β-Lactoglobulin Peptide Fragments Conjugated with Caffeic Acid Displaying Dual Activities for Tyrosinase Inhibition and Antioxidant Effect

Article abstract of DOI:10.1021/acs.bioconjchem.8b00050

The regulation of tyrosinase activity and reactive oxygen species is of great importance for the prevention of dermatological disorders in the fields of medicine and cosmetics. Herein, we report a strategy based on solid-phase peptide chemistry for the sy

Full text of DOI:10.1021/acs.bioconjchem.8b00050

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#-Lactoglobulin Peptide Fragments Conjugated with Caffeic Acid
Displaying Dual Activities for Tyrosinase Inhibition and Antioxidant Effect
Jin-Kyoung Yang, Eunjin Lee, In-Jun Hwang, DaBin Yim, Juhee Han, Yoon-Sik Lee, and Jong-Ho Kim
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00050 • Publication Date (Web): 13 Mar 2018
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Bioconjugate Chemistry
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βꢀLactoglobulin Peptide Fragments Conjugated with
Caffeic Acid Displaying Dual Activities for Tyrosinase
Inhibition and Antioxidant Effect
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JinꢀKyoung Yang1, Eunjin Lee2, InꢀJun Hwang1, DaBin Yim1, Juhee Han1, YoonꢀSik Lee2,
and JongꢀHo Kim1,*
1Department of Chemical Engineering, Hanyang University, Ansan 426-791, Republic of
Korea
2School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744,
Republic of Korea
*To whom correspondence should be addressed: kjh75@hanyang.ac.kr.
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Abstract
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The regulation of tyrosinase activity and reactive oxygen species is of great importance for
the prevention of dermatological disorders in the fields of medicine and cosmetics. Herein,
we report a strategy based on solidꢀphase peptide chemistry for the synthesis of βꢀ
lactoglobulin peptide fragment/caffeic acid (CA) conjugates (CAꢀPeps) with dual activities of
tyrosinase inhibition and antioxidation. The purity of the prepared conjugates, CAꢀMHIR,
CAꢀHIRL, and CAꢀHIR significantly increased to 99%, as acetonideꢀprotected CA was
employed in solidꢀphase coupling reactions on Rink amide resins. The tyrosinase inhibitory
activities of all CAꢀPep derivatives were higher than the activity of kojic acid, and CAꢀMHIR
exhibited the highest tyrosinase inhibition activity (IC50 = 47.9 ꢀM). Moreover, CAꢀPep
derivatives displayed significantly enhanced antioxidant activities in the peroxidation of
linoleic acid as compared to the pristine peptide fragments. All CAꢀPep derivatives showed
no cytotoxicity against B16ꢀF1 melanoma cells.
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Tyrosinase, a copperꢀcontaining enzyme, catalyzes the hydroxylation of monophenols
and oxidation of oꢀdiphenols to produce reactive precursors that are essential for the
formation of melanin pigments in melanocytes.1, 2 Melanin pigments are known to protect
skin cells from damage caused by ultraviolet radiation.3 However, the excessive accumulation
of melanin pigments may cause dermatological disorders such as melasma, lentigo, and
freckle.4 Moreover, tyrosinase is known to be involved in neuromelanin formation and
neurodegeneration related to Parkinson’s disease.5, 6 Hence, the regulation of tyrosinase
activity for the prevention of hyperpigmentation is of great importance in the field of
cosmetics and medicine.
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Several efforts have been directed for the development of tyrosinase inhibitors via
natural resources and synthetic routes.7 Kojic acid (KA), one of the wellꢀknown tyrosinase
inhibitors, chelates the copper atom at the active site of tyrosinase.8 Fungal metabolites, plant
polyphenols, and hydroquinone derivatives that inhibit tyrosinase activity have been
developed.9 However, these inhibitors are thought to be mutagenic and cytotoxic to
mammalian cells and melanocytes.10ꢀ12 In addition, the cell penetration of KA and
hydroquinone inhibitors was reported to be poor, thereby limiting their widespread use in
cosmetic and pharmaceutical industries. Therefore, there is an unmet need for the
development of novel tyrosinase inhibitors with high activity and reduced adverse effects.
