Content uploaded by Liu Bo
Author content
All content in this area was uploaded by Liu Bo on Mar 15, 2016
Content may be subject to copyright.
ORIGINAL ARTICLE Aquaculture
Antibacterial properties of anthraquinones extracted
from rhubarb against Aeromonas hydrophila
Chunxia Lu •Hongxin Wang •Wenping Lv •
Pao Xu •Jian Zhu •Jun Xie •Bo Liu •
Zaixiang Lou
Received: 14 November 2010 / Accepted: 28 February 2011 / Published online: 13 April 2011
ÓThe Japanese Society of Fisheries Science 2011
Abstract Antibacterial properties of crude extract from
rhubarb and its major bioactive compounds against Aero-
monas hydrophila were assayed. Major bioactive com-
pounds (anthraquinone derivatives) in rhubarb collected
from different cultivation areas were determined by ultra-
performance liquid chromatography (UPLC); the antibac-
terial activity [minimum inhibitory concentration (MIC)]
of rhubarb was positively related to the anthraquinone
content (r=0.9306, P\0.01). The MIC values of five
anthraquinones against A. hydrophila were found to be in
the range 50–200 lg/ml. Action-mode studies showed that
anthraquinones (emodin) inhibits cellular functions by
binding to cell DNA after penetrating the cell membrane,
resulting in cell death. The present study suggests that
anthraquinones extracted from rhubarb have potential use
as antimicrobials for control of A. hydrophila.
Keywords Rhubarb Aeromonas hydrophila UPLC
Antimicrobial property
Introduction
Aeromonas hydrophila is a Gram-negative rod-shaped
bacterium belonging to the Aeromonidae, a family that is
widely distributed in fresh water, sewage-contaminated
water, sludge, soil, and foods. A. hydrophila is an impor-
tant bacterial fish pathogen and is associated with several
diseases of fish, such as hemorrhagic septicemia, fin and
tail rot, and epizootic ulcerative syndrome [1,2]. These
diseases have caused high mortality in freshwater fish,
resulting in extensive losses around the world. Antibiotics
and chemotherapeutics used to control these diseases
can result in the development of drug-resistant bacteria,
environmental pollution, and residues in fish. With the
increasing demand for organic aquaculture, there has been
growing interest in using natural products in aquaculture to
prevent diseases for their lesser side-effects than antibiotics
[3–6].
Rhubarb is an important Chinese herbal medicine
(called Dahuang) and has been widely used as a plant
medicine for treatment of blood stagnation, constipation,
and mental and renal disorders, as well as a purgative
agent, in China for a long time. In rhubarb, anthraquinone
derivatives [emodin, chrysophanol, rhein, aloe-emodin, and
physcion (Fig. 1) and their glucosides] are thought to be
the major active components, having many different bio-
logical and pharmacological properties such as antioxidant
[7], antibacterial [8], antiviral [9], antifungal [10], anti-
atherosclerotic [11], and anticancer activities [12]. Due to
their biological effects, increasing attention is being paid to
these compounds. Recent literature has shown that rhubarb
can promote nonspecific immune system functions in fish
and prawns to prevent pathogenic infection, mitigate the
negative effects of crowding stress, and promote growth [6,
13]. None of these previous studies, however, screened
C. Lu H. Wang (&)W. Lv Z. Lou
State Key Laboratory of Food Science and Technology, School
of Food Science and Technology, Jiangnan University, 214122
Wuxi, People’s Republic of China
e-mail: whx200720082009@yahoo.cn
P. Xu J. Zhu J. Xie B. Liu
Freshwater Fisheries Research Center, Chinese Academy
of Fishery Sciences, Key Open Lab for Genetic Breeding of
Aquatic Animals and Aquaculture Biology, Ministry of
Agriculture, Shanshui East Road No. 9, 214081 Wuxi,
People’s Republic of China
123
Fish Sci (2011) 77:375–384
DOI 10.1007/s12562-011-0341-z
antibacterial activity of rhubarb and its major components
against A. hydrophila in vitro. Meanwhile, there have been
few reports and discussion on the mechanisms of action of
antimicrobial components.
Therefore, the aims of the present work are: (1) to
investigate the antibacterial activities of crude extract from
rhubarb and its major components against A. hydrophila,
(2) to detect the contents of five anthraquinones in rhubarb
collected from different cultivation areas, and (3) to
investigate the mechanism of action of anthraquinones
against A. hydrophila.
