Oxidative stress caused by UV-generated singlet oxygen is the most significant
factor influencing human skin pigmentation (Hensley and
Floyd, 2002). The proinflammatory effects from oxidative stress in keratinocytes
lead to increased secretion of α-melanocyte stimulating hormone (α-MSH)
acting on melanocortin 1 receptor (MC1R) in neighboring melanocytes. Such stimulation
results upregulation of melanogenic proteins such as tyrosinase, leading to
increased melanin production. Moreover, the oxidative stress increases the activity
of protease activated receptor-2 (PAR-2) which stimulates distribution of melanosomes
and their uptake into keratinocytes (Seiberg et al.,
2000a). Accordingly, appropriate antioxidant compounds might reduce skin
There is increasing demand for active ingredients derived from natural sources,
which are perceived as safe and effectiveness. Plant phenols and polyphenols
are promising naturally-occurring compounds which are capable of reducing oxidative
stress (Bravo, 1998) and thus prevent pigmentation when
applied on skin (Cos et al., 1998). Indeed, this
has recently been shown that some phenolics can reduce skin pigmentation via
tyrosinase inhibition. For examples, catechin and epicatechin gallate can inhibit
tyrosinase activity by chelating the cupric ion within its active site (Kim
et al., 2004).
Tamarind (Tamarindus indica L., family Leguminosae) grows wild in Thailand
and its fruit pulps have been widely used as cosmetics purpose for many centuries.
In addition, various health benefits of the tamarind seed coat have also been
reported (Siddhuraju, 2007; Pumthong,
1999) but any effects on skin pigmentation remains unexplored. Extracts
of tamarind seed coat contain many polyphenols, including catechin, procyanidin
B2, epicatechin, procyanidin trimer, procyanidin tetramer, procyanidin pentamer
and procyanidin hexamer (Sudjaroen et al., 2005)
and thus may influence melanogenic and melanosomogenesis function activity.
Therefore, the present study aimed to assess the effect of tamarind seed coat
extract on melanin production in α-MSH-stimulated B16-F1 mouse melanoma
cells. To gain some insight into its action, tyrosinase and PAR-2 activities
in primary human skin cell were also investigated. The inhibitory effects obtained
support the idea that extract of tamarind seed coat might be a useful skin-hypopigmenting
MATERIALS AND METHODS
Materials: Ethyl acetate was purchased from LabScan Asia, Co. Ltd.,
Bangkok, Thailand. Folin-Ciocalteu reagent, alpha-melanocyte-stimulating hormone
(α-MSH), catechin, Triton x-100, L-DOPA, ethylene diamine tetraacetic acid
(EDTA), Dulbeccos Modified Eagles
Medium (DMEM, low glucose), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES) and sulfinpyrazone were purchased from Sigma-Aldrich, Inc., Missouri,
USA. Kojic acid was purchased from Sigma-Aldrich, Steinheim, Germany. Fetal
Bovine Serum (FBS), serum-free-keratinocyte medium (SFM) with supplements and
trypsin (0.25%) were purchased from GIBCO, Paisley, UK. Fluo-3 AM was purchased
from Molecular Probes Inc., Oregon, USA.
Preparation of the extract from tamarinds seed coat: The seeds
of tamarind were purchased from local market in Petchaboon Province, Thailand.
The studies were performed between the time periods of June 2009 to October
2010. The seeds were heated in a hot air oven at 140°C for 45 min, cooled
and cracked to separate the outside brown layer. Only brown-red seed coats were
collected and these were then ground into fine powder (Siddhuraju,
2007). Liquid extraction with ethyl acetate was used for seed coat extraction.
After tannin was removed by Sephadex LH20 (Sigma-Aldrich, Inc., Missouri, USA)
column, the extract solution was dried under vacuum (Pumthong,
1999). The resultant extract was stored in a tight-amber glass at 4°C
for further studies.
Quantification of total phenol in the extract: Phenolic content of the
extract was determined using Folin-Ciocalteu assay (Singleton
et al., 1999). Total phenolic content was expressed as Catechin Equivalents
(CE) (mg g-1 dry extract).