Short peptides and their derivatives are promising alternatives for biocompatible
tyrosinase inhibitors.13ꢀ17 A milk protein βꢀlactoglobulin was reported to suppress
18, 19
melanogenesis in melanocytes
and its peptide fragments such as YFYPEL,
WYSLAMAA, YVEEL, and MHIRL were found to be the primary sites involved in
tyrosinase inhibition.20, 21 These peptide fragments may serve as good precursors for
derivatization to improve the tyrosinase inhibition activity. In particular, the development of
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short peptide derivatives with multiꢀfunctional activities, including tyrosinase inhibition, may
be of great interest in the fields of medicine, biology, and cosmetics.
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Caffeic acid (CA) is an organic compound bearing catechol and acrylic functional groups,
which is known to display an antioxidant activity.22, 23 Thereby, CA has been used to
scavenge reactive oxygen species (ROS) in various biological systems.24 As ROS are
involved in both physiological and pathophysiological processes,25, 26 it is required to keep a
balance between their generation and exhaustion in biological systems using antioxidants like
CA. However, CA is very susceptible to autoxidation via the conversion of oꢀdiphenol into oꢀ
quinone and chemical oxidation, limiting its derivatization to produce a variety of CA
derivatives. Hence, it is desired to develop an effective synthetic method for the facile
preparation of CA derivatives with multiple bioactivities, including tyrosinase inhibition
activity and antioxidant effect.
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Herein, we report a synthetic approach based on solidꢀphase peptide chemistry for the
conjugation of tetraꢀ and triꢀpeptide fragments of βꢀlactoglobulin with CA to obtain CAꢀPep
conjugates with tyrosinase inhibition and antioxidant activities. The use of diolꢀprotected CA
significantly improved the purity of CA/peptide conjugates (CAꢀPeps). In addition, the
antioxidant activity as well as the tyrosinase inhibition effect of CAꢀPeps were explored in
this work. Furthermore, the cytotoxicity of CAꢀPeps against B16ꢀF1 melanoma cells was
examined.
To synthesize CAꢀPeps, solidꢀphase Fmoc chemistry on Rink amide resins was employed
27. When unprotected CA was coupled to the tetraꢀpeptide (MHIR) synthesized on Rink
amide resins using Fmocꢀamino acids, several side products were observed in the
chromatogram, as evident from HPLC analysis (Figure S1a). To improve the purity of desired
CAꢀPeps, the catechol group of CA was protected using DMP and TsOH to produce
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acetonideꢀprotected CA (CA[acetonide]) after esterification of the carboxylic acid of CA
(Steps (1) and (2) in Figure 1a). A white powder of CA(acetonide)ꢀOMe was obtained at 55%
yield. After the hydrolysis of CA(acetonide)ꢀOMe to produce CA(acetonide)ꢀOH, it was
coupled to tetraꢀpeptides (MHIR
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a)
(1)
(2)
CA(acetonide)-OMe, 55%
CA methyl ester (CA-OMe)
71%
Caffeic acid (CA)
(3)
CA(acetonide)-OH, 21%
(7)
(4) – (5)
(6)
Rink amide
AM resin
(0.47 mmol/g)
(8)
b)
CA-HIR
CA-HIRL
CA-MHIR
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Figure 1. Synthesis of acetonideꢀprotected CA and CAꢀPep derivatives. a) Synthesis of
acetonideꢀprotected caffeic acid (CA[acetonide]ꢀOH). (1) SOCl2 (2 equivalent) and DMAP
(0.2 equivalent) incubated in anhydrous MeOH for overnight at RT. (2) DMP (4 equivalent)
and TsOH (0.05 equivalent) dissolved in benzene with reflux for 3 h. (3) LiOH (3 equivalent)
treated with THF/H2O (2:1) in ice bath for 24 h, and each step was purified by column
chromatography. Solidꢀphase synthesis of caffeoylꢀcontaining peptides. (4) FmocꢀLꢀamino
acid (3 equivalent), BOP (3 equivalent), HOBt (3 equivalent), and DIPEA (6 equivalent)
dissolved in NMP for 3 h. (5) 20% piperidine/NMP (v/v) for 50 min. (6) Repeat (4ꢀ5), (7)
CA(acetonide)ꢀOH (3 equivalent), BOP (3 equivalent), HOBt (3 equivalent), and DIPEA (6
equivalent) were treated in NMP for 3 h. (8) Cleavage cocktail: 95% TFA, 3% anisole, 1%
TIPS, 1% DODT/DCM (v/v) for 2 h and diethyl ether precipitation. R: side chain of amino
acid. b) Structure of synthesized CAꢀpeptide derivatives.