Materials and methods
Microorganisms and chemicals/reagents
Aeromonas hydrophila TPS-30, BSK-10 were obtained from
Zhejiang Institute of Freshwater Fisheries, and A. hydro-
phila IB101, JG101, 4LNS301, CCH201, LNB101, CG101
were obtained from the Freshwater Fisheries Research
Center, Chinese Academy of Fishery Sciences. Eight rhu-
barb samples were purchased in Chinese markets near pro-
duction areas of Rheum species. Emodin, chrysophanol,
rhein, aloe-emodin, and physcion (with purity [99%) were
obtained from Kemiou Chemical Reagent Company
(Shanghai, China). A Spin Column Genomic DNA isolation
kit was purchased from Bio Basic Inc., Canada. A Cell
Apoptosis PI detection kit was purchased from Nanjing
KeyGen Biotech. Co. Ltd., China. Ethidium bromide (EB)
was obtained from Amersco Inc. (Solon, OH, USA). UPLC-
grade methanol was purchased from Sigma–Aldrich (St.
Louis, MO, USA). All other reagents (Sinopharm Chemical
Reagent Co., Ltd., Shanghai, China) were of analytical
grade.
Extraction of anthraquinones
Dried rhubarb was ground into fine powder and passed
through a sieve (60 mesh). Rhubarb powder (10.0 g) was
mixed with 100 ml 80% ethanol, and extraction was car-
ried out at 80°C for 2 h and repeated three times. The
extracts were combined, filtered, and then concentrated
using a rotary evaporator at 40°C under vacuum and
lyophilized using a freeze-dryer (LGJ-10D; Four-Ring
Science Instrument Beijing Co., Ltd., China). The freeze-
dried sample of crude extract was stored at 4°C until use.
UPLC analysis
Ultra-performance liquid chromatography (UPLC) analy-
ses were carried out using an UPLC apparatus equipped
with a Waters Acquity PDA detector (Waters, USA) and an
Acquity UPLCTM BEH C18 column (100 mm 92.1 mm,
particle size 1.7 lm; Waters, USA). The column oven
temperature was fixed at 45°C. The eluents were: A, water
0.1% formic acid; B, acetonitrile/methanol (20:80, v/v).
The gradient program was as follows: 10–30% B (15 min),
30–100% B (18 min) at constant flow of 0.3 ml/min. The
peaks of the anthraquinone compounds were monitored at
280 nm. UV–Vis absorption spectra were recorded online
from 200 to 600 nm during UPLC analysis.
Extraction of A. hydrophila genomic DNA
Genomic DNA from A. hydrophila TPS-30 was extracted
using the Spin Column Genomic DNA isolation kit. The
purity of the extracted DNA was checked by the absor-
bance ratio A
260
/A
280
(OD
260
/OD
280
=1.83). DNA con-
centration was determined from the absorbance at 260 nm
(A
260
=1.0 OD for 50 lg/ml) using a UV-2100 spectro-
photometer (UNIC) [14].
Antibacterial activity (MIC)
The antimicrobial activities of the rhubarb extracts and its
major components (anthraquinone derivatives) were
determined by using a twofold microdilution broth method
[15]. A. hydrophila were grown to mid-log phase in LB
broth for 16 h at 37°C. Twofold serial dilutions of 80 llof
test samples were transferred to test-tubes to final con-
centrations of 6400, 3200, 1600, 800, 400, 200, 100, 50, 25,
Fig. 1 Structures of five anthraquinones
376 Fish Sci (2011) 77:375–384
123
12.5, 6.25, and 0 lg/ml, which were previously filled with
1900 ll LB medium. Bacterial suspension (20 ll) was then
added to each test-tube to final concentration of 10
6
col-
ony-forming units (CFU)/ml. Test-tubes were incubated at
37°C for 24 h. After incubation, microbial growth was
determined by estimating the increased turbidity of each
well, measured at 630 nm using a UV-2100 spectropho-
tometer microplate reader (UNIC). The MIC was calcu-
lated from the highest dilution showing complete inhibition
of the tested strain. All analysis was carried out in tripli-
cate, and the median value of each triplicate was used for
data analysis.
Bacterial membrane permeability
Aeromonas hydrophila TPS-30 was grown to mid-log
phase in LB for 16 h at 37°C, and cells were collected,
washed, and resuspended in 1 ml deionized water (absor-
bance at 630 nm was adjusted to 0.2). The emodin sample
solution (10 ll) of 2 MIC concentrations was added in test-
tubes, and incubated at 37°C for various times. Then, the
cell suspensions were centrifuged at 10000 rpm for
10 min, and the supernatants were diluted at 100-fold [16].
The amount of released K
?
was measured by atomic
absorption spectrometer (Spectr AA 220; VARIAN, USA).
