Antioxidant activity of a new C-glycosylflavone from the leaves of Ficus microcarpa

Article abstract of DOI:10.1016/j.bmcl.2010.12.025

By bioactive-guided fractionation of methanol extract of the Ficus microcarpa leaves, one new C-glucosylflavone, ficuflavoside (1), one new megastigmane glycoside, ficumegasoside (8), and twelve known compounds including flavonoids (2-6), phenylpropanoids (7), megastigmanes (9-11) and sterol derivatives (12-14) were isolated. Their chemical structures were elucidated by mass, 1D, and 2D NMR spectroscopies. The antioxidant activities of these compounds were measured using the oxygen radical absorbance capacity methods. Compounds 1-6 exhibited potent antioxidant activities of 6.6-9.5 μM Trolox equivalents at the concentration of 2.0 μM. The results indicated 2, 3, and 5 having meaningful reducing capacity of copper (I) ions concentration of 6.1-8.4 μM.

Full text of DOI:10.1016/j.bmcl.2010.12.025

Bioorganic & Medicinal Chemistry Letters 21 (2011) 633–637
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antioxidant activity of a new C-glycosylflavone from the leaves
of Ficus microcarpa
Phan Van Kiem b, Nguyen Xuan Cuong b, Nguyen Xuan Nhiem a,b, Vu Kim Thu b, Ninh Khac Ban b,
Chau Van Minh b, Bui Huu Tai a,b, Truong Nam Hai c, Sang Hyun Lee d, Hae Dong Jang d, Young Ho Kim a,
⇑
a College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea
b Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam
c Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam
d Department of Food and Nutrition, Hannam University, Daejeon 305-811, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
By bioactive-guided fractionation of methanol extract of the Ficus microcarpa leaves, one new C-glucosylf-
lavone, ficuflavoside (1), one new megastigmane glycoside, ficumegasoside (8), and twelve known com-
pounds including flavonoids (2–6), phenylpropanoids (7), megastigmanes (9–11) and sterol derivatives
(12–14) were isolated. Their chemical structures were elucidated by mass, 1D, and 2D NMR spectrosco-
pies. The antioxidant activities of these compounds were measured using the oxygen radical absorbance
Received 26 September 2010
Revised 6 December 2010
Accepted 7 December 2010
Available online 10 December 2010
capacity methods. Compounds 1–6 exhibited potent antioxidant activities of 6.6–9.5
lents at the concentration of 2.0 M. The results indicated 2, 3, and 5 having meaningful reducing capac-
ity of copper (I) ions concentration of 6.1–8.4 M.
lM Trolox equiva-
Keywords:
Ficus microcarpa
Moraceae
l
l
Ó 2010 Elsevier Ltd. All rights reserved.
Ficuflavoside
Ficumegasoside
Antioxidant
Oxygen radical absorbance capacity
Natural antioxidants function as free radical scavengers, reduc-
ing agents, chelators of pro-oxidant metals, or as quenchers of
singlet oxygen. Natural antioxidants are also present in many
health-promoting supplements.1 Consumption of foods rich in nat-
ural antioxidants has been reported as being protective against
certain types of cancer2 and coronary disease,3 and may also re-
duce the risk of cardiovascular and cerebrovascular events. These
actions of antioxidants have been attributed to their ability to scav-
enge free radicals, thereby reducing oxidative damage of cellular
biomolecules such as lipids, proteins, and nucleic acids.4 Oxygen
radical absorbance capacity (ORAC) is a method of measuring anti-
oxidant capacities in biological samples in vitro. It has been re-
cently accepted as a standard method to analyze the antioxidant
potential of active substances of plants.5,6 The ORAC assay involves
the completion of free radicals activities as a mean of quantization,
and combines both the extent of inhibition and the length of inhi-
bition time of free radical action by antioxidants into a single quan-
tity. The ORAC assay provides important information regarding the
antioxidant capacity of various biological samples from pure com-
pounds such as phenolic acids and flavonoids to complex matrices
such as tea, fruits, vegetables, and animal tissues.5
Ficus (Moraceae) constitutes one of the largest species of flow-
ering plants with about 800 genera of deciduous trees, hemi-
epiphytes shrubs and climbers occurring in tropical and subtropi-
cal regions of both hemispheres. The genus is remarkable for the
large variation in the habits of its species.7 Ficus microcarpa L.f.