Effects of extract on melanogenesis in B16-F1 (mouse melanoma cell)
Cells and treatments: This study was designed to primarily screen the extract
for inhibitory action on melanin production by using B16-F1 (ATCC No. CRL-6323,
American Type Culture Collection, Virginia, USA) mouse melanoma cells, a well-known
model for determination of melanogenesis inhibitory activity (Donsing
et al., 2008; Siegrist and Eberle, 1986).
B16-F1 cells were initially cultured in a 25 cm2 flask with DMEM
supplemented with 10% FBS incubated at 37°C with a humid atmosphere containing
5% CO2. The medium was changed every two days and the number of passages
did not exceed 6.
To determine the effect of the extract on melanin production in α-MSH-stimulated
melanogenesis, the cell suspension was transferred from a 25 cm2
flask into a 24-well plate (1x105 cells per well). The old medium
was then replaced with 1 mL DMEM. Two protocols were tested (i) 1 nM α-MSH
(Morandini et al., 1998) was present for 48 h
and at 24 h, 50-200 μg mL-1 catechin equivalents was also added.
(ii) 50-200 μg mL-1 catechin equivalents was present throughout
the 48 h and 1 nM α-MSH added after the first 24 h. In addition, 50 μg
mL-1 kojic acid, a well-known lightening compound was also tested
using the same protocols. Each experiment was performed in triplicate. Control
wells contained cells stimulated with 1 nM α-MSH but no extract.
Melanin content assay: The melanin content assay was performed as previously
described with slight modifications (Donsing et al.,
2008; Mun et al., 2004). The assay based
on destroying the retractile cells leaving behind the melanin granules, which
could then be quantified spectrophotometrically. The cells within the wells
were trypsinized and washed twice with Phosphate Buffer Saline (PBS), air-dried,
dissolved in 200 μL of 1 N NaOH, heated at 80°C for 1 h and then cooled.
The melanin content was measured at 490 nm using a microplate reader (Spectra
Count®; Perkin Elmer Inc., Boston, Massachusetts, USA). The content
of melanin was calculated by comparing the averaged absorbance of the 3 wells
with that of the control cells and expressed as percentage.
Melanogenetic activity of the extract in human primary cells
Isolation of cells: Co-culture of isolated keratinocytes and melanocytes
were used as an in vitro model in this study. Keratinocytes and melanocytes
were isolated from foreskin tissues coming from humans aged≤3 years. Tissues
were incubated in 10 mL of 5% dispase solution at 4°C for 24 h and the epidermal
layer was then separated from the tissue using forceps. This epidermal layer
was cut into small pieces (2x2 mm) by using surgical blade and then incubated
in 4 mL of 0.25% trypsin solution at 37°C and under 5% CO2 for
20 min. Four milliliter of DMEM medium with 10% FBS were used to stop trypsin
reaction. The cell suspension containing both keratinocytes and melanocytes
was centrifuged at 1,500 rpm for 5 min and cells maintained in serum-free medium
with supplements under air/5% CO2 and at 37°C.
Determination of tyrosinase inhibitory activity from keratinocyte/ melanocyte
co-cultures: To determine inhibition of tyrosinase, the modified method
of (Huang et al., 2005) was used. A suspension
of cells from the primary cultures was transferred from a 25 cm2 flask
into a 24-well plate (1x104 cells per well) and then incubated with
the extract (50-500 μg mL-1) or kojic acid (0.5-500 μg
mL-1) at 37°C under 5% CO2 for 72 h. The cells were
washed with ice-cold PBS and then lysed by adding 0.5 mL PBS pH 6.8 containing
1% triton x-100 with sonication. A cell-free supernatant was collected by centrifugation
and tested for the tyrosinase activity. Ninety micro litters of each sample
solution adjusted to equal protein value were added in a 96-well plate. After
incubation at room temperature for 5 min, 10 μL substrate (10 mM L-DOPA)
was added to each well. After a further 30 min of incubation, the optical densities
of the L-DOPA oxidation product, dopachrome, produced by tyrosinase were measured
at 475 nm with the Spectra Count microplate reader. The concentration of the
extract giving 50% inhibition (IC50) was determined from plot of
percent inhibition against log concentration of the extract or kojic acid using
Prism (GraphPad, California, USA). Percent inhibition was calculated by using
the following equation:
|A(treatment) : Absorbance intensity of extract-treated
|A(control) : Absorbance intensity of untreated
The study was performed in three batches of keratinocyte-melanocyte co-cultures
isolated from one-skin tissues.
