全 文 ：Received date:2004-01-05
Foundation item:National natural science foundation of china(30070885)
Biography:Lou Hong-xiang(1965-), male (Han nationality), a native of Cangshan , Linyi , Ph.D., Dean and P ro fesso r
(the supervisor of doctoral students),main research interest lies in natural products , Tel:(0531)8382019 , Fax:(0531)8382373 ,
E-mail:louhongxiang@sdu.edu.cn.
Article ID:1005-0108(2004)04-0202-07
Identification of stilbenoids from the roots of
Vitis amurensis and their antioxidative effects
LOU Hong-xiang , ZHAO Yan ,REN Dong-mei , Fan Pei-hong , Ji Mei
(School of Pharmacy , Shandong Universi ty , Jinan 250012 , China)
Abstract:Aim To identify the chemical constituents from the roots o f Vitis amurensis and evaluate their
antioxidative effects.Methods The constituents were isolated and purified through column chromato-
g raphy of silica gel , Toyopearl HW-40 , Sephadex LH-20 or preparative HPLC.Their structures and
stereochemistry have been established on the basis of spectral evidence and preparation of chemical
derivatives.The antioxidative effects w ere de termined by the DPPH radical scavenging test and reducing
power test.The anti-g lycation effects were also evaluated.Results Four stilbene derivatives were isolated
tog ether with two triterpenes and high contents of salicylic acid.Their inhibitory effects on protein ad-
vanced g lycation end products(AGEs)formation as w ell as fer ric reducing/antioxidant pow er , free radical
scavenging activities were prominent , and the anti-glycation effects have a good co rrelation w ith the an-
tio xidant effects.Conclusion These stilbenes have potential in the therapeutic use.
Key words:medicinal chemistry;iso lation;identification;chromatog raphy;Vitis amurensi Rupr;stil-
benoids;antio xidation;DPPH radical scavenging;AGE inhibitory effects
CLC number:R914　　　Document code:A
　　More than 150 resveratrol oligomers have been
isolated and their structures elucidated[ 1 ,2] .The in-
teresting biological activities recently found in stil-
bene derivatives such as induction of apoptosis in
colon cancer[ 3] , blood sugar reduction[ 4] , antibacte-
rial , antifungal[ 1] , HIV inhibito ry and cy toto xic[ 5]
effects revealed the importance of the plants con-
taining stilbenoids as resources fo r the development
of new drugs.Vitis amurensis is widely dist ributed
in China , whose fruits are edible and can be used
for w ine fermentation.The roots of V.amurensis
are used as a folk medicine of ant ialgesic ,heptopro-
tective , anti-rheumatic effects and can alleviate the
complicat ions of diabetic patients
[ 6] .
In our continuous studies on the natural phe-
nolic compounds and their biological signif i-
cance[ 7] , from the roots of V .amurensis , four
resveratrol stilbenoids(3 ～ 6)were isolated together
with two t ri terpenes(1 ,2)and high contents of sali-
cy lic acid(7).Their structures and stereochemist ry
were established on the basis of spectral evidence
and chemical derivatives preparat ion.Their po ten-
tial inhibitory effects on protein advanced g lycat ion
end products(AGEs)formation as w ell as ferric re-
ducing/ant ioxidant pow er , f ree radical scavenging
activi ties were tested.These compounds exhibited
considerable inhibitory activity on AGEs formation
and free radical scavenging.TheAGEs inhibitory effects
were found to have a good correlation with the reducing
power and free radical scavenging ability.
1　EXPERIMENT
1.1　Plant materials
The roots and the rhizomes of Vit is amurensi
Rupr w ere collected at Mountain Lushan(Zibo ,
Shandong Province)in April 2000 and identified by
第 14 卷　第 4 期
2004 年8月　总 60期
中 国 药 物 化 学 杂 志
Chinese Journal o f Medicinal Chemistry
Vol.14　No.4　p.202
Aug.2004 Sum 60
DOI :10.14142/j.cnki.cn21-1313/r.2004.04.003
Professor Li Yunyao at the department of pharma-
cognosy of Shandong University and a voucher
specimen w ith a voucher number V-0132 is de-
posited there in the herbarium.
