Novel highly oxygenated and B-ring-seco-ent-diterpene glucosides from the seeds of Prinsepia utilis

Article abstract of DOI:10.1016/j.tet.2015.10.055

Two novel highly oxygenated and one rare B-ring cleave ent-kaurane diterpene glucosides, named prinsoside A (1), B (2) and C (3), respectively, were isolated from an ethanolic extract of the seeds of Prinsepia utilis. To our knowledge, prinsoside B (2) was the most highly oxygenated ent-kaurane diterpene glucoside discovered in nature. Their structures were elucidated by various spectroscopic methods including 1D, 2D NMR and HR-ESI-MS analyses, with the absolute configurations clarified by CD measurements on their corresponding diterpenoid aglycones obtained by enzymatic hydrolysis with β-glucosidase. Compounds 1-3 exhibited weak α-glucosidase inhibitory activities.

Full text of DOI:10.1016/j.tet.2015.10.055

Tetrahedron 71 (2015) 9415e9419
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Novel highly oxygenated and B-ring-seco-ent-diterpene glucosides
from the seeds of Prinsepia utilis
,
b
,
,
b
,
,
a,
Qiao Zhang a y, Hong-Xin Liu a y, Hai-Bo Tan a , Sheng-Xiang Qiu
*
*
a Program for Natural Product Chemical Biology, Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical
Garden, Chinese Academy of Sciences, Guangzhou, 510650, People’s Republic of China
b University of Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two novel highly oxygenated and one rare B-ring cleave ent-kaurane diterpene glucosides, named
prinsoside A (1), B (2) and C (3), respectively, were isolated from an ethanolic extract of the seeds of
Prinsepia utilis. To our knowledge, prinsoside B (2) was the most highly oxygenated ent-kaurane di-
terpene glucoside discovered in nature. Their structures were elucidated by various spectroscopic
methods including 1D, 2D NMR and HR-ESI-MS analyses, with the absolute configurations clarified by CD
Received 8 September 2015
Received in revised form 16 October 2015
Accepted 20 October 2015
Available online 21 October 2015
measurements on their corresponding diterpenoid aglycones obtained by enzymatic hydrolysis with
glucosidase. Compounds 1e3 exhibited weak -glucosidase inhibitory activities.
Ó 2015 Elsevier Ltd. All rights reserved.
b-
Keywords:
Rosaceae
Prinsepia utilis
Diterpene glucoside
Prinsosides A, B and C
a
1. Introduction
sodiated molecular ion [MþNa]þ determined at m/z 583.2706
(calcd for C27H44O12Na, 583.2833) in the high-resolution electro-
spray ionization mass spectroscopy (HR-ESI-MS). In the 1H NMR
spectrum, a typical anomeric proton was observed at dH 4.33 (1H, d,
J¼7.8 Hz), in tandem with a distinct signal set assignable to a hexose
unit (dC 105.1, 78.0, 77.8, 75.2, 71.6 and 62.7) observed in the 13C
NMR spectrum, implying the presence of a glucopyranosyl moiety
by a comparison with the corresponding data reported in the lit-
Genus Prinsepia (Rosaceae) encompasses five species, which are
mainly distributed in the Himalayan mountain district with four
species growing in China,1 among which Prinsepia utilis Royle and
Prinsepia sinensis Oliver ex Bean are used, both as traditional Chinese
herbal medicines2 to treat a variety of disorders such as skin prob-
lems, fracture, rheumatism and inflammation.3 A few phytochemical
studies have been done on the stems and leaves of Prinsepia which
revealed the presence of triterpenoids, hemiterpenes, flavonoids and
hydroxynitrile glucosides.4e7 However, little is known on the
chemical constituents of the fruits besides essential oils. Thus, we
conducted an extensive phytochemical investigation on the seeds of
P. utilis, which led to the isolation of two new highly oxygenated ent-
kaurane diterpene glucosides (1 and 2), and one rarely B ring-cleaved
kauranoid diterpene glucoside (3). Hydrolysis of the kaurane diter-
pene glucosides gave rise to three new kaurane diterpenoid agly-
cones. This report describes the structural characterization of the
erature.8 Moreover, a
b-D-configuration was also suggested based
on the relatively large coupling constant of the anomeric proton
(J¼7.8 Hz), the chemical shift of the anomeric carbon (dC 105.1), and
biogenesis consideration.
