Bioactive glycosides from Salacia cochinchinensis

Article abstract of DOI:10.1016/j.carres.2019.107777

Four new triterpene glycosides, named salaciacochinosides A-D (1–4) were isolated from the 90% ethanol extract of Salacia cochinchinensis, together with five known compounds 2α,3β,23-trihydroxyurs-12,18-dien-28-oic acid 28-O-β-D-glucopyranoside (5), racemiside (6), alangiplatanoside (7), acantrifoside E (8), and syringin (9). The structures of the four new triterpenoids were characterized by chemical methods and MS, IR, 1D and 2D NMR spectral analyses. The α-glucosidase inhibitory activities of the nine compounds were assessed, compounds 6 and 7 showed remarkable α-glucosidase inhibitory activities, with IC₅₀ values of 0.44 and 0.75 μM, respectively. Compounds 1–5 exhibited moderate α-glucosidase inhibitory activities, and compounds 8 and 9 showed none α-glucosidase inhibitory activity in our current experiments.

Full text of DOI:10.1016/j.carres.2019.107777

Journal Pre-proof
Bioactive glycosides from Salacia cochinchinensis
Hui-Mei You, Jia-Wen Zhao, Yu-Xing Jing, Juan-Rong Zhang, Wei Wang, Yun-Tao
Jiang, Ai-Xue Zuo, Jun-Ting Fan, Li-Zhu Zhang, Min Zhou, Zhi-Yong Jiang
PII:
S0008-6215(19)30261-7
DOI:
https://doi.org/10.1016/j.carres.2019.107777
CAR 107777
Reference:
To appear in: Carbohydrate Research
Received Date: 22 April 2019
Revised Date: 27 July 2019
Accepted Date: 13 August 2019
Please cite this article as: H.-M. You, J.-W. Zhao, Y.-X. Jing, J.-R. Zhang, W. Wang, Y.-T. Jiang, A.-X.
Zuo, J.-T. Fan, L.-Z. Zhang, M. Zhou, Z.-Y. Jiang, Bioactive glycosides from Salacia cochinchinensis,
Carbohydrate Research (2019), doi: https://doi.org/10.1016/j.carres.2019.107777.
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Graphical Abstract

Contents
Page 1
Title page
Abstract
Text
Page 2
Page 3
1
Page 14-15
Page 16-17
Page 17
Page 18
Page 19
Page 20
Table 1. H-NMR data of compounds 1-4.
1
3
Table 2. C-NMR data of compounds 1-4.
Table 3. The α-glucosidase inhibitory activities of compounds 1-9.
Fig. 1. The structures of compounds 1-9.
1
1
Fig. 2. Selected HMBC and H- H COSY correlations of compound 1-4
Fig. 3. Key ROESY correlations of compounds 1 and 2.

Bioactive glycosides from Salacia cochinchinensis
a
a
a
a
a
a
Hui-Mei You, Jia-Wen Zhao, Yu-Xing Jing, Juan-Rong Zhang, Wei Wang, Yun-Tao Jiang, Ai-Xue
b
c
a
a,
a,
Zuo, Jun-Ting Fan, Li-Zhu Zhang, Min Zhou, * Zhi-Yong Jiang *
a
Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission &
Ministry of Education, Yunnan Minzu University, Chenggong New District, Kunming, Yunnan, China
6
50500
b
Yunnan University of Traditional Chinese Medicine, Chenggong New District, Kunming, Yunnan,
China 650500
c
Department of Pharmceutical Analysis, School of Pharmcy, Nanjing Medical University, Nanjing,
China 211166
Corresponding author:
Prof. Dr. Zhi-Yong Jiang.
Tel: +86 871 65926940; E-mail: jiangzy2010@163.com
1

Abstract
Four new triterpene glycosides, named salaciacochinosides A-D (1 - 4) were isolated from the
9
2
0% ethanol extract of Salacia cochinchinensis, together with five known compounds
α,3β,23-trihydroxyurs-12,18-dien-28-oic acid 28-O-β-D-glucopyranoside (5), racemiside (6),
alangiplatanoside (7), acantrifoside E (8), and syringin (9). The structures of the four new triterpenoids
were characterized by chemical methods and MS, IR, 1D and 2D NMR spectral analyses. The
α-glucosidase inhibitory activities of the nine compounds were assessed, compounds 6 and 7 showed
remarkable α-glucosidase inhibitory activities, with IC50 values of 0.44 and 0.75 µM, respectively.
Compounds 1 - 5 exhibited moderate α-glucosidase inhibitory activities, and compounds 8 and 9
showed none α-glucosidase inhibitory activity in our current experiments.
Keywords: Salacia cochinchinensis; triterpene glycosides; salaciacochinosides A-D; α-glucosidase
2

