全 文 ：Antioxidation properties and mechanism of action of
dihydromyricetin from Ampelopsis grossedentata
ZHANG You-sheng1 ,2＊ , NING Zheng-xiang1 , YANG Shu-zhen3 , WU Hui1
(1 .College of Food and Biological Engineering , South China University of Science and Technology , Guangzhou 510641 , China;
2 .Food College of Hunan Agricultural University , Changsha 410128 , China;
3 .College of Horticulture , Northwest Science and Technology University of Agriculture and Foresty , Yanglin 712100 , China)
Abstract:Aim　To assess the antioxidative properties and the mechanism of action of dihydromyricetin
(DMY)from Ampelopsis grossedentata.Methods　The antioxidative properties of DMY were measured by
scavenging 1 ,1-diphenyl-2-picrylhydrazyl radical(DPPH)and inhibiting lipid peroxidation induced by FeSO4-
edetic acid in linoleic acid.The mechanism of antioxidative properties of DMY was tested by measuring the
chelating activities of DMY for Fe
2+
with ultraviolet spectrum (UV)method.Results　The specific absorption
of DPPH radical solution at 517 nm was reduced 73.3%-91.5%when DMY was added into the reaction
solution in the concentration range from 0.01% to 0.04%.DMY was shown to greatly inhibit the increase of
lipid peroxidation(LPO)values in linolei acid system catalyzed by FeSO4-edetic acid.The reaction rates(A532
·min-1)of lipid peroxidation were 0.002 1-0.000 4 in the concentration range from 0.01% to 0.04% and
the inhibition activities of DMY was found to be in a concentration-dependent manner.The mechanism of
antioxidative properties of DMY was chelating Fe
2+
in the Fe
2+-dependent lipid peroxidation system.
Conclusion　DMY showed great antioxidative effect and would be a good natural antioxidant.
Key words:dihydromyricetin;lipid peroxidation;antioxidation;chelating
CLC number:R282.71;R963　　　Document code:A　　　Article ID:0513-4870(2003)04-0241-04
显齿蛇葡萄中二氢杨梅树皮素的抗氧化作用及其机制
张友胜1, 2＊ , 宁正祥1 , 杨书珍3 , 吴　晖1
(1.华南理工大学 食品与生物工程学院 , 广东 广州 510641;2.湖南农业大学 食品学院 , 湖南 长沙 410128;
3.西北农林科技大学 园林学院 , 陕西 杨林 712100)
摘要:目的　研究从显齿蛇葡萄植物中提取和纯化的二氢杨梅素(DMY)的抗氧化作用及其抗氧化机制。方法
用 DPPH 作稳定自由基测量DMY对其清除效果;用FeSO4-依他酸引发亚酸油过氧化 , 用硫代巴比妥酸法测量 DMY对
过氧化产物的抑制率;用紫外法测 DMY对 Fe2+的络合作用。结果　DMY对稳定自由基 DPPH 的清除率高达 73.3%
～ 91.5%;DMY(0.01%～ 0.04%)可抑制亚油酸过氧化 ,其作用效果等同或优于 TBHQ 。DMY在 FeSO4-依他酸引发的
亚油酸过氧化体系中的抗氧化机制为络合 Fe2+ ,阻止由 Fe2+引发的亚油酸过氧化。结论　DMY 有较好的抗氧化效
果 ,有可能成为一个很好的天然抗氧化剂 。
关键词:二氢杨梅素;脂质过氧化;抗氧化作用;络合
Received date:2002-04-22.
Foundat ion item:China postdoctoral science foundation(2002031285);
Guangdong science foundation(020842).
＊Corresponding author　Tel:86-20-87114606 ,
E-mai l:youshengzhang@263.net
　　Dihydromyricetin (3 ,3′, 4′, 5 ,5′, 7-hexahydroxy-2 ,
3-dihydroflavanol , DMY), extracted and purified from
the tender stem and leaves of Ampelopsis grossedentata
(Hand-Mazz) W.T.Wang[ 1] , is an important
dihydroflavonol and was shown to have many bioactive
·241·药学学报 Acta Pharmaceutica Sinica 2003 , 38(4):241-244
DOI :10.16438/j.0513-4870.2003.04.001
functions , such as relieving cough , removing sputum ,
protecting-lung inflammation , reducing the danger of
EtOH , protecting lung cancer , etc[ 2] .The content of
DMY was more than 27%in the tender stem and leaves of
this species , especially , more than 40% in the
cataphyll
[ 3, 4] .However , there is no information about the
effect of DMY on antioxidative properties.In the present
study , the effects of DMY on these aspects were
examined.