1.2　General experimental procedures
Melting points were measured on Mocro-X4
(uncorrected), NMR spect ra w ere recorded on
DRX-500 spect rometer wi th cryo-probe.UV spec-
tra w ere recorded on a Shimadzu UV 2200 spec-
trometer.The ESI-MS was performed on an API
4000 mass spectrometer(Canada).For ESI-MS ,
samples were dissolved in MeCN-H2O(1∶1 , V∶V)
and int roduced into the mass spectrometer by con-
tinuous flow injection at 5 μL/min and a needle
voltage of +4.5 kV.IR spect ra w ere measured
w ith Nicolet NEXUS 470 FT-IR spectrometer.Sil-
ica gel 60(100 ～ 200 mesh , Qingdao), Sephadex
LH-20(Pharmacia),ODS(100 ～ 200 mesh , Tianjin
Chemical Co.), and Toyopearl HW-40(fine g rade ,
Tosoh)were used fo r CC.Kieselgel 60 F 254(Merck)
was used as analy tical TLC.
Semi-preparative HPLC separations were achieved
on a 10 μm RP-18 column(prep-LC 25 mm Module
column ,Waters , USA).Linear g radient elution w as
used starting f rom 40%(volume percent , φ)to
60%MeOH(φ)in 5% aq.MeCN(φ)(over 60 min
at a f low rate of 6.2 mL/min.The UV trace was
recorded at 280 nm.Peak fractions w ere collected
manually , concentrated on a rotavapor and then
lyophilized.The purified ext racts and isolated stil-
benes were analyzed by analytical HPLC with a 5 μm
C-18 Luna(250 mm×4.6 mm;DIKMA , USA)us-
ing 50%MeOH(φ)in 1% aq.HCOOH(φ)as mo-
bile phase at 1.0 mL/min.On-line UV spectra of
stilbenes were recorded with a diode-array detector
(HP-1100 , USA).Preparative HPLC was carried
out on Waters 600 ～ 996.
1.3　Extraction and isolation of phenolic ingredients
The dried roo ts of Vitis amurensis(5.2 kg)
were powdered and ex tracted with boiling 95%
EtOH(φ)twice(60 L ×2),1 h each.The combined
ex traction w as evaporated at reduced pressure.The
residue(548 g)was then ext racted successively w ith
ethyl acetate(2 L ×5), n-butanol(1 L ×5).After
evapo ration of the solvent , the ethyl acetate ext ract
(272 g)was subjected to silica gel(1.1 kg)column
chromatography eluted w ith a g radient system of
petroleum ether(60 ～ 90 ℃)and acetone w ith in-
creasing polarity to give eleven fractions(Fr.A-K).
Fr.D (11.2 g)(eluted w ith the mixture of
petroleum ether and acetone at 97∶3 , V∶V), was
further purified over silica gel(80 g), and eluted
with the mixture of hexane and ethy l acetate
(97∶3 , V∶V)to afford compounds 1(1.10 g)and 2
(3.25 g).Compound 3(4.50 g)was obtained as
colorless needles f rom Fr.H(38 g)(fraction ob-
tained by elution w ith the mixture of pet roleum
ether and acetone at 6∶4 , V∶V).The phenolic Fr.
I(27 g)(eluted w ith the mix ture of petroleum ether
and acetone at 5∶5 , V∶V), af ter being further sep-
arated over Toyopearl HW-40(500 mL)and eluted
with the mixture of MeOH and w ater(3∶7 , V∶
V), af forded four subfractions(Sub-fr.1 ～ 4).Sub-
fr.1(1.6 g)was further purified over Sephadex
LH-20(100 g)and eluted with MeOH and w ater(4∶6 ,
V∶V)afforded 3(200 mg)and 6(610 mg).Sub-f r.
3(2.6 g), af ter being preliminarily purified over
Sephadex LH-20(100 g)and eluted wi th MeOH
and w ater(4∶6 , V∶V), was purif ied over semi-
preparat ive HPLC.Compound 4(490 mg)and 5
(190 mg)and 6(120 mg)were af forded.The n-bu-
tanol ex tract ,when w orking out of the solvent , the
salicy lic acid 7(16 g)was obtained.The purity of
the isolated compounds was mo re than 96% by
analytical HPLC except 5(the purity was about
90%).
