Improved, rapid and efficient synthesis of polymethoxyflavones under microwave irradiation and their inhibitory effects on melanogenesis

Article abstract of DOI:10.3987/COM-03-9891

The microwave-assisted methylation of 2,5-dihydroxy-1,3-dimethoxybenzene with (CH₃O)₂SO₂ for 5 min gave easily 1,2,3,5-tetramethoxybenzene, which was converted into pentamethoxybenzene. The microwave-assisted Friedel-Crafts acylation of pentamethoxybenzene in the presence of In(CF₃SO₃)₃ gave pentamethoxyacetophenone for 15 min under solvent-free conditions in high yield. The microwave-assisted cyclization reaction of the diketones, which were synthesized from the acetophenone via three steps under microwave irradiation, gave the desired polymethoxyflavones for 1.5-3 min in high yields. The polymethoxyflavones showed inhibitory effects on melanogenesis in a human melanoma.

Full text of DOI:10.3987/COM-03-9891

HETEROCYCLES, Vol. 60, No. 12, 2003, pp. 2775 - 2784
Received, 18th August, 2003, Accepted, 9th October, 2003, Published online, 14th October, 2003
IMPROVED, RAPID AND EFFICIENT SYNTHESIS OF POLY-
METHOXYFLAVONES UNDER MICROWAVE IRRADIATION AND
THEIR INHIBITORY EFFECTS ON MELANOGENESIS
Masao Tsukayama,a* Yasuhiko Kawamura,a Takaaki Ishizuka,a Shinji
Hayashi,b and Fumihito Toriib
aDepartment of Chemical Science and Technology, Faculty of Engineering, The
University of Tokushima, Minamijosanjima-cho, Tokushima 770-8506, Japan
bNOF Corporation Oleochemicals Research Laboratory, 1-56 Ohama-cho,
Amagasaki 660-0095, Japan
Abstract - The microwave-assisted methylation of 2,5-dihydroxy-1,3-
dimethoxybenzene with (CH3O)2SO2 for 5 min gave easily 1,2,3,5-
tetramethoxybenzene, which was converted into pentamethoxybenzene.
The
microwave-assisted Friedel-Crafts acylation of pentamethoxybenzene in the
presence of In(CF3SO3)3 gave pentamethoxyacetophenone for 15 min under
solvent-free conditions in high yield.
The microwave-assisted cyclization
reaction of the diketones, which were synthesized from the acetophenone via
three steps under microwave irradiation, gave the desired polymethoxyflavones
for 1.5-3 min in high yields.
The polymethoxyflavones showed inhibitory
effects on melanogenesis in a human melanoma.
Flavones are widely distributed in nature and have various biological properties.1
The flavones
contained in Citrus species have been found to exhibit antitumor, antioxidant and antiinflammatory
activities.2 Among these compounds, Nobiletin (3’,4’,5,6,7,8-hexamethoxyflavone) and tangeretin
(4’,5,6,7,8-pentamethoxyflavone) in Citrus aurantium have shown antiallergic activity and a cell-growth
inhibitory effect,3 while we have recently extracted nobiletin and tangeretin as a small amount of
components from the pericarps of Citrus aurantium tachibana (Makino) Tanaka and have discovered that
both these compounds also suppressed tyrosinase activity.4
been receiving much attention in the fields of preventive medicine and pharmaceutics, as well as in food
science and cosmetics in recent years. While polymethoxyflavones such as nobiletin have been
prepared using a variety of methods,5 many of the procedures are somewhat unsatisfactory with regard to
operational simplicity, reaction times, yields or cost of the reagents. Microwave irradiation has been
Not surprisingly, flavone derivatives have