Materials and methods
　　Materials　The materials of Ampelopsis grossedentata
were collected and identified by doctor ZHANG You-
sheng in Guangdong province.A voucher specimen has
been deposited at the Botany Herbarium of South China ,
the Chinese Academy of Sciences.DMY , a kind of white
needle crystals , was extracted and purified according to
the reference
[ 5] .TBHQ(tertiary butylhydroquinone)were
purchased from Guangzhou Chemical Factory.Other
reagents were of AR.
Extraction and purification of DMY 　Dried
tender stems and leaves of Ampelopsis grossedentata were
extracted twice with 95% ethanol or water at reflux
conditions , 30 minutes each time.The extracts were
combined and evaporated under reduced pressure.
Purification of DMY was carried out by re-crystallization
using the different solubility between boiled water and
cold water.According to the different times of re-
crystallization , DMY of different purities were obtained.
Measurement of DMY purity　An HPLC system
(Beckman , USA)was used to measure the purity of
DMY.Nova-Pak C18 column(150mm×3.9mm ID)was
used and the mobile phase was methanol-water-phosphoric
acid(36∶64∶0.1).The flow rate was 1 mL·min-1.The
effluent was monitored at 294 nm and the injection volume
was 10μL[ 5] .
Stable free radical scavenging potency of DMY　
Ethanol solution(2 mL)containing various concentration
of DMY was added to ethanol solution of 0.5 mol·L-1 1 ,
1-diphenyl-2-picrylhydrazyl radical(DPPH)1 mL.After
being mixed 30 min , the absorbance at 517 nm in the
mixed solution was recorded in order to examine the
scavenging potency of DPPH radical
[ 6] .The scavenging
potency is expressed as P =[(A0-Ax) A0] ×100%,
where A0 and Ax are the absorbance values of controls and
samples , respectively.
Determination of antioxidative activity of
DMY　Linoleic acid(2 mol·L-1 , in 95% ethanol)0.1
mL was added into KH2PO 4 buffer (20 mmol·L-1 , pH
6.8)1.3 mL and 0.1mL of different samples or KH2PO4
buffer 0.1 mL for control.After incubated at 37 ℃ for 5
min , FeSO4-edetic acid 0.5 mL was added.At intervals
of 30 min aliquots were taken out , 20% trichloroacetic
acid 0.5 mL and 0.67% TBA 1.0 mL were added and
simultaneously 2% butylated hydroxytoluene 0.05 mL
added to prevent further peroxidation of linoleic acid
during heating.The mixture was heated for 10 min in a
boiling water bath and the tubes were then cooled and
centrifuged(3 500 r·min-1 , 15 min).The absorption of
supernatant was read at 532 nm for the determination of
malondiadehyde(MDA)[ 7] .The antioxidative activity was
expressed by reaction rate (A532·min-1), the bigger the
values of A532·min-1 are , the weaker the antioxidative
activities are.
Measurement of chelating activity of DMY 　
Chelating activity for Fe
2+
by DMY was measured using
UV-spectrum.Firstly , the UV absorption spectrum of
DMY in buffer solution was measured , then , FeSO4 was
added into DMY solution and spectrum was recorded at
the same condition
[ 8] .
Results
1　Stable free radical scavenging potency of DMY
DPPH is a stable nitrogen-centered free radical
agent.The change of DPPH in absorbance was used to
evaluate the ability of free radical scavengers (test
compounds).The more the absorbance reduced , the
stronger the scavenging effects on hydroxyl and
superoxygen free radicals the test compounds exhibited.
From Table 1 , it can be seen that the specific absorption
of DPPH radical solution at 517 nm was significantly
reduced by 73.3%-91.5%when DMY was added into
the reaction solution in the concentration range from
0.01% to 0.04%, although TBHQ reduced the
absorption by 75.5%-82.7%in the concentration range
0.01%-0.02%(the biggest concentration allowed for
TBHQ is 0.02%, so 0.03%and 0.04%treatments were
not measured).The reductive potency of DMY or TBHQ
against the specific absorption of DPPH radical was in a
concentration-dependent manner.The reductive potency
of DMY was less than that of TBHQ when the
concentration was 0.01% or 0.02% weight , but the
reductive potency of DMY was better than that of TBHQ
when the concentration was 0.02% or 0.04% weight.
