Melanin is reported to have a protoprotective function in human skin but humans
are still conscious about their skin color because the undesirable skin discoloration
or hyperpigmentation can cause aesthetic problems. Because melanin formation
is an important factor to human skin color development, the inhibition of melanin
formation may result in a reduction of skin darkness. The formation of melanin
in the human body is reduced by several mechanisms, including anti-oxidation,
direct tyrosinase inhibition, melanin inhibition of migration from cell to cell
and hormonal activities, etc (Slominski et al., 2004).
Tyrosinase is a rate limiting enzyme associated with melanin synthesis in melanocytes.
It is a copper-containing monooxygenase and catalyzes two major reactions: monophenolase,
the hydroxylation of L-tyrosine and diphenolase, the oxidation of L-DOPA (3,4-dihydroxyphenylalanine).
Inhibition of tyrosinase (monophenolase and diphenolase) activity will decrease
the melanin synthesis (Dubey et al., 2006).
Present previous research found that out of the 23 plant species screened,
Intsia palembanica (Local name: Merbau) is a species which has the most
potent activity as tyrosinase inhibitor (Batubara et
al., 2010). I. palembanica is extensively used as furniture and
building material in place of native wood (Imamura et
al., 1974). Some ethnic groups in Indonesia use the wood of Merbau as
a medicine. For instance, Maluk ethnic group in Sumbawa Island used it to treat
impotency (Sangat et al., 2000). This plant has
only 48.2% survival rate (Hassan et al., 2007)
and has low growth increments (Affendy et al., 2009).
It is interesting to know the active compounds in Merbau which has activity
as a tyrosinase inhibitor, particularly as skin whitener. Therefore, the aim
of this study is to obtain active compounds from Merbau as a skin whitener based
on tyrosinase inhibitory activity and melanin cell growth inhibition on B16
MATERIALS AND METHODS
Plant material: I. palembanica was collected from Samarinda, East Kalimantan, Indonesia. Identification and voucher specimen (No. FHT.LA.12.9 p) was deposited at the Wood Anatomy Laboratory, Faculty of Forestry, Mulawarman University, East Kalimantan, Indonesia.
|| Structure of Isolated Compounds
Extraction and isolation of (-)-robidanol, (+)-epirobidanol and compound 1: I. palembanica was dried and ground before submitting to methanol extraction. Briefly, 100 g I. palembanica wood meal was macerated with 5 L methanol for 12 h three times. The extracts were filtered using Whatman filter paper (No. 2) and concentrated in vacuo at 30°C using a rotary evaporator to obtain 6.7 g extract (6.7 % yield based on dried sample).
Part of the extract (3 g) was fractionated using n-hexane, EtOAc and water to result to n-hexane soluble part, EtOAc soluble part and aqueous soluble part. EtOAc soluble part (1.4 g) was separated by silica gel column chromatography with hexane, ethyl acetate and methanol as the developing solvents and resulted to 26 fractions. Fraction 4-8 were eluted with ethyl acetate and gave a mixture of (-)-robidanol, (+)-epirobidanol, 4-dehydroxyrobidanol and some other compounds. Further purification was conducted using preparative HPLC with reversed phase column Inertsil ODS-3 (GL Sciences 10 mm id x 200 mm) monitored at 280 nm. The solvent system used was as follows: a gradient program for 45 minutes from 5 to 100% of methanol in TFA 0.05% (in water) at flow rate of 3 mL min-1. This separation step gave crude (-)-robidanol, (+)-epirobidanol and 4-dehydroxyrobidanol (Fig. 1). Repeated subjection to preparative HPLC resulted in a (-)-robidanol (15.8 mg), (+)-epirobidanol (47.4 mg) and 4-dehydroxyrobidanol (10.5 mg). The structures of the compounds were determined by comparison of their spectroscopic data with those reported in the literature. 1H- and 13C- NMR were recorded with a JEOL ECP 600 MHZ spectrometer with TMS as the internal reference and chemical shifts expressed in δ (ppm). Homonuclear 1H-1H COSY and heteronuclear HMBC correlation were analyzed using the same instrument. Mass data was measured by direct injection in Shimadzu GCMS-QP5050A (gas chromatography-mass spectrometer).