(Moraceae) is widely distributed as an ornamental plant and is
one of the most common street trees in warm climates.8 The dried
leaves, aerial roots, and barks from F. microcarpa have been used as
folk herbs for perspiration, alleviating fever, and relieving pain in
Vietnam.8 Previous phytochemical and biological investigations
of F. microcarpa have yielded a number of triterpenoids,9–11 flavo-
noids,12 monoterpenoids, and other class compounds.13,14 In our
screening project for antioxidant agents from natural sources, we
found F. microcarpa to possess antioxidant effects. As part of our
continuing research to evaluate the biological activities of this
plant, here we report the isolation, structural elucidation, and eval-
uation of the antioxidant activity of one new flavonoid, one new
megastigmane glycoside and twelve known compounds from the
methanol extract of the F. microcarpa leaves.
Dried leaves of F. microcarpa15 were extracted with methanol
and then fractionated with chloroform, ethyl acetate and water.
From these extracts and by using combined chromatographic sep-
arations, two new and twelve known compounds were isolated.16
Their structures were elucidated using physicochemical and spec-
troscopic methods.
⇑
Corresponding author. Tel.: +82 42 8215933; fax: +82 42 8236566.
E-mail address: yhk@cnu.ac.kr (Y.H. Kim).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.025

634
P. V. Kiem et al. / Bioorg. Med. Chem. Lett. 21 (2011) 633–637
Compound 1 was obtained as a yellow amorphous powder. Its
supported by the good agreement of 13C NMR chemical shifts for
the sugar moieties with those of abrusin 200-O-apioside.19
Consequently, the structure of 1 was determined to be 8-C-(200-
O-b-D-apiofuranosyl)-b-D-glucopyranosyl apigenin, a new com-
pound named ficuflavoside.
basic ion peak at m/z 565.1 [M+H]+ was observed in the positive-
ion electrospray ionization mass spectrometry (ESI-MS), and
HR-ESI-MS analysis revealed the molecular formula to be
C
26H28O14, with a cluster ion peak at m/z 565.1568 [M+H]+ (calcd
for C26H29O14: 565.1557). The 1H NMR spectrum of 1 (in metha-
nol-d4) showed the following signals: four aromatic protons were
at dH 6.84 (2H, d, J = 8.4 Hz) and 7.88 (2H, d, J = 8.4 Hz), assigning
to the AA0BB0 coupling system in B ring, one singlet proton of A ring
was at dH 6.16 (1H, s), and two anomeric protons were at dH 4.87
(1H, d, J = 9.0 Hz) and 5.03 (1H, d, J = 1.2 Hz), suggesting the
appearance of two sugar units. The 13C NMR and DEPT spectra re-
vealed 26 carbon signals, of which, 15 signals were assigned to a
flavone aglycone and 11 signals were assigned to two monosaccha-
ride moieties. The heteronuclear multiple bond correlations
(HMBC) cross peaks from H-3 (dH 6.49) to C-10 (dC 123.7), C-2 (dC
166.7), C-4 (dC 183.1), and C-10 (dC 105.7); from H-6 (dH 6.16) to
C-5 (dC 162.7), C-7 (dC 164.1), C-8 (dC 105.4), and C-10 (dC 105.7)
confirmed that the two singlet protons were at C-3 and C-6,
Compound 8 was obtained as a white amorphous powder and
its molecular formula was determined to be C24H38O11 by ESI-MS
at m/z 525 [M+Na]+ (positive) and HR-ESI-MS at m/z 525.2317
[M+Na]+ (calcd for C24H38O11Na 525.2312). The 1H NMR spectrum
