Cyanogenetic glycosides and simple glycosides from the linseed meal

Article abstract of DOI:10.1016/j.fitote.2015.08.008

Three new cyanogenetic triglycosides linustatins A-C (1-3), and two new simple glycosides linustatins D and E (4 and 5) were isolated from the 70% ethanol extract of flaxseed meal (Linum usitatissimum L.). Their structures were elucidated on the basis of spectroscopic analysis and chemical evidence. All of the isolates showed moderate activities against aldose reductase and weak activities against α-glucosidase, DPP-IV, and FBPase at the same concentrations as the positive control drugs.

Full text of DOI:10.1016/j.fitote.2015.08.008

Fitoterapia 106 (2015) 78–83
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Cyanogenetic glycosides and simple glycosides from the linseed meal
Qing-Yun Yang, Li Song, Ji-Fa Zhang, Zhu-Fang Shen, Quan Liu, Shuai-Nan Liu,
Wen-Sheng Zheng ⁎, Chun-Suo Yao ⁎
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, and Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of
Education, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 June 2015
Received in revised form 14 August 2015
Accepted 17 August 2015
Available online 22 August 2015
Three new cyanogenetic triglycosides linustatins A–C (1–3), and two new simple glycosides linustatins D and E
(4 and 5) were isolated from the 70% ethanol extract of flaxseed meal (Linum usitatissimum L.). Their structures
were elucidated on the basis of spectroscopic analysis and chemical evidence. All of the isolates showed moderate
activities against aldose reductase and weak activities against α-glucosidase, DPP-IV, and FBPase at the same
concentrations as the positive control drugs.
©
2015 Elsevier B.V. All rights reserved.
Keywords:
Linum usitatissimum L.
Flaxseed meal
Cyanogenetic glycoside
Linustatin
1
. Introduction
glucosides must be conducted systematically to develop and utilize
flaxseed meal. In previous paper [7], our research group reported four
new compounds from the flaxseed meal. On the basis of the above
research, continuous research on the flaxseed meal resulted in the isola-
tion of five new compounds linustatins A–E (1–5) (Fig. 1). Among them,
compounds 1, 2, and 3 are new cyanogenetic triglycosides, and com-
pounds 4 and 5 belong to new simple glycoside derivatives. Pharmaco-
logical tests indicated that all of the isolates present moderate activities
against aldose reductase and weak activities against α-glucosidase,
DPP-IV, and FBPase at the same concentrations as the positive control
drugs.
Several plants contain compounds capable of liberating hydrogen
cyanide during hydrolysis. This ability, which is known as cyanogenesis,
has been observed for centuries in plants such as apricots, peaches,
and other important food plants. Most cases of cyanide poisoning are
caused by consuming plants belonging to the families of Rosaceae,
Leguminosae, and Euphorbiaceae or members of the genus Sorghum.
While some cases of cyanide poisoning are accidental, large numbers
of people are exposed daily to low concentrations of cyanogenic com-
pounds that are present in the food they eat. Cyanogenic glycosides,
which liberate hydrogen cyanide when sugar is removed, are responsi-
ble for this cyanophoric capability [1]. Flaxseed (Linum usitatissimum L.),
an herbaceous plant belonging to the Linaceae family, is an important
industrial crop worldwide; this crop is grown for its fiber and oilseed
2. Experimental details
2.1. General experimental procedures
[
2]. Recent pharmacological research shows that flaxseed exhibits
strong anti-cancer, immunodepression, antioxidant, hypoglycemic,
and hypolipidemic effects [3]. The maximum lignan concentration in
flaxseed is 3% (w/w); thus, flax is one of the richest edible sources of
lignans [4]. In addition, flaxseed meal is rich in cyanogenic compounds.
However, only a few studies on cyanogenic compounds (e.g., linamarin,
lotaustralin, linustatin, neolinustatin, and amygdalin) in flaxseed meal
have been reported [5,6], largely because of difficulties associated with
isolating and purifying these compounds and the inherent instability
of aglycones during hydrolysis. Therefore, research on cyanogenetic
Optical rotations were measured on a P2000 polarimeter (JASCO,
Tokyo, Japan). UV spectra were obtained on a JASCO P650 spectro-
photometer. IR spectra were obtained on a Nicolet 5700 FT-IR micro-
scope instrument (FT-IR microscope transmission). 1D and 2D NMR
1
spectra were respectively acquired at 600 MHz for H NMR and
150 MHz for 13C NMR on a Bruker AVANCE III HD 600 MHz (Bruker
4
Corporation, Germany) in MeOH-d , and solvent peaks were used as
references. ESIMS and HRESIMS data were obtained using an AccuToFCS
JMST100CS spectrometer (Agilent Technologies, Ltd., Santa Clara, CA,
USA). Column chromatography (CC) was performed using silica gel
(
200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, People's Re-
⁎
Corresponding authors.
