全 文 ：天然产物研究与开发 Nat Prod Res Dev 2012，24:747-753
文章编号:1001-6880(2012)06-0747-07
Received May 23，2011;Accepted October 21，2011
Foundation Item:This work was supported by National Innovative Ex-
periment Project for College Students (No:091049715)．
* Corresponding author Tel:86-013667148461;E-mail:xyzg198891 @
163． com
构树黄酮对小鼠学习记忆的影响
熊燕飞，吕 波* ，李政政，舒思敏，郑 浩
武汉理工大学生物科学与技术系，武汉 430070
摘 要:Morris水迷宫在啮齿目动物的空间学习与记忆的研究中被广泛使用。研究表明摄食抗氧化剂能够增强
空间学习与记忆能力。本文目的在于研究构树黄酮对昆明小鼠的空间学习与记忆能力的影响。用构树黄酮固
体脂质纳米粒对小鼠灌胃 4 周，然后进行 Morris水迷宫测试。与对照组相比，实验组小鼠的各项指标均有显著
改善。这表明构树黄酮能显著增强小鼠的空间学习与记忆能力。同时研究还表明构树黄酮对小鼠的生长发育
没有影响。
关键词:Morris水迷宫;构树黄酮固体脂质纳米粒;抗氧化
中图分类号:Q599 文献标识码:A
Effect of Flavonoids from Broussonetia papyrifera on
Learning and Memory in Mice with Morris Water Maze Test
XIONG Yan-fei，LV Bo* ，LI Zheng-zheng，SHU Si-min，ZHENG Hao
Department of Biological Science and Technology，Wuhan University of Technology，Wuhan 430070，China
Abstract:The Morris Water maze (MWM)is a widely used device to investigate spatial learning and memory in ro-
dents． Previous studies involving antioxidant showed that spatial learning and memory were improved afforded by dietary
antioxidants． The present study aimed at investigating the efficacy of flavonoids from Broussonetia papyrifera on spatial
acquisition and probe trial performance in KM mice． 16 young adult male KM mice were randomly separated into 2
groups，a control (CG)and an experimental group (EG)． Mice underwent gavage with normal saline (0． 9%)and fla-
vonoid-loaded solid lipid nanoparticles (FSLN for short)from B． papyrifera (5 mg /kg /day)with the same volume for 4
weeks，respectively． Then，the MWM test was performed． Our results showed that flavonoids extracted from B． papyrifera
significantly augmented the spatial learning and reference memory of mice in the experimental group，which may be as-
cribed to decreasing the overproduction of free radicals and therefore，retarding oxidative damage． Interestingly，swimming
speed of the mice sharply increased latterly albeit there was no difference between the EG and CG． This study also dem-
onstrated that FSLN treatment had no adverse marginal impact on the growth and development of the mice． Collectively，
this work indicated that flavonoids from B． papyrifera could improve the spatial learning and reference memory in young
adult male KM mice without obvious side effect and be used as potential drug for health care．
Key words:Morris water maze;flavonoid-loaded solid lipid nanoparticles (FSLN) ;antioxidants
Introduction
The Morris Water maze (MWM)is a widely used de-
vice to investigate spatial learning and memory in ro-
dents，which is strongly correlated with hippocampal
synaptic plasticity and N-methyl-D-aspartic acid (NM-
DA)receptor function． And it was developed by Rich-
ard Morris［1］ in which rats or other rodent animals
learned to escape from water onto a submerged plat-
form． He delineated the basic procedures in 1984［2］．
Nowadays，it has become one of the most frequently
used methods in cognitive neuroscience［3］ with diverse
methodological variations．
Flavonoids comprise the most common group of poly-
phenolic compounds in human diet (fruits，vegetables，
tea and wine，etc．) and were found ubiquitous in
plants［4］． They are active ingredients of many drugs，es-
pecially Chinese herbal medicine． Flavonoids of differ-
ent kinds are versatile． Some of them are potent in trea-
ting cognitive decline and memory loss． And，as an an-
tioxidant，they contain one or more phenolic hydroxyl
groups． Some researchers believe that it is this feature
that enable flavonoids to reduce the number of free rad-
icals in the brain and thereby，neuronal damage is re-
tarded and cognitive and memory are resumed． Howev-
er，not everyone agrees with this hypothesis．
There are abundant resources of B． papyrifera in China
as well as other parts of the world． Most of the wood fi-
ber is for papermaking． And，extracting flavonoids from
B． papyrifera to produce drugs and health products be-
fore that is valuable． This way，it is able to achieve full
utilization of natural resources． Studies have been done
with regard to the biochemical functions of antioxidants
by dietary，including vitamins E and C ［5］，spinach，
strawberry and blueberry extracts ［6］，etc． But in the
present study，flavonoids were extracted and purified
