全 文 ：Study on the Inhibitory Mechanism of Sophora japonica N-
hexane Extract on Microcystis aeruginosa
ZHOU Xiao-jian1，2，XIA Jie1，JIN Cui-li1，2，MIAO Li1，2，DONG Kun-ming1，2，FENG Ke1，2*
1． College of Environmental Science and Engineering，Yangzhou University，Yangzhou 225127;2． Marine Science and Technology Institute，
Yangzhou University，Yangzhou 225127
Abstract ［Objective］The research aimed to analyze the inhibitory mechanism of Sophora japonica n-hexane extract which significantly inhibi-
ted Microcystis aeruginosa in the prior research． ［Method］S． japonica n-hexane extract was used to treat M． aeruginosa． By inspecting chloro-
phyll a content，protein content，cell membrane permeability and superoxide dismutase (SOD)activity，the inhibitory mechanism of S． japonica
n-hexane extract on M． aeruginosa was analyzed initially． ［Result］S． japonica n-hexane extract destroyed the cell membrane system of M．
aeruginosa，and increased the cell membrane permeability． The contents of chlorophyll a and protein respectively declined to 10% and 50% of
that in the control group after cultivated for 7 d，which indicated the photosynthetic reaction system of M． aeruginosa was destroyed． In addition，
under the effect of S． japonica n-hexane extract，SOD activity of M． aeruginosa increased in the early period and decreased in the latter period．
［Conclusion］The possible inhibitory mechanism of S． japonica n-hexane extract on M． aeruginosa was destroying the cell membrane to increase
the membrane permeability;destroying the photosynthetic reaction system to decrease the contents of photosynthetic pigment and protein;mak-
ing SOD activity showing the phased variation．
Key words S． japonica;M． aeruginosa;N-hexane extract;Inhibitory mechanism
Received:June 30，2011 Accepted:September 20，2011
Supported by National Natural Science Foundation of China(41076097，
41006097，41106113);Science and Technology Research Key Project
of Chinese Ministry of Education(211065);Natural Science Foundation
of Jiangsu Province，China (BK2010322);Open Research of Jiangsu
Key Laboratory of Environmental Material and Environmental Engineer-
ing (K090027，K090025，K090026，K090028); New Century Talent
Project of Yangzhou University，China．
* Corresponding author． E-mail:fengke@yzu． edu． cn
For the eutrophication of water body，the fulminant prolif-
eration of algae was induced，and the water bloom was
formed． It has been the worldwide major environmental issue．
Moreover，the toxic cyanobacteria bloom had expanding and
spreading tendency，whether in frequency or scope［1 －2］． One
of major dominant species in cyanobacteria bloom was Micro-
cystis aeruginosa． Besides the outbreak of M． aeruginosa wa-
ter bloom induced that the aquatic organism died of hypoxia，a
large number of algae clogged the water body filtration sys-
tem，which caused human drinking water lacked． Even more
serious was that it could secrete toxin into water，which not
only directly harmed fishes，human and animals，but also was
the induction factor of liver cancer［3 －4］． Therefore，the control
of M． aeruginosa was important topic of water bloom treat-
ment research．
The organism generates direct or indirect inhibitory or
promoting effect on others by releasing chemical substances
into the environment，which is called as the allelopathy effect．
It has been one of methods controlling algae water bloom in
recent years［5］． The allelochemicals is secondary metabolites
of organism，is easy to decompose in the natural environ-
ment，and cant bring potential persistent ecological hazard．
Therefore， it has good application prospect［6］． In recent
years，the type and source range of active substance which
control toxic algae were increasing and enlarging． The corre-
lated research showed that the allelochemicals from straw and
rootstock of plant could inhibit the growth of cyanobacteria［7］．
Ikawa et al． reported that C14 －C18 fatty acids could inhibit the
growth of Chlorella pyrenoidosa［8］． Nymphoides peltatum of
floating-leaved plants could secrete active substance and in-
hibit the growth of M． aeruginosa［6］． The allelochemicals of
Asparagus officinalis could destroy the submicroscopic struc-