In the 13C NMR data (Table 1), a total of 27 carbon signals, be-
sides the signals arising from the glucopyranosyl moiety, were
observed, including three methyls (dC 51.9, 32.6, 17.0), seven
methylenes (dC 74.0, 41.6, 40.9, 34.7, 27.1, 20.0, 19.2), six methines
(dC 82.7, 79.1, 72.1, 52.5, 49.3, 45.0), four quaternary carbons (dC 80.7,
52.3, 45.5, 41.8), and one carbonyl carbon (dC 180.2). A closer in-
new compounds and their
a-glucosidase inhibitory activities.
spection revealed that the 13C NMR data was similar to that re-
ported for the known diterpene glucoside ent-6a,7a,16b,17-
tetrahydroxykauran-19-oate 16-O-b-D
-glucopyranoside.9 The only
2. Results and discussion
difference is that compound 1 has one more hydroxylated 13C sig-
nal, which also accounts for its mass spectral data.
Prinsoside A (1) was obtained as a white needle crystal. The
The five hydroxyl groups were ascribed to C-6, 7, 15, 16 and 17
(Fig. 1), respectively, through interpretation of the COSY and HMBC
spectral data. Briefly, the analysis of 1He1H COSY correlations of 1
established three coupling segments as of C-1/C-2/C-3 (a), C-5/C-6/
molecular formula was established to be C27H44O12, based on the
* Corresponding authors. Tel./fax: þ86 20 37081190; e-mail address: sxqiu@scbg.
ac.cn (S.-X. Qiu).
y
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.tet.2015.10.055
0040-4020/Ó 2015 Elsevier Ltd. All rights reserved.

9416
Q. Zhang et al. / Tetrahedron 71 (2015) 9415e9419
Table 1
10; H-7 and C-15; Me-18 and C-3, C-4, C-5; as well as Me-20 and C-
1H and 13C NMR data of 1e3
1, C-5, C-9, C-10. Furthermore, the conclusive evidence supporting
the presence of hydroxyl groups at C-6, C-7, C-15 and C-16 was
derived from the HMBC correlations between H-6 and C-4, C-8, C-
10; H-7 and C-15; H-15 and C-7, C-17. In addition, the observation of
HMBC correlation between H-10 and C-17 verified the location of
the glucopyranosyl moiety at C-17. The relative configuration of 1
was deduced by the analysis of ROESY correlations (Fig. 3). The key
ROESY correlations between H-6 and H-7 indicated that the two
Position
1
2
3
dC
dH (J in Hz) dC
dH (J in Hz)b dC
dH (J in Hz)b
a
b
a
a
1a
1b
2a
2b
3a
3b
4
41.6 1.84, m
0.93, m
19.2 1.63, m
1.47, m
40.9 2.30, m
1.13, m
45.5
52.5 1.95, d,
(11.1)
72.1 4.23, dd,
(11.1, 2.2)
79.1 3.80, m,
(2.2)
52.3
49.3 1.59, m
41.8
20.0 1.80, m
1.43, m
27.1 1.70, m
1.49, m
45.0 2.18, m
34.7 1.80, m
1.64, m
82.7 3.39, m
35.0 1.75, m
1.59, m
19.0 1.47, m
30.5 1.85, m
1.57, m
38.3 2.13, m
1.27, m
32.1 2.05, m
1.31, m
42.4
protons were cofacial and arbitrarily assigned as
while H-5 was assigned as -orientation. The correlation between
H-6 and H-7, H-12b, Me-20 and between H-7 and H-6, H-12b, H-
14b confirmed Me-20 to be -oriented. In addition, the ROESY
correlation between H-5, H-1 and H-15 and between H-15 with H-
17b revealed that the OH group at C-15 and C-16 was of -orien-
tation. Thus, 1 was shown to be a glucoside with methyl ent-
,7 ,15 ,16 ,17-pentahydroxykauran-19-oate as the aglycone,
a-orientation,
44.9
b
5
50.6 1.91, d
(11.3)
71.4 4.28, d,
55.8 1.93, m
6
7
100.8 5.67, s
207.3 9.80, s
a
(11.3, 1.9)
79.1 3.77, d,
(1.9)
a
8
9
10
52.7
49.5 1.87, m
46.8
20.8 2.96, m
1.42, m
27.4 1.60, m
1.52, m
45.1 2.16, m
35.6 1.80, m
1.60, m
59.6
49.0 2.09, m
40.6
20.7 1.84, m
1.77, m
26.3 1.78, m
1.66, m
46.5 2.30, m
32.1 2.41, m
1.71, m
47.5 1.86, m
1.60, m
6a
a
b
b
which linked to a glucosyl unit by a glycosidic bond via the hydroxyl
at C-17.