Salacia cochinchinensis, a perennial shrub about one or two meters high, is mainly distributed in
south-east Asia such as China, Vietnam, and Cambodia. In Xishuangbanna, China, the roots and leaves
of S. cochinchinensis had been used for the treatment of diabetes and inflammation by the indigenes. In
consideration that many plants of the Salacia genus had been reported to possess antidiabetic, antiobese
1
-4
and anti-inflammatory activity, the S. cochinchinensis was also tested for the antidiabetic activity in
our previous bioassay experiment. Results suggested the 90% ethanol extract of S. cochinchinensis
possessed remarkable α-glucosidase inhibitory activity. As far as we know, there was no investigation
on S. cochinchinensis reported. With the aim to find active chemicals from this medicinal plant, the S.
cochinchinensis was phytochemically investigated and nine compounds were isolated from the title
plant. By chemical methods and comprehensive analysis of the IR, MS, HRESIMS, 1D and 2D NMR
spectral data, all the isolates were identified as 2α,3α,24-trihydroxyurs-12-en-28-oic acid
2
2
8-O-β-D-glucopyranosyl-(1→2)-β–D-glucopyranoside
(salaciacochinoside
A,
1),
α,3α,23-trihydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranosyl-(1→2)-β–D-glucopyranoside
(
(
salaciacochinoside B, 2), 2α,3β,23-trihydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranosyl-
1→2)-β–D-glucopyranoside (salaciacochinoside C, 3), 2α,3α,24-trihydroxylup-12,20(29)-dien-28-oic
acid
28-O-β-D-glucopyranosyl-(1→2)-β–D-glucopyranoside
(salaciacochinoside
D,
4),
5,6
7
2
α,3β,23-trihydroxyurs-12,18-dien-28-oic acid 28-O-β-D- glucopyranoside (5), racemiside (6),
8
9
10
alangiplatanoside (7), acantrifoside E (8), syringin (9) (Figure 1). Compounds 1 - 4 were new
triterpene glycosides. All compounds were assayed for their α-glucosidase inhibitory activities; results
suggested compounds 6 and 7 showed rermarkable α-glucosidase inhibitory activities, with IC50 values
of 0.44 and 0.75 µM, respectively, and compounds 1 - 5 possessed moderate α-glucosidase inhibitory
activity. Herein we reported the isolation, structural elucidation and α-glucosidase inhibitory activity of
3

all isolates.
Compound 1 was obtained as a white amorphous powder. Its molecular formula was deduced as
-
C H O by the HRESIMS at m/z 811.4488 [M-H] (C H O , calcd. 811.4480). The IR spectrum
4
2
68 15
42 67 15
-1
showed the absorption bands at 3348, 1735, 1647 cm , suggesting the presence of hydroxyl, carbonyl
1
and olefinic functions in compound 1. In the H NMR spectrum (Table 1), an olefinic proton at δ 5.43
(
1H, brs, H-12) was observed, besides a typical doublet methine at δ 2.50 (1H, d, J = 11.2 Hz, H-18),
six methyl signals at δ 1.63 (3H, s, H-23), 1.08 (3H, s, H-27), 1.07 (3H, s, H-26), 0.97 (3H, s, H-25),
0
.92 (3H, d, J = 6.4 Hz, H-29), 0.88 (3H, d, J = 6.2 Hz, H-30), suggesting that compound 1 was a
urs-12-en-28-oic acid derivative. The anomeric proton signals at δ 6.16 (1H, d, J = 8.1 Hz, H-1') and
5
.66 (1H, d, J = 7.7 Hz, H-1") indicated the presence of two β-linkaged sugar moieties in compound 1.
1
3
The C NMR spectrum (Table 2) displayed 42 carbon signals, of which an ester carbonyl at δ 176.3
(
C-28), two olefinic carbon signals at δ 125.8 (C-12), 138.7 (C-13) were clearly visible, besides two
anomeric carbon signals at δ 93.6, 104.9. The NMR data of the aglycone of compound 1 was
11
comparable to those of methyl 2α, 3α, 24- trihydroxyurs-12- en- 28- oate, indicating compound 1 also
contained an aglycone of 2α, 3α, 24- trihydroxyurs-12- en- 28- oic acid. After hydrolysis of 1 with 8%
HCl in MeOH, glucose was identified by a comparison with the authentic sugar sample on TLC
(
developed by n-BuOH/AcOH/H O 4:1:5, upper layer; and PhOH/H O 4:1). The absolute
2 2
configuration of D-glucose was finally confirmed by GC analysis of the trimethylsilyl L-cysteine
1
3
12
derivative. The identical C NMR data of the sugar region with those of asteryunnanoside B, in
combination with the HMBC correlations (Figure 2) between H-1' (δ 6.16) and C-28 (δ 176.3), H-1" (δ
5
.66) and C-2' (δ 79.4), established the existence of β-D- glucopyranosyl-(1→2)-β–D-glucopyranosyl
at C-28. The 2α, 3α hydroxyl groups were also verified by the ROESY (Figure 3) correlations of
4