recently used for a variety of applications including organic synthesis,6 and the new microwave irradiation
methodology could also be applicable to the synthesis of polymethoxyflavones. Improvement of
polymethoxyflavone synthesis methods would further the continuation of our work and facilitate
examinations of biological activities of these and other related compounds. We wish to report for the
first time on the efficient synthesis of 3’,4’,5,6,7,8-hexamethoxyflavone (nobiletin) (1), 4’,5,6,7,8-
pentamethoxyflavone (tangeretin) (2) and 5,6,7,8-tetramethoxyflavone (3) under microwave-assisted
conditions, and on the inhibitory effects of the obtained compounds on melanogenesis in a human
melanoma.
RESULTS AND DISCUSSION
The catalytic hydrogenation of 2,6-dimethoxy-1,4-benzoquinone7 over Raney nickel gave 2,5-dihydroxy-
1,3-dimethoxybenzene (4). Microwave-assisted methylation of 4 with (CH3O)2SO2 in the presence of
K2CO3 gave the tetramethoxybenzene (5) for 5 min in minimal acetone in 84% yield. The reaction of 5
with acetyl chloride in the presence of AlCl3 for 1.5 h gave easily tetramethoxyacetophenone (6), which
was partially demethylated with AlCl3 in acetonitrile to give 2’-hydroxyacetophenone (7) in 83% yield.
However, the published method gave 7 (66%) and 3’-ethoxy-2’-hydroxy-4’,6’-dimethoxyacetophenone
(7 %) at 30 ˚C for 6 h.8
the published method in reaction time, operational simplicity, yield and absence of 3’-ethoxy-2’-hydroxy-
4’,6’-dimethoxyacetophenone formation. Microwave-assisted acetylation of 5 with acetyl chloride in
Therefore, the above-mentioned improved synthesis of 6 and 7 is superior to
the presence of In(CF3SO3)3 (2%)9 gave the desired acetophenone (6) as a major product and 2’-acetoxy-
3’,4’,6’-trimethoxyacetophenone, which was readily converted into the 2’-hydroxycetophenone (7), as a
minor product for 6 min under solvent-free conditions.
The oxidative rearrangement of 7 with 6%
H2O2 gave 2,3-dihydroxybenzene (8),10 which was readily converted into pentamethoxybenzene (9) by the
(CH3O)2SO2-K2CO3 method. Microwave-assisted acetylation of 9 with acetyl chloride in the presence
of In(CF3SO3)3 (0.5%) for 15 min without solvent gave pentamethoxyacetophenone (10) in 75% yield,
and no by-product was obtained.
The pentamethoxyacetophenone (10) had already been prepared in
low yield by an alternative method.7 The described microwave-assisted Friedel-Crafts acetylation was
found to proceed quite rapidly and efficiently in spite of the existence of many methoxy groups (Scheme
1).

The partial demethylation of 10 with AlCl3 in acetonitrile was carried out to give 2’-hydroxyacetophenone
(11) in 83% yield. The above results revealed that the microwave-assisted acetylation of
polymethoxybenzenes in the presence of In(CF3SO3)3 is an excellent method for the synthesis of
polymethoxyacetophenones because of rapid reaction, high yield and solvent-free conditions.
The esterification of 11 with benzoyl chloride (12) in minimal pyridine was carried out for 4 min under
microwave irradiation to give benzoate (13) in 75% yield.
13 in the presence of KOH was carried out in minimal pyridine for 4 min under microwave irradiation to
give easily the desired 1,3-diketone (14). The microwave-assisted cyclization reaction of 14 with
The Baker-Venkataraman rearrangement of
concd H2SO4 in minimal acetic acid was achieved for 1.5 min to give the desired tetramethoxyflavone (3)
in 81% yield (Scheme 2).
In the same manner as in the case of 13, the esterification of 11 with 4-methoxybenzoyl chloride (15)
gave 4-methoxybenzoate (16) for 2.5 min under microwave irradiation.
crude benzoate (16) with KOH in pyridine gave the desired diketone (17) for 5 min under microwave
irradiation. The microwave-assisted cyclization reaction of 17 with concd H2SO4 was achieved in
The rearrangement of the
acetic acid for 3 min to give the desired pentamethoxyflavone (2) in 60% yield via three steps from 11.
The esterification of 11 with 3,4-dimethoxybenzoyl chloride (18) also gave easily 3,4-dimethoxybenzoate
(19) for 2 min under microwave irradiation.
In the same manner as in the case of 2, the rearrangement
of the crude dimethoxybenzoate (19) into diketone (20), followed by the cyclization reaction of 20 was
achieved for 3 min under microwave irradiation to give the desired hexamethoxyflavone (1) in 63% yield
via three steps from 11.