11a
11b
12a
12b
13
14a
14b
15a
15b
16
82.7 3.65, s
82.5 3.70, m
80.7
81.0
81.2
17a
74.0 4.14, d,
(10.3)
74.0 4.12, d,
(10.3)
74.1 4.24, d,
(10.3)
17b
3.63, d,
3.64, d,
3.60, d,
(10.3)
(10.3)
(10.3)
18
19
32.6 1.46, s
180.2
32.3 1.45, s
180.0
30.6 1.40, s
184.3
20
OMe
Glc-10
17.0 0.87, s
51.9 3.72, s
105.1 4.33, d,
(7.8)
13.2 0.98, s
51.2 3.60, s
105.1 4.33, d,
(7.8)
22.6 0.73, s
105.2 4.32, d,
(7.8)
Glc-20
Glc-30
Glc-40
Glc-50
Glc-6’a
77.8 3.40, m
75.2 3.26, m
71.6 3.30, m
78.0 3.30, m
62.7 3.89, br d,
(11.1)
77.9 3.39, m
75.2 3.25, m
71.6 3.31, m
78.0 3.30, m
62.7 3.89, br d,
(11.1)
77.9 3.38, m
75.3 3.24, m
71.7 3.29, m
78.1 3.30, m
62.8 3.89, dd,
(1.3, 11.7)
Fig. 2. Key 2D NMR correlations of 1e3.
Glc-6’b
3.67, dd,
3.67, dd,
3.68, br d,
(1.3, 11.1)
(1.3, 11.1)
(11.7)
a
Measured in CD3OD at 125 MHz.
In CD3OD at 500 MHz.
b
Fig. 3. Key ROESY correlations of 1and 2.
To determine the absolute configuration of the glucosyl linkage,
efforts were made with an aim to obtain the free aglycone and
glycone moieties. Unexpectedly, 1 (as well as 2 and 3) was found to
be resistant to acidic hydrolysis with mineral acids even at high
concentration and temperature (refluxed in 6 N HCl for 6 h), pre-
sumably due to the steric hindrances of this rigid molecule. How-
ever, the enzymatic hydrolysis underwent readily by soluble
b-
glucosidase to give -glucose as determined by TLC comparison
D
with an authentic sample, which further confirmed the
figuration of glucosyl moiety.
b-D-con-
Furthermore, the enzymatic hydrolysate was subjected to
chromatographic purification to afford an aglycone (1a), which was
characterized as methyl-6b,7b,15a,16a-tetrahydroxykauran-19-
Fig. 1. Structures of compounds 1e3.
oate by spectroscopic methods (NMR and ESI-MS). The CD spec-
trum of both 1 and 1a displayed a similar negative (lmax 223 nm)
Cotton effect to that of ent-kaur-16-en-19-oic acid which also
exhibited a negative (lmax 216 nm) Cotton effect,10 suggesting 1
possessed an ent-kaurane molecular skeleton. Taken together, 1
C-7 (b) and C-12/C-13/C-14 (c) (Fig. 2). In the HMBC spectrum, the
correlations between H-14 and C-9, C-12, C-15, C-16; H-11 and C-8,
C-13; H-13 and C-11, revealed the presence of a bicyclo [3.2.1] oc-
tane system (rings C and D). Rings A and B were assigned based on
the HMBC correlations between H-1 and C-20, C-3; H-6 and C-8, C-
was
concluded
as
methyl
ent-6a,7a,15b,16b,17-
pentahydroxykauran-19-oate 17-O-
b
D
-glucopyranoside.

