全 文 ： 2009年 3月 第 7卷 第 2期 Chin J Nat Med Mar. 2009 Vol. 7 No. 2 123

Chinese Journal of Natural Medicines 7 (2009) 0123−0128
doi: 10.3724/SP.J.1009.2009.00123
Chinese
Journal of
Natural
Medicines







LC-DAD/MS/MS Detection of Macrocyclic
Bisbibenzyls from the Liverwort Reboulia
hemisphaerica and the Cell-based Screening
of Their Microtubule Inhibitory Effects
GAO Jian1, LI Xia1, 2∆, LV Bei-Bei1, SUN Bin1, ZHU Chang-Jun3, LOU Hong-Xiang1*
1School of Pharmaceutical Sciences, Shandong University, Jinan 250012;
2School of Oceanology, Shandong University, Weihai 264209;3School of Medicine, Shandong University, Jinan 250012, China
Available online: 20 Mar 2009
【ABSTRACT】 AIM: To investigate the microtubule inhibitory active macrocyclic bisbibenzyls from the liverwort Reboulia hemi-
sphaerica. METHODS: LC-DAD/MS/MS was used to detect macrocyclic bisbibenzyls from the microtubule depolymerization active
extract of R. hemisphaerica. Preparative HPLC was applied for the isolation and purification of compounds, the structures of which
were identified by spectroscopic technology. The antimicrotubule activities of these compounds were examined on human tumor cell
line HeLa (cervical carcinoma) stably expressing EYFP-tubulin. RESULTS: Eleven bisbibenzyls were detected and eight of them were
obtained and were further identified to be isomarchantin C (1), riccardin C (2), neomarchantin A (3), marchantin A (4), isoriccardin C
(5), riccardin B (6), plagiochin E (7), and marchantin C (8). Compounds 4 and 8 showed strong microtubule depolymerization activi-
ties. CONCLUTION: Compounds 3-7 have been isolated from this plant for the first time. This is also the first report that bisbibenzyl
is the novel class of natural molecule having antimicrotubule activity.
【KEY WORDS】 Reboulia hemisphaerica; LC-DAD/MS/MS; Bisbibenzyls; Microtubule
【CLC Number】 R284; R965.1 【Document code】 A 【Article ID】1672-3651(2009)02-0123-06

The bryophytes, taxonomically divided into Musci
(mosses), Hepaticae (liverworts) and Anthocerotae (horn-
worts), are morphologically placed between the algae and the
pteridophytes (fern). There are about 24 000 species of the
spore-forming terrestrial green plants in the world. Bisbiben-
zyls are plant metabolites mainly found in liverworts from
genera such as Riccardia, Marchantia, Plagiochila, Reboulia
and Preissia[1]. They are categorized into three main struc-
tural types, which are made up of macrocyclic rings linked
via one biphenyl ether bond (A type), two biphenyl ether
bonds (B type), or one biphenyl ether and one biphenyl bond
(C type), respectively. These types of bis bibenzyls are

【Received on】 2008-10-19
【Foundation Item】 This project was supported by the National
Natural Science Foundation of China (No. 30730109) and the Shan-
dong Provincial Postdoctoral Special Innovative Projects Foundation,
China (No. 200703063)
【 *Corresponding author 】 LOU Hong-Xiang: Prof., Tel:
86-531-88382012, Fax: +86-531-88382019, Email: louhongxiang@
sdu.edu.cn
∆Co-first author
diversified by additional functions (e.g. hydroxyl and meth-
oxyl) on both the benzene nucleus, ethylidene bridge, and the
ways in which aromatic rings are linked[2]. These aromatic
compounds reportedly have versatile biological activities,
which include antioxidative, anticancer and antifungal activi-
ties as well as inhibitory effects on cyclooxygenase and
5-lipoxygenase[3-7]. Marchantin C, a representative bisbiben-
zyl from liverworts, was certified to show significant cyto-
toxicity against P-388 leukemia cell lines and induce apop-
tosis of human glioma A172 cells in the previous studies[8,9].
The LC-DAD/MS/MS method was developed and vali-
dated for screening bisbibenzyls in bryophyte crude extracts
at sub-ppm levels in our previous study[2,10]. Herein, we
adopted this method to screen bisbibenzyls from the R. hemi-
sphaerica crude extract, which had antiproliferation effects
on tumor cells and microtubule depolymerization activities in
the preliminary test, and eleven bisbibenzyls were detected
with the proposed structures relating to type B and C. To
further identify the structures of these bisbibenzyls, prepara-
tive HPLC was applied and afforded eight known bisbiben-
zyls: isomarchantin C (1), riccardin C (2), neomarchantin A
GAO Jian, et al. /Chinese Journal of Natural Medicines 2009, 7(2): 123−128
124 Chin J Nat Med Mar. 2009 Vol. 7 No. 2 2009年 3月 第 7卷 第 2期