E-mail addresses: zhengwensheng@imm.ac.cn (W.-S. Zheng), yaocs@imm.ac.cn
public of China) and a Pharmadex LH-20 column (Amersham Biosciences,
Inc., Shanghai, China). Preparative TLC separation was performed using
(
C.-S. Yao).
http://dx.doi.org/10.1016/j.fitote.2015.08.008
367-326X/© 2015 Elsevier B.V. All rights reserved.
0

Q.-Y. Yang et al. / Fitoterapia 106 (2015) 78–83
79
Fig. 1. Structures of compounds 1–5.
high-performance silica gel TLC plates (HSGF254, glass-precoated,
Yantai Jiangyou Silica Gel Development Co., Ltd., Yantai, China).
HPLC separation was performed on an experimental setup consisting
of a Waters 515 pump, Waters 2487 dual λ absorbance detector, and
Knauer Smartline RI detector 2300 with a YMC semi-preparative
C18-packed column (250 mm × 10 mm i.d., 5 μM). TLC was conduct-
ed using glass-precoated silica gel GF-254 plates. Spots were visual-
of CHCl
3
/MeOH/H
2
O (v:v:v = 50:5:0.5–5:5:0.5) to yield six subfractions
of LW-VIII-B2-a1 to LW-VIII-B2-a6. Fraction LW-VIII-B2-a4 (32 mg) was
loaded on a Sephadex LH-20 column using MeOH/H O (v:v = 1:1) as
the eluent to produce a subfraction (12 mg) that was subsequently
purified by semi-preparative Rp-HPLC using MeOH/H O (v:v = 10:90)
as the eluent to yield compound 5 (6.88 mg). Fraction LW-VIII-C
(1.239 g) was subjected to a Sephadex LH-20 column using a mobile
2
2
ized under UV light or by spraying with 7% H
followed by heating.
2
SO
4
in 95% EtOH
2
phase of MeOH/H O (v:v = 1:1) to produce three subfractions, namely,
LW-VIII-C1 to LW-VIII-C3. Subfraction LW-VIII-C1 (906 mg) was further
separated on an ODS column (40–63 μm, 4.0 × 20 cm) and eluted using
2
.2. Plant material
The linseed meal (L. usitatissimum L.) was provided by Gansu Puyuan
3 2
a gradient of increasing CH CN (0%–10%) in H O to provide three
subfractions, namely, LW-VIII-C1-1 to LW-VIII-C1-3. Subfraction
LW-VIII-C1-1 (30 mg) was then purified by semi-preparative Rp-HPLC
(column, Rp-18, 250 × 10 mm, 5 μm) and eluted using MeCN/1‰TFA/
Pharmaceutical Technology Co., Ltd. (Gansu Province, People's Republic
of China, May 2009) and identified by Prof. Wang-zhi Song (Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking
Union Medical College, Beijing, 100050, China). A voucher specimen
H
2
O (v:v = 2:98) to afford compounds 1 (1.6 mg), 2 (5.83 mg), and 3
(5.18 mg).
Fraction LW-V (193.0 mg) was subjected to silica gel CC (mesh
2
45–75 μm, 1.5 × 20 cm) and eluted using CHCl /MeOH/H O
(
No. ID-S-2540) was deposited at the Herbarium of the Department of
3
Medicinal Plants, Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing 100050,
China.
(v:v:v = 85:5:0.5–5:5:0.5) to afford nine subfractions, namely, LW-
V-A to LW-V-I. Compound 4 (4.92 mg) was obtained by separating
LW-V-H (30.6 mg) on a semi-preparative Rp-HPLC and elution using
2
MeOH/H O (v:v = 10:90).
2
.3. Extraction and isolation
Linustatin A (2-[(O-β-D-glucopyranosyl-(1 → 4)-O-β-D-glucopyranosyl-
1 → 6)-β-D-glucopyranosyl)oxy]-2-methyl-(2R)-butanenitrile, 1).