Q. Zhang et al. / Tetrahedron 71 (2015) 9415e9419
9417
Prinsoside B (2) was obtained as a yellow gum with the mo-
lecular formula C27H44O13 based on the quasi-molecular ion
[MþNa]þ measured at m/z 599.2651 (calcd for C27H44O13Na,
599.2782) by positive mode HR-ESI-MS. The high similarity be-
tween the 1H and 13C NMR spectra of 1 and 2 (Table 1) indicated
that 2 is also an ent-kaurane diterpenoid glucoside, with a structure
very similarly to 1 except for the presence of an extra hydroxyl
group. A closer inspection of the 13C NMR spectra of 2 in compar-
ison to 1 revealed that the downfield shifts were observed at C-1
(from dC 41.6 to 82.7), C-2 (from dC 18.4 to 30.5) and C-10 (from dC
40.6 to 46.8 ppm), suggesting that the hydroxyl group of compound
2 was located at C-1. The HMBC spectrum provided further evi-
dence for the conclusion above, based on several key correlations
(Fig. 2.) such as H-1 to C-3 and to C-5, and Me-20 to C-1. The relative
br s/dC 104.7 in coccinin], as well as on C-17 [dH 4.24, d, J¼10.3 Hz,
3.60, d, J¼10.3 Hz/dC 74.1 in 3; while dH 6.30, 2H, br s/dC 65.7 in
coccinin], suggesting that the glucosyl moiety in 3 was linked to C-
17, rather than to C-6 as in coccinin, which was further confirmed
by the conclusive correlation between anomeric proton (dH 4.32, d)
and the methylene signal (dC 74.1) in the HMBC of 3 (Fig. 2). Thus,
the structure of 3 should be a diterpenoid glucoside composed of an
unusual B-ring seco-kaurane framework. Similarly to compounds 1
and 2, the absolute configuration of the kaurane diterpene and the
glucosyl moiety was determined by means of enzymatic hydrolysis
and CD measurements. Enzymatic hydrolysis of 3 with
dase afforded -glucose and the aglycone 3a, which was shown to
be ent-6 ,16 ,17-trihydroxy-7,19-dioxo-6,19-epoxy-6,7-seco-kaur-
b-glucosi-
D
b
a
ane based on NMR and HR-ESI-MS spectral analysis as well as the
configuration of the hydroxyl at C-1 was deduced to be
based on ROESY spectrum wherein conclusive correlation perks
were observed between H-1 and H-5, H-9.
a
-oriented
negative Cotton effect (ꢀ3.74, lmax 233 nm) observed in the CD
spectrum. Taken together, 3 was elucidated as ent-6b,16a,17-
trihydroxy-7-oxokauran-19,6-olide 17-O-b D-glucopyranoside.
In analogy with 1, compound 2 was subjected to enzymatic hy-
drolysis with b-glucosidase to afford D-glucose and the aglycone 2a.
All of the NMR data was accountable with the assigned structure and,
as expected, 2a showed a negative Cotton effect (lmax 226 nm),
confirming that 2 (2a) had an ent-kaurane backbone. Therefore, 2 was
A plausible biogenetic pathway was proposed as shown in
Scheme 1 to account for the biosynthesis of prinsoside AeC in this
plant. Briefly, ent-kaur-16-en-19-oic acid was assumed to be the
biogenetic precursors for prinsoside A and B. They undergo selec-
tive allylic oxidation, methylene oxidation and dihydroxylation to
yield the key intermediate i, which in turn transform into prinso-
side A upon glycosylation and methylation. The further oxidation of
prinsoside A would generate prinsoside B. Prinsoside C ought to be
also generated from ent-kaur-16-en-19-oic acid. The selective oxi-
dation and dihydroxylation would induce the hydroxyl groups and
established as methyl ent-1b,6a,7a,15b,16b,17-hexahydroxykauran-
19-oate 17-O- -glucopyranoside.
b-D
Prinsoside C (3) was obtained as a yellow gum, and its molecular
formula, C26H40O11, was assigned based on negative mode HR-ESI-
MS spectrum wherein a pseudo-molecular ion [MꢀH]ꢀ was mea-
sured at m/z 527.2500 (calcd for C26H39O11, 527.2571), which in-
dicated seven degrees of unsaturation. The 1H NMR data displayed
signals indicative of two methyl groups (dH 0.73, s and 1.40, s),
give rise to the natural diterpenoid ent-6a,7a,16b,17-tetrahydroxy-
kauranoic acid.9 Then, the further B ring-cleavage followed by
intramolecular hemiacetalation would transform it to prinsoside C.
