全 文 ： 2013 年 7 月 第 11 卷 第 4 期 Chin J Nat Med Jul. 2013 Vol. 11 No. 4 411

Chinese Journal of Natural Medicines 2013, 11(4): 04110414
doi: 10.3724/SP.J.1009.2013.00411
Chinese
Journal of
Natural
Medicines








Isolation of cytotoxic compounds from the seeds of
Crataegus pinnatifida
LI Ling-Zhi1, PENG Ying2*, NIU Chao1, GAO Pin-Yi3, HUANG Xiao-Xiao1, MAO Xin-Liang4,
SONG Shao-Jiang1, 5*
1 School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China;
2 School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China;
3 College of Chemical Engineering, Shenyang University of Chemical Technology, Shenyang 110142, China;
4 College of Pharmaceutical Science, Soochow University, Suzhou 215123, China;
5Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical University), Ministry of Education，Shenyang
110016, China
Available online 20 July 2013

[ABSTRACT] AIM: To study the chemical constituents and bioactivity of the seeds of Crataegus pinnatifida. METHODS: The
chemical constituents were isolated and purified by macroporous adsorptive resin D101, silica gel, and ODS column chromatography,
and preparative HPLC. Their structures were elucidated on the basis of spectroscopic methods. In addition, the cytotoxic activities of
compounds 1−4 were investigated on OPM2 and RPMI-8226 cells. RESULTS: Four compounds were obtained and their structures
were identified as (7S, 8S)-4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-(hydroxymethyl)ethoxy]-3, 5-dimethoxybenzaldehyde
(1), (+)-balanophonin (2), erythro-guaiacylglycerol-β-coniferyl aldehyde ether (3), buddlenol A (4). CONCLUSION: Compound 1 is
a novel norlignan, while compounds 1−4 exhibited marginal inhibition on the proliferation of OPM2 and RPMI-8226 cells.
[KEY WORDS] Crataegus pinnatifida; Norlignans; Cytotoxic activity
[CLC Number] R284.1 [Document code] A [Article ID] 1672-3651(2013)04-0411-004

1 Introduction
Crataegus pinnatifida Bunge (common name hawthorn)
is a member of the Rosaceae family. The genus is widely
distributed throughout the northern temperate regions of the
world with approximately 1 000 species, primarily in Asia,
Europe, and America [1]. Hawthorn fruits have long been used
in traditional Chinese medicine and European herbal medi-
cine [2]. Pharmacological and toxicological studies have
demonstrated that the extract of hawthorn has many health
benefits, including being cardiovascular protective, hypoten-

[Received on] 21-May-2012
[Research funding] This project was supported by the China Na-
tional Fundamental Fund of National Science Talent Training Base
in pharmacy (No. J1103606), the Scientific Research Starting Foun-
dationfor Doctors of Liaoning Province of China (No. 20121106),
the Foundation from the Project of Education Department of Liaon-
ing Province, China (No. L2012358) and Innovative Research Team
of the Ministry of Education and Program for Liaoning Innovative
Research Team in University
[*Corresponding author] SONG Shao-Jiang: Prof., Tel: 86-24-
23986510, E-mail: songsj99@gmail.com; PENG Ying: Associate
prof., Tel: 86-24-23986361, E-mail: yingpeng99@yahoo.com.cn
These authors have no conflict of interest to declare.
sive, hypocholesterolemic, and lowering serum cholesterol
[3-4]. Previous studies on the chemical constituents of haw-
thorn led to the isolation of diverse compounds, including
flavonoids, terpenoids, and organic acids [5]. In order to ex-
amine the bioactivity of C. pinnatifida, the seeds of hawthorn
were subjected to phytochemical investigation resulting in
the isolation of a novel norlignan 1, together with three
known compounds 2–4 which were isolated from this genus
for the first time. Their chemical structures were elucidated
by their physicochemical properties and spectral data, in-
cluding 1D, 2D NMR, HR-TOF-MS and CD spectra (Fig. 2).
In addition, the cytotoxic activities of compounds 1−4 were
investigated on OPM2 and RPMI-8226 cells. Compounds
1−4 exhibited marginal activity in the model cell systems
used.
2 Experimental
2.1 General experimental procedures
Mass spectra were determined on a HR-TOF-MS: Mi-
croTOF spectrometer (Bruker Daltonics, CA). NMR spectra
were recorded on a Bruker ARX-600 spectrometer with TMS
as internal standard in chloroform-d or dimethyl sulfoxide-d6
(DMSO-d6). Circular dichroism (CD) was measured using a
LI Ling-Zhi, et al. / Chinese Journal of Natural Medicines 2013, 11(4): 411414
412 Chin J Nat Med Jul. 2013 Vol. 11 No. 4 2013 年 7 月 第 11 卷 第 4 期