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and HIRL) or a triꢀpeptide (HIR) on Rink amide resins using solidꢀphase Fmoc chemistry to
produce three different types of CAꢀPeps (Figure 1b). After the cleavage of the products from
the resin using an acid solution containing water as the carbocation scavenger, the purity of
CAꢀMHIR was determined to be over 99% (Figure S1b). CAꢀMHIR was completely
converted to DODT adduct (Figure S1c) when an acid solution containing DODT as the
scavenger was used in the cleavage step, as confirmed by ESIꢀMS. This side product may be
obtained via 1,4ꢀaddition of a thiol compound to α,ßꢀunsaturated ketones.28, 29 These results
reveal that the protection of the catechol moiety of CA and use of the proper scavenger during
the course of the reaction are essential for the synthesis of highꢀpurity CAꢀPeps.
This optimized reaction enabled us to synthesize highꢀpurity CAꢀMHIR, CAꢀHIRL, CAꢀHIR,
MHIR, HIRL, and HIR (Table 1). The molecular weights of the products were confirmed by
ESIꢀMS.
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Tyrosinase is the key enzyme that catalyzes the following two oxidation steps in
melanogenesis: hydroxylation of tyrosine to LꢀDOPA and the subsequent oxidation of Lꢀ
DOPA to Lꢀdopaquinone, which is cyclized into dopachrome. The inhibition activity of three
CAꢀPeps, two tetrapeptides, and one tripeptide against mushroom tyrosinase was assessed by
measuring the characteristic absorption of dopachrome formed in the oxidation and
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cyclization reactions of L
ꢀDOPA (2.5 mM) at 475 nm.30
As shown in Figure S2, the formation of dopachrome from LꢀDOPA was significantly
suppressed in the presence of 50 ꢀM of CAꢀMHIR. Moreover, no dopachrome was formed
upon the treatment of LꢀDOPA solution with higher concentration of CAꢀMHIR (100 ꢀM).
These results clearly suggest that CAꢀMHIR was able to effectively inhibit the catalytic
activity of tyrosinase. To compare the inhibitory activity of CAꢀPep derivatives and peptides,
their inhibition efficacies were measured as a function of concentration (Figure 2a). All CAꢀ
Pep
Table 1. Purity and Molecular Weights of Peptides and CAꢀPep Derivatives
Compounds
Purity (%)
Mass ([M+H]+)
Calculated
Found
717.5
555.5
699.7
537.5
586.6
424.4
CAꢀMHIR
MHIR
>99
>99
95
717.3
555.3
699.7
537.3
586.6
424.2
CAꢀHIRL
HIRL
94
CAꢀHIR
HIR
94
>99
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a)
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0
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CA-MHIR
MHIR
CA-HIRL
HIRL
CA-HIR
HIR
KA
0
100
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400
500
Concentration (ꢀM)
100
75
50
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b)
-25
c)
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ꢀ
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M
M
M
60
40
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0
-0.2 0.0
0.2
0.4
0.6
0.8
1.0
1/[L-DOPA] (mM)-1
Figure 2. Tyrosinase inhibition activity of CAꢀPep derivatives. a) Tyrosinase inhibitory
activity of CAꢀPep derivatives, pristine peptides, and KA as a function of concentration. b)
Quantitative comparison of tyrosinase inhibitory activity at 100 ꢁM of each compound. A
total of 2.5 mM LꢀDOPA and 100 ꢁg/mL tyrosinase were incubated at 25°C for 10 min. c)
LineweaverꢀBurk plot of tyrosinase inhibition reaction in the presence of CAꢀMHIR at
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various concentrations.
