Kaempferol glycosides from the twigs of Cinnamomum osmophloeum and their nitric oxide production inhibitory activities

Article abstract of DOI:10.1016/j.carres.2012.10.008

In the present study, ethanolic extract of twigs from Cinnamomum osmophloeum led to isolate nine kaempferol glycosides including two new kaempferol triglycosides that were characterized as kaempferol 3-O-β-d-xylopyranosyl-(1→2)-α-l-arabinofuranosyl-7-O-α-

Full text of DOI:10.1016/j.carres.2012.10.008

Carbohydrate Research 364 (2012) 49–53
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Kaempferol glycosides from the twigs of Cinnamomum osmophloeum
and their nitric oxide production inhibitory activities
⇑
Huan-You Lin, Shang-Tzen Chang
School of Forestry and Resource Conservation, National Taiwan University, Taipei 106, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 August 2012
Received in revised form 2 October 2012
Accepted 10 October 2012
Available online 22 October 2012
In the present study, ethanolic extract of twigs from Cinnamomum osmophloeum led to isolate nine
kaempferol glycosides including two new kaempferol triglycosides that were characterized as kaempfer-
ol 3-O-b-
O-b- -xylopyranosyl-(1?2)-
these compounds were assigned by the application of 1D and 2D NMR spectroscopy and other tech-
niques. Among these nine compounds, kaempferol 7-O- -rhamnopyranoside (9) revealed inhibitory
effect against LPS-induced production of nitric oxide in RAW 264.7 macrophages with an IC50 value of
41.2 M. It also slightly reduced PGE2 accumulation by 26% at the concentration of 50 M.
D
-xylopyranosyl-(1?2)-
a
-
L-arabinofuranosyl-7-O-
a-L-rhamnopyranoside (1) and kaempferol 3-
D
a
-L
-rhamnopyranosyl-7-O-
a-L
-rhamnopyranoside (2). The structures of
a-L
Keywords:
Kaempferol glycosides
Cinnamomum osmophloeum
Lauraceae
l
l
Ó 2012 Elsevier Ltd. All rights reserved.
Anti-inflammation
1. Introduction
study is to focus on the isolation and structural assignation of nine
kaempferol glycosides from n-butanolic fraction of 70% ethanolic
extract of C. osmophloeum twigs. Among them, compounds 1 and
2 (Fig 1) are new compounds, and compounds 3, 4 and 6 are
obtained for the first time from this species. In addition, these nine
compounds were evaluated for their inhibitory effects against lipo-
polysaccharide (LPS)-induced nitric oxide production in RAW
264.7 macrophages.
Cinnamomum osmophloeum Kaneh. (Lauraceae) is an endemic
tree that grows in natural hardwood forest at an elevation between
400 and 1500 m in Taiwan. Traditionally, the leaves of C. osmophlo-
eum were utilized in folk medicine for the treatments of
inflammation, intestinal infections, astringent, diuretic, and dia-
betic complications.1 Many preceding investigations manifested
that the essential oil from leaves and twigs of this plant exhibited
various medicinal and biological properties such as sweet
constituents, anti-bacterial, anti-fungal, anti-cancer, anti-termitic,
anti-mite, anti-mildew, mosquito larvicidal, anti-pathogenic, anti-
inflammatory, anti-red imported fire ant, and anti-hyperuricemic
activities.2–16 Moreover, despite the essential oil use, phytochemi-
cal studies for the secondary metabolites demonstrated several
bioactive compounds from different plant parts of C. osmophloeum.
From its leaves, four anti-inflammatory and three insulin-mimetic
kaempferol glycosides were identified.18,19 From its heartwood and
roots, six lignanoids including two cytotoxic lignan feruloyl esters
were isolated.20 And from its twigs, an antioxidant kaempferol gly-
coside was isolated.17 These above literature reports encouraged
phytochemists to investigate the secondary metabolites of C.
osmophloeum.
2. Results and discussion
Compound 1 was obtained as a pale yellow amorphous solid.
