全 文 ：黑蕊虎耳草中岩白菜素没食子酸酯类及其对
丙型肝炎丝氨酸蛋白酶的抑制作用?
左国营1 , 2 , 李正全1 , 陈丽蓉1 , 何红平3 , 徐筱杰1
??
(1 北京大学化学与分子工程学院 , 养生堂天然药物实验室 , 北京 100871; 2 昆明总医院 , 云南 昆明 650032;
3 中国科学院昆明植物研究所西部植物化学与植物资源国家重点实验室 , 云南 昆明 650204 )
摘要 : 研究了黑蕊虎耳草 ( Saxifraga melanocentra) 中岩白菜素衍生物的化学和生物活性 , 从其地上部分分
离纯化得到一个新的岩白菜素没食子酸酯 , 主要通过 1 维和 2 维核磁共振波谱鉴定结构为 11-氧-(4-氧甲基
没食子酰 ) 岩白菜素 [11- O - (4- O-methylgalloyl) bergenin (1 ) ] , 该化合物对丙型肝炎丝氨酸蛋白酶 (HCV
NS3 serine protease) 具有抑制活性 , 其 IC50 为 0 .32 mg?mL。
关键词 : 抗丙型肝炎 ; 黑蕊虎耳草 ; 11-氧- (4-氧甲基没食子酰 ) 岩白菜素 ; IC50
中图分类号 : Q 946 文献标识码 : A 文章编号 : 0253 - 2700 (2007) 04 - 486 - 03
Gallic Acid Esters of Bergenin from Saxifraga melanocentra
(Saxifragaceae) and Their Inhibition Against
HCV NS3 Protease
ZUO Guo-Ying
1 , 2
, LI Zheng-Quan
1
, Chen Li-Rong
1
, HE Hong-Ping
3
, XU Xiao-Jie
1 * *
( 1 Collegeof Chemistry and Molecular Engineering, Yangshengtang Nature MedicineLaboratory, Peking University,
Beijing 100871 , China; 2 Kunming General Hospital , Kunming 650032 , China; 3 The State Key Laboratory
of Phytochemistry and Plant Resources in West China, Kunming Instituteof Botany,
Chinese Academy of Sciences, Kunming 650204 , China)
Abstract: To study the chemical and bioactive characteristics of bergenin derivatives which were purified from Saxifraga
melanocentra Franch ., anewgallic acidester of bergenin, 11- O- (4 - O-methylgalloyl) bergenin (1) was isolatedfromthe
aerial parts of Saxifraga melanocentra Franch ., and its structure was established mainly on the basis of 1D and 2D NMR
spectroscopic analysis . It showed weak inhibitory activity against HCV NS3 serine protease with IC50 of 0 .32 mg?ml .
Key words: HCV NS3 serine protease; Saxifraga melanocentra; 11- O - (4 - O-methylgalloyl ) -bergenin; IC50
Bergenin ( Chinese named yan-baicai-su) , a fa-
mous antitussive agent which has been used clinically
for manyyears, is amajor constituent fromthegenusof
Bergenia and Saxifraga (Saxifragaceae) and the other
plant species . It showed a variety of biological activi-
ties, including antioxidant, hepatoprotective ( Kim et
al . 2000 ) , antiarrhythmic (Pu et al . 2002) , anti-ul-
cer (Goel et al . 1997) and anti-HIV (Piacente et al .
1996) . In the courseof our screening natural products
as Hepatis C virus ( HCV) inhibitors, we studied the
active constituents of Saxifraga melanocentra Franch
previously ( Zuo et al . 2005a) . Further investigation
of its active constitution led us to isolate a new berge-
nin derivative, 11- O -( 4 - O-methylgalloyl ) bergenin
1 , and four known ones, i . e . 11- O -( 3 , 4 -di- O-
methylgalloyl) bergenin 2 ( Jia et al . 1995) , 11- O-
云 南 植 物 研 究 2007 , 29 (4) : 486～488
Acta Botanica Yunnanica

?
