Diterpene glycosides from the ethanol extract of Diplopterygium rufopilosum

Article abstract of DOI:10.1080/10286020.2015.1010523

A phytochemical investigation on the 70% EtOH extract from the dry fronds of Diplopterygium rufopilosum afforded two new labdane-type diterpene glycosides, (3β,13S)-3-O-β-d-glucopyranosyl-13-O-l-rhamnopyranosyl-labda-8(17),14-diene (1) and (3β,13S)-3-O-l-rhamnopyranosyl-13-O-[6-O-acetyl-β-d-glucopyranosyl-(1 → 4)-2-O-acetyl-l-rhamnopyranosyl]-labda-8(17),14-diene (2). Their structures were determined on the basis of chemical method and spectroscopic analyses, including 1D and 2D NMR (COSY, HMQC, HMBC, and NOESY) and HR-ESI-MS analyses.

Full text of DOI:10.1080/10286020.2015.1010523

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Diterpene glycosides from the ethanol
extract of Diplopterygium rufopilosum
Jiang Hua, Yan Songb, Hui Lic, Xia Maoa & Xiao-Dong Shia
a College of Biological Resources and Environment Science, Qujing
Normal University, Qujing 655011, China
b Department of Pharmacy, 455 Hospital of People's Liberation
Army, Shanghai 200052, China
c College of Physics and Electronic Engineering, Qujing Normal
University, Qujing 655011, China
Published online: 24 Feb 2015.
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To cite this article: Jiang Hu, Yan Song, Hui Li, Xia Mao & Xiao-Dong Shi (2015) Diterpene glycosides
from the ethanol extract of Diplopterygium rufopilosum, Journal of Asian Natural Products
Research, 17:3, 262-267, DOI: 10.1080/10286020.2015.1010523
To link to this article: http://dx.doi.org/10.1080/10286020.2015.1010523
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Journal of Asian Natural Products Research, 2015
Vol. 17, No. 3, 262–267, http://dx.doi.org/10.1080/10286020.2015.1010523
Diterpene glycosides from the ethanol extract of Diplopterygium
rufopilosum
Jiang Hua*, Yan Songb, Hui Lic, Xia Maoa and Xiao-Dong Shia
aCollege of Biological Resources and Environment Science, Qujing Normal University, Qujing
655011, China; bDepartment of Pharmacy, 455 Hospital of People’s Liberation Army, Shanghai
200052, China; cCollege of Physics and Electronic Engineering, Qujing Normal University, Qujing
655011, China
(Received 2 December 2014; final version received 19 January 2015)
A phytochemical investigation on the 70% EtOH extract from the dry fronds of
Diplopterygium rufopilosum afforded two new labdane-type diterpene glycosides,
(3b,13S)-3-O-b-D-glucopyranosyl-13-O-a-L-rhamnopyranosyl-labda-8(17),14-diene
(1) and (3b,13S)-3-O-a-L-rhamnopyranosyl-13-O-[6-O-acetyl-b-D-glucopyranosyl-
(1 ! 4)-2-O-acetyl-a-L-rhamnopyranosyl]-labda-8(17),14-diene (2). Their structures
were determined on the basis of chemical method and spectroscopic analyses, including
1D and 2D NMR (COSY, HMQC, HMBC, and NOESY) and HR-ESI-MS analyses.
Keywords: Diplopterygium rufopilosum; Gleicheniaceae; diterpene glycosides;
labdane
1. Introduction
D. rufopilosum systematically and resulted
in the isolation of two new labdane-type
diterpene glycosides, (3b,13S)-3-O-b-D-
glucopyranosyl-13-O-a-L-rhamnopyrano-
syl-labda-8(17),14-diene (1) and (3b,13S)-
3-O-a-L-rhamnopyranosyl-13-O-[6-O-
acetyl-b-D-glucopyranosyl-(1 ! 4)-2-O-
acetyl-a-L-rhamnopyranosyl]-labda-8
(17),14-diene (2) (Figure 1). This paper
deals with the isolation and structure
elucidation of the two new diterpene
glycosides on the basis of the spectro-
scopic analysis and acid hydrolysis.
