Two novel steroidal alkaloid glycosides from the seeds of lycium barbarum

Article abstract of DOI:10.1002/cbdv.201000293

Two novel steroidal alkaloid glycosides, lycioside A (1) and lycioside B (2) were isolated from the seeds of Lycium barbarum. Their structures were determined by various spectroscopic analyses. Compounds 1 and 2 showed inhibitory activities with the IC₅₀ values of 75.3 and 72.8I?M against rat intestinal sucrase, and 63.4 and 59.1I?M against rat intestinal maltase. Copyright

Full text of DOI:10.1002/cbdv.201000293

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
2277
Two Novel Steroidal Alkaloid Glycosides from the Seeds of Lycium barbarum
a
b
a
a
c
b
d
by Kun Wang ) ), Tatsunori Sasaki ), Wei Li* ), Qin Li ), Yinghua Wang ), Yoshihisa Asada ),
a
a
Hiroyoshi Kato ), and Kazuo Koike )
a) Faculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan
(
e-mail: liwei@phar.toho-u.ac.jp)
b) Ningxia Institute for Drug Control, Yinchuan 750004, P. R. China
c) Laboratory Center, The Fourth Affiliated Hospital, China Medical University, Shenyang 110005,
P. R. China
d) Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda,
Chiba 278-8510, Japan
Two novel steroidal alkaloid glycosides, lycioside A (1) and lycioside B (2) were isolated from the
seeds of Lycium barbarum. Their structures were determined by various spectroscopic analyses.
Compounds 1 and 2 showed inhibitory activities with the IC50 values of 75.3 and 72.8 mm against rat
intestinal sucrase, and 63.4 and 59.1 mm against rat intestinal maltase.
Introduction. – Lycium barbarum L. (Solanaceae) is a perennial bush widely
distributed in southeastern Europe and eastern Asia, and mainly cultivated in the
northwest areas of P. R. China [1][2]. Various parts of the plant, including fruits, root
barks, and leaves, have been used in Traditional Chinese Medicine (TCM). The fruits,
known as wolfberry or ꢀGojiꢁ berry, have a high popularity on the novel food market,
and were used for prevention and treatment of various chronic diseases, such as
diabetes, cancer, and hyperlipidemia [1][3][4]. Chemical studies on the fruits have
demonstrated the presence of flavonoids, carotenoids, and polysaccharides with a large
variety of biological activities [1][5–7]. The seeds account for a quarter of the total
weight of the dried fruits, and, in most cases, the fruits and seeds are consumed
together. However, few chemical studies have been conducted so far on the seeds of L.
barbarum; however, sterols, triterpenes, and fatty acids have been reported from the
seeds of a related species, L. chinense [8–10]. As a part of our studies on bioactive
compounds from TCM [11–13], we investigated on the seeds of L. barbarum. Here, we
report the isolation and structural elucidation of two novel steroidal alkaloid
glycosides, named lycioside A (1) and lycioside B (2), from the seeds of L. barbarum.
The inhibitory activities of 1 and 2 against rat intestinal sucrase and maltase were also
evaluated.
Results and Discussion. – The MeOH extract of the seeds of L. barbarum was
partitioned between BuOH and H O. The BuOH-soluble portion was subsequently
2
separated by silica gel, ODS column chromatography and reversed-phase HPLC to
provide lyciosides A and B (1 and 2, resp.) (Fig. 1).
