Cytotoxic phenolic glycosides from boschniakia himalaica

Article abstract of DOI:10.1007/s10600-012-0308-z

A new lignan glycoside, 9-acetyl lanicepside B (1), was isolated from the whole plant of Boschniakia himalaica, along with five known compounds, lanicepside A (2), plantainoside D (3), plantamajoside (4), (-)-woonenoside XI (5), and syringin (6). The new compound's structure was established on the basis of detailed 1D and 2D NMR spectroscopic analysis and chemical evidence. 9-Acetyl lanicepside B (1) showed weak in vitro cytotoxic activities against A549 and P388 with IC50 values of 64.7 μM and 72.5 7μM, respectively. Syringin (6) showed moderate in vitro cytotoxic activities against A549 and HL-60 with IC50 values of 32.5 μM and 41.8 μM, respectively.

Full text of DOI:10.1007/s10600-012-0308-z

Chemistry of Natural Compounds, Vol. 48, No. 4, September, 2012 [Russian original No. 4, July–August, 2012]
CYTOTOXIC PHENOLIC GLYCOSIDES FROM Boschniakia himalaica
1
3
2*
Xinzhou Yang, Jinquan Yuan, and Jinfu Wan
UDC 547.972
A new lignan glycoside, 9-acetyl lanicepside B (1), was isolated from the whole plant of Boschniakia
himalaica, along with five known compounds, lanicepside A (2), plantainoside D (3), plantamajoside (4),
(
–)-woonenoside XI (5), and syringin (6). The new compoundꢀs structure was established on the basis of
detailed 1D and 2D NMR spectroscopic analysis and chemical evidence. 9-Acetyl lanicepside B (1) showed
weak in vitro cytotoxic activities againstA549 and P388 with IC values of 64.7 ꢁM and 72.5 ꢁM, respectively.
50
Syringin (6) showed moderate in vitro cytotoxic activities against A549 and HL-60 with IC50 values of
2.5 ꢁM and 41.8 ꢁM, respectively.
3
Keywords: Boschniakia himalaica, lignan glycosides, phenolic glycosides, cytotoxicity.
Boschniakia himalaica Hook. f. et. Thomson (Orobanchaceae) is a parasitic herb growing on the roots of the genus
Rhododendron (Betulaceae) and distributed widely in Yunnan, Sichuan, and Tibet, China [1]. This plant, served traditionally
as a decoction, has been one of the commonly-used folk medicines in the minority nationality areas of Yunnan Province.
Pharmacological studies show that this plant exhibits a variety of activities, including antitussive, anti-inflammatory, and
antioxidative activities, and is also used to relieve abdominal pain and syndromes such as gastralgia [2, 3]. Previous chemical
studies of B. himalaica have led to the isolation of lignans, lignan glucosides, triterpenoids, and sterols [4, 5]. To depict the
chemical constituents related to its bioactivities, a further investigation on the title plant was carried out. On the basis of the
traditional usage-decoction, the polar fraction was selected for detailed isolation. Herein, we reported the isolation and structural
elucidation of a new lignan glycoside, 9-acetyl lanicepside B (1), along with five known compounds, lanicepside A (2) [6],
plantainoside D (3) [7], plantamajoside (4) [8], (–)-woonenoside XI (5) [9], and syringin (6) [10]. All compounds were evaluated
for their cytotoxic activities against three tumor cell lines, A549, HL-60, and P388.
O
HO
HO
H
3
C
7'
'
4'
9
OH
OGlc
OMe
O
1'
HO
8
8
3
'
6
1
9'
OMe
7
O
O
4
GlcO
HO
3
OMe
1
OMe
2
OGlc
OH
R
2
MeO
GlcO
MeO
OMe
OH
OH
O
R
1
O
OH
OH
O
HO
HO
HO
OH
O
OH
OMe
3
, 4
: R = OH, R = OCOCH=CH-C H (OH)
2
5
OH
6
3
1
2
6
3
4
: R = OCOCH=CH-C H (OH) , R = OH
1 6 3 2 2
1
) College of Pharmacy, South-Central University for Nationalities, 708 Min-Zu Road, 430074, Wuhan, P. R. China;
2
) Yunnan Baiyao group Co. LTD, 51 Xiba Road, 650032, Kunming, P. R. China, fax: +86 27 67841196, e-mail:
yxzyxz01@hotmail.com; 3) Guangxi Institute of Medicinal Plants, 189 Chang-Gang Road, 530023, Nanning, P. R. China.
Published in Khimiya Prirodnykh Soedinenii, No. 4, July–August, 2012, pp. 502–504. Original article submitted July 23, 2011.
0
0
009-3130/12/4804-0555 2012 Springer Science+Business Media, Inc.
555

