Melanogenic inhibition and toxicity assessment of flavokawain A and B on B16/F10 melanoma cells and zebrafish (Danio rerio)

Article abstract of DOI:10.3390/molecules25153403

Excessive production of melanin implicates hyperpigmentation disorders. Flavokawain A (FLA) and flavokawain B (FLB) have been reported with anti-melanogenic activity, but their melanogenic inhibition and toxicity effects on the vertebrate model of zebrafish are still unknown. In the present study, cytotoxic as well as melanogenic effects of FLA and FLB on cellular melanin content and tyrosinase activity were evaluated in α-MSH-induced B16/F10 cells. Master regulator of microphthalmia-associated transcription factor (Mitf) and the other downstream melanogenic-related genes were verified via quantitative real time PCR (qPCR). Toxicity assessment and melanogenesis inhibition on zebrafish model was further observed. FLA and FLB significantly reduced the specific cellular melanin content by 4.3-fold and 9.6-fold decrement, respectively in α-MSH-induced B16/F10 cells. Concomitantly, FLA significantly reduced the specific cellular tyrosinase activity by 7-fold whilst FLB by 9-fold. The decrement of melanin production and tyrosinase activity were correlated with the mRNA suppression of Mitf which in turn down-regulate Tyr, Trp-1 and Trp-2. FLA and FLB exhibited non-toxic effects on the zebrafish model at 25 and 6.25 μM, respectively. Further experiments on the zebrafish model demonstrated successful phenotype-based depigmenting activity of FLA and FLB under induced melanogenesis. To sum up, our findings provide an important first key step for both of the chalcone derivatives to be further studied and developed as potent depigmenting agents.

Full text of DOI:10.3390/molecules25153403

molecules
Article
Melanogenic Inhibition and Toxicity Assessment of
Flavokawain A and B on B16/F10 Melanoma Cells and
Zebrafish (Danio rerio)
Nurshafika Mohd Sakeh 1 , Nurliyana Najwa Md Razip 1 , Farah Idayu Mohd Ma’in 1,
Mohammad Nazri Abdul Bahari 1, Naimah Latif 1, Muhammad Nadeem Akhtar 2,
Zetty Norhana Balia Yusof 1 and Syahida Ahmad 1,
*
1
Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia,
UPM Serdang , Serdang 43400, Selangor, Malaysia; shafikams@gmail.com (N.M.S.);
najwabiochem@gmail.com (N.N.M.R.); farahidayu1991@gmail.com (F.I.M.M.);
nazrischolar@gmail.com (M.N.A.B.); naimah_latif@yahoo.com (N.L.); zettynorhana@upm.edu.my (Z.N.B.Y.)
Faculty of Industrial Sciences & Technology, Universiti Malaysia Pahang, Lebuhraya Tun Razak, Gambang,
Kuantan 26300, Pahang, Malaysia; nadeem@ump.edu.my
2
*
Correspondence: syahida@upm.edu.my; Tel.: +603-89466699; Fax: +603-89467590
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Alexandru Mihai Grumezescu
Received: 19 May 2020; Accepted: 2 July 2020; Published: 28 July 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Excessive production of melanin implicates hyperpigmentation disorders. Flavokawain
A (FLA) and flavokawain B (FLB) have been reported with anti-melanogenic activity, but their
melanogenic inhibition and toxicity effects on the vertebrate model of zebrafish are still unknown.
In the present study, cytotoxic as well as melanogenic effects of FLA and FLB on cellular melanin
content and tyrosinase activity were evaluated in α-MSH-induced B16/F10 cells. Master regulator of
microphthalmia-associated transcription factor (Mitf) and the other downstream melanogenic-related
genes were verified via quantitative real time PCR (qPCR). Toxicity assessment and melanogenesis
inhibition on zebrafish model was further observed. FLA and FLB significantly reduced the
specific cellular melanin content by 4.3-fold and 9.6-fold decrement, respectively in α-MSH-induced
B16/F10 cells. Concomitantly, FLA significantly reduced the specific cellular tyrosinase activity by
7-fold whilst FLB by 9-fold. The decrement of melanin production and tyrosinase activity were
correlated with the mRNA suppression of Mitf which in turn down-regulate Tyr, Trp-1 and Trp-2.
FLA and FLB exhibited non-toxic effects on the zebrafish model at 25 and 6.25 µM, respectively.
Further experiments on the zebrafish model demonstrated successful phenotype-based depigmenting
activity of FLA and FLB under induced melanogenesis. To sum up, our findings provide an important
first key step for both of the chalcone derivatives to be further studied and developed as potent
depigmenting agents.
Keywords: α-MSH; B16/F10 melanoma; chalcone; flavokawain; melanogenesis; zebrafish
1. Introduction
Impairment of mechanisms regulating melanogenesis may cause overproduction of melanin,
resulting in various hyperpigmentation disorders. In addition to excessive exposure to ultraviolet
radiation (UVR), post-inflammatory melanoderma occurring in healing after inflammation [
involuntaryactivationofalpha-melanocytestimulatinghormone( -MSH)receptorsbyadrenocorticotropic
hormone (ACTH) associated to Addison’s disease may result in hyperpigmentation [ ]. Melanocortin
1 receptor (MC1R), a G-protein-coupled receptor located in the membrane of melanocytes is responsible
in regulating skin pigmentation [ ]. Both -MSH and ACTH are melanocortins derived from proteolytic
cleavage of pro-opiomelanocortin (POMC) which are endogenous agonists for MC1R [ ]. Activation
1–3] and
α
3,4
5
α
3,6
Molecules 2020, 25, 3403; doi:10.3390/molecules25153403
www.mdpi.com/journal/molecules