of 8 (methanol-d4) showed signals for three tertiary methyl groups
at d 0.98, 1.00, and 1.91 (each 3H, s), one secondary methyl group
at d 1.26 (3H, d, J = 6.6 Hz), and two anomeric protons at dH 4.32
(1H, d, J = 7.8 Hz) and 4.94 (1H, d, J = 1.2 Hz) suggesting the pres-
ence of two sugar units, as listed in Table 1. The 13C NMR and DEPT
spectra revealed 24 carbon signals, 13 of which were assigned to a
megastigmane aglycone moiety and the remaining 11 assigned to
the two monosaccharide moieties. The aglycone of 8 was con-
cluded to be 9-hydroxy-4,7-megastigmadien-3-one.20 The HMBC
cross peaks from H-4 (dH 5.86) to C-3 (dC 202.1), C-5 (dC 166.0),
C-6 (dC 56.6), and C-13 (dC 23.8); from H-13 (dH 1.91) to C-4 (dC
126.1), C-5 (dC 166.0), and C-6 (dC 56.6) confirmed that a double
bond was at C-4/C-5, ketone and tertiary methyl groups were at
C-3 and C-5, respectively. On the other hand, the chemical shift
of C-1 (dC 37.1), C-5 (dC 166.0), C-6 (dC 56.6), and C-7 (dC 129.1)
in the aglycone confirmed the stereochemistry at C-6 to be
R-configuration by comparing with the corresponding data
respectively. Acid hydrolysis of 1 revealed D-apiose (identified as
trimethylsilyl (TMS) derivatives by a gas chromatography meth-
od).17 Furthermore, the 13C NMR spectral data of 1 showed the
presence of one b-D-apiofuranose and one C-b-D-glucopyranose
moieties.18 The HMBC between the glc H-100 (dH 4.87) and C-7 (dC
164.1), C-8 (dC 105.4), C-9 (dC 158.5) of the aglycone, between
api H-1000 (dH 5.03) and glc C-200 (dC 77.4), between glc H-200 (dH
4.13) and api C-1000 (dC 111.2) were observed (see Fig. 2). These
observations indicated the sequence of sugar linkages of 1 and
sugar moiety located at C-8 of the aglycone, which was also
of the (6S,7E,9S)-9-hydroxy-4,7-megastigmadien-3-one 9-O-b-D-
glucopyranoside21 [dC values for C-1 (37.5), C-5 (165.9), C-6
(57.3), and C-7 (131.8)] and salvionoside A22 ([dC values for C-1
Table 1
The 1H and 13C NMR data of compounds 1 and 8 in CD3OD
Position
1
Position
8
a
b
a
b
dC
dH mult. (J in Hz)
dC
dH mult. (J in Hz)
Aglycone
Aglycone
2
3
166.7
103.5
—
1
2
37.1
48.2
—
6.49 (s)
2.02 (d, 16.8)
2.42 (d, 16.8)
—
5.86 (s)
—
3.80(d, 9.0)
5.63 (dd, 9.0, 15.0)
5.73 (dd, 6.0, 15.0)
3.34 (m)
1.26 (d, 6.6)
0.98 (s)
4
5
6
7
8
9
183.1
162.7
99.3
—
—
3
4
5
6
7
8
9
10
11
12
13
202.1
126.1
166.0
56.6
129.1
138.0
77.4
21.1
27.6
28.0
23.8
6.16 (s)
—
—
—
—
—
7.88 (d, 8.4)
6.84 (d, 8.4)
—
6.84 (d, 8.4)
7.88 (d, 8.4)
164.1
105.4
158.5
105.7
123.7
130.1
117.0
162.6
117.0
130.1
10
10
20
30
1.00 (s)
1.91 (s)
40
50
60
8-C-Glc
100
200
300
400
500
600
9-O-Glc
73.8
77.4
80.9
72.2
82.9
63.0
4.87 (d, 9.0)
4.13 (t, 9.0)
3.53 (t, 7.5)
3.53 (t, 7.5)
3.33 (m)
10
20
30
40
50
60
102.5
75.1
77.9
71.8
76.7
67.9
4.32 (d, 7.8)
3.15 (t, 7.8)
3.32c
3.25 (t, 9.0)
3.36 (m)
3.70 (d, 11.4)
3.90 (dd, 2.0, 11.4)
3.56 (dd, 6.0, 10.8)
3.95c
Api (1?2)Glc
AAra (1?6)Glc
1000
2000
3000
4000
111.2
77.8
80.6
74.7
5.03 (d, 1.2)
3.67 (d, 1.2)
—
3.04 (d, 9.6)
2.45 (d, 9.6)
3.29 (d, 11.4)
3.18 (d, 11.4)
100
200
300
400
109.9
83.2
78.9
85.9
4.94 (d, 1.2)
3.96c
3.78 (m)
3.93 (m)
5000
65.8
500
63.1
3.62 (dd, 5.4, 10.8)
3.72 (dd, 3.6, 10.8)