The genus Diplopterygium (Gleichenia-
ceae) is endemic and widely distributed in
the tropical and subtropical regions of
Africa, Asia, and the northwest part of
Australia. Among 40 species, 9 species of
Diplopterygium naturally occur in China
[1]. Diplopterygium contains diverse types
of natural products, including diterpenes,
diterpene glycosides, triterpenoids, ecdy-
steroids, flavonoids, flavone glycosides, and
phenylpropanoid glycosides [2–9]. The
diterpenes and diterpene glycosides have
been reported as the characteristic constitu-
ents of Diplopterygium. In previous studies
on the chemical constituents of Diplopter-
ygium rufopilosum, we have reported the
isolation of diterpene glycosides and ecdy-
steroids from the plant source [9,10].
In continuation of our search for bioactive
and structurally interesting substances, the
presentstudywasundertakentoexamine the
70% ethanol extract of the dry fronds of
2. Results and discussion
Compound 1, a white amorphous powder
exhibited a molecular ion peak at m/z
613.3586 [M 2 H] þ in the high-resolution
mass spectrometry, which corresponded to
the molecular formula C32H54O11 with six
degrees of unsaturation. The IR spectrum
of 1 exhibited the strong absorption for
*Corresponding author. Email: hujiang@ustc.edu
q 2015 Taylor & Francis

Journal of Asian Natural Products Research
263
O
16
OAc
12
13
O
2''
3''
O
11
1''
5''
OH
20
17
14
15
O
OH
1
4
2
3
9
6'
8
OH
4''
10
HO
O
O
4'
5
5'
2'
AcO
HO
HO
O
6''
HO
HO
7
O
O
O
HO
6
HO
1'
3'
18
OH
HO
19
OH
OH
1
2
Figure 1. The structures of compounds 1 and 2.
hydroxyl groups (3427 cm21) and double
bonds (1631 cm21). The 1H and 13C NMR
spectra of 1 showed the characteristic
resonances of a diterpene and two hexoses
(Table 1). Acidic hydrolysis of 1 with 2%
HCl afforded D-glucose and L-rhamnose,
which were identified directly by the GC
analysis of their trimethylsilyl imidazole
derivatives [11]. The trans diaxial coup-
ling constant (J ¼ 8.0 Hz) of the anomeric
proton indicated the b-configurations of
the glucopyranosyl unit, while the equa-
torial and axial relationship of H-l00 and
H-200 (J ¼ 1.3 Hz) indicated the a-con-
figuration of the rhamnopyranosyl unit
(Table 1). In addition to the signals of
sugar moieties, the diterpene moiety of 1
contained four CH3 groups (dC 28.7, 23.4,
16.8, and 15.1), six CH2 groups (dC 42.1,
39.4, 38.5, 27.7, 25.0, and 18.9), four
olefinic C-atoms (dC 149.5, 143.9, 115.7,
and 107.6), two CH groups (dC 59.4 and
49.5), two quarternary C-atoms (dC 40.6
and 40.3), an oxygenated methane (dC
90.9), and an oxygenated quaternary
carbon (dC 80.9) according to the NMR
spectra, which suggested that it was a
labdane-type diterpene. The aglycone (1a)
obtained by the acid hydrolysis of 1 was
determined as 3b-hydroxy-13-epimanool,
a common aglycone of the labdane-type
glycosides of Diplopterygium, by direct
comparison of the 1H NMR data with
those of authentic samples (3b-hydroxy-
13-epimanool and 3b-hydroxy-13-man-
ool). The signals of H-17a appeared at dH
4.51 in 1a and 3b-hydroxy-13-epimanool,
but at dH 4.45 in 3b-hydroxy-13-manool
[9,12,13]. The HMBC correlation between
the anomeric protons (dH 4.31 and 4.83) of
the glucopyranosyl and rhamnopyranosyl
units with C-3 (dC 90.9) and C-13 (dC
80.9) of the aglycone revealed that the
sugar moieties were located at C-3 and C-
13, respectively (Figure 2). The relative
configuration of 1 was deduced from its
ROESY spectrum. The ROESY corre-
lations of H-3 with H-1a and H-5
confirmed that H-3 was a-oriented. From
a biogenetic perspective, 3b-hydroxy-13-
epimanool should be the aglycone of 1
because it was isolated from the same
18
genus, and its specific rotation (½aꢀD 219
(MeOH; c ¼ 0.65)) coincided with the
reported value. Thus, the structure of 1 was
determined as (3b,13S)-3-O-b-D-gluco-
pyranosyl-13-O-a-L-rhamnopyranosyl-
labda-8(17),14-diene.