Lycioside A (1) was obtained as an amorphous white powder. In positive-ion mode
þ
HR-ESI-MS exhibited a pseudo-molecular-ion peak at m/z 950.5097 ([MþNa] ),
ꢂ 2011 Verlag Helvetica Chimica Acta AG, Zꢃrich

2
278
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Fig. 1. Chemical structures of 1 and 2
suggesting the molecular formula of C H NO . The IR spectrum exhibited
4
7
77
17
ꢀ
1
ꢀ1
13
absorptions for a OH (3339 cm ) and an amide group (1639 cm ). The C-NMR
spectrum (Table 1) displayed 47 C-atom resonances including those for three sugar
1
anomeric C-atoms at d(C) 105.2, 101.9, and 99.6. In the H-NMR spectrum (Table 1),
resonances corresponding to the anomeric H-atoms were observed at d(H) 6.22, 5.13,
and 4.97. By acid hydrolysis, d-glucose and l-rhamnose were determined as the sugar
components. Starting from the anomeric H-atoms, all sugar resonances were assigned
1
1
by detailed analysis of H, H-COSY, TOCSY, HMQC, HMBC, and NOESY spectra,
evidencing the presence of two b-d-glucopyranosyl and one a-l-rhamnopyranosyl
moieties. The sugar sequence was established by analysis of the following HMBC
correlations: from d(H) 6.22 (HꢀC(1)(Rha)) to d(C) 77.6 (C(2)(Glc)), from d(H) 5.13
(
HꢀC(1)(Glc’)) to d(C) 82.2 (C(4)(Glc)), and from d(H) 4.97 (HꢀC(1)(Glc)) to d(C)
7
7.3 (C(3)). Furthermore, the presence of the acetamide moiety was confirmed by
characteristic resonances at d(C) 170.0 (C(28)), and d(H) 8.38 (NH) and 2.09
Me(29)). Excluding the sugar and acetamide moieties, the aglycon part of 1 contained
7 C-atoms. The characteristic resonances, including those of three tertiary Me groups
at d(H) 1.64 (Me(21)), 0.87 (Me(19)), 0.71 (Me(18)), a secondary Me group at d(H)
.00 (Me(27)), and of olefinic C-atoms at d(C) 152.3 (C(22)) and 103.7 (C(20)),
(
2
1
1
1
suggested the presence of a steroid skeleton [14]. The H, H-COSY spectrum was
carefully analyzed with HꢀC(1) signal and the Me doublet at d(H) 1.00 (J¼6.6), the

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
2279
latter attributable to Me(27) as the starting points. The successive correlations starting
from HꢀC(1) and terminating at HꢀC(17), and those staring from Me(27) ending at
HꢀC(23) were shown in Fig. 2. Moreover, the HMBC correlations from d(H) 2.30
(
(
H ꢀC(23)) to d(C) 152.3 (C(22)), and from d(H) 1.64 (HꢀC(21)) to d(C) 103.7
a
C(20)) supported the location of the double bond, i.e., C(20)¼C(22). The relative
configurations of the aglycon part were established on the basis of detailed NOESY
spectrum analysis (Fig. 3). NOESY Correlations HꢀC(3)/HꢀC(5), HꢀC(5)/HꢀC(9),
HꢀC(9)/HꢀC(14), HꢀC(14)/HꢀC(16), and HꢀC(14)/HꢀC(17) indicated that these H-
atoms were all a-oriented. The NOESY correlations HꢀC(8)/Me(18), and Me(18)/
Me(19) allowed these to be assigned as b. As a consequence, the relative configuration
for each ring junction, A/B, B/C, and C/D are trans, and D/E is cis. The acetamido group
at C(26) was confirmed by the HMBC correlations from the amide H-atom signal at
d(H) 8.38 (NH) to that of the C¼O C-atom at d(C) 170.0 (C(28)) and the signal at 45.6
(
C(26)), and from the signal at d(H) 2.09 (HꢀC(29)) to that at d(C) 170.0 (C(28)). The
absolute configuration at C(25) was not determined and needs further investigation.
Thus, the structure of lycioside A (1) was established as N-[(3b,5a)-3-({O-b-d-
glucopyranosyl-(1!4)-O-[a-l-rhamnopyranosyl-(1!2)]-b-d-glucopyranosyl}oxy)fu-
rost-20(22)-en-26-yl]acetamide.