Intracellular calcium and PAR-2 activity: PAR-2 activity was assessed
by the rise in intracellular calcium (Ca2+) when released from the
endoplasmic reticulum by the action of PAR-2 (Bohm et
al., 1996). Briefly, the co-cultured cells were collected by using EDTA-containing
calcium-free isotonic PBS. The cells were suspended in DMEM containing 10%FBS
and 200 μL of this was added to black 96 well-plates. 0.25 mM sulfinpyrazone
and 1 μM fluo-3 AM were then added. The mixture was incubated at room temperature
for 20-25 min and the cells were then washed twice. Two hundred microliters
of assay buffer (150 mM NaCl, 3 mM KCl, 1.5 mM CaCl2, 20 mM HEPES,
10 mM glucose and 0.25 mM sulfinpyrazone) (untreated group), assay buffer plus
10 μM trypsin (as a PAR-2 activator) or assay buffer plus 200 μg mL-1
extract were added and incubated for a further 60 min.
Fluorescence was excited at 485 nm and emission measured at 530 nm using a
multimode detector (Beckman Coulter Inc., California, USA). The emission signal
was adjusted to 100% using the untreated cells and intracellular free calcium
expressed as a percentage assuming that this is linearly proportional to calcium
at low concentrations (<500 nM). The study was performed in three batches
of keratinocyte-melanocyte co-culture cells isolated from one-skin tissues.
Cell viability assay: Viability following treatment was determined by
counting the number of trypan blue positive cells in each well under x100 microscope.
The study was performed in triplicate to obtain average number of viable cells.
Statistic analysis: All quantitative data reported here are expressed
as mean±SD of samples for each treatment. Students
unpaired t-test was used for comparison between two groups. The p<0.05 was
The appearance and phenolic content of the extract: The crude extract
from tamarind seed coat after evaporation process was a brownish powder, as
shown in Fig. 1. The percent yield of the extract obtained
was 25.4±1.2% w/w. As determined by Folin-Ciocalteu assay, the amount
of the phenolic compounds contained in the extract was 85.6±0.9 mg CE
Effects of extract on melanogenesis in B16-F1 (mouse melanoma cell):
The melanogenesis activity of B16-F1 cells was investigated by determining content
of the melanin produced by cells. The extract showed the dose-dependent inhibition
and protection of melanin production of B16-F1 cells stimulated by α-MSH,
as shown in Fig. 2. Furthermore, the extract did not affect
viability of cells within the range of concentrations tested, as shown in Fig.
|| The appearance of tamarind seed coat crude extract
As compared to the α-MSH-stimulated cells without extract treatment, the
percentage of melanin reduction was about 20-32% in the cells treated with the
extract at high concentration (150-200 μg mL-1) after being
stimulated with α-MSH (inhibition condition) whereas the melanin reduction
was about 42-59% in the cells treated with the extract at the similar concentrations
before being stimulated with α-MSH (protection condition). For kojic acid
(50 μg mL-1), which was used as a reference inhibitor, the decrease
in melanin content was observed at about 50% in both inhibition and protection
Melanogenetic activity of the extract in human primary cells: In this
study, the co-culture of keratinocyte-melanocyte without any melanogenesis stimulator
was used in order to mimic the melano-epidermal activity.
||Tamarind seed coat extract reduced melanin production. B16-F1
melanoma cells were treated with the extract at concentrations in range
of 50-200 μg mL-1 after (inhibition, I) or before (protection,
P) being stimulated with α-MSH. Each bar represents Mean±SD
of triplicate study. *p<0.05 and **p<0.01 denote significant differences
when compared to control (Students t-test)
From the results obtained, IC50 value as determined by plotting
between the log concentration of the test agent and percent inhibition of dopachrome
formation was 152.1±10.2 μdg mL-1 for the extract whereas
that of the kojic acid was 33.3±2.5 μg mL-1 (Fig.