1
1
H-2/Me-25 and H-3/Me-25. The other HMBC, H- H COSY (Figure 2) and ROESY (Figure 3)
correlations allowed the full proton and carbon assignments of compound 1. Consequently, the
structure of compound 1 was determined as 2α,3α,24-trihydroxyurs-12-en- 28-oic acid 28-O-β-D-
glucopyranosyl-(1→2)-β–D-glucopyranoside and named as salaciacochinoside A (1).
Compound 2 was obtained as a white amorphous powder and assigned the molecular formula
+
+
C H O by the positive HRESIMS at m/z 835.4450 [M+Na] (C H O Na , calcd. 835.4456). Its IR
4
2
68 15
42 68 15
-1
spectrum showed hydroxyl, carbonyl and olefinic functions at 3440, 1735, and 1645 cm , respectively.
Hydrolysis of 2 with 8% HCl in MeOH liberated D-glucose, identified by a comparison with the
1
authentic sugar sample and further confirmed by GC analysis. The H NMR spectrum (Table 1)
presented an olefinic proton signal at δ 5.41 (1H, br.s, H-12), a doublet methine at δ 2.48 (1H, d, J =
11.3 Hz, H-18), together with four singlet methyls [δ 1.10 (H-26), 1.05 (H-27), 0.95 (H-25), 0.77
(
H-24)] and two doublet methyls [δ 0.89 (3H, d, J = 6.3 Hz, H-29); 0.85 (3H, d, J = 6.1 Hz, H-30)],
suggesting compound 2 was also a urs-12-en-28-oic acid derivatives. The anomeric proton signals at δ
6
.18 (1H, d, J = 8.2 Hz, H-1') and 5.72 (1H, d, J = 7.8 Hz, H-1") suggested there were two β-linked
1
3
sugar units in compound 2. The C NMR spectrum (Table 2) of compound 2 exhibited 42 carbon
signals. The carbon signals due to the sugar moieties were identical with those of 1, implying
compound 2 contained the same sugar linkage as compound 1. Compound 2 differed from 1 mainly in
the aglycone unit, where a hydroxy linked at C-23 instead of a hydroxyl at C-24 in compound 1. The
structure of the aglycone was determined as 2α,3α,23-trihydroxyurs-12-en-28-oic acid by comparing
1
3
13
the C NMR data with those of methyl 2α,3α,23-trihydroxyurs-12-en-28-oate, and further verified by
the ROESY (Figure 3) correlations of H-2/H-25, H-3/H-25, and H-23/H-5. Accordingly, the structure
5

of
compound
2
was
deduced
as
2α,3α,23-trihydroxyurs-12-en-28-oic
acid
2
8-O-β-D-glucopyranosyl-(1→2)-β–D-glucopyranoside and named as salaciacochinoside B (2).
Compound 3 was obtained as a white amorphous powder with a molecular formula of C H O
68 15
42
-
deduced by the HRESIMS at m/z 811.4476 [M-H] (C H O , calcd. 811.4480). The IR spectral
42
67 15
1
showed almost the same absorptions as those of compounds 1 and 2. In the H NMR spectrum (Table
1
), the characteristic proton signals for a 12-en ursane triterpene aglycone [δ 5.23 (1H, brs, H-12), 2.20
(
1H, d, J = 11.2 Hz, H-18), 0.97 (3H, d, J = 6.1 Hz, H-30), 0.90 (3H, d, J = 6.1 Hz, H-29), 1.12, 1.05,
13
0
.83, 0.70 (each 3H, all s, for H-27, 25, 26, 24, respectively)] were displayed. A comparison of the C
14
NMR (Table 2) data of 3 with those of asiaticoside E indicated that both compounds shared a same
aglycone of 2α,3β,23-trihydroxyurs-12-en-28-oic acid. Acidic hydrolysis of 3 liberated D-glucose, .
The evidence that the NMR data ascribable to sugar moieties were essentially identical to those of
compounds 1 and 2, illustrated compound 3 also contained a (1→2) linkaged diglucopyranosyl at C-28.
This deduction was further proved by the HMBC cross-peak between H-1' (δ 5.42) and C-28 (δ 178.0),
H-1" (δ 4.76) and C-2' (δ 78.7). Consequently, the structure of compound 3 was deduced as
2
α,3β,23-trihydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranosyl-(1→2)-β–D-glucopyranoside and
named as salaciacochinoside C (3).
Compound 4 was obtained as a white amorphous powder and had the molecular formula of
+
+
C H O revealed by the HRESIMS at m/z 833.4292 [M+Na] (C H O Na , calcd. 833.4299). On
4
2
66 15
42 66 15
1
hydrolysis of 4, D-glucose was detected by a comparison with the authentic sugar sample. The H
NMR (Table 1) displayed three olefinic proton signals at δ 5.43 (1H, brs, H-12), 4.76 (1H, br.s, H -29),
a
4
.72 (1H, br.s, H -29), besides five singlet methyls and two anomeric protons ascribable to β-D-glucose
b
1
3
units. The C NMR spectrum showed 42 carbon resonances, of which two glucosyl anomeric carbon
6