1
13
TABLE 1. H (600 MHz) and C NMR (150 MHz) Data for Compound 1 (methanol-d , ꢄ, ppm, J/Hz)
4
C atom
ꢄH
ꢄC
C atom
ꢄH
ꢄC
1
2
3
4
5
6
7
8
9
134.1
111.7
149.6
148.0
116.6
120.9
85.9
7ꢀ
8ꢀ
9ꢀꢂ
4.50 (d, J = 7.7)
2.54 m
3.93 (dd, J = 9.3, 7.6)
4.27 (dd, J = 9.3, 4.3)
3.85 s
76.6
50.8
71.6
6.92 (d, J = 1.6)
ꢅ
6.77 (d, J = 8.1)
6.79 (dd, J = 8.1, 1.6)
4.55 (d, J = 8.4)
2.12 m
3-OMe
3ꢀ-OMe
57.2
50.7
21.2
3.84 s
2.05 s
COCH
3
51.5
64.3
COCH
Glc
1ꢀꢀ
3
172.2
3.20 m
3
.30 m
4.85 (d, J = 7.7)
3.48 m
103.2
75.4
78.3
71.8
78.6
63.0
1
2
3
4
5
6
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
140.0
112.7
151.4
147.9
118.5
121.1
2ꢀꢀ
3ꢀꢀ
4ꢀꢀ
5ꢀꢀ
6.96 (d, J = 1.8)
3.46 m
3.39 m
3.39 m
7.11 (d, J = 8.5)
6.85 (dd, J = 8.5, 1.8)
6ꢀꢀ
3.66 (dd, J = 12.0, 4.7)
3.85 m
TABLE 2. Cytotoxic Activity of Compounds 1–6 (IC , ꢁM; mean ꢆ SD, n = 3)
5
0
Cell lines
HL-60
Cell lines
Compound
Compound
A549
P388
A549
HL-60
P388
1
2
3
4
64.7 ꢆ 4.3
90.5 ꢆ 5.7
> 100
> 100
> 100
> 100
> 100
72.5 ꢆ 3.9
N.d.
> 100
> 100
5
6
89.7 ꢆ 3.7
32.5 ꢆ 5.1
6.8 ꢆ 1.7
> 100
41.8 ꢆ 4.7
13.6 ꢆ 2.1
85.1 ꢆ 5.6
92.6 ꢆ 7.2
0.8 ꢆ 0.03
b
a
Colchicine
b
N.d.
_
_____
a
b
Colchicine as positive control; N.d.: not determined.
Dried powdered whole plant of B. himalaica (500 g) were extracted with 95% EtOH. The crude EtOH extract was
subjected to repeated column chromatography over polyamide resin and silica gel, and preparative HPLC as well as preparative
TLC, to afford compounds 1–6.
2
0
Compound 1, obtained as light yellow powder, [ꢂ] –19.0ꢃ (c 0.1, MeOH), has the molecular formula C H O ,
D
28 36 13
+
13
determined on the basis of a quasi-molecular ion [M + Na] at m/z 603.2059 generated by HR-ESI-MS, C NMR, and DEPT
spectra. The UV spectrum showed absorption maxima at 220 and 276 nm, and the IR spectrum showed the presence of
–
1
–1
–1
hydroxyl groups (3366 cm ), ester carbonyl groups (1745 cm ), and aromatic moieties (1605, 1517 cm ), indicating a
phenolic moiety.
1
The H NMR spectrum (Table 1) displayed signals of three aromatic protons at ꢄ 6.92 (d, J = 1.6 Hz), 6.79 (dd,
J = 8.1, 1.6 Hz), and 6.77 (d, J = 8.1 Hz), assignable to one set of ABX system, as well as three other aromatic protons at
1
ꢄ 7.11 (d, J = 8.5 Hz), 6.96 (d, J = 1.8 Hz), and 6.85 (dd, J = 8.5, 1.8 Hz), ascrible to another set of ABX system. The H NMR
spectrum also displayed the presence of two oxygenated methylenes, one at ꢄ 3.20 (m) and 3.30 (m), the other at ꢄ 4.27
(
dd, J = 9.3, 4.3 Hz) and 3.93 (dd, J = 9.3, 7.6 Hz), two oxygenated methines at ꢄ 4.55 (d, J = 8.4) and 4.50 (d, J = 7.7), two
methines at ꢄ 2.54 (m) and 2.12 (m), an acetyl methyl group at ꢄ 2.05 (s), correlated with an ester C=O at ꢄ 172.2 in the
C
HMBC spectrum, as well as an anomeric proton at ꢄ 4.85 (d, J = 7.7 Hz), and oxygenated methine and methylene protons
ascribed to a sugar moiety. All these resonances were almost identical with those of lanicepside B [6], except for an additional
acetyl group. Complete acid hydrolysis with HCl yielded D-glucose, which was determined by comparison with the derivatives
of authentic samples by GC analysis. On the basis of the above partial structures, the molecular framework was constructed by
the HMBC experiment. The HMBC correlations between the anomeric proton at ꢄ 4.85 and C-4 at ꢄ 148.0 confirmed that the
H
C
glucose unit was located at C-4. Another HMBC long-range correlation between H-9 at ꢄ 3.20 and the C=O moiety at
H
ꢄC 172.2 confirmed that the acetyl unit was located at C-9.
556