Compound 2 was obtained as a white
amorphous powder. Its molecular formula
C42H68O17 was determined by analysis of
1H, 13C, and DEPT NMR data and verified
by HR-ESI-MS at m/z 879.4148 [M
þ Cl]2, which revealed nine unsaturation
degrees. Inspection of the NMR spectra of
2 established the presence of a diterpene,
three hexose moieties and two acetyl
groups. The resemblance of the NMR
spectra (Table 1) to those of 1 suggested
that 2 was a derivative of 3b-hydroxy-13-
epimanool. Acid hydrolysis of 2 with 1 M
HCl in dioxane–H2O (1:1) gave the
aglycone, D-glucose, and L-rhamnose
which were identified directly by the GC
analysis [11]. The glucopyranosyl and
rhamnopyranosyl units were elucidated as

264
J. Hu et al.
Table 1. 1H and 13C NMR spectral data of compounds 1 and 2 (1H: 500 MHz; 13C: 125 MHz, in
CD3OD).
dH (mult., J in Hz)
dC
No.
1
2
No.
1
2
1ax
1eq
2ax
2eq
3ax
5
1.17 (ddd, 13.2, 13.0, 4.0) 1.20 (ddd, 13.2, 13.0, 4.0)
1
2
3
4
5
6
7
8
9
38.5 (t)
25.0 (t)
90.9 (d)
40.6 (s)
49.5 (d)
27.7 (t)
39.4 (t)
149.5 (s)
59.4 (d)
40.3 (s)
18.9 (t)
42.1 (t)
80.9 (s)
37.9 (t)
24.6 (t)
89.7 (d)
40.0 (s)
55.8 (d)
27.3 (t)
38.8 (t)
149.2 (s)
57.8 (d)
39.9 (s)
18.4 (t)
41.8 (t)
80.2 (s)
1.78 (ddd, 13.0, 4.0, 3.4)
1.64–1.68 (overlapped)
1.91–1.93 (m)
1.82 (ddd, 13.0, 4.0, 3.4)
1.23–1.25 (m)
1.79–1.80 (m)
3.18 (dd, 13.2, 4.5)
1.13 (dd, 13.5, 4.0)
1.39–1.41 (m)
3.20 (dd, 13.2, 4.5)
1.15 (dd, 13.5, 4.0)
1.65–1.67 (m)
6ax
6 eq 1.64–1.68 (overlapped)
1.95–1.97 (m)
2.02 (ddd, 13.5, 13.0, 4.0) 2.06 (ddd, 13.5, 13.0, 4.0)
7ax
7eq
9
2.39 (ddd, 13.0, 4.0, 3.4)
1.53 (t, 7.0)
1.37–1.39 (m)
1.58–1.60 (m)
1.28 (td, 13.5, 7.0)
1.71 (td, 13.5, 7.0)
5.77 (dd, 17.0, 10.5)
5.17 (dd, 17.0, 1.5)
5.22 (dd, 10.5, 1.5)
1.33 (s)
2.42 (ddd, 13.0, 4.0, 3.4)
1.62 (t, 7.0)
10
11
12
13
14
15
16
17
18
19
20
10
11a
11b
12a
12b
14
15a
15b
16
17a
17b
18
19
20
10
1.46–1.48 (m)
1.61–1.63 (m)
1.32 (td, 13.5, 7.0)
1.75 (td, 13.5, 7.0)
5.86 (dd, 17.0, 10.5)
5.20 (dd, 17.0, 1.5)
5.25 (dd, 10.5, 1.5)
1.38 (s)
143.9 (d) 143.4 (d)
115.7 (t)
23.4 (q)
107.6 (t)
28.7 (q)
16.8 (q)
15.1 (q)
106.7 (d)
75.5 (d)
78.1 (d)
71.8 (d)
77.8 (d)
62.7 (t)
96.8 (d)
83.3 (d)
72.5 (d)
75.0 (d)
69.8 (d)
18.9 (q)
–
115.6 (t)
23.2 (q)