signals at δ 93.7 and 104.8 were displayed. The characteristic terminal double bond at δ 153.4 (C-20,
6,15
C), 105.1 (C-29, CH ) suggested compound 4 might be a lupine-type triterpene derivative,
which
2
was finally corroborated by the HMBC correlations between H-29 and C-19, C-30; H-18 and C-20;
1
1
H-21 and C-20. The H- H COSY correlations (Figure 2) of H-18/H-19, H-19/H-21, H-21/H-22 also
13
supported the presence of five member ring E in compound 4. In consideration that the C NMR data
of rings A-D in compound 4 were almost the same as compound 1, it can be concluded that the
compound 4 had an aglycone of 2α,3α,24-trihydroxylup-12-en-28-oic acid, which was ascertained by
1
1
the HMBC and H- H COSY correlations indicated in Figure 2. Compound 4 possessed the same
diglucosyl connections as 1-3 by a comparison of the sugar NMR data with those of 1-3. Lastly, the
structure of compound
4
was elaborated as 2α,3α,24-trihydroxylup-12-en-28- oic acid
2
8-O-β-D-glucopyranosyl-(1→2)-β–D-glucopyranoside and named as salaciacochinoside D (4).
The other compounds were identified as 2α,3β,23-trihydroxyurs-12,18-dien-28-oic acid
5
,6
7
8
9
2
8-O-β-D-glucopyranoside (5), racemiside (6), alangiplatanoside (7), acantrifoside E (8), syringin
1
0
(
9) by comparison the NMR data with those reported in literatures.
All isolates were assayed for their α-glucosidase inhibitory activities. Results were shown in
tables 3. Compounds 6 and 7 exhibited remarkable α-glucosidase inhibitory activities, with IC50 values
of 0.44 and 0.75 µM, respectively. Compounds 1-5 possessed moderate α-glucosidase inhibitory
activities, with IC50 values ranging from 1.01 to 4.72 µM, and compounds 8 and 9 showed no
α-glucosidase inhibitory activity in our experiment. The above results suggested the lignan and
triterpene glycosides might be the bioactive constituents. Further investigation on this plant is still
ongoing.
In conclusion, nine glycosides, including four new triterpene glucosides, were isolated from S.
7

cochinchinensis. All chemicals were tested for their α-glucosidase inhibitory activities and two lignan
glycosides were found to possess notable α-glucosidase inhibitory activity. The triterpene glycosides
also showed moderate inhibiting α-glucosidase activity. Our results suggested further investigation on
this medicinal plant is necessary.
1
1
. Experimental
.1 General experiment procedures
Column chromatography (CC): silica gel (200-300 mesh; Qingdao Marine Chemical Inc., China);
Lichrospher Rp-18 gel (40-63µ; Merck, Germany); MCI gel CHP-20P (70-150 µm, Mitsubishi
Chemical Corporation, Tokyo, Japan). Semi-preparative HPLC was performed on an Agilent 1260
liquid chromatography with a Venusil XBP C18 (10 × 250 mm, 5µm) column. GC experiments were
conducted on an Agilent 7890A equipment. Optical rotations were carried out on a HORIBA SEPA-300
High Sensitive Polarimeter. IR spectra were measured on a Bio-Rad FTS-135 spectrometer with KBr
-1
pellets, ν in cm . HRESIMS data were obtained on an Agilent QTOF 6545 LC/MS. NMR spectra were
1
13
recorded on a Bruker AM-400 ( H/ C, 400 MHz/100 MHz) spectrometer, and chemical shifts were
given in δ (ppm) with TMS as internal reference. The authentic sugar samples (D--glucose and
L-glucose) and Acarbose (positive control) were obtained from Sigma–Aldrich Chemical Co. (St. Louis,
MO, USA). Fractions were monitored by TLC and spots were visualized by heating silica gel plates
sprayed with 8% H SO in EtOH.
2
4
1
.2. Planta material
The branches and leaves of S. cochinchinensis were collected from Xishuangbanna in
September, 2015, and were identified as Salacia cochinchinensis by Dr. Shi-Shun Zhou from
Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. A voucher specimen
8

(
TSYJ-2015057) was deposited in the Key Laboratory of Chemistry in Ethnic Medicinal Resources,
State Ethnic Affairs Commission & Ministry of Education, Yunnan Minzu University.
1
.3. Extraction and isolation
The air-dried branches and leaves of S. cochinchinensis (10.0 kg) were powdered and
extracted with 90% ethanol under reflux for three times, 2 hours each time. After concentrated in
vacuo, the extract was suspended in water and successively partitioned with petroleum ether, EtOAc
and n-BuOH to give petroleum ether (A). EtOAc (B), n-BuOH (C) and aqueous (D) fractions.
The n-BuOH (C) (150 g) fraction was then subjected to silica gel chromatography column (CC)
and eluted with gradient CHCl /MeOH/H O (95:5:0, 90:10:0, 85:15:1, 80:20:2, 70:30:5) to give four
3
2
fractions (Frs.C.1-4). The Fr.C.3 (18 g, eluted by CHCl /MeOH/H O 85:15:1) was subjected on a silica
3
2
gel CC with a gradient elution of CHCl /MeOH/H O (90:10:0, 85:15:1) to afford four sub-fractions
3
2
(
Frs.C.3.1-3.4). The Fr.C.3.2 (4.1 g) was performed to a MCI CC (MeOH/H O 60:40 to 100: 0) and
2
further purified by HPLC with an eluent of MeOH/H O (65:35, flow rate: 2ml/min) to yield
2
compounds 6 (7.3 mg), 8 (28 mg) and 9 (22 mg). The Fr.C.3.3 (3.8 g) was separated on a MCI CC
(
MeOH/H O 40:60 to 100: 0) to yield three sub-fractions (Frs.C.3.3.1-3.3.3). Fr.C.3.3.1 (0.5 g) was
2
purified by HPLC (MeOH/H O, 68:32, flow rate: 2 ml/min) to afford compounds 4 (18.7 mg), 5 (12
2
mg) and 7 (14.3 mg). Fr.C.3.3.2 (1.01 g) was successively performed on a silica gel CC and further
purified by Rp-18 CC (MeOH/H O 60:40) to yield compounds 1 (10.5 mg), 2 (23 mg), 3 (15 mg).
2
1
.4. Identification
21.3
-1
Salaciacochinoside A (1): white amorphous powder; [α]D +10.5 (c 0.20, MeOH); IR (KBr) cm :
1
13
3
348, 2996, 1735, 1647, 1068; H and CNMR (C D N, 400 MHz and 100 MHz) data, see Tables 1
5 5
-
-
-
and 2; ESI (neg.): m/z 811 [M - H] ; HRESIMS (neg.): m/z 811.4488 [M - H] , (calcd for C H O ,
42
67 15
9