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derivatives (CAꢀMHIR, CAꢀHIRL, and CAꢀHIR) exhibited higher inhibition activities than
pristine peptides (MHIR, HIRL, and HIR). Note that CA itself had no tyrosinase inhibitory
activity. This result reveals that the conjugation of CA to the peptides was very effective in
improving their tyrosinase inhibitory activities. Notably, CAꢀMHIR exhibited higher
inhibitory activity (75 ± 7%) than KA (52 ± 6%) (Figure 2b).
We calculated the half maximal inhibitory concentrations (IC50) of CAꢀPeps, pristine
peptides, and KA (Table S1). As expected, all CAꢀPeps exhibited lower IC50 values than
pristine peptides. CAꢀMHIR had the lowest IC50 value of 47.9 ꢀM, which was five times
lower than that of pristine MHIR. Thus, CAꢀMHIR had the highest potency to inhibit
tyrosinase activity. We speculate that CA conjugation may improve the accessibility of the
peptides to tyrosinase to induce deformation of the active site. Based on IC50 values, the
tyrosinase inhibitory activity was reported in the following order:
CAꢀMHIR (47.9 ꢀM) > CAꢀHIR (154.8 ꢀM) > CAꢀHIRL (166.2 ꢀM) > KA (201.7 ꢀM) >
HIRL (218.8 ꢀM) > MHIR (257.1 ꢀM) > HIR (none)
Subsequently, we investigated inhibition kinetics of CAꢀMHIR to understand the
mechanism underlying tyrosinase inhibitory activity. As shown in Figure 2c, inhibition
kinetics was measured at three different concentrations of CAꢀMHIR and the Lineweaverꢀ
Burk plots were drawn to extract a Michaelis constant (Km) and a maximum velocity (Vmax).
Vmax value for the inhibition reaction of CAꢀMHIR decreased from 0.10 and 0.073 to 0.056
nm·min ꢀ1 as the concentration of CAꢀMHIR increased from 25 and 50 to 75 ꢁM, whereas Km
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value remained constant (2.0 mM). These kinetic parameters reveal that CAꢀMHIR is a nonꢀ
competitive inhibitor for tyrosinase. Therefore, CAꢀMHIR may bind to sites other than the
active site of tyrosinase for oxidation, leading to the deformation of the three dimensional
structure of the active site.
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Antioxidant activities of CAꢀPep derivatives were investigated using the linoleic acid
oxidation assay with FTC, a common method for the evaluation of antioxidant activity in
hydrophobic environment.31, 32 In this assay, the peroxidation of linoleic acid in Tween 20
emulsion spontaneously occurs in the presence of molecular oxygen, resulting in an increase
in the absorption of the solution at 500 nm. The antioxidant activities of CAꢀPeps and pristine
peptides were examined by measuring the absorbance at 500 nm over time (Figure 3). We
hardly observed any increase in the absorbance of the solution containing CAꢀMHIR,
indicating that the peroxidation of linoleic acid was prevented by CAꢀMHIR (Figure 3a).