BioLogic spectrophotometer. Silica gel (200−300 mesh,
Qingdao Marine Chemical Co., China); Sephadex LH-20
(25−100 µm, Greenherbs Science and Technology Develop-
ment Co., Ltd. China); MCI gel (CHP20P, 75–150 µm, Mit-
subishi Chemical Corporation, Japan) and reversed-phase C18
silica gel (60–80 µm, Merck, Germany) were used for col-
umn chromatography, and silica gel GF254 (Qingdao Marine
Chemical Co., China) was used for TLC. Solvents were of
industrial purity and distilled prior to use.
2.2 Plant material
The seeds of C. pinnatifida were collected in November,
2009 from Tianjin, China, and identified by Prof. LU Jin-Cai
of the Department of Pharmacognosy, Shenyang Pharmaceu-
tical University. A voucher specimen (No. CPTJ0911) was
deposited in the Natural Products Laboratory of Shenyang
Pharmaceutical University, Shenyang, China.
2.3 Extraction and isolation
Dried and powdered seeds (15 kg) of C. pinnatifida were
extracted with EtOH (70% V/V) three times, and the extracts
were combined and concentrated, followed by suspension in
water. The water layer was extracted with EtOAc, affording
326 g of the residue, which was subjected to silica gel
(200−300 mesh) chromatography eluting with an increasingly
polar mixture of CHCl2-MeOH (100 : 0 to 1 : 1) to afford six
fractions (Fr. 1−6). Fr. 3 (45 g) was subjected to an MCI gel
column (MeOH/H2O 0 : 100 → 100 : 0), and further purified
by silica gel (petroleum ether/EtOAc 100 : 1 → 1 : 1),
Sephadex LH-20 (MeOH/H2O 80 : 20), and reversed-phase
silica gel (MeOH/H2O 40 : 60 → 80 : 20) to yield compounds
1 (15 mg) and 2 (16 mg). Fr. 4 (55 g) was applied to silica gel
(CHCl2/MeOH 100 : 1 to 2 : 1) chromatography, then puri-
fied by MCI gel (MeOH/H2O 10 : 90 → 80 : 20), re-
versed-phase silica gel (MeOH/H2O 40 : 60 → 80 : 20), Se-
phadex LH-20 (MeOH/H2O 80 : 20), and semipreparative
HPLC to give compounds 3 (26 mg) and 4 (14 mg).