?? ?Author for correspondence; E-mail : xiaojxu@chem.pku. edu. cn; Tel : 86 - 10 - 62757456
Received date: 2006 - 10 - 11 , Accepted date: 2006 - 11 - 28
作者简介 : 左国营 (1963 - ) 男 , 博士 , 主要从事植物化学的研究。 ?
Foundation item: NSFC 30472147
Fig . 1 Structures of compounds 1 - 5
galloylbergenin 3 ( Yoshida et al . 1982 ) , 11- O -( 4-
hydroxy benzoyl ) bergenin 4 ( Fuji et al . 1996 ) and
bergenin 5 (Taneyama et al . 1983) . The present pa-
per describes the structural elucidationof the newgallic
acid ester of bergenin ( Fig . 1 ) . Compounds 1 - 5
showed weak to moderate inhibitory activities against
HCV NS3 serine protease (Table 1) .
Compound 1 was obtained as colorless needles,
whosemolecular formula C22 H22 O13 was established by
HRESIMS spectrum ( m?z 493 .1057 [M-l ] - , calcd .
for C22 H21 O13 493 .1060) . The IR absorptions at3390 ,
3250 , 1726 , 1704 , 1608 , 1464 cm- 1 suggested the
presence of hydroxyl and carbonyl groups . Its 13 C NMR
spectra exhibited characteristics of a bergenin moiety,
including a lactone group (δ 163 .7 ) , two phenolic
hydroxyl groups (δ151 .7 and 148.9 ) and a phenoxy-
Table 1 Inhibitory activities of compounds 1 - 5
against HCV NS3 serine proteasea
compounds IC50 ?( mg?ml)
1 ?0 g. 32±0 M. 08
2 ?> 1 ?. 00
3 ?0 g. 07±0 M. 02
4 ?> 1 ?. 00
5 ?0 g. 56±0 M. 13

a The results are the mean values of triplicate tests .
methyl group (δ141 .3 , 60 .6) , five tertiary oxygenat-
ed and a second oxygenated linkages, which were
closely similar to those of compound 5 . Its 1 H NMR
spectrumshowed two methoxyl singlets atδ3 .88 (s,
Me) and 3 .89 (s, Me) , whose attached carbons were
overlapped and exhibited strongly atδ60 .6 in the 13 C
NMR spectrum . This suggested the two methoxyl
groups were in the similar chemical situation . There
were three aromatic singlets at δ 7 .09 , 7 .17 and
7 .17 , of which the latter two were also overlapped to
double approximately their height in comparison with
the former singlet . The same phenomenon was further
observed by their corresponding signals atδ 110 .3 ,
110 .0 and 110 .0 in the 13 C NMR spectrum, of which
two carbonyl signals atδ163 .7 and 166 .5 were desig-
nated to C - 6 andC - 1′, respectively .Thepresenceof
amethylated galloyl moiety, to which the C - 11 meth-
ylene was attached by an ester linkage, was suggested
by the HMBC correlation of H - 11 with carbon in the
ester, and a methoxyl group was attached to C - 4′of
thegalloyl moiety, also suggested by the HMBC corre-
lationof them . Therefore, the structure of Compound 1
was elucidated as 11- O -( 4- O-methylgalloyl ) berge-
nin . All the signals of protons and carbons of 1 were
assigned unambiguously through analysis of the spectra
of DEPT, HSQC and HMBC (Fig . 2 ) , and compared
with the correspondingdataof compound 3 . Identifica-
tionof compounds 2 - 5 were performed by analysis of
their spectraof MS, IR , NMR and compared with lite-
ratures and an authentic sample of bergenin (5) .