However, pristine MHIR exhibited no antioxidant activity. Thus, CA conjugation to the
peptide imparts antioxidant and tyrosinase inhibitory activities to the resulting peptide
conjugate. Similar results were observed for CAꢀHIRL and CAꢀHIR, showing significant
enhancement in the antioxidant activity after CA conjugation (Figure 3b and 3c). The extent
of the inhibition of lipid peroxidation (%Pi) in the presence of CAꢀPep derivatives was
calculated when the absorbance of the control reached approximately 1. As shown in Figure
3d, the antioxidant activity of CAꢀMHIR was more than four times higher than that of MHIR.
In addition, both CAꢀHIRL and CAꢀHIR exhibited twoꢀthree times higher antioxidant activity
than pristine peptides. The antioxidant activity of CAꢀPep derivatives was higher than CA,
but lower than BHA, a wellꢀknown antioxidant for preventing lipid peroxidation. As BHA
has been known as a potential carcinogenic and toxic agent,33 CAꢀPep derivatives would be
more beneficial for applications in various biological systems. The order of the antioxidant
activity of CAꢀPeps based on %Pi values is as given below:
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BHA (86 ± 1) > CAꢀMHIR (79 ± 0.2) > CAꢀHIR (78 ± 0.8) > CAꢀHIRL (76 ± 0.3) > CA (73
± 1) >> HIRL (32 ± 6) > HIR (26 ± 5) > MHIR (ꢀ23 ± 4)
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Figure 3. Antioxidant activities of CAꢀPep derivatives in terms of linoleic acid peroxidation.
The absorbance of peroxidized linoleic acid at 500 nm over time was recorded in the
presence of a) CAꢀMHIR and MHIR, b) CAꢀHIRL and HIRL, and c) CAꢀHIR and HIR. d)
Efficacy of CAꢀPep derivatives at 100 ꢀM concentration for the inhibition of lipid
peroxidation. As a negative control, 25 ꢀL of methanol was added instead of CAꢀPep
derivatives.
The biological activities of peptides, proteins, and their derivatives have been widely
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explored, however, most of them exhibited a single activity.34ꢀ37 Although some molecules
were reported to have a dual activity on tyrosinase inhibition and antioxidant effect, their dual
activity was inferior to that of CAꢀPeps.38, 39 In addition to the superior dual activity of CAꢀ
Peps, the synthetic method for CAꢀPeps was more effective and versatile to produce diverse
bioactive conjugates than the extractionꢀbased methods for the aforementioned products.
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The cytotoxicity of CAꢀPep derivatives and pristine peptides was examined in B16ꢀF1
melanoma cells using MTT assay. CAꢀPep conjugates displayed no noticeable cytotoxicity at
100 ꢁM concentration (Figure 4). In addition, pristine peptides as well as CA exhibited no
cytotoxicity against melanoma cells. This result clearly suggests that CAꢀPep derivatives may
be applied for the regulation of tyrosinase catalytic activity and scavenging of reactive
oxygen species in various biological systems.
In conclusion, highꢀpurity CAꢀPep derivatives were successfully synthesized by coupling
CA(acetonide)ꢀOH to βꢀlactoglobulin peptide fragments on resin supports for tyrosinase
inhibition and antioxidant effects. All CAꢀPep derivatives showed much higher tyrosinase
inhibition than pristine peptides and KA. In particular, CAꢀMHIR exhibited the highest
inhibitory activity. Moreover, CAꢀPep derivatives showed excellent antioxidant activities in
the lipid peroxidation assay and dispalyed no cytotoxicity against B16 melanoma cells. The
synthetic approach for designing CAꢀPep derivatives may be applicable for the synthesis of
various CAꢀfunctionalized molecules. In addition, the utility of CAꢀPep derivatives with dual
activities may be extended to cosmetics and medicines to inhibit tyrosinase and scavenge
reactive oxygen species.
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Figure 4. Cytotoxicity of CAꢀPep derivatives and peptides against B16 melanoma cells. A
total of 100 ꢁM of each compound was incubated with cells for 72 h at 37°C.
Supporting Information
Synthetic procedures and characterizations for all the CAꢀPep derivatives, in vitro assay for
the evaluation of tyrosinase inhibition and antioxidant activity of CAꢀPep derivatives, and a
cytotoxicity test are available free of charge on the ACS Publications website at.