Fig. 2. 1H, H-COSY (—) and key HMBC (H!C) correlations of 1 and 2
1

2
280
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Table 1. 1H- and 13C-NMR Data of 1 and 2 (at 500 and 125 MHz, resp., in (D )pyridine; d in ppm
5
relactive to TMS, J in Hz)
Position
1
1
2
d(H)
d(C)
d(H)
d(C)
1.58–1.62 (m), 0.80–0.83 (m)
37.2
1.59 (m),
37.1
0
.80 (td, J¼4.2, 13.8)
2
3
4
5
6
7
8
9
2.04–2.07 (m), 1.78–1.81 (m)
3.90–3.92 (m)
1.88–1.92 (m), 1.65–1.70 (m)
0.89–0.93 (m)
1.16–1.18 (m), 0.87–0.90 (m)
1.52–1.55 (m)
1.37–1.40 (m)
29.9
77.3
34.4
46.7
29.0
32.6
35.1
54.5
35.9
21.4
2.03–2.07 (m), 1.77–1.79 (m)
3.92–3.94 (m)
29.8
77.2
34.3
46.5
28.8
32.3
35.1
54.3
35.8
21.1
1.89–1.92 (m), 1.64–1.67 (m)
0.91 (dd, J¼3.4, 9.9)
1.13–1.15 (m)
1.52–1.55 (m), 0.82–0.84 (m)
1.42 (br. dd, J¼3.2, 8.7)
0.52 (td, J¼3.2, 12.3)
0.50–0.56 (m)
1
1
0
1
1.45–1.49 (m),
1.43–1.45 (m),
1
.26 (br. dd, J¼3.4, 12.6)
1.72 (ddd, J¼2.1, 5.1, 12.6),
.10 (ddd, J¼2.1, 3.0, 5.1)
1.23 (qd, J¼4.2, 8.9, 12.8)
1.68 (ddd, J¼2.2, 4.5, 12.8),
1.05 (ddd, J¼2.2, 2.5, 4.5)
1
2
39.9
39.9
1
1
1
1
1
1
1
1
2
2
2
2
3
4
5
6
7
8
9
0
1
2
3
43.8
54.8
34.4
84.6
64.7
14.4
12.4
103.7
11.8
41.0
56.2
32.0
81.3
64.2
16.4
12.3
40.3
16.0
112.5
30.7
0.84–0.86 (m)
2.10–2.12 (m), 1.42–1.43 (m)
4.77–4.82 (m)
2.44 (d, J¼10.0)
0.71 (s)
0.98 (dd, J¼3.2, 8.7)
1.94–1.98 (m), 1.34–1.37 (m)
4.43–4.45 (m)
1.71–1.73 (m)
0.81 (s)
0.87 (s)
0.86 (s)
2.23–2.26 (m)
1.17 (d, J¼6.9)
1.64 (s)
152.3
23.7
2.27–2.32 (m), 2.19–2.25 (m)
2.00 (br. d, J¼4.6),
1
.80 (dd, J¼2.3, 7.1)
24
25
26
27
28
29
1.82–1.85 (m), 1.44–1.47 (m)
1.85–1.88 (m)
3.40–3.46 (m), 3.33–3.38 (m)
1.00 (d, J¼6.6)
32.4
33.5
45.6
17.9
170.0
23.1
1.74–1.77 (m), 1.32–1.34 (m)
1.82–1.85 (m)
29.1
34.2
45.7
17.7
169.9
23.0
3.47–3.52 (m), 3.32–3.35 (m)
1.00 (d, J¼6.6)
2.09 (s)
2.10 (s)
NH
8.38 (t, J¼6.2)
8.38 (t, J¼6.2)
2
2-MeO
3.25 (s)
47.1
Glca)
1
2
3
4
5
6
4.97 (d, J¼7.3)
4.17–4.20 (m)
99.6
77.6
76.3
82.2
77.7
62.1
4.98 (d, J¼7.2)
4.18–4.20 (m)
99.5
77.5
76.1
82.0
77.6
61.9
3.92 (t, J¼6.9)
3.90 (t, J¼6.9)
4.20–4.22 (m)
4.22–4.25 (m)
4.24–4.27 (m)
4.25–4.27 (m)
4.52 (br. s), 4.28–4.30 (m)
4.53 (dd, J¼3.5, 10.1),
4.30–4.32 (m)
Glc’a)
1
2
3
5.13 (d, J¼7.8)
4.07 (t, J¼7.8)
4.21–4.24 (m)
105.2
75.0
78.3
5.13 (d, J¼8.1)
4.06 (t, J¼8.1)
4.22–4.24 (m)
105.1
74.9
78.2

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
2281
Table 1 (cont.)
