Mushroom tyrosinase inhibitors from Aloe barbadensis Miller

Article abstract of DOI:10.1016/j.fitote.2012.09.028

Two new chromones, 5-((S)-2a€-oxo-4a€-hydroxypentyl)- 2-(β-glucopyranosyl-oxy-methyl)chromone (1) and 5-((S)-2a€-oxo- 4a€-hydroxypentyl)-2-methoxychromone (2), together with four known analogues, 8-C-glucosyl-7-O-methyl-(S)-aloesol (3), isoaloeresin D (4)

Full text of DOI:10.1016/j.fitote.2012.09.028

Fitoterapia 83 (2012) 1706–1711
Contents lists available at SciVerse ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Mushroom tyrosinase inhibitors from Aloe barbadensis Miller
⁎
⁎
Xiaofang Wu, Sheng Yin, Jiasheng Zhong, Wenjing Ding, Jinzhi Wan , Zhiyong Xie
School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, 510006, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 July 2012
Received in revised form 25 September 2012
Accepted 30 September 2012
Available online 7 October 2012
Two new chromones, 5-((S)-2′-oxo-4′-hydroxypentyl)-2-(β-glucopyranosyl-oxy-methyl)
chromone (1) and 5-((S)-2′-oxo-4′-hydroxypentyl)-2-methoxychromone (2), together
with four known analogues, 8-C-glucosyl-7-O-methyl-(S)-aloesol (3), isoaloeresin D (4),
8-C-glucosyl-(R)-aloesol (5), and aloesin (6) were isolated from the aqueous extract of Aloe
barbadensis Miller. Their structures were determined on the basis of spectroscopic evidences (1-D
and 2-D NMR, HRMS, UV, and IR data), chemical methods and the literature data. The Mosher's
method was applied to establish the absolute configuration of compounds 1 and 2. The inhibitory
effects of these chromones on the activity of mushroom tyrosinase were examined, and compound
Keywords:
Aloe barbadensis Mill
Chromone
6 was identified as a noncompetitive tyrosinase inhibitor with an IC50 value of 108.62 μg·mL−1
.
Tyrosinase inhibitory activity
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
structural elucidation and mushroom tyrosinase inhibitory
activity assay of six isolates are described.
Aloe barbadensis Mill, a member of Asphodelaceae (Liliaceae)
family, is a short-stemmed succulent herb widely distributed in
Europe, Asia and southern parts of North America [1]. Of over
300 Aloe species, A. barbadensis has long been used in traditional
medicine for the treatment of various diseases, and also used as
raw materials of cosmetics and health foods [2,3]. Among its
previously investigated chemical constituents [4], chromones
and their derivatives have been reported to have multiple
biological properties, for example, anticancer [5], antibacterial
[6], antioxidant [7], anti-inflammatory [8] and mushroom
tyrosinase inhibitory activity [9]. In a search for bioactive
components from A. barbadensis, we found that the aqueous
extract of this plant exhibited mushroom tyrosinase inhib-
itory activity. Phytochemical investigation has led to the
isolation of two new chromones (1 and 2) and four known
analogues including 8-C-glucosyl-7-O-methyl-(S)-aloesol (3),
isoaloeresin D (4), 8-C-glucosyl-(R)-aloesol (5), and aloesin (6)
from the aqueous extract of A. barbadensis. Herein, the isolation,
2. Experimental
2.1. Generals
Melting points were determined using a WRS-2A melting
point apparatus and are uncorrected. Optical rotations were
measured on a Perkin Elmer digital polarimeter. Ultraviolet
(UV) spectra were recorded using a Shimadzu UV2457 spectro-
photometer and infrared (IR) spectra were obtained with a
Bruker Tensor 37 spectrophotometer. High-resolution mass
spectra (HRMS) were acquired on a Shimadzu LCMS-IT-TOF,
and MS data were measured on an Agilent 1200 series
LC-MS/MS system consisting of a quaternary pump, a vacuum
degasser, an autosampler, a thermostat column and a multimode
electrospray ionization/APCI spray chamber. 1-D (1H, 13C, DEPT)
and 2-D (COSY, HMBC, HSQC) NMR spectra were recorded on a
Bruker AVANCE 400 spectrometer and chemical shifts (δ) were
given in ppm and were referenced to the CD3OD signals (δH 3.30,
δC 49.0). AB-8 macroporous resins (manufactured by Nankai
University Chemical Industry Factory, Tianjin, China) were
used for column chromatography. Thin layer chromatography
(TLC) analysis was carried out on silica gel plates (Marine
Chemical Ltd., Qingdao, China). Preparative medium pressure
liquid chromatography (MPLC) was carried out on an Eyela
⁎
Corresponding authors at: School of Pharmaceutical Sciences, Sun Yat-Sen
University, 132 Waihuan Rd. East, Guangzhou Higher Education Mega Center,
Guangzhou, 510006, PR China. Tel.: +86 13326660067, +86 13726805787;
fax: +86 2039943040.
