Three new monoterpene glycosides from Sibiraea laevigata (L.) Maxim

Article abstract of DOI:10.1016/j.phytol.2017.01.002

Three new compounds, 3,7-dimethy-7-methoxy-3-octene-5-one-1-O-β-D-glucopyranoside (1), 3,7-dimethy-7-methoxy-3(Z)-octene-5-one-1-O-β-D-glucopyranoside (2) and 3,7-dimethy-3-hydroxy-6-octene-5-one-1-O-β-D-glucopyranoside (3), together with fourteen known c

Full text of DOI:10.1016/j.phytol.2017.01.002

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Phytochemistry Letters 19 (2017) 176–180
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Short communication
Three new monoterpene glycosides from Sibiraea laevigata (L.) Maxim
Jian-Qiang Zhaoa,1, Yan-Ming Wangb,1, Qi-Lan Wanga, Yun Shaoa, Yan-Ping Shib,
a,
Li-Juan Meia, , Yan-Duo Tao
*
*
a
Key Laboratory of Tibetan Medicine Research of CAS and Key Laboratory for Tibetan Medicine of Qinghai Province, Northwest Institute of Plateau Biology,
Chinese Academy of Sciences, Xining 810008, PR China
Key Laboratory of Chemistry of Northwestern Plant Resources of CAS and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of
b
Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China
A R T I C L E I N F O
A B S T R A C T
Article history:
Three new compounds, 3,7-dimethy-7-methoxy-3-octene-5-one-1-O-b-D-glucopyranoside (1), 3,7-
Received 13 October 2016
Received in revised form 30 December 2016
Accepted 3 January 2017
Available online xxx
dimethy-7-methoxy-3(Z)-octene-5-one-1-O-b-D-glucopyranoside (2) and 3,7-dimethy-3-hydroxy-6-
octene-5-one-1-O- -glucopyranoside (3), together with fourteen known compounds (4–17) were
b-D
isolated from the leaves and shoots of S. laevigata. The structures of the new compounds were elucidated
on the basis of extensive spectroscopic analysis, including one- and two-dimensional NMR, as well as
Keywords:
Sibiraea laevigata
Monoterpene glycosides
mass spectral data. All isolates were evaluated for their
activities. The results demonstrated that 3,7-dimethyl-3(Z),6-ocatdien-5-one-1-O-
sitosteryl -glucoside (17) exhibited -glucosidase inhibitory effects with IC50 values of 220.0 and
113.0 M, respectively.
a-glucosidase inhibitory and antioxidant
b-D-glucoside (7) and
b
-
D
a
a
-Glucosidase inhibitory activity
m
Antioxidant activity
© 2017 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
1. Introduction
et al., 2010; Xia et al., 2011). Chemical constituent studies on S.
angustata reveal that monoterpene glycosides are its main
characteristic components, and these showed hypolipidemic and
weight-loss effects (Ito et al., 2009; Wang et al., 2013; Li et al., 2010,
2015). For example, sibiskoside, a monoterpene glucoside from S.
angustata, significantly affected the plasma levels of total
cholesterol, triglycerides, and blood glucose (Ito et al., 2009).
Previous bioactivity and phytochemical investigations of “Liucha”
focused on S. angustata. Only Lai et al. reported the composition
and antioxidant activity of the essential oil from aboveground parts
of S. laevigata (Lai et al., 2016). In addition, we previously reported
new monoterpenes from the EtOAc fraction of S. laevigata (Zhao
et al., 2016). To identify the chemical constituents of “Liucha”, we
conducted a phytochemical study on the n-BuOH fraction of S.
laevigata. Herein, we reported the separation procedure and the
structural elucidation of the monoterpene glycosides and other
compounds from S. laevigata. To explore the health benefits of
“Liucha” is used by Tibetan people to make tea as a treatment
for dyspepsia and epigastric fullness after eating. Liucha is
obtained from two species of Sibiraea (Rosaceae), S. laevigata
and S. angustata, which are endemic to the plateau region between
2000 and 4000 m above sea level. S. laevigata and S. angustata
mainly grow in Tibet, Qinghai and Gansu of China and S. angustata
is also distributed in the Sichuan and Yunnan provinces (Editorial
Committee of Chinese flora, 1974). The leaf shape of these two
species is similar to that of a willow leaf, which is the source of the
name of “Liucha” (willow tea). In addition, people have believed
that drinking “Liucha” can offer health benefits. It has been
commonly known that livestock such as cattle and goats have their
spirits raised and their skins and hair become silky after taking
“Liucha.” Meanwhile, they would also lose weight, suggesting a
potential anti-obesity effect of “Liucha”. Thus, Liu and co-workers
carried out experiments to explore the anti-obesity effect of the
water extract of S. angustata. The results showed that the water
extract of S. angustata can not only regulate lipid metabolism in
obese rats but also inhibit the increase of rats body weight (Wang
“Liucha”,
a-glucosidase inhibitory and antioxidant activities of all
of the isolates were also tested.