Acknowledgement
This study was supported by the research fund of Hanyang University (HYꢀ2017ꢀP).
Conflict of interest
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The authors have no conflict of interest regarding the publication of this paper.
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References
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(1) Ko, H.ꢀH., Chiang, Y.ꢀC., Tsai, M.ꢀH., Liang, C.ꢀJ., Hsu, L.ꢀF., Li, S.ꢀY., Wang, M.ꢀC.,
Yen, F.ꢀL., and Lee, C.ꢀW. (2014) Eupafolin, a skin whitening flavonoid isolated from Phyla
nodiflora, downregulated melanogenesis: Role of MAPK and Akt pathways. J.
Ethnopharmacol. 151, 386ꢀ393.
(2) Hearing, V. (1993) Unraveling the melanocyte. American journal of human genetics 52, 1.
(3) Brenner, M., and Hearing, V. J. (2008) The protective role of melanin against UV damage
in human skin. Photochem. Photobiol. 84, 539ꢀ549.
(4) Piamphongsant, T. (1998) Treatment of melasma: a review with personal experience. Int.
J. Dermatol. 37, 897ꢀ903.
(5) Xu, Y., Stokes, A. H., Freeman, W. M., Kumer, S. C., Vogt, B. A., and Vrana, K. E. (1997)
Tyrosine mRNA is expressed in human substantia nigra. Mol. Brain Res. 45, 159ꢀ162.
(6) Gaeta, A., MolinaꢀHolgado, F., Kong, X. L., Salvage, S., Fakih, S., Francis, P. T.,
Williams, R. J., and Hider, R. C. (2011) Synthesis, physical–chemical characterisation and
biological evaluation of novel 2ꢀamidoꢀ3ꢀhydroxypyridinꢀ4 (1H)ꢀones: Iron chelators with
the potential for treating Alzheimer’s disease. Bioorg. Med. Chem. 19, 1285ꢀ1297.
(7) Peng, L.ꢀH., Liu, S., Xu, S.ꢀY., Chen, L., Shan, Y.ꢀH., Wei, W., Liang, W.ꢀQ., and Gao, J.ꢀ
Q. (2013) Inhibitory effects of salidroside and paeonol on tyrosinase activity and melanin
synthesis in mouse B16F10 melanoma cells and ultraviolet Bꢀinduced pigmentation in guinea
pig skin. Phytomedicine 20, 1082ꢀ1087.
(8) ElꢀShanawany, A., Mostafa, M. E., and Barakat, A. (2005) Fungal populations and
mycotoxins in silage in Assiut and Sohag governorates in Egypt, with a special reference to
14
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Page 15 of 23
Bioconjugate Chemistry
1
2
3
4
characteristic Aspergilli toxins. Mycopathologia 159, 281ꢀ289.
5
6
7
8
9
(9) Kim, Y.ꢀJ., and Uyama, H. (2005) Tyrosinase inhibitors from natural and synthetic sources:
structure, inhibition mechanism and perspective for the future. Cell. Mol. Life Sci. 62, 1707ꢀ
1723.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(10) Curto, E. V., Kwong, C., Hermersdörfer, H., Glatt, H., Santis, C., Virador, V., Hearing, V.
J., and Dooley, T. P. (1999) Inhibitors of mammalian melanocyte tyrosinase: in vitro
comparisons of alkyl esters of gentisic acid with other putative inhibitors. Biochem.
Pharmacol. 57, 663ꢀ672.
(11) Wei, C., Huang, T., Fernando, S., and Chung, K. (1991) Mutagenicity studies of kojic
acid. Toxicol. Lett. 59, 213ꢀ220.
(12) SMITH, C. J., O'HARE, K. B., and ALLEN, J. C. (1988) Selective Cytotoxicity of
Hydroquinone for Melanocyte- Derived Cells is Mediated by Tyrosinase Activity but
Independent of Melanin Content. Pigment Cell Melanoma Res 1, 386ꢀ389.