E-mail addresses: jinzhiwan2004@yahoo.com.cn (J. Wan),
xiezy2074@yahoo.com (Z. Xie).
0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.fitote.2012.09.028

X. Wu et al. / Fitoterapia 83 (2012) 1706–1711
1707
instrument (Tokyo, Japan) consisted of a VSP-3050 pump,
UV-9000 UV–vis detector and DC-1500 fraction collector, using
an Eyela column (300×20 mm i.d.) filled with Chromatorex
SMB ODS (20–40 μm, Fuji Silisia Ltd., Nagoya, Japan). Analytical
HPLC was carried out on a LC-20AT Shimadzu liquid chromato-
graph with a Nucleodur 100–5 C18 column (250×4.6 mm,
5 μm), connected with an SPD-M20A diode array detector
(DAD) and an Alltech 3300 evaporative light scattering detector
(ELSD). Absorbance of mushroom tyrosinase assay was mea-
sured on a FlexStation 3 microplate reader (Molecular Devices,
USA) and analyzed using a SoftMax Pro 5 software (Molecular
Devices, USA).
was filtered through a membrane filter and injected for a
reversed-phase HPLC–DAD–ELSD analysis. The HPLC system
using CH3CN (B) and H2O (A) as mobile phase was run with a
gradient program at 1 mL·min−1 (15%B–20%B, 0–10 min; 20%B,
10–20 min), flow rate of mobile phase, UV detection wave-
length, drift tube temperature, nitrogen flow-rate and gain were
set at 1.0 mL·min−1, 254 nm, 85 °C, 2.0 L·min−1 and 8. HPLC
analysis of the hydrolysate from compound 1 revealed the
presence of its aglycone (Rt=12.94 min) and glucopyranose
(Rt=2.50 min); their retention time was identical with that of
compound 2 and standand glucopyranose. The hydrolysate and
standand glucopyranose were also spotted on an analytical silica
gel TLC plate [the plate was developed with n-BuOH–HOAc–H2O
(3:1:1, v/v/v), sprayed aniline-oxalic acid solution for visualiza-
tion]. The hydrolysate from compound 1 exhibited a dark yellow
spot (Rf=0.51) which was identical with that observed for
standand glucopyranose.
2.2. Plant material
The dried A. barbadensis powder was purchased from
Yunnan Yuanjiang Evergreen Biological Co., Ltd. (Yuxi, China),
and authenticated by Prof. Xin-jun Xu, School of Pharmaceu-
tical Sciences, Sun Yat-Sen University, P. R. China. A voucher
specimen (Batch no: 20120301) was deposited in School of
Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou,
China.
2.5. Preparation of (R) and (S)-MTPA Esters (2a and 2b) of 2
Compound 2 (7.0 mg) was dissolved in 500 μL of dry
pyridine and stirred at room temperature (rt) for 10 min.
