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Saewan, N.; Koysomboon, S.; Chantrapromma, K.J. Med. Plant. Res. 2011, 5(6), 1018-1025.
Prepared by: Hershey Y. Jopia
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Outline
Introduction
Objective of the study
Materials & Methods and Results
Discussion
Conclusion
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Introduction
Tyrosinase is a copper-containing monooxygenase,which catalyzes the first two reactions of melanin
synthesis,(1) hydroxylation of L-tyrosine to L-3,4-
dihydroxyphenylalanine (L-DOPA), and (2) oxidation of
L-DOPA to dopaquinone (Seo et al ., 2003).
Cosmetic agents that inhibit tyrosinase activity or
that block melanogenic pathways leading to skin
lightening have been the subject of many researches(Kim et al ., 2002; Shimizu et al ., 2000; Son et al .,
2000).
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Introduction
Hydroxylated flavonoids are good target compoundsfor tyrosinase inhibitors because they share structural
similarities with the natural substrate for tyrosinase
(Jeong et al ., 2009).
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Objective of the study
The study aimed to examine the flavonoids
from B. balsamifera DC leaves on the
inhibition of mushroom tyrosinase as well as
anti-cancer activity against three human KB,MCF-7 and NCI-H187 cell lines.
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Materials & Methods and Results
Extraction
Leaves of B. balsamifera DC (5.2kg)
Air-dried, ground, and extracted with ethanol
Extract was evaporated to dryness
Ethanolic extract (102.9g) was suspended in water and
re-extracted with hexane and ethylacetate
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Materials & Methods and Results
Extraction
Fractions were evaporated to give hexane(41.1g),
ethylacetate(36.9g), and water(22.7g) extracts
Extracts were dissolved in dimethyl sulfoxide (DMSO)
and then was subjected to mushroom tyrosinase
assay
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Table 1. The concentration of tyrosinase inhibition activity of
extracts of Blumea balsamifera DC leaves (n=3)
Extracts IC50 (mg/mL)
Hexane extract 0.319±0.015
Ethyl acetate extract 0.206±0.037
Water extract 0.345±0.017
Paper mulberry extract 0.157±0.023
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Discussion
Paper mulberry extract, a natural skinlightening cosmetics, was chosen as positive
control. The ethyl acetate extract showed the
most potent anti-tyrosinase activity.
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Materials & Methods and Results
Separation Ethylacetateextract
F3F2F1 F8F7F4 F6 F9F5
421,3,6,8,
97
5
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Materials & Methods and Results Fraction F2 was subjected to Flash CC with hexane-
ethyl acetate(4:1) and purified by TLC with samesolvent(1:1)
Fraction F4 was purified by flash CC with the same
solvent(9:1) and subsequently by flash CC with DCM-ethyl acetate-acetone(18:1:1)
Fraction F5 was applied to a silica gel flash CC withhexane-ethyl acetate(1:1)
Fraction F7 was purified by sephadex LH-20 withethanol and then by TLC with DCM-ethylacetate-acetone(7:2:1)
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Materials & Methods and Results
Melting points were determined on a electrothermalmelting point apparatus, UV-Vis spectra were
measured with a Biochrom Libra S22 UV/Vis
spectrophotometer.
1H and 13C NMR were recorded using Bruker FTNMR
Ultrashield spectrometer, and chemical shifts were
recorded in parts per million(δ) with TMS as the
internal reference.
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Figure 1. Structures of the isolated compounds.
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Figure 1. Structures of the isolated compounds.
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Materials & Methods and Results
Measurement of anti-tyrosinase activityextracts were dissolved in dimethyl sulfoxide (DMSO) at 1.0
mg/ml
diluted to different concentrations using DMSO
Diluted w/ 1800µl of 0.1M sodium phosphate(pH 6.8) and
1000µl of L-3,4-dihydroxyphenylalanine(L-DOPA)
Addition of 100µl of mushroom tyrosinase soln(138 units)
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Materials & Methods and Results
dopachrome formation was measured using UV-Vis
Spectrometer at 475nm for 6 mins.
% Tyrosinase-inhibition activity was calculated and is expressed
as IC50
% Tyrosinase inhibition=[A –(B –C)]/A *100
where: A=absorbance of control treatment
B=absorbance of test sample treatment
C=absorbance of test sample blank treatment
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Materials & Methods and Results
Determination of mode of tyrosinaseinhibition
Assay was varied, L-DOPA concentrations(0.5, 1.0, 1.5,
and 2.5 mM)
Kinetic constants(Km and Vmax) were determined by
the Lineweaver-Burk plot of the reciprocal of the
reaction rate(µmol/min)-1 versus the reciprocal of
substrate concentration (mM)-1
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Materials & Methods and Results
Determination of Inhibition Constant
Assay was varied, L-DOPA concentration(0.5, 1.0, 1.5,
and 2.5 mM) and inhibitor(0, 20, 40, 60, and
80µg/mL)
Inhibition constants were obtained by the second plots
of the apparent Michaelis-Menten constant (Kmapp)
versus conc. of inhibitor and calculated as:
Kmapp=(Km/Ki)[l]+Km
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Table 2. The concentration of tyrosinase inhibition activity and mode of
inhibition of isolated compounds (n=3).