a
b
c
150 MHz.
600 MHz.
Overlapped signal. Assignments were done by HMQC, HMBC, and 1H–1H COSY experiments, Glc:
D-glucopyranosyl, Api:
D-apiofuranosyl, Ara: L-arabinofuranosyl.

P. V. Kiem et al. / Bioorg. Med. Chem. Lett. 21 (2011) 633–637
635
(37.1), C-5 (165.8), C-6 (56.7), and C-7 (128.7)]. Moreover, the con-
figuration at C-9 was identified to be R-configuration by comparing
chemical shifts at C-8 (dC 138.0), C-9 (dC 77.4), and C-10 (dC 21.1) of
the aglycone with the corresponding data of the salvionoside A22
[dC values for C-8 (138.1), C-9 (dC 76.5), and C-10 (dC 20.9)] and
salvionoside B22 [dC values for C-8 (136.8), C-9 (dC 74.8), and
C-10 (dC 22.2)]. The 13C NMR spectral data of 8 showed the pres-
ence of one
ties.18 Furthermore, acid hydrolysis of 8 revealed
-glucose as sugar components (identified as TMS derivatives by
a-
L
-arabinofuranose and one b-
D
-glucopyranose moie-
L
-arabinose and
D
a gas chromatography method).17 The HMBC between the glc H-
10 (dH 4.32) and C-9 of the aglycone (dC 77.4), between ara H-100
(dH 4.94) and glc C-60 (dC 67.9), between glc H-60 (dH 3.56 and
3.95) and ara C-100 (dC 109.9) were observed (see Fig. 2). These
observations indicated the sequence of sugar linkages of 8 and
sugar moiety located at C-9 of the aglycone, which was also sup-
ported by the good agreement of 13C NMR chemical shifts for the
sugar moieties with those of megastigmane glycosides from
Erythroxylum cuneatum.18 Consequently, the structure of 8 was
determined to be (6R,7E,9R)-9-hydroxy-4,7-megastigmadien-3-
Figure 2. Selected HMBC spectrum of compounds 1 and 8.
10
8
1 µg/mL
2 µg/mL
6
one 9-O-[
a-
L
-arabinofuranosyl-(1?6)-b-
D
-glucopyranoside],
a
new compound named ficumegasoside.
The other compounds were characterized as (+)-catechin (2),23
(ꢀ)-epicatechin (3),23 isovitexin (4),24 luteolin 6-C-b-
D-glucopyran-
4
oside (5),25 isosaponarin (6),26 syringin (7),27 (3S,5R,6R,7E,9S)-
megastigman-7-ene-3,5,6,9-tetraol (9),28 bridelionoside B (10),29
dihydroalangionoside A (11),30 b-sitosterol (12),31 daucosterol
2
(13),31 and b-sitosterol 3-O-(60-octadecanoyl) b-
D-glucopyranoside
(14)32 (see Fig. 1) on the basis of spectral data and chemical evi-
dence, which were in good agreement with those reported in the
literature. Of these, dihydroalangionoside A (11) was isolated from
nature for the first time. Compounds 4–7, 9, 10, and 14 were iso-
lated from this genus for the first time.