ture of Scrippsiella trochoidea［9］． The water extracts of Sar-
gassum thunbergii and Symphyocladia latiuscula had inhibitory
effects on the growth of Phaeodactylum tricornutum［10］． Our
prior work also found that the extracts of three terrestrial
plants (Magnolia grandiflora，S． japonica and Buxus sinica)
could effectively inhibit the growth of M． aeruginosa［11］． After
the absolute ethanol extract of S． japonica leaf was allocated by
n-hexane，ethyl acetate，n-butanol and water in turn，the extract
of n-hexane phase had significant inhibitory effect on the growth
of M． aeruginosa． Under the effects of 25 and 50 mg/L of
n-hexane components，the inhibitory rates of M． aeruginosa
growth respectively reached 75% and 90% after 7 d［12］．
On the basis of prior research，we further studied n-hex-
ane extract of S． japonica． Under the treatment of n-hexane
extract，the chlorophyll a content，protein content，cell mem-
brane permeability and SOD activity of M． aeruginosa were
measured． The inhibitory mechanism of S． japonica n-hexane
extract on M． aeruginosa was initially analyzed．
Materials and Methods
Materials
S． japonica was collected from the suburb in Yangzhou
City，Jiangsu Province． M． aeruginosa was bought from Wu-
han Institute of Hydrobiology，Chinese Academy of Sciences．
The numbering is FACHB-905．
Cultivation of M． aeruginosa
The cultivation of M． aeruginosa used BG11 medium［13］．
After the activation of algae，the cultivation was carried out in
the light incubator． The culture temperature was 25 ℃，and
the ratio of light∶dark was 12 h∶12 h．
Preparation of S． japonica n-hexane extract
350 g S． japonica leaves were put into a jar with 500 ml
of absolute ethanol for 48 h． Extraction were repeated three
Agricultural Science ＆ Technology，2011，12(10):1543 －1546
Copyright 2011，Information Institute of HAAS． All rights reserved． Resources and Environment
DOI:10.16175/j.cnki.1009-4229.2011.10.001
times，and the extracted liquids were merged． It was concen-
trated by the rotary evaporator at 30 ℃ until the volume didnt
change． 300 ml of distilled water was added into the extract．
Then，it was set into 1 000 ml of separatory funnel． The equal
volume of n-hexane was added，then the liquid separated into
layers． The upper layer was n-hexane phase with light green
color． The extracted liquid of n-hexane phase was concentrat-
ed by the rotary evaporator at 30 ℃． Then，n-hexane extract
was obtained．
Measuring methods of physiological indexes of M． aerug-
inosa
The n-hexane extract of S． japonica was dissolved in
DMSO and was prepared into 10 g/L of mother solution，
which was added into the algae solution at logarithmic growth
stage，making the final concentration of extract 50 mg/L． The
same volume of DMSO was added into the control． There
were 3 parallel samples．
The measurement of chlorophyll a of M． aeruginosa used
acetone method［14］，and the measurement of protein content
used coomassie brilliant blue method［15］． SOD activity used
the method of Stewert et al．［16］，and the cell membrane per-
meability used the method of Xie Tian et al［17］． The proper
amount of algae solution was set in a certain amount of doub-
le-distilled water for several hours． After centrifugated，the
optical density at 264 nm was measured and expressed with
OD264 nm /［g(FW)·h］．
Results and Analyses
Influence of S． japonica n-hexane extract on chlorophyll
a content of M． aeruginosa
The n-hexane extract had obvious inhibitory effect on
chlorophyll a content (Fig． 1)． The rise trend of chlorophyll a
content in the control group was obvious． It increased from
250 mg/L at the beginning of experiment to 502 mg/L on the
seventh day． In the whole experiment process，the concentra-
tion of chlorophyll a doubled． But in the treated group，the
chlorophyll a content continuously declined． On the seventh
day，it was only 38 mg/L as the minimum value． The concen-
tration did not reach 1 /10 of that in the control group．
Fig． 1 Influence of S． japonica n-hexane extract on chlorophyll
a content of M． aeruginosa
Influence of Sophora japonica n-hexane extract on pro-
tein content of M． aeruginosa
The protein content of M． aeruginosa was obviously in-
hibited by S． japonica n-hexane extract (Fig． 2)． The protein