2. Results and discussion
Multistep column chromatography separation of the n-BuOH
layer from the 95% ethanol extract of the young leaves and shoots
of S. leavigata led to the isolation of seventeen compounds (1–17),
including three new monoterpene glycosides (1–3) (Fig. 1).
* Corresponding authors.
E-mail addresses: meilijuan111@163.com (L.-J. Mei), soqiang.happy@163.com
(Y.-D. Tao).
1
The authors contributed equally to this paper.
http://dx.doi.org/10.1016/j.phytol.2017.01.002
1874-3900/© 2017 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.

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J.-Q. Zhao et al. / Phytochemistry Letters 19 (2017) 176–180
177
Fig. 1. Chemical structures of compounds 1–3.
Compound 1 was obtained as light yellow gum with a chemical
formula defined as C17H30O8 according to HRESIMS. The IR
spectrum of compound 1 showed absorption bands for hydroxy
(3400 cmꢀ1) group, carbonyl (1679 cmꢀ1) group conjugated with
a Z-isomer of 1 (Maldonado et al., 1998). A NOESY experiment
(Fig. 2) also confirmed the result through the correlations between
H-4 and H-10. Thus, compound 2 was identified as 3,7-dimethy-7-
methoxy-3(Z)-octene-5-one-1-O-b-D-glucopyranoside. The struc-
double bond, and olefinic (1614 cmꢀ1
)
group. The 1H NMR
tures of compounds 1 and 2 showed a high resemblance to the
structure of sibiraglycoside K, which was isolated from the other
source of “Liucha”, S. angustata. The only difference between the
structures was an additional methoxy group in 1 and 2 (Yao et al.,
2016).
Compound 3 was also obtained as light yellow gum and was
assigned a molecular formula of C16H28O8, which was determined
through HRESIMS to be the same as sibiraglycosides I (4) and J (5).
The IR spectrum of 3 also showed presence of a hydroxy
(3408 cmꢀ1) group, conjugated carbonyl (1672 cmꢀ1) group, and
olefinic (1613 cmꢀ1) group. Inspection of the 13C NMR spectrum of
3 revealed the presence of a carbonyl group (dC 202.4), a double
bond (dC 157.3 and 126.4), an oxygen-bearing quaternary carbon
spectrum showed the presence of protons in the double bond
[
[
dH 6.32 (1H, s), H-4], methoxy [dH 3.25 (3H, s)] group, vinyl methyl
dH 2.15 (3H, s)] group, gem-dimethyl [dH 1.26 (6H, s)] group and an
anomeric proton [dH 4.3 (1H, d, J = 7.8 Hz)]. The 13C NMR spectrum
suggested the presence of seventeen carbon signals, and these
signals were further interpreted through an HSQC spectrum,
indicating the presence of a carbonyl, a trisubstituted double bond,
three methylene groups, including one oxygen-bearing methylene,
three methyls, a methoxy group, and six carbons attributed to a
sugar moiety. The proton sequence of (O)-CH2(H-1)-CH(H-2) and
the sugar unit were confirmed by the corresponding 1H-1H COSY
cross-peaks. The sugar moiety was identified as
by acid hydrolysis analysis, by comparison of the 13C NMR data
with those of -glucopyranose reported in the literature (Yao
b-D-glucopyranose
(dC 72.5), an oxymethylene (dC 67.0), and carbon signals due to a
b
-D
sugar unit. In addition, the NMR spectrum of 3 closely resembled
those of sibiraglycoside I (4) and J (5), indicating that 3 was an
isomeride of 4 and 5 (Yao et al., 2016). The structure of 3 was
deduced through 2D NMR correlations. The hydroxyl group was
attached at C-3, which was supported by HMBC correlations from
H-1 to C-3 and C-10. HMBC correlations from the methyls (dH 2.12
and 1.91) and H-4 to the tertiary carbon (dC 126.4) of the double
bond, and from the olefinic proton and H-4 to the carbonyl
supported the presence of an isobutenyl group that was connected
with the carbonyl. Using this data, along with the correlations from
the singlet methyl to C-2 and C-4, the planar structure of 3 was
et al., 2016) and by the large coupling constant of the anomeric
proton. The abovementioned structural units were further con-
structed through inspection of the HMBC spectrum. The correla-
tions from H-1 to C-3, from H-2 and H-6 to C-4, from H-8 and H-9 to
C-6, from H-4 and H-6 to C-5, and from H-10 to C-2 were consistent
with the structure of an acyclic monoterpene, as shown in Fig. 2.