Position
1
2
d(H)
d(C)
d(H)
d(C)
4
5
6
4.27 (t, J¼9.0)
71.3
78.5
62.2
4.27–4.29 (m)
3.97–3.99 (m)
4.45–4.48 (m),
4.33–4.35 (m)
71.1
78.4
62.0
3.97–4.00 (m)
4.47 (dd, J¼2.3, 12.0),
4.33 (dd, J¼6.6, 12.0)
Rhaa)
1
2
3
4
5
6
6.22 (s)
101.9
72.4
72.8
74.1
69.4
18.7
6.22 (s)
4.74 (br. s)
4.56 (dd, J¼3.2, 9.2)
4.32 (t, J¼9.2)
4.88–4.92 (m)
1.75 (d, J¼6.0)
101.8
72.3
72.6
74.0
69.3
18.5
4.74 (dd, J¼1.4, 3.2)
4.56 (dd, J¼3.2, 9.4)
4.32 (t, J¼9.4)
4.88–4.93 (m)
1.75 (d, J¼6.2)
a) Glc, glucopyranosyl; Rha, rhamnopyranosyl.
Fig. 3. Key NOE correlations ($) of the aglycone moieties of 1 and 2
Lycioside B (2) was also obtained as an amorphous white powder. Its molecular
formula was determined as C H NO by the pseudo-molecular-ion peak at m/z
4
8
81
18
þ
1
9
82.5361 ([MþNa] ) in positive-ion mode HR-ESI-MS. Comparing H- and
1
3
C-NMR data (Table 1) of 2 and 1, superimposable resonances assignable to the
sugar part, acetamido moiety, and A, B, C, D ring systems of the aglycon part, and
differences of the resonances of the E ring were observed. Namely, the resonances of
olefinic C-atoms at d(C) 152.3 and 103.7 in 1 were replaced by the signals of an O-
substituted quaternary C-atom at d(C) 112.5 and of a CH C-atom at d(C) 40.3 in 2,
together with that of a new MeO group (d(H) 3.25 (MeO), d(C) 47.1 (MeO))). The
1
1
observed H, H-COSY correlations HꢀC(20)/HꢀC(17) and HꢀC(21)/HꢀC(20), as

2
282
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
shown in Fig. 2, were new. The HMBC correlation d(H) 3.25 (MeO) to d(C) 112.5
C(22)) evidenced the MeO group at C(22). The NOESY correlations between
(
HꢀC(17)/HꢀC(21) and HꢀC(21)/MeO suggested these protons to be a-oriented. Thus,
lycioside B (2) was determined as N-[(3b,5a,22b)-3-({O-b-d-glucopyranosyl-(1!4)-
O-[a-l-rhamnopyranosyl-(1!2)]-b-d-glucopyranosyl}oxy)-22-methoxyfurostan-26-yl]-
acetamide.
Lyciosides A and B (1 and 2, resp.) are the first two examples of furosten-type
steroidal alkaloid glycosides. The use of MeOH in the isolation protocol suggests that
compound 2 might be an artifact of 1. Compounds 1 and 2 are also the first two
examples of steroidal alkaloid glycosides from L. barbarum.