Bioactivity tests: Bioactivity tests were performed for 3 isolated compounds and 6 other compounds which were reportedly contained in Merbau such as (+)-catechin, (-)-epicatechin, quercetine, robinetin, ampelopsin and 3,7,3, 5-tetrahydroxyflavone. Bioactivities were analyzed for tyrosinase activity inhibitor, antioxidants and inhibition of melanin cell growth in B16 cell.
Inhibition of tyrosinase activity (monophenolase) and DOPA auto-oxidation
(diphenolase): This assay was performed using methods as described earlier
(Curto et al., 1999; Nerya
et al., 2003). Extracts were dissolved in DMSO (dimethyl sulphoxide)
to a final concentration of 20 mg mL-1. The extract stock solutions
were then diluted to 600 μg mL-1 in 50 mM potassium phosphate
buffer (pH 6.5).
The extracts were tested at the concentrations ranging from 7.8 to 2000.0 μg mL-1. Kojic acid, which was used as positive control was also tested at concentrations from 7.8 to 500.0 μg mL-1. In 96-well plate, 70 μL of each extract dilution was combined with 30 μL of tyrosinase (333 Units mL-1 in phosphate buffer) in triplicate. After incubation at room temperature for 5 min, 110 μL of substrate (2 mM L-tyrosine or 12 mM L-DOPA) was added to each well. Incubation lasted for 30 min at room temperature. Optical densities of the wells were then determined at 510 nm with multi-well plate reader. The concentration of plant extract at which half the original tyrosinase activity is inhibited (IC50), was determined for each plant extract.
Melanin synthesis test on B16 cell
Cell culture: Murine B16 melanoma cells were purchased from Riken Cell Bank
(Tsukuba, Japan). The cells were cultured in Dulbeccos modified Eagles
medium (DMEM) that was supplemented with 10 % (v/v) fetal bovine serum, 100
Unit mL-1 of penicillin and 100 μg mL-1 of streptomycin,
at 37°C in a humidified, CO2-controlled (5%) incubator.
Melanogenic assays: Cells (4x104 cells) were inoculated on
10 cm dishes and compound treatment began 24 h after seedling. Each compound
in DMSO was added to the cell cultures with a final concentration of 0.5% v/v.
The cells were harvested 48 h later and the melanin content was determined in
triplicate for each treatment (Ohguchi et al., 2005).
Cells were washed with phosphate-buffered saline (PBS) and dissolved in 1 N
NaOH for 1 h at 60°C. The absorbance was measured at 405 nm. The cell proliferation
was determined by the trypan blue exclusion test. Cell proliferation was shown
in percentage values. Each percentage value in the treated cells was calculated
with respect to that in the control cells.
Statistical analysis: Data of IC50 were expressed as Mean±SD. The significant differences between groups were assessed by one-way ANOVA followed by comparisons of the groups with a control using Dunnetts test, p<0.05 was considered as significant.
RESULTS AND DISCUSSION
Identification of compounds: The separation guided by bioassay resulted
three isolated compounds from I. palembanica namely, (-)-robidanol, (+)-epirobidanol
and 4-dehydroxyrobidanol based on NMR data. The structures of isolated
compounds are shown in Fig. 1. (-)-robidanol, white powder;
1H-NMR (600MHz, CD3OD) δ:2.68, 3.06 (each 1H, dd,
J = 3.4, 16.5 Hz, H-4), 4.13 (1H, dd, J = 1.9, 6.8 Hz, H-3), 4.79(1H, d, J =
6.8 Hz, H-2), 6.30 (1H, d, J=2.0Hz, H-8), 6.32 (1H, dd, J = 8.2, 2.0Hz, H-6),
6.48 (2H, s, H-2 and H-6), 6.86 (1H, d, J = 8.2Hz, H-5); 13C-NMR
(150MHz, CD3OD):32.6 (C-4), 66.5 (C-3), 78.7 (C-2), 102.6 (C-8),
105.6 (C-2 and C-6),108.2 (C-6), 110.5 (C-4a), 130.1 (C-1),
130.3 (C-5), 132.3 (C-4), 145.4 (C-3 and C-5), 155.2 (C-8a),
156.4 (C-7).; EIMS m/z: 290 [M+].