8
11.4480).
15.5
-1
Salaciacochinoside B (2): white amorphous powder; [α]D +18.4 (c 0.25, MeOH). IR (KBr) cm :
1
13
3
340, 2998, 1735, 1645, 1067; H and C NMR (C D N, 400 MHz and 100 MHz) data, see Tables 1
5 5
+
+
and 2; ESI (pos.): m/z 835 [M+Na] ; HRESIMS (pos.): m/z 835.4450 [M+Na] (calcd for
+
C H O Na , 835.4456).
4
2
68 15
21.6
-1
Salaciacochinoside C (3): white amorphous powder; [α]D +25.7 (c 0.20, MeOH). IR (KBr) cm :
1
13
3
338, 1736, 1651, 1065; H and C NMR (CD OD, 400 MHz and 100 MHz) data, see Tables 1 and 2;
3
-
-
-
ESI (neg.): m/z 811 [M - H] ; HRESIMS (neg.): m/z 811.4476 [M - H] (calcd for C H O ,
42
67 15
8
11.4480).
21.0
-1
Salaciacochinoside D (4): white amorphous powder; [α]D +47.8 (c 0.21, MeOH). IR (KBr) cm :
1
13
3
380, 1734, 1646, 1062; H and C NMR (C D N, 400 MHz and 100 MHz) data, see Tables 1 and 2;
5 5
+
+
+
ESI (pos.): m/z 833 [M + Na] ; HRESIMS (+): m/z 833.4292 [M + Na] (calcd for C H O Na ,
42
66 15
8
1
1
33.4299).
.5. Sugar identification
.5.1. Acidic hydrolysis of compounds 1-4.
Each solution of compounds 1-4 (each 3 mg) in a mixture of MeOH (1.0 mL) and 8% HCl (1.0
mL) was stirred at reflux for 4 h. The hydrolysate was allowed to cool, diluted 2-fold with H O, and
2
then extracted with EtOAc (3 × 2 mL). The aqueous layer was neutralised with 2 M ammonium
hydroxide and concentrated in vacuo to give a residue in which D-glucose was identified by
comparison with authentic sugar sample (n-BuOH/AcOH/H O 4:1:5, upper layer; PhOH/H O, 4:1) on
2
2
TLC (sprayed with aniline phthalate reagent, followed by heating).
1
.5.2. Determination of absolute configuration of D-glucose
1
0

After being dried over P O for 48 h, the above-mentioned aqueous residue was dissolved in
2
5
anhydrous pyridine (1.0 mL), and 5 mg of L-cysteine methyl ester hydrochloride was added. The
mixture was stirred at 60 °C for 2 h. The reaction mixture was then concentrated in vacuo to furnish a
dry residue. 0.5 mL of N-trimethylsilylimidazole was added, and the reaction mixture was heated at
6
0 °C for 1 h. Subsequently, the mixture was partitioned between n-hexane and H O (1:1; v/v) and the
2
n-hexane extract was directly subjected to GC analysis under the following conditions: capillary
column, HP-5 (30 × 0.25 mm, with a 0.25 µm film, Dikma); detection, FID; detector temperature,
-1
2
80 °C; injection temperature, 250 °C; initial temperature 160 °C, raised to 280 °C at 5 °C·min with
the final temperature being maintained for 10 min; N gas as carrier. By comparing the retention times
2
(
Rt.) of the derivatives with those of authentic sugars [D-glucose (Rt. 19.8 min), and L-glucose (Rt.
2
0.7 min)] prepared in a similar way, D-glucose (Rt. 19.8 min) from 1 - 4 were detected.
.6. α-Glucosidase inhibitory assay
1
16
The α-glucosidase inhibitory activity was determined using the procedure previous reported in
9
6-well plates with acarbose (purity > 99%, purchased from Sigma) as a positive control. Briefly, each
well of the plates contained 40 µL of 2 mM 4-nitrophenyl α-D-glucopyranoside (purchased from TCI)
in 100 mM potassium phosphate buffer (pH 7.0) and 5 µL of sample in DMSO. The reaction was
initiated by the addition of 5 µL of the enzyme solution (0.30 µunits/ml from Bacillus
stearothermophilus). The plates were incubated at 37 ℃ for 20 min and the absorbance was measured
with a Spectra Max Plus plate reader. The increased absorbance (∆A) was compared with that of the
control to calculate the inhibition.
Inhibition (%) = (∆Acontrol –∆Asample) ⁄∆Acontrol
Acknowledgments
11