Fig. 1 Structures of macrocyclic bisbibenzyls 1-11

(3), marchantin A (4), isoriccardin C (5), riccardin B (6),
plagiochin E (7), and marchantin C (8) as determined by
spectroscopic measurements (Fig. 1).
Microtubules are one of the major dynamic components
of the eukaryotic cytoskeleton, which participate in a variety
of cell functions including the development and maintenance
of cell shape, reproduction and division, signaling, intracel-
lular transportation and cell movement[11,12]. Micro-
tubule-targeted drugs become one of the most effective and
potential agents in cancer chemotherapy[13]. We established
an easy way to identify microtubule depolymerization active
compounds by employing the EYFP-tubulin HeLa cells,
which can be directly observed the cellular microtubules
morphology under fluorescence microscopy. Among the eight
bisbibenzyls (1-8), marchantin A (4) and marchantin C (8) are
found to have strong microtubule deploymerization activities.
1 Experimental
1.1 General
NMR spectra were recorded on a Bruker Avance DRX
600 spectrometer operating at 600 MHz for 1H-NMR and 150
MHz for 13C NMR with Me4Si as internal standard in CDCl3.
Reverse phase preparative HPLC was performed on an
Agilent 1100 system equipped with a G1310A isopump, a
G1322A degasser, and a G1314 UV detector at 280 nm;
Column: ZORBAX SB-C18 (9.4 mm × 250 mm, 5 µm).
Column chromatography was performed on Silica gel
(200–300 mesh, Qingdao Marine Chemical Factory),
Sephadex LH-20 (Pharmacia), and MCI gel (CHP20P,
75–150 µm, Mitsubishi Chemical Industries, Ltd). Precoated
silica gel GF254 plates (Qingdao Marine Chemical Factory)
were used for TLC. Melting points were determined on a
XT-4 apparatus and were uncorrected.
LC-DAD/MS/MS screening bisbibenzyls were analyzed
by Thermo AccELA HPLC coupled to a LTQ-Orbitrap mass
spectrometry system operating in electrospray ionization
mode (Thermo Fisher Scientific, Bremen, Germany). The
mobile phases were (A) 100% water with 0.1% formic acid,
and (B) 100% acetonitrile with 0.1% formic acid at a flow
rate of 0.7 mL·min−1. The LC conditions were 50% B during
0-25 min, a linear gradient from 50% to 65% B during 25-30
min, ramped from 65% to 75% B during 30-40 min, and then
rose from 75% to 90% during 40-50 min. The column used
was a Phenomenex Luna C18 (4.6 mm × 150 mm, 5 µm). To
reduce the interferences, the UV detection wavelength was
set at 280 nm, and the injection volume was chosen 15 µL.
The 50% eluant was sprayed into the mass spectrometer at +5
kV with nebulizer, sheath and sweep gases set at 70, 30 and 5
arbitrary units, respectively, and desolvation of the solvent
droplets was further aided by setting the heated capillary
temperature at 300 °C. Samples were detected by full scan
mass analysis from m/z 200-650 at a resolving power of 60
000 (at m/z 400, FWHM; 1-s acquisition). A data-dependent
program was used in the liquid chromatography/ tandem
mass spectrometry analysis so that the [M + H]+ ions were
selected as the precursor ions for HCD fragmentation to pro-
duce MS/MS spectra. The collision-induced dissociation
(CID) energy was adjusted to 26%. The isolation width of the
precursor ions was 3.0 Th.
1.2 Plant material
R. hemisphaerica was collected in Kunming, Yunnan
Province, China, in July 2006, and was identified by Prof.
Xuesen Wen, School of Pharmaceutical Sciences, Shandong
University, Jinan, China. A voucher specimen (No. 20060714)
was deposited at the Department of Natural Products Chem-
istry, School of Pharmaceutical Sciences, Shandong Univer-
sity, Jinan, China.
1.3 Extraction , isolation and LC-DAD/MS/MS analysis
R. hemisphaerica was air-dried and ground mechanically
to a homogeneous powder. The powder (860 g) was extracted
with MeOH (3 × 3 L) by maceration for 2 d. The combined
MeOH extracts were filtered and evaporated in vacuo to fur-
GAO Jian, et al. /Chinese Journal of Natural Medicines 2009, 7(2): 123−128
2009年 3月 第 7卷 第 2期 Chin J Nat Med Mar. 2009 Vol. 7 No. 2 125