The molecular formula of 1 was revealed to be C31H36O18 by the
HRESIMS data (m/z 735.1519 ([M+K]+)). The IR spectrum exhibited
absorption bands of a hydroxyl group at 3358 cmꢀ1, a conjugated
carbonyl group at 1660 cmꢀ1, a phenyl group at 1598 cmꢀ1, and
a glycosidic nature at 1178 and 1065 cmꢀ1. The UV spectra in
methanol and with shift reagents of 1 were consistent with 3,
7-di-O-substituted flavonols with free hydroxyl groups at 5 and
40-positions.22 The 1H NMR spectrum (Table 1) of 1 displayed six
aromatic protons, that is, four protons as two ortho-coupled
doublets at dH 7.92 (2H, d, J = 8.7 Hz, H-20,60) and 6.91 (2H, d,
J = 8.7 Hz, H-30,50) with characteristics for an A2B2 system that con-
firmed the 10,40-disubstituted structure of B-ring and two protons
as meta-relation at dH 6.70 (br s, H-8) and 6.42 (br s, H-6) of A-ring
decided by HSQC and HMBC spectra. In addition, three anomeric
protons [dH 5.70 (br s, H-100), 5.56 (br s, H-10000), and 4.43 (d,
J = 9.0 Hz, H-1000)] and three anomeric carbons [dC 108.2 (C-100),
104.7 (C-1000), and 99.8 (C-10000)] of 1 were observed by 1H and 13C
NMR (Table 1), as well as HRESIMS data, indicating the presence
of triglycosidic structure with one hexose and two pentoses.
Previously, Chua et al. demonstrated that an ethanolic extract of
C. osmophloeum twigs possessed excellent antioxidant activities.
However, only a kaempferol glycoside with moderate antioxidant
activities was found.17 This result interested us to further study
the chemical constitutions of C. osmophloeum twigs. The aim of this
⇑
Corresponding author. Tel.: +886 2 3366 4626; fax: +886 2 2365 4520.
E-mail address: peter@ntu.edu.tw (S.-T. Chang).
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.10.008

50
H.-Y. Lin, S.-T. Chang / Carbohydrate Research 364 (2012) 49–53
existence of a rhamnose. The 13C NMR of compound 1 exhibited
OH
4'
upfield shift for C-3 and C-7 by 1.9 and 2.1 ppm, compared to
unsubstituted kaempferol that usually appears at dC 137.1 (C-3)
and 165.6 (C-7),23 revealing glycosidation at both positions. Among
O
O
9
2
1'
α-L-Rhap
7
them, the diequatorial coupling anomeric proton at dH 5.56 (H-10000
)
4
10
O
R
was attributed to O-a-L-rhamnopyranosyl moiety directly linked to
5
the C-7 position by the HMBC correlation between H-10000/C-7 and
NOESY correlations between H-10000/H-6 and H-8 (Fig. 2). Moreover,
OH
O
a
-configuration of the rhamnopyranose was identified by NOESY
correlations of H-10000/H-20000, and H-30000/H-5. Another anomeric pro-
Compounds
R
ton at dH 5.70 (H-100) and extremely downfield glycosidic carbons
signals [dC 91.0 (C-200) and 87.3 (C-400)] were assigned as O-
a-L-ara-
1
2
3
4
5
6
7
8
9
β-D-Xylp-(1 2)-α-L-Araf
β-D-Xylp-(1 2)-α-L-Rhap
β-D-Glcp-(1 2)-α-L-Araf
α-L-Rhap-(1 2)-α-L-Araf
β-D-Apif-(1 2)-α-L-Araf
β-D-Glcp-(1 2)-α-L-Rhap
β-D-Glcp-(1 4)-α-L-Rhap
α-L-Rhap
binofuranosyl moiety, which were the spectral characters of arab-
inofuranose and has been published by Nakano et al.24 This
assignation can also be confirmed by comparing the 13C NMR of
1 with a known compound kaempferol 3-O-b-
D
-glucopyranosyl-
(1?2)- -arabionpyranosyl-7-O-
a
-
L
a-L-rhamnopyranoside which
revealed data of dC 77.1 (C-200) and 68.8 (C-400), moreover the arab-
inofuranose moiety revealed more downfield glycosidic signals at
C-200 and C-400 than arabionpyranosyl structure.29 In addition,
HMBC correlation between H-100/C-3 proved that O-
a-
L-arabinofur-
anosyl moiety was attached to the C-3 position. The
a-configura-
tion of the arabinofuranosyl moiety was proved by NOESY of
H-1000/H-100, H-300, and H2-500.