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of MC1R triggers signaling cascade of cyclic adenosine monophosphate (cAMP) pathway through
stimulation of adenylate cyclase which leads to activation of protein kinase A (PKA). Activated
PKA further phosphorylates cAMP-responsive element binding (CREB) transcription factor which
in turn, promotes transcriptional activation of microphtahlmia-associated transcription factor (Mitf) [7].
Mitf gene plays important role as master regulator of melanogenesis. It is essentially associated with the
regulation of three other melanogenic-related genes namely tyrosinase (Tyr), tyrosinase-related protein
1 (Trp-1) and tyrosinase-related protein 2 (Trp-2) via E-box of Mitf basic-helix-loop-helix-leucine-zipper
(bHLH-LZ) [8,9]. Melanin, represented as eumelanin, pheomelanin, neuromelanin, and mixed
melanin are the end products of complex metabolic steps that initiated from amino acid l-tyrosine
precursor [10]. Tyrosinase (EC 1.14.18.1) plays a crucial role as key regulatory as well as rate-limiting
enzyme in converting l-tyrosine into substrates that finally form melanin. Dopachrome tautomerase
which is abbreviated as Trp-2 functions in isomerization of intermediate 5,6-dihydroxyindole (DHI) to
5,6-dihydroxyindole-2-carboxylic acid (DHICA) [11]. Subsequently, Trp-1 also known as DHICA oxidase,
converts DHICA to indole-5,6-quinone-carboxylic acid, catalyzing eumelanogenesis [12,13].
Abnormalities of skin tone due to pigmenting disorders results in formidable emotional and social
problems [14
,15]. The earliest established depigmenting agent, hydroquinone demonstrated strong
depigmentation effect [16
,17], but also raised the concerns of carcinogenicity and mutagenicity [18],
and was known to permanently caused damage to melanocytes [19]. Kojic acid and arbutin which
are structurally similar to l-DOPA and l-tyrosine, were later discovered as tyrosinase inhibitors
by chelating copper in tyrosinase’s active site [20–22]. However, only arbutin is widely being
used by cosmetic consumers since kojic acid was reported to promote hypersensitivity in skin [23],
hepatocarcinogenesis [24] and mutagenic [25]. Hence, the elevation of public concern on drugs safety
has augment the development of effective yet harmless depigmenting agents.
Toxicology assessment requires large, laborious, time consuming and expensive preclinical trials
utilizing in vivo models such as mice and guinea pigs. On the contrary, zebrafish (Danio rerio) model
has been identified as an ideal model organism for large screening due to their smaller in size, easy to
maintain, readily reproducible in laboratories, externally and rapidly developing transparent embryos
permitting live observations with high throughput chemical screening [26]. The zebrafish model
has also been extensively used as a resort to toxicity assessment of drug compounds [27–29] and
melanogenesis studies since the lower vertebrate is highly comparable to human melanogenesis [30].
The presence of melanin pigments on surface of zebrafish larvae allow ready observation in testing
percutaneous effect of depigmenting agents [31]. Moreover, screening of embryotoxicity of drugs or
small molecules involves rapid and simple procedure whereby compounds diluted in embryo media
is readily absorbed through skin and gills during early stage of embryo [32,33].
Flavokawain A (FLA) and flavokawain B (FLB) were naturally occurring kava chalcones first extracted
from Kava (Piper methysticum), a plant grown in the Pacific Islands [34]. FLA and FLB were then reported to
havesignificanteffectsasanti-cancer[35,36], antinociceptive[37]andanti-inflammatory[38,39]. FLAandFLB
have been reported to have anti-tyrosinase activity in our previous study [40]. However, the depigmenting
mechanisms on B16/F10 cells have only been reported on FLB, but not on FLA. Thus, in the present study
the anti-melanogenic activity of FLA and FLB were evaluated on α-MSH-induced B16/F10 melanoma cells
by validating the expression level of Mitf, Tyr, Trp-1 and Trp-2. Toxicity assessments (embryotoxicity and
teratogenicity) and in vivo phenotype-based depigmenting effects were also performed on zebrafish model
to further elucidate FLA and FLB as potent yet safe depigmenting agents.
2. Results
2.1. Cytotoxic Effect of Chalcone Derivatives on B16/F10 Cells
The toxicity effects of FLA and FLB on α-MSH-induced B16/F10 cells are illustrated as in Figure 1.
In the present study, a commercialized depigmenting agent (namely arbutin) was also evaluated for
its cytotoxic effect. Both FLA and FLB showed no significant toxicity effect towards induced B16/F10

Molecules 2020, 25, 3403
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cells after incubation for 72 h up to concentration 25
µ
M with cell viability of 86.66%
M, FLA and FLB showed significant cytotoxic
±
1.78 (FLA) and
85.81%
effect with cell viability decreased to 85.26%
demonstrated that FLA showed lesser cytotoxic effect than FLB. On the other hand, arbutin (positive
control) showed no significant cytotoxic effect with more than 90% of cell viability at 50 M. Control
was set as α-MSH-induced B16/F10 in 0.1% DMSO which serve as carrier solvent of compounds.
±
0.78 (FLB). At highest concentration of 50
µ
±
1.35 and 80.41%
±
1.75, respectively. The results
µ
Figure 1. Cell viability of
Toxicity effects of FLA and FLB were determined with respect to induced control by MTT assay.
FLA and FLB were tested for cytotoxic effect at different concentrations of 0.78–50 M. Arbutin (50 M)
was used as positive control. All values are the mean S.E.M. of three independent experiments. * p <
α-MSH induced B16/F10 cells after 72 h treatment with FLA and FLB.
µ
µ
±
0.05 and ** p < 0.01 were considered significantly different to α-MSH-induced control group.
2.2. Inhibitory Effect of Chalcone Derivatives on Cellular Melanin Content
Melanin is the final product of a complete melanin biosynthesis pathway. A potent depigmenting
agent is described with respect of decrement in melanin formation. The depigmenting effect of both
FLA and FLB on
-MSH results in significant elevation of cellular melanin production. In order to study the decrement
production of cellular melanin content, induced B16/F10 cells were treated with FLA and FLB by 4-fold
serial dilution of compounds (25, 6.25 and 1.56 M). Results demonstrated high extent of depigmenting
effect of FLA (0.84 0.06 g melanin/ g protein) and FLB (0.38 0.03 g melanin/ g protein) at
highest concentration of 25 M. In the absence of compounds, -MSH-induced B16/F10 cells produced
3.75 0.05 g melanin/ g protein. It was also found that both compounds showed inhibition towards
α-MSH-induced B16/F10 cells is described as in Figure 2. Induced melanogenesis by
α
µ
±
µ
µ
±
µ
µ
µ
α
±
µ
µ
cellular melanin production in dose-dependent manner. Interestingly, cellular melanin content of
B16/F10 cells were reduced albeit at the lowest concentration of 1.56 µM.