nish a crude extract (27 g), which was suspended in H2O, and
then extracted successively with hexane (5 × 0.5 L), Et2O (5
× 0.5 L), and BuOH (5 × 0.5 L). The microtubule depoly-
merization test showed that Et2O-soluble fraction is the active
fraction. Further analysis by LC-DAD/MS/MS revealed the
presence of eleven bisbibenzyls. The Et2O-soluble extract (13
g), was subjected to column chromatography over silica gel
(hexane/acetone), Sephadex LH-20 (CHCl3/MeOH 1:1), MCI
gel (MeOH/H2O 4:1), and Preparative HPLC (MeOH/H2O
70:30, flow rate 1.8 mL·min−1) to yield compounds 1-8.
1.4 Cell-based activity
1.4.1 Plasmid, cell culture and reagents
Plasmid of pEYFP-tubulin was constructed as previ-
ously reported[11]. HeLa cell line stably expressing
EYFP- α-tubulin was generated by a transient transfection of
the pEYFP-α- tubulin plasmid into HeLa cells using Li-
pofectamine 2000 (Invitrogen Inc), followed by selection
culture with G418. Finally, EYFP-tubulin cells, the stable
cell line expressing EYFP-tubulin, were maintained in nor-
mal medium (DMEM medium supplemented with 10% fetal
bovine serum at 37 °C and 5% CO2 in a humidified incuba-
tor) supplemented with 0.2 mg·mL−1 G418. DAPI, 3-(4,
5-dimethylthiazol)-2, 5-diphenyltetrazolium bromide (MTT),
were purchased from Sigma Co, USA. Vincristine (VCR)
and Paclitaxel (Tax) were purchased from Zhejiang Haimeng
Pharmaceutical Factory and Beijing Xiehe Pharmaceutical
Factory (China), respectively.
1.4.2 MTT and fluorescence microscopy assay
EYFP-tubulin cells were seeded into 96-well plates at
(3-5)×103/well. Various concentrations of the purified mac-
rocylcic bisbibenzyls derivatives were subsequently added
and incubated for 24, 48 and 72 h. The cytotoxicity of these
compounds was evaluated by the MTT assay. Before MTT
assays, microtubule changes can be observed under fluores-
cent microscopy.
1.4.3 Immunofluorescence analysis
Immunofluorescence analysis was performed with the
compounds examined targeting to microtubules comparison
with other positive drugs. In brief, cells were seeded onto
coverslips in 24-well plates and incubated with various com-
pounds for 24 h. After that, cells were stained with DAPI (0.5
µg·mL−1) for 1 h at room temperature. After washing for
three times, cells were mounted with FluoroGuard (Bio-Rad,
Richmond, CA) and photographed using a fluorescent mi-
croscope.
2 Results
2.1 LC-DAD-MS/MS screening and structure elucidation of
bisbibenzyls from R. hemisphaerica
Since bisbibenzyls are the main components in the spe-
cies of Reboulia genera, the rapid screening and structural
elucidation of the compounds from R. hemisphaerica were
performed by investigation of the fragmentation pathways of
bisbibenzyls according to the method proposed in our previ-
ous paper[2,10]. As a result, from the antimicrotubule active
extract of R. hemisphaerica, a total of eleven bisbibenzyls
were detected by employing LC-DAD-MS/MS method,
which was applied to guide further separation of the eight
bisbibenzyls, as well as for further screening of bioactive
substances in this plant.
By comparing the retention time of the eight compounds
purified by the LC-DAD-MS/MS guided separation with the
crude extracts, we found that two unobtained bisbibenzyls
(10, 11) had lower content as shown in Figs. 2 and 3. Their
rational structures were also proposed according to the frag-
mentation pathway in our previous work[2]. The mass spectra
and the structures were shown in Fig. 4, 1. Take compound
11 as an example, the ions at m/z 211, 227 indicate that it
belongs to B type bisbibenzyls with two ether bonds in the
structure. The ions at m/z 347, 335, which are 94 and 106
mass units less than the [M + H]+ at m/z 441, are formed by
the loss of A and B ring, respectively. The appearance of ions
at m/z 255 and 241 which contain A, C ring or B, D ring in
the fragment ions, indicates that there are two hydroxyl
groups in B/C ring, and m/z 239, 225 suggests that there is
only one hydroxyl group in the other half part of skeleton.
The ions at m/z 423, 199, 137 were also found in the mass
spectrum of compound 11.