Fig. 1 Structures of compounds 1−4

3 Results and Discussion
Compound 1 was obtained as a colorless oil (MeOH).
[α]20D 12.7 (c 0.38, MeOH). CD (c 0.15, MeOH) Δε (nm): −4.39
(285), The HR-TOF-MS data gave the molecular formula
C19H22O8 (m/z 401.120 9 [M + Na]+, Calcd. 401.120 7). The 1H
NMR (CDCl3) spectrum of 1 showed signals assignable to an
aldehyde proton δ 9.89 (1H, s), an AMX system at δ 6.97 (1H,
br s), 6.88 (1H, d, J = 8.4 Hz), 6.96 (1H, dd, J = 8.4, 1.2 Hz),
two aromatic protons δ 7.18 (2H, br s), a methylene group at δ
3.60 (1H, dd, J = 12.9, 3.0 Hz), 3.25 (1H, br s), two methines at
δ 4.04 (1H, m) and 5.08 (1H, d, J = 8.4 Hz), and three methoxy
groups at δ 3.98 (6H, s) and 3.89 (3H). The 13C NMR spectrum
of 1 exhibited three methoxy groups (δC 56.6, 56.6, and 56.1),
a methylene (δC 60.8), two methines (δC 74.1 and 89.4), an
aldehyde carbon (δC 190.8), and twelve aromatic carbons (δC
131.6, 109.8, 146.6, 145.6, 114.5, 120.4, 140.9, 153.7, 106.7,
132.6, 106.7, and 153.7) indicating the presence of two phenyl
groups. Further demonstration of the planar structure of 1 (Fig.
3) was obtained from the HMBC experiment. In the HMBC
spectrum, key long-range correlations of H-2 with C-1, C-3,
C-6 and C-7, of H-5 with C-1 and C-3, of H-6 with C-1, C-2
and C-7, of H-7 with C-1, C-2, C-6, C-8 and C-9, of H-3′ with
C-1′, C-2′, C-4′ and C-5′, of H-7′ with C-3′, C-4′ and C-5′, and
of the three methoxy proton signals with C-2′, C-6′, C-3, were
observed, respectively. The absolute configuration of C-7 in 1
was clarified by the CD spectrum (Fig. 2) which showed a
negative Cotton effect at 285 nm (Δε −4.39), indicating that the
absolute configuration of C-7 is S [6], the S orientation of C-8
was supported by the coupling constants observed with δ 5.08
(1H, d, J = 8.4 Hz, H-7) and 4.04 (1H, m, H-8) [7]. According
to the above analysis, compound 1 was deduced as (7S,
8S)-4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-(hydroxy
methyl)ethoxy]-3, 5-dimethoxybenzaldehyde, a novel norlig-
nan (Table 1).



Fig. 2 CD spectrum of compound 2


Compound 2 Brown needles (MeOH). [α]20 D +2.6 (c
0.80, MeOH), ESI-MS m/z 379 [M + Na]+, 1H NMR (300
MHz, CDCl3) δ: 6.90 (3H, br s, H-2, 5, 6), 7.04 (1H, br s,
H-2′), 7.13 (1H, s, H-6′), 7.42 (1H, d, J = 15.6 Hz, H-7′), 6.60
(1H, dd, J = 7.8 Hz, 15.6 Hz, H-8′), 9.64 (1H, d, J = 7.8 Hz,
H-9′), 3.69 (1H, m, H-8), 3.97 (2H, m, H-9), 3.87 (3H, s,
-OCH3), 3.93 (3H, s, -OCH3). 13C NMR (75 MHz, CDCl3) δ:
132.2 (C-1), 108.7 (C-2), 146.7 (C-3), 145.9 (C-4), 114.4
(C-5), 118.1 (C-6), 89.0 (C-7), 53.0 (C-8), 63.9 (C-9), 129.0
LI Ling-Zhi, et al. / Chinese Journal of Natural Medicines 2013, 11(4): 411414
2013 年 7 月 第 11 卷 第 4 期 Chin J Nat Med Jul. 2013 Vol. 11 No. 4 413