Type-2 diabetes mellitus has become one of the most serious worldwide health
problems [15]. Mammalian a-glucosidase is the key enzyme catalyzing the final step in
the digestive process of carbohydrates. Inhibition of a-glucosidase has been demon-
strated to be an effective therapeutic target for Type-2 diabetes mellitus [16][17]. Since
the MeOH extract and BuOH fraction used in this study showed inhibitory activities
against rat intestinal sucrase and maltase, 1 and 2 were also evaluated for these
activities. Lyciosides A and B (1 and 2, resp.) exhibited moderate inhibitory activities
with IC values of 75.3ꢁ3 and 72.8ꢁ1.5 mm, respectively, against rat intestinal sucrase,
50
and 63.4ꢁ0.4 and 59.1 ꢁ 0.2 mm against maltase, respectively, while the corresponding
values of the positive control of acarbose were 11.2ꢁ0.3 and 8.7ꢁ0.2 mm, respectively
(
Table 2). Steroidal alkaloids and their glycosides occurring in numerous Solanum
species are known to possess a large variety of bioactivities including antitumor [18],
antifungal [19], antiviral [20], and embryotoxic activeties [21], but to the best of our
knowledge, the present study is the first report of steroidal alkaloid glycosides with a-
glucosidase inhibitory activities.
Table 2. a-Glucosidase Inhibitory Activities of Extracts and Compounds
Sample
IC50a)
Sucrase
Maltase
b
b
MeOH Extract
BuOH Fraction
Lycioside A (1)
Lycioside B (2)
0.95ꢁ0.05 )
0.57ꢁ0.02 )
b
b
0.98ꢁ0.03 )
0.84ꢁ0.08 )
75.3ꢁ3.3
72.8ꢁ1.5
11.2ꢁ0.3
63.4ꢁ0.4
59.1ꢁ0.2
8.7ꢁ0.2
c
Acarbose )
a) Values are meansꢁS.D. , n ¼ 3, IC50 in mm. b) IC50 in mg/ml. ) Positive control.
c
Experimental Part
General. HPLC Separations: JASCO PU-2080 HPLC system equipped with a Shodex RI-101
differential refractometer detector and a YMC-Pack Pro C18 column (150 mmꢂ20 mm i.d.; particle size,
5
mm); flow rate, 0.8 ml/min. Column chromatography (CC): silica gel (silica gel 60N, Kanto Chemical
Co., Inc., Tokyo, Japan), and ODS (100–200 mesh, Chromatorex DM1020T ODS, Fuji Silysia Chemical
Ltd., Aichi, Japan). TLC: Kieselgel 60 F254 plates (E. Merck, USA). Optical rotations: JASCO DIP-370
ꢀ
1
digital polarimeter in a 0.5-dm length cell. IR Spectra: JASCO FT/IR-300E spectrometer; KBr; ˜n in cm
.
1
13
ESI-MS and HR-ESI-MS: JEOL JMS-T100LPAccuTOF LC-plus mass spectrometer. H- and C-NMR

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
2283
spectra: JEOL ECP-500 spectrometer, with TMS as the internal reference, and chemical shifts in d
ppm].
Plant Material. The seeds of L. barbarum were collected in October 2006, in Guyuan City, Ningxia
[
Hui Autonomous Region, P. R. China, and were identified by Prof. Shirui Xing (Ningxia Institute for
Drug Control, P. R. China). A voucher specimen (TH320) has been deposited with the Department of
Pharmacognosy, Faculty of Pharmaceutical Sciences, Toho University.
Extraction and Isolation. The seeds of L. barbarum (1.0 kg) were extracted by means of ultrasonic-
assisted method with MeOH (5ꢂ3 l, each 1 h) at r.t. After evaporation of the solvent, the resulting
residue (120.9 g) was partitioned between BuOH and H O (each 1 l). The BuOH extract (11.2 g) was
2
subjected to CC (silica gel; CHCl /MeOH/H O 100 :0 :0, 98 :2 :0, 95 :5 :0, 9 :1:0, 60 :20 :3, 6 :4 :1) to
3
2
give six fractions. Fr. 4 (117.3 mg) was subjected to CC (ODS; 50% MeOH), and then separated by
HPLC (70% MeOH) to give 1 (9.0 mg; t 24.1 min), whereas Fr. 5 (1.7 g) was separated by HPLC (60%
R
MeOH) to give 2 (18.0 mg; tR 19.8 min).