Fig . 2 Key HMBC correlations for 1
Experimental
Melting pointswere determined usinga Kofler micro-melting
point apparatus and are uncorrected . Optical rotations were de-
termined on a Horiba SEPA-300 polarimeter . 1 H and 13 C NMR
spectra were determined on a Bruker ARX400 spectrometer at
400 and100 MHz, respectively, in CD3 OD, withSiMe4 as inter-
nal standard; chemical shifts are given as ( ppm) values . IR
spectra were recorded on a Nicolet Magana-IR750 . Electrospray
ionization and time of flight Mass spectra ( ESI-TOF-MS) were
taken on aMariner Biospectrometry workstation . TLC plateswere
made with polyamide film ( China) , and spots were detected by
ultraviolet (UV) and by spraying with FeCl3 ( 5% in ethanol )
reagent . Polyamide ( 100 - 200 mesh, China) and Sephadex .
LH-20 ( Amersham Pharmacia Biotech) was used for column
chromatography ( C . C) . The organic solvents used wereof ana-
lytical grade . Solvents in the extracts were evaporated under re-
7844 期 ZUO Guo-Ying et al .: Gallic Acid Esters of Bergenin from Saxifraga melanocentra (Saxifragaceae) . . .
duced pressure at below40℃ .
The aerial parts of S. melanocentra were bought from the
Qinghai Institute of Tibetan Medicines ( Qinghai Province, Chi-
na) in October, 2002 , where a voucher specimen has been de-
posited .
Part of the experimental materials and methods have been
reported previously (Zuo et al . 2005b) . The powdered aerial
part of S. melanocentra ( 1 kg) were extracted with ethanol
(80 % , 4 L×3) at roomtemperatureand concentrated to dryness
invacuumtogive acrudeextract (205 g) . Theextractwas parti-
tioned between H2 O ( 600 mL) andpetroleumether (60 - 90℃ ) ,
chloroform, ethyl acetate and n-butanol (250 mL×3 each) suc-
cessively . The ethyl acetate layer part ( 20 g) having the most
potent inhibitory activity for HCV NS3 serineprotease by ca80%
at 100 mg?mL was fractionated over silicagel C . C . with solvent
systems as ethyl acetate-methanol-H2 O (10∶1∶0 - 7∶1 .5∶1) to
give four active fractions ( Frs-1-4 ) . The more active fraction
(Fr-3 , 4 .5 g) was further isolated by polyamide C . C . (ethyl
acetate-methanol-H2 O (120∶13∶5 - 77∶13∶10 ) ) to give com-
pounds 1 - 5 (50 mg, 20 mg, 16 mg, 22 mg, 3 . 4 g) , respective-
ly (Fig . 1) .
Compound 1 , colorless needles (MeOH ) , mp: 142 -
144℃ ; [α] 20D : + 39.3°( c, 0 . 5 , EtOH) ; EIMS m?z ( % ) :
494 (3) , 328 ( 4 ) , 208 ( 14 ) , 184 ( 12 ) , 167 ( 22 ) , 138
(63 ) , 121 ( 100) , 93 ( 26) ; HRESIMS: ( m?z 493 .1057 [M-
l ] - , calcd . for C22 H21 O13 493 .1060 ) . IRνKBrmax cm- 1 : 3390 ,
3250 (OH ) , 2852 ( CH2 ) , 1726 , 1704 ( C = O) , 1608 , 1527
(aromatic) , 1464 , 1349 , 1236 (aromatic C-O) , 864 ( C-H ) ;
1 H-NMR (400 MHz, CD3 COCD3 , ( ppm, J Hz) : δ7 .17 (s,
2H, H - 2′, 6′) , 7 . 09 (s, 1H , H - 7) , 5 . 17 ( d, 1H, J =
10 .4 , H - 10b) , 4 . 94 ( d, 1H, J = 2 .0 , H - 11α) , 4 . 45 ( d,
1H, J = 5 .6 H - 11β) , 4 . 16 (m, 1H, H - 4a) , 4 . 13 (m, 1H,
H - 2) , 3 . 96 ( m, 1H, H - 4 ) , 3 . 89 (s, 3H, C-9-OC - 苩H 苩3 ) ,
3 . 88 (s, 3H , C-4′-OC - ?H ?3 ) , 3 . 72 ( m, 1H , H - 3) ; 13 C-NMR
(100 MHz, CD3 COCD3 , δppm) : 166 .5 ( s, - 菒COO-methylgal-
loyl) , 163 .7 (s, C - 6) , 151.7 (s, C - 8) , 151 .3 ( s, C - 3′,
5′) , 148 .9 (s, C - 10) , 141 .3 (s, C - 9) , 140 .7 (s, C - 4′) ,
126 .0 ( s, C - 1′) , 119 .5 (s, C - 6a) , 116.7 ( s, C - 10a) ,
110 .3 ( d, C - 7 ) , 110 .0 ( d, C - 2′, 6′) , 80 .7 ( d, C - 4a) ,
80 .1 ( d, C - 2) , 75 .3 ( d, C - 4 ) , 74 .1 ( d, C - 10b) , 71 .5
(d, C - 3 ) , 64.3 ( t, C - 11) , 60.6 ( q, C-9-O - 薀CH3 , C-4′-
O - ?CH3 ) .