0
MeOH extract
CHCl3 fraction
EtOAc fraction
Water fraction
Fractions
Figure 3. Peroxyl radical-scavenging activity (Trolox equivalent,
l
M) of extracts.
The antioxidant capacities of methanol extract and other frac-
tions (chloroform, ethyl acetate and water fractions) from the
leaves of F. microcarpa were tested by ORAC assay (see Fig. 3).33
Trolox was used as a control standard and prepared fresh daily.
The results showed that methanol extract showed significant per-
The ORAC value is calculated by dividing the area under the sample curve by the
area under the Trolox curve, with both areas being corrected by subtracting the area
under the blank curve. One ORAC unit is assigned as the net area of protection
provided by Trolox at a final concentration of 1 lM. The area under the curve of the
sample is compared to the area under the curve for Trolox, and the antioxidant
value is expressed in micromoles of Trolox equivalent per liter. The results
represent the mean S.D. of values obtained from three measurements.
oxyl radical-scavenging activity with 2.6 lM Trolox equivalent (TE)
Figure 1. Structures of isolated compounds (1–14) from the leaves of F. microcarpa.

636
P. V. Kiem et al. / Bioorg. Med. Chem. Lett. 21 (2011) 633–637
at the concentration of 2.0
l
g/mL. The ethyl acetate fraction exhib-
100
80
60
40
20
0
1 µM
2 µM
ited potent antioxidant activity followed by water fraction and
chloroform fraction with values of 8.8, 6.4, and 3.0 M TE at the
concentration of 2.0 g/mL, respectively. Subsequently, all isolates
from F. microcarpa were measured by ORAC assay at the concentra-
tion of 1.0 and 2.0 M (see Fig. 4). With regard to peroxyl
l
l
l
radical-scavenging activity, six flavonoids (1–6) exhibited potent
antioxidant activity compared with that of positive control, Trolox.
Among them, isosaponarin (6) was reported antioxidant activity
for the first time, isovitexin (4) was evaluated antioxidant activity
using ORAC method for the first time. However, catechin (2),
epicatechin (3), and luteolin 6-C-b-D-glucopyranoside (5) was
reported antioxidant activity using ORAC method.34,35 Another
compounds as phenylpropanoid (7), megastigmanes (8–11), and
sterol derivatives (12–14) showed weak or no activity. Our results
suggest that hydroxyl groups on aromatic rings may contribute to
peroxyl radical-scavenging activity by donating hydrogen atoms to
peroxyl radicals. We next tested reducing capacity of all com-
pounds by measuring the concentration of Cu (I) ions reduced from
Cu (II) ions.36 The results indicated 2, 3, and 5 having meaningful
Cu2+
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Compounds
Figure 6. Metal chelating activity of compounds 1–14. The results represent the
mean S.D. of values obtained from three measurements.
having two hydroxyl groups at C-3 and C-4 in B ring appear as a
strong antioxidant to donate their hydrogens or electrons to per-
oxyl radicals and Cu (II) ions, respectively. Although compounds
1, 4, and 6 showed potent peroxyl radical-scavenging activity, they
appear to have low reducing capacity compared to compounds 2, 3,
and 5. This result may conclude that the hydrogen donating
activity of compounds 1, 4, and 6 is strong, but their single elec-
tron transferring activity is weak due to one hydroxyl group in B
ring resulting in unfavorable electron transfer to Cu (II) ion.