For preparation of the (R)-MTPA ester (2a) of 2, 50 μL of
(R)-(−)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride
(MTPA-Cl) was added to the reaction vial, and the mixture was
stirred at rt for 16 h. Completion of the reaction was monitored
by LC/MS. The reaction mixture was dried in vacuo and
redissolved in CH3OH, and purification by analytical ODS HPLC
using 85% CH3CN in H2O provided (R)-MTPA ester of compound
2 (2a, 6.5 mg).
2.3. Extraction and isolation
The dried A. barbadensis powder (about 50 g) was extracted
five times with H2O under ultrasonication at ambient temper-
ature and filtered. The filtrate was concentrated under the
reduced pressure to give a crude aqueous extract (about 20 g).
The aqueous extract was then subjected to AB-8 resin column
chromatography eluted with a gradient of EtOH–H2O (15:85 to
55:45, v/v) to give 3 major fractions 1–3. Fraction 2 was further
purified on RP-C18 MPLC using CH3OH–H2O (26:74, v/v; flow
rate: 10 mL·min−1) as mobile phase to afford new compounds
1 (58.1 mg) and 2 (66.5 mg). Fraction 1 was then separated on
a RP-C18 MPLC eluted with CH3OH–H2O (26:74, v/v; flow rate:
10 mL·min−1) to obtain compounds 6 (70.2 mg), 3 (90.2 mg)
and 5 (45.0 mg). Compound 4 (2.8 g) was obtained from
fraction 3 using RP-C18 MPLC with the mobile phase of CH3OH–
H2O (33:67, v/v; flow rate: 20 mL·min−1).
Compound (1, Fig. 1): 5-((S)-2′-oxo-4′-hydroxypentyl)-2-
(β-glucopyranosyl-oxy-methyl)chromone; slightly white amor-
phous powder; mp 167.4 °C; [α]2D0 −43.12° (c 1.09 , H2O); UV
(MeOH) λmax nm (log ε): 225 (4.32), 249 (3.96), 302 (3.86); IR
bands (KBr) νmax cm−1: 3401, 2966, 1712, 1652, 1605, 1480;
HRMS (ESI) calcd. for C21H26O10 [M−H]− 437.1453, found
437.1448; 1H and 13C NMR spectral data see Table 1.
Compound 2a: white, amorphous solid; 1H NMR data
(400 MHz, CD3OD) δ 7.69 (t, J=7.9, H=7), 7.55 (d, J=7.0,
H-8), 7.06 (d, J=7.4, H-6), 6.22 (s, H-3), 5.63 (m, H-4′), 5.36
(s, H-9), 4.26 (d, J=17.0, H-1a′), 4.02 (d, J=17.1, H-1b′),
3.28 (s, O-CH3), 3.20 (s, O-CH3), 3.13 (dd, J=7.9, 17.5, H-3a′),
3.00 (dd, J=4.5, 17.7, H-3b′), 1.43 (d, J=6.2, H-5′); ESI-MS
m/z 709.1 [M+H]+
.
In an analogous way, (S)-MTPA ester (2b) of compound 2
was obtained from (S)-(+)-MTPA-Cl similarly to 2a. (S)-MTPA
ester of compound 2 was purified on an ODS analytical column
using 85% CH3CN in H2O as eluent to obtain 2b (7.0 mg).
Compound 2b: white, amorphous solid; 1H NMR data
(400 MHz, CD3OD) δ 7.71 (t, J=7.9, H=7), 7.55 (d, J=8.0,
H-8), 7.16 (d, J=7.3, H-6), 6.26 (s, H-3), 5.60 (m, H-4′), 5.36
(q, J=14.12, 14.12, 14.15, H-9), 4.36 (d, J=17.1, H-1a′), 4.17
(d, J=17.1, H-1b′), 3.60 (s, O-CH3), 3.47 (s, O-CH3), 3.18 (dd,
J=8.36, 17.8, H-3a′), 3.00 (dd, J=4.2, 17.8, H-3b′), 1.33 (d,
Compound (2, Fig. 1): 5-((S)-2′-oxo-4′-hydroxypentyl)-
2-methoxychromone; yellowish amorphous powder; mp
121.0 °C; [α]2D0 −22.09° (c 0.86, MeOH); UV (MeOH) λmax
nm (log ε): 225 (4.10), 248 (3.76), 302 (3.68); IR bands
(KBr) νmax cm−1: 3347, 2963, 1718, 1643, 1603, 1480;
HRMS (ESI) calcd. for C15H16O5 [M−H]− 275.0925, found
275.0917; 1H and 13C NMR spectral data see Table 1.