Scheme 1. Proposed biogenetic pathway of compounds 1e3.
a hemiacetal proton (dH 5.67, br s), a glucopyranosyl moiety (dH
4.32, d, J¼7.8 Hz), and an aldehyde proton (dH 9.80, s). This, in
tandem with its 13C NMR spectrum composed of 26 carbon signals
consisting of two methyl groups (dC 22.6 and 30.6), eight methylene
groups (dC 19.0, 20.7, 26.3, 32.1, 32.1, 35.0, 47.5 and 74.1), three
methine carbons (dC 46.5, 49.0 and 55.8), one hemiacetal carbon (dC
100.8), four quaternary carbons (dC 40.6, 42.4, 59.6 and 81.2), and
two carbonyl carbons (dC 184.3 and 207.3), in addition to a gluco-
pyranosyl moiety (dC 105.1, 75.3, 78.1, 71.7, 77.9, 62.8), indicated
Compounds 1e3 and 1ae3a exhibited weak a-glucosidase in-
hibitory (IC50>1.0 mM), inferior to the reference compound acar-
bose with an IC50 value of 0.371 mM.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured on a PerkineElmer 341 po-
larimeter. UV spectra were recorded in MeOH on a PerkineElmer
Lambda 35 UVevis spectrophotometer. 1D and 2D NMR spectra
were recorded on a Bruker Advance-500 spectrometer with TMS as
internal standard. HR-ESI-MS data were obtained on a Bruker Bio
TOF IIIQ mass spectrometer. All solvents were of analytical grade
that 3 was similar in structure to coccinin, namely ent-6
b
,16
a,17-
trihydroxy-7,19-dioxo-6,19-epoxy-6,7-seco-kaurane 6-O-
b-D-glu-
copyranoside, which was previously isolated from Phaseolus coc-
cineus.11 The only difference between 3 and coccinin was observed
on the NMR signals at C-6 [dH 5.67, br s/dC 100.8 in 3; while dH 6.30,

9418
Q. Zhang et al. / Tetrahedron 71 (2015) 9415e9419
(Shanghai Chemical Plant, Shanghai, People’s Republic of China).
Silica gel (200e300 mesh) was used for column chromatography,
and precoated silica gel GF254 plates (Qingdao Haiyang Chemical
Plant, Qingdao, People’s Republic of China) were used for TLC. C18
reversed-phase silica gel (150e200 mesh, Merck), MCI gel (CHP20P,
(silica gel, CHCl3/MeOH, 8:1, v/v) to afford aglycones 1a (5.92 mg),
2a (6.80 mg), respectively. The reaction mixtures of 3 were con-
centrated in vacuo and purified with flash column chromatography
(silica gel, CHCl3/MeOH, 12:1/10:1, v/v) to afford aglycone 3a
(8.0 mg). The aglycone of each compound was identified by NMR
data analysis, including 2D NMR data (see Supplementary data),
because no spectroscopic data had been reported in literature.
75e150
mm, Mitsubishi Chemical Industries Ltd.), and Sephadex
LH-20 gel (Amersham Biosciences) was also used for column
chromatography. TLC spots were visualized under UV light and by
dipping into 5% H2SO4 in alcohol followed by heating.
3.8. 1a
20
3.2. Plant material
White amorphous solid; [
(MeOH) lmax (log
a]
¼ꢀ37.1 (c 0.38, MeOH); UV
D
3
) 206.4 (3.15) nm; CD
D
3
(c 0.005, MeOH) ꢀ3.84
(224); 1H and 13C NMR, see Table 2; ESI-MS m/z 421 [MþNa]þ; HR-
The mature fruits of P. utilis were collected in 2012 in Lijiang City,
Yunnan Province of China. The identity of the plant material was
verified by Dr. Fa-Guo Wang of South China Botanical Garden
(SCBG) and a voucher specimen was deposited at the Laboratory of
Natural Product Chemistry Biology, SCBG.
