全 文 ：Chemical Constituents of Pteris multifida and Their Inhibitory
Effects on Growth of Rat Prostatic Epithelial Cells in vitro
QIN Bo
1 , ZHU Da-Yuan1＊ , JIANG Shan-Hao1 , XIANG Gui2 , LENG Ying1 , GU Zhi-Ping1 ,
WANG Yi-Qing2 , SHAO Xue-Feng2
1 StateKey Laboratory of Drug Research , Shanghai Instituteof Materia Medica , Shanghai Institute of Biological Sciences , Chinese Academy
of Sciences , Shanghai 201203;
2 Taicang People s Hospital , Taicang 215400 , P .R .China
【ABSTRACT】　AIM:To study the active chemical constituents of Pteris multifida against growth of rat prostatic epithelial
cells(PECs).METHODS:Column chromatographies of macroporous resin D-1400 , Sephadex LH-20 , Rp-18 , silica gel
were used for the isolation of compounds , and spectroscopic techniques(NMR , IR, UV andMS)were used for the structural
identification.The inhibitory activities of compounds on rat PECs growth were evaluated by MTT method under serum-free con-
ditions with or without growth factors added to the culture medium.RESULTS:22 compounds were isolated from acetone and
20% ethanol extracts of the titled plant.Among them , luteolin , palmitic acid and apigenin 4′-O-α-L-rhamnopyranoside
showed close effect against growth of rat PECs in vitro with epristeride.CONCLUSION:Pteroside P′(1)was identified to
be a new C14 sesquiterpene glycoside;luteolin , palmitic acid and apigenin 4′-O-α-L-rhamnopyranoside may be the main active
components of Pteris multifida against benign prostatic hypertrophy.
【KEY WORDS】　Pteris multifida;Prostatic epithelial cells;Benign prostatic hyperplasia;Pteroside P′
【CLCNumber】　R284.1　　【Document code】　A　　【Ariticle ID】　1672-3651(2006)06-0428-04
【Received on】　2006-09-19
【＊Corresponding Author】　Zhu Da-Yuan , Professor of phytochemistry
and medicinal chemistry , Tel:(86)-21-50806728 , Fax:(86)-21-
50807088 , E-mai l:dyzhu@mai l.shcnc.ac.cn
1　Introduction
The whole plant of Pteris multifida Poir.(Polypo-
diaceae)is a traditional Chinese medicine for the treat-
ment of enteritis , hepatitis , bacterial dysentery , hae-
matemesis , haematuria , tonsillitis , parotitis , and
eczema.[ 1] Previous chemical investigation on this plant
resulted in the isolation of diterpenoids
[ 2] , sesquiter-
penoids and flavonoids[ 3 ,4] .In the clinical practice of
Taicang People s Hospital , the decoction of P.multi-
fida was found to be able to attenuate symptoms of be-
nign prostatic hypertrophy.In order to discover the ac-
tive components of this plant , we conducted chemical
constituents investigation , leading to the isolation of one
new C14 sesquiterpenoid , pteroside P′(1)and other
four ones , three diterpenoids , nine flavonoids , four or-
ganic acids and one sterol from the 20%EtOH and ace-
tone extracts.Bioactive evaluation showed that apigenin
4′-O-α-L-rhamnopyranoside (3), luteolin (4) and
palmitic acid(5)could potently inhibit the growth of rat
prostatic epithelial cells(PECs)with or without growth
factors in the culture medium , while apigenin 7-O-α-
D-glucopyranosyl-4′-O-α-L-rhamnopyranoside (2)and
behenic acid(6)had also moderate activity.
2　Results and discussion
2.1　Structural Determination
The known compounds , 2[ 5] , 3[ 5] , 4[ 6] , 5[ 7] ,
6[ 8] ,2β ,16β ,18-trihydroxy-ent-kaurane(7)[ 9] , luteolin
3′-O-β-D-glucopyranoside (8)[ 10] , luteolin 7-O-β-D-
428　　Chin J Nat Med　Nov.2006　Vol.4　No.6 　2006年 11月　第 4卷　第 6期
glucopyranoside (9)[ 10] , and apigenin 7-β-D-glucopy-
ranoside (10)[ 10] , pterosin C (11)[ 11] , pterosin P
(12)[ 12] , pterosin N (13)[ 13] , 2β , 16β-dihydroxy-ent-
kaurane (14)[ 14] , 2β , 15β-dihydroxy-ent-kaur-16-ene
(15)[ 13] , apigenin(16)[ 10] , naringenin(17)[ 15] , eri-
odictyol(18)[ 16] , β-sitosterol (19)[ 17] , p-hydroxyben-
zoic acid(20)[ 18] , cerotic acid(21)[ 17] , and pteroside
P (22)[ 13] were identified by comparing their spectro-
scopic data (optical rotation , MS , 1H and 13C NMR)
with those reported in the cited literatures.