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Figure 2. Effect of chalcone derivatives on melanin production of
Cellular melanin content after treated with ( ) FLA and ( ) FLB. Both compounds were tested at
different concentrations (25, 6.25 and 1.56 M) and incubated for 72 h. Arbutin (50 M) was used as
positive control. All values are the mean S.E.M. of three independent experiments. ** p < 0.01 and
α-MSH-induced B16/F10 cells.
A
B
µ
µ
±
*** p < 0.01 were considered significantly different to α-MSH-induced control group.
2.3. Inhibitory Effect of Chalcone Derivatives on Cellular Tyrosinase Activity
Tyrosinase plays a major role in the initial biosynthesis reaction of melanogenesis. Melanogenic
inhibition targeting on tyrosinase has been a major interest in developing new depigmenting agent.
The melanogenic effect of chalcone derivatives was evaluated on cellular tyrosinase activity of
α
-MSH-induced B16/F10 cells (Figure 3). In the study, enzyme activity was measured by using
l-DOPA as enzyme substrate while cells were treated in 4-fold serial dilution of compounds dosages
descending from 25 to 1.56 M. The induced tyrosinase activity of cells was observed to decline
tremendously with the treatment of FLA (1.23 0.04 U/ g protein) and FLB (0.96 0.02 U/
protein) at highest concentration of 25 M. In the absence of compounds, -MSH-induced B16/F10
cells exhibited 8.64 0.15 U/ g protein of tyrosinase activity. It was also demonstrated that enzyme
µ
±
µ
µ
±
µ
µg
µ
α
±
µ
µ
inhibitory activity of FLA and FLB exhibited a dose-dependent manner in which significant inhibition
was evident up to 6.25 µM.

Molecules 2020, 25, 3403
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Figure 3. Effect of chalcone derivatives on cellular tyrosinase activity of
Cellular tyrosinase activity after treated with ( ) FLA and ( ) FLB. Both compounds were tested at
different concentrations (25, 6.25 and 1.56 M) and incubated for 72 h. Arbutin (50 M) was used
as positive control. All values are the mean S.E.M. of three independent experiments. A value of
*** p < 0.001, was considered significantly different to α-MSH-induced control group.
α-MSH-induced B16/F10 cells.
A
B
µ
µ
±
2.4. Down-Regulation Effect of Chalcone Derivatives on Tyr, Trp-1, Trp-2 and Mitf Genes Expression in
B16/F10 Cells
Mitf as well as Tyr, Trp-1, and Trp-2 genes are well-studied to regulate majorly in melanogenesis.
The effect of FLA and FLB on mechanism of melanin biosynthesis pathway was elucidated on Tyr,
Trp-1, Trp-2 and Mitf genes expression (Figure 4). In the present study, B16/F10 cells were exposed to
various concentrations of chalcone derivatives (1.56, 6.25 and 25
Trp-1 and Trp-2 genes in B16/F10 cells was observed after treatment with
a dose-dependent manner of gene suppression was observed on Tyr, Trp-1, Trp-2 and Mitf genes
after treatment with FLA and FLB. At highest concentration of 25 M, both FLA and FLB showed
suppression on Tyr gene with 0.20 0.01 and 0.15 0.04-fold expression respectively. Suppression
on the master regulator of Mitf gene was also found significant on both FLA and FLB at highest
concentration of 25 M with 0.41 0.07 and 0.32 0.03-fold expression, respectively. The other
melanogenic-related genes of Trp-1 and Trp-2 demonstrated a similar pattern of gene suppression by
both FLA and FLB. However, the genes were only significantly downregulated up to 6.25 M by FLB.
Arbutin at 50 M was set as positive control and showed significant downregulation on all genes.
µM). Induced expression of Mitf, Tyr,
α
-MSH for 72 h. Interestingly,
µ
±
±
µ
±
±
µ
µ

Molecules 2020, 25, 3403
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In the assay, induced group (control) was set at 1.00-fold expression and the expression of all genes of
interest was normalized in the reference of endogenous genes transcript amount (GAPDH and ß-actin).
Figure 4. Effect of chalcone derivatives on the mRNA expression of melanogenic-related genes:
tyrosinase (Tyr), tyrosine-related protein 1 and 2 (Trp-1 and -2) and microphthalmia-associated
transcription factor (Mitf). B16/F10 cells (2
for 72 h in the presence or absence of FLA and FLB. Total RNA was extracted, and mRNA expression
×
105 cells/well) were treated with 10 nM of
α-MSH
was analyzed by RT-qPCR. The fold expression results shown in figure were normalized by GAPDH
and ß-actin mRNA levels. Data are expressed as the mean
of ** p < 0.01 and *** p < 0.001 were considered significantly different to α-MSH-induced control group.
±
SEM of three separate experiments. A value
2.5. Zebrafish Toxicity Assessment
3-isobutyl-1-methylxanthine (IBMX), a chemical compound was used in the experiment to increase
melanin production regulated by the same pathway as -MSH [41 42]. Figure 5 showed the survival
rates of zebrafish post-treated with 10-fold serially diluted FLA and FLB (0.78–50 M) in 0.1% DMSO
with the presence of IBMX inducer at 100 M. The induced group which consisted of zebrafish embryo
in embryo media with 0.1% DMSO and IBMX (100 M) demonstrated 100% survival rate until the end
α
,
µ
µ
µ
of testing period. Twelve embryos were selected and incubated at 9–144 hours-post-fertilization (hpf).
The survival rate of zebrafish was concluded based on the presence of heart beat visual and the
absence of teratogenic effect. Based on the heart beat visual, the findings demonstrated high survival
rate of zebrafish with higher than 80% when treated with FLA from 0.78–25
0.78–12.5 M. However, teratogenic effect has been detected on zebrafish treated with FLB at 12.5
Hence, the optimal concentrations of FLA and FLB for melanogenic inhibitory tests were determined
at 25 M and 6.25 M, respectively. The LC50 values calculated for FLA and FLB were at 45 M and
25 µM, respectively.
µM while FLB from
µ
µM.
µ
µ
µ