In o rder to further purify 5 , peracety lation of
the phenolic compound 5 was carried out by dis-
solving 5(30 mg)in the mix ture of py ridine and an-
hydrous acetic acid(2∶1 , V∶V , 1mL)and stirred at
room temperature overnight.The reaction mix ture
was poured into iced w ater(25 mL)and the precipi-
tation was obtained and crystallized w ith acetone ,
compound 5a(28 mg)was obtained.
1.4　Identification of the isolated compounds
The st ructures of lupeol(1),betulinic acid(2),
reseverat rol(3)and salicylic acid(7)were identified
(Figure 1)by NMR techniques and by comparison
2034 期 LOU Hong-xiang et al:Identification of stilbenoids from the roots of Vit is amurensis and their antioxidative effect s
w ith previous literature[ 8] o r direct comparison with authentic samples.
Fig.1　The chemical structures isolated from the roots of V.amurensis
　　(-)-cis-Vitisin B(4).White pow der and eas-
ily become yellow brow n when being exposed to air
in an aqueous solution for a long time.[ α] 25D -85°
(c 0.5 ,MeOH).The ESI-MS gave the molecular
w eight at m / z:907.3(MH+/ z).The 1H-NMR
spectrum show ed three pai rs of mutually coupled
aliphatic protons assignable to three dihydrofuran
rings[ δ:5.30(1H ,d , J =5.0Hz ,H-7a),4.27(1H ,
d , J =5.0 Hz , H-8a);5.44(1H , d , J =5.5 Hz , H-
7c), 4.22(1H ,d , J =5.5 Hz ,H-8c);5.21(1H ,d , J
=6.0 Hz , H-7d), 3.85(1H , d , J =6.0 Hz , H-
8d)] , six sets of ortho-coupled aromatic hydrogens
[ δ:7.12(2H , d , J =8.5 Hz , H-2a , H-6a), 6.76
(2H ,d , J =8.5 Hz ,H-3a , H-5a);7.01(2H , d , J =
9.0 Hz ,H-2d ,H-6d),6.73(2H ,d , J =8.0 Hz ,H-
3d ,H-5d);6.60(2H , d , J =8.5 Hz , H-2c , H-6c),
6.54(2H ,d , J =8.5 Hz ,H-3c ,H-5c)] , two sets of
meta-coupled aromatic protons[ δ:6.17(1H ,d , J =
1.5 Hz ,H-12b),6.20(1H ,d , J =1.5 Hz , H-14b);
6.28(1H ,d , J =2.0 Hz , H-12c), 6.11(1H , d , J =
2.0 Hz ,H-14c)] , two sets of AX2-type meta-cou-
pled pro tons[ δ:5.97(2H ,d , J =2.0 Hz ,H-10a ,H-
14a),6.06(1H , t , J =2.0 Hz ,H-12a);5.93(2H ,d ,
J =2.0 Hz ,H-10d ,H-14d), 6.09(1H , t , J =2.0 Hz ,
H-12d)] and trisubstituted benzene ring [ δ:6.54
(1H ,d ,J =1.5 Hz ,H-2b), 6.56(1H ,d , J =8.0 Hz ,
H-5b), 6.91(1H , dd , J =8.0 , 2.0 Hz , H-6b)] .
One cis-double bond protons at 5.96(1H , d , J =
12.0 Hz ,H-7b), 6.06(1H , d , J =12.0 Hz , H-8b)
were also assigned by the signals in 1H-NMR.The
structure of compound 4 was finally decided to be
(-)-cis-Vitisin B by HMBC and NOESY experi-
ments.All the NMR data w ere in accordance w ith
those in the li terature[ 9 ,10] .
The API-MS of 5 gave a molecular w eight at
m/ z :907.3(MH+/z), 1H-NMR of 5 was resem-
bled w ith that of 4 except the protons at δ:6.52
(1H ,d , J=17.0 Hz)and 6.68(1H ,d , J =17.0 Hz)in
1H-NMR which assigned to a t rans-double bond.