White amorphous powder; [α] = −26.9 (c 0.13, MeOH); IR
D
(
2
0
Linseed meal (4.8 kg) was dried, ground and extracted three times
with 10 L of 70% EtOH/H
5 min at room temperature. The combined aqueous EtOH extracts
were evaporated to near dryness under vacuum, and the resulting
residue (500 g) was suspended in H O (5 L). The insoluble solid was re-
moved, and the water-soluble fraction was successively partitioned
using petroleum ether (4 × 2 L) and EtOAc (4 × 2 L). The water solution
was then absorbed on a macroporous resin and successively eluted with
2
O under ultrasonication. Each extraction lasted
νmax: 3406, 2924, 2597, 2515, 1720, 1670, 1622, 1546, 1436, 1131,
−
1
1
13
4
801, 669, 602, 473 cm ; H-NMR (CD
(CD OD, 150 MHz) spectroscopic data, see Tables 1 and 2; (+)-
ESIMS m/z 608 [M + Na] , (−)-ESIMS m/z 584 [M-H] , 620
3
OD, 600 MHz) and C-NMR
3
+
−
2
−
+
[M + Cl] ; HR-ESI-MS [M + Na] m/z 608.2169 (calcd for
39NO16Na, 608.2161).
Linustatin B (2-[(O-β-D-glucopyranosyl-(1 → 6)-O-β-D-glucopyranosyl-
(1 → 6)-β-D-glucopyranosyl)oxy]-2-methyl-(2R)-butanenitrile, 2).
23
C H
H
2
O, 40% EtOH/H
Exactly 45 g of the 40% EtOH/H
80 cm × 10 cm, 200–300 mesh) and eluted using a mixtures of CHCl
MeOH with increasing polarity (v:v = 4:1, 3:1, 7:3, 2:1, 3:2, 1:1, 2:3, 3:7,
2
O, 60% EtOH/H
2 2 2
O, 70% EtOH/H O, and 90% EtOH/H O.
White amorphous powder; [α]2
0
2
O fraction was subjected to silica gel CC
D
= −26.8 (c 0.49, MeOH); IR
(
3
/
ν
max: 3406, 2890, 2519, 1675, 1433, 1380, 1291, 1203, 1167, 1068,
−
1
1
899, 837, 800, 721, 666, 604 cm
and 13C-NMR (CD
OD, 150 MHz) spectroscopic data, see Tables 1 and
2; (+)-ESIMS m/z 608 [M + Na] , (−)-ESIMS m/z 584 [M-H] , 620
3
; H-NMR (CD OD, 600 MHz)
1
:4, 1:9, 3 L each) to yield ten fractions, namely, LW-I to LW-X.
Fraction LW-VIII (6.684 g) was loaded on an MPLC with an ODS
3
+
−
−
+
column (40–63 μm, 4.0 × 50 cm) and eluted using a gradient of increas-
ing MeOH (v:v = 95:5, 50:50, 0:100) in H O to produce three
[M + Cl] ; HR-ESI-MS [M + Na] m/z 608.2175 (calcd for
39NO16Na, 608.2161).
Linustatin C (2-[(O-β-D–glucopyranosyl-(1 → 6)-O-β-D-glucopyranosyl-
(1 → 6)-β-D-glucopyranosyl)oxy]-2-methyl-propionitrile, 3). White
2
23
C H
subfractions, LW-VIII-A to LW-VIII-C. Subfraction LW-VIII-B (4.0 g)
was applied to an ODS column (40–63 μm, 4.0 × 50 cm) and eluted
2
0
using MeOH/H
2
O (v:v = 0:100, 5:95, 10:90, 15:85, 20:80, 25:75,
amorphous powder; [α]
D
= −27.1 (c 0.43, MeOH); IR νmax: 3390,
3
0:70, 50 mL each) to afford seven subfractions, LW-VIII-B1 to LW-
2975, 2894, 1677, 1434, 1368, 1203, 1138, 1076, 1049, 880, 841,
−
1
1
13
VIII-B7. Fraction LW-VIII-B2 (1.208 g) was further separated on a silica
801, 722, 625 cm
3
; H-NMR (CD OD, 600 MHz) and C-NMR
gel column (mesh 45–75 μm, 1.5 × 20 cm) and eluted using a mixture
(CD OD, 150 MHz) spectroscopic data, see Tables 1 and 2; (+)-ESIMS
3

80
Q.-Y. Yang et al. / Fitoterapia 106 (2015) 78–83
Table 1
Linustatin D (2-methyl-2,3-dihydroxybutyl-3-O-β-D-glucopyranoside,
1
H NMR spectroscopic data of compounds 1–5.