J=6.3, H-5′); ESI-MS m/z 731.0 [M+Na]+
.
2.6. Determination of mushroom tyrosinase inhibition activity
The mushroom tyrosinase inhibition activity of all tested
compounds, using L-DOPA as substrate, was measured according
to the method of Lin et al. [10] with slight modification.
Mushroom tyrosinase and L-DOPA used for the bioassay
were each manufactured at Worthington Biochemical Corp.
(Lakewood, NJ, USA) and Boston Biomedical Inc. (Boston,
MA, USA). Phosphate used for preparing buffer was pur-
chased from Tianjin Damao Chemical Reagent Factory (Tianjin,
China). Mushroom tyrosinase, L-DOPA and tested samples were
prepared by dissolving in 1/15 mol·L−1 Na2HPO4–NaH2PO4
2.4. Hydrolysis of compound 1
About 1.0 mg of 1 was dissolved in 1.0 mL of 20% aqueous
HCl solution, and left at 70 °C for about 4 h with constant
stirring. The hydrolysate was dried in vacuum at 45 °C and
re-dissolved by methanol–water (1:1, v/v) solution, which

1708
X. Wu et al. / Fitoterapia 83 (2012) 1706–1711
buffer (pH 6.8). Reaction mixtures containing 50 μL of
OH
O
2 mmol·L−1 of L-DOPA, 50 μL of phosphate buffer and
50 μL of different concentrations of tested compounds
were added in 96 well microtiter plates, following by
adding 50 μL of 0.2 mg·mL−1 of mushroom tyrosinase.
Then, the absorbance variations accompanying the oxida-
tion of the substrate (L-DOPA) were recorded using a
FlexStation 3 microplate reader at 475 nm under a constant
temperature of 37 °C. Average velocity (v) of the oxidation
of the substrate (L-DOPA) was determined from the linear
slope of curve. Arbutin was used as positive control.
Inhibitory effects of tested samples on the enzyme activity
were represented as % of relative enzyme activity=vt/v×100,
where vt=average velocity of the oxidation of the substrate
with tested sample and v=average velocity of the oxidation of
the substrate without tested sample.
4'
2'
1'
O
4
5'
3'
5
OH
4a
1a
3
6
6''
4''
5''
O 1''
2
O
HO
7
O
HO
2'' OH
9
8
3''
1
(1)
OH
O
4'
2'
1'
O
3'
5'
5
4
4a
1a
3
6
2
HO
7
O
8
9
1
(2)
O
CH3
2.7. Determination of the inhibition type of compound 6 on
mushroom tyrosinase
OH
CH3
The kinetic behavior of mushroom tyrosinase during the
oxidation of L-DOPA by compound 6 was studied according
to the method of Cho et al. [11]. Mushroom tyrosinase (50 μL;
0.2 mg·mL−1) was incubated with 50 μL of various concen-
trations of enzyme substrate and 50 μL of phosphate buffer,
and then 50 μL of different concentrations of tested sample
were simultaneously added to the reaction mixtures. The
absorbance variations from these studies were used to
generate Lineweaver–Burke plots to determine the inhibition
type of aloesin on mushroom tyrosinase, and concentrations
of compound 6 for curves 0–5 were 0, 100, 200, 300, 400,
500 μg·mL−1, respectively. Kinetic parameter (Km) of the
tyrosinase activity was calculated by linear regression from
Lineweaver–Burk plots. Inhibition constant (Ki) was calcu-
lated by Dixon graphical chart with 1/v versus the concen-
tration of compound 6, and concentrations of L-DOPA for
curves 1–5 were 0.5, 0.7, 1.0, 1.4, 2.0 mmol·L−1, respectively.