This work was financially supported by the National Natural Science Foundation of China (NSFC
No. 81560708, 81860620), the Reserve Talent Project of Middle & Young Academic Leaders in
Yunnan Province (2015HB050), and the Open Fund (MZY1503) from Key Laboratory of Chemistry in
Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, Yunnan Minzu
University. The authors appreciated the staff of the analytical group of the State Key Laboratory of
Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of
Sciences, for the measurements of all spectra.
Supplementary data
1
13
1
1
Supplementary data (HRESIMS, H, C-NMR, HSQC, HMBC, H- H COSY, ROESY spectra of
compounds 1-4) associated with this article can be found.
References
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4
2
1
3
4
1
5
6
7
. Matsuda, H.; Murakami, T.; Yashiro, K.; Yamahara, J.; Yoshikawa, M. Chem. Pharm. Bull. 1999,
7, 1725-1729.
. Kawazoe, K.; Shimogai, N.; Takaishi, Y.; Rao, K. S.; Imakura, Y. Phytochemistry 1997, 44,
569-1573.
. Joshi, B. S.; Kamat, V. N.; Viswanathan, N. Tetrahedron 1973, 29, 1365-1374.
. Yoshikawa, M.; Shimoda, H.; Nishida, N.; Takada, M.; Matsuda, H. J. Nutrition 2002, 132,
819-1824.
. Durham, D. G.; Liu, I. X.; Richards, R. M. E. Phytochemistry 1996, 42, 505–508.
. Li, W.; Fu, H.W.; Bai, H.; Sasaki, T.; Kato, H.; Koike, K. J. Nat. Prod. 2009, 72, 1755–1760.
. Khan, S.; Riaz, N.; Afza, N.; Malik, A.; Regman, A.U.; Iqbal, L.; Lateef, M. J. Asia Nat. Prod. Res.
1
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9
1
009, 11, 44-48.
. Otsuka, K.; Kashima, N.; Nakamotoa, K. Phytochemistry 1996, 42, 1435-1438.
. Liu, Y.; Wang, Z.Y.; He, W.J.; Tan, N.H.; Yin, Z.Q. Acta Pharm. Sin. 2015, 50, 475-479.
0. Yang, M.C.; Lee, K.H.; Kim, K.H.; Choi, S.U.; Lee, K.R. Arch Pharm. Res. 2007, 30, 1067-1074.
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1
1
1
1
1
2. Shao, Y.; Zhou, B. N.; Lin, L. Z.; Cordell, G. Phytochemistry 1995, 38, 1487–1492.
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6. Ma, C.M.; Hattori, M.; Daneshtalab, M.; Wang, L.L. J. Med. Chem. 2008, 51, 6188-6194.
1
3

1
Table 1. The H-NMR (400 MHz) spectral data of compounds 1-4.
a
a
b
a
position
1
1
2
3
4
1
1
.92-1.94 (1H, m)
.83-1.85 (1H, m)
1.91-1.95 (1H, m)
1.81-1.84 (1H, m)
1.93-1.98 (1H, m)
1.24-1.32 (1H, m)
1.92-1.97 (1H, m)
1.80-1.84 (1H, m)
2
3
5
4.43-4.46 (1H, m)
4.61 (1H, brs)
4.24-4.28 (1H, m)
4.15 (1H, m)
3.68-3.73 (1H, m)
3.31-3.35 (1H, m)
1.25-1.32 (1H, m)
4.41-4.47 (1H, m)
4.62 (1H, m)
1.75-1.81 (1H, m)
2.01-2.04 (1H, m)
1.75-1.82 (1H, m)
1.61-1.68 (1H, m)
1.03-1.09 (1H, m)
1
.42-1.50 (1H, m)
1
1
.62-1.68 (1H, m)
.38-1.42 (1H, m)
1.55-1.59 (1H, m)
1.24-1.31 (1H, m)
6
1
1
.70-1.73 (1H, m)
.59-1.62 (1H, m)
1.62-1.66 (1H, m)
1.28-1.35 (1H, m)
7
9
1.60-1.63 (2H, m)
1.58-1.64 (2H, m)
1.85-1.89 (1H, m)
2.03-2.05 (2H, m)
5.43 (1H, brs)
1.84-1.90 (1H, m)
2.01-2.03 (2H, m)
5.41 (1H, brs)
2.38-2.41 (1H, m)
1.29-1.34 (1H, m)
2.27-2.30 (1H, m)
2.01-2.04 (1H, m)
1.64-1.68 (1H, m)
1.96-2.02 (2H, m)
5.23 (1H, brs)
1.04-1.09 (1H, m)
0.96-1.02 (1H, m)
1.82-1.90 (1H, m)
1.98-2.06 (2H, m)
5.43 (1H, brs)
1.32-1.38 (1H, m)
1.21-1.24 (1H, m)
2.27-2.35 (1H, m)
2.21-2.25 (1H, m)
11
1
1
2
5
2
.39 (1H, m)
.30-1.34 (1H, m)
.26 (1H, brd, 11.7)
.07 (1H, m)
1
2
1
6
1.82-1.88 (2H, m)
2
1
1
2
8
9
0
2.50 (1H, d, 11.2)
1.34-1.42 (1H, m)
0.93-1.11 (1H, m)
2.48 (1H, d, 11.3)
1.32-1.37 (1H, m)
0.84-0.90 (1H, m)
2.20 (1H, d, 11.2)
1.40 (1H, m)
2.61 (1H, d, 11.8)
2.35-2.41 (1H, m)
-
0.93-0.97 (1H, m)
1
1
1
1
.34-1.40 (1H, m)
.0-1.2 (1H, m)
.75-1.80 (1H, m)
.83-1.90 (1H, m)
1.29-1.34 (1H, m)
1.10-1.23 (1H, m)
1.81-1.86 (1H, m)
1.68-1.72 (1H, m)
1.50-1.54 (1H, m)
1.28-1.32 (1H, m)
1.78-1.82 (1H, m)
1.59-1.64 (1H, m)
3.50 (1H, d, 11.1)
3.27 (1H, d, 11.1)
2.28-2.34 (1H, m)
2.10-2.17 (1H, m)
1.81-1.83 (1H, m)
1.99-2.04 (1H, m)
2
2
2
2
1
2
3
4
3
3
.84 (1H, d, 10.8)
.69 (1H, d, 10.8)
1.63 (3H,s)
1.64 (3H, s)
4
.08 (1H, m)
4.08 (1H, d, 9.1)
3.71 (1H, d, 10.9)
0.77 (3H, s)
0.70 (3H, s)
3
.71 (1H, d, 10.0)
2
2
5
6
0.97 (3H, s)
0.95 (3H, s)
1.10 (3H, s)
1.05 (3H, s)
0.83 (3H, s)
0.97 (3H, s)
1.06 (3H, s)
1.07 (3H, s)
1
4