Compound 1 was obtained as white amorphous
powder.Its molecular formula C20H28O8 was deduced
from the molecular ion peak m/z at 396.180 1 in the
HR-EI-MS.The IR absorption revealed the presence of
hydroxyl(3 334 cm-1)and carbonyl(1 680 cm-1).Up-
on acid hydrolysis , 1 yielded a D-glucose and agly-
cone.The latter had the same R f value with that of 12
in the TLC experiment.The 1H NMR spectrum of 1
showed an anomeric proton at δ4.29 (1H , d , J =
7.7 Hz), demonstrating the β-configuration of the sug-
ar.The 13C NMR spectrum of 1(Table 1)showed 14
carbon signals for the aglycone unit , which were re-
solved into one carbonyl , one penta-substituted aromatic
ring , one methine , four methylenes , and two methyl
carbons through the DEPT experiments.In contrast to
the
13
C NMR data of 12 , the mere difference is that δ
63.4(12-CH2)and 147.6(5-C)of 12 were replaced
by 68.1(CH2)and 143.5(C)of 1 , respectively , in-
dicating the glucose being attached to C-12 of the agly-
cone.The HMBC cross-peaks of H2-12/C-1′substanti-
ated this structure.Thus , 1was structurally established
to be pterosin P 12-O-β-D-glucopyranoside , i .e.,
1H-inden-1-one , 5-[( 1-β-D-glucopyranosyloxy )
methyl]-2 ,3-dihydro-6-(hydro-xyethyl)-2 ,7-dimethyl.
2.2　Biological activities
The inhibitory activities of compounds obtained
from P.multifida on rat PECs growth were evaluated
under serum-free conditions with or without growth fac-
tors added to the culture medium.The compounds were
applied at concentrations of 180 and 360 μmol·L-1 ,
and epristeride was used as a positive control.There
was no significant effect of the growth factors on the in-
hibitory activity of the compounds 3 , 4 and 5 inhibited
the growth of rat PECs in serum-free medium with or
without growth factors , with activity comparable with
epristeride (Table 1).6 and 16 also inhibited cell
growth , although their efficacy was weaker than that of
epristeride.In addition , 2 inhibited the growth of PECs
in medium containing the growth factors.
Table 1　 Inhibition on rat PECs growth in vitro by com-
pounds 2 ～ 6 , 16
Compounds
With growth factors
(μmol·L-1)
180 360
No growth factors
(μmol·L-1)
180 360
Epristeride 55.6 100 53.3 95.9
2 54.6 95.5 / /
3 76.3 100 60.4 81.1
4 79.7 99.3 79.3 89.7
5 63.0 93.9 38.0 93.1
6 50.0 76.5 52.2 57.3
16 50.0 50.0 41.3 43.5
3　Experimental
3.1　General experimental procedures
Melting points were determined with an X-4micro-
melting point apparatus and are uncorrected.Optical ro-
tations were measured using a Perkin-Elmer 241MC po-
larimeter.IR spectra were taken on a 10DX-FTS spec-
trometer with KBr pellets.EI-MS and FAB-MS were
recorded on a MAT-711 and a ZAB-HS spectrometer ,
respectively.HR-EI-MS and HR-ESI-MS were analyzed
on a MAT-711 and a 7.0 TESLA FTMS high resolution
spectrometer , respectively.NMR spectra were run on a
Bruker AM-400 instrument with TMS as an internal
standard.Macroporous resin D-1400 (Yangzhou Phar-
maceutical Factory , Yangzhou , China), Sephadex LH-
20 (Pharmacia Biotech AB , Uppsala , Sweden), Rp-18
(Greenherbs Science &Technology Development Co.,
Ltd., Beijing , China), silica gel (200 ～ 300 , 400
mesh , Qingdao Haiyang Chemical Group Co., Qing-
dao , China)were used for column chromatography
(CC).
3.2　Plant material
The air-dried whole plant of P.multifida Poir.
was purchased from Xuhui Chinese Medicinal Flake
Factory , Shanghai , China , in March of 2001 , and i-
dentified by Prof.Dao-feng Chen of Department of
Pharmacognosy , School of Pharmacy , Fudan Universi-
ty , Shanghai , China.The voucher specimens are de-
posited in the Herbarium of our institute.
