Study of Tyrosinase inhibitors
from natural sources
CMLS, Cell. Mol. Life Sci. 62 (2005)1707-1723
Presented by Hoi Young Kim
Organic & Natural Product Chemistry Lab
Contents
Melanin
Biosynthetic pathway of melanin
Function of melanin
Tyrosinase
Mechanism of tyrosinase action
Tyrosinase inhibitors
Natural and synthetic sources
Applicaton
New Development of
Tyrosianse inhibitors
Organic & Natural Product Chemistry Lab
Melanin
Melanin is one of the most widely distributed pigments and is found in
bacteria, fungi, plants and animals. It is a heterogeneous polyphenol-like
biopolymer with a complex structure and color varying from yellow to
black.
melanine
keranocyte
melanoine
melanocyte
Melanocyte
Organic & Natural Product Chemistry Lab
Biosynthetic pathway of melanin
Tyrosinase
(Monophenolase)
HO
COOH
NH2
HO
Tyrosinase
(Diphenolase)
COOH
O
NH2
HO
O
L-DOPA
Tyrosine
COOH
NH2
Dopaquinone
HO
Glutathoione
or
cysteine
COOH
NH2
HO
S
H2N
COOH
Cysteinyldopa
HO
- CO2
N
H
HO
DHI
Tyrosinase
O
O
-
O
HO
COOH
+
NH
Dopachrome
COOH
N
H
Leukodopachrome
HO
N
H
Indole-5,6-quinone
Oxidase
HO
N
H
HO
COOH
NH2
N
Tautomerase
S
O
COOH
O
HO
DHICA
O
1,4-Benzothiazinylalanine
N
H
Indole-5,6-quinonecarboxylic acid
Eumelanin
Eumelanin
Pheomelanin
Pheomelanin
Mixed-melanins
Mixed-melanins
Organic & Natural Product Chemistry Lab
Melanin Function
Several types
- Eumelanin (predominant in human)
- Pheomelanin
Functional bio-macromolecule found throughout nature:
- Increases that live and competition
- protect the skin from ultraviolet(UV)
- Detoxification for cell toxicity substances
Melanin overproduction:
bring about freckles, hyperpigmentation and cancer
Melanin is formed through a series of oxidative reactions involving the
amino acid tyrosine in the presence of tyrosinase
Organic & Natural Product Chemistry Lab
Tyrosinase (EC 1. 14. 18. 1)
Phenolase, Monopheol oxidase, Catechol oxidase, etc.
Copper-containing enzyme.
Catalyzes two distinct reactions of melanin synthesis
Tyrosinase
(Monophenolase)
HO
COOH
Monophenolase activity
HO
NH2
HO
COOH
NH2
L-DOPA
Tyrosine
Tyrosinase
(Diphenolase)
Diphenolase activity
HO
HO
COOH
NH2
L-DOPA
O
O
COOH
NH2
Dopaquinone
Organic & Natural Product Chemistry Lab
Classification and properties of tyrosinase
Table 1. Properties of Well-Characterized Tyrosinases
source
Number of Molecular weight
subunits Of subunit (kDa)
Spectroscopy of oxy form
Absorption (nm) CD (nm)
Streptomyces glaucescens
(Eubacteria)
1
30.9
345
640
345
470
575
740
Neurospora crassa
(Fungi)
1
46
345
425
600
345
520
600
750
Agaricus bisporus
(Fungi; mushroom)
2
13.4
43
345
600
353
Beta vulgaris
(Plant; spinach-beet)
1
40
345
-a
Human melanocyte
(Animal)
1
66.7
-a
-a
a
No data available.
Organic & Natural Product Chemistry Lab
Mechanism of tyrosinase action
Diphenolase
Catalytic cycle
Monophenolase
Catalytic Cycle
Figure . Catalytic cycles of the hydroxylation of monophenol and oxidation of o-quinone by tyrosinase
Organic & Natural Product Chemistry Lab
Tyrosinase inhibitors from natural sources
Structure of tyrosinase inhibitors
Organic & Natural Product Chemistry Lab
Tyrosinase inhibitors from natural sources
Mushroom Tyrosinase inhibitory activities of the Flavonoids
Compound
No.