The HMBC correlation from H-10 to C-1 illustrated that the
b-D-
glucopyranose was located at C-1, and the correlation from the
methoxy protons to C-7 confirmed that the methoxy group was
located at C-7. Hence, the planar structure of 1 was determined to
be 3,7-dimethy-7-methoxy-3-octene-5-one-1-O-
b
-
D
-glucopyra-
established as 3,7-dimethy-3-hydroxy-6-octene-5-one-1-O-b-D-
noside (Fig. 1). The identification of the Z/E isomer for compound
1 was made from a NOESY experiment (Fig. 2). NOESY correlations
between H-2 and H-4 confirmed the E-configuration of 1.
glucopyranoside. It was noted that the 13C NMR spectra of
compound 3 recorded at room temperature exhibited the doubling
of some signals. This observation could result from the R/S
configuration of the hydroxyl group at C-3. Because there was no
large, sterically hindering group on the hydroxyl, the molecular
could be very flexible and thus we could not separate the two
enantiomorphs. Thus, the structure of compound 3 could be only
elucidated as a planar structure.
Compound 2 showed a pseudo-molecular ion [M+Na]+ at m/z
385.1872 in the HRESIMS spectrum consistent with the molecular
formula C17H30O8, which is the same as that of 1. In addition, the
UV, IR, and NMR spectral features of 2 were similar with those of 1.
Thus, 2 was identified as an isomer of 1. Compared with the same
proton in 1, the H-2 proton in 2 was shielded. Conversely, the
carbon of C-2 was deshielded in 2, indicating that compound 2 was
The other known compounds were identified as sibiraglyco-
side I (4) (Yao et al., 2016), sibiraglycoside J (5) (Yao et al., 2016),
Fig. 2. Key HMBC, NOESY, and 1H-1H COSY correlations of compounds 1–3.

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178
J.-Q. Zhao et al. / Phytochemistry Letters 19 (2017) 176–180
3,7-dimethyl-3(E),6-ocatdien-5-one-1-O-
b
-D-glucoside
(6)
3.3. Extraction and isolation
(Takeda et al., 1990), 3,7-dimethyl-3(Z),6-ocatdien-5-one-1-O-
-glucoside (7) (Takeda et al., 1990), 3,7-dimethyl-2(E),6-
ocatdien-5-one-1-O- -glucoside (8) (Takeda et al., 1990),
sibiraglucoside G (9) (Wang et al., 2013), sibiraglucoside H (10)
(Wang et al., 2013), sibiskoside (11) (Ito et al., 2009), kodemario-
side A (12) (Yoshida et al., 2010), kodemarioside E (13) (Yoshida
et al., 2010), cimicifugolide A (14) (Ma et al., 2013), (3S)-4-
b
-D
The air-dried and powdered young leaves and shoots of S.
leavigata (9.5 kg) were extracted with 95% ethanol under reflux.
After removal of the organic solvent, the ethanol extract was
suspended in water and then extracted successively with EtOAc
and n-BuOH. The n-BuOH fraction (300 g) was subjected to a silica
gel CC, eluting with a CHCl3-MeOH-H2O gradient system (9:1:0.1–
6:4:1, v/v/v), to give four fractions (Fr. 1–4). Fr. 1 (80 g) was
separated by MCI gel CHP 20P CC, eluting with a gradient of
aqueous MeOH (elution with 10%-90% of methanol) to yield
fractions 1a–1c. Fraction 1a was subjected to a silica gel CC, eluting
with an isocratic system of CHCl3-MeOH-H2O (9:1:0.1) to yield
compounds 16 (6 mg) and 17 (51 mg). Fraction 1b was further
subjected to prep-HPLC using column DAC-HB50 to obtain
fractions 1b-1 and 1b-2. Compounds 1 (22 mg) and 2 (51 mg)
were obtained from fraction 1b-1 through purification on an
XCharge C18 column (elution with 14% MeCN with 0.2% FA for
20 min, with a flow rate of 15 mL/min). Using the same purification
method, compound 8 (33 mg) was obtained from fraction 1b-2.