Table 2 The inhibitory rates of compounds 1-4 against RPMI-8226 and OPM2 cells ( x ± s)
Compound RPMI-8226 cells (24 h) 50 µmol·L−1 100 µmol·L−1
RPMI-8226 cells (48 h)
50 µmol·L−1 100 µmol·L−1
RPMI-8226 cells (72 h)
50 µmol·L−1 100 µmol·L−1
OPM2 cells (48 h)
500 µmol·L−1
1 / / / 84.6 ± 1.8
2 44.3 ± 4.1 52.9 ± 3.8 70.6 ± 0.1 76.0 ± 0.3 84.7 ± 0.7 92.0 ± 10.3 87.5 ± 1.7
3 34.5 ± 2.1 44.5 ± 4.1 59.0 ± 0.8 70.9 ± 0.1 73.5 ± 1.2 86.2 ± 17.8 84.0 ± 2.1
4 32.9 ± 3.8 42.7 ± 2.7 53.0 ± 3.1 69.0 ± 0.2 59.2 ± 3.0 77.6 ± 27.5 61.0 ± 3.8



Fig. 3 Key HMBC correlations of compound 3

Table 1 NMR data for compound 1 (600 MHz, CDCl3)
Positions δH (J in Hz) δC HMBC (H-C)
1 131.6
2 6.97 (br s) 109.8 C-1,C-3,C-6,C-7
3 146.6
4 145.6
5 6.88 (d, 8.4) 114.5 C-1,C-3
6 6.96 (dd, 8.4, 1.2) 120.4 C-1,C-2,C-7
7 5.08 (d, 8.4) 74.1 C-1, C-2, C-6, C-8,
8 4.04 (m) 89.4 C-7
9 3.60 (dd, 12.9, 3.0), 3.25 (br s) 60.8 C-7
1′ 140.9
2′ 153.7
3′ 7.18 (s) 106.7 C-1′, C-2′, C-4′, C-5′
4′ 132.6
5′ 7.18 (s) 106.7 C-1′, C-3′, C-4′, C-6′
6′ 153.7
7′ 9.89 (s) 190.8 C-3′, C-4′, C-5′
3-OCH3 3.89 (3H, s) 56.1 C-3
2′- OCH3 3.98 (3H, s) 56.6 C-2′
6′- OCH3 3.98 (3H, s) 56.6 C-6′