2
2
3
7
9
0
1.08 (3H, s)
1.05 (3H, s)
1.12 (3H, s)
1.06 (3H, s)
4
4
.76 (1H, brs)
.72 (1H, brs)
0.92 (3H, d, 6.4)
0.88 (3H, d, 6.2)
0.89 (3H, d, 6.3)
0.85 (3H, d, 6.1)
0.90 (3H, d, 6.1)
0.97 (3H, d, 6.1)
1.05 (3H, s)
Glc-
1
2
3
4
5
′
′
′
′
′
6.16 (1H, d, 8.1)
4.42-4.48 (1H, m)
4.28-4.34 (1H, m)
4.18-4.28 (1H, m)
3.90-3.96 (1H, m)
6.18 (1H, d, 8.2)
4.46-4.52 (1H, m)
4.27-4.33 (1H, m)
4.22-4.30(1H, m)
3.93-3.98 (1H, m)
5.42 (1H,d,8.0)
3.78-3.83 (1H, m)
3.33-3.39 (1H, m)
3.35-3.43 (1H, m)
3.31-3.65 (1H, m)
6.15 (1H, d, 8.2)
4.41-4.47 (1H, m)
4.25-4.32 (1H, m)
4.15-4.21 (1H, m)
3.90-3.95 (1H, m)
4.42-4.47 (1H, m)
4.34-4.38 (1H, dd, 11.7, 4.4）
4
4
.41-4.46 (1H, m)
.32-4.36 (1H, m)
4.42-4.45 (1H, m)
4.35-4.39 (1H, m)
3.75-3.81 (1H, m)
3.65-3.70 (1H, m)
6
′
Glc-
1
2
3
4
5
″
″
″
″
″
5.66 (1H, d, 7.7)
4.06-4.14 (1H, m)
4.22-4.28 (1H, m)
4.11-4.16 (1H, m)
3.99-4.04 (1H, m)
5.72(1H, d, 7.8)
4.12-4.15 (1H, m)
4.24-4.28 (1H, m)
4.11-4.15 (1H, m)
4.02-4.06 (1H, m)
4.76 (1H, d, 8.0)
3.21-3.26 (1H, m)
3.33-3.39 (1H, m)
3.11-3.17 (1H, m)
3.33-3.39 (1H, m)
5.64 (1H, d, 7.7)
4.05-4.14 (1H, m)
4.21-4.27 (1H, m)
4.10-4.15 (1H, m)
3.98-4.04 (1H, m)
4
4
.63 (1H, brd, 11.4)
.42-4.48 (1H, m)
4.69 (1H, dd, 11.6, 2.7)
4.37-4.43 (1H, m)
3.89 (1H, dd, 11.2, 2.1)
3.61-3.65 (1H, m)
4.62-4.64 (1H, m)
4.41-4.47 (1H, m)
6
″
a
b
δ in ppm, J in Hz. Measured in C D N. Measured in CD OD.
5
5
3
1
5