Moreover, all compounds showed very weak metal chelating activ-
ity.37 This suggested antioxidant activity of these compounds is not
due to their metal chelating activities with transition metal ions
(see Fig. 6).
reducing capacity of copper (I) ions of 6.1–8.4
lM, at the concen-
tration of 2 M (see Fig. 5). From the results of antioxidant activi-
l
ties of all compounds, we may conclude compounds 2, 3, and 5
12
1 µM
2 µM
10
8
6
4
2
0
Acknowledgments
This work was financially supported by Vietnam National Foun-
dation for Science
& Technology Development (Project No:
104.01.31.09) and Priority Research Center Program through the
National Research Foundation of Korea (NRF) funded by the
Ministry of Education, Science and Technology (2009-0093815),
Republic of Korea. The authors are grateful to Institute of Chemis-
try, VAST and KBSI for recording the NMR and mass spectra.
1
2
3
4
5
6
7
8
9
10 11 12 13 14
Compounds
Figure 4. Peroxyl radical-scavenging activity (Trolox equivalent,
pounds (1–14).
lM) of com-
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P. V. Kiem et al. / Bioorg. Med. Chem. Lett. 21 (2011) 633–637
637
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28. Takeda, Y.; Okada, Y.; Masuda, T.; Hirata, E.; Shinzato, T.; Takushi, A.; Yu, Q.;
Otsuka, H. Chem. Pharm. Bull. 2000, 48, 752.
29. Sueyoshi, E.; Liu, H.; Matsunami, K.; Otsuka, H.; Shinzato, T.; Aramoto, M.;
Takeda, Y. Phytochemistry 2006, 67, 2483.
(50:1?1:1, v/v) to obtain fractions F2A–F2D. The F2B fraction was
chromatographed on an YMC RP-18 column eluting with MeOH–H2O (10:1,
v/v) to yield 2 (6.3 mg) and 3 (12.0 mg). The F2C fraction was chromatographed
on a silica gel column eluting with CHCl3–MeOH (7:1, v/v) to give fractions
F2C1–F2C3. The F2C1 traction was chromatographed on an YMC RP-18 column
eluting with MeOH–water (5:1, v/v) to yield 4 (5.0 mg) and 5 (11.5 mg). The
F2D fraction was chromatographed on a silica gel column eluting with CH2Cl2–
MeOH–H2O (6:1:0.07, v/v/v) to yield 7 (6.0 mg). The water soluble fraction F3
was chromatographed on a Diaion HP-20P column (Mitsubishi Chem. Ind. Co.,
Tokyo, Japan) eluting with water containing increasing concentrations of
MeOH (0%, 25%, 50%, 75%, and 100% MeOH) to give four fractions, F3A–F3D. The
F3B fraction was chromatographed on a silica gel column eluting with CHCl3–
MeOH–H2O (5:1:0.1, v/v/v) to give fractions, F3B1–F3B3. The F3B2 fraction was
30. Otsuka, H.; Kamada, K.; Ogimi, C.; Hirata, E.; Takushi, A.; Takeda, Y.
Phytochemistry 1994, 35, 1331.
31. Chang, I. M.; Yun, H. S.; Yamasaki, K. Korean J. Pharmacogn. 1981, 12, 12.
32. Jung, K.; Chin, Y. W.; Kim, Y. C.; Kim, J. Arch. Pharm. Res. 2005, 28, 1381.
33. The ORAC assay was carried out on a Tecan GENios multi-functional plate
reader with fluorescent filters using excitation and emission wavelengths of
485 and 535 nm, respectively. In the final assay mixture, 40 nM fluorescein
chromatographed on
a silica gel column eluting with CH2Cl2–MeOH–H2O
(5:1:0.1, v/v/v) to yield the new compound 8 (4.5 mg). The F3B3 fraction was
chromatographed on a silica gel column eluting with CH2Cl2–acetone–H2O
(1:2.5:0.1, v/v/v) to yield the new compound 1 (4.0 mg). The F3D fraction was
chromatographed on a silica gel column using CH2Cl2–MeOH–H2O (6:1:0.05, v/
v/v) to give fractions F3D1–F3D4. The F3D2 fraction was further separated on
an YMC RP-18 column eluting with acetone–MeOH–H2O (1:1:2, v/v/v) to yield
10 (5.0 mg) and 11 (8.0 mg). The F3D4 fraction was separated on an YMC RP-18
column eluting with MeOH–H2O (1:2, v/v) to yield 6 (5.5 mg).