Molecules 2020, 25, 3403
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A)
B)
Figure 5. Toxicity effect of chalcone derivatives on zebrafish embryos. Embryos were incubated in
seven different concentrations (0.78 M–50 M) of ( ) FLA and ( ) FLB. Control was set as embryos
µ
µ
A
B
treated in embryo media with 0.1% DMSO. The survival rates were recorded every 24 h until 144 h by
using inverted microscope (Nikon TS100). Twelve fertilized eggs at 9 h post fertilization were selected
for each concentration tested and control. Each value in the table is represented as mean
±
S.E.M (n = 4).
2.6. Zebrafish Phenotype-Based of Melanogenic Inhibition by FLA and FLB
Synchronized embryos collected at 9 hpf were treated with FLA and FLB at 25 µM and 6.25 µM,
respectively (Figure 6). Uninduced zebrafish group demonstrated normal melanogenesis of zebrafish
in embryo media with the presence of 0.1% DMSO. Induced zebrafish group treated with IBMX
at 100
200 M (positive control), melanin production of zebrafish was successfully inhibited from 9 to 96 hpf.
After PTU-washed off and treatment with IBMX at 96 hpf, the zebrafish pigmentation of positive control
was found recovered at 144 hpf. Similarly, pigmentation of zebrafish was inhibited with PTU (200 M)
for FLA and FLB treatment groups from 9 to 96 hpf. The zebrafish depigmenting of both groups was
revoked by substitution of PTU with IBMX (100 M) melanin inducer. The melanin recovery exhibited
µM exhibited excessive production of melanin. In the presence of phenylthiourea (PTU) at
µ
µ
µ
by zebrafish groups treated with FLA and FLB after PTU-washed off and IBMX-induced at 96 hpf,
was lower as compared to both positive and negative controls. The results indicate the ability of both
compounds to inhibit and/or reduce the zebrafish melanogenesis.

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Figure 6. Phenotype-based melanogenesis inhibition of chalcone derivatives on zebrafish model.
Synchronized zebrafish embryos were collected and maintained with ( ) embryo media with 0.1%
DMSO (uninduced) and ( ) IBMX (100 M) in embryo media with 0.1% DMSO (induced). Zebrafish
embryos ( ) were treated with PTU (200 M) from 9–96 hpf. Treatment of ( ) FLA at 25 M and
) FLB at 6.25 M was carried out at 96 hpf after PTU-washed off and treated simultaneously with
IBMX at 100 µM.
A
B
µ
C
–
E
µ
D
µ
(
E
µ
3. Discussion
Researchers have been addressing tyrosinase inhibition as crucial target to reduce excessive production
of melanin which causes hyperpigmentation [43 44]. Flavonoids are a group of most studied polyphenols
with the ability of chelating copper ions in tyrosinase’s active site [45 46]. According to the new classification
of polyphenols (Phenol-Explorer Version 3.6 database, www.phenol-explorer.eu), chalcones are considered
as a subclass of flavonoids [47]. In addition to the anti-tyrosinase property [40 48 49], chalcones and their
,
,
,
,
derivatives have been reported to possess various biological and pharmacological activities including
anti-inflammatory, antipyretic, analgesic, bactericidal, insecticidal, anti-fungal anticancer and antioxidant [50].
Flavokawains used in the study are a class of chalcones found on kava plants, which were synthesized
by base catalyzed Claisen–Schmidt condensation (Figure 7A). In our preliminary studies, anti-tyrosinase
activity and structural activity relationship of FLA (IC50 = 14.26
have been evaluated [40]. The findings are consistent with reports that suggested 4-subtituted methoxy
group in B ring of FLA significantly contributes to better anti-tyrosinase activity of chalcones [48 51].
±
0.08
µM) and FLB (IC50 = 14.38
±
0.12 µM)
,
Tyrosinase is a copper-containing enzyme which plays a crucial role as rate-regulator of melanin synthesis
pathway whereby it catalyzes the hydroxylation of l-tyrosine precursor to l-DOPA and further oxidized
to dopaquinone [52]. It was postulated that the amount of tyrosinase activity, is equal to the amount of
melanin synthesized [53]. Due to this vital fact, development of new whitening agent has been targeting
tyrosinase in the aim to combat various melanogenic-related diseases. It was suggested that the prominent
inhibitory activities of chalcone on tyrosinase were due to its resemblance structure with the substrates of
tyrosinase, namely tyrosine and l-DOPA (Figure 7B).