Lycioside A (¼ N-[(3b,5a)-3-({O-6-Deoxy-a-l-mannopyranosyl-(1!2)-O-[b-d-glucopyranosyl-
(
1!4)]-b-d-glucopyranosyl}oxy)furost-20(22)-en-26-yl]acetamide; 1). Amorphous white powder.
2
5
1
[
a]
D
¼–27 (c¼0.10, MeOH). IR: 3339, 2948, 2836, 1634, 1455, 1261, 1083, 1026, 804. H- and
13
þ
þ
C-NMR: see Table 1. ESI-MS (pos.): 950.6 ([M þ Na] ). HR-ESI-MS (pos.): 950.5097 ([M þ Na] ,
þ
C H NaO ; calc. 950.5089).
47
77
17
Lycioside B (¼ N-[(3b,5a,22b)-3-({O-6-Deoxy-a-l-mannopyranosyl-(1!2)-O-[b-d-glucopyrano-
syl-(1!4)]-b-d-glucopyranosyl}oxy)-22-methoxyfurostan-26-yl]acetamide; 2). Amorphous white pow-
2
5
1
der. [a]
D
¼ ꢀ49 (c¼0.10, MeOH). IR: 3406, 2946, 2836, 1634, 1454, 1250, 1100, 1026, 883. H- and
13
þ
þ
C-NMR: see Table 1. ESI-MS (pos.): 982.5 ([M þ Na] ). HR-ESI-MS (pos.): 982.5362 ([M þ Na] ,
þ
C H NaO ; calc. 982.5352).
48
81
18
Acid Hydrolysis and Determination of the Absolute Configuration of Sugars in 1 and 2. Acid
hydrolysis of compounds 1 and 2 (each 1.0 mg) was carried out by the same procedure as reported in [11].
Briefly, acid hydrolysis was carried out in 1m HCl (dioxane/H O 1:1; 1.0 ml) at 1008 for 1 h under Ar.
2
After the soln. was extracted with AcOEt (1.0 mlꢂ3), the aq. layer was evaporated to give the sugar
fraction. The soln. of the sugar fraction in pyridine (0.1 ml) was added to a soln. of 0.08m l-cysteine
methyl ester hydrochloride in pyridine (1.5 ml) and kept at 608 for 1.5 h. The residue was
trimethylsilylated with 1-(trimethylsilyl)-1H-imidazole (0.1 ml) for 2 h, and then partitioned between
hexane and H O (0.3 ml each). The GC/MS analyses of the hexane extracts and those of derivatives from
2
d-glucose, l-glucose and l-rhamnose standards, showed the presence of d-glucose and l-rhamnose in 1
and 2, resp.
Rat Intestinal Sucrase and Maltase Inhibitory Activity. The sucrase and maltase inhibitory activities of
the isolated compounds were determined by the same procedure as described in [11]. Briefly, a-
glucosidase prepared from rat intestinal acetone powder (Sigma-Aldrich Japan Co., Tokyo, Japan) was
used for sucrase and maleate inhibition assay. The reaction mixtures containing the above enzyme soln.
(
0.1 ml), substrate soln. (0.1 ml), samples of various concentrations in MeOH (0.01 ml), and 56 mm
maleate buffer (0.04 ml) were incubated for 60 min at 378, and heated at 1028 for 10 min to stop the
reaction. The glucose release was determined in a 96-well plate using a glucose assay kit (Glucose CII-test
Wako, Wako Pure Chem. Co., Osaka, Japan), based on the glucose oxidase/peroxidase method. Acarbose
was used as positive control with the IC50 value of 11.2ꢁ0.3 mm to sucrase and 8.7ꢁ0.2 mm to maltase, as
determined using the same assay system.
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Received October 14, 2010