R3'
R8
R7
R2'
R4'
O
R6
R5'
R3
R5
O
R6'
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
R3
OH
R5
R6
OH
OH
OH
OH
OH
R7
R8
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
R2'
R3'
R4'
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
R5'
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH OH
OH OH
OH
OH
IC50
R6' (μM)
0.07
3.49
8.51
8.88
12.78
12.95
17.48
24.10
59.00
59.00
74.01
98.33
99.26
118.00
137.67
139.49
174.67
174.68
177.00
181.67
222.02
236.00
277.54
314.23
393.32
Organic & Natural Product Chemistry Lab
Tyrosianse inhibitors from synthetic sources
Structure of tyrosinase inhibitors
Organic & Natural Product Chemistry Lab
Plant Source : Sophora flavescens
Plant taxonomy
- Leguminous
- Herbaceous perennial plant
Pharmacological reserch
- Antipyretic
- Antibiotic
- Diuretic
- Antifungal
- Anthelmintic - Antitumor
Main component
- Flavonoids
- Quinolizidine alkaloids
- Triterpenoid
Organic & Natural Product Chemistry Lab
Reported compounds from Sophora flavescens
OH
OH
HO
HO
O
O
OH
HO
HO
O
OMe
OMe O
OH
HO
O
HO
OH
OMe O
OMe O
O
O
OMe
HO
O
O
O
OH
O
OH
OH O
OH
HO
HO
OH
OH O
O
OH
O
OH
OMe O
HO
HO
O
OH
OH O
OH O
OH
HO
OH
OH
OH O
HO
O
MeO
OMe
O
O
O
O
O
OMe O
Organic & Natural Product Chemistry Lab
Isolated compounds from Sophora flavescens
OH
HO
HO
O
O
HO
O
OH
OH
OH
OH
OMe
OH O
O
OMe O
Compound 2
Sophoraflavanone G
Compound 1
Kushenol
Compound 3
(2S)-2’-methoxykurarinone
OH
HO
OH
HO
O
HO
O
OH
OH
OMe O
OMe O
Compound 4
Kurarinone
Compound 5
Kurarinol
Organic & Natural Product Chemistry Lab
Tyrosinase inhibition compounds from Sophora flavescens
OH
HO
OH
O
HO
O
OH
OH
HO
HO
O
OH
OH O
OH
OMe O
2. Sophoraflavanone G
OMe O
4. Kurarinone
5. Kurarinol
Compound
IC50 (μM)
Ki (μM)
Inhibition type
1
>30
-
-
2
2
77.0
Noncompetitive
3
>93
-
-
4
0.1
4.1
Noncompetitive
5
0.05
45.0
Competitive
Arbutin
10
-
-
Kojic acid
15
-
-
Organic & Natural Product Chemistry Lab
Enzyme kinetic analysis
Lineweaver-Burk plot
Lineweaver-Burk plot
Figure. Lineweaver-Burk plots for inhibition
of Kurarinone on mushroom tyrosinase
for catalysis of L-tyroine at 30℃, pH 6.8
Figure. Lineweaver-Burk plots for inhibition
of Kurarinol on mushroom tyrosinase for
catalysis of L-tyroine at 30℃, pH 6.8
Organic & Natural Product Chemistry Lab
Conclusion
Tyrosinase inhibition mechanism stydy.
SAR (Structure Activity Relationship) from Flavonoids
R3'
R8
R7
R2'
R4'
O
R6
R5'
R3
R5
O
R6'
Active site
- Hydroxyl groups at the R7 and R4' sites
in the A and B rings.
- Dihydroxyl group in the A and B rings.
Isolated of potent tyrosinase inhibitors from Sophora flavescens
- Sophoraflavaone G (IC50 =2μM , noncompetitive inhibition)
- Kurarinone (IC50 = 0.1μM, noncompetitive inhibition)
- Kurarinol (IC 50 = 0.05μM, competitive inhibition)
Development of Whitening agent
Organic & Natural Product Chemistry Lab