Molecules 2020, 25, 3403
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A)
R
B)
H3CO
OCH3
OH
O
Chalcone derivatives
OH
OH
OH
NH2
NH2
HO
HO
O
O
L-DOPA
Tyrosine
Figure 7. Synthesis of chalcone derivatives and chemical structures of compounds. (
A) Synthesis
of Flavokawain A (FLA) and flavokawain B (FLB). ( ) Skeleton structure of chalcone resembles the
B
skeleton structures of tyrosinase substrates which are tyrosine and l-DOPA.
In the early efforts to determine the potential of FLA and FLB as safe and reliable depigmenting agents,
the cytotoxic effects of both compounds were characterized on
α-MSH-induced B16/F10 melanoma cells.
It was shown that FLA and FLB did not exert cytotoxic effects until at 25
µM which allow further
characterization of depigmenting activity on B16/F10 cells. Interestingly the functional property of both
FLA and FLB as depigmenting compounds were successfully elucidated through in vitro study on B16/F10
cells revealing reduced melanin production in a dose-dependent manner. Jeong et al. [54] has previously
reported the melanogenesis inhibition activity of FLB and FLC, but the FLA was found to exhibit no
melanin inhibitory activity. Although the cellular melanin content was tested similarly in MSH-activated
B16 melanoma cells, the authors did not determine the cellular tyrosinase inhibition activity of FLA
and FLB. In this study, further in vitro evaluation of melanogenic inhibition by FLA and FLB was carried
out by evaluating the inhibitory activity on cellular tyrosinase. Treatment of FLA and FLB has clearly
showed decrement of
α-MSH-induced cellular tyrosinase activity in B16/F10 cells. Our findings were able
to correlate the successful inhibition of cellular tyrosinase activity with the reduction of cellular melanin

Molecules 2020, 25, 3403
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content. At transcriptional level, our findings showed that both FLA and FLB significantly suppressed the
melanogenic master regulator of Mitf gene expression which in turn suggesting the down-regulation of
other melanogenic-related genes including Tyr, Trp-1 and Trp-2 [55]. The successful suppression of Mitf
determines the effectiveness of FLA and FLB as potent depigmenting agents since Mitf is essential in
melanin-bearing pigment cells across species and tissue types [55
implication of FLA and FLB in mechanistic inhibition on cAMP/ -MSH mechanism pathway. Interestingly,
both in vitro results suggested FLB as a better anti-melanogenic candidate as compared to FLA.
-MSH is one of bioactive melanocortin family peptides which derived from larger precursor
peptide of POMC that is present in melanocytes and keratinocytes [57]. It was well-known that
the minute concentration of -MSH interacts simultaneously through cAMP second messenger via
MC1R, which will then promote melanogenesis [58]. Under normal circumstances, over-exposure
of UVR may activate excessive amount of -MSH which results in the induced melanogenesis [ ].
However, the pigmentary hormone of -MSH has been reported to interfere normal biological function
causing increased metabolic rate and cardiovascular dynamics in vertebrate [59 60]. Extracellular
stimulus of IBMX results in hyperpigmentation by inducing cAMP which activates similar pathway
caused by -MSH [41 61]. Hence, IBMX was used as melanogenic inducer agent in the zebrafish
model to further evaluate the phenotype-based toxicity and depigmenting activity of FLA and FLB.
FLA demonstrated no toxicity on zebrafish embryo at 25 M, which was the same concentration when
tested in B16/F10 cells. However, FLB was only found non-toxic at 6.25 M when tested on zebrafish
,56]. The outcome has suggested the
α
α
α
α
3,7
α
,
α
,
µ
µ
embryo. The toxicity effect of FLA and FLB on zebrafish model may be influenced by factors including
absorption and exposure time [26]. The higher toxicity effect of FLB as compared to FLA was in line
with other reports [62,63], and zebrafish has become a more powerful approach as toxicological model
with high genetic homology to mammals [64]. Intriguingly, the melanogenic inhibition of both FLA
and FLB was also found effective on zebrafish vertebrate model even though the concentration used
for FLB treatment was 2-fold lower than tested in B16/F10 cells. Both compounds were able to inhibit
the production of melanin in zebrafish in the presence of IBMX melanin inducer agent.
In summary, FLA and FLB demonstrated no toxicity effect in B16/F10 cells and zebrafish at 25
and 6.25 µM, respectively. Our findings showed that both compounds were also efficient in inhibiting
melanogenesis up to molecular level by suppressing the expression patterns of master regulator Mitf
which concomitantly decreased the expression of other melanogenic-related genes including Tyr, Trp-1
and Trp-2. Elucidation of the downstream regulations by the depigmenting agents as well as its
elaborated toxicity assessment are important as the first step in the development of whitening agents
to treat hyperpigmentation diseases. Zebrafish toxicological model was utilized to further evaluate
the toxicity assessment of FLA and FLB in association with the depigmenting effect. Our findings
offer the refinement of both compounds as reliable and competent melanogenic inhibitors in zebrafish
model. To sum up, both chalcone derivatives of FLA and FLB have the potential to be developed as
depigmenting agents for cosmetic or health purposes.
4. Materials and Methods
4.1. General Procedure for Preparation of Analogues
FLA and FLB were synthesized by Claisen-Schmidt condensation of respective acetophenone
and benzaldehyde [40]. An effective straight-forward synthesis of desired chalcone derivatives were
illustrated as in Figure 7A. Products were finally purified by using flash column chromatography,
with ratio of ethylacetate:hexane of 1:1. FLA and FLB were then treated with MeOH to yield yellow
recrystallized compounds. Compounds were characterized based on spectroscopic techniques such as
IR, UV, EI-MS and NMR data, consistent as previously reported [40].