H3C
O
O
OH
O
HO
OH
OH
(3)
O
CH3
OH
CH3
OH
H3C
O
O
O
O
HO
O
OH
OH
(4)
3. Results and discussion
O
CH3
Compound 1 was obtained as slightly white amorphous
powder. The HRESIMS displayed the molecular ion peak at m/z
437.1448 [M−H]− (calcd. 437.1453) corresponding to the
molecular formula C21H26O10, which was consistent with the
NMR data. The IR spectrum exhibited absorption bands for
hydroxyl groups (3401 cm−1), carbonyl group (1712 cm−1),
and aromatic moiety (1652, 1605 and 1480 cm−1). The UV
maximum absorption (λmax 225, 248 and 303 nm) displayed a
chromone ring feature [12]. Analysis of 1D-NMR spectra data of
1 (Table 1) further displayed the presence of a chromone
skeleton [one olefinic proton singlet at δH 6.47 (s, H-3); three
aromatic protons at δH 7.18 (d, J=7.2, H-6), 7.69 (t, J=7.9, H-7)
and 7.51 (d, J=8.4, H-8); four quaternary sp2 carbons at δC
122.7 (C-4a), 159.2 (C-1a), 137.3 (C-5) and 166.7 (C-2),
showing no HSQC correlation with any protons; a conjugated
carbonyl carbon at δC 182.1(C-4)] [13], a β-linked glucosyl
moiety [an anomeric proton at δH 4.48 (d, J=7.7, H-1″); δC
104.0, 74.9, 77.9, 71.4, 77.7 and 62.6], a 2-oxo-4-hydroxypentyl
group [a methyl (δC 23.6, C-5′); two methylenes (δC 50.7 and
52.6, C-1′ and C-3′); a hydroxyl-bearing methine (δC 65.2, C-4′)
and a carboxyl (δC 210.1, C-2′)] [14], and an oxygenated
OH
H3C
O
OH
OH
OH
O
HO
OH
(5)
O
CH3
O
H3C
O
OH
OH
O
HO
OH
OH
(6)
Fig. 1. The structures of compounds 1–6.

X. Wu et al. / Fitoterapia 83 (2012) 1706–1711
1709
Table 1
13C (100 MHz) and 1H NMR (400 MHz) data of compound 1a and 2b.
0.00
0.00
O
O
+0.02
+0.10
OR
Carbon
1
2
δC
δH mult. (J in Hz)
δC
δH mult. (J in Hz)
+0.04
2
3
4
5
6
7
8
1a
4a
9
166.7
111.0
182.1
137.3
130.8
135.1
119.1
159.2
122.7
67.8
170.4
109.5
182.0
137.7
130.6
134.8
119.0
159.3
122.9
61.5
+0.05
+0.06
6.47, s
6.41, s
-0.10
-0.03
+0.10
+0.15
7.18, d (7.2)
7.69, t (7.9)
7.51, d (8.4)
7.24, d (7.3)
7.74, t (7.9)
7.56, d (8.5)
OR
O
2a,R=(R)-MTPA
2b,R=(S)-MTPA
4.82, d (15.0)
4.69, d (15.0)
4.36, d (17.3)
4.26, dc(17.3)
4.58, s
1′
50.7
50.7
4.45, d (17.3)
4.34, dc(17.3)
Fig. 3. ΔδSR values (ΔδSR=δS−δR) obtained for (S)- and (R)-MTPA esters of 2.