1
The relative stereochemistry of 1 was established on the basis of H NMR data and ROESY experiments. The strong
cross peaks of H-7 to H -9 and H-9ꢀꢂ, H -9 to H-8ꢀ and H-7, and H-8ꢀ to H-9ꢀꢂ and H -9 indicated that H-7, H -9, and H-8ꢀ had
2
2
2
2
ꢂ orientations, similar to those of lanicepsides A and B [6], tinosposide B [11], and tanegosides A, B, and C [12]. The relative
configuration of H-7ꢀ was determined as having a ꢅ orientation by comparison of the chemical shift and coupling constant of
H-7ꢀ with those of lanicepsides Aand B [6], tinosposide B [11], and tanegool [13], further supported by the ROESY correlation
between H-7ꢀ and H-9ꢅ. Thus, the structure of compound 1 was identified as shown and named 9-acetyl lanicepside B.
Compounds 2–6 were identified as lanicepside A (2) [6], plantainoside D (3) [7], plantamajoside (4) [8], (–)-woonenoside
XI (5) [9], and syringin (6) [10] by comparing their physical and spectral data with the data reported in the literature.
All compounds isolated form this herb were evaluated against three tumor cell lines, A549, HL-60, and P388 (Table 2).
9
7
3
-Acetyl lanicepside B (1) showed weak in vitro cytotoxic activities against A549 and P388 with IC50 values of 64.7 ꢁM and
2.5 ꢁM, respectively. Syringin (6) showed moderate in vitro cytotoxic activities against A549 and HL-60 with IC values of
2.5 ꢁM and 41.8 ꢁM, respectively.
50
EXPERIMENTAL
General Experimental Procedures. Optical rotations were measured on a Perkin–Elmer polarimeter 341. IR spectra
–
1
were determined on a Nicolet Magna FT-IR 750 spectrometer (ꢇ , cm ). The NMR spectra were recorded on Bruker DRX-400
max
13
1
NMR and Varian UNITYINOVA 600 spectrometers for H and C NMR. The chemical shifts are given on the ꢄ (ppm) scale
with tetramethylsilane as an internal standard, and coupling constants (J) are in Hz. The ESI-MS and HR-ESI-MS data were
recorded on a Q-TOF Micro LC-MS-MS mass spectrometer. Preparative and semipreparative HPLC system: two PrepStar
SD-1 solvent delivery modules, a ProStar UV-Vis 320 detector and a ProStar 701 fraction collector (Varian, Walnut Creek,
CA, USA); a LinChrospher 100 RP-18 (Merck, Darmstadt, Germany) column (220 ꢈ 25 mm i.d.; particle size 5 ꢁm) for
TM
preparative isolation; a XTerra C column (3.5 ꢁm; 250 ꢈ 10 mm, waters) for semi-preparative isolation. Commercial silica
18
gel (Qing Dao Hai Yang Chemical Group Co., 200–300 and 400–600 mesh) was used for column chromatography. Precoated
Si gel plates (Yan Tai Zi Fu Chemical Group Co., G60 F-254) were used for analytical TLC.
Plant Material. The whole plant of B. himalaica were collected in Dali County, Yunnan Province, P. R. China in
September, 2006 and identified by Prof. Shukun Chen of Kunming Institute of Botany, ChineseAcademy of Sciences, Kunming,
P. R. China. A voucher specimen (20060913B) is deposited in the Herbarium of South-Central University for Nationalities,
Wuhan, P. R. China.
Extraction and Isolation. The air-dried whole plant of B. himalaica (200 g) were percolated with 95% aq. EtOH
(
1 L ꢈ 3) at room temperature. The EtOH extract was filtered and concentrated under reduced pressure. Then the concentrated
extract was suspended in H O and partitioned with n-BuOH (1 L ꢈ 3). The n-BuOH fraction (8 g) was subjected to CC over
2
polyamide resin (300 g) and eluted with 0%, 20, 40, 60, and 80% aq. EtOH in a step manner. The fraction eluted with 20% aq.
EtOH was applied to a silica gel column (CHCl –MeOH–H O 9:3:0.2) to afford subfractions 1.1–1.6. Subfraction 1.2 was
3
2
submitted to prep. TLC (CHCl –MeOH–H O 9:3:0.2) to yield 1 (2 mg) and 2 (5 mg). Subfraction 1.3 was submitted to
3
2
semipreparative HPLC (H O–MeOH 95% : 5%ꢉ75%:25%, 35 min) to give 3 (7 mg, 9.6 min) and 4 (8 mg, 10.9 min). Subfraction
2
1
.4 was subjected to CC over silica gel and then preparative TLC (CHCl –MeOH–H O 8:3:0.2) to give 5 (11 mg) and 6 (13 mg).
3 2
2
5
9
-Acetyl Lanicepside B (1). Light yellow powder; [ꢂ] –19.0ꢃ (c 0.1, MeOH). UV (MeOH, ꢊmax, nm): 220, 276.
D
–1
1
13
IR (ꢇmax, film, cm ): 3366, 2926, 1745, 1605, 1517, 1465, 1264, 1227, 1161, 1068, 1025, 817. H and C NMR, see Table 1;
HR-ESI-MS m/z 603.2059 [M + Na] (C H O Na, calcd 603.2054).
+
28
36 13
Cytotoxicity Assay. Cell lines A549 (human lung carcinoma), HL-60 (human promyelocytic leukemia), and P388
(
murine leukemia) were purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA). The cytotoxic
assay was performed as previously described [14]. Briefly, three cell lines suspended in RPMI 1640 containing 10% FBS were
4
seeded at 1 ꢈ 10 cells (100 ꢁL) per well in a flat 96-well plate and incubated in a humidified atmosphere of 5% CO in air at
2
3
7ꢃC. After 24 h, the medium containing different concentrations of compounds 1–6 was added, and 0.1% DMSO was used as
solvent control. After that, the cells were fixed with EtOH–H O (95:5, v/v), stained with crystal violet solution, and lysed with
2
a solution of 0.1 N HCl in MeOH. The absorbance in control and drug-treated wells was measured in an automated microplate
reader (Bio-Rad 550) at 550 nm.
557