from the fiber of B． papyrifera to evaluate the function
of these bioactive substances for the first time． For
there’s no data in support of a direct relationship be-
tween the consumption of flavonoids and behavioural
outcomes at present．［4］ Then，the flavonoids-loaded
solid lipid nanoparticles (FSLN)were prepared． This
is also one of the crucial points of innovation of this
study． FSLN were administered to the mice by gavage
and their spatial cognition and memory were decided u-
sing the MWM test． The reason why we didn’t use fla-
vonoids solution directly was that we found that poor
water-soluble flavonoids could not be absorbed easily
by mice in the previous study． It brought about the re-
sult that the experimental group (EG)and the control
group (CG)animals had no difference． The prepara-
tion of FSLN contributed to improve flavonoids’bio-
availability to perform its biological functions［7］． Be-
sides，the procedure of preparation was relatively sim-
ple． Meanwhile，we preliminary checked the side effect
of FSLN through measuring the change of body weight
and indexes of organs during the treatment． The present
study will extend our acquaintance with flavonoids from
B． papyrifera and lay the foundation for the comprehen-
sive utilization of B． papyrifera resource．
Materials and Methods
The preparation of FSLN
The raw material for flavonoids preparation was the
roots of B． papyrifera which were collected in Jiangxia
district，Wuhan，China． Then，flavonoids were extracted
and purified by solvent endosmosis with ultrasonic
treatment． The method of‘emulsion evaporation-solidi-
fication at low temperature’ which was optimized
through orthogonal design，as depicted by CHENG Zhi-
gang (2005) ，was used here to prepare FSLN in which
stearic acid (SA)and phosphatidylchline (PC)were
used as carriers． The final scheme was presented as fol-
lows． During the optimization，entrapment rate，drug-
loading rate and average particle size were used as ba-
rometers．
Flavonoids from B． papyrifera (17%，by mass) ，SA
(43%)and PC (12%)were filled into a small beak-
er． Then acetone was also loaded on the mixture，mak-
ing the ratio between the weight of flavonoids and the
volume of acetone equal to 2． Next，the mixture was al-
lowed for ultrasonic treatment until the substances were
dissolved． Put it in water bath (the temperature of the
water was 65 ℃)for at least 5 minutes． During this
process，the beaker was sealed． The organic phase was
obtained and was injected into the aqueous phase at 65
℃ with syringe． The ratio between the weight of fla-
vonoids and the volume of the aqueous phase was 1．
The aqueous phase was agitated at 1500 rpm for 3
hours during which PEG400 were loaded (11%)as
approach emulsifier． In this process，acetone would e-
vaporate and the system would be concentrated． After-
wards，the concentrated system was injected into the
water phase which had the same volume and stirring
speed as the former． After that，add PEG400 (17%)as
approach stabilizer． Then，continued to stir for at least 2
hours． Finally，colloidal suspension of FSLN was ob-
tained．
Experimental animals and feeding medicine by
gavage
Sixteen young adult (4 weeks old;21-25 g)male KM
mice (level SPF)were obtained from Hubei Research
Center of Laboratory Animal (China)． All mice were
847 Nat Prod Res Dev Vol. 24
group-housed (lights on 14 h /off 10 h) in standard
mouse cages under conventional laboratory conditions
with food and water available ad libitum． Room temper-
ature and relative humidity were 24-28 ℃ and 45-
70%，respectively［8］． Animals were divided into 2 e-
qual groups randomly and were treated by gavage with
either normal saline (0． 9%) (the CG)or flavonoids-
loaded solid lipid nanoparticles from B． papyrifera (the
EG) (5 mg /kg /day)with the same volume for 4
weeks． The body weight of all mice was weighted and
noted every weekend at the same time． Then，the mice
began 6 days of Morris water maze test． Finally，the
mice were dissected． Their livers，spleens，kidneys and
testicles were removed and were also weighted and re-
corded． Every effort had been made to minimize the an-
imals’suffering．
Equipement setup
A black circular pool (100 cm diameter × 40 cm high)
was filled with water (22 ℃)． And the water was kept
opaque with ink (100 ml per day)． The pool was divid-
ed into 4 equal-sized quadrants (north-east，north-