3.3　Extraction and isolation
5 kg of dried and powdered whole plant of P .
multifida was extracted with 20%EtOH at room tem-
perature.The crude extract (395 g)was subjected to
CC over D-1400 macro resin eluting with H2O , 50%
and 95%EtOH.The last two were combined and chro-
matographed on Sephadex LH-20 column with MeOH
and MeOH/CHCl3(70/30 , V/ V) as eluents.The
MeOH fraction was further divided into fractions A1-4
through silica gel column with gradient CHCl3-MeOH as
　2006年 11月　第 4卷　第 6期 Chin J Nat Med　Nov.2006　Vol.4　No.6　429　
eluents.Fr.A2 was isolated by a RP-18 CC with
MeOH-H2O (90∶ 10)as eluent to yield 1 (55 mg)
and 22(52 mg);Fr.A3 afforded 3(18 mg)after pu-
rification of silica gel CC washed with CHCl3-MeOH-H2
O(80∶20∶1);2(127mg)was obtained from Fr.A4
via silica gel CC (CHCl3-MeOH-H2O , 80∶20∶2).
The MeOH/CHCl3(70/30)fraction was divided into
fractions B1-3 through silica gel column(CHCl3-MeOH-
H2O , 80∶20∶1).Fr.B1 afforded 7(15 mg)after
purified with silica gel CC eluting with CHCl3-Me2O(85∶15).Fr.B2was purified with silica gel CC elut-
ed with CHCl3-MeOH-H2O (80∶20∶1)and yielded
10(92mg).Fr.B3 afforded 8(73mg)and 9(68 mg)
after repeated purification of silica gel CC (CHCl3-
MeOH-H2O , 80∶20∶2).
In addition , 10 kg of P .multifida was extracted
three times with acetone.The combined extracts were
concentrated in vacuum and subjected to silica gel CC
eluted with gradient CHCl3-Me2CO(1∶0 ～ 0∶1)to
yield fractions C1-6.Fr.C1 yielded 6(15 mg)and 21
(17 mg)after purification by silica gel column(n-hex-
ane-acetone , 90∶10).From Fr.C2 , 5(12 mg)and
19(35 mg)were isolated through silica gel CC washed
with n-hexane-acetone (90∶10).Fr.C3 furnished 14
(12 mg)and 15(8 mg)after isolated by silica gel CC
(n-hexane-acetone , 80∶20).Fr.C4 was purified by
repeated CC of Sephadex LH-20(MeOH-CHCl3 , 7∶3)
and silica gel (CHCl3-Me2CO , 85 ∶15-70 ∶30)to
provide 11(15 mg), 12 (22 mg), and 13 (18 mg).
Fr.C5 was purified with silica gel CC washed with
CHCI3-MeOH (90∶10)and gave 17(17 mg)and 18
(6 mg).Fr.C6 was repeatedly purified with silica gel
CC eluted with CHCl3-MeOH (90∶10 ～ 85∶15)to
yield 4(25 mg), 16(34 mg)and 20(10 mg).
Pteroside P′(1)White amorphous powder , mp
219-221 ℃, [α] 25D +17(c 0.12 , MeOH).IR(KBr)ν
3 334(OH), 2 912 , 1 680(O=C), 1 599 , 1 440 , 1
385 , 1 105 , 1 078 , 1 034 , 893;1H NMR(400MHz ,
DMSO-d6):7.50(1H , s , H-4), 5.16(1H , d , J =
13.0 Hz , H-12b), 4.96 (1H , d , J =13.0 Hz , H-
12a), 4.29(1H , d , J =7.7 Hz , H-1′), 3.70(1H ,
d , J =6.0 Hz , H-6′a), 3.50(1H , d , J =6.0 Hz ,
H-6′b), 3.46 (2H , t , J =7.6 Hz , H-14), 3.26
(1H , m , H-3a), 3.12(1H , m , H-5′), 3.11(1H ,
m , H-3′), 3.10(1H , m , H-4′), 3.09(1H , m , H-
2′), 2.85(2H , t , J =7.5 Hz , CH2-13), 2.60(1H ,
m , H-2), 2.59(3H , s , Me-15), 2.58(1H , m , H-
3b), 1.16(3H , d , J =7.1 Hz , Me-11);13C NMR
(400 MHz , DMSO-d6):209.7(s , C-1), 152.1 (s ,
C-9), 143.5(s , C-5), 136.7 (s , C-7), 135.1 (s ,
C-6), 132.4(s , C-8), 123.8(d , C-4), 102.3(d ,
C-1′), 77.0(d , C-5′), 76.7(d , C-3′), 73.6 (d ,
C-2′), 70.1(d , C-4′), 68.1 (t , C-12), 61.1 (t ,
C-5′), 60.3(t , C-14), 42.1(d , C-2), 33.4(t , C-
3), 31.2(t , C-13), 16.2(q , C-11), 13.0(q , C-
15);FAB-MS (positive mode)m/ z (rel.int.):419
([ M+Na] +)(8), 397([M+H] +)(1), 435([M+
K] +(5), 117 (31);EI-MS m/z (rel.int.):396
(M+ , 15), 366 (M-2Me)(10), 234 (25), 217
(85), 203(35), 186 (100), 175(20), 161 (18);
HR-EIMS([M] + m/z = 396.180 1 , calcd.396.