The third sugar moiety with diaxial coupling anomeric proton
H
resonated upfield at dH 4.43 (H-1000) and was assigned to the O-b-
D-
xylosepyranosyl moiety involved in the interglycosidic linkage,
Figure 1. Structures of compounds 1–9.
and its pyranosidic conformation and b-configuration were estab-
lished by NOESY correlations between H-1000/H-3000, H -5000, and
a
Besides, fourteen glycosidic protons located among dH 4.42–
3.18 ppm together with a coupled doublet methyl group at dH
1.25 (d, J = 6.2 Hz, H-60000) were observed, which establish the
H-2000/H-4000 (Fig. 2). By comparing the 13C NMR data of
1
with kaempferol 3-O-
a-
L
-arabinofuranosyl-7-O-a-L
-rhamnoside,24
Table 1
1H (500 MHz) and 13C NMR (125 MHz) data of compounds 1 and 2 in methanol-d4, chemical shift in ppm, J in Hz
Position
1
2
13C
1H
13C
1H
2
3
4
5
6
7
8
159.7 s
135.2 s
180.0 s
162.8 s
100.6 d
163.5 s
95.6 d
159.7 s
137.0 s
179.8 s
163.0 s
100.6 d
163.5 s
95.6 d
6.42 br s
6.70 br s
6.42 d (2.0)
6.68 d (2.0)
9
10
10
20, 60
30, 50
40
107.3 s
157.9 s
122.4 s
132.1 d
116.6 d
161.7 s
107.5 s
158.0 s
122.3 s
131.8 d
116.7 d
161.8 s
7.92 d (8.7)
6.91 d (8.7)
7.76 d (8.7)
6.93 d (8.7)
3-O-
a
-
L
-Araf
3-O-
a
-
L
-Rhap
100
200
300
400
500
600
108.2 d
91.0 d
77.2 d
87.3 d
62.6 t
5.70 br s
103.1 d
82.6 d
72.1 d
73.6 d
71.9 d
17.7 q
200-O-b-
107.7 d
75.2 d
77.8 d
71.0 d
67.1 t
5.42 br s
4.42 ma
4.20 br d (3.3)
3.83 dd (9.6, 3.3)
3.33 t (9.6)
3.68 ma
4.08 dd (5.6, 2.7)
3.82 ma
3.52 ma, 3.48 ma
0.99 d (6.2)
200-O-b-
104.7 d
74.8 d
77.8 d
71.1 d
67.1 t
D
-Xylp
D-Xylp
1000
2000
3000
4000
5000
4.43 d (9.0)
3.18 t (9.0)
4.29 d (7.7)
3.19 dd (8.9, 7.7)
3.33 t (9.0)
3.30 ma
3.48 ma
3.41 ma
3.81 ma, 3.21 t (11.3)
3.68 ma, 3.07 t (11.0)
7-O-
a
-
L
-Rhap
7-O-a-L-Rhap
10000
20000
30000
40000
50000
60000
99.8 d
71.6 d
72.1 d
73.6 d
71.3 d
18.1 q
5.56 br s
4.04 br s
3.84 ma
99.8 d
71.7 d
71.9 d
73.6 d
71.3 d
18.1 q
5.55 br s
4.02 br s
3.83 dd (9.6, 3.3)
3.48 t (9.6)
3.60 ma
3.48 ma
3.59 ma
1.25 d (6.2)
1.25 d (6.2)
a
Overlapping with other singles.

H.-Y. Lin, S.-T. Chang / Carbohydrate Research 364 (2012) 49–53
51
anosyl-(1?2)-
(5),24 kaempferol 3-O-b-
anosyl-7-O-
-rhamnopyranoside (6),18 kaempferol 3-O-b-
pyranosyl-(1?4)- -rhamnopyranosyl-7-O- -rhamnopyranoside
(7),25 kaempferitrin (8),26 and kaempferol 7-O-
-rhamnopyrano-
a
-
L
-arabinofuranosyl-7-O-
a
-
L
-rhamnopyranoside
-rhamnopyr-
-gluco-
D-glucopyranosyl-(1?2)-
a-L
a-L
D
a-L
a-L
a-L
side (9),14 by comparing their IR, MS, and NMR data with those
reported in the literature. Among them, compounds 3, 4, and 6
were isolated from this species for the first time. The structural
characteristics of these isolated kaempferol triglycosides was the
interglycosidic connection at C-200 position between the 3-O-glyco-
side and the followed sugar moiety except for compound 7.