Fraction 1c was purified on an XCharge C18 column (elution with
10–40% MeCN with 0.2% FA for 60 min with a flow rate of 15 mL/
min) to yield compounds 14 (11 mg) and 15 (7 mg). Fr. 2 (54 g) was
separated by MCI gel CHP 20P CC, eluting with 90% aqueous MeOH
to give Fr. 21 (27 g), followed by preparative HPLC on a DAC-HB50
column (elution with 30–50% MeOH with 0.2% FA for 60 min with a
flow rate of 60 mL/min) to yield fractions 2a and 2b. Fraction 2b
was further purified on an XCharge C18 column (elution with 10–
30% MeCN with 0.2% FA for 60 min with a flow rate of 15 mL/min) to
yield compounds 4 (22 mg) and 5 (51 mg). Fr. 3 (37 g) was purified
by prep-HPLC using a DAC-HB50 column (elution with 30–60%
MeOH with 0.2% FA for 60 min with a flow rate of 60 mL/min) to
give nine fractions (Fr. 3a-3i). Fraction 3b was purified on an XAqua
C-18 column (elution with 10–30% MeCN with 0.2% FA for 50 min
with a flow rate of 15 mL/min) to get compounds 3 (22 mg), 6
(13 mg), and 7 (15 mg). Using the same purification method,
compounds 11 (37 mg) and 13 (17 mg) were obtained from
fractions 3d and 3f, respectively. Fraction 3 h was purified on an
XAqua C-18 column (elution with 23% MeCN with 0.2% FA with a
flow rate of 15 mL/min) to yield compound 12 (25 mg). Compounds
9 (13 mg) and 10 (21 mg) were obtained from the purification of
fraction 3i on an XCharge C-18 column (elution with 10–30% MeCN
with 0.2% FA for 40 min with a flow rate of 15 mL/min).
b-D
a
-hydroxy-3-(2-hydroxyethyl-idene)-5-
enyl)-dihydrofuran-2-one (15) (Ma et al., 2013), 4-(
anosyloxy)-3-(3-methyl-2-butenyl) benzoic acid (16) (Bilia et al.,
1993), and sitosteryl -glucoside (17) (Sakakibara et al., 1983)
b
-(2-methyl-prop-1-
b-D-glucopyr-
b-D
through comparison of NMR data with those reported in the
literature.
All compounds isolated from S. laevigata were evaluated for
their a-glucosidase inhibitory effect and antioxidant activity using
ABTS and DPPH radical scavenging assays. The results showed that
compounds 9, 10, 12, and 13 displayed radical scavenging effect
against ABTS radical with SC50 values of 25.3, 27.3, 39.3 and
31.6
effect against DPPH radical with SC50 values of 28.3, 25.1, 31.6, and
30.8 M, respectively. Compounds 7 and 17 exhibited -glucosi-
dase inhibitory effects with IC50 values of 220.0 and 113.0 M,
respectively. Acarbose was used as positive control with the IC50
values of 182.9 M.
mM, respectively. They also showed potent radical scavenging
m
a
m
m
3. Materials and methods
3.1. General experimental procedures
IR spectra were recorded on
a
Bruker Tensor-27 FT-IR
spectrometer (Bruker Corporation, Germany) with KBr pellets.
Optical rotations were acquired on Jasco P-1020 automatic
polarimeter (Jasco Inc, Easton, MD, USA). UV spectra were
measured on a Shimadzu UV2401A ultraviolet-visible spectro-
photometer (Shimadzu Corporation, Kyoto, Japan). HRESIMS
were recorded on an API Qstar Pulsa LC/TOF spectrometer.
NMR spectra were obtained on
spectrometer, using TMS as an internal standard. Chemical shifts
were reported in units of (ppm), and coupling constants (J) were
a Bruker Avance III-600
d
expressed in Hz. Column chromatography (CC) was carried out
over silica gel (200–300 mesh, Qingdao Haiyang Chemical Co.,
Qingdao, China) and MCI gel CHP 20P (75–150 mm, Tokyo, Japan).
Pre-coated silica gel plates (Qingdao Haiyang Chemical Co.,
Qingdao, China) were used for TLC. TLC detection was done under
UV light (254 nm and 365 nm) and by spraying the plates with a
10% sulfuric acid ethanol solution followed by heating. An Agilent
series 1200 (Agilent Technologies) was used for analytical HPLC.