Molecules 2020, 25, 3403
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4.1.1. (E)-1-(20-Hydroxy-40,60-dimethoxyphenyl)-3-(4-methoxyphenylprop-2-en-1-one: (FLA)
Yellow needles crystals. Yield 82.4%. m.p 110–112 ◦C. (Molecular formula C18H18O5). IR (KBr):
1
1654 (C=O), 3200 (Ar C-H str.), 1590, 1589, 1476, (C=C), 1202 (C-O str.), 850 cm−1. H-NMR (CDCl3,
500 MHz):
H-2, 6), 7.49 (d, 2H, H-3, 5), 6.87 (br s, 1H, H-5’), 6.02 (br s, 1H, H-3’), 3.93 (s, 3H, OMe, C-6’), 3.85 (s,
3H, OMe, C-4’), 3.82 (s, 3H, OMe, C-4). 13C-NMR (CDCl3, 125 MHz):
192.6 (C=O), 168.7 (C2’), 166.2
(C4’), 163.1 (C6’), 160.9 (C4), 141.6 (C ), 130.1(C2, C6), 127.5 (C1), 125.4 (C ), 114.3 (C3, C5), 93.56 (C3’),
91.4 (C5’), 55.79 (OCH3), 55.53 (OCH3), 55.35 (OCH3). EI-MS m/z 298.33.
δ 13.6 (s, 1H, OH), 8.02 (d, 1H, J = 15.5 Hz, Hβ), 7.81 (d, 1H, J = 15.5 Hz, Hα), 7.73 (brd, 2H,
δ
β
α
4.1.2. (E)-1-(20-Hydroxy-40,60-dimethoxyphenyl)-3-phenylprop-2-en-1-one:(FLB)
Yellow needles crystals: Yield 82.4%, m.p. 102–104 ◦C. (Molecular formula C17H16O4). IR (KBr):
1
3060 (Ar C-H str.), 1642 (C=O), 1589, 1588, 1478, (C=C ring), 1202 (C-O) cm−1. H-NMR (CDCl3, 500
MHz):
6), 7.48 (d, 3H, H-3, 4, 5), 6.98 (br s, 1H, H-5’), 6.21 (br s, 1H, H-3’), 3.93 (s, 3H, OMe, C-6’), 3.85 (s, 3H,
OMe, C-4’). 13C-NMR (CDCl3, 125 MHz):
193.2 (C=O), 168.9 (C2’), 166.7 (C4’), 162.5 (C6’), 142.51
(C ), 141.0 (C4), 132.9 (C1), 130.2 (C2, C6), 128.1 (C3, C5), 126.6 (C ), 108.14 (C1’), 93.6 (C3’), 90.94
δ 14.3 (s, 1H, OH), 7.92 (d, 1H, J = 15.0 Hz, Hβ), 7.80 (d, 1H, J = 15.0 Hz, Hα), 7.64 (brd, 2H, H-2,
δ
β
α
(C5’), 55.53 (OCH3), 55.7 (OCH3). EI-MS m/z 284.23.
4.2. Cell Culture and Treatment
The B16/F10 murine melanoma cells were purchased from the American Type Culture Collection
(ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco’s Modified Eagle‘s Medium
(DMEM, Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS) (Gibco,
Life Technologies, Waltham, MA, USA), 2 mM l-Glutamine, 4.5 g/L glucose and antibiotics 100 U/mL
of penicillin/streptomycin (Gibco, Life Technologies, Carlsbad, CA, USA). The cell line was cultured in
75 cm2 flask (TPP, Trasadingen, Switzerland) at 37 ◦C supplied with 5% carbon dioxide (CO2) in fully
humidified air. B16/F10 melanoma was adherent cells whereby cells are attached to the surface of flask.
The culture medium was changed every alternate day. Cells were harvested by trypsinization using
1 TrypLE Express (Gibco, Life Technologies, Carlsbad, CA, USA). The subcultures were prepared by
×
trypsinization and the sub cultivation ratio was between 1:2 to 1:4 (culture:media). Cells were seeded
with appropriate seed density for 24 h and then treated with various concentrations of drugs in the
presence of 10 nM α-MSH (melanin inducer). Blank control was set as cells in culture media DMEM
only. Cells were harvested after 72 h post-treatment (hpt).
4.3. Cell Viability Test
Cell viability test using MTT assay was performed in order to examine the viability of cells upon
treatment with different concentrations of samples. This assay depends on the ability of cells to reduce
the MTT reagent (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) to a purple formazan
product. Cells were seeded (1
×
105 cell/well) in 96-well plate (TPP, Trasadingen, Switzerland) for
24 h and replaced with new culture media containing different concentrations of drugs. Mixed media
was removed 72 hpt, and replaced with 5 mg/mL MTT reagent in phosphate buffer saline (PBS).
Cells containing MTT solution were incubated further for 4 h before the solution was removed and
cells were dissolved in 100% DMSO. Dissolved purple formazan crystals were measured at 570 nm in
spectrophotometer (SpectraMax, Plus 384, Molecular Devices, Inc., San Jose, CA, USA). Control was
set as cells induced with α-MSH and 0.1% DMSO. Cells survival rate were calculated as follows: Cell
viability (%) = (Asample/Acontrol) × 100%.
4.4. Determination of Cellular Melanin Content
To determine the ability of samples in attenuating the excessive production of cellular melanin,
measurement of melanin content in
α-MSH-induced B16/F10 cells was conducted according to