All these nine compounds were tested for their inhibitory effects
against LPS-stimulated NO generation in RAW 264.7 macrophages.
Compound 9 inhibited LPS-induced NO production with an IC50 va-
lue of 41.2
of compounds 1–8 were less than 10%. Quercetin was used as posi-
tive control with an IC50 value of 7.4 M. In addition, MTT assay re-
vealed no significant cytotoxic effects (over 90% cells survival) on
cells treated with compounds 1–9 at concentrations up to 100 M.
lM. However, at the dosage of 100 lM, inhibitory effects
Figure 2. Key HMBC, NOESY, and COSY correlations of compounds 1 and 2.
l
attachment of this xylose to the C-200 position of the 3-O-
a-
L-arabi-
l
nofuranosyl moiety was determined based on downfield shift signal
of C-200 by 7.5 ppm and upfield shift signals of C-100 and C-300 by 1.8
and 1.7 ppm (in pyridine-d5), respectively. In addition, that the xylo-
Furthermore, inhibitory effect of compound 9 against LPS-stimulated
PGE2 accumulation in RAW 264.7 macrophages was also examined.
Table 2 shows the result of examination. As there was no any treat-
ment of drug and sample (vehicle), the PGE2 accumulation was
72.7 pg/mL. Then, treatment of LPS to cells caused PGE2 production
up to 655.6 pg/mL. Compound 9 slightly inhibited the PGE2 accumu-
lation against LPS-stimulated in RAW 264.7 macrophages. At the
pyranose was attached to the C-200 position of
a-L-arabinofuranosyl
moiety was proved by a pair of HBMC correlations between H-1000/
C-200 and H-200/C-1000. A combination of 1D and 2D NMR (COSY,
NOESY, HSQC, and HMBC) data permitted the complete assignment
of every 1H and 13C NMR signals of compound 1 (Table 1). The sugar
composition and aglycone were determined to be arabinose, xylose,
rhamnose, and kaempferol by TLC analyzing the acid hydrolyte of 1.
The absolute configurations of the component monosaccharides
concentration of 50 lM, compound 9 decreased PGE2 production
to 483.9 pg/mL, indicating 26% inhibitory effect.
3. Experimental part
were determined as
L
-arabinose,
D
-xylose, and
L-rhamnose
respectively, by GCMS analysis with the retention times of their cor-
responding trimethylsilylated thiazolidine derivatives.21 According
to the above evidences, compound 1 was determined as kaempferol
3.1. General methods
Column chromatography (CC) separations were carried out by
using silica gels SI 60 (40–63
3-O-b-
D-xylopyranosyl-(1?2)-a-L-arabinofuranosyl-7-O-a-L-rha-
lm) and Lichroprep RP-18 gels (40–
mnopyranoside.
63 m) supplied by Merck, Darmstadt, Germany. The column
l
Compound 2 was isolated as pale yellow amorphous solid. The
molecular formula was revealed as C32H38O18 by the HRESIMS data
(m/z 711.2151 ([M+H+])), combining with the UV, IR, and 13C NMR
spectral data (Table 1), suggested that 2 was a 3,7-disubsitiuted
kaempferol triglycoside as well as 1. The 1H NMR of 2 exhibited
two downfield shift anomeric protons [dH 5.42 (br s, H-100) and
5.55 (br s, H-10000)] as well as two coupled doublet methyl groups
[3H, dH 0.99 (d, J = 6.2 Hz, H-600) and d(H) 1.25 (3H, d, J = 6.2 Hz,
H-60000)], which were deduced for two rhamnoses connecting to C-
3 and C-7 positions, respectively. In addition, HMBC correlations
between H-100/C-3 and H-10000/C-7 confirmed the above assumption.