Preparative HPLC (prep-HPLC) was done on a Hanbon liquid
chromatograph (Hanbon Sci & Tech, Jiangsu, China). Prep-HPLC
3.4.
a-Glucosidase assay
The
Musa et al. (Yilmazer-Musa et al., 2012). 0.26 U of
from Saccharomyces cerevisiae were diluted with 0.1 M phosphate
buffer consisting of Na2HPO4 and KH2PO4 (pH 6.5). The 25 L test
compound and 25 -glucosidase were preincubated in 96-well
plates at 37 ꢃC for 15 min. The reaction was initiated by adding
50 L of 0.3125 mM pNPG as substrate. The plate was incubated for
an additional 15 min at 37 ꢃC, followed by the reaction being
stopped by adding 50 L of 0.2 M Na2CO3. All test compounds were
a
-glucosidase inhibition assay was adapted from Yilmazer-
a
-glucosidase
m
columns DAC-HB50, XAqua C-18 and XAmide (10
m
m, 100 Å,
mL a
250 mm ꢁ 20 mm, Acchrom, Beijing, China) were used in for
compound separation. 1,1-Diphenyl-2-picrylhydrazyl free radical
(DPPH, purity >97%), 2,20-Azinobis-(3-ethylbenzthiazoline-6-sul-
m
fonate) (ABTS, purity >98%) and p-nitrophenyl
a-D
-glucopyrano-
m
side (pNPG, purity ꢂ98%) were obtained from Sigma (St. Louis, MO,
dissolved in DMSO, then diluted with buffer. The reaction was
monitored by the change of absorbance at 405 nm using a
Dimension RxL Max clinical chemistry system Enspire MP150
(Siemens Healthineers).
USA).
3.2. Plant material
The young leaves and shoots of S. leavigata were collected in
Huzhu City, Qinghai Province, China in August 2011 and were
identified by Professor Lijuan Mei. A voucher specimen (No.
0317608) was deposited in the Key Laboratory of Tibetan Medicine
Research (Northwest Institute of Plateau Biology, Chinese Academy
of Science).
3.5. DPPH radical scavenging assay
The DPPH assay was performed as described in previous paper
(Gao et al., 2010), and ascorbic acid was used as the positive
control. Reaction mixtures containing an ethanol solution of
200 mM DPPH (100 mL) and 2-fold serial dilutions of the sample

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J.-Q. Zhao et al. / Phytochemistry Letters 19 (2017) 176–180
179
Table 1
1H NMR and 13C NMR spectroscopic data of compounds 1–3 (CD3OD).
Pos.
1
1
2
3
dH ppm, mult. (J in Hz)
dC
dH ppm, mult. (J in Hz)
dC
dH ppm, mult. (J in Hz)
dC
3.75, m
68.3
3.71, m
69.2
3.69, m
67.0
4.08, dd (9.8, 6.5)
2.50, t (6.5)
4.00, dd (16.5, 7.1)
2.91, td (7.1, 1.8)
4.06, ddt (13.8. 9.9, 6.8)
1.89, m
2
3
4
41.9
156.4
127.4
35.3
157.4
127.8
41.9
72.52/72.49
50.02/50.14
6.32, s
2.64, s
6.29, s
2.62, s
2.66, dd (15.0, 7.1)
2.68, dd (15.0, 3.1)
5
6
7
8
201.8
54.2
76.2
25.9
25.9
19.8
104.4
75.1
78.1
71.7
78.0
62.8
201.4
54.1
76.1
25.7
25.7
26.5
104.3
75.1
78.3
71.8
78.0
62.9
202.4
126.4
157.30/157.26
20.8
27.7
27.75/27.62
104.1
75.1
78.1
71.6
77.9
6.23, s
1.26, s
1.26, s
2.15, s
4.30, d (7.8)
1.26, s
1.26, s
1.99, s
2.12, s
1.91, s
1.25/1.26, s
4.30, d (7.8)
3.15, m
9
10
10
20
30
40
50
60
4.31, d (7.8)
3.18, t (8.4)
3.38, m
3.30, overlapped
3.29, overlapped
3.69, dd (12.3, 2.7)
3.88, d (12.3)
3.24, s
3.18, t (8.2)
3.37, dd (12.1, 5.6)
3.29, overlapped
3.30, overlapped
3.69, dd (11.5, 2.7) 3.89, d (11.5)
3.35, m
3.26, overlapped
3.26, over;apped
3.66, m
62.7
3.86, d (13.9)
OCH3
3.25, s
49.9
49.8
solutions were placed in a 96 well microplate and incubated at
room temperature for 30 min. After incubation, the absorbance
was read at 517 nm and the scavenging activity was determined by
following equation: % scavenging activity = [Ablank ꢀ Asample]/Ablank
ꢁ 100. The SC50 value was obtained and was defined as the
concentration of sample required to scavenge 50% of DPPH
radicals.