west，south-east and south-west)． A circular escape
platform (10 cm diameter × 28 cm high)was placed at
a fixed position in the centre of the north-east quadrant
(quadrant 1) ，submerged approximately 1． 5 cm below
the surface of the water． And constant room temperature
(26 ℃)was achieved by airconditioner． Then，the
maze was placed in a lab with ample surrounding visual
cues which were remained fixed throughout the test． To
obscure distal cues，the lab got curtains． The tracking
software was Morris Microsoft 1. 0． And tracking soft-
ware was sensitive to light reflections from the water
surface． Whereupon，room lighting was indirect during
the test． In addition，experimenter stood behind a visual
barrier waiting for the mice to perform the task［9］．
Morris Water maze procedures
The mice were accustomed to the new environment for
2 days before the test started． Every day，test began at 9
am． The spatial acquisition phase consisted of five days
with four trials per day，which started at four distinct
positions with the mice being released facing the side-
wall in a semi-random order［4］(Tab． 1)and were the
same for all mice on all four trials on a given day with a
30 minutes inter-trial interval． If a mouse did not reach
the escape platform within 60 s，the experimenter would
guide the animal to the goal where it had to remain for
20 s before being picked up and returned to its cage．
24 h after the last spatial acquisition day，a probe trial
was performed ． The escape platform was removed from
the maze and the mice were given 120 s to swim freely，
starting at a novel start position (south-west quadrant)
opposite the platform quadrant．
Table 1 Morris water maze start positions
Acquisition
Day Trial 1 Trial 2 Trial 3 Trial 4
1 NW W S SE
2 W S SE NW
3 S SE NW W
4 SE NW W S
5 NW W S SE
6 (probe) SW
Statistics and analysis
Learning curves representing latency (time from start to
goal)and path length［10］ were compared with repeated
measure analysis of variance (RM-ANOVA)． Latency
or path length were used as measures，while days as
within-subject factor(s)and groups as between-subject
factor(s)． Distance in the target quadrant，first bearing
and site crossings were used as indexes of memory
here． As well，time in the target quadrant was divided
by time in the opposite quadrant to figure out a coeffi-
cient—T target /T opposite． Higher coefficient was
considered as an index of reference memory retention．
Independent-Samples T Test was employed for compari-
son of the difference between treatments． For all of the
analyses of variance or means，the null hypothesis was
that there was not a significant difference between the
results obtained with distinct treatment． Statistical ana-
lyses were performed with SPSS 17． 0． All data were ex-
pressed as mean ± Stand Error of the Mean (SEM)．
P-levels ＜ ． 05 was considered significant．
Results
The preparation of FSLN
The entrapment rate and drug-loading rate of FSLN
947
Vol. 24 XIONG Yan-fei，et al:Effect of Flavonoids from Broussonetia papyrifera on
Learning and Memory in Mice with Morris Water Maze Test
were 82． 67% and 33． 73%，respectively． In addition，
the average particle size of FSLN was 461． 3 nm，meas-
ured by Zetasizer Nano Nano-size-Zeta potential instru-
ment (Malvern Instruments Ltd，UK．)
Spatial acquisition
As shown in Fig． 1，all mice improved their perform-
ance during the spatial acquisition phase，as was indi-
cated by a significant efficacy of day on latency (RM-
ANOVA;F(4，11)= 24． 073，P ＜ 0． 001)and path
length (F(4，11)= 9． 159，P ＜ 0． 01)． Fig． 1(A)also
illustrates the two groups performed differently (RM-
ANOVA;F(1，14)= 468． 950，P ＜ 0． 005)． Analogous
results (Fig． 1(B) )were obtained for path length
(RM-ANOVA;F(1，14)= 395． 658，P ＜ 0． 01)． And
the latency was significantly shorter in the EG than in
the CG，as was the case for path length． In sum，daily
treatment with FSLN from B． papyrifera for 4 weeks did
improve mice’s spatial acquisition． Average swimming
speed of the mice during the test was shown in Fig． 1
(C)．
Fig. 1 Acquisition phase performance of young adult
KM mice
Each data point represented mean ± SEM averaged across four trials
per day． Escape latency and path length were shown in fig． 1(A)
and fig． 1(B) ，respectively． Fig． 1(C)represented the changes of