20
4
). White amorphous powder; [α]
D
= −8.4 (c 0.41, MeOH); IR νmax:
No.
1
1
2
3
4
5
3359, 2980, 2923, 1725, 1675, 1452, 1382, 1316, 1255, 1175, 1078, 1040,
−
1
1
9
6
49, 892, 866, 755, 652, 612, 583, 524 cm
;
3
H-NMR (CD OD,
1.11 (s)
3.53 (q, 6.6)
1.11 (s)
3.57 (q, 6.6)
00 MHz) and 13C-NMR (CD
OD, 150 MHz) spectroscopic data, see
3a
1.82
1.83
1.68 (s)
3
+
(dt, 7.4,14.1) (dt, 7.3,14.6)
Tables 1 and 2; (+)-ESIMS m/z 289 [M + Na] , (−)-ESIMS m/z 265
−
+
3b
1.90
1.92
[
2
M-H] ; HR-ESI-MS [M + Na] m/z 289.1267 (calcd for C11
89.1258).
Linustatin E (2-methyl-2,3-dihydroxybutyl-3-O-β-D-glucopyranosyl-
(1 → 6)-β-D-gluco pyranoside, 5). White amorphous powder;
22 7
H O Na,
(dt, 7.4,14.1) (dt, 7.3,14.6)
4
5
1
2
3
4
5
6
1.03 (t, 7.4)
1.56 (s)
1.03 (t, 7.3)
1.57 (s)
4.57 (d, 7.8) 4.64 (d, 7.8) 4.35 (d, 8.4)
3.15 (m)
3.32 (m)
3.26 (m)
3. 42 (m)
3.71
1.66 (s)
1.18 (d, 6.0)
1.11 (s)
1.18 (d, 6.6)
1.11 (s)
′
′
′
′
′
4.56 (d, 7.8)
3.15 (m)
3.32 (m)
3.32 (m)
3. 43 (m)
4.36 (d, 7.8)
3.16 (m)
3.28 (m)
3.30 (m)
3.40 (m)
4.07 (dd,
12.0, 2.4)
3.73
2
0
3.22 (m)
3.34 (m)
3.31 (m)
3.47 (m)
3.50 (m)
3.15 (m)
3.28 (m)
3.24 (m)
3.20 (m)
3.80 (dd,
12.0, 2.4)
3.61
(dd, 12.0, 5.4) (dd, 12.0, 6.0)
4.35 (d, 7.8)
3.16 (m)
[α]
D
= −24.2 (c 0.57, MeOH); IR νmax: 3388, 2979, 2913, 1649,
−1
1
589, 1446, 1379, 1288, 1171, 1077, 1041, 949, 899, 621 cm
;
1
13
H-NMR (CD
3 3
OD, 600 MHz) and C-NMR (CD OD, 150 MHz)
spectroscopic data, see Tables 1 and 2; (+)-ESIMS m/z 451
′a 3.71
+
−
−
(
dd, 11.4, 6.0) (dd, 11.4, 4.8)
′b 4.09 4.10
dd, 11.4, 1.8) (dd, 11.4, 1.8)
[M + Na] , (−)-ESIMS m/z 427 [M-H] , 463 [M + Cl] ; HR-ESI-
+
6
4.5 (m)
MS [M + Na] m/z 451.1800 (calcd for C17
.4. Absolute configurations of the sugars for compounds 1, 2, 3, 4, and 5
The absolute configurations of glucose were determined according
32
H O12Na, 451.1786).