This showed that DMY would be a good scavenger to
interact with the nitrogen-centered stable free radical.
·242· 药学学报 Acta Pharmaceutica Sinica 2003 , 38(4):241-244
Table 1　Effect of dihydromyricetin on scavenging
1 ,1-diphenyl-2-picrylhydrazyl radical(%)
Sample
Concentration %
0.01 0.02 0.03 0.04
Dihydromyricetin(DMY) 73.3 81.4 87.7 91.5
Tertiary butylhydroquinone 75.5 82.7 - -
The content of DMY was more than 99%
2　Determination of antioxidative activity of DMY
Fe
2+-depedent lipid peroxidation is thought to play a
central role in pathologically relevant oxy-radical-induced
tissue damage in vitro.Reagents that inhibit Fe2+-
dependent lipid peroxidation may represent a rational
approach to the management of oxy-radical-diseases.
Table 2 shows that DMY can greatly inhibit the increase of
LPO values in linolei acid system catalyzed by FeSO 4-
edetic acid , the reaction rate(A532·min-1)was 0.002 1
- 0.000 4 , especially , with the increase of the
concentration of DMY (0.01%-0.04%)added , the
antioxidative activities of DMY were shown to be in a
concentration-dependent manner.TBHQ , a common and
effective antioxidant , was used in the same system as
DMY and showed stronger inhibition activities than DMY
in the same concentrations.But , the antioxidative
activities of DMY were stronger than those of TBHQ ,
while DMY reached 0.04% and TBQH was equal to
0.02%.
Table 2 　Reaction rate (A532 ·min-1) of
dihydromyricetin for scavenging peroxidation
(LPO)
Sample
Concent ration %(wt)
0.01 0.02 0.03 0.04
Dihydromyricetin(DMY) 0.002 1 0.001 8 0.001 4 0.000 4
Tertiary butylhydroquinne 0.001 9 0.001 5 - -
The content of DMY was more than 99%
3　Chelating activity of DMY
According to the reference
[ 5] , neither DMY nor
FeSO4 showed absorption peak at 355 nm , but when
FeSO4 was added to DMY solution , the new solution
showed an absorption peak at 355 nm.The peak should
be a Fe
2+-DMY chelating compound according to the
reference
[ 8](Figure was not given).
Discussion
　　Free radicals are reactive chemical species that differ
from other compounds in that they have unpaired electrons
in their outer orbital.They are capable of damaging
cellular components and lead to various diseases , such as
inflammation , cancer , aging , ischemia-reperfusion , etc.
In order to scavenge the excessive free radicals , searching
for safe and effective antioxidants from botanical resources
has become the focus of attention in recent years.More
and more investigation indicated that most of the
flavonoids existing in various plants possess antioxidative
function , which are related to their special chemical
structure including the number and position of the
phenolic hydroxy groups in their molecule.In general ,
The phenolic hydroxy groups at C-3 of the C-ring and C-
5 , C-7 of the A-ring are important for the scavenging of
hydroxyl free radicals , while the ortho-phenolic hydroxy
groups at C-3′and C-4′of the B-ring are important for
scavenging of superoxygen anion free radicals.DMY ,
posseses six phenolic hydroxy groups , especially ,
posseses OH groups at C-3 in the C-ring , and at C-5 and
C-7 in the A-ring and ortho-phenolic hydroxyls in the B-
ring , posses available chemical structure with high
scavenging activity of hydroxyl and superoxide free
radicals (Figure 1).So , it expresses high scavenging
activities of DPPH radical and posses a unique activity to
inhibit lipid peroxidation in this experiment.
Figure 1　Chemical structure of dihydromyricetin
　　According to Lu et al[ 9] , most flavonoid compounds
show dual characteristics of antioxidative and prooxidative
effect.On the one hand , they could scavenge hydroxy
and superoxygen anion free radicals.On the other hand ,
they possess the proxidative funcation which depended on
the concentration.However , in this experiment , DMY
only showed the antioxidative activity but prooxidative
activity in the concentration range of 0.01%-0.04%.