106.3 (t)
28.4 (q)
16.6 (q)
14.9 (q)
96.2 (d)
83.7 (d)
72.6 (d)
74.7 (s)
69.0 (s)
18.1 (q)
105.5 (d)
79.4 (d)
72.8 (d)
74.8 (d)
72.5 (d)
17.9 (q
106.3 (d)
75.2 (d)
77.9 (d)
71.5 (d)
74.4 (d)
64.4 (t)
172.9 (s)
20.8 (q)
171.0 (s)
19.2 (q)
4.56 (d, 1.5)
4.83 (d, 1.5)
1.04 (s)
0.82 (s)
0.70 (s)
4.63 (d, 1.5)
4.86 (d, 1.5)
1.08 (s)
0.85 (s)
0.73 (s)
5.17 (d, 1.6)
3.90–3.92 (m)
3.74–3.76 (m)
3.42–3.44 (m)
3.85–3.87 (m)
1.22 (d, 6.0)
20
30
40
4.31 (d, 8.0)
3.18–3.20 (m)
3.41–3.43 (m)
3.25–3.27 (m)
3.35–3.37 (m)
3.64 (dd, 13.0, 5.6)
3.85 (dd, 13.0, 2.6)
4.83 (d, 1.3)
3.89–3.91 (m)
3.80–3.82 (m)
3.42–3.44 (m)
3.75–3.77 (m)
1.21 (d, 6.0)
–
50
20
60
30
100
40
200
50
300
60
400
–
4.86 (d, 1.6)
500
100
600
200
3.92–3.94 (m)
3.79–3.81 (m)
3.59–3.61 (m)
3.77–3.79 (m)
1.23 (d, 6.0)
1000
20000
30000
40000
50000
60000
CvO
CH3
CvO
CH3
300
–
–
–
–
–
–
–
–
400
500
600
1000
2000
3000
4000
5000
6000
4.57 (d, 8.0)
–
–
–
–
3.18–3.20 (m)
3.39–3.41 (m)
3.29–3.31 (m)
3.79–3.81 (m)
4.25 (dd, 12.0, 5.6)
4.37 (dd, 12.0, 2.0)
2.09 (s)
–
–
CH3
–
b- and a-configurations by the coupling
constants of their anomeric protons,
respectively (Table 1). In the HMBC
spectrum (Figure 2), the correlation
between the anomeric proton (dH 4.57) of
the glucopyranosyl unit and C-400 (dC 74.8)
of the rhamnopyranosyl unit identified a
glucopyranosyl (1 ! 4) rhamnopyranosyl

Journal of Asian Natural Products Research
265
O
O
O
O
O
OH
O
OH
OH
O
O
HO
HO
HO
HO
HO
O
O
O
O
HO
O
HO
O
HO
OH
OH
OH
1
2
Figure 2. Key HMBCs (
) of compounds 1 and 2.
linkage. The sugar chain was linked to
C-13 of the aglycone by the HMBC
correlation of the anomeric proton (dH
4.86) of the rhamnopyranosyl unit and C-
13 (dC 80.2) of the aglycone. The HMBC
correlation of the anomeric proton (dH
5.17) of another rhamnopyranosyl unit
with C-3 (dC 89.7) of the aglycone
suggested that the rhamnopyranosyl unit
linked at C-3 position (Figure 2). Further-
more, the C-2 and C-6 locations of the
acetyl groups in the rhamnopyranosyl unit
and glucopyranosyl unit were confirmed
by the HMBC correlations of H-200 (dH
3.93) and H-6000 (dH 4.37 and 4.25) with the
carbonyl carbons (dC 171.0) and (dC
172.9) of the acetyl groups, respectively.
