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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-3,4dihydroxyphenylalanine (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 compounds for 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.

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

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

Table 1. The concentration of tyrosinase inhibition activity of extracts of Blumea balsamifera DC leaves (n=3) Extracts Hexane extract Ethyl acetate extract Water extract IC50 (mg/mL) 0.3190.015 0.2060.037 0.3450.017

Paper mulberry extract

0.1570.023

Discussion
Paper mulberry extract, a natural skin lightening cosmetics, was chosen as positive control. The ethyl acetate extract showed the most potent anti-tyrosinase activity.

Materials & Methods and Results


Separation
Ethylacetate extract

F1

F2

F3

F4

F5

F6

F7

F8

F9

1,3,6,8, 9

5 7

Materials & Methods and Results


Fraction F2 was subjected to Flash CC with hexaneethyl acetate(4:1) and purified by TLC with same solvent(1:1) Fraction F4 was purified by flash CC with the same solvent(9:1) and subsequently by flash CC with DCMethyl acetate-acetone(18:1:1) Fraction F5 was applied to a silica gel flash CC with hexane-ethyl acetate(1:1) Fraction F7 was purified by sephadex LH-20 with ethanol and then by TLC with DCM-ethylacetateacetone(7:2:1)

Materials & Methods and Results


Melting points were determined on a electrothermal melting 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() with TMS as the internal reference.

Figure 1. Structures of the isolated compounds.

Figure 1. Structures of the isolated compounds.

Materials & Methods and Results


Measurement of anti-tyrosinase activity
extracts were dissolved in dimethyl sulfoxide (DMSO) at 1.0 mg/ml
diluted to different concentrations using DMSO Diluted w/ 1800l of 0.1M sodium phosphate(pH 6.8) and 1000l of L-3,4-dihydroxyphenylalanine(L-DOPA) Addition of 100l of mushroom tyrosinase soln(138 units)

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(BC)]/A *100 where: A=absorbance of control treatment B=absorbance of test sample treatment C=absorbance of test sample blank treatment

Materials & Methods and Results


Determination of mode of tyrosinase inhibition
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(mol/min)-1 versus the reciprocal of substrate concentration (mM)-1

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 80g/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 Dihydroquercetin-4-methyl ether (1) Dihydroquercetin-7,4-dimethyl ether (2) 5,7,3,5-Tetrahydroxyflavanone(3) Blumeatin (4) Quercetin (5) Rhamnetin (6) Tamarixetin (7) Luteolin (8) Luteolin-7-methyl ether(9) Kojic acid Arbutin IC50 (mM)SD 0.1150.013 0.1620.042 0.4230.049 0.6240.029 0.0960.004 0.1070.017 0.1440.004 0.2580.015 0.3500.002 0.0440.005 0.2330.025 Mode of Inhibition Competitive Competitive Competitive * Competitive Competitive * Non-competitive Non-competitive -

* Unable to establish

Discussion
The presence of methoxyl at the C-8 position tended to 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 antityrosinase 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 isolated flavonoids might be due to chelating with copper in the active center of tyrosinase.

Materials & Methods and Results


Determination of copper chelation
Reaction mixture containing 1800Lof 0.1 M phosphate buffer(pH 6.8), 1000Lof DI water, 100Lof 0.14mM CuSO4 soln and 100Lof 0.050mM inhibitor soln
Incubated at 25C for 30min

UV-Vis absorption spectra were measured at 240540nm

Materials & Methods and Results


Determination of ability to chelate copper in the enzyme
Reaction mixture containing 1800L of 0.1 M phosphate buffer(pH 6.8), 1000Lof DI water, 100L of mushroom tyrosinase soln and 100Lof 0.050mM inhibitor soln Incubated at 25C for 30min UV-Vis absorption spectra were measured at 240540nm

Table 3. The shift UV-Vis spectra of isolated compounds by adding Cu2+ and tyrosinase
Compounds Dihydroquercetin-4-methyl ether (1) Dihydroquercetin-7,4-dimethyl ether (2) 5,7,3,5-Tetrahydroxyflavonone (3) Shift by Cu2+ (nm) 325 -> 325 326 -> 327 322 -> 318 Shift by tyrosinase (nm) 325-> 315 326 -> 313 322 -> 321

Blumeatin (4)
Quercetin (5) Rhamnetin (6) Tamarixetin (7) Luteolin (8) Luteolin -7-methyl ether (9)

324 -> 321


371 -> 440 373 -> 435 375 -> 359 350 -> 399 348 -> 396

324 -> 320


371 -> 367 373 -> 325 375 -> 353 350 -> 316 358 -> 315

Discussion
The UV-Vis spectra of dihydroflavonols(1 and 2) and flavanones(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 isolated flavonols chelated with copper in tyrosinase.

Table 4. Biological activity of flavonoids isolated from the leaves of B. balsamifera DC.
Compounds Dihydroquercetin-4-methyl ether (1) Dihydroquercetin-7,4-dimethyl ether (2) 5,7,3,5-Tetrahydroxyflavonone (3) Blumeatin (4) Quercetin (5) Cytotoxicity (IC50, g/mL)

KBa
Inactive 17.09 Inactive 47.72 Inactive

MCF7b
Inactive Inactive Inactive Inactive Inactive

NCI-H187c
Inactive 16.29 29.97 Inactive 20.59

Luteolin-7-methyl ether (9)


Tamoxifen Doxorubicine Ellipticine

17.83
0.222 0.553

Inactive
4.03 9.65 -

5.21
0.073 1.39

a=Oral cavity cancer; b=Breast cancer; c=Small cell lung cancer.

Discussion
Compounds 2 and 9 show moderate toxicity against the 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-tyrosinase activity. Compounds 2, 4, and 9 were active against the KB cells, all are inactive against the MCF7cells, while compounds 2, 3, 5, and 9 exhibited activity against the NCI-H187 cells. These findings have ecological and economic significance for application of B. balsamifera DC leaves extract as skin lightening agent in cosmetic industry.