(
1
2
3
4
5
6
″
″
″
″
″
4.39 (d, 7.8)
3.21 (m)
3.50 (m)
3.50 (m)
3.38 (m)
4.34 (d, 7.8) 4.37 (d, 7.8)
2
3.15 (m)
3.32 (m)
3.26 (m)
3.47 (m)
3.70
3.22 (m)
3.34 (m)
3.31 (m)
3.57 (m)
3.50 (m)
3.28 (m)
3.20 (m)
3.20 (m)
3.81 (dd,
12.0, 2.4)
3.61 (dd,
12.0, 6.0)
to a reported procedure [8]. Compound 1 (1 mg) was stirred in 1 M
HCl (1 mL) at 100 °C for 1 h. The reaction mixture was dried under
vacuum. Then water was added to the mixture, and the acidic solution
was evaporated to remove HCl. After drying under vacuum, the residue
was dissolved in pyridine (0.5 mL) containing L-cysteine methyl
ester hydrochloride (2 mg) and heated at 60 °C for 1.5 h. o-
Toylisothiocyanate (2 μL) was subsequently added, and the mixture
was heated at 60 °C for 2 h. Each reaction mixture was directly
analyzed by HPLC [consisting of a Waters 515 pump, Waters 2487
dual λ absorbance detector, and Knauer Smartline RI detector 2300
with a COSMOSIL packed column C18-AR II (250 mm × 4.6 mm i.d.,
″a 3.80 (m)
(
dd, 11.4, 4.8)
4.08
dd, 11.4, 1.8)
4.31 (d, 7.8) 4.37 (d, 7.8)
6
″b 3.80 (m)
4.15 (m)
(
1
2
3
4
5
6
‴
‴
‴
‴
‴
4.35 (d, 7.8)
3.18 (m)
3.32 (m)
3.24 (m)
3.30 (m)
3.15 (m)
3.32 (m)
3.26 (m)
3.22 (m)
3.62 (dd,
12.0, 5.4)
3.81
3.22 (m)
3.41 (m)
3.31 (m)
3.28 (m)
3.68 (dd,
12.0, 4.8)
3.30
‴a 3.60 (dd,
2.0, 5.4)
‴b 3.81
1
6
(dd, 12.0, 1.8) (dd, 12.0, 2.4) (br d, 10.2)
5
μM); temp, 35 °C; flow, 0.8 mL/min; eluate, CH
3 2
CN-H O (25:75)
1
1
4
Measured in MeOH-d at 600 MHz, with assignments confirmed by H– H COSY, NOESY,
HMQC and HMBC.
containing 50 mM H PO ] at 250 nm. The reaction conditions for D-
3
4
and L-glucose were the same as described above. Retention times of
authentic sugar derivatives, D-glucose (11.72 min), and L-glucose
(
10.58 min) were used for comparison with those from reaction mix-
tures. A peak at 11.63 min of the sugar derivative from 1 coincided
with the derivative of D-glucose. The absolute configurations of the
sugars for 2–5 were identified using the same method as 1.
+
−
−
m/z 594 [M + Na] , (−)-ESIMS m/z 570 [M-H] , 606 [M + Cl] ; HR-
+
ESI-MS [M + Na] m/z 594.2005 (calcd for C22H37NO16Na, 594.2005).
2.5. Assessment of inhibitory activities of α-glucosidase, lipase, DPP-IV,
aldose reductase, and FBPase
Table 2
1
3
C NMR spectroscopic data of compounds 1–5.
Detailed procedures for conducting assays for α-glucosidase, lipase,
DPP-IV, aldose reductase, and FBPase inhibition are provided in a pre-
vious study [9–13]. Acarbose, orlistat, INDP-2, epalrestat, and FBPI377-6
were used as positive controls.
No.
1
2
3
4
5
1
2
3
4
5
121.6
76.1
35.1
121.7
76. 1
34.9
8.86
24.3
100.5
74.8
78.0
71.6
76.9
70.0
104.9
75.1
77.8
71.6
77.2
70.5
104.9
75.2
77.7
71.6
77.9
62.7
122.2
72.5
28.6
27.4
26.0
74.3
85.9
17.6
24.3
106.4
75.7
78.1
71.6
78.0
62.7
26.1
74.3
85.5
17.6
24.4
106.1
75.1
77.9
71.5
77.2
69.8
104.9
75.6
77.9
71.6
78.0
62.7
8.82
3. Results and discussion
24.2
100.5
74.8
77.9
71.3
77.3
69.9
104.6
75.0
75.9
80.6
76.2
61.8
104.4
74.9
77.9
71.4
78.1
62.4
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
′
′
′
′
′
′
″
″
″
″
″
″
‴
‴
‴
‴
‴
‴
100.9
74.8
78.0
71.5
77.0
69.9
104.9
75.1
77.8
71.6
77.0
70.1
105.0
75.1
77.7
71.6
77.9
62.7
Compound 1 was isolated as a white amorphous powder. HR-ESI-MS
+
results showed a pseudomolecular ion [M + Na] at m/z 608.2169
(
calcd., 608.2161), which agrees with the molecular formula C23
H39NO16.
The IR spectrum of compound 1 shows the presence of a hydroxyl group
−
1
−1
1
at 3525 and 3406 cm , and a cyano group at 2515 cm . The H NMR
spectrum of compound 1 (Table 1) shows one methyl signal at δ 1.56
3H, s, H-5) and one ethyl signals at δ 1.03 (3H, t, J = 7.4 Hz, H-4), 1.82
1H, dt, J = 7.4, 14.1 Hz, H-3a), and 1.90 (1H, dt, J = 7.4, 14.1 Hz, H-3b).