(13) Kwak, S.ꢀY., Noh, J.ꢀM., Lee, S., Lee, S. H., Jeon, S.ꢀj., Kim, H.ꢀI., Kim, J.ꢀH., and Lee,
Y.ꢀS. (2014) Effect of alphaꢀresorcylic acid–lꢀphenylalanine amide on collagen synthesis and
matrix metalloproteinase expression in fibroblasts. Bioorg. Med. Chem. Lett. 24, 742ꢀ745.
(14) Kwak, S. Y., Yang, J. K., Kim, J. H., and Lee, Y. S. (2013) Chemical modulation of
bioactive compounds via oligopeptide or amino acid conjugation. Pept. Sci. 100, 584ꢀ591.
(15) Kwak, S.ꢀY., Yang, J.ꢀK., Choi, H.ꢀR., Park, K.ꢀC., Kim, Y.ꢀB., and Lee, Y.ꢀS. (2013)
Synthesis and dual biological effects of hydroxycinnamoyl phenylalanyl/prolyl hydroxamic
acid derivatives as tyrosinase inhibitor and antioxidant. Bioorg. Med. Chem. Lett. 23, 1136ꢀ
1142.
(16) Noh, J. M., Kwak, S. Y., Kim, D. H., and Lee, Y. S. (2007) Kojic acid–tripeptide amide
as a new tyrosinase inhibitor. Pept. Sci. 88, 300ꢀ307.
(17) Noh, J.ꢀM., and Lee, Y.ꢀS. (2011) Inhibitory activities of hydroxyphenolic acid–amino
15
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Page 16 of 23
1
2
3
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acid conjugates on tyrosinase. Food Chem. 125, 953ꢀ957.
5
6
7
(18) Nakajima, M., Shinoda, I., JIMA, Y. S., Miyauchi, H., Fukuwatari, Y., and Hayasawa, H.
8
9
(1996) K- Casein Suppresses Melanogenesis in Cultured Pigment Cells. Pigment Cell
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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32
33
34
35
36
37
38
39
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Melanoma Res 9, 235ꢀ239.
(19) Nakajima, M., Shinoda, I., Mikogami, T., Iwamoto, H., HASHIMOTO, S. I., Miyauchi,
H., Fukuwatari, Y., and Hayasawa, H. (1997) β- Lactoglobulin Suppresses Melanogenesis in
Cultured Human Melanocytes. Pigment Cell Melanoma Res 10, 410ꢀ413.
(20) Li, Y. W., Li, B., He, J., and Qian, P. (2011) Structure–activity relationship study of
antioxidative peptides by QSAR modeling: the amino acid next to C- terminus affects the
activity. J. Pept. Sci. 17, 454ꢀ462.
(21) HernándezꢀLedesma, B., Dávalos, A., Bartolomé, B., and Amigo, L. (2005) Preparation
of antioxidant enzymatic hydrolysates from αꢀlactalbumin and βꢀlactoglobulin. Identification
of active peptides by HPLCꢀMS/MS. J. Agric. Food. Chem. 53, 588ꢀ593.
(22) Chen, J. H., and Ho, C.ꢀT. (1997) Antioxidant activities of caffeic acid and its related
hydroxycinnamic acid compounds. J. Agric. Food. Chem. 45, 2374ꢀ2378.
(23) Gülçin, Đ. (2006) Antioxidant activity of caffeic acid (3, 4ꢀdihydroxycinnamic acid).
Toxicology 217, 213ꢀ220.
(24) Gülçin, Đ. (2006) Antioxidant activity of caffeic acid (3, 4ꢀdihydroxycinnamic acid).
Toxicology 217, 213ꢀ220.
(25) Gill, S. S., and Tuteja, N. (2010) Reactive oxygen species and antioxidant machinery in
abiotic stress tolerance in crop plants. Plant physiology and biochemistry 48, 909ꢀ930.
