A new steroidal glycoside from the seeds of Hyoscyamus niger

Article abstract of DOI:10.1080/14786419.2013.805331

A new steroidal glycoside hyoscyamoside G (1), together with two known analogues hyoscyamoside E (2) and hyoscyamoside F₁ (3), was isolated from the seeds of Hyoscyamus niger. The structure of 1 was established as (22R,24Z)-1α,3β,7β,22,26-pentakishydroxylergost-22-O-β-d- gulcopyranosyl-5,24-diene-26-O-β-d-glucopyranoside, by means of chemical and spectroscopic methods including HRESI-MS, 1D and 2D NMR. In vitro, compound 2 showed cytotoxicity against human lung cancer cell H460 with IC₅₀ value of 66 g/mL.

Full text of DOI:10.1080/14786419.2013.805331

Natural Product Research, 2013
Vol. 27, No. 21, 1971–1974, http://dx.doi.org/10.1080/14786419.2013.805331
A new steroidal glycoside from the seeds of Hyoscyamus niger
Wenna Zhang, Wei Zhang, Jianguang Luo and Lingyi Kong*
Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang,
Nanjing 210009, P.R. China
(
Received 17 December 2012; final version received 27 April 2013)
A new steroidal glycoside hyoscyamoside G (1), together with two known analogues
hyoscyamoside E (2) and hyoscyamoside F1 (3), was isolated from the seeds of
Hyoscyamus niger. The structure of 1 was established as (22R,24Z)-1a,3b,7b,22,26-
pentakishydroxylergost-22-O-b-D-gulcopyranosyl-5,24-diene-26-O-b-D-glucopyrano-
side, by means of chemical and spectroscopic methods including HRESI-MS, 1D and
2D NMR. In vitro, compound 2 showed cytotoxicity against human lung cancer cell
H460 with IC50 value of 66 mg/mL.
Keywords: Hyoscyamus niger; steroidal glycoside; Solanaceae; cytotoxicity
1
. Introduction
The genus Hyoscyamus (Solanaceae) is well known for its anticholinergic properties and
alkaloidal constituents (Chinese Pharmacopoeia Commission 2010). However, the presence of
some non-alkaloidal constituents, such as anolides, lignanamides (Ma et al. 2002), tyramine
derivatives and steroidal glycosides (Lunga et al. 2008a, 2008b) has also been reported in
Hyoscyamus niger L. (Li et al. 2011). We earlier reported the lignanamides from the seeds of
H. niger (Zhang et al. 2012). In our continuing search for bioactive constituents from H. niger,
three steroidal glycosides were obtained. Herein, we report the isolation and structural
elucidation of a new steroidal glycoside (1).
2
. Results and discussion
2
D
5
Hyoscyamoside G (1) was obtained as white amorphous powder, ½aꢀ 242 (c 0.09, CH OH),
3
–
negative-ion HRESI-MS (m/z, 785.4329, [M 2 H] ) revealed the molecular formula of
1
compound 1 to be C H O (calcd for 785.4328). The H NMR spectrum showed signals due
to four tertiary methyl groups at d 0.78, 1.02, 1.84, 1.86; a secondary methyl groups at d 1.04
4
0 66 15
(
[
d, J ¼ 6.5 Hz); one olefinic proton (d 5.74, d, J ¼ 4.5 Hz), together with two anomeric protons
d 4.26 (d, J ¼ 7.5 Hz), 4.29 (d, J ¼ 7.5 Hz)]. Moreover, one set of oxygenated methylene
protons at d 4.13, 4.42 (each 1H, ABq, J ¼ 10.5 Hz) indicated the presence of –CH O–
2
group. The DEPT spectrum showed 40 carbon signals, of which 28 could be assigned to the
signals of the aglycone composed of five methyl groups (d 12.1, 13.5, 18.2, 18.9, 19.3), one
hydroxymethyl (d 71.3), four oxygenated methine carbons (d 66.0, 66.7, 73.6, 83.2) and two
1
13
double bonds (d 127.4, 128.1, 134.6, 144.3). The full assignments of H NMR and C NMR
signals were accomplished by a combination of HSQC, HMBC and ROESY experiments
(
Figure 1). The NMR data of the aglycone were similar with those of cilistol v isolated from
*
Corresponding author. Email: cpu_lykong@126.com
q 2013 Taylor & Francis