2′
3′
4′
5′
1″
2″
3″
4″
5″
6″
210.1
52.6
65.2
23.6
104.0
74.9
77.9
71.4
77.7
62.6
209.0
52.8
65.3
23.7
2.84, m
2.90, m
4.29, mc
4.36, mc
The extensive analyses of 1D- and 2D-NMR data of compound 2
and acid hydrolysis of compound 1 suggested that 2 should be
the aglycone of compound 1. The absolute configuration of the
hydroxy-bearing carbon (C-4′) of the 2-oxo-4-hydroxypentyl
chain in 2 was established by the Mosher's method [15,16].
Compound 2 (the aglycone of compound 1) was treated
with (R)-(−)- and (S)-(+)-α-methoxy-α-(trifluoromethyl)-
phenylacetyl chloride (MTPA-Cl) in dry pyridine separately to
yield (R)- and (S)-MTPA ester derivatives 2a and 2b, respectively.
All proton signals of the two Mosher diastereoisomeric
esters were assigned by a HSQC experiment, and 1H NMR
chemical shift values of the (S)-MTPA esters (2b) were
subtracted from the corresponding values of the (R)-MTPA
esters (2a) (Fig. 3). Positive ΔδSR (δS −δR) values were
found for H-3a′ and H-3b′ (+0.05 and +0.06, respectively),
while a negative ΔδSR value was found for H-5′CH3 (−0.10).
Following the MTPA rules, these data indicated the S-
configuration for the hydroxy-bearing carbon (C-4′) in com-
pound 2. Thus, the structure of compound 2 was elucidated as
5-((S)-2′-oxo-4′-hydroxypentyl)-2-methoxychromone, and
the S-configuration of the hydroxy-bearing carbon (C-4′) in 1
(glucoside derivative of compound 2) could also be established.
Four known compounds (3–6) were identified by comparing
their physical and spectral data with literature data. They were
8-C-glucosyl-7-O-methyl-(S)-aloesol (3) [12], isoaloeresin D (4)
[12], 8-C-glucosyl-(R)-aloesol (5) [17] and aloesin (6) [12].
Preliminary screening with mushroom tyrosinase revealed
that the aqueous extract of A. barbadensis inhibited L-DOPA
1.24, d (6.2)
4.48, d (7.7)
3.26, m
3.36, m
3.31, m
3.29, m
3.87, d (11.8)
3.68, m
1.32, d (6.4)
a
Recorded in (CD3OD+D2O), J in Hz, δ in ppm.
Recorded in (CD3OD), J in Hz, δ in ppm.
Overlapping signals.
b
c
methylene (δC 67.8, C-9). The aforementioned data suggested
that compound 1 was a chromone glucoside derivative. 2D-NMR
experiments established the structure of compound 1(Fig. 2).
The HMBC correlations from H-1′ to C-4a, C-6, and C-5, and from
H-6 to C-1′ indicated that 2-oxo-4-hydroxypentyl was attached
at C-5 of chromone ring. The oxygenated methylene was
connected to C-2 of chromone ring by HMBC correlations of
H-9/C-2 and C-3, and H-3/C-9. The attachment of the
glucosyl moiety to C-9 was made by the HMBC correlations
of H-9/C-1″ (anomeric carbon) and H-1″ (anomeric proton)/C-9.
Comparison of acid hydrolysis products of compound 1
with standard glucopyranosyl confirmed the presence of
glucopyranosyl in 1. Thus, compound 1 was identified as
5-(2′-oxo-4′-hydroxypentyl)-2-(β-glucopyranosyl-oxy-methyl)
chromone.
Compound 2, yellowish amorphous powder, had a molec-
ular formula C15H16O5 as determined by HRESIMS (m/z found
275.0917 [M−H]−, calcd. 275.0925). The IR, UV, 1H and 13C
NMR spectra of 2 (Table 1) were similar to those of 1, except for
the absence of the signals for glucosyl moiety in 2, indicating
that 2 was a de-glucosyl derivative of 1. The molecular formula
of compound 2 showed 162 mass units less than that of
compound 1, which supported the assumption made above.