mice’s swimming speeds during the MWM test． Asterisks indicated
significant difference (P levels ＜ 0． 05 are considered significant．)
between groups in a particular day． * P ＜ 0． 05．
There was significant difference in terms of days (RM-
ANOVA;F(4，48)= 41． 159;P ＜ 0． 001)but no in
terms of average speed between the two groups (RM-
ANOVA;F(1，12)= 0． 513;P = 0． 488)． The swim-
ming speed increased greatly in day four and five．
Swimming speed of the two groups was compared day
after day with Independent-Samples T Test． P values
were 0． 800，0． 981，0． 327，0． 990 and 0． 389，respec-
tively． The result demonstrated that FSLN treatment had
no marginal impact on the ability of swimming or loco-
motor of the mice．
Probetrial
Distance in the target quadrant，first bearing，site cross-
ings and T target /T opposite were used as indexes of
better reference memory． As shown in Table 2，the two
groups had significant difference (Independent-Sam-
ples T Test;P ＜ 0． 001 for distance in the target quad-
rant;P ＜ 0． 05 for first bearing;P ＜ 0． 01 for site cross-
ing;P ＜ 0． 01 for T target /T opposite)． The experi-
mental group showed an obvious preference for the tar-
get quadrant，or the escape platform area． When site
crossings were taken into account，the animals in the
EG crossed more often through the escape platform area
than in the CG． Probe trial results indicated better ref-
erence of memory of the experimental group．
Table 2 Probe trial results
Group the CG the EG
Distance in the target quadrant 232． 68 ± 16． 74 414． 85 ± 24． 58＊＊＊
First bearing 36． 94 ± 5． 04 15． 29 ± 4． 59*
Site crossings 3． 12 ± 0． 40 7． 75 ± 0． 99＊＊
T target /T opposite 0． 57 ± 0． 10 1． 21 ± 0． 12＊＊
All data was given as mean ± SEM． Asterisks indicated significant differ-
ence (P-levels ＜ 0． 05 are considered significant)between pairs of means
as achieved by Independent-Samples T Test． * P ＜ 0． 05;＊＊ P ＜
0. 01;＊＊＊ P ＜ 0． 001．
Change in body weights and indexes of organs
All mice were growing during the treatment with FSLN
for their body weights increased (RM-ANOVA;F(4，
11)= 116． 864;P ＜ 0． 001) ，as shown in Fig． 2． Body
weights of the mice from the two groups were compared
every week (Independent-Samples T Test;P = 0． 734
for the initial week;P = 0． 604 for the first week;P =
0． 582 for the second week;P = 0． 773 for the third
week;P = 0． 828 for the fourth week)． Fig． 2 also indi-
057 Nat Prod Res Dev Vol. 24
cated that change in body weights between the two
groups had no significant difference (RM-ANOVA;F
(1，14)= 0． 022;P = ． 883)． It meant that FSLN treat-
ment had no impact on the development and growth of
the mice．
Fig. 2 Change in body weights of the mice during the
FSLN treatment
The unit of body weight was g． Week zero was when the mice were
bought from Hubei Research Center of Laboratory Animal． Each data
point represent mean ± SEM．
To ascertain it，weights of different organs were ob-
tained． However，absolute weights were not used here
for individual differences existed． To resolve this prob-
lem，indexes of organs were introduced． It was the ratio
between a kind of organs and the body weight of the
mouse then． The indexes could reflect the weights of or-
gans from different mice． Liver，spleen，kidney and tes-
ticle were chosen for they were easy to separate and ob-
tain accurate weights． As shown in Fig． 3，relative
weights of different organs between the two groups had
no significant difference (Independent-Samples T Test;
t = 1． 714 and P = 0． 130 for liver;t = 0． 996 and P =
0． 366 for spleen;t = 1． 189 and P = 0． 273 for kidney;
t = 1． 445 and P = 0． 192 for testicle)． The result dem-
onstrated again that FSLN treatment would not affect
the development and growth of the mice．
Fig. 3 Change in indexs for organs of the mice
White column stood for the EG and column with slash represented
the CG． All data was giver as mean ± SEM．
Discussion
In this study，FSLN were prepared to maintain the ab-
sorbance of flavonoids． It did make it when the results
were compared with outcomes of our previous study
(unpublished data)． The finding also echoed previous
researches．［11］ Mice in the EG did better than mice in
the CG in the MWM test． Escape latency and path
length were significantly short in the EG than in the
CG． Similar results were obtained in the probe trial． So，
cognitive aging of mice was slowed or even reversed by