(C-1′), 112.2 (C-2′), 144.8 (C-3′), 151.5 (C-4′), 128.1 (C-5′),
119.4 (C-6′), 153.1 (C-7′), 126.4 (C-8′), 193.6 (C-9′), 56.0
(-OCH3), 56.0 (-OCH3). Compound 2 was characterized as
(+)-balanophonin by comparison of the physical and spectral
data with the literature [8].
Compound 3 Brown oil. 1H NMR (300 MHz,
DMSO-d6): δ 9.61 (1H, d, J = 7.8 Hz, -CHO),7.62 (1H, d, J =
15.6 Hz, H-7), 6.81 (1H, dd, J = 7.8 Hz, 15.6 Hz, H-8), 7.36
(1H, br s, H-2′), 7.23 (1H, d, J = 8.4 Hz, H-6′), 7.11 (1H, d, J
= 8.4 Hz, H-5), 6.97 (1H, br s, H-2), 6.77 (1H, d, J = 8.4 Hz,
H-6), 6.69 (1H, d, J = 8.4 Hz, H-5′), 4.72 (1H, d, J = 4.5 Hz,
H-7′), 4.44 (1H, br s, H-8′), 3.83 (3H, s, -OCH3), 3.73 (3H, s,
-OCH3), 3.60 (1H, dd, J = 3.3 Hz, 11.4 Hz, H-9a′), 3.30 (1H,
dd, J = 6.6 Hz, 11.4 Hz, H-9b′). Compound 3 was character-
ized as erythro-guaiacylglycerol-β-coniferyl aldehyde ether
by comparison of the physical and spectral data with the
literature [8].
Compound 4 Brown oil. ESI-MS m/z 605 [M + Na]+.
1H NMR (300 MHz, DMSO-d6): δ 7.07 (1H, s, H-2), 7.14
(1H, s, H-6), 7.43 (1H, d, J = 15.9 Hz, H-7), 6.81 (1H, dd, J =
7.8 Hz, 15.6 Hz, H-8), 9.65 (1H, d, J = 7.8 Hz, H-9), 6.70
(2H, br s, H-2′, 6′), 5.71 (1H, d, J = 6.9 Hz, H-7′), 3.72 (1H,
m, H2-8′), 4.01, 3.95 (each 1H, m, H-9′), 6.95 (1H, br s,
H-2′′), 6.87 (1H, d, J = 7.8 Hz, H-5′′), 6.76 (1H, br d, J = 7.8
Hz, H-6′′), 4.99 (1H, m, H-7′′), 4.12 (1H, m, H2-8′′), 3.94,
3.51 (each 1H, m, H-9′′), 3.96 (3H, s, -OCH3), 3.78 (3H, s,
-OCH3), 3.76 (6H, s, -OCH3). 13C NMR (75 MHz, CDCl3) δ:
128.7 (C-1), 112.6 (C-2), 145.2 (C-3), 151.6 (C-4), 129.1
(C-5), 118.3 (C-6), 153.2 (C-7), 126.9 (C-8), 193.9 (C-9),
137.4 (C-1′), 103.4 (C-2′), 153.8 (C-3′), 135.1 (C-4′), 153.8
(C-5′), 103.4 (C-6′), 89.1 (C-7′), 53.4 (C-8′), 64.3 (C-9′),
131.5 (C-1′′), 108.7 (C-2′′), 146.9 (C-3′′), 145.2 (C-4′′), 114.5
(C-5′′), 119.0 (C-6′′), 72.8 (C-7′′), 87.4 (C-8′′), 60.8 (C-9′′),
56.4 (-OCH3), 56.3 (-OCH3), 56.5 (-OCH3), 56.5 (-OCH3).
Compound 4 was characterized as buddlenol A by
comparison of the physical and spectral data with the
literature [9].
4 Cytotoxicity Assay
The colorimetric method using 3-(4, 5-dimethylthiazol-
2-yl)-2, 5-diphenyltetrazolium bromide (MTT) [10-11] was
used to investigate the inhibitory effect of compounds 1−4 on
OPM2 and RPMI-8226 cells (Table 3). The compounds to be
tested were dissolved in DMSO, and the final concentration
of DMSO in the culture medium was maintained at less than
0.1% (V/V). Cultivated human cells were seeded in a 96-well
plate with 1×104 cells/well. The plate was incubated with the
compounds for 24−72 h. MTT solution (2.5 mg·mL−1 in PBS)
was added (10 µL/well), and the plate was incubated for an
additional 4 h at 37 °C. The formazan crystals that were
produced were dissolved in 100 μL of buffer (20% SDS, 1
mol·L−1 HCl), and the optical density of the solution was
measured at 490 nm using a microplate reader. The per cent
cytotoxic activity was determined by comparison with the
control (DMSO).
Compounds 1−4 exhibited marginal cytotoxic activity
against OPM2 cells and the inhibitory rates were (61.0 ±
3.8)%−(87.5 ± 1.7)% (500 μmol·mL–1) after 48 h, while
compounds 2−4 exhibited weak cytotoxic activity against
RPMI-8226 cells in a time-dependent manner (24, 48 and 72
h) and the inhibitory rates are presented in Table 2.

LI Ling-Zhi, et al. / Chinese Journal of Natural Medicines 2013, 11(4): 411414
414 Chin J Nat Med Jul. 2013 Vol. 11 No. 4 2013 年 7 月 第 11 卷 第 4 期