content of control group maintained about 11． 5 mg/g(FW)
and was stable in the whole experiment process． But the pro-
tein content of treated group showed continuous decline from
the second day． It was only 5． 7 mg/g(FW)on the seventh
day and was about 1 /2 of that in the control group．
Fig． 2 Influence of S． japonica n-hexane extract on protein
content of M． aeruginosa
Influence of S． japonica n-hexane extract on cell mem-
brane permeability of M． aeruginosa
The n-hexane extract made the cell membrane permea-
bility of M． aeruginosa greatly increased (Fig． 3)． The cell
membrane permeability of control group was stable in the
whole experiment process，while it showed gradual increase
in the treated group． It reached peak value on the fourth day
and was five times of that in the control group． After that，the
cell exosmosis rate slightly declined． It illustrated that the n-
hexane extract had strong damaging effect on the integrity of
cell membrane． Because a large number of intracellular mate-
rials penetrated outside，making the content of intracellular
material decreased，the exosmosis rate slightly declined．
Fig． 3 Influence of S． japonica n-hexane extract on cell mem-
brane permeability of M． aeruginosa
Fig． 4 Influence of S． japonica n-hexane extract on SOD
activity of M． aeruginosa
Influence of S． japonica n-hexane extract on SOD activity
of M． aeruginosa
SOD activity of M． aeruginosa was affected by n-hexane
extract． It presented as promoting effect in the prior period of
experiment and inhibitory effect in the latter period (Fig． 4)．
SOD activity of control group was stable． But SOD activity of
4451 Agricultural Science ＆ Technology Vol． 12，No．10，2011
treated group rose and reached the peak value on the fourth
day． Then，it declined sharply． SOD is protective enzyme of cell
and priority substance which cleans free radicals in the organ-
ism． When SOD activity has stress increase，it has protection
effect on cell． SOD activity declined in the latter period of experi-
ment，meaned that the cell suffered substantial damage．
Conclusions and Discussions
The growth of phytoplankton depends on photosynthesis．
The chlorophyll a is major photosynthetic pigment of algae
light response system (PSI and PSII)． The chlorophyll mole-
cule which locates at reaction center transforms light energy
into chemical energy． Therefore，the chlorophyll a content can
be as the index which evaluates the photosynthesis capacity of
algae［6］． S． japonica n-hexane extract made the chlorophyll a
content of M． aeruginosa greatly declined． It indicated that the
photosynthetic capacity of algae was seriously affected． Maybe
it was because that n-hexane extract inhibited the synthetic
process of chlorophyll， or accelerated the decomposition
process of chlorophyll［18］． The chlorophyll was destroyed，which
illustrated that the light reaction system was the important acting
site of allelopathic substance on M． aeruginosa cell． The struc-
ture or function of algae photosynthetic system was affected，
which was one of inhibitory mechanisms of S． japonica n-hex-
ane extract on algae growth［19 －20］．
The protein is important biological macromolecule． It is
not only structural component of organism，but also is catalyst
(enzyme)of intracellular metabolic reaction． The algae cell
has many kinds of proteins which include every kind of en-
zyme，protein of photosynthetic system，phycobiliprotein and
so on［19］． When affected by environmental stress，the cell
may synthesize stress protein to confront the environment．
But in this research，S． japonica n-hexane extract made the
protein content of M． aeruginosa continuously declined． It in-
dicated that the cellular structure was irreversibly damaged．
The protein accumulation decreased even stopped． Mean-
while，a large number of enzymes which joined in every kind
of physiological activity were decomposed，or the synthesis
was inhibited． These variations would cause that the cellular
metabolic function declined or disordered until the cell died． In
addition，in the algae cell，the chlorophyll a mainly combined
with protein，and existed in the thylakoid membrane of chloro-