Fig. 2 The total ion (A) and LC-DAD (B) chromatograms of
bisbibenzyls from the crude extract of R. hemisphaerica


Fig. 3 The LC-DAD chromatograms of crude extract and
eight bisbibenzyls (1-8) isolated from R. hemisphaerica
GAO Jian, et al. /Chinese Journal of Natural Medicines 2009, 7(2): 123−128
126 Chin J Nat Med Mar. 2009 Vol. 7 No. 2 2009年 3月 第 7卷 第 2期


Fig. 4 The mass spectra of unconfirmed bisbibenzyls (as shown in Fig. 1) from the crude extract of R. hemisphaerica

These eight purified bisbibenzyls were identified as
isomarchantin C, riccardin C, neomarchantin A, marchantin
A, isoriccardin C, riccardin B, plagiochin E, and marchantin
C on the basis of a comparison of their 1H NMR and 13C
NMR spectroscopic data with those previously published
(Fig. 1)[1].
2.2 MTT and fluorescence microscopy assay
Microtubules play an important role in multiple cellular
processes including cell division. In this study, we described
a cell-based screening system to identify active compounds
of interfering cell microtubule formation and mitotic pro-
gression with EYFP-tubulin HeLa cells. In this screening
system, MTT assays combined with fluorescence micros-
copy analysis were performed to determine cytotoxicity and
anti-microtubule effects of small natural molecules. Cell
viability of the eight compounds for 48 h was shown in Fig 5.
IC50 values (concentration resulting in 50% inhibition of cell
growth) were calculated from plotted results using untreated
cells as 100%. As shown in Fig. 5, compounds 4-8 showed
some cytotoxicity with IC50 22.6, 42.2, 41.6, 32.7, 23.2
µmol·L−1 respectively, and the effects become stronger with
longer incubation time. Furthermore, morphological altera-
tions of microtubule filaments in cells and the progression of
cell mitosis were examined under fluorescence microscopy.
As shown in Fig. 6 in comparison with the no treatment cells,
we found weaker fluorescence intensity in the cells pre-
treated with the controlled microtubule inhibitors VCR and
the compound 4. Compound 8 represented microtubule de-
polymerization in the macrograph. Representative images
(original magnification: 20 ×) were shown in Fig. 6.


Fig. 5 Cytotoxicity of compounds 1-8 on EYFP-tubulin cells.
Cytotoxicity of compounds 1-8 on EYFP-tubulin cells were
examined by MTT method. The cells were treated with vari-
ous concentrations of compounds 1-8. Cells viability rate
were calculated as control 100%. Data were expressed as
x ± s. from three independent experiments.


Fig. 6 Cellular microtubules and mitosis analysis of EYFP-tubulin cells treated with different compounds. In comparison with no
treatment cells, weaker fluorescence intensity was observed in the cells pretreated with tubulin inhibitors such as VCR, Tax and
compounds (4, 8). Representative images (magnification: 20 ×) were shown.
GAO Jian, et al. /Chinese Journal of Natural Medicines 2009, 7(2): 123−128
2009年 3月 第 7卷 第 2期 Chin J Nat Med Mar. 2009 Vol. 7 No. 2 127

2.3 Immunofluorescence analysis
Active candidates screened from the above results were
performed with immunofluorescence staining analysis with
the use of the standard fixation. The microtubule network
can be easily observed under fluorescence microscope. Ad-
ditionally, at the same time, the ratio of cells in M phase can
also be measured from DNA staining with DAPI (Fig. 7). It
should be noted that tubule active candidates identified from
this assay are consistent with previous active cell-based
screening assay.