1H-NMR spectral data for the synthetic hexa- and pentamethoxyflavones (1 and 2) were identical with
those of natural nobiletin and tangeretin, respectively. Each melting point of the synthetic compounds
(1 and 2) was not lowered by each admixture with natural nobiletin and tangeretin. On the basis of
these results, structures of natural nobiletin and tangeretin in the pericarps of Citrus aurantium tachibana
(Makino) Tanaka were unequivocally established to be 3’,4’,5,6,7,8-hexamethoxyflavone and 4’,5,6,7,8-
pentamethoxyflavone, respectively.
The new synthetic methodology under microwave-enhanced conditions also is environmentally friendly
and results in high yields of the desired pentamethoxyacetophenone and polymethoxyflavones.
Inhibitory effects on melanogenesis in a human melanoma cell line, HM3KO,11 were carried out at
different concentrations of the synthetic polymethoxyflavones (1, 2 and 3), and the results are shown in
Figure 1.
These compounds suppressed melanin production according to dosedependence.
Nobiletin (1) and tangeretin (2) suppressed up to 50-61% of the melanin production at a concentration of
10 µM, but the tetramethoxyflavone (3) showed as much as 77% inhibition at the same concentration.
The inhibitory effects of polymethoxyflavones on melanogenesis in HM3KO were consistent with those
of natural nobiletin and tangeretin. On the other hand, polymethoxyflavones did not affect the cell
growth at the concentration of less than 10 uM.
From these results, the flavones containing more

methoxy groups were found to show higher inhibitory effects on melanogenesis.
Polymethoxyphenols
(pentamethoxyacetophenone and pentamethoxybenzene) except for polymethoxyflavones showed no
inhibitory effect. From these results, hexa- and pentamethoxyflavones (1 and 2) are utilizable as an
agent for the treatment of skin pigmentation such as spots and freckles induced by UV light.
The improved, rapid synthesis of polymethoxyflavones under microwave irradiation would be useful for
various examinations of biological activities, because the synthesis of these compounds is both simple
and efficient.
EXPERIMENTAL
The melting points were measured on a Yanaco MP-J3 micro melting-point apparatus and are uncorrected.
The 1H-NMR spectra were measured with a JEOL EX400 spectrometer (400 MHz), using
tetramethylsilane as internal standard (δ, ppm). Elemental analyses were performed with a Yanaco
CHN corder model MT-5.
A domestic microwave oven (Sanyo Co., 650 W and 2.45 GHz, modified
properly by fitting a condenser and a thermosensor through the holes made in the roof) was used as a
reaction apparatus. Column chromatography and thin-layer chromatography (TLC) were carried out on
Kieselgel 60 (70-230 mesh) and with Kieselgel 60 F-254 (Merck).
2,6-Dimethoxy-1,4-hydroquinone (4)
2,6-Dimethoxy-1,4-benzoquinone (20.18 g, 0.12 mol), prepared by published method,7) was hydrogenated
over Raney Ni (6.23 g) in methanol (200 mL) at rt with stirring until the uptake of hydrogen ceased.
The resulting compound was chromatographed over a short cilica gel column (AcOEt : hexane = 1:1 as a
solvent) to give the hydroquinone (4)7 (19.32 g, 96%) as brown needles, mp 164-165 ˚C, from AcOEt).
1,2,3,5-Tetramethoxybenzene (5)
A mixture of 4 (20.1 g, 0.12 mol), (CH3O)2SO2 (28.4 mL, 0.30 mol) and K2CO3 (83 g, 0.60 mol) in
acetone (200 mL) was refluxed at 70 ˚C for 3 h with stirring.
The resulting compound was purified by
silica gel column chromatography (AcOEt:hexane =1:1) to give 57 (17.8 g, 75% from 2,6-dimethoxy-1,4-
1
benzoquinone) as colorless needles, mp 32-33 ˚C (crystallized from AcOEt-hexane); H-NMR (CDCl3)
δ 3.79 (6H, s, OCH3 x 2), 3.85 (6H, s, OCH3 x 2), 6.15 (2H, s, Ar-H x 2).
1,2,3,5-Tetramethoxybenzene (5) (under microwave irradiation).
A mixture of the hydroquinone (4) (5 g, 29.4 mmol), (CH3O)2SO2 (11.2 mL, 0.12 mol) and K2CO3 (20 g,
0.15 mol) in acetone (24 mL) was stirred at 73 ˚C for 5 min (1 min x 5 with intermissions of 20 sec in
between) under microwave irradiation.
The resulting compound was purified by silica gel column
chromatography to give 5 (4.6 g, 84%) as a colorless oil.
2’,3’,4’,6’-Tetramethoxyacetophenone (6)
To a mixture of 5 (3.01 g, 15.18 mmol) and AlCl3 (6.71 g, 50.32 mmol) in ether (50 mL) was gradually