(+)-epirobidanol. White powder 1H-NMR (600MHz, CD3OD): δ:2.64, 2.84 (each 1H, dd, J = 7.6, 15.8Hz, H-4), 3.97 (1H, dd, J = 2.0, 6.9 Hz, H-3), 4.60 (1H, d, J = 6.9Hz, H-2), 6.26 (1H, d, J = 2.1Hz, H-8), 6.30 (1H, dd, J = 8.2, 2.1Hz, H-6), 6.36 (2H, s, H-2 and H-6), 6.83 (1H, d, J = 8.2Hz, H-5); 13C-NMR (150 MHZ, CD3OD): 31.3 (C-4), 67.5 (C-3), 81.6 (C-2), 102.3 (C-8), 105.7 (C-2 and C-6), 108.1 (C-6), 111.2 (C-4a), 129.9 (C-5), 130.3 (C-1), 130.0 (C-5), 132.7 (C-4), 145.6 (C-3 and C-5), 154.8 (C-8a), 156.6 (C-7). EIMS m/z: 290 [M+].
4-dehydroxyrobidanol. White powder, 1H-NMR (600 MHZ, CD3OD): δ: 2.69, 3.08 (each 1H, dd, J = 2.8, 13.7Hz, H-4), 4.20 (1H, dd, J = 1.9, 6.8 Hz, H-3), 4.56 (1H, d, J = 1.9 Hz, H-2), 6.18(1H, t, J = 2.0, H-4), 6.32 (1H, dd, J = 2.0, 8.2Hz, H-6), 6.33 (1H, d, J = 2.0Hz, H-8), 6.43 (2H, d, J = 2.0Hz, H-2& 6), 6.80 (1H, d, J = 8.2Hz, H-5); 13C-NMR (150 MHZ, CD3OD): 32.6 (C-4), 66.4 (C-3), 78.0 (C-2), 101.4 (C-4), 102.7 (C-8), 108.3 (C-6), 104.9 (2C, C-2 and 6), 109.0 (C-4a), 130.3 (C-5), 141.0 (C-1), 155.0 (C-8a), 156.0 (C-7), 157.0 (2C, C-3 and 5); EIMS m/z: 274[M+]
Structure of three compounds were proved by homonuclear corellation data (COSY)
and heteronuclear corellation data (HMBC). The importance data was found in
HMBC NMR data. The HMBC data was especially usefull in determining the placement
|| Tyrosinase IC50 values of pure compounds from
|*Data given as mean±standard deviation of triplicate
tests. IC50 data followed by the same letter are not significantly
different according to Dunnetts test, p<0.05. Filed to achieve
50% inhibition at maximum concentration of 2500 μg mL-1
Proton from B-ring (H-2 and 6) were correlated to carbon no 2 in
C-ring and also correlated to carbon No. 1, 2, 3, 4, 5
and 6 in B-ring.
Bioactivity tests: The tyrosinase inhibitory activity of pure compounds, 6 pure compounds reported in I. palembanica and positive control (kojic acid) are shown Table 1. The results showed that (-)-robidanol was the most active compounds with IC50 (monophenolase: 8.7 μM and diphenolase: 26.6 μM). 4-dehydroxyrobidanol was the next prospective compounds with IC50 (monophenolase: 15.2 μM and diphenolase: 50.0 μM). These two isolated compounds had activity better than kojic acid as positive control.
Almost all of the compounds reached IC50 before the maximum concentration 2500 μg mL-1. Only 3,7,3,5-tetrahydroxyflavone failed to achieve 50% inhibition at maximum concentration. For diphenolase activity, only 4 compounds achieved 50% inhibition ((-)-robidanol, (+)-epirobidanol, 4-dehydroxyrobidanol and ampelopsin).