(26) Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T., Mazur, M., and Telser, J. (2007) Free
radicals and antioxidants in normal physiological functions and human disease. Int. J.
Biochem. Cell Biol. 39, 44ꢀ84.
(27) Merrifield, R. B. (1963) Solid phase peptide synthesis. I. The synthesis of a tetrapeptide.
16
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Page 17 of 23
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
J. Am. Chem. Soc. 85, 2149ꢀ2154.
(28) Harris, P. W., Kowalczyk, R., Yang, S. H., Williams, G. M., and Brimble, M. A. (2014)
An important side reaction using the thiol, 3, 6 dioxa 1, 8 octanedithiol (DODT), in 9
fluorenylmethoxycarbonyl based solid phase peptide synthesis. J. Pept. Sci. 20, 186ꢀ190.
(29) Lin, W., Yuan, L., Cao, Z., Feng, Y., and Long, L. (2009) A Sensitive and Selective
-
-
-
-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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27
28
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30
31
32
33
34
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36
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39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
-
Fluorescent Thiol Probe in Water Based on the Conjugate 1, 4- Addition of Thiols to α, β-
Unsaturated Ketones. Chem. Eur. J. 15, 5096ꢀ5103.
(30) Mason, H. S. (1948) The chemistry of melanin III. Mechanism of the oxidation of
dihydroxyphenylalanine by tyrosinase. Journal of Biological Chemistry 172, 83ꢀ99.
(31) Göçer, H., and Gülçin, Đ. (2011) Caffeic acid phenethyl ester (CAPE): correlation of
structure and antioxidant properties. International Journal of Food Sciences and Nutrition 62
,
821ꢀ825.
(32) Gülçin, Đ. (2011) Antioxidant activity of eugenol: A structure–activity relationship study.
Journal of medicinal food 14, 975ꢀ985.
(33) Ito, N., Hirose, M., Fukushima, S., Tsuda, H., Shirai, T., and Tatematsu, M. (1986)
Studies on antioxidants: their carcinogenic and modifying effects on chemical carcinogenesis.
Food and Chemical Toxicology 24, 1071ꢀ1082.
(34) Chen, H.ꢀM., Muramoto, K., Yamauchi, F., and Nokihara, K. (1996) Antioxidant activity
of designed peptides based on the antioxidative peptide isolated from digests of a soybean
protein. J. Agric. Food. Chem. 44, 2619ꢀ2623.
(35) Mendis, E., Rajapakse, N., Byun, H.ꢀG., and Kim, S.ꢀK. (2005) Investigation of jumbo
squid (Dosidicus gigas) skin gelatin peptides for their in vitro antioxidant effects. Life Sci. 77
,
2166ꢀ2178.
(36) Schurink, M., van Berkel, W. J., Wichers, H. J., and Boeriu, C. G. (2007) Novel peptides
with tyrosinase inhibitory activity. Peptides 28, 485ꢀ495.
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5
6
(37) Chen, Q.ꢀX., and Kubo, I. (2002) Kinetics of mushroom tyrosinase inhibition by
quercetin. J. Agric. Food. Chem. 50, 4108ꢀ4112.
7
8
9
(38) Kato, N., Sato, S., Yamanaka, A., Yamada, H., FUWA, N., and NOMURA, M. (1998)
Silk protein, sericin, inhibits lipid peroxidation and tyrosinase activity. Biosci. Biotechnol.
Biochem. 62, 145ꢀ147.
10
11
12
13
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15
16
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21
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46
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48
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50
51
52
53
54
55
56
57
58
59
60
(39) Zhuang, Y., Sun, L., Zhao, X., Wang, J., Hou, H., and Li, B. (2009) Antioxidant and
melanogenesis- inhibitory activities of collagen peptide from jellyfish (Rhopilema
esculentum). J. Sci. Food Agric. 89, 1722ꢀ1727.
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