4). Percent inhibition of the extract at the highest concentration used
(500 μg mL-1) was 94.7% where as that of kojic acid was 96.6%.
Although, the extract showed lower inhibitory activity, it (at high concentrations,
200-500 μg mL-1) did not affect the viability and morphology
of human keratinocyte-melanocyte cells, as shown in Fig. 5.
Intracellular calcium and PAR-2 activity: The present study was designed
to examine the possibility that tamarinds seed coat extract could also
affect pigmentation by inhibiting the PAR-2 activity. The results from fluorescence
values of staining calcium expressed as a percentage are shown in Fig.
||The viability of B16-F1 mouse melanoma cells treated with
tamarind seed coat extract at concentrations in range of 50-200 μg
mL-1 for 24 h. Each bar represents Mean±SD of triplicate
|| Percent inhibition of tyrosinase activity by, (a) Tamarind
seed coat extract and (b) Kojic acid. The study was performed in three batches
of keratinocyte-melanocyte co-culture cells isolated from one-skin tissues
||The viability and morphology of human keratinocyte-melanocyte
co-culture treated with 200 μg mL-1 (open bar) of tamarind
seed coat extract and untreated (control) (filled bar) at day 0 and day
3. Each bar represents Mean±SD of study from three batches of keratinocyte-melanocyte
||Tamarind seed coat extract and intracellular calcium related
to PAR-2 activity. Trypsin (TRP, 10 μM) was used as a PAR-2 activator.
Each bar represents Mean±SD of three batches of keratinocyte-melanocyte
co-culture cells. **p<0.01 denotes significant differences when compared
to untreated cells (Students t-test)
The activation by TRP significantly promoted PAR-2 activity (p<0.01) as
compared to the untreated group. Tamarind seed coat extract at any concentration
used did not alter PAR-2 activity.
Plant phenols and polyphenols constitute an important group of naturally-occurring
antioxidants by virtue of the fact that the phenolic group can stabilize free
radicals which there is much supporting evidence for an antioxidative benefit
to skin (Cherniack, 2010). The fruit pulps of tamarind
are used as medicinal plant for centuries. Even though the seed coat possesses
antioxidant activity and other health beneficial effects, the activities of
bioactive compounds extracted from dry heated tamarind seed coat in hypopigmentation
purpose remain unexplored. In this study, the crude extract from tamarind seed
coat contained 85.6±0.9 mg phenolics expressed as catechin equivalents
per gram extract which accords with a previous study (Soong
and Barlow, 2004). The previous study showed that the extract showed antioxidant
scavenging activity on a variety of oxidants in a dose dependent manner (50-200
μg mL-1) (Siddhuraju, 2007). Therefore,
this concentration range was used in the present study.
The potential of any substance that can improve hyperpigmentation is reducing
total melanin content in the skin. To prove the potential of tamarind seed coats
extract in reducing melanin content, mouse melanoma cell line (B16-F1) is a
proper model because its released melanin can be clearly observed spectrophotometrically.
In the presence of extract (50-200 μg mL-1), there was a concentration
dependent reduction in melanin production, which depended on the protocol used.
The results show that extract dose-dependently caused some reduction in melanin
content of B16-F1 melanoma cells pre-stimulated by α-MSH. Furthermore,
the extract had no clear effect on cell viability within the range of concentrations
tested. This indicated that the reduction of melanin production was not due
to cell death. Interestingly, as compared to the α-MSH-stimulated cells
without extract treatment, the percentage of melanin reduction was about 20-32%
in the cells treated with the extract at high concentration (150-200 μg
mL-1) after being stimulated with α-MSH (inhibition condition)
whereas the melanin reduction was about 42-59% in the cells treated with the
extract at the similar concentrations before being stimulated with α-MSH
(prevention condition). For kojic acid (50 μg mL-1), which was
used as a reference inhibitor, the decrease in melanin content was observed
at about 50% in both inhibition and protection conditions. Our findings indicate
that the protection effect of the extract was greater than the inhibition effect.