ACKNOWLEDGMENT
The work was financially supported by the Talented Faculty Foundation of South-Central University for Nationalities
YZ211002 to X. Z. Yang).
(
REFERENCES
1
.
G. J. Xu, H. X. He, L. S. Xu, and R. Y. Jin, The Chinese Materia Medica (Zhonghua Benchao), Chinese Medicinal
Science & Technology Publishing House, Beijing, Vol. 7, 1999, p. 506.
2
3
.
.
Z. Y. Wu, The List of Seed Plants in Yunnan, Yunnan Peopleꢀs Publishing House, Kunming, 1984, p. 1633.
Yunnan Yao Cai Co. Ltd, The List of Traditional Chinese Medicines in Yunnan, Science Publishing Press, Beijing,
1
993, p. 500.
4
5
.
.
Y. S. Chen and S. B. Shang, J. Integr. Plant Biol., 34, 878 (1992).
L. H. Liu, J. X. Pu, J. F. Zhao, S. X. Mei, X. D. Yang, Y. B. Wang, H. B. Zhang, and L. Li, Chin. Chem. Lett., 15,
4
3 (2004).
6
7
8
9
.
.
.
.
Z. W. Zhou, S. Yin, X. N. Wang, C. Q. Fan, H. Li, and J. M. Yue, Helv. Chim. Acta, 90, 951 (2007).
T. Miyase, M. Ishino, C. Akahori, A. Ueno, Y. Ohkawa, and H. Tanizawa, Phytochemistry, 30, 2015 (1991).
Y. Shoyama, M. Matsumono, and I. Nishioka, Phytochemistry, 25, 1633 (1986).
K. Yoshikawa, H. Kinoshita, and S. Arihara, Nat. Med., 51, 244 (1997).
10.
11.
12.
13.
14.
M. S. Kamel, Phytochemistry, 62, 1247 (2003).
W. Li, K. Koike, L. Liu, L. Lin, X. Fu, Y. J. Chen, and T. Nikaido, Chem. Pharm. Bull., 52, 638 (2004).
F. Abe and T. Yamauchi, Chem. Pharm. Bull., 38, 2143 (1990).
F. A. Macias, A. Lopez, R. M. Verela, A. Torres, and J. M. G. Molinillo, J. Agric. Food Chem., 52, 6443 (2004).
T. Tengchaisri, R. Chawengkirttikul, and N. Rachaphaew, Cancer Lett., 133, 169 (1998).
558