1
972
W. Zhang et al.
Figure 1. Structure of compound 1 with selected HMBC (H–C) correlations.
Solanum cilistum (Zhu et al. 2001) with the exception of the carbon signal d 66.0 (C-7) instead of
d 32.3, indicating an additional OH group at C-7, which was further confirmed by the HMBC
correlation between H-6 (d 5.74) and C-7 (d 66.0). From the anomeric protons of each
monosaccharide moiety, all the protons within each spin system were assigned by HMBC and
ROESY analyses (Figure 1). The b-configuration of the anomeric proton of the glucopyranosyl
residue was assigned based on its JH1–H2 value (J ¼ 7.5 Hz). Acid hydrolysis of 1 liberated
D-glucose, which was identified by comparison Thin-layer chromatography (TLC) analysis and
opticalrotationdispersionwithauthenticglucose.Theaglyconewasdeducedasanewcompoundon
the basis of 1D and 2D NMR data. The linkages of sugars at C-22 and C-26 were determined by
0
00
HMBC correlations between H-1 (d 4.26) and C-22 (d 83.2) and H-1 (d 4.29) and C-26 (d 71.3),
respectively. Thus, the plain structure for 1 is presented as shown. The geometry at the double bond
atC-24wasdeducedasZonthebasisofROESYbetweenmethylgroupsatC-27andC-28(Zhuetal.
2
001). Since the proton signals at C-1, C-3 displayed as a broad singlet (W1/2 ¼ 2.0 Hz), multiplet
W1/2 ¼ 23Hz), thestereoconfigurationsofthe hydroxylgroupsatC-1,C-3wereconcludedtobea,
b, respectively (Zhu et al. 2001). Furthermore, the ROESY correlations of H-9 (d 1.94) with H-14(d
.55) and H-7 (d 3.74), and of H-8 (d 1.48) with H-18 (d 0.78) and H-19 (d 1.02) suggested that B/C
(
1
and C/D trans ring junctions as usual steroids (Lu et al. 2011) and the 7-OH in b-orientation. The
multiplicity of H-22 as a broad double triple with couplings of about 9.0 and 3.5 Hz was consistent
with a proton adjacent to one methylene and one methane. This is very similar to the appearance of
H-22 in steroids with straight chain reported from Solanaceae family (Waiss et al. 1993). The
configuration at C-22 was thus deduced as R based on identical biogenesis. Therefore, compound 1
was formulated as (22R,24Z)-1a,3b,7b,22,26-pentakishydroxylergost-22-O-b-D-glucopyranosyl-
5
,24-diene-26-O-b-D-glucopyranoside (Figure 1), and named hyoscyamoside G.
The structures of the known compounds were identified as hyoscyamoside E (2) (Lunga et al.
2
008b) and hyoscyamoside F (3) (Lunga et al. 2008b) by comparison of their spectroscopic data
1
to previously reported values.
Compounds 1–3 were tested for in vitro cytotoxic activity against the HCT116 (human
colon cancer), H460 (human lung cancer) and SMMC-7721 (human hepatoma cancer) cells. The
result showed that compound 2 exhibited moderate cytotoxic activities against human lung
cancer cell H460, with IC50 of 66 mg/mL. Fluorouracil was used as a positive control with IC50
of 12 mg/mL.