added acetyl chloride (1.49 g, 18.98 mmol) in ether (10 mL) and the whole mixture was stirred at 0 ˚C for
a further 1.5 h. To the reaction mixture was added 6% HCl (50 mL), and then the resultant mixture was
stirred for 30 min at rt. The resulting compound was extracted with ether and washed with 5% aqueous
NaOH and water, and dried (Na2SO4). The resulting compound gave the acetophenone (6)7 (3.66 g) as
a pale paste, which was used for the next experiment without purification.
2’-Hydroxy-3’,4’,6’-trimethoxyacetophenone (7)
A mixture of 6 (3.66 g) and AlCl3 (6.31 g, 47.3 mmol) in CH3CN (20 mL) was stirred at 70 ˚C for 1 h.
To the reaction mixture was added 6% HCl (50 mL) and the resultant mixture was stirred at 60 ˚C for 30
min.
The resulting compound was recrystallized from methanol to give the 2’-hydroxyacetophenone
1
(7)8 (2.85 g, 83% from 5) as pale yellow prisms, mp 112-114 ˚C; H-NMR (CDCl3) δ 2.62 (3H, s,
CH3CO), 3.82 (3H, s, OCH3), 3.90 (3H, s, OCH3), 3.94 (3H, s, OCH3), 5.97 (1H, s, Ar-H), 13.81 (1H, s,
OH).
2’,3’,4’,6’-Tetramethoxyacetophenome (6) (under microwave irradiation)
A mixture of 5 (1.5 g, 7.65 mmol), CH3COCl (1.08 mL, 15.1 mmol) and In(CF3SO3)3 (75 mg, 0.13 mmol)
was stirred for 6 min (1 min x 6 with intermissions of 1 min in between) at 75-90 ˚C under microwave
(350 W) irradiation.
The resulting compound was chromatographed over a silica gel column to give
the acetophenone (6) (0.963 g, 53%) and 2’-acetoxy-3’,4’,6’-trimethoxyacetophenone (0.13 g, 6%),
which was hydrolyzed with 10% aqueous NaOH (5 mL) at rt for 30 min to give the 2’-
hydroxyacetophenone (7) (90 mg, 80% from AcOEt-hexane), mp 112-114 ˚C.
2,3-Dihydroxy-1,4,5-trimethoxybenzene (8)
To a mixture of 7 (15.0 g, 66.31 mmol) and 5% aqueous NaOH (67.5 mL, 67 mmol) in methanol (130
mL) was drop by drop added 6% H2O2 (135 mL, 0.20 mol) at 3-5 ˚C for 1 h, and then the whole mixture
was stirred at rt for 1 h.
was removed under reduced pressure. The residue was extracted with ether and washed with water and
dried (Na2SO4).
The resulting compound gave the dihydroxybenzene (8)7,10 (7.77 g, 59%) as pale
After the reaction mixture was acidified with 10% HCl, the organic solvent
yellow needles (mp 90-92 ˚C; part of which was crystallized from hexane-benzene), which was used for
the next experiment without purification.
1,2,3,4,5-Pentamethoxybenzene (9)
A mixture of 8 (7.76 g, 38.76 mmol), (CH3O)2SO2 (17.0 mL, 0.18 mol) and K2CO3 (53.9 g, 0.96 mol) was
refluxed in acetone (150 mL) at 60 ˚C for 1.5 h with stirring.
The resulting compound gave the
pentamethoxybenzene (9)7 (7.52 g) as a colorless solid, which was used for the next experiment without
purification.
2’,3’,4’,5’,6’-Pentamethoxyacetophenone (10)