1803).
3.4　Acid hydrolysis of 1
Compound 1(1.5 mg)was dissolved in 1 N HCl
(0.5 mL)and stirred at 80 ℃ for 4 h.After cooling ,
the reaction mixture was diluted with H2O and extracted
twice with EtOAc.From the EtOAc layer , the aglycone
was detected by TLC by comparison with 12.The water
layer was concentrated by blowing with N2.The residue
was dissolved in 1-(trimethylsilyl)-imidazole and pyri-
dine (0.2 mL)by stirring at 60 ℃ for 5 min.After
drying the solution with a stream of N2 , the residue was
separated by water and CH2Cl2(1 mL , 1∶1 , V/ V).
The CH2Cl2 layer was analyzed by GC using an L-Chi-
rasil-Val column(0.32 mm ×25m).Temperatures of
the injector and detector were 200 ℃.A temperature
gradient system was used for the oven , starting at 100
℃ for 1 min and increasing up to 180°C at a rate of 5
°C permin.A peak of the hydrolysate of 1 was detected
at 14.72 min.Whereas retention times for D-glucose
after being treated simultaneously with 1-(trimethylsi-
lyl)-imidazole in pyridine was 14.74 min.
3.5 Bioassay for inhibition on rat PECs growth in vitro
Immature male Sprague-Dawley rats(40 days old)
were purchased from SIPPR/BK (Shanghai , China).
Prostates were excised from the rats , cut into pieces and
digested with 200 IU·mL-1 collagenase and 100 μg·
mL-1 DNAase at 37℃ for 18 h[ 19] .Dispersed PECs
were washed and seeded at a density of 2.5×105 cells/
well into 24-well plates in serum-free McCoy s 5A
medium and cultured in 5%CO2 at 37℃ for 3 days.
Then , the inhibitory effects of the compounds on the
growth of the PECs were examined with or without
growth factors in the culture medium.The growth factors
used in the experiment were insulin (10 μg·mL-1),
epidermal growth factor (10 ng·mL-1), transferrin (5
μg·mL-1), prolactin (100 ng·mL-1)and testosterone
(0.1 μmol·L-1).Cultured rat PECs were incubated
with the different compounds at concentrations of 180
and 360 μmol·L-1.In the vehicle control group , cells
430　　Chin J Nat Med　Nov.2006　Vol.4　No.6 　2006年 11月　第 4卷　第 6期
were incubated with medium containing 0.1%
dimethylsulfoxide , which has no effect on cell growth.
Epristeride was used as the positive control.After 3 d of
treatment with the compounds or the positive control , an
MTT colorimetric assay was performed to evaluate the
effect of the samples on the rat PECs growth[ 20] .
Briefly , cells were incubated with MTT solution(1 mg·
mL
-1)for 4 h at 37 ℃.Then , 10%SDS-5% isobu-
tanol-0.01 mol·L-1 HCl was added to dissolve MTT-for-
mazan crystals.After 12 h , absorbance was recorded at
570 nm.Inhibition was expressed as the percent de-
crease in the OD value in the compound treated group
compared to the vehicle control group.All experiments
were performed at least three times.
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凤尾草的化学成分及其对体外大鼠前列腺上皮细胞
增生的抑制效应
秦　波1 ,朱大元1＊ ,蒋山好1 ,向　贵2 ,冷　颖1 ,顾芝萍1 ,汪意青2 , 邵雪峰2
1中国科学院 上海生命科学研究院 上海药物研究所 新药研究国家重点实验室 , 上海 201203;
2太仓市人民医院 ,江苏 215400
【摘　要】　目的:研究凤尾草抑制前列腺上皮细胞增生的活性化学成分。方法:大孔树脂 、凝胶 、正相及反相硅胶柱
层析方法用于化合物的分离;红外 、紫外 、核磁和质谱等方法用于鉴定化合物结构。化合物对大鼠前列腺上皮细胞的抑制
活性用MTT 方法测定。结果:从凤尾草的丙酮和 20%乙醇提取物中 ,分离并鉴定了 22 个化合物 , 其中化合物木犀草素 , 棕
榈酸和芹菜素 4′-O-α-L-鼠李糖苷显示与爱普列特相比拟的大鼠前列腺上皮细胞增生体外抑制效应。 结论:化合物 ptero-
side P′(1)是一个新的 C14的倍半萜苷;化合物木犀草素 、棕榈酸和芹菜素 4′-O-α-L-鼠李糖苷可能是凤尾草能抑制良性前列
腺增生的主要活性成分。
【关键词】　凤尾草;前列腺上皮细胞;良性前列腺增生;Pteroside P′
　2006年 11月　第 4卷　第 6期 Chin J Nat Med　Nov.2006　Vol.4　No.6　431