The anomeric configuration of two rhamnopyranoyl groups was
fractions were monitored by thin layer chromatography (TLC) on
precoated Merck Silica gel 60F254 aluminum plates; the spots were
detected by using a UV lamp (254 nm) and by spraying with 10%
H2SO4 in EtOH followed by heating at 105 oC for 5 min. IR spectra
were obtained in KBr disks from a Bio-rad FTS-40 spectrophotome-
ter. UV spectra were recorded by Jasco V-550 spectrometer. Specific
optical rotations were measured by HORIBA SEPA-300 optical rota-
tion spectra-photometer. High performance liquid chromatography
(HPLC) was conducted by Jasco model PU-980 instrument and the
detector employed was Jasco MD-910 photodiode array at 260
and 350 nm wavelengths. The HPLC columns were Phenomenex
Luna RP-18, Synergi Fusion-RP, and Luna Silica (2) semipreparative
assigned as
a-configuration because of NOESY correlations of H-
columns (250 ꢁ 10 mm id, 5
lm particle size for RP-18 and Silica
100/H-200, H-300/H-500, H-10000/H-20000, and H-30000/H-50000. The third sugar
was elucidated as xylose based on the same evidences mentioned
in compound 1 section. Furthermore, the interglycosidic conforma-
tion of xylopyranosyl moiety and 3-O-rhamnopyranosyl moiety
was confirmed by HMBC correlation pairs for H-1000/C-200 and H-
and 4 m for Fusion-RP), and a flow rate of 4.0 mL/min was em-
l
ployed. Electrospray ionization mass spectrometry (ESIMS) data
were collected using a VG Platform Electrospray ESIMS, and high
resolution electrospray ionization mass spectrometry (HRESIMS)
was obtained from Finnigan MAT-958 mass spectroscopy. 1D and
2D NMR data (including HSQC, HMBC, COSY, and NOESY spectra)
were recorded by using a Bruker Avance-500 spectrometer operat-
ing at 500.1 and 125.7 MHz, respectively. Compounds were ana-
lyzed in methanol-d4. Chromatography-Mass spectrometry (GCMS)
analysis was carried out on a Trace GC chromatography, coupled
with an ion trap mass spectrometer PoLaris Q (Austin, TX, USA).
200/C-1000. Then, this xylopryanosyl group was assigned as
a-config-
uration because of NOESY correlations of H-1000/H-3000, H-5a000, and
H-2000/H-4000. GCMS analysis result deduced that the three sugars
of compound 2 were two
compound 2 was assigned as kaempferol 3-O-b-
(1?2)- -rhamnopyranosyl-7-O- -rhamnopyranoside.
L
-rhamnoses and one
D-xylose. Therefore,
D-xylopyranosyl-
a
-L
a-L
Seven known kaempferol glycosides were also isolated from
ethanolic extracts of twigs from C. osmophloeum Kaneh.
3.2. Plant material
These were characterized as kaempferol 3-O-b-
syl-(1?2)- -arabinofuranosyl-7-O- -rhamnopyranoside (3),
Kaempferol 3-O- -rhamnopyranosyl-(1?2)- -arabinofurano-
syl-7-O- -apiofur-
-rhamnopyranoside (4),30 kaempferol 3-O-b-
D-glucopyrano-
a
-L
a-L
The twigs of a 13-year-old Cinnamomum osmophloeum Kaneh.
were collected at the end of July 2004 from the Taiwan Sugar
a-
L
a-L
a-
L
D

52
H.-Y. Lin, S.-T. Chang / Carbohydrate Research 364 (2012) 49–53
Table 2
Inhibitory effect of compound 9 against LPS-induced PGE2 production (pg/mL) in RAW 264.7 macrophages
Compounds
Concentration (
10
lM)
Vehicle
50
20
2
0
9
483.9 8.3
32 1.5
587.1 10.6
41.8 2.0
631.9 12.0
55.1 1.3
626.1 15.9
75.4 3.0
655.6 13.2
72.7 2.3
Indomethacina
a
Positive control. Each experiment was performed three times, and the data were averaged (n = 3).
Company Research Center located in central Taiwan. The
authenticity of the species was confirmed by Dr. Yen-Ray Hsui of
the Taiwan Forestry Research Institute and voucher specimens
(CO1001) were deposited at the laboratory of wood chemistry
(School of Forestry and Resource Conservation, National Taiwan
University). Diameter of the twigs selected was below 1.5 cm.