The relative configuration was established
by a ROESY experiment. The ROESY
correlations of H-1a/H-3/H-5 confirmed
the a-oriented configuration of H-3.
Thus, the structure of 2 was determined
as (3b,13S)-3-O-a-L-rhamnopyranosyl-
13-O-[6-O-acetyl-b-D-glucopyranosyl-
(1 ! 4)-2-O-acetyl-a-L-rhamnopyrano-
syl]-labda-8(17),14-diene.
Magna FT-IR 750 spectrophotometer using
KBr disks (American Laboratory Trading,
East Lyme, CT, USA). NMR spectra were
recorded on Bruker AM-500 and Varian
Inova NMR spectrometers (Varian Medical
Systems, Salt Lake City, UT, USA). The
chemical shift (d) values are given in ppm
with TMS as the internal standard, and
coupling constants (J) are in Hz. FAB-MS
and HR-ESI-MS were recorded on a VG
AutoSpec-3000 mass spectrometer (VG
Instruments, London, UK). Column chro-
matographies were carried out using silica
gel (200–300 mesh and H60, Qingdao
Haiyang Chemical Group Corporation,
Qingdao, China), MCI gel CHP20P (75–
150 mm, Mitsubishi Chemical Industries,
Tokyo, Japan), and Sephadex LH-20
(Pharmacia Biotech AB, Uppsala, Sweden)
as packing material. TLC was carried out
on precoated silica gel GF254 plates (Yantai
Chemical Industries, Yantai, China), and
the TLC spots were viewed at 254 nm and
visualized using 5% sulfuric acid in alcohol
containing 10mg/ml vanillin. Analytical
HPLC was performed on a Waters 2690
instrument with a 996 PAD (photodiode
array detector) coupled with an Alltech
ELSD 2000 detector. Semipreparative
and preparative HPLC were performed
on a Varian SD1 instrument with a 320
single-wave detector (Varian Medical
Systems). Their chromatographic separ-
ations were carried out on C-18 columns
(250 £ 10 mm, 5 mm, Waters, Milford,
MA, USA; 220 £ 25mm, 10 mm, Merck,
3. Experimental
3.1. General experimental procedures
UV spectra were performed on a UV-1700
spectrophotometer (Shimadzu Corporation,
Tokyo, Japan). Optical rotations were
taken on a Perkin-Elmer 341 polarimeter
(PerkinElmer Corporation, Waltham, MA,
USA). IR spectra were recorded on Nicolet

266
J. Hu et al.
Darmstadt, Germany), using a gradient
solvent system consisting of H2O and
MeOH, with a flow rate of 3.0 and
15.0ml/min, respectively.
250 £ 20 mm) using MeOH/H2O (20:80,
15.0 ml/min) as the mobile phase to afford
2 (51 mg, 20.40 min).
3.3.1. (3b,13S)-3-O-b-D-
Glucopyranosyl-13-O-a-L-
rhamnopyranosyl-labda-8(17),14-diene (1)
3.2. Plant material
The fronds of D. rufopilosum were
collected in the Caojian Town, Yunnan
Province of China, in July 2013 and
identified by one of the authors (X. Mao).
A voucher specimen (No. DR20130701) is
deposited in the Herbarium of the College
of Biological Resources and Environment
Science, Qujing Normal University, Quj-
ing, Yunnan Province, China.
White amorphous powder; C32H54O11;
23:3
½aꢀD 221.80 (c ¼ 0.029, MeOH); UV
(MeOH) lmax (log 1) 203 (3.75) nm; IR
(KBr) nmax 3427, 2937, 1744, 1631,
1
1060 cm21; for H NMR (500 MHz) and
13C NMR (125 MHz) spectral data, see
Table 1; FAB-MS (neg.): m/z 613
[M 2 H]2; HR-ESI-MS: m/z 613.3586
[M 2 H]2 (calcd for C32H53O11,
613.3588).
3.3. Extraction and isolation
The fronds of D. rufopilosum (5 kg) were
ground into powder and extracted with
70% EtOH (10 l £ 3) at room temperature.