3.8. Spectroscopic data
3,7-dimethy-7-methoxy-3(E)-octene-5-one-1-O-
anoside (1), Light yellow gum; [
]25.7 D: ꢀ25.14 (c 0.012, CH3OH);
IR (KBr) vmax (cmꢀ1): 3400, 2973, 2933, 1679, 1614, 1380 and 1077;
b-D-glucopyr-
a
UV (MeOH) lmax nm (log e
): 195 (3.60), 242 (3.96), 387 (1.47); 1H
NMR and 13C NMR data see Table 1; HRESIMS: m/z 385.1825 [M
+Na]+ (calcd. 385.1833).
3.6. ABTS+ radical scavenging assay
3,7-dimethy-7-methoxy-3(Z)-octene-5-one-1-O-
anoside (2), Light yellow gum; [
]25.7 D: ꢀ47.00 (c 0.02, CH3OH);
IR (KBr) vmax (cmꢀ1): 3420, 2972, 2931, 1679, 1611, 1379 and 1075;
b-D-glucopyr-
a
The ABTS radical cation decolorization assay was carried out as
previously reported (Re et al.,1999). ABTS was dissolved in water to
make a 7 mM solution. The ABTS radical cation (ABTSꢄ+) was
produced by reacting ABTS stock solution with 2.45 mM potassium
persulfate (final concentration) and allowing the mixture to stand
in the dark at room temperature for 12–16 h before use. The ABTSꢄ+
solution was diluted with ethanol to give an absorbance of
0.700 ꢅ 0.020 at 734 nm. Aliquots of the solutions containing the
UV (MeOH) lmax nm (log e
): 203 (3.79), 240 (3.87), 323 (2.34); 1H
NMR and 13C NMR data see Table 1; HRESIMS: m/z 385.1827 [M
+Na]+ (calcd. 385.1833).
3,7-dimethy-3-hydroxy-6-octene-5-one-1-O-
b-D-glucopyra-
noside (3), Light yellow gum; IR (KBr) vmax (cmꢀ1): 3408, 2971,
2927, 2721, 1672, 1631, 1379 and 1078; UV (MeOH) lmax nm (log e):
195 (3.67), 240 (3.98), 323 (2.50); 1H NMR and 13C NMR data see
test compounds (20
culture plates. And then 180
m
L) were added into each well of 96-well cell
Table 1; HRESIMS: m/z 371.1653 [M+Na]+ (calcd. 371.1676).
m
L of the diluted ABTSꢄ+ solution
(A734nm = 0.700 ꢅ 0.020) was added to the plates. The absorbance
reading was taken after mixing for 5 min. The optical density was
measured at 734 nm on a microplate reader. All determinations
were carried out in triplicate. The percentage inhibition of
absorbance at 734 nm is calculated, and the IC50 value was
calculated.
Acknowledgements
This work was supported by the Significant Science and
Technological Project of Qinghai Province (2014-GX-A3A) and
the Project of Discovery, Evaluation and Transformation of Active
Natural Compounds, Strategic Biological Resources Service Net-
work Programme of Chinese Academy of Sciences (ZSTH-027).
3.7. Acid hydrolysis of compounds 1–3
Appendix A. Supplementary data
The acid hydrolysis of compounds 1–3 was performed as
described previously (Zhao et al., 2013). Compound 1–3 (5 mg) in
2 M HCl (2 mL) were heated at 80ꢃ C using a water bath for 8 h. The
reaction mixture was partitioned between CHCl3 and H2O three
times. The aqueous layer was neutralized by base and then
concentrated to dryness to give a saccharide residue. The solution
of the sugar residue together with the standard, D/L glucose, in
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
phytol.2017.01.002.
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supernatants (4
ides of compounds 1–3 were determined to be
comparing of the retention times of their corresponding deriva-
tives with those of standard D/L glucose.
m
L) were then analysed by GC. The monosacchar-
-glucose by
D