Compounds IC50 (mM)±
SD Mode of Inhibition
Dihydroquercetin-4’-methyl ether (1) 0.115±0.013 Competitive
Dihydroquercetin-7,4’-dimethyl ether (2) 0.162±0.042 Competitive
5,7,3’,5’-Tetrahydroxyflavanone(3) 0.423±0.049 Competitive
Blumeatin (4) 0.624±0.029 *
Quercetin (5) 0.096±0.004 Competitive
Rhamnetin (6) 0.107±0.017 Competitive
Tamarixetin (7) 0.144±0.004 *
Luteolin (8) 0.258±0.015 Non-competitiveLuteolin-7-methyl ether(9) 0.350±0.002 Non-competitive
Kojic acid 0.044±0.005 -
Arbutin 0.233±0.025 -
* Unable to establish
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Discussion
The presence of methoxyl at the C-8 position tendedto reduce the anti-tyrosinase activity as indicated by
the results: (1)>(2),(3)>(4 ),(5)>(6),(7),(8)>(9).
In addition, (2) exhibited comparatively weaker anti-tyrosinase activity than (7), which suggests that the
presence of the C2-C3 double bond is also essential
for tyrosinase inhibitor ability.
The tyrosinase inhibitory activity of the isolatedflavonoids might be due to chelating with copper in
the active center of tyrosinase.
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Materials & Methods and Results
Determination of copper chelation
Reaction mixture containing 1800µLof 0.1 M phosphate
buffer(pH 6.8), 1000µLof DI water, 100µLof 0.14mM
CuSO4 soln and 100µLof 0.050mM inhibitor soln
Incubated at 25°C for 30min
UV-Vis absorption spectra were measured at 240-
540nm
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Materials & Methods and Results
Determination of ability to chelate copper inthe enzyme
Reaction mixture containing 1800µL of 0.1 Mphosphate buffer(pH 6.8), 1000µLof DI water, 100µLof mushroom tyrosinase soln and 100µLof 0.050mM
inhibitor soln
Incubated at 25°C for 30min
UV-Vis absorption spectra were measured at 240-540nm
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Table 3. The shift UV-Vis spectra of isolated compounds by adding
Cu2+ and tyrosinase
CompoundsShift by Cu2+
(nm)Shift by
tyrosinase (nm)
Dihydroquercetin-4’-methyl ether (1) 325 -> 325 325-> 315
Dihydroquercetin-7,4’-dimethyl ether (2) 326 -> 327 326 -> 313
5,7,3’,5-Tetrahydroxyflavonone (3) 322 -> 318 322 -> 321
Blumeatin (4) 324 -> 321 324 -> 320
Quercetin (5) 371 -> 440 371 -> 367
Rhamnetin (6) 373 -> 435 373 -> 325
Tamarixetin (7) 375 -> 359 375 -> 353
Luteolin (8) 350 -> 399 350 -> 316
Luteolin -7-methyl ether (9) 348 -> 396 358 -> 315
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Discussion
The UV-Vis spectra of dihydroflavonols(1 and 2) andflavanones(3 and 4), showed no significant shift by
adding Cu2+ and by incubation of the enzyme which
suggests that those flavonoids compete with
substrate to combine with free enzyme without
chelating copper in enzyme pathway.
While the spectra of flavonols(5-7) exhibited
hypsochromic shift, it suggests that the isolatedflavonols chelated with copper in tyrosinase.
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Table 4. Biological activity of flavonoids isolated from the leaves of
B. balsamifera DC.
CompoundsCytotoxicity (IC50, µg/mL)
KBa MCF7b NCI-H187c
Dihydroquercetin-4’-methyl ether (1) Inactive Inactive Inactive
Dihydroquercetin-7,4’-dimethyl ether (2) 17.09 Inactive 16.29
5,7,3’,5’-Tetrahydroxyflavonone (3) Inactive Inactive 29.97
Blumeatin (4) 47.72 Inactive Inactive
Quercetin (5) Inactive Inactive 20.59
Luteolin-7-methyl ether (9) 17.83 Inactive 5.21
Tamoxifen - 4.03 -
Doxorubicine 0.222 9.65 0.073
Ellipticine 0.553 - 1.39
a=Oral cavity cancer; b=Breast cancer; c=Small cell lung cancer.
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Discussion
Compounds 2 and 9 show moderate toxicity againstthe KB cells, whereas, compound 4 exhibited weak
activity.
All compounds were inactive against MCF7 cells.
Compounds 2, 3, and 5 showed moderate activity
against NCI-H187 cells, while compound 9 exhibited
strong activity.
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Conclusion
Nine flavonoids isolated from the leaves of B.Balsamifera DC showed moderate of anti-tyrosinaseactivity.
Compounds 2, 4, and 9 were active against the KB
cells, all are inactive against the MCF7cells, whilecompounds 2, 3, 5, and 9 exhibited activity againstthe NCI-H187 cells.
These findings have ecological and economic
significance for application of B. balsamifera DCleaves extract as skin lightening agent in cosmeticindustry.