After removal of EtOH under reduced
pressure, the aqueous brownish syrup (1 l)
was suspended in H2O (l l) and then
partitioned with ethyl acetate (1 l £ 3) to
afford 63 g AcOEt-soluble fraction. The
AcOEt-soluble fraction was further frac-
tionated through a silica gel column,
eluting with gradient CHCl3/MeOH
(from 100:1 to 1:1) to yield fractions 1–6
according to TLC analysis. Fraction 4
(2.1 g) was applied to an ODS MPLC
column and eluted with MeOH–H2O
(20:80, 30:70, 40:60, each 500 ml) to
afford four subfractions (Fr. 4-1 to 4-4). Fr.
4-2 (MeOH–H2O, 387 mg) was chromato-
graphed by a Sephadex LH-20 column
eluting with MeOH/CHCl3 (50:50) and
purifed by a preparative RP-HPLC (ODS
column, 250 £ 20 mm) using MeOH/H2O
(25:75, 15.0 ml/min) as the mobile phase
to give 1 (40 mg, 21.20 min). Fraction 5
(1.2 g) was applied to an ODS MPLC
column eluting with MeOH/H2O (20:80,
30:70, 40:60, each 500 ml) to yield two
subfractions (Fr. 5-1 and Fr. 5-2). Sub-
fraction 5-2 (268 mg) was purified by
a preparative RP-HPLC (ODS column,
3.3.2. (3b,13S)-3-O-a-L-
Rhamnopyranosyl-13-O-[6-O-acetyl-b-D-
glucopyranosyl-(1 ! 4)-2-O-acetyl-a-L-
rhamnopyranosyl]-labda-8(17),14-diene(2)
White amorphous powder; C42H68O17;
23:3
½aꢀD 228.67 (c ¼ 0.031, MeOH); UV
(MeOH) lmax (log 1) 204 (3.56) nm; IR
(KBr) nmax 3430, 2937, 1745, 1631,
1
1060 cm21; for H NMR (500 MHz) and
13C NMR (125 MHz) spectral data, see
Table 1; ESI (neg.): m/z 843 [M 2 H]2;
HR-ESI-MS: m/z 879.4148 [M þ Cl]2
(calcd for C42H68O17Cl, 879.4145).
3.3.3. 3b-Hydroxy-13-epimanool (1a)
23:3
C20H34O2; ½aꢀD
þ6.5 (c ¼ 1.110,
1
CHCl3); H NMR (500 MHz): d 3.22 (dd,
J ¼ 11.5, 4.5 Hz, H-3), 5.88 (dd, J ¼ 17.5,
10.7 Hz, H-14), 5.02 (dd, J ¼ 10.6, 1.4 Hz,
H-15a), 5.18 (dd, J ¼ 17.5, 1.4 Hz, H-
15b), 1.25 (s, H-16), 4.51 (d, J ¼ 1.5, H-
17a), 4.81 (d, J ¼ 1.5, H-17b), 0.96 (s, H-
18), 0.74 (s, H-19), 0.66 (s, H-20); 13C
NMR (125 MHz): d 37.1 (C-1), 27.9 (C-2),
78.9 (C-3), 39.2 (C-4), 54.7 (C-5), 24.0 (C-
6), 38.2 (C-7), 148.1 (C-8), 57.0 (C-9),
39.6 (C-10), 17.9 (C-11), 41.3 (C-12), 73.6
(C-13), 145.3 (C-14), 111.7 (C-15), 27.7

Journal of Asian Natural Products Research
(C-16), 107.0 (C-17), 28.4 (C-18), 15.5 (C- Disclosure statement
19), 14.5 (C-20); ES-MS (neg.): m/z 306.
267
No potential conflict of interest was reported
by the authors.
3.4. Acid hydrolysis of 1 and 2
Funding
A solution of compound 1 or 2 (about
10.0 mg) in 1 M HCl (dioxane–H2O, 1:1,
1 ml) was heated at 958C for 2 h based on a
previous reference [11]. After cooling, the
reaction mixture was neutralized by
passage through an Amberlite IRA-93ZU
(Organo, Tokyo, Japan) column and
subjected to silica gel chromatography
using a gradient mixture of CHCl3–MeOH
(19:1; 9:1; 1:1) to give an aglycone
fraction and a sugar fraction (3.0 mg).