Compound 5 can be decided to be trans-vintisin B
by combinational analysis of its
1
H-NMR and 13C-
NMR data.Because some impurities inf luenced the
spectral interpretat ion clearly , the peracetylat ion
derivative of 5 w as prepared.The nona-acetylated
trans-vitisin B 5a was obtained af ter acetylation of
5 and its structure w as finally decided to be t rans-
vi tisin B[ 11] by the 2DNMR experiment.
Ampelopsin A(6).White pow der , [ α] 25D =+
187°(c 0.78 , MeOH).The ESI-MS gave the
molecular weight of 6 at m /z :471.2(MH+/ z)
corresponding to the molecular fo rmula C28H22O7 ,
suggesting that 6 was a resverat rol dimmer.One
group of mutually coupled aliphatic protons was
assignable to a dihydrofuran ring[ δ:5.70(1H ,d , J
=11.1 Hz , H-7a), 4.02(1H , d , J =11.1 Hz , H-
8a)] , the other group of mutually coupled aliphat ic
protons[ δ:5.38(1H , d , J =5.1 Hz , H-7b), 5.39
(1H , d , J =5.1 Hz , H-8b)] could be deduced to
connect w ith two carbons at δ:44.0(C-7b)and
71.6(C-8b)by the HMQC spect ra.Two g roups of
204 中 国 药 物 化 学 杂 志 14卷
o rtho-coupled aromat ic protons[ δ:7.02(2H , d , J =
8.5 Hz ,H-2a , H-6a),6.68(2H , d , J =8.5 Hz , H-
3a ,H-5a)and 6.82(2H , d , J =8.5 Hz , H-2b , H-
6b),6.59(2H ,d , J =8.5 Hz ,H-3b ,H-5b)] as w ell
as the tw o 3 , 5-dihydroxyphenyl groups[ δ:6.31
(1H ,d , J=2.2 Hz ,H-12a),6.10(1H ,d , J =2.2 Hz ,
H-14a), 6.11(1H , d , J =2.1 Hz , H-12b), 6.53
(1H , d , J =2.1 Hz , H-14b)] suggested the four
benzene ring s in the st ructure.The positions of the
protons and the stereochemist ry of 6 were further
identified by the HMBC and NOESY experiments
and its st ructure w as finally identified to be am-
pelopsin A.All the NMR data were in accordance
w ith those in the reference
[ 12] .
1.5　Determination of the effects on DPPH radi-
cal(1 ,1-diphenyl-2-picrylhydrazyl)
The test sample in the solution w as added to a
methanolic solution(1 mL)of DPPH radical(final
concentration of DPPH was 250 μmol/ L).The
mixture was shaken vigorously and lef t to stand for
30 min.The absorbance of the resulting solut ion
w as measured at 517 nm and compared to the ab-
sorbance of DPPH in the absence of the sample so-
lution.The low er the absorbance at 517 nm repre-
sents the higher DPPH scavenging act ivity .The
percent inhibi tion of each sample w as calculat-
ed[ 13] .All analyses were run in three replications
and averaged.
1.6　Inhibition test on AGE formation in vi tro
The inhibition test was carried out according to
previously reported[ 14] .Samples of the test compounds
1 mL(dissolved in phosphate buffer ,pH 7.4)and 1 mL
of the bovine serum albumin(BSA)(1.0 mg/mL in
phosphate buffer , pH 7.4)were incubated w ith
1 mL of 0.5 mol/L glucose in 50 mmol/L phos-
phate buffer , pH 7.4 , fo r 40 h at 55 ℃.The pro-
duction of fluorescent AGE(Advanced protein Gly-
cation End product)products w ere monito red by as-
sessing the formation of the novel glucose-derived
fluorescence using excitation and emission w ave-
lengths of fluorescence 337 nm and 430 nm , respec-
tively , in the presence or absence of the sample so-
lution.The percent inhibi tions were calculated.All
analy ses were run in three replications and aver-
aged.
1.7　Determination of reducing power
The reducing power of different phenolic com-
pounds was determined according to the method of Oy-
aizu[ 15] .Samples(2.5 mL)were mixed w ith 2.5 mL
phosphate buf fer(0.2 mol/L ,pH 6.6)and 2.5 mL
potassium ferricy anide(0.03 mol/ L).The mix ture
was then incubated at 50 ℃ for 20 min , 2.5 mL
trichloroacetic acid(0.6 mol/L)was added and the
mixture w as centrifuged at 4000 r/min fo r 5 min.