H
(
H
(
C
Two quaternary C signals at δ 76.1 and 121.6 ppm were observed in
the 13C NMR and DEPT spectra (Table 2); Combination of these results
−1
with the signal at 2515 cm in IR spectrum, suggests the presence of 2-
methyl-2-hydroxyl-butanenitrile moieties. The presence of three
anomeric proton signals at δ
J = 7.8 Hz, H-1″), and 4.35 (1H, d, J = 7.8 Hz, H-1‴) in the H NMR
spectrum as well as three anomeric C signals at δ 100.5, 104.6, and
104.4 ppm and 15 characteristic C signals at δ 62.0–90.0 ppm in
H
4.56 (1H, d, J = 7.8 Hz, H-1′), 4.39 (1H, d,
1
Measured in MeOH-d
4
at 150 MHz, with assignments confirmed by DEPT, HMQC
and HMBC.
C
13
C
C

Q.-Y. Yang et al. / Fitoterapia 106 (2015) 78–83
81
Fig. 2. Significant HMBC correlations of compounds 1, 2, and 3.
NMR spectrum indicates the presence of three β-D-glucopyranose groups
in compound 1. Comparison of the NMR data of 1 and neolinustatin
reported in the literature [6] demonstrated that the two compounds
exhibited similar structures, and the major difference between them was
the replacement of a C-4″ hydroxyl in neolinustatin by a D-glucopyranose
unit in compound 1. These evidences suggested that compound 1 was a
cyanogenetic glucoside with three D-glucopyranoses. In the HMBC
spectrum (Fig. 2), correlations of H-1′/C-2; H-3, H-4, and H-5/C-2; H-1″/
produced D-glucose. This result, together with the coupling constants
(J = 7.8, 7.8, 7.8 Hz) of the anomeric protons and chemical shifts
(δ 100.5, 104.9, 104.9) of the anomeric carbons, confirmed the presence
C
of β-D-glucopyranose in compound 2. Thus, compound 2 was determined
as 2-[(O-β-D-glucopyranosyl-(1 → 6)-O-β-D-glucopyranosyl-(1 → 6)-β-
D-glucopyranosyl)oxy]-2-methyl-butanenitrile, and named linustatin B.
Compound 3 was isolated as an amorphous powder. Its molecular
formula was determined as C22H37NO16 from the positive HR-ESI-MS
+
2
C-6′; H -6′/C-1″; H-1‴/C-4″; and H-4″/C-1‴, together with their corre-
data at m/z 594.2005 [M + Na] (calcd. for C22
H37NO16Na, 594.2005).
sponding chemical shifts, verified the connections of C-2 and C-1′, C-1″
and C-6′, and C-1‴ and C-4″. The coupling constants (J = 7.8, 7.8, 7.8 Hz)
The IR spectrum of 3 showed the presence of hydroxyl group at
−
1
−1
3390 cm
and cyano group at 2502 cm . Comparison of the NMR
of the anomeric protons and chemical shifts (δ
C
100.5, 104.6, 104.4)
data (Tables 1 and 2) of compounds 3 and 2 indicated that the two
compounds had an identical sugar chain; the major difference between
them was the replacement of aglycone of 2-methyl-2-hydroxyl-
of the anomeric C demonstrated that three sugar moieties should be β-
anomeric configuration [14]. Hydrolysis of compound 1 in the presence
of acid produced D-glucose; This result, together with its NMR data,
demonstrated that all three glycosyl groups were D-glucopyranoses.
Thus, compound 1 was determined as 2-[(O-β-D-glucopyranosyl-
butanenitrile in compound 2 by 2-methyl-propionitrile [δ
H
1.66 (3H, s,
C-1 (122.2), C-2 (72.5),
C-3 (28.6), and C-4 (27.4) in 13C NMR] in compound 3. The correlations
of H-1′/C-2, H-1″/C-6′, H -6′/C-1″, H-1‴/C-6″, and H -6″/C-1‴ in the
1
H-4) and 1.68 (3H, s, H-3) in H NMR, and δ
C
(
1 → 4)-O-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranosyl)oxy]-2–
methyl-butanenitrile, and subsequently named linustatin A.