Table 2
Mushroom tyrosinase inhibition of compounds 1–6.
Compounds
Relative enzyme activitya (%)
1
N
2
N
3
N
4
5
6
N
1H-1H COSY
HMBC
91.33
27.30
91.49
86.62
Aqueous extract
Arbutinb
N, no inhibition.
a
The relative enzyme activity of tested samples were measured at the
concentration of 500 μg·mL−1 on the mushroom tyrosinase.
b
Fig. 2. Key 1H–1H COSY and HMBC correlations for compound 1.
Positive control.

1710
X. Wu et al. / Fitoterapia 83 (2012) 1706–1711
60
40
20
0
60
5
4
3
1
2
50
40
30
20
10
0
3
4
5
2
1
0
-200
-100
0
100
200
300
400
500
600
-2.0
-1.0
0.0
1.0
2.0
-10
caloesin/(µg·ml-1)
[cs]-1/(mmol·L-1 -1
)
Fig. 5. Dixon plots for inhibition of compound 6 on mushroom tyrosinase.
Fig. 4. Lineweaver–Burk plots for inhibition of compound 6 on mushroom
tyrosinase for the catalysis of L-DOPA.
extract of A. barbadensis Miller. All the isolates were tested on
mushroom tyrosinase assay, and compound 6 appeared to be the
key tyrosinase inhibitor in the aqueous extract of A. barbadensis
Miller and has great potential to be developed into an anti-
browning agent for food products and skin whitening agent for
cosmetics.
oxidation (relative enzyme activity was 91.49%). Consequently,
six chromones isolated from the aqueous extract were tested on
mushroom tyrosinase assay in which L-DOPA was the substrate,
and arbutin was the positive control (Table 2). Compound 6
strongly inhibited the enzyme activity and was more potent
than positive control (arbutin); compound 5 showed slightly
inhibitory activity and compounds 1–4 had no activity on
mushroom tyrosinase. Some possible structure–activity rela-
tionship could be inferred from tyrosinase inhibitory assay
results: (1) compounds 5 and 6, the chromone derivatives with
a 7-hydroxy at the chromone ring, showed inhibitory effect on
mushroom tyrosinase; (2) when the 7-hydroxyl group at the
chromone ring was absent or methylated, the compounds,
such as compounds 1–4, became inactive against mushroom
tyrosinase; (3) compound 6 with 2-oxopropyl group at C-2
showed remarkable enzyme inhibitory activity compared with
compound 5 with 2-hydroxypropyl group at C-2, and it
inhibited the enzyme activity to a greater degree than the
positive control (arbutin); (4) these studies suggested that
both 7-hydroxyl group and 2-oxopropyl group of the chromone
affected the tyrosinase inhibitory activity, with the 2-oxopropyl
group playing a more important role, possibly. Additionally, the
inhibition kinetics of compound 6 analyzed by the Lineweaver–
Burk plots (Fig. 4 and Table 3) indicated that compound 6 was a
noncompetitive inhibitor of mushroom tyrosinase with an IC50
value of 108.62 μg·mL−1 because the vertical axis intercept
(1/Vm) enlarged with increasing of the inhibitor's concentration
but with a common the horizontal axis intercept (−(1/Km)),
then the Km was equal to 0.78 0.03 mmol·L−1; and the
equilibrium constant for inhibitor binding with the free enzyme
(Ki) obtained from the horizontal axis intercept (−Ki) in Dixon
Acknowledgment
This work was supported by the science and technology
projects of Zhuhai municipal (PC20051072) and projects of
Guangdong Province Administration of Traditional Chinese
Medicine (1050163).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.fitote.2012.09.028.
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Table 3
Inhibition constants of compound 6 for mushroom tyrosinate.
IC50*(μg·mL−1
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108.62
Km (mmol·L−1
)
0.78 0.03
130.88 3.90
Reversible
Ki (μg·mL−1
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Inhibition
Inhibition type
Noncompetitive
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⁎
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