Natural Product Research
1973
3
3
. Experimental
.1. General
Optical rotations were measured with a JASCO P-1020 polarimeter (Jasco, Tokyo, Japan); IR
spectra (KBr disks) were recorded on a Bruker Tensor 27 spectrometer (Bruker, Karlsruhe,
Germany). NMR spectra were measured in CD OD at 303 K on Bruker ACF-500 NMR
3
1
13
instruments ( H NMR: 500 MHz and C NMR: 125 MHz), with tetramethylsilane as internal
standard. Electrospray ionisation (ESI) and high-resolution ESI mass spectral data were acquired
on an Agilent 1100 series LC/MSD ion trap mass spectrometer. All solvents used were of
analytical grade (Jiangsu Hanbang Science and Technology Co., Ltd, Nanjing, China). Column
chromatography was performed on silica gel H (200–300 mesh, Qingdao Marine Chemical
Industry, Qingdao, China), D-101 macroporous resin (Bonchem, Cangzhou, China), MCI gel
CHP-20 (Mitsubishi, Tokyo, Japan) and ODS (40–63 mm, FuJi, Aichi, Japan). TLC was
performed on precoated silica gel GF254 plates (Qingdao Marine Chemical Co. Ltd) and
detected by spraying with 10% H SO in EtOH (v/v).
2
4
3
.2. Plant material
The seeds of H. niger were purchased from local market in Bozhou, Anhui Province, China, in
September 2010. The botanical identification was made by Prof. Min-jian Qin, Department of
Medicinal Plants, China Pharmaceutical University. A voucher specimen (No. 100705) is
deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University.
3
.3. Extraction and isolation
Air-dried seeds of H. niger (15 kg) were ground and extracted with 95% EtOH under reflux.
After concentration of the solution, the obtained crude extract (1 kg) was suspended in water, and
then partitioned with petroleum ether and CHCl . Subsequently, the H O layer (22 g) was
3
2
subjected to macroporous resin D101 column chromatography using a gradient of EtOH in H O
2
(
H O, 70:30) was subjected to an MCI gel, eluting with CH OH–H O (30:70–100:0, gradient
0:100, 30:70, 70:30, 100:0, v/v) to afford four fractions (AA–AD). The fraction AC (EtOH–
2
3
2
system) to obtain fractions of AC1–AC4. The subfraction AC2 (1 g) was further separated to
ODS column chromatography and medium-pressure preparation liquid chromatography
CH OH–H O (40:60–100:0, gradient system) to give rise to compound 1 (7.0 mg). The
3
2
subfraction AC3 (2.5 g) was isolated to column chromatography on silica gel eluting with
mixtures of CHCl –CH OH–H O (70:25:5) to afford compounds 2 (28.0 mg) and 3 (54.5 mg).
3
3
2
3
.4. Determination of cytotoxic activities
Compounds 1–3 were evaluated for cytotoxic activity against HCT116 (human colon cancer),
SMMC-7721 (human hepatoma cancer) and H460 (human lung cancer) cells by an
3
-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as described in the
literature (Lu et al. 2009). The concentration of test compounds that gave 50% inhibition of
cell growth was expressed as the IC50 value. Fluorouracil was used as a positive control, and the
experiments were conducted for three independent replicates.
3
.5. Hyoscyamoside G (1)
2
D
5
–1
(cm ): 3439,
max
White amorphous powder (CH OH); ½aꢀ 242 (c 0.09, CH OH); IR (KBr) n
3
3
–
–
2
932, 1076, 1046; ESI-MS: m/z 785 [M 2 H] , 605 [M 2 H–Glu–H O] ; HRESI-MS: m/z
2
–
1
[
M 2 H] calcd for C H O , 785.4328; found: 785.4329. H NMR (500 MHz, CD OD): d 3.84
40 65 15 3