This implies that the extract competes with both the downstream activation of
α-MSH and the upstream control of melanogenesis perhaps including α-MSH-MC1R
Other plant extracts and their bioactive constituents have been explored previously
for tyrosinase inhibitory activity (Yoon et al.,
2011; Momtaz et al., 2008). Polyphenols,
such as epicatechin, epigallocatechin and epicatechin-3-gallate, isolated from
plants proved to be effective inhibitors of tyrosinase. Thus polyphenols in
our extract may also inhibit tyrosinase and/or inhibit L-DOPA auto-oxidation.
Here, keratinocyte and melanocyte were co-cultured without any melanogenesis
stimulator thus mimicking in vivo melano-epidermal activity using dopachrome
formation from L-DOPA. The extract was clearly less potent (IC50
152.1±10.2 μg mL-1 compared to kojic acid (33.3±2.5
μg mL-1) although the maximum effects were similar (94.7% for
extract and 96.6% kojic acid). The morphology of both the keratinocytes and
melanocytes appeared to be unaffected by the extract (200 μg mL-1).
Furthermore, the cell counts were unchanged in the culture conditions suggesting
that the reduced tyrosinase activity did not result from cell death or reduced
cell replication. This might include chelation of copper ion within the tyrosinase
or the phenolic hydroxyls binding to the enzyme causing stearic hindrance (Yoon
et al., 2011). For kojic acid treated group, we found that kojic
acid at high concentrations (≥100 μg mL-1) caused changes
in cell morphology and such changes might influence the tyrosinase amount and
The melanogenesis steps in skin include melanin synthesis and transportation
of melanin from melanocyte to keratinocyte. For this reason, the present study
was designed to examine the possibility that tamarinds seed coat extract
could also affect pigmentation by inhibiting the PAR-2 activity. PAR-2 is expressed
mainly in keratinocyte and increased activity of PAR-2 in keratinocytes causes
increasing uptake and distribution of melanosomes by keratinocytes in the epidermis.
Keratinocyte-melanocyte co-culture was therefore used to mimic the physiological
situation happening when melanocytes and keratinocytes co-operate for the transfer
of melanins. The natural PAR-2 activator (trypsin, TRP) was used to determine
the receptor activity and also consistency of keratinocyte-melanocyte distribution
of each batch of isolated co-culture. The results from fluorescence values of
staining calcium expressed as a percentage are shown in Fig. 6.
As similar to another study (Paine et al., 2001),
the activation by TRP significantly promoted PAR-2 activity (p<0.01) as compared
to the untreated group. Tamarinds seed coat extract at any concentration
used did not alter PAR-2 activity. Although the direct inhibitory effect of
the extract on PAR-2 was not observed, it cannot not be concluded that the extract
does not affect melanosome transfer. Recent studies indicate that the skin depigmentation
of soymilk and the soybean-derived serine protease inhibitors is not directly
involved with receptor inhibition but correlates with blocking action of trypsin,
resulting in a decreased transfer of melanosome (Seiberg
et al., 2000b; Paine et al., 2001).
Therefore, further study should be performed to determine depigmentation by
extract via inhibition of the PAR-2 pathway.
In conclusion, this study clearly showed that melanogenesis could be inhibited
by an extract of the seed coat of tamarind in both a melanoma cell line (B16-F1)
and in primary cells from human melanocytes co-cultured with physiological partners,
keratinocytes. The extract was also able to completely inhibit tyrosinase prepared
from the human keratinocyte/melanocyte cultures over the same concentration
range. This extract can form the basis of further purification and refinement
with the ultimate goal of creating skin depigmentating products.
Financial and facility supports from the Center of Excellence for Innovation
in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of Education
are gratefully acknowledged. In addition, we also thank Dr. C. Norman Scholfield
for useful discussions.