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Saewan, N.; Koysomboon, S.; Chantrapromma, K.J. Med. Plant. Res. 2011, 5(6), 1018-1025.

Prepared by: Hershey Y. Jopia



Outline

Introduction

Objective of the study

Materials & Methods and Results

Discussion

Conclusion



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).



Introduction

Hydroxylated flavonoids are good target compoundsfor tyrosinase inhibitors because they share structural

similarities with the natural substrate for tyrosinase

(Jeong et al ., 2009).



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.




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




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



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



Discussion

Paper mulberry extract, a natural skinlightening cosmetics, was chosen as positive

control. The ethyl acetate extract showed the

most potent anti-tyrosinase activity.




Separation Ethylacetateextract

F3F2F1 F8F7F4 F6 F9F5

421,3,6,8,

97

5



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)




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.



Figure 1. Structures of the isolated compounds.




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)




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




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




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



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



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.




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




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



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



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.



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.



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.



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.

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