Inhibitory activities of compounds 1 - 5 against HCV NS3
serine protease were determined inour laboratory by ELISA (Ta-
ble1 ) . A peptide substrate with an acetyl group at N-terminus
and a biotin at C-terminus was hydrolyzed by NS3 protease into
product with a freeamino moiety at N-terminus . Theproductwas
immobilized and the free amino moiety was analyzed (Takeshita
et al . 1997 ; Zuo et al . 2005a) .
Acknowledgements : Spectra data were recorded by the Analyti-
cal Center of Peking University and analytical groupof Laboratory
of Phytochemistry and Kunming Institute of Botany, the Chinese
Academy of Sciences, which is gratefully acknowledged .
References:
Fuji ?M , Miyaichi Y , Tomimori T, 1996 . Studies onNepalese crudedrugs
on the phenolic constituents of the rhizome of Bergenia ciliata
( Haw .) sternb . [ J ] . Nat Med, 50 : 404—408
Goel ?RK , Maiti RN, ManickamM et al . 1997 . Antiulcer activity of nat-
urally occurringpyrano-coumarin and isocoumarins and their effect on
prostanoid synthesis using humancolonic mucosa [ J ] . Indian J Exp
Biol , 35 : 1080—1083
J ia ?ZH , Mitsunaga K , Koike K et al . 1995 . New bergenin derivatives
from Ardisia crenata [ J ] . Nat Med, 49 : 187—189
Kim ?HS, LimHK , Chung MW et al . 2000 . Hepatoprotective effects of
bergenin, a majorconstituent of Mallotus japonicus, on carbon tetra-
chloride- intoxicated rats [ J ] . J Ethnopharmacol , 72 : 469—474
Piac ?ente S, Pizza C, DeTommasi N et al . 1996 . Constituents of Ardisia
japonica and their in vitro anti-HIV activity [ J ] . J Nat Prod, 59 :
565—569
Pu H ?L , Huang X , Zhao JH et al . 2002 . Bergenin is the antiarrhythmic
principle of Fluggea virosa [ J ] . Planta Med, 68 : 372—374
Take ?shitaN , Kakiuchi N , Kanazawa T et al . 1997 . An enzyme- linked
immunosorbent assay for detecting proteolytic activity of hepatitis C
virus protease [ J ] . Anal Biochem, 247: 242—246
Tane ?yama M, Yoshida S, Kobayashi M et al . 1983 . Isolation of norber-
genin from Saxifraga stolonifera [ J ] . Phytochemistry, 22 : 1053—
1054
Yosh ?ida T, Seno K , Takama Y et al . 1982 . Bergenin derivatives from
Mallotus japonicus [ J ] . Phytochemistry, 21 : 1180—1183
Zuo ?GY , Li ZQ, Chen LR et al . 2005a . In vitro anti-HCV activities of
Saxifraga melanocentra and its related polyphenolic compounds [ J ] .
Antivir ChemChemother, 16 : 393—398
Zuo ?GY , Zhang ZJ , Chen LR et al . 2005b . Chemical constituentsof Ti-
betan herbal medicine Saxifraga melanocentra [ J ] . Acta Bot Yun-
nan, 27 : 691—694
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