1
3
Table 2. The C NMR (100 MHz) data of compounds 1-4.
a
a
b
a
Position
1
2
3
4
1
2
3
4
5
6
7
8
9
43.3(t)
66.3(d)
74.1(d)
45.2(s)
49.5(d)
18.8(t)
34.0(t)
40.5(s)
48.2(d)
38.5(s)
24.0(t)
125.8(d)
138.7(s)
42.5(s)
29.3(t)
24.4(t)
48.3(s)
53.4(d)
39.3(d)
39.2(d)
30.9(t)
36.5(t)
23.8(q)
65.1(t)
17.3(q)
17.5(q)
23.8(q)
176.3(s)
17.4 (q)
21.3(q)
42.8(t)
66.3(d)
79.0(d)
41.8(s)
43.4(d)
18.3(t)
33.2(t)
40.3(s)
48.1(d)
38.3(s)
23.7(t)
125.7(d)
138.7(s)
42.5(s)
29.3(t)
24.3(t)
48.3(s)
53.3(d)
39.2(d)
39.2(d)
30.8(t)
36.4(t)
71.2(t)
17.7(q)
17.2(q)
17.6(q)
23.8(q)
176.3(s)
17.4(q)
21.3(q)
48.0 (t)
69.7(d)
78.2(d)
44.1(s)
48.2(d)
19.0(t)
33.6(t)
41.0(s)
49.0(d)
38.9(s)
24.5(t)
126.8(d)
139.4(s)
43.3(s)
29.9(t)
24.8(t)
49.5(s)
54.1(d)
40.3(d)
40.3(d)
31.7(t)
37.3(t)
66.3(t)
13.9(q)
17.7(q)
17.9(q)
24.2(q)
178.0(s)
17.6(q)
21.6(q)
43.3(t)
66.2(d)
74.2(d)
45.1(s)
49.5(d)
18.8(t)
34.0 (t)
40.4(s)
48.2(d)
38.5(s)
23.9(t)
126.2(d)
138.3(s)
42.5(s)
29.2(t)
24.4(t)
48.5(s)
55.5(d)
37.5 (d)
153.4(s)
32.5(t)
38.6(t)
23.8(q)
65.1(t)
17.3(q)
17.5(q)
23.6(q)
175.7(s)
105.1 (t)
16.4(q)
1
0
11
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Glc-
1
2
3
4
5
6
′
′
′
′
′
′
93.6(d)
79.4(d)
78.9(d)
70.8(d)
79.1(d)
62.2(t)
93.6(d)
79.2(d)
78.9(d)
70.7(d)
79.0(d)
62.0 (t)
93.9(d)
78.7(d)
78.5(d)
70.8(d)
78.7 (d)
62.3(t)
93.7(d)
79.3(d)
78.8(d)
70.8(d)
79.1(d)
62.2(t)
Glc-
1
″
″
104.9(d)
76.0(d)
104.9(d)
76.1(d)
103.9(d)
75.8(d)
104.8(d)
76.0(d)
2
1
6

3
4
5
6
″
″
″
″
78.3(d)
72.7(d)
78.3(d)
63.7(t)
78.3 (d)
72.6(d)
78.3(d)
63.6(t)
78.1(d)
72.5(d)
77.9 (d)
63.6 (t)
78.4(d)
72.7(d)
78.3(d)
63.8(t)
a
b
Measured in C D N. Measured in CD OD.
5
5
3
Table 3. The α-glucosidase inhibitory activities of compounds 1-9.
-1
Compounds
IC ±RSD (n=3)/(µmol·L )
50
1
2.32±1.5%
3.46±2.1%
2.66±3.0%
1.01±1.6%
4.72±2.2%
0.44±2.0%
0.75±1.8%
>20
2
3
4
5
6
7
8
9
>20
Acarbose
0.12±2.5%
1
7

30
29
12
1
1
O
2
5
26
O
28
H
O
1
H
H
HO
HO
HO
HO
HO
HO
O
O
O
2
7
HO
H
HO
HO
H
O
H
O
2
4
23
O
HO
OH
OH
HO
OH
OH
OH
1'
OH
OH
HO
O
HO
HO
O
O
O
O
O
OH
OH
3
1
OH
OH
OH
OH
2
1
"
OH
OH
OH
29
2
0
19
3
0
22
OMe
OH
O
1
H
HO
O
O
HO
HO
O
HO
HO
OH
O
O
CH3
HO
OMe
OMe
H
23
O
HO
24
H
OH
OH
O
OH OH
OH
MeO
HO
OH
OH
OH
HO
O
5
6
4
O
OH
OH
OH
OH
OH
OH
HO
MeO
O
MeO
O
OH
O
HO
HO
OMe
O
O
OO
OH
OH
OH
OH
OH
OMe
MeO
MeO
OH
OH
OH
O
O
8
7
9
OH
OH OH
Figure 1. The structures of compounds 1-9.
1
8

1
9

H
H
O
H
H
H
H
O
HO
HO
H
HO
O
H
HO
O
HO
HO
H
H
O
O
O
OH
OH
OH
HO
OH
OH
HO
HO
O
O
O
OH
OH
OH
OH
OH
OH
1
2
H
H
Figure 3. Key ROESY correlations of compounds 1 and 2.
2
0

Highlights
1
.Nine glycosides were isolated from the branches and leaves of S. cochinchinensis.
. Four new triterpene glucosides were identified by chemical methods and MS, IR, 1D
2
and 2D NMR spectral analyses.
. All isolates were assayed for their α-glucosidase inhibitory activities.
3