was used as
a target of free radical attack with 20 mM 2,2-azobis
dihydrochloride (AAPH) as a peroxyl radical generator in a peroxyl radical-
scavenging capacity (ORACROOÅ) assay. Trolox (1 mM) was used as a control
standard and prepared fresh daily. The analyzer was programmed to record the
fluorescence of fluorescein every 2 min after addition of AAPH or H2O2–CuSO4.
All fluorescence measurements were expressed relative to the initial reading.
Final results were calculated based on the difference in the area under the
fluorescence decay curve between the blank and each sample. ORACROOÅ was
expressed as micromoles of Trolox equivalents (TE). One ORAC unit is
equivalent to the net protection area provided by 1 mM of Trolox.
34. Wolfe, K. L.; Liu, R. H. J. Agric. Food Chem. 2008, 56, 8404.
17. Each compound (1 and 8, 2.0 mg) was dissolved in 1.0 N HCl (dioxane–H2O,
1:1, v/v, 1.0 mL) and then heated to 80 °C in a water bath for 3 h. The acidic
solution was neutralized with silver carbonate and the solvent thoroughly
driven out under N2 gas overnight. After extraction with CHCl3, the aqueous
layer was concentrated to dryness using N2 gas. The residue was dissolved in
0.1 mL of dry pyridine, and then
L
-cysteine methyl ester hydrochloride in
35. Hoyweghen, L. V.; Karalic, I.; Calenbergh, S. V.; Deforce, D.; Heyerick, A. J. Nat.
Prod. 2010, 73, 1573.
pyridine (0.06 M, 0.1 mL) was added to the solution. The reaction mixture
was heated at 60 °C for 2 h, and 0.1 mL of trimethylsilylimidazole solution was
added, followed by heating at 60 °C for 1.5 h. The dried product was
partitioned with n-hexane and H2O (0.1 mL, each), and the organic layer
was analyzed by GC: Column: column of SPB-1 (0.25 ꢁ 30 m); detector FID,
column temp 210 °C, injector temp 270 °C, detector temp 300 °C, carrier gas He
(2.0 mL/min). The retention times of persilylated glucose, apiose, and arabinose
were founded to be 14.11, 6.70, and 9.16 min, respectively, when compared
with the standard solutions prepared by the same reaction from the standard
36. The 40
lL of different concentrations of the compounds in distilled water were
mixed with 160
lL of the mixture containing 0.5 mM CuCl2 and 0.75 mM
neocuproine in 10 mM phosphate buffer, pH 7.4. The absorbance was
measured with a micro-plate reader at 454 nm for 1 h. Increased absorbance
of the reaction mixture indicates increased reducing capacity.
37. One hundreds micro-liters of different concentration of the compounds were
mixed with 100
mixture solution was added to 100
l
L
of 0.1
l
M
CuSO4. After one hundreds micro-liters of
L of 0.1 M calcein, the fluorescence of
l
l
monosaccharides. The retention times of persilylated
-apiose, -apiose, -arabinose, and -arabinose were 14.11, 14.26, 6.70, 6.95,
4.72, and 9.16 min, respectively.
D
-glucose,
L
-glucose,
mixture solution was measured using a Tecan GENios multi-functional plate
reader with fluorescent filters (excitation wavelength: 485 nm and emission
filter: 535 nm) and compared to the fluorescence intensity of control which
contained only calcein.
D
L
D
L
18. Kanchanapoom, T.; Sirikatitham, A.; Otsuka, H.; Ruchirawat, S. J. Asian Nat.
Prod. Res. 2006, 8, 747.