The result of melanin synthesis inhibitor is showed Fig. 3. The three isolated compounds had cell viability between 95-140% which not significantly different compared to cell in 0.5% DMSO. Melanin synthesis activities of the three isolated compounds were depended on their concentration. (+)-epirobidanol had inhibition activity at high concentration (100 μM), while at lower concentration had acceleration activity (1.6 μM). (-)-robidanol gave melanin synthesis inhibition activity at all concentration applied. Different with the two isolated compounds, 4-dehydroxyrobidanol gave melanin acceleration activity at all concentration applied.
Isolated compounds from I. palembanica: Bioassay-guided separation
of Merbau extracts led to the purification of 3 compounds. Three flavanol compounds
were isolated: (-)-robidanol, (+)-epirobidanol and 4-dehydroxyrobidanol.
(-)-robidanol and (+)-epirobidanol are flavanol compounds which had been found
in some fruits, like Gleditschia triacanthos (Weinges,
1964), strawberry (Lopes-da-Silva et al., 2007),
red grape (Makris et al., 2008) and Annona
senegalensis (Potchoo et al., 2008).
Among all the flavanol which isolated, 4-dehydroxyrobidanol was found
to be a novel compound and its structure was elucidated by NMR data. 4-dehydroxyrobidanol
gave molecular ion peak at m/z 274 [M+], consistent with molecular
formula of C15H14O5. In its proton NMR spectrum,
the signal for ring A and the aliphatic signal were the same with (-)-robidanol.
The differences were found only in signal of ring B. It has signal δ at
6.18 ppm with J about 2 Hz and coupling with 2 proton signals δ
at 6.43 ppm with the same coupling constant. The data also proved by proton-proton
correlation (COSY) data. This data mean that B rings had no hydroxyl group at
C-4. It is an unusual flavonoids pattern especially in ring B but the
same pattern had been reported to exist in the same species of I. palembanica
(Imamura et al., 1974). They reported a new
type of flavonol in the shake of Merbau wood. The new type flavonol was 3,7,3,5-tetrahydroxyflavone,
a pale yellow microcrystals.
Other flavonoids isolated from Merbau belong to 2 groups, flavanols and flavonols.
The flavonoid belonging to flavanol group were (+)-catechin and (-)-epicatechin
while those belonging to the flavonol group were robinetin, myricetin, fisetin,
quercetin, naringenin, ampelopsin, leucocyanidin and piceatannol. On this paper,
we used robinetin, ampelopsin, 3,7,3,5-tetrahydroxyflavone which
were isolated previously together with quercetin, (+)-catechin and (-)-epicatechin
from Merck for the activity tests (Fig. 2c, b).
Tyrosinase inhibitory activity: The tyrosinase inhibitory activity of pure compounds is shown in Table 1 with kojic acid as positive control. The data reported in IC50 values are the concentrations which can inhibit 50% activity compared to the control. The results showed that isolated flavanol compounds were more active to inhibit tyrosinase activity compared to flavonol compounds either in monophenolase or diphenolase.
Many reported flavonoid compounds had the capability to inhibit tyrosinase
activity but those of compounds from the flavanol group were still limited.
(-)-epigallocatechin is a compound from the flavanol group which has been reported
to have a monophenolase inhibitory activity (IC50 0.035 mM) (No
et al., 1999). A study about the flavanol group reported (+)-catechin
as a cofactor or substrate for tyrosinase (Kubo et al.,
||Flavonol and catechin compounds reportedly isolated from I.
Present results showed that the IC50 of (-)-epicatechin was about
0.092 mM for monophenolase and (+)-catechin inhibited the tyrosinase activity
for monophenolase with IC50 of about 0.077 mM.
