaria hydropiper (whole plant). These extracts showed extremely high free radical
scavenging activity compared to BHT (di-t-butyl hydroxytoluene) (SC50 =25.4 µg/ml),
which was used as a positive control (Figure 1). Most radical scavengers that exist in a
natural substance are phenolic or poly-phenolic compounds, such as flavonoids or an-
thocyanins. Their antioxidative activity is based on the inhibition of free radical transfer
mechanisms. Therefore, it is suggested that 12 extracts with superior free radical scav-
enging capacity have certain levels of compounds, such as flavonoids or anthocyanins,
and it is also believed that radical scavenging activity results from the presence of those
compounds.
ELASTASE ACTIVITY INHIBITION
The 60 plant extracts, obtained from Jeju Island and prepared at concentrations of 10,
50, and 100 µg/ml, were tested for their inhibitory effect on porcine pancreatic elastase.
The resuits are summarized in Table III. Seveu exi..iaci.s, Typhu orie ztalis (seed), P€rsicaria
hydropiper (whole plant), Capsicum annum (whole plant), Nymphaea tetragona (rhizome),