Molecules 2020, 25, 3403
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Chan et al. [65] with minor modifications. Cells were seeded (2
culture plates with 3 mL of medium prior to different concentrations of drugs for 72 h. At the end of
×
105 cells/well) for 24 h in 6-well
cell treatment, cells were washed twice with PBS and harvested at 15,000
×
g for 15 min. The pellets
obtained were lyzed with 1 mL of 1 N NaOH in 10% DMSO and incubated at 80 ◦C for 1 h to dissolve the
melanin. Absorbance was read at 475 nm by microplate reader (Sunrise, Tecan, Switzerland). Melanin
content was evaluated from standard curve (0.78–100
synthetic melanin (Sigma, St. Louis, MO, USA).
µg/mL) prepared from authentic standard of
4.5. Determination of Cellular Tyrosinase
To examine the inhibitory effect of samples on tyrosinase activity, cellular tyrosinase activity
α-MSH-induced B16/F10 was determined by employing the method outlined by Lin et al. [66],
of
with minor modifications. Tyrosinase activity in cells was evaluated by measuring the oxidation rate
of l-DOPA. Cells were seeded (2
105 cells/well) for 24 h in 6-well culture plates with 3 mL of medium
×
and harvested at 72 hpt. Cells were washed twice with ice-cold PBS then lysed in lysate buffer of
20 mM phosphate buffer (pH 6.8) containing 1 mM PMSF and 1% Trion X-100. Membrane-bound
tyrosinase was released from melanosomes with the presence of detergent. Cells were then disrupted
by using sonicator with 30 kHz frequency wave in 15 s sonication burst cycles on ice. Lysates were
then clarified by centrifugation at 15,000
g at 4 ◦C for 15 min. Protein content in supernatant was
×
determined by using Bradford assay (Bio-Rad Laboratories Inc, Hercules, CA, USA) with bovine
serum albumin (BSA, Sigma, USA) used as protein standard. Cellular tyrosinase assay was measured
by adding 0.4 µg of cell lysate protein into reaction mixture of 50 mM of phosphate buffer (pH 6.8)
and 2.5 mM of l-DOPA. After incubation of 15 min, absorbance readings were monitored at 475 nm
by spectrophotometer (Sunrise, Tecan, Switzerland). The amount of dopachrome formation was
calculated using the Beer–Lambert Law applying the molar extinction coefficient of dopachrome
value 3600 M−1.cm−1. One unit of tyrosinase activity is equivalent to the total enzyme (tyrosinase)
that catalyzes the formation of 1 µmol of dopachrome per min. The specific tyrosinase activity was
evaluated by normalizing protein content in the reaction.
4.6. Quantitative Real-Time PCR (RT-qPCR) of Tyr, Trp-1, Trp-2 and Mitf
FLA- and FLB-treated of
α-MSH-induced B16/F10 melanoma cells were harvested and centrifuged
at 300
×
g for 5 min in 4 ◦C. Total RNA was extracted according to manufacturer’s protocol of RNeasy
Plus Mini kit (Qiagen, Hilden, Germany). cDNA was synthesized according to protocol by iScript
Reverse Transcription Supermix for RT-qPCR (Bio-Rad Laboratories Inc, Hercules, CA, USA). Real-Time
PCR was performed as according to manufacturer’s protocol of iTaq Universal SYBR Green Supermix
(Bio-Rad, Hercules, CA, USA). Reaction mixtures included 0.5
µM of both forward and reverse
primers, cDNA template (0.1 g RNA) and 2 iTaq Universal SYBR Green Supermix. Standard curves
µ
×
of each targeting gene were conducted by performing ten-fold serial dilutions of template cDNA.
All genes of interest were amplified using similar the◦rmal cycling conditions as follows; 95 ◦C for
3 min (DNA Polymerase activation) followed by 95 C for 10 s (DNA denaturation) and 30 s of
specific annealing/extension temperatures (Tyr at 60 ◦C, Trp-1 at 52 ◦C, Trp-2 at 52 ◦C, Mitf at 60 ◦C,
ß-actin at 58 ◦C and GAPDH at 58 ◦C) with 40 cycles for each gene expression analysis. Real-time
PCR analysis was carried out by normalizing expression ratio using Livak method of 2−(∆∆Ct)
Fold differences between Ct value of control samples with corresponding gene of interest was calculated
as ∆∆Ct = Ct
− ∆Ct control samples. Both ß-actin and GAPDH were used as reference genes
in the experiment. All samples were run in triplicate and values represent the mean
fold difference. Sequences of forward and reverse primers were listed as in Table 1.
[67].
∆
treated sample
±
SD of normalized

Molecules 2020, 25, 3403
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Table 1. Sequences of Tyr, Trp-1, Trp-2 and Mitf used in RT-qPCR.
Gene
Primer Sequences
Accession Number
Forward: TTG CCA CTT CAT GTC ATC ATA GAA TAT T
Reverse: TTT ATC AAA GGT GTG ACT GCT ATA CAA AT
Tyr
NM_011661.5
NM_031202.3
NM_010024.3
NM_008601.3
NM_008084.3
NC_007393.5
Forward: GCT GCA GGA GCC TTC TTT CTC
Reverse: AAG ACG CTG CAC TGC TGG TCT
Trp-1
Trp-2
Forward: GGA TGA CCG TGA GCA ATG GCC
Reverse: CGG TTG TGA CCA ATG GGT GCC
Forward: TAC AGA AAG TAG AGG GAG GAG GAC TAA G
Reverse: CAC AGT TGG AGT TAA GAG TGA GCA TAG CC
Mitf
Forward: ACC ACA GTC CAT GCC ATC AC
Reverse: TCC ACC ACC CTG TTG CTG TA
GAPDH
ß-actin
Forward: ACC GTG AAA AGA TGA CCC AG
Reverse: TAC GGA TGT CAA CGT CAC AC
Tyr, Tyrosinase; Trp-1 and -2, Tyrosinase related protein 1 and 2; Mitf, Microphthalmia-associated transcription
factor; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.
4.7. Zebrafish Husbandry and Embryos Collection
The present in vivo studies were conducted in obligations to policies of Institutional Animal
Care and Use Committee of Universiti Pura Malaysia (IACUC/AUP-R-24/2014). Wild-type shortfin
phenotype of adult zebrafish (Danio rerio), was purchased from Danio Assay Laboratories Sdn.
Bhd. (Malaysia) and bred in-house at Department of Biochemistry, Faculty of Biotechnology and
Biomolecular Sciences, UPM. Male and female adult zebrafish at the ratio of 1:2 were let to acclimatized
in a recirculating dechlorinated freshwater aquarium system with controlled conditions for a week at
28
±
1 ◦C on a 14 h light:10 h dark photoperiod cycle to induce spawning [68]. Adult zebrafish was
fed twice daily with brine shrimp (Artemia salina, San Francisco Bay Brand, San Francisco, CA, USA)
in the morning and flake foods in the evening. The collection container was placed with artificial
aquatic plants to imitate spawning sites [69] and fertilized eggs were collected 1 h post light onset [70].
Embryos were washed once with distilled water and twice with embryo media (15 mM NaCl, 0.5 mM
KCl, 1 mM MgSO4, 0.15 mM KH2PO4, 0.05 mM Na2HPO4, 1 mM CaCl2 and 0.7 mM NaHCO3, pH 7.0).
Successfully fertilized embryos with intact chorion membranes and reached gastrulation stage 50%
epiboly at approximately 5 to 9 hpf [71] were selected under dissecting microscope (Leica Zoom 2000)
at magnification of 40×.
4.8. Zebrafish Toxicity Assay of FLA and FLB
The toxicity effects of FLA and FLB during the embryonic stage of zebrafish were evaluated on
parameters such as spontaneous tail coiling (24 hpf), heartbeat activity (48 hpf), mortality (72 hpf)
and morphological deformities throughout the exposure time [28,29]. Twelve fertilized eggs at 9
hpf were selected for each concentration tested and control (n = 96). For the calculation of LC50,
the OECD guidelines for Fish Embryo Acute Toxicity Test (TG 236) were used [72]. Dead embryos
were identified and removed immediately to avoid any contamination during exposure time when it
forms coagulation, non-detachment of tail, lack of somite formation as well as absence of heartbeat [72].
Mortality and teratogenic effect of drugs on zebrafish embryos were observed daily between 48 to 144
hpf by using inverted microscope (Nikon Eclipse TS 100 Shinagawa, Japan). The number of zebrafish
embryo’s tail coiling was counted within 1 min period. Cardiac toxicity was evaluated by measuring
heart rates of zebrafish embryos in natural lateral recumbent position within 1 min period without
anesthesia to avoid bias heartbeat scoring [73,74]. Zebrafish larvae were not fed during the experiment.