ESI-MS m/z 421.2199 [MþNa]þ, calcd for C21H34O7Na, 421.2305.
Table 2
1H and 13C NMR data of 1ae3a
Position 1a
2a
3a
dH (J in Hz)d dC
35.0 1.75, m
1.69, m
dH (J in Hz)d
a
c
c
dHb (J in Hz) dC
3.3. Extraction and isolation
dC
41.6 1.76, m
0.91, m
1a
1b
2a
2b
3a
3b
4
81.4 3.27, m
The air-dried powder of P. utilis fruits (20 kg) was exhaustively
extracted with EtOH (3ꢁ20 L). The resulting EtOH extract (1.15 kg)
was suspended in H2O and further partitioned into three fractions
as follows: petroleum ether (A, 800 g), EtOAc (B, 100 g), and H2O (C,
270 g). Subsequently, fraction (Fr.) C was subjected to resin D101
eluting with a gradient of increasing EtOH in H2O (0%e95%) to af-
ford Fr. A1eA4. Fr. A3 (8 g, EtOH/H2O, 50:50) was subjected to col-
umn chromatography (silica gel; CHCl3/MeOH, 10:1/1:1, v/v) to
afford Fr. A31eA34. Fr. A33 purified by Sephadex LH-20, (CHCl3/
MeOH, 1:3), RP-18 (MeOH/H2O, 40:60/90:10, v/v), and repeated
silica gel column afforded 2 (1.5 g). Fr. A4 (12 g, EtOH/H2O, 90:10)
was subjected to column chromatography (silica gel; CHCl3/MeOH,
15:1/2:1, v/v) to afford Fr. A41eA45. Fr. A43 was chromatographed
on a Sephadex LH-20 column (CHCl3/MeOH,1:1) and RP-18 (MeOH/
H2O, 60:40/90:10, v/v) then purified by repeated silica gel column
to afford 3 (1.5 g). Fr. A44 was subsequently purified by Sephadex
LH-20 (CHCl3/MeOH, 1:3) and repeated silica gel column to afford 1
(170 mg).
18.4 1.50, m
29.1 1.74, m
1.43, m
43.1 2.02, m
1.15, m
43.7
49.2 1.79, d,
(11.3)
19.0 1.44, m
40.1 2.30, m
1.13, m
44.6
51.7 2.29, d,
(11.1)
70.8 4.63, dd,
(11.1, 2.2)
78.6 4.30, m,
(2.2)
51.5
48.2 1.81, m
40.6
19.3 1.93, m
1.39, m
26.5 1.81, m
1.50, m
32.1 2.07, m
1.32, m
42.5
5
55.8 1.94, m
6
7
70.0 4.15, d,
100.9 5.67, s
207.3 9.80, s
(10.8, 1.9)
78.1 3.63, d,
(1.9)
51.2
48.0 1.74, m
45.4
19.4 2.85, m
1.28, m
8
9
10
59.7
49.0 2.09, m
40.6
11a
11b
12a
12b
13
14a
14b
15a
15b
16
20.8 1.83, m
26.1 1.43, m
26.4 1.70, m
43.9 2.52, m
34.2 2.09, m
1.69, m
43.1 2.01, m
34.3 1.68, m
1.48, m
46.2 2.26, m
32.2 2.42, m
1.70, m
47.8 1.83, m
1.60, m
82.0 4.02, s
81.0 3.53, m
3.4. Prinsoside A (1)
80.7
80.3
82.0
17a
65.7 4.09, m
64.5 3.55, m
66.1 3.75, d,
(11.4)
3.67, d,
(11.4)
30.6 1.44, s
184.3
20
White amorphous solid; [
a
]
¼ꢀ55.9 (c 0.51, MeOH); UV
3
D
(MeOH) lmax (log
3
) 311.3 (2.00) nm; CD
D
(c 0.051, MeOH) ꢀ4.26
17b
(223); 1H and 13C NMR, see Table 1; ESI-MS m/z 583 [MþNa]þ; HR-
ESI-MS m/z 583.2706 ([MþNa]þ, calcd for C27H44O12Na, 583.2833).