3.3.1. Kaempferol 3-O-b-
D
-xylopyranosyl-(1?2)-a-L-
arabinofuranosyl-7-O-a-L-rhamnopyranoside (1)
Pale yellow amorphous powder; ½a D29
ꢀ242.8 (c 2.08, MeOH);
ꢂ
UV (MeOH) kmax (loge) 265 (4.36), 352 (4.27); +NaOMe 278, 391;
+NaOAc 264, 349; +NaOAc/H3BO3 265, 353; +AlCl3 269, 303 (sh),
345, 388 (sh); +AlCl3/HCl 273, 303 (sh), 346, 388 nm; IR (KBr) mmax
3358, 2924, 1660, 1598, 1208, 1178, 1065, 961, 809 cmꢀ1 1H and
;
3.3. Extraction and isolation
13C NMR see Tables 1 and 2, respectively. ESIMS positive mode m/z
(rel. int.) 697 [M+H]+ (24), 433 [(M+H)-Xyl-Ara]+ (43), 287 [(M+H)-
Xyl-Ara-Rha]+ (24) and negative mode m/z (rel. int.) 695 [MꢀH]ꢀ
(100), 549 [M–H-Rha]ꢀ (15). HRESIMS positive mode m/z
735.1519 [M+K]+ (calcd for 735.1539; C31H36O18K+) and 719.1849
[M+Na]+ (calcd for 719.1799; C31H36O18Na+); HRESIMS negative
mode m/z 695.1847 [MꢀH]ꢀ (calcd for 695.1823; C31H35O18ꢀ).
GCMS analysis of sugar components, tR: 13.13, 14.35, and
13.13 min. (See Supplementary data).
The air-dried twigs of C. osmophloeum (2.02 kg) were cut into
small pieces and extracted with 70% ethanol at ambient tempera-
ture for seven days. The combined extracts were decanted, filtered,
and then concentrated by rotary evaporator to obtain a dark brown
syrup about 374 g (18.5% based on the dry weight of the twigs).
The crude extracts were suspended in H2O, and then partitioned
with n-hexane (Hex), ethyl acetate (EtOAc), and n-butanol (BuOH)
successively to yield the Hex (4.9%), EtOAc (12.0%), BuOH (50.0%),
H2O (18.0%), and water insoluble (15.0%) fractions. The BuOH frac-
tion was subjected to RP-CC by eluting with MeOH/H2O (gradient
elution was performed by changing from 20/80 to 100/0). After
separating the BuOH fraction, seventeen subfractions (BU1-BU17)
were collected by analyzing the TLC and combining its similar
patterns.
BU6 was subjected to silica gel CC eluted with CH2Cl2 to 50%
EtOH in CH2Cl2 and divided into seven fractions (BU6a to BU6g).
BU6b was separated and purified by RP-HPLC with linear gradient
of MeOH/H2O (40/60 to 55/45) in 30 min to give compound 8
(152 mg, retention time = 14.9 min). BU6c was separated and puri-
fied by RP-HPLC with linear gradient of MeOH/H2O (35/65 to 20/
3.3.2. Kaempferol 3-O-b-D-xylopyranosyl-(1?2)-a-L-
rhamnopyranosyl-7-O-a-L-rhamnopyranoside (2)
Pale yellow amorphous powder; ½a D29
ꢀ180.9 (c 2.53, MeOH); UV
ꢂ
(MeOH) kmax (loge) 266 (4.37), 354 (4.25); +NaOMe 278, 391; +NaO-
Ac 265, 350; +NaOAc/H3BO3 266, 352; +AlCl3 270, 302 (sh), 344, 391
(sh); +AlCl3/HCl 275, 302 (sh), 347, 387 nm; IR (KBr) max 3429, 2964,
m
2928, 1656, 1602, 1262, 1089, 1038, 968, 806 cmꢀ1; 1H and 13C NMR
see Tables 1 and 2, respectively. ESIMS m/z (rel. int.) 749 [M+K]+ (93),
711 [M+H]+ (45), 433 [(M+H)-Xyl-Rha]+ (66), 287 [(M+H)-Xyl-
(2 ꢁ Rha)]+ (34); HRESIMS positive mode m/z 711.2151 [M+H]+
(calcd for 711.2136; C32H39O18+); GCMS analysis of sugar compo-
nents, tR: 13.12 and 14.35 min. (See Supplementary data).