1
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W. Zhang et al.
(
H, brs, H-1), 1.75 (H, m, H-2), 2.04 (H, m, H-2), 3.93 (H, m, H-3), 2.34 (H, m, H-4), 2.37 (H, m, H-
4
), 5.74 (H, d, J ¼ 4.5 Hz, H-6), 3.74 (H, t, J ¼ 4.5 Hz, 7.5 Hz, H-7), 1.94 (H, m, H-9), 1.52 (H, m,
H-11), 1.58 (H, m, H-11), 1.25 (H, m, H-12), 2.06 (H, m, H-12), 1.84 (H, m, H-16), 1.22 (H, m,
H-16), 1.24 (H, m, H-17), 0.78 (3H, s, H-18), 1.02 (3H, s, H-19), 2.18 (H, m, H-20), 1.04 (3H, d,
J ¼ 6.5 Hz, H-21), 3.91 (H, br dt, J ¼ 9.0 Hz, 3.5 Hz, H-22), 2.02 (H, m, H-23), 2.48 (H, dd,
J ¼ 11.0 Hz, 18.0 Hz, H-23), 4.13(H, d, J ¼ 10.5 Hz, H-26), 4.42 (H, d, J ¼ 10.5 Hz, H-26), 1.86
0
00
(
3H, s, H-27), 1.84 (3H, s, H-28), 4.26 (H, d, J ¼ 7.5 Hz, H-1 -Glc), 4.29 (H, d, J ¼ 7.5 Hz, H-1 ’-
3
1
Glc). C NMR (500 MHz, CD OD): d 73.6 (C-1), 39.1 (C-2), 66.7 (C-3), 42.4 (C-4), 144.3 (C-5),
3
1
5
27.4 (C-6), 66.0 (C-7), 39.0 (C-8), 35.1 (C-9), 43.3 (C-10), 21.0 (C-11), 40.6 (C-12), 43.7 (C-13),
0.7 (C-14), 28.7 (C-15), 25.3 (C-16), 54.4 (C-17), 12.1 (C-18), 18.9 (C-19), 42.1 (C-20), 13.5
(
(
(
C-21), 83.2 (C-22), 34.5 (C-23), 128.1 (C-24), 134.6 (C-25), 71.3 (C-26), 19.3 (C-27), 18.2
0
0
0
0
0
0
C-28), 105.7 (C-1 , 22-O-Glc), 75.9 (C-2 ), 77.9 (C-3 ), 71.8 (C-4 ), 78.0 (C-5 ), 62.9 (C-6 ), 103.3
00
0
0
00
00
00
00
C-1 , 26-O-Glc), 75.1 (C-2 ), 78.0 (C-3 ), 71.8 (C-4 ), 78.0 (C-5 ), 62.9 (C-6 ).
3
.5.1 Acid hydrolysis of 1
Compound 1 (2.0 mg) was treated in 1 M H SO (dioxane: H O, 1:1, 2 mL) at 1008C for 2 h. The
2
4
2
acid aqueous layer was neutralised with BaCl to give a BaSO precipitate. After filtering,
2
4
the aqueous was concentrated under reduce pressure to dryness. TLC analysis compared with the
authentic glucose (n-BuOH–HOAc–H O, 4:1:5, v/v/v, upper layer) and optical rotation
2
2
5
dispersion ½aꢀD þ 52.6 (c 0.03, H O) indicated the presence of D-glucose.
2
Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1–S8.
Acknowledgements
We acknowledge the financial support by the National Natural Science Foundation of China (No.
8
1073009) and the Fund of the Scientific Innovation Program of Postgraduates in Jiangsu Province. The
project was also funded by the Priority Academic Program Development of Jiangsu Higher Education
Institutions.
References
Chinese Pharmacopoeia Commission. 2010. Chinese pharmacopoeia. Vol. 1. Beijing: Chemical Industry Press, p. 41.
Li J, Shi J, Yu XW, Sun JK. 2011. Chemical and pharmacological researches on Hyoscyamus niger. Chin Herb Med.
3:117–126.
Lu YY, Luo JG, Huang XF, Kong LY. 2009. Four new steroidal glycosides from Solanum torvum and their cytotoxic
activities. Steroids. 74:95–101.
Lu YY, Luo JG, Kong LY. 2011. Steroidal alkaloid saponins and steroidal saponins from Solanum surattense.
Phytochemistry. 72:668–673.
Lunga I, Bassarello C, Kintia P, Shvets S, Piacente S, Pizza C. 2008a. Steroidal saponins from the seeds of Hyoscyamus
niger L. Nat Prod Commun. 3:731–734.
Lunga I, Kintia P, Shvets S, Bassarello C, Piacente S. 2008b. Steroidal saponins from the seeds of Hyoscyamus niger L.
Chem J Moldova. Gen Ind Ecol Chem. 3:89–93.
Ma CY, Liu WK, Che CT. 2002. Lignanamides and nonalkaloidal components of Hyoscyamus niger seeds. J Nat Prod.
65:206–209.
Waiss AC, Jr, Elliger CA, Haddon WF, Benson M. 1993. Insect inhibitory steroidal saccharide esters from Physalis
peruviana. J Nat Prod. 56:1365–1372.
Zhu XH, Ando J, Takagi M. 2001. Six new with anolide-type steroids from the leaves of Solanum cilistum. Chem Pharm
Bull. 49:161–165.
Zhang WN, Luo JG, Kong LY. 2012. Phytotoxicity of lignanamides isolated from the seeds of Hyoscyamus niger. J Agric
Food Chem. 60:1682–1687.