There are many mechanisms of antioxidation for
flavonoids.It can be seen from this experiment that DMY-
Fe
2+
chelates are sufficiently stable and DMY is able to
inhibit the Fe
2+-dependent lipid peroxidation in vitro
through chelating Fe
2+.So , DMY also exhibit their
therapeutic potential by the same mechanism in vivo.
References:
[ 1] Zhou TD , Zhou XX.Isolation , structure determination and
pharmacological activity of flvanonol from Ampelopsis
grossedentata [ J] .Chin Pharm J(中国药学杂志), 1996 ,
·243·ZHANG You-sheng , et al:Antioxidation properties and mechanism of action of dihydromyricetin from Ampelopsis grossedentata
31(8):458-461.
[ 2] Zhong ZX , Qin JP , Chen XF , et al.Hypoglycemic effect of
ampelopsis on diabetic rats induced by steptozotocin [ J] .
Guangxi Sci(广西科学), 2000 , 7(3):203-205.
[ 3] Zhang YS , Yiang WL , Xi HP.Chemical constituent of
essential oil in Ampelopsis grossedentata [ J] .J Hunan Agric
Univ(湖南农业大学学报), 2001 , 27(5):100-101.
[ 4] Zhang YS , Yiang WL , Xi HP.Study on nutrition constituents
in Ampelopsis grossedentata [ J] .Food Sci (食品科学),
2001 , 27(5):100-101.
[ 5] Tian SL , Zhang YS , Yang WL, et al.Measurement of
dihydromyricetin in Ampelopsis grossedentata by RP-HPLC
[ J] .J Hunan Agric Univ(湖南农业大学学报), 2002 , 28
(1):32-35.
[ 6] Kyoichi O , Minako T , Shinichi H , et al.Tea catechins
inhibit cholesterol oxidation accompanging oxidation of low
density lipoprotein in vitro [ J] .Comp Biochem Physiol
(C), 2001 , 128(5):153-164.
[ 7] Li J , Ge RC , Zheng RL, et al.Antioxidative and chelating
activities of phenylpropanoid glycosides from Pedicularis
striata [ J] .Acta Pharmacol Sin , 1997 , 18(1):77-80.
[ 8] Afanas′ev IB , Dorozhko AV , Kostyuk VK , et al.Chelating
and free radical scavenging mechanisms of inhibitory action of
rutin and quercetin in lipid peroxidation [ J] .Biochem
Pharmacol , 1989 , 38(3):1763-1769.
[ 9] Lu JF , Li T , Jin D , et al.Preliminary study of dual
characteristics of antioxidation and prooxidation of flavonoids
[ J] .Chin Pharmaceu Sci , 1999 , 8(1):15-19.
第八届全国生化药理学术讨论会及中国药理学会
第七届 Servier奖颁奖大会第二轮通知
(2003年 6月 20-24日 ,青岛)
我会征集如下内容的论文:(1)药物与生物大分子(包括酶 、受体 、膜和核酸)的相互作用:(2)药物的生化作用机理;(3)
药物代谢和药酶;(4)毒素 、激素和递质的生化药理;(5)细胞因子的生化药理;(6)生化药剂的开发研究;(7)生物工程技
术在药理学研究中的应用;(8)生化药理学的新技术 、新方法;(9)生化药理边缘学科及其他内容。
请将论文写成 500字以内的论文摘要(包括研究目的 、结果 、结论和重要数据 , 不附图表)或 1000 字以内的综述 , 一式两
份 ,请注明姓名 、单位 、通讯地址 、邮编和电话。
会议期间将举办青年优秀论文评比 , 条件如下:论文作者 35 岁以下(1968 年6 月20 日以后出生);应在国内独立或在导师
指导下完成。论文署名不超过 2人 , 并有单位推荐及介绍信。要求论文全文及 500 字以内摘要 2 份 ,会议期间由本人宣读。
所有论文摘要要求用计算机打印在 A4纸上 , 并于 2003年 3 月 31 日以前连同审稿 、出版费 50 元寄至北京先农坛一号(邮
编100050)中国医学科学院药物研究所 程桂芳教授。会议将论文摘要登载在中国药理学通讯上。会议邀请北京 、上海 、南京 、
台湾 、美国等地药理学专家教授在会议期间做专题或大会报告。
中国药理学会生化药理专业委员会
2003年 3 月 7 日
·244· 药学学报 Acta Pharmaceutica Sinica 2003 , 38(4):241-244