Compound 2 exhibited the molecular formula C23
2
2
HMBC spectrum (Fig. 2) substantiated the connections between C-1′
and C-2, C-1″ and C-6′, and C-1‴ and C-6″. In addition, hydrolysis of com-
pound 3 in the presence of acid produced the same D-glucose obtained
from compound 2. These above evidences confirm the presence of β-
D-glucopyranose in 3. Therefore, compound 3 was determined as 2-
[(O-β-D–glucopyranosyl-(1 → 6)-O-β-D-glucopyranosyl-(1 → 6)-β-D-
glucopyranosyl)oxy]-2-methyl-propionitrile, and named linustatin C.
Compound 4 was obtained as an amorphous powder, and its mole-
H
39NO16, as
+
indicated by the HR-ESI-MS results (m/z 608.2175 [M + Na] ; calcd.
for C23
H39NO16Na, 608.2161) and NMR data. Its IR spectrum showed a
−
1
−1
hydroxyl group at 3406 cm
and a cyano group at 2515 cm . The
NMR data of 2 (Tables 1 and 2) were similar to those of compound 1,
which showed the presence of a 2-methyl-2-hydroxyl-butanenitrile
moiety and three β-D-glucopyranose moieties. This suggested that
13
compound 2 could be an isomer of 1. The C NMR data of the sugar
cular formula was C11
289.1267 [M + Na] (calcd., 289.1258). The IR spectrum of compound
4 exhibits the presence of a hydroxyl group at 3359 cm and saturated
H
22
O
7
based on the positive HRESIMS data at m/z
+
moiety of compounds 1 and 2 were compared, and the downfield values
−
1
at C-6′ and C-6″ (i.e., δ
C
69.9 and 70.0 for C-6′, δ
C
61.8 and 70.5 for C-6″)
−
1
were observed; which suggested that three β-D-glucopyranose moieties
were linked through C-1‴/C-6″ and C-1″/C-6′. This association was also
confirmed by the HMBC correlations (Fig. 2) of H-1″ with C-6′, H
aliphatic protons at 2980, 2924, and 1382 cm . Signals at δ
J = 6.0 Hz, H-4), 1.11 (3H, s, H-1), 1.11 (3H, s, H-5), and 3.53 (1H, q, J =
6.0 Hz, H-3) and δ 26.0, 24.3, 17.6, 85.9, and 74.3 in the NMR spectrum
of compound 4 (Tables 1 and 2) indicate the presence of a 2-methyl-2,3-
dihydroxybutyl moiety. Anomeric proton and C signals at δ 4.35 (1H, d,
J = 8.4 Hz, H-1′) and δ 106.4 ppm, together with five characteristic C
H
1.18 (3H, d,
2
-6′
C
with C-1″, H-1‴ with C-6″, and H -6″ with C-1‴. The HMBC correlations
2
of C-2 with H-1′, H-3, H-4, and H-5 indicated that C-2 was connected
to C-1′. In addition, hydrolysis of compound 2 in the presence of acid
H
C
Fig. 3. Significant HMBC correlations of compounds 4 and 5.

82
Q.-Y. Yang et al. / Fitoterapia 106 (2015) 78–83
Fig. 4. The proposed biosynthetic pathway for the cyanogenic glucosides 1–3.
signals from δ
C
62.7 ppm to 78.1 ppm, confirm the presence of a glycosyl
possible pathways for cyanogenic glucosides 1, 2, and 3 are proposed
and outlined in Fig. 4. The first step catalyzed by P450 proceeds via two
successive N-hydroxylations of the amino group of the parent amino
acids (L-isoleucine for 1 and 2, L-valine for 3), followed by decarboxylation
and dehydration. The formed aldoxime is subsequently converted to
an α-hydroxynitrile through the action of a second cytochrome p450.
This reaction involves an initial dehydration reaction that forms a nitrile
and is followed by hydroxylation of the alpha carbon to generate a
cyanohydrin. Glycosilation of the cyanohydrin catalyzed by a UDPG-
glycosyltranferase is the final step for the synthesis of cyanogenetic glu-
cosides. However, it seems difficult to rationalize the biogenetic origin
of simple glycosides 4 and 5 by the amino pathway since the absence
of cyano group. As far as them, biosynthesis through glycosilation of al-
iphatic alcohol is probably a reasonable interpretation, although more
investigation needs to be done.
group. In the HMBC spectrum (Fig. 3) correlations of H-1′ with C-3 and
H-3 with C-1′ indicate that C-1′ is connected to C-3. Hydrolysis of com-
pound 4 in the presence of an acid produced D-glucose. This result, to-
gether with the corresponding NMR data, demonstrates the presence
of β-D-glucopyranose. Therefore, compound 4 was identified as 2-
methyl-2,3-dihydroxybutyl-3-O-β-D-glucopyranoside according to the
1
1
results of DEPT, H– H COSY, HMBC, and HSQC experiments; this com-
pound was subsequently named linustatin D.