The aglycone fraction was purified by
silica gel CC eluting with hexane–Me2CO
(4:1) to give 3b-hydroxy-13-epimanool.
The solutions of those sugar parts obtained
as described above in pyridine (2 ml) were
added to L-cysteine methyl ester hydro-
chloride (1.5 mg) and kept at 608C for 1 h
each. Then trimethylsilyl imidazole
(1.5 ml) was added to the reaction mixture
and kept at 608C for 30 min. Supernatant
was subjected to GC analysis under the
following conditions: column temperature:
180/2808C, programmed increase: 38C/
min, carrier gas: N2 (1 ml/min), injector
and detector temperature: 2508C, injection
volume: 4 ml, split ratio: 1/50. Configur-
ation identification of D-glucose and L-
rhamnose was carried out by comparison
with its derivative’s retention time. Reten-
tion time in GC of standard D/L-glucose
and L/D-rhamnose derivatives was
19.450/19.856 and 15.849/16.312 min,
respectively. By comparing with the
retention time of the authentic sugars in
the form of derivatives under the same
condition, the sugar moieties of com-
pounds 1 and 2 were determined to be
D-glucose and L-rhamnose. All chemical
reagents and standard sugars were pur-
chased from Sigma-Aldrich Corporation
(St Louis, MO, USA).
The above research was made by the grant from
the Scientific Planning Project of the Applied
Basic Research of Yunnan Province [grant
number S2012FZ0005], the Developing Key
Subject of Ecology of Qujing Normal Univer-
sity and Qujing Kechuang Enterprise Incu-
bation Center.
References
[1] X.F. Jin and B.Y. Ding, Acta Bot. Yunnan.
30, 9 (2008).
[2] M.M. Li, K. Wang, J. He, L.Y. Peng,
X.Q. Chen, X. Cheng, and Q.S. Zhao, Nat.
Prod. Bioprospect. 3(2), 38 (2013).
doi:10.1007/s13659-012-0022-3.
[3] X. Fang, X. Lin, S. Liang, W.D. Zhang,
Y. Feng, and K.F. Ruan, J. Asian Nat.
Prod. Res. 14(12), 1175 (2012). doi:10.
1080/10286020.2012.739615.
[4] P.T. Zhang, J. He, G. Xu, L. Tu, N. Guo,
Y. Zhao, L.Y. Peng, and Q.S. Zhao, Acta
Bot. Yunnan. 31(2), 183 (2009). doi:10.
3724/SP.J.1143.2009.09020.
[5] M.M. Li, K. Wang, X.Q. Chen, L.Y. Peng,
X. Cheng, and Q.S. Zhao, Acta Bot.
Yunnan. 36, 559 (2009).
[6] H. Ageta, K. Iwata, and Y. Otake, Chem.
Pharm. Bull. 11(3), 407 (1963). doi:10.
1248/cpb.11.407.
[7] T. Kariyone and H. Ageta, Yakugaku
Zasshi. 79, 105 (1959).
[8] T. Kariyone and H. Ageta, Yakugaku
Zasshi. 79, 47 (1959).
[9] J. Hu, X.D. Shi, X. Mao, H. Li, J.G. Chen,
and J.Y. Shi, Phytochem. Lett. 8, 73
(2014). doi:10.1016/j.phytol.2014.02.003.
[10] J. Hu, X.Q. Chen, and Q.S. Zhao, Helv.
Chim. Acta 94(6), 1085 (2011). doi:10.
1002/hlca.201000337.
[11] S. Hara, H. Okabe, and K. Mihashi, Chem.
Pharm. Bull. 35(2), 501 (1987). doi:10.
1248/cpb.35.501.
[12] W.R. Abraham, Phytochemistry 36(6),
1421 (1994). doi:10.1016/S0031-9422
(00)89734-4.
[13] K. Shiojima, M. Suzuki, H. Aoki, and
H. Ageta, Chem. Pharm. Bull. 43(1), 5
(1995). doi:10.1248/cpb.43.5.