In a 50 mL round-bottomed flask were placed 9 (10.33 g, 45.28 mmol), CH3COCl (6.50 mL, 91.4 mmol)
and In(CF3SO3)3 (128.4 mg, 0.22 mmol). The whole mixture was stirred by a magnetic stirrer for
uniform mixing and after that the mixture was stirred for 15 min (20 sec x 45 with intermissions of 30 sec
in between) under microwave irradiation. The reaction mixture was poured into ice water and then was
extracted with ethyl acetate, after which the extract was washed with 3% aqueous NaOH, then water, and
dried (Na2SO4).
The resulting compound was chromatographed over a silica gel column to give the
1
acetophenone (10)7 (9.20 g, 75%, crystallized from hexane) as pale needles, mp 42-45 ˚C; H-NMR
(CDCl3) δ 2.50 (3H, s, CH3CO), 3.84 (6H, s, OCH3 x 2), 3.88 (6H, s, OCH3 x 2), 3.95 (3H, s, OCH3).
2’-Hydroxy-3’,4’,5’,6’-tetramethoxyacetophenone (11)
A mixture of 10 (5.49 g, 20.29 mmol), AlCl3 (13.51 g, 101.31 mmol) and CH3CN (20 mL) was stirred at
60 ˚C for 30 min.
The resulting compound was extracted with ether, washed with sodium carbonate
and water, and dried (Na2SO4). The obtained compound was chromatographed over a short silica gel
column to give the 2’- hydroxyacetophenon (11)7 (4.31 g, 83%) as a pale yellow oil.
2’-Benzoyloxy-3’,4’,5’,6’-tetramethoxyacetophenone (13)
A mixture of 11 (1.01 g, 3.95 mmol), benzoyl chloride (0.54 mL, 4.65 mmol) and pyridine (1.2 mL) was
stirred for 2 min (1 min x 2 with an intermission of 1 min in between) under microwave irradiation.
The whole mixture was extracted with ether, and the extract was washed with 6% HCl and water, and
dried (Na2SO4).
The resulting compound was recrystallized from methanol to give the ester (13) (1.05
1
g, 74%) as pale brown needles, mp 78-79 ˚C; H-NMR (CDCl3) δ 2.48 (3H, s, CH3CO), 3.82 (3H, s,
OCH3), 3.90 (3H, s, OCH3), 3.98 (3H, s, OCH3), 7.50 (2H, t, J=8.1 Hz, Ar-H x 2), 7.63 (1H, m, Ar-H),
8.11-8.17 (2H, m, Ar-H x 2).
1-(2-Hydroxy-3,4,5,6-tetramethoxyphenyl)-3-phenyl-1,3-propanedione (14)
A mixture of 13 (2.75 g, 7.13 mmol), KOH (2.07 g, 35.64 mmol) and pyridine (15 mL) was stirred for 4
min (1 min x 4 with intermissions of 1 min in between) under microwave irradiation.
mixture was poured into a solution of ice-hydrochloric acid and stirred for 30 min at rt.
The whole
The mixture
was slightly acidified with 3% hydrochloric acid and extracted with ether, after which the extract was
washed with water and dried (Na2SO4). The resulting compound gave the desired diketone (14) (2.20
g) as a pale brown oil, which was used for the next experiment without purification.
5,6,7,8-Tetramethoxyflavone (3)
A mixture of 14 (2.20 g, 6.11 mmol), concd H2SO4 (2 mL) and acetic acid (15 mL) was stirred for 1.5 min
(1 min + 0.5 min with intermissions of 1 min in between) under microwave irradiation.
mixture was poured into ice water and extracted with CHCl3, after which the extract was washed with 3%
aqueous NaOH, then water, and dried (Na2SO4). The resulting compound was recrystallized from
The reaction