18
19
32.2 1.73, s
178.4
30.9 1.32, s
178.6
20
OMe
16.6 0.90, s
51.0 3.67, s
11.8 0.86, s
50.5 3.67, s
22.6 0.73, s
3.5. Prinsoside B (2)
a
Measured in pyridine-d5 at 125 MHz.
In pyridine-d5 at 500 MHz.
In CD3OD at 125 MHz.
20
Yellow gum; [
a
]
¼ꢀ30.0 (c 1.1, MeOH); UV (MeOH) lmax (log
3
)
b
c
D
291.0 (3.48) nm; CD
D
3
(c 0.01, MeOH) ꢀ9.58 (204); 1H and 13C
NMR, see Table 1; ESI-MS m/z 577 [MþH]þ; HR-ESI-MS m/z
d
In CD3OD at 500 MHz.
577.2839 [MþH]þ, calcd for C27H44O13, 577.2782.
3.9. 2a
3.6. Prinsoside C (3)
20
White amorphous solid; [
(MeOH) lmax (log
a
]
D
¼ꢀ27.1 (c 0.25, MeOH); UV
20
3
) 279.0 (2.69), 221.2 (3.07) nm; CD D 3 (c 0.016,
Yellow gum; [
a
]
D
¼ꢀ34.7 (c 0.13, MeOH); UV (MeOH) lmax (log
MeOH) ꢀ9.3 (226); 1H and 13C NMR, see Table 2; ESI-MS m/z 437
[MþNa]þ; HR-ESI-MS m/z 437.2152 [MþNa]þ, calcd for C21H34O8Na,
437.2254.
3
) 228.5 (3.34), 280.3 (3.05) nm; CD
D
3
(c 0.013, MeOH) ꢀ1.18 (226);
1H and 13C NMR, see Table 1; ESI-MS m/z 527 [MꢀH]ꢀ; HR-ESI-MS
m/z 527.2500 [MꢀH]ꢀ, calcd for C26H40O11, 527.2571.
3.7. Enzymatic hydrolysis of 1e3
3.10. 3a
20
A solution of each sample in H2O (6 mL) was individually
White amorphous solid; [
lmax (log
a
]
D
¼ꢀ9.8 (c 0.48, MeOH); UV (MeOH)
3
hydrolysed with crude
b
-Glucosidase (30 mg, from almonds, Sol-
3
) 274.4 (2.89) nm; CD
D
(c 0.017, MeOH) ꢀ3.74 (233); 1H
abio) at 37 ꢂC for 72 h. Reaction mixtures of 1 and 2 were con-
centrated in vacuo and purified with flash column chromatography
and 13C NMR, see Table 2; ESI-MS m/z 367 [MþH]þ; HR-ESI-MS m/z
367.2097 [MþH]þ, calcd for C20H31O6, 367.2042.

Q. Zhang et al. / Tetrahedron 71 (2015) 9415e9419
9419
3.11.
a
-Glucosidase inhibition assay
positive control. The blank was set by adding phosphate buffer
instead of the -glucosidase using the same method. Inhibition rate
a
The
1ae3a) was determined spectrophotometrically in
microtiter plate based on p-nitrophenyl- -glucopyranoside
(PNPG) as a substrate following the method described in the liter-
ature with slight modifications.12,13 In brief,
-glucosidase (20 L,
0.5 U/mL) and various concentrations (20, 10, 5, 2.5, 1.25 mM, 8 L)
of tested compounds in DMSO were mixed with 67 mM phosphate
buffer (112 L, pH 6.8) at room temperature for 10 min. The test
blank was set by adding phosphate buffer instead of the -gluco-
sidase using the same method. Reactions were initiated by the
addition of 5.0 mM PNPG (20 L). The reaction mixture was in-
cubated for 15 min at 37 ꢂC in a final volume of 160
L. Then, 0.2 M
Na2CO3 (80 L) was added to the incubation solution to stop the
a
-glucosidase inhibitory activity of six compounds (1e3 and
(%)¼[(ODnegative controlꢀODblank)ꢀ(ODtestꢀODtest blank)]/(ODnegative
blankꢀODblank)ꢁ100%. IC50 values of the samples were calculated
using the Microsoft Office Excel 2007 and SPSS Statistics 17.0.
a
96-well
a-D
a
m
Acknowledgements
m
This work was financially supported by National Natural Science
Foundation of China (No. 30973635, 81373293).
m
a
m
Supplementary data
m
m
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tet.2015.10.055.
reaction. The activities were detected in a 96-well plate, and the
absorbance was determined at 405 nm (for p-nitrophenol). The
negative control and negative blank was set by adding neat DMSO
instead of the sample via the same way as the test and test blank.