Fig. 7 Immunofluorescence staining analysis of EYFP-tubulin cells treated with different compounds. EYFP-tubulin cells were
incubated with 20 µmol·L−1 of compounds (4, 8) for 24 h, and then fixed and stained with DAPI (blue). The cellular microtubule
distribution was analyzed by a fluorescent microscope at magnification of 100 ×, 0.5 µmol·L−1 of VCR, 0.05 µmol·L−1 of Tax and
same amount of DMSO were used as controls. Representative images were shown.

3 Discussion
The macrocyclic bisbibenzyls, exclusively isolated from
liverworts, have been reported to have diverse biological
activities including cytotoxic[9], antioxidative[1], antimicrobial
and antifungal effects[5,8,14]. In order to quickly find this class
of compounds and their biological significance, we estab-
lished an easy way to throughput screening the bisbibenzyls
by using the LC-DAD-MS/MS method, and anti-microtubule
active candidates by observing the microtubules morphology
of EYFP-tubulin HeLa cells. By combination use of
LC-DAD-MS/MS and the cell-based bioassay, from the an-
timicrotubule active fraction of R. hemisphaerica, eleven
bisbibenzyls were detected and eight were purified, two of
them were found to be the active compounds ascribed for the
microtubule depolymerization activity. This is the first time
to prove that bisbibenzyl is the novel class of natural mole-
cule with antimicrotubule activities.
LC-DAD/MS/MS method is an easy and convenient way
for the high-throughput screening (HTS) of bisbibenzyls in
bryophytes, and was proved to be an economic way to avoid
repeated isolation and structural determination for known
compounds. Based on the fragmentation pathways of the A-,
B-, and C-type of bisbibenzyls in LC-DAD/MS/MS, eleven
bisbibenzyls can be preliminarily determined from the an-
timicrotubule active extract of R. hemisphaerica and eight of
them were further confirmed by comparison with those puri-
fied compounds. The three unconfirmed structures were also
proposed accordingly.
EYFP-tubulin HeLa cells stably expressing EYFP- tubu-
lin fusion protein provided a possible way to identify active
compounds involved in microtubule formation and mitotic
progression[15]. The antiproliferation assay of these purified
bisbibenzyls can be achieved by MTT. Before MTT, by ob-
serving the morphological change under the fluoroscence
microscope and further confirming by immunofluoroscent
staining assay, the microtubule depolymerising molecules can
be easily detected. From the eight screened compounds, two
were found to be active possibly. This provides an easy way
to identify bisbibenzyls and identify the antimicrotubule ac-
tive molecules from liverworts by combination use of
LC-DAD-MS/MS and cell-based screening.
Microtubule plays an important role in multiple cellular
process of cell cycle, which makes it a favorite target of anti-
tumor compounds. The antimicrotubule activities of these
bisbibenzyls uncovered the mechanism of their antiprolifera-
tion in one aspect. The underneath mechanism of these com-
pounds and their possible application of bisbibenzyl deriva-
tives need to be investigated in the future.
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LC-DAD/MS/MS检测石地钱中大环双联苄及其微管抑制活性的
细胞筛选
高 健 1, 李 霞 1, 2∆, 吕蓓蓓 1, 孙 斌 1, 朱长军 3, 娄红祥 1*
1山东大学药学院, 济南 250012;
2山东大学威海分校, 威海 264209;
3山东大学医学院, 济南 250012
【摘 要】 目的：研究苔类植物石地钱中具有微管抑制活性的大环双联苄类化合物。方法：在具有微管抑制活性的石地钱
提取部分中, 利用 LC-DAD/MS/MS方法检测其中的大环双联苄, 通过制备高效液相分离和纯化此类化合物、光谱方法鉴定其结
构, 利用稳定表达 EYFP-tubulin的 HeLa细胞株考察其抑制微管活性。结果：LC-DAD/MS/MS检测到 11个双联苄类化合物, 经
分离纯化得到其中 8个并鉴定为：异地前素 C (1)、片叶苔素 C (2)、新地前素 A (3)、地前素 A (4)、异片叶苔素 C (5)、片叶苔
素 B (6)、羽苔素 E (7)和地前素 C (8)。化合物 4和 8 具有显著的抑制微管解聚活性。结论：化合物 3-7 均为首次从该植物中分
得, 并首次报道联苄类化合物具有微管抑制活性。
【关键词】 石地钱; LC-DAD/MS/MS; 双联苄; 微管

【基金项目】 国家自然科学基金(No. 30730109)与山东省博士后创新基金(No. 200703063)