plast． The decreases of these proteins in photosynthetic sys-
tem and the great decline of photosynthetic pigment content
would inevitably lead that the photosynthetic capacity of cell
greatly declined．
The cell membrane is barrier and channel which control
substance exchange of cell and external environment，and is
also the place of many kinds of enzymatic reactions． The sta-
bility of cellular plasma membrane plays important roles in
substance transportation， energy exchange， information
transfer，maintaining concentration difference of intracellular
and extracellular ions，active transportation of substance［21］．
The n-hexane extract destroyed the integrity of membrane． It
showed as that a large number of nonelectrolyte penetrated
outside． This kind of destroy effect on cellular inner mem-
brane system was one of important inhibitory manners of allel-
opathic substance on algae［9，22］．
It is a common phenomenon that organism generates
active oxygen radical under the stress condition． Under the
normal situation，organism can clean it by the union effect of
antioxidase to reduce the damage［23］． SOD is just the key en-
zyme of antioxidation which uses radical as the substrate． It
catalyzes superoxide anion into H2O2 ． Then，CAT catalyzes
H2O2 into H2O and O2． When the organism is affected by mild
stress，SOD activity will rise and strengthen the scavenging
capacity on active oxygen． When the organism is affected by
severe environmental stress，the balance of antioxidant sys-
tem in cell is destroyed，and SOD activity will be inhibited．
The active oxygen in vivo accumulates，which induces organ-
ism injury［24］． In the prior period of S． japonica n-hexane ex-
tract treating M． aeruginosa，SOD activity increased． But as
the treated time prolonged，the cell generated excessive ac-
tive oxygen，which surpassed the capacity threshold of antiox-
idant system in cell． SOD activity was inhibited，and the cell
was seriously damaged． The variation rule of SOD activity
was consistent with the phased and temporal changes of SOD
activity，which was found by Song Chao et al［25］．
S． japonica n-hexane extract is mixture of many kinds of
low-polarity constituents，and has many inhibitory action sites
on M． aeruginosa． It illustrates that maybe the inhibitory effect
is the result of comprehensive effect of many different sub-
stances． Seen from the above analyses，the possible inhibitory
mechanism of S． japonica n-hexane extract on M． aeruginosa
was，destroying cell membrane，which made the membrane
permeability increased; destroying photosynthetic system，
which caused the contents of photosynthetic pigment and pro-
tein decreased;making SOD activity showing phased varia-
tion． In further research，we will continue to study the type
and structure of allelochemicals in S． japonica n-hexane ex-
tract，separate and purify the active chemical．
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Responsible editor:SONG Ping Responsible proofreader:
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WU Xiao-yan
龙爪槐正己烷提取物对铜绿微囊藻抑制机理的研究(摘要)
周晓见1，2，夏洁1，靳翠丽1，2，缪莉1，2，董昆明1，2，封克1，2* (1．扬州大学环境科学与工程学院，江苏扬州 225127;2．扬州
大学海洋科学与技术研究所，江苏扬州 225127 )
［目的］对前期研究发现的具有显著抑制铜绿微囊藻的龙爪槐正己烷提取物进行抑制机理分析。
［方法］采用龙爪槐正己烷提取物处理铜绿微囊藻，通过考察铜绿微囊藻的叶绿素 a含量、蛋白质含量及细胞膜透性和 SOD活性，初步分析
龙爪槐提取物对铜绿微囊藻抑制作用的机理。
［结果］龙爪槐正己烷提取物破坏铜绿微囊藻的细胞膜系统，增加细胞膜透性;叶绿素 a 和蛋白质含量在培养 7 d 后分别减少为对照组的
10%和50%左右，对藻细胞的光合反应系统造成破坏;另外，藻细胞的 SOD活性在龙爪槐正己烷粗提物作用下，在初期呈现为应激性上升，在
后期表现为抑制的阶段性变化。
［结论］龙爪槐正己烷提取物抑制铜绿微囊藻的可能机理是:破坏细胞膜，使膜通透性增大;破坏光合反应系统，造成光和色素和蛋白质含量
减少;SOD活性发生阶段式变化。
关键词 龙爪槐;铜绿微囊藻;正己烷提取物;抑制机理
基金项目 国家自然科学基金项目(41076097、41006097、41106113);教育部科学技术研究重点项目(211065);江苏省自然科学基金(BK2010322);江
苏省环境材料与环境工程重点实验室开放课题(K090027、K090025、K090026、K090028);扬州大学“新世纪”人才工程支持项目。
作者简介 周晓见(1976 － )，男，安徽安庆人，副教授，博士，从事水环境生物学研究。* 通讯作者，E-mail:fengke@ yzu． edu． cn。
收稿日期 2011-06-30 修回日期
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2011-09-20
(From page 1534)
［结论］上述结构特征为 TLR9发挥生物学功能奠定了基础，同时也为进一步研究新疆野生盘羊 TLR9基因提供了理论依据。
关键词 新疆野生盘羊(天山亚种) ;TLR9;基因克隆;序列分析
作者简介 马长宾(1982 － )，男，山东莘县人，硕士，从事动物疫病防疫工作，E-mail:cbinma@163． com。* 通讯作者，教授，博士，从事临床兽医学研
究，E-mail:sym@ shzu． edu． cn。
收稿日期 2011-07-27 修回日期 2011-09-20
6451 Agricultural Science ＆ Technology Vol． 12，No．10，2011