Compared to (+)-catechin and (-)-epicatechin which only had monophenolase inhibition activity, (-)-robidanol, (+)-epirobidanol and 4-dehydroxyrobidanol had inhibition activities against tyrosinase either for monophenolase or diphenolase. The most active tyrosinase inhibitor found on (-)-robidanol with IC50 for monophenolase of about 8.7 μM and diphenolase about 26.6 μM. Its activity is about 4 times compared to kojic acid as positive control. When we compared the chemical structure of (-)-robidanol and (-)-epigallocatechin, (-)-robidanol has no hydroxyl group on C-5. This data indicated the absence of hydroxyl group on C-5, probably can increase the tyrosinase inhibitory activity. The other interesting data was the absence of hydroxyl groups on C-4 of 4-dehydroxyrobidanol which can decrease the tyrosinase inhibitory activity about two times compared to (-)-robidanol.
|| Cell viability and % melanin synthesis of isolated compounds
For flavonol groups found in I. palembanica, the most active flavonol compound was ampelopsin which had IC50 value not significantly different with kojic acid as positive control on monophenolase activity. The other flavonol compounds did not show IC50 data on diphenolase. Interestingly, 3,7,3,5-tetrahydroxyflavone did not show inhibition activity for tyrosinase for both monophenolase and diphenolase, while 4-dehydroxyrobidanol which has the same B-ring pattern showed some inhibition activity.
Melanin synthesis on B16 cell and cell viability: Another approach for
a whitening agent is to inhibit melanin synthesis and it called as melanogenesis.
Kim et al. (2010) stated that melanogenesis is
a well known physiological response of human skin which mainly caused by ultraviolet
irradiation and some genetic factors. Many natural products searched for anti-melanogenesis
such as from Neolitsea aciculate (Kim et al.,
2010) and sea cucumber (Stichopus japonipus) (Yoon
et al., 2010).
Our research focus on the compounds isolated from I. palembanica. Based on the color of cell which subjected with (-)-robidanol was lighter compared to blank cell. Compared with (-)-robidanol, 4-dehydroxyrobidanol cannot change the color of cell. It can be concluded that 4-dehydroxyrobidanol cannot inhibit the synthesis of melanin.
The melanin synthesis inhibition and cell viability of B16 cell in the presence of isolated compounds are shown in Fig. 3. All the isolated compounds were not toxic to the cell because cell viability is almost the same with the blank and a little bit increasing compared to blank cell (Fig. 3). This result showed that the compounds were safe to use for cell assay
The melanin synthesis inhibition of each compound was different from each other. (-)-robidanol still showed the most active compound as a whitening agent with the melanin synthesis inhibition approach. (-)-robidanol inhibit melanin synthesis of about 46.2% at 100 μM concentration. This activity was higher compared to kojic acid as positive control which only inhibited about 44.0 % at 12 mM concentration.
(+)-epirobidanol at 100 μM concentration had the activity to inhibit the melanin synthesis of about 21.0% or about 45.0% activity of (-)-robidanol. The difference in the activity may due to the differential activity of each compound to inhibit tyrosinase. Compared to (+)-epirobidanol and (-)-robidanol, 4-dehydroxyrobidanol did not inhibit melanin synthesis. 4-dehydroxyrobidanol increased the melanin synthesis on B16 cell of about 47.0%.
Three flavanol compounds were isolated from Intsia palembanica, namely (-)-robidanol, (+)-epirobidanol and 4-dehydroxyrobidanol. Isolated compounds along with 6 other flavonoid compounds were analyzed for tyrosinase inhibition and inhibition of melanin cell growth in B16 cell. (-)-Robidanol was the best compound as a whitening agent based on tyrosinase and melanin synthesis inhibitory activities.
We express our gratitude to the Faculty of Forestry Mulawarman University, East Kalimantan, Indonesia for preparation of the samples. We also thank Ms. Mary Grace Saldajeno for grammatical correction of the manuscript. This work was supported by Higher Education Directorate of the National Education Department of Republic of Indonesia (Hibah kompetitif penelitian untuk publikasi internasional No: 4472H/PP/DP2M/VI/2010) and the Chemistry of Natural Products Laboratory, Gifu University, Japan.