Compound 5 was obtained as a white amorphous powder, and its
molecular formula was determined as C17
H
32
O
12 based on the positive
HRESIMS data at m/z 451.1800 [M + Na] (calcd. for C17 12Na,
51.1786). The IR spectrum of compound 5 demonstrates the presence
+
32
H O
4
−
1
of a hydroxyl group at 3388 cm
at 2979, 2913, and 1379 cm . The NMR data (Tables 1 and 2) of com-
and saturated aliphatic protons
−
1
pound 5 are similar to those of compound 4, in which the presence of a
The inhibitory activities of compounds 1–5 against α-glucosidase
(40 μM), lipase (10 μM), DPP-IV (10 μM), aldose reductase (10 μM),
and FBPase (10 μM) were evaluated. Isolates of compounds 1, 2, 3, 4,
and 5 showed moderate activities against aldose reductase with in-
hibition rates of 30.0%, 35.5%, 30.0%, 30.0%, and 28.4%, respectively
(epalrestat was used as the positive control, Table 3). In addition, all
compounds showed weak activities against α-glucosidase, DPP-IV, and
FBPase at the same concentration as the positive control drugs
(i.e., acarbose, INDP-2, epalrestat, and FBPI377-6, Table 3). All of the
compounds were no inhibitory effects on lipase at the same concentra-
tion as the positive drug orlistat.
2
-methyl-2,3-dihydroxybutyl moiety as well as a β-D-glucopyranose
moiety was observed. The remaining characteristic NMR data of com-
pound 5 [δ 4.35 (d, J = 7.8 Hz, H-1″) and δ 104.9, 75.6, 77.9, 71.6,
8.0, and 62.7] suggest the presence of another β-D-glucopyranose
H
C
7
1
3
moiety. The C NMR data of the sugar moiety in compounds 4 and 5
were compared, and the C-6′ in the β-D-glucopyranose group of
C
compound 5 was observed at a lower field (δ 69.8). This result suggests
that C-1″ of the second β-D-glucopyranose group is linked to C-6′ of the
first β-D-glucopyranose. HMBC correlations (Fig. 3) of H-1″ with C-6′
2
and H -6′ with C-1″ confirm this assumption, while HMBC correlations
of H-3 with C-1′ and H-1′ with C-3 indicate that the sugar chain is linked
to C-3. Hydrolysis of compound 5 in the presence of an acid produced
Conflict of interest
D-glucose; this result, together with the NMR data, demonstrates that
both glycosyl groups of compound 5 are β-D-glucopyranose moieties.
Finally, compound 5 was determined as 2-methyl-2,3-dihydroxybutyl-
The authors declare no conflict of interest.
3
-O-β-D-gluco pyranosyl-(1 → 6)-β-D-glucopyanoside and named
linustatin E.
From biogenesis point of view, cyanogenic glucosides are amino
Acknowledgment
This work was financially supported by National Mega-project for
Innovative Drugs (grant No. 2012ZX09301002-001-003). We are grateful
to the Department of Instrumental Analysis, Institute of Materia Medica,
Chinese Academy of Medical Sciences and Peking Union Medical College
for measuring the IR, UV, NMR, and MS spectra.
acid-derived secondary plant metabolites. Five protein amino acids,
valine, leucine, isoleucine, phenylalanine, and tyrosine, are considered
to be the primary precursors of their aglycones. The biosynthetic pathway
is catalyzed by two membrane-bound multifunctional cytochrome P450s
and an apparently soluble glucosyltransferase [15,16]. Therefore, the
Appendix A. Supplementary data
Table 3
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.fitote.2015.08.008.
Assessment of α-glucosidase, lipase, DPP-IV, aldose reductase, and FBPase inhibitory activity
(%) of compounds 1–5.
b
Lipasea DPP-IVa Aldose reductasea FBPasea
Compounds α-glucosidase
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3
4
6.6
9.8
29.5
11.5
6.6
3.5
0.0
3.2
0.0
0.0
18.6
8.1
14.0
22.1
14.0
30.0
35.5
30.0
30.0
28.4
13.1
23.3
18.2
23.3
19.9
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