The upper layer of the 5 mL of the solution was
then mixed w ith 5 mL of distilled water and 1 mL
of ferric chloride(0.006 mol/L).The absorbance
was measured at 700 nm.Increased abso rbance of
the reaction mix ture indicated increased reducing
power.
2　RESULTS
In the experiment , four resverat rol stilbenoids
(3 ～ 6)were isolated together wi th two triterpenes
(1 , 2)and high contents of salicy lic acid(7).From
the roots of V .amurensis , the resveratrol dim-
mers , t rimers and tetramers have been reported pre-
viously[ 4 ,16 , 17] .This is the f irst time to repo rt the
vi tisin B and its stereochemical isomer cis-vitisin B
from this plant.Their potential inhibitory effects to
protein advanced glycation end products(AGEs)forma-
tion as well as ferric reducing/antioxidant power , f ree
radical scavenging activities were tested.
2.1　DPPH radical-scavenging activities
The DPPH radical-scavenging activi ty of the
isolated st ilbene polyphenols is show n in Figure 2
and expressed as the percentage reduction of the
initial DPPH absorption by the test compound.Cat-
echin w as used as a posi tive control.
Figure 2 show s that reseverat rol and i ts
oligomeric derivatives(3 ～ 6)are excellent DPPH
radical-scavengers.When the phenol hydro xyl
groups were acetylated(5a), no DPPH scavenging
effects w ere observed.And under this condition , no
effect on f ree radical scavenging effects was ob-
served for salicy lic acid.The o rder of the potency of
these compounds according to their correspond IC50
are:catechin>3>4 ,5 ,6 >5a ,7.
2054 期 LOU Hong-xiang et al:Identification of stilbenoids from the roots of Vit is amurensis and their antioxidative effect s
Fig.2　The scavenging effect of stilbenoids from Vitis
amurensis on DPPH radical.(catechin was used as a control)
2.2　Reducing power
Among the six tested compounds , catechin as a
control , the reducing power of resverat rol and its
oligomers was excellent(Figure 3).According ly ,
when the phenolic OH was acetylated , the reducing
pow er decreased greatly.Also no obvious reducing
pow er w as observed for salicylic acid.The same po-
tency order of these st ilbenoids as the f ree radical
scavenging ef fects was found.
Fig.3　The reducing power of stilbenoids from Vitis
amurensis(catechin was used as a control)
2.3　AGEs inhibition
The inhibi tory effects of resveratrol and its
oligomer derivatives on BSA(Sigma , USA)glyca-
tion w ere tested by the fluorescence method and
showed that resverat rol and phenolic stilbenes have
potent inhibi tory effects to the formation of AGEs
(Figure 4).The ef fects had a good correlation w ith
the reducing pow er and free radical scavenging ability
except salicylic acid.Salicylic acid was found to have
some inhibitory effect on the AGEs formation.
Fig.4　The AGE inhibition ef fects of stilbenoids from Vitis
amurensis(catechin was used as a control)
3　DISCUSSION
There is now increasing interest in antioxidant
activi ty of phytochemicals.Antioxidants and free
radical scavenging agents are believed to play a very
important role in the body defense system against
reactive oxygen species(ROS),which are the harm-
ful byproducts generated during the normal cell
aerobic respiration[ 18] .The 1 , 1-dipheny l-2-picryl-
hydrazyl(DPPH)radical is a stable radical wi th a
maximum abso rption at 517 mm that can readily
undergo reduction by an antioxidant .Because of the
ease and convenience of this reaction , i t now has
widespread use in the free radical-scavenging activity
assessment.According to the test results , we can find
that the reducing power had a good correlation with the
free radical scavenging effects.The reducing power
might be due to their hydrogen-donating ability.
Exposure of proteins to reducing sugars like
glucose results in the non-enzymatic g lycat ion
which fo rms reversible Schif f bases and Amadori
compounds[ 19] .A series of further complex molecu-
lar rearrangements then yields irreversible AGEs.