References
[1] Zhang ZS, Ho WKK, Huang Y, et al. Hypocholesterolemic
activity of hawthorn fruit is mediated by regulation of
cholesterol-7α-hydroxylase and acyl CoA: cholesterol
acyltransferase [J]. Food Res Int, 2002, 35(9): 885–891.
[2] Chang Q, Zuo Z, Chow MSS, et al. Effect of storage
temperature on phenolics stability in hawthorn (Crataegus
pinnatifida var. major) fruits and a hawthorn drink [J]. Food
Chem, 2006, 98(3): 426–430.
[3] Zhang ZS, Chang Q, Zhu M, et al. Characterization of
antioxidants present in hawthorn fruits [J]. J Nutr Biochem,
2011, 2(3): 144–152.
[4] Yao M, Ritchie HE, Brown-Woodman PD. A reproductive
screening test of hawthorn [J]. J Ethnopharmacol, 2008, 118
(1): 127–132.
[5] Song SJ, Li LZ, Gao PY, et al. Terpenoids and hexenes from
the leaves of Crataegus pinnatifida [J]. Food Chem, 2011,
129(3): 933–939.
[6] Wu LJ. Practical spectroscopy techniques for organic com-
pounds analysis[M]. Beijing: People’s Medical Publishing
House, 2007: 64–66.
[7] Morikawa T, Tao J, Ueda K, et al. Medicinal Foodstuffs.
XXXI. Structures of new aromatic constituents and
inhibitors of degranulation in RBL-2H3 cells from a
Japanese folk medicine, the stem bark of Acer nikoense [J].
Chem Pharm Bull, 2003, 51(1): 62–67.
[8] Lee DY, Song MC, Yoo KH, et al. Lignans from the fruits of
Cornus kousa Burg. and their cytotoxic effects on human
cancer cell lines [J]. Arch Pharm Res, 2007, 30(4): 402–407.
[9] Yang XW, Zhao PJ, Ma YL, et al. Mixed lignin-neolignans
from Tarenna attenuata [J]. J Nat Prod, 2007, 70(4):
521–525.
[10] Tao YZ, Zhang Y, Zhang L. Chemical modification and
antitumor activities of two polysaccharide-protein complexes
from Pleurotus tuber-regium [J]. Int J Biolog Macromol,
2009, 45(2): 109–115.
[11] Wei DD, Wang JS, Zhang Y, et al. A new phenylpropanoid
glycoside from the fruits of Illicium simonsii [J]. Chin J Nat
Med, 2012, 10(1): 20–23.

山楂核中具有抗肿瘤活性化合物的分离
李玲芝 1, 彭 缨 2*, 牛 超 1, 高品一 3, 黄肖霄 1, 毛新良 4, 宋少江 1, 5*
1沈阳药科大学中药学院，沈阳 110016;
2沈阳药科大学药学院，沈阳 110016;
3沈阳化工大学化学工程学院，沈阳 110142;
4苏州大学医学部药学院, 苏州 215123；
5沈阳药科大学基于靶点的药物设计与研究教育部重点实验室，沈阳 110016

【摘 要】 目的：对山楂核的化学成分及生物活性进行研究。方法：运用大孔吸附树脂 D101，硅胶，ODS 和制备
高效液相色谱等方法分离化合物, 通过多种波谱方法进行结构鉴定。此外，还对化合物进行了 OPM2 和 RPMI-8226 两
组细胞株的细胞毒活性测试。结果：从山楂核中得到 4 个化合物：(7S, 8S)-4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)
-1-(hydroxymethyl)ethoxy]-3, 5-dimethoxybenzaldehyde (1), (+)-balanophonin (2), erythro-guaiacylglycerol-β-coniferyl
aldehyde ether (3), buddlenol A (4)。结论：化合物 1 为一新降木脂素。化合物 2−4 为属内首次分离得到。活性测试结果
表明化合物 1−4 的抗肿瘤活性不明显。
【关键词】 山楂; 降木脂素; 生物活性

【基金项目】 国家基础科学人才培养基金本科生“能力提高（科研训练）项目”(No. J1103606), 辽宁省博士启动科研基
金(No. 20121106)，辽宁省教育厅一般项目(No. L2012358)教育部创新团队发展计划和辽宁省高校创新团队支持计划资助