Molecules 2020, 25, 3403
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4.9. Phenotype-Based Depigmenting Test of FLA and FLB on Zebrafish
Phenotype-based melanogenesis inhibition of zebrafish embryos treated with FLA and FLB were
determined following reported method [75]. To observe depigmenting of FLA and FLB on zebrafish,
fertilized embryos at 9 hpf were selected and arrayed in 96 well-plate with single embryo per well.
Control was set as embryos media containing 0.1% DMSO. Embryos treated with IBMX at 100
set as negative control. Positive control was set as embryos maintained in 200 L of embryo media
containing 100 M of PTU from 9 to 96 hpf. Treatment of the zebrafish with FLA and FLB was carried
out at 96 hpf after PTU was washed off and embryos were treated simultaneously with 100 M IBMX
to induce melanogenesis. PTU was used to suppress melanogenesis in zebrafish without adverse
toxicity [76 77]. IBMX was used as better melanogenic inducer agent in zebrafish model instead of
-MSH to mimic cAMP signaling pathway in melanogenesis omitting any effects of -MSH towards
zebrafish [30 78]. Inhibition of melanin by FLA and FLB in 0.1% DMSO was observed every 48 h until
µM was
µ
µ
µ
,
α
α
,
144 hpf. The developmental phenotypes of the experimental embryos were observed every 24 h for
144 h by using inverted microscope Nikon Eclipse TS100 and were imaged.
4.10. Statistical Analysis
All the results were presented as the mean
±
standard error of mean (S.E.M) of three independent
experiments. Differences between groups were determined by using one-way analysis of variance
(ANOVA) followed by Dunnett test. Values of * p < 0.05, ** p < 0.01 and *** p < 0.001 were considered
significant. All graphs were generated by using GraphPad Prism version 5.0 (GraphPad Software, Inc.,
San Diego, CA, USA).
Author Contributions: Conceptualization, M.N.A., Z.N.B.Y. and S.A.; methodology, N.M.S., N.N.M.R., F.I.M.M.
and N.L.; software, N.M.S., M.N.A.B. and S.A.; validation, N.M.S. and S.A.; formal analysis, S.A.; investigation,
N.M.S.; resources, M.N.A. and S.A.; data curation, N.M.S. and S.A.; writing—original draft preparation, N.M.S.;
writing—review and editing, N.M.S. and S.A.; visualization, N.M.S., N.N.M.R. and F.I.M.M.; supervision, S.A.;
project administration, M.N.A. and Z.N.B.Y.; funding acquisition, M.N.A. and S.A. All authors have read and
agreed to the published version of the manuscript.
Funding: This project was supported by Research University Grant Scheme (RUGS-initiative 2), Universiti Putra
Malaysia (05-02-12-2137RU) and the University Malaysia Pahang (internal grant No. RDU 120373).
Acknowledgments: The authors appreciatively acknowledge to Laboratory of Immunotherapeutic and Vaccine,
Institute of Bioscience, Universiti Putra Malaysia (UPM) for providing the necessary support for the study.
Nurshafika Mohd Sakeh, Mohammad Nazri Abdul Bahari and Naimah Latif were Master students who received
UPM scholarship under Graduate Research Fellowship (GRF) scheme.
Conflicts of Interest: All authors declared no conflict of interest.
Ethical Standard: In vivo studies on zebrafish were conducted in obligations to policies of Institutional Animal
Care and Use Committee of Universiti Putra Malaysia (IACUC/AUP-R-24/2014).
Abbreviations
ACTH
α-MSH
bHLH-LZ
BSA
adrenocorticotropic hormone
alpha-melanocyte stimulating hormone
basic-helix-loop-helix-leucine-zipper
Bovine serum albumin
DHI
Dihydroxyindole
DHICA
DMEM
DMSO
EDTA
FBS
Dihydroxyindole carboxylic acid
Dulbecco’s Modified Eagle’s Medium
Dimethyl sulfoxide
Ethylenediaminetetraacetic acid
Foetal bovine serum
(E)-1-(20-Hydroxy-40,60-dimethoxyphenyl)-3-(4-methoxyphenylprop-2-en-1-one
FLA

Molecules 2020, 25, 3403
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FLB
(E)-1-(20-Hydroxy-40,60-dimethoxyphenyl)-3-phenylprop-2-en-1-one
GAPDH
IBMX
l-DOPA
Mitf
MTT
NMR
PTU
Glyceraldehyde-3-phosphate dehydrogenase
3-isobutyl-1-methylxanthine
l-3,4-dihydroxyphenylalanine
Microphthalmia associated transcription factor
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide
Nuclear magnetic resonance
Phenylthiourea
qPCR
Trp-1
Trp-2
Tyr
Quantitative real-time polymerase chain reaction
Tyrosinase related protein-1
Tyrosinase related protein-2
Tyrosinase
UVR
Ultraviolet radiation
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