80) in 40 min to give compound
5 (1082 mg, retention
time = 20.7 min). BU6d was separated by RP-HPLC with linear gra-
dient of MeOH/H2O (30/70 to 45/55) in 40 min and then purified
by RP-HPLC with linear gradient of acetonitrile/H2O (40/60 to 60/
40) in 30 min to give compound 4 (132 mg, retention time = 14.5 -
min). BU6e was separated by RP-HPLC with linear gradient of
MeOH/H2O (30/70 to 60/40) in 40 min to give 2 fractions, BU6e-1
and BU6e-2. BU6e-1 was purified by RP-HPLC with linear gradient
of acetonitrile/H2O (40/60 to 60/40) in 30 min to give compound 3
(241 mg, retention time = 13.4 min). BU6e-2 was separated by NP-
HPLC with linear gradient of EtOH/CH2Cl2 (20/80 to 60/40) in
60 min to give two fractions, BU6e-2a and BU6e-2b. BU6e-2b
was purified by RP-HPLC with linear gradient of MeOH/H2O (40/
60 to 50/50) in 20 min to afford compound 6 (22 mg, retention
time = 17.8 min). BU6e-2a was separated by Fusion-RP-HPLC
with linear gradient of MeOH/H2O (60/40 to 50/50) in 30 min
to give two fractions, BU6e-2a-1 and BU6e-2a-2. BU6e-2a-2 was
purified by RP-HPLC with linear gradient of MeOH/H2O (40/60 to
3.4. Acid hydrolysis of compounds 1 and 2
Compounds 1 and 2 (5 mg each) in 1 M HCl (2 mL) were heated
at 80 °C and refluxed for 4 h. After cooling, the reaction mixture
was extracted with EtOAc to offer organic phase and water phase.
The organic phase was concentrated by rotary evaporator and
examined by TLC (EtOAc/n-Hexane = 4/1) with authentic sample
(Rf = 0.52). The water phase was dried by rotary evaporator and
then redissolved in MeOH and subjected to TLC (CHCl3/MeOH/
H2O = 6/4/1) with authentic samples (Rf = 0.35, 0.26, 0.53, and
0.40 for arabinose, glucose, rhamnose, and xylose, respectively).
The color developing agent for sugar analysis was p-anisaldehyde
in acidic ethanol (p-anisaldehyde/H2SO4/EtOH = 1/1/18).
3.5. Determination of sugar absolute configurations21
Solutions of compounds 1 and 2 (1 mg each) in 1 M HCl (1 mL)
were stirred at 80 °C for 4 h. After cooling, the solution was evap-
orated under N2. The residue was dissolved in anhydrous pyridine
50/50) in 20 min to afford compound
2 (21 mg, retention
time = 17.9 min). BU6e-2a-1 was separated by Fusion-RP-HPLC
with linear gradient of MeOH/H2O (60/40 to 50/50) in 30 min to
(100 lL), 0.1 M L-cysteine methyl ester hydrochloride (200 lL in
give compound
1
(25 mg, retention time = 19.2 min) and
7
dry pyridine) was added, and the mixture was warmed at 60 oC
for 1 h. The trimethylsilylation reagent HMDS-TMCS (hexamethyl-
(21 mg, retention time = 20.6 min). Compounds 9 (33 mg, reten-
tion time = 17.2 min) was isolated and purified from BU8 by RP-
HPLC with linear gradient of MeOH/H2O (20/80 to 80/20) in
30 min.
disilazane/trimethylchlorosilane/pyridine, 2/1/10; 200
added, and warming at 60 °C was continued for another 30 min.
The precipitate was centrifuged off, and the supernatant (1 L)
lL) was
l

H.-Y. Lin, S.-T. Chang / Carbohydrate Research 364 (2012) 49–53
53
was subjected to GCMS analysis to identify the sugar. GCMS condi-
m);
oven temperature program: 180–300 °C at 4 °C/min; injection tem-
perature: 250 °C; carrier gas: He at flow rate of 0.8 mL/min. The
retention times (tR) of authentic samples were 13.16, 17.01, 14.37,
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