In diabetes melli tus(DM), prolonged hyperglyce-
mia superdrives this reaction and AGEs accumulate
in the ci rculating blood and in various tissues[ 19] .A
hypo thesis that the interactions between AGE and
206 中 国 药 物 化 学 杂 志 14卷
receptor for AGE(RAGE)are the majo r causes of
diabetic v ascular derangement and diabetes compli-
cations have been test if ied from experiments[ 20 ,21] .
Hyperglycemia also results in oxidative st ress ,
which may play a role in the generation of int racel-
lular AGEs , and the inhibition of oxidant pathw ay s
prevents int racellular format ion of AGEs.The dis-
covery of the inhibito rs of the fo rmation of AGEs
w ill provide one impo rtant way to the treatment of
diabetic complicat ions.From the above tests , we
can f ind that these stilbenoids either in their
monomeric or in oligomeric states have potent ef-
fects on f ree radical scavenging and ferric reducing
capacity as well as the AGEs inhibi tion activities ,
these results were also verified by our recent exper-
iment in st reptozotocin -induced DM mice.But the
oligomerization can reduce its antioxidative poten-
cy .The phenolic hydroxyls in the st ructure should
play a key role in these biolog ical tests.When the
phenolic hydroxy ls were acetylated , their act ivit ies
disappeared.No free radical scavenging effect was
show n fo r salicylic acid in our experiment , but still
some inhibitory ef fect was found wi th the AGEs
formation.This is because the formation of AGEs
w as not to tally an oxidative process ,but a complex-
ity potent ial pathw ay[ 22] .These results underline
the importance of antioxidants in diabetes and indi-
cate the possibility of the therapeutic use of these
phenolic compounds fo r the prevention of diabetic
complicat ions.
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蛇葡萄根中艹氐类化合物的鉴定及其抗氧化作用
娄红祥 ,赵 艳 ,任冬梅 ,范培红 ,季 梅
(山东大学药学院 ,山东 济南 250012)
摘　要:目的 对蛇葡萄根中的化学成分及其抗氧化活性进行研究。方法 采用硅胶柱色谱 、Toyopearl HW-40 柱色谱 、
Sephadex LH-20 柱色谱及半制备高效液相分离纯化 ,通过波谱解析及衍生物制备确定结构 , 并进行 DPPH 自由基捕获 、
还原 、抗糖化试验。结果 分离鉴定了 4 个艹氐类化合物 , 均具有较强的抗氧化 、抗糖化能力。结论 这些艹氐类化合物具有
治疗应用前景。
关键词:药物化学;分离;鉴定;色谱法;蛇葡萄;艹氐类;抗氧化;DPPH 自由基捕获;AGE 抑制
(上接第 201页)
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3-哌啶甲酸和四氢烟酸的 4 ,4-二芳基-3-丁烯衍生物的
三维定量构效关系(比较分子力场分析)研究
郑剑斌1 ,闻 韧1 ,罗小民2 ,蒋华良2
(1.复旦大学药学院药物化学教研室 ,上海 200032;2.中国科学院上海药物研究所 ,上海 201203)
摘　要:目的 研究 3-哌啶甲酸和四氢烟酸的 4 , 4-二芳基-3-丁烯衍生物结构和对 γ-氨基丁酸摄取抑制活性之间的关系。
方法使用三维定量构效关系(3D-QSAR)———比较分子场分析(CoMFA)方法进行构效关系研究。结果 交叉验证系数 q2
和非交叉验证系数 r2 分别为 0.726 和 0.986 , 方差比 F 为 117.562 ,标准估计误差(SEE)为 0.062。结论 这些数值表明
所得的 CoMFA 模型有实际意义 , 并且对 3-哌啶甲酸和四氢烟酸的 4 , 4-二芳基-3-丁烯衍生物抑制活性具有较好的预测
能力。
关键词:药物化学;三维定量构效关系;比较分子场分析;3-哌啶甲酸衍生物;四氢烟酸衍生物;GABA 摄取抑制剂
ZHENG Jian-bin et al:3D-QSAR study on 4 , 4-diaryl-3-butenyl derivatives
of nipeco tic acid and guvacine w ith comparative molecular field analysis
208 中 国 药 物 化 学 杂 志 14卷