increasing the intake of antioxidants，i． e． flavonoids
from B． papyrifera． Interestingly，the swimming speed of
the mice was stable in the first three days and sharply
increased in the fourth day． In theory，difference did
exist for mice of different lines in terms of swimming
speed and didn’t for mice of the same line in case the
treatment doesn’t have impact on the motor organs of
experimental animals． As demonstrated by RM-ANO-
VA，there was no difference between the two groups
when it came to average swimming speed． Changes in
speeds could be partially accounted for by the fact that
the mice had learned to locate the escape platform and
avoid strolling． If a mouse couldn’t find the platform
all the while，the swimming speed would decrease due
to fatigue． However，other possibilities couldn’t be ex-
cluded easily． In simple terms，this should be taken into
consideration when conducting similar experiments．
Antioxidants，in particular those from fruit and vegeta-
bles，can enhance mice’s MWM performance ［12］． Fla-
vonoids are antioxidants，but not only antioxidants． Pre-
vious studies have proved that flavonoids from particu-
lar families were able to permeate the blood-brain bar-
rier (BBB)and might have a multiplicity of direct and
indirect effects that could profoundly affect a variety of
neuronal parameters that lead to alterations in motor
and cognitive behavior ［12］． Therefore，there are at least
four different points of view for the moment． Firstly，fla-
vonoids，as antioxidants，can eliminate reactive oxygen
species or free radical which relate to the aging process
in the brain and retard oxidative damage［13］． The sec-
ond one is related with neurogenesis． Formation of new
neurons in different areas of the brain has shown to
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Vol. 24 XIONG Yan-fei，et al:Effect of Flavonoids from Broussonetia papyrifera on
Learning and Memory in Mice with Morris Water Maze Test
continue throughout life［14］． However，neurogenesis de-
creases with age［15］． Flavonoids may actually increase
neurogenesis． This hypothesis highlights the plasticity of
brain． Additionally，to varying degrees flavonoids may
have a direct efficacy on cell signaling［16］． Whilst，
some researchers hold the perspective that flavonoids’
effects on the peripheral and cerebral vascular system
may also lead to enhancements in cognitive perform-
ance through increased brain blood flow［4］． Hence，
Flavonoid-rich (especially flavanols)foods (e． g． co-
coa，tea and soy etc．) can improve cerebrovascular
blood flow and surrogate makers of cardiovascular func-
tions in humans［17］．
However，it’s not clear yet if they are independent of
each other． Maybe the molecular mechanism of them is
closely linked，such as through maintaining the number
and quality of synaptic connections in the brain to some
extent［12］．
At the conclusion of the experiment，the animals were
dissected and their blood，brains and livers were re-
moved to testify enzyme activity (unpublished re-
sults) ，including antioxidant enzymes (such as SOD，
CAT，GST，etc．)and enzymes in the nervous system
(such as MAO，TcHE，etc．)． And our findings clearly
showed that enzyme activity was increased in the EG，
compared to the CG． So，the present findings consist
with the first hypothesis．
Furthermore，differences in body weights and organs
were compared by performing a Independent-Samples T
Test． And the results demonstrated that the uptake of
the FSLN didn’t make any difference to the growth and
development of the mice in terms of body weights and
indexes of organs as shown in fig． 2 and fig． 3．
In conclusion，learning and memory of KM mice can be
augmented by flavonoids from B． papyrifera which are
the potential drugs for health care． However，there are a
number of questions remaining to be resolved． Most no-
tably，lecithin was used as carrier of FSLN in the pres-
ent study． But some studies have found that fatty acids
could play a similar role［18］． We will take this point in-
to account in herein further studies and make some rel-
evant researches．
Acknowledgements The authors would like to grate-
fully acknowledge the assistance of Liu H． and Dong
X．(Wuhan University of Technology)in offering guid-
ance with regard to the MWM test and data analysis．
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