Acarbose was utilized as positive control. Inhibition rate (%)¼
References and notes
1. Gu, C. Z.; Bartholomew, B. Flora of China; Science /Missouri Botanical Garden:
Beijing/St. Louis, 2003, Vol. 9, pp 389e391.
[(ODnegative
controlꢀODblank)ꢀ(ODtestꢀODtest
blank)]/(ODnegative
blankꢀODblank)ꢁ100%. IC50 values of the samples were calculated
2. Editorial Board of ‘Zhonghua Bencao’. State Administration of Traditional Chinese
Medicine of the People’s Republic of China, Zhonghua Bencao; Shanghai Science
and Technology: Shanghai, China, 1999, Vol. 4, pp 193e195.
3. Jiangsu New Medical College. Dictionary of Traditional Chinese Medicines;
Shanghai People’s: Shanghai, 1977; 1239e1239.
4. Guan, B.; Peng, C. C.; Zeng, Q.; Cheng, X. R.; Yan, S. K.; Jin, H. Z.; Zhang, W. D.
Planta Med. 2013, 79, 365e368.
5. Xu, Y. X.; Yao, Z.; Hu, J. Y.; Teng, J.; Takaishi, Y.; Duan, H. Q. J. Asian Nat. Prod. Res.
2007, 9, 637e664.
6. Guan, B.; Li, T.; Xu, X. K.; Zhang, X. F.; Wei, P. L.; Peng, C. C.; Fu, J. J.; Zeng, Q.;
Cheng, X. R.; Zhang, S. D.; Yan, S. K.; Jin, H. Z.; Zhang, W. D. Phytochemistry 2014,
105, 135e140.
7. Rai, V. K.; Gupta, S. C.; Singh, B. Biol. Plant. 2003, 46, 121e124.
8. Qiu, S. X.; Cordell, G. A.; Kumar, B. R.; Rao, Y. N.; Ramesh, M.; Kokate, C.; Rao, A.
V. N. A. Phytochemistry 1998, 50, 485e491.
9. Kim, K. H.; Choi, S. U.; Lee, K. R. J. Nat. Prod. 2009, 72, 1121e1127.
10. Scott, A. I.; Wrixon, A. C. Tetrahedron 1971, 27, 4787e4819.
11. Yamashita, M.; Kinjo, J.; Ito, Y.; Kajimoto, T.; Marubayashi, N.; Ueda, I.; Nohara, T.
Chem. Pharm. Bull. 1990, 38, 2905e2906.
12. Feng, J.; Yang, X. W.; Wang, R. F. Phytochemistry 2011, 72, 242e247.
13. Li, W.; Fu, H. W.; Bai, H.; Sasaki, T.; Kato, H.; Koike, K. J. Nat. Prod. 2009, 72,
1755e1760.
using the Microsoft Office Excel 2007 and SPSS Statistics 17.0.
The
1ae3a) was determined spectrophotometrically in
microtiter plate based on p-nitrophenyl- -glucopyranoside
(PNPG) as a substrate following the method described in the liter-
ature with slight modifications.12,13 In brief,
-glucosidase (20 L,
0.5 U/mL) and various concentrations (20, 10, 5,2.5, 1.25 mM) of
tested compounds (120 L) in 67 mM phosphate buffer (pH 6.8)
were mixed at room temperature for 10 min. Reactions were ini-
tiated by the addition of 5.0 mM PNPG (20 L). The reaction mixture
was incubated for 15 min at 37 ꢂC in a final volume of 160
L. Then,
0.2 M Na2CO3 (80 L) was added to the incubation solution to stop
a-glucosidase inhibitory activity of six compounds (1e3 and
a
96-well
a-D
a
m
m
m
m
m
the reaction. The activities were detected in a 96-well plate, and the
absorbance was determined at 405 nm (for p-nitrophenol). The
negative blank was set by adding phosphate buffer instead of the
sample via the same way as the test. Acarbose was utilized as