1
methanol to give the flavone (3) (1.69 g, 81%) as colorless needles, mp 107-108 ˚C; H-NMR (CDCl3)
δ 3.96 (6H, s, OCH3 x 2), 4.03 (3H, s, OCH3), 4.11 (3H, s, OCH3), 6.70 (1H, s, 2-H), 7.54 (3H, m, Ar-H x
3), 7.94 (2H, m, Ar-H x 2); Anal. Calcd for C19H18O6: C, 66.66; H, 5.30. Found: C, 66.58; H, 5.36.
2’-(4-Methoxybenzoyloxy)-3’,4’,5’,6’-tetramethoxyacetophenone (16)
A solution of 11 (1.98 g, 7.73 mmol) and anisoyl chloride (15) (1.61 g, 9.41 mmol) in pyridine (5 mL)
was stirred for 1.5 min (1 min + 0.5 min with an intermission of 1 min in between) under microwave
irradiation. The reaction mixture was worked up in the same manner as in the case of the benzoate (13)
to give the anisoate (16) (3.37 g) as a pale yellow solid, which was used for the next examination without
purification.
1-(2-Hydroxy-3,4,5,6-tetramethoxyphenyl)-3-(4-methoxyphenyl)-1,3-propanedione (17)
A mixture of the crude 16 (3.37 g), KOH (1.38 g, 24.59 mmol) and pyridine (10 mL) was stirred for 5
min (1 min x 5 with intermissions of 1 min in between) under microwave irradiation.
The reaction
mixture was worked up in the same manner as in the case of 13 to give the crude diketone (17) (3.10 g) as
a pale brown solid, which was used for the next experiment without purification.
4’,5,6,7,8-Pentamethoxyflavone (2)
A solution of the crude 17 (3.10 g) and concd H2SO4 (2 mL) in acetic acid (15 mL) was stirred for 3 min
(1 min x 3 with intermissions of 1 min in between) under microwave irradiation.
The reaction mixture
was worked up in the same manner as in the case of 3 to give the flavone (2)4 (1.75 g, 60% via three steps
from 11) as a pale brown solid, which was recrystallized from methanol as colorless needles, mp 150-151
1
˚C; H-NMR (CDCl3) δ 3.89 (3H, s, OCH3), 3.95 (6H, s, OCH3 x 2), 4.03 (3H, s, OCH3), 4.10 (3H, s,
OCH3), 6.61 (1H, s, 2-H), 7.03 (2H, d, J=8.8 Hz, Ar-H x 2), 7.88 (2H, d, J=8.8 Hz, Ar-H x 2); Anal.
Calcd for C20H20O7: C, 64.51; H, 5.41. Found: C, 64.57; H, 5.44.
2’-(3,4-Dimethoxybenzoyloxy)-3’,4’,5’,6’-tetramethoxyacetophenone (19)
A solution of 11 (2.15 g, 8.39 mmol) and fresh 3,4-dimethoxybenzoyl chloride (18) (1.61 g, 9.41 mmol)
in pyridine (10 mL) was stirred for 2 min (1 min x 2 with an intermission of 1 min in between) under
microwave irradiation. The reaction mixture was worked up in the same manner as in the case of 13 to
give the dimethoxybenzoate (19)5a (3.78 g) as a pale brown oil, which was used for the next experiment
without purification.
1-(2-Hydroxy-3,4,5,6-tetramethoxyphenyl)-3-(3,4-dimethoxyphenyl)-1,3-propanedione (20)
A mixture of the crude 19 (3.78 g), KOH (2.40 g, 42.7 mmol) and pyridine (30 mL) was stirred for 6 min
(1 min x 6 with intermissions of 1 min in between) under microwave irradiation.
The reaction mixture
was worked up in the same manner as in the case of 13 to give the crude diketone (20)5a (4.58 g) as a pale
brown solid, which was used for the experiment without purification.

3’,4’,5,6,7,8-Hexamethoxyflavone (1)
A solution of the crude 20 (4.58 g) and concd H2SO4 (3 mL) in acetic acid (30 mL) was stirred for 3 min
(1 min x 3 with intermissions of 1 min in between) under microwave irradiation.
The reaction mixture
was worked up in the same manner as in the case of 3 to give the desired hexamethoxyflavone (1)4, 5a
(3.53 g) as pale yellow needles, which were recrystallized from ethyl acetate to afford colorless needles
(2.03 g, 63% via 3 steps from 11), mp 136-137 ˚C; 1H-NMR (CDCl3) δ 3.96 (6H, s, OCH3 x 2), 3.97 (3H,
s, OCH3), 3.98 (3H, s, OCH3), 4.03 (3H, s, OCH3), 4.11 (3H, s, OCH3), 6.63 (1H, s, 2-H), 7.00 (1H, d,
J=8.6 Hz, Ar-H), 7.42 (1H. s, Ar-H), 7.57 (1H, d, J=8.6 Hz, Ar-H); Anal. Calcd for C21H22O8: C, 62.68;
H, 5.51. Found: C, 62.70; H, 5.54.
Cell culture.
Human melanoma cells (HM3KO) were maintained in Dulbecco’s modified Eagle
medium containing 10% fetal bovine serum, 100,000 U/L benzylpenicillin potassium and 100 mg/L
kanamycin sulfate.
Melanin assay.
cultured. After incubation for 9 h, the cells were treated with various concentrations of test reagents.
Three days later, the medium was changed and the test reagents at the same concentration were added to
the medium. The cells were cultured for an additional 2 days, after which they were harvested by
trypsinization, washed with phosphate-buffered saline (PBS), suspended in 1 mL of PBS, and the number
of them was counted. The cells were washed with 5% trichloroacetic acid aqueous solution,
Melanoma cells were seeded in 85 mm dishes at a density of 4 x 105 cells per dish and
ethanol/ether (3/1) and ether in turn, and dissolved in 1 mL of 2 M NaOH. Inhibitory activity of
melanogenesis was calculated by measuring absorbance at 410 nm of the solution.
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