Transmission electron microscopy
Exponential-phase bacteria were treated with 2 MIC of
emodin for 4 h at 37°C. Cells were harvested by centri-
fugation and washed twice with deionized water. After
treatment, the bacterial pellets were fixed with 2.5% buf-
fered glutaraldehyde for 1 h. The cells were then post-fixed
in 1% buffered osmium tetroxide for 1 h, stained en bloc
with 1% uranyl acetate, dehydrated in graded ethanol
concentrations, and subsequently embedded in spur resin.
The buffer used was 0.1 M sodium cacodylate (pH 7.4).
Thin sections were prepared on Formvar copper grids and
stained with 2% uranyl acetate and lead citrate [17]. Pen-
icillin was used as positive controls, and double-distilled
water as negative controls. Microscopy was performed
with a transmission electron microscopy (H-7000; Hitachi,
Japan) under standard operating conditions.
Flow cytometric analysis
Membrane integrity after emodin treatment was deter-
mined by flow cytometric analysis using propidium iodide
(PI) as a probe [18]. A. hydrophila TPS-30 was grown to
log phase in LB and mixed with emodin at concentration of
2 MIC for 4 h at 37°C. Cells were washed three times with
phosphate-buffered saline (PBS), and resuspended at con-
centration of 10
6
CFU/ml in the same buffer. The emodin-
treated cells were incubated in PI solution (50 lg/ml final
concentration) for 30 min at 37°C, followed by removal of
unbound dye through excessive washing with PBS. PI was
excited at 488 nm using an argon laser, and the resulting
fluorescence emission was collected through a 660-nm
long-pass filter. Penicillin was used as positive controls,
and double-distilled water as negative controls. Flow
cytometry analysis was conducted using a FACScan
instrument (Calibur, BO, USA).
Fluorescence measurement
The fluorescence spectrum of emodin in the absence and
presence of A. hydrophila genomic DNA was measured
using a F-7000 fluorophotometer (Hitachi, Japan) at room
temperature with excitation at 290 nm (kex =290 nm).
The change of fluorescence spectra from 360 to 560 nm
was measured as increasing concentrations (0, 5.0, 10.0 and
20 lg/ml) of DNA were added to emodin with a fixed
concentration (of 200 lg/ml) at room temperature [19].
Tris–HCl buffer (10 mM, pH 7.2) was used as blank
solution for all samples.
Competitive binding of emodin and EB with bacterial
DNA
Fluorescence measurements of competitive binding assays
were carried out using a F-7000 fluorophotometer (Hitachi,
Japan). DNA was dissolved in 2 ml Tris–HCl buffer
(10 mM, pH 7.2) to final concentration of 10 lg/ml; 10 ll
EB (2 lM) solution was added to the DNA solution, and
the EB–DNA solution was placed in a thermostated water
bath at 37°C for 10 min. Varying concentrations of emodin
(0, 50, 100, and 200 lg/ml) were then added to the EB–
DNA solution, and the fluorescence spectra were measured
for each test solution after 30 min of incubation at 37°C.
The solutions were excited at 535 nm, and spectra were
recorded from 550 to 720 nm [20,21]. Tests were per-
formed in a 1-cm-path-length quartz cell.
KI fluorescence quenching
The experiment was performed according to methods
described by Guo et al. [19] and Song et al. [22], with
some modifications. The concentration of emodin was
adjusted to 200 lg/ml; potassium iodide (KI) was dis-
solved in Tris–HCl buffer (10 mM, pH 7.2) to final con-
centrations of 0, 1, 2, 4, 6, 8, and 10 mmol. Varying
concentrations of KI were then added to emodin-containing
solutions in the absence or presence of DNA (10 lg/mL).
Emission spectra were scanned from 360 to 560 nm with
fixed excitation wavelength of 290 nm. Data were plotted
according to the Stern–Volmer equation [23]
Fish Sci (2011) 77:375–384 377
123
F0=F¼1þKSV½Q;
where F
0
and Fare the fluorescence intensities in the
absence and the presence of the DNA, respectively. K
SV
is
the Stern–Volmer quenching constant, and [Q] is the con-
centration of quencher.
Results
Determination of anthraquinones in rhubarb
Ultra-performance liquid chromatography analysis results
of the crude extract of rhubarb are shown in Table 1and
Fig. 2. The total contents of five anthraquinones obtained
from rhubarb from different cultivation areas ranged from
5.87 ±0.30 to 24.86 ±0.81 mg/g. The antibacterial
activity (MIC) of rhubarb was positively related to the
anthraquinone content (r=0.9306, P\0.01).
Antibacterial activity of anthraquinones
The antibacterial activities (MIC) of five anthraquinones
are shown in Table 2. The MIC values of the five anthra-
quinones against A. hydrophila ranged from 50 to 200 lg/ml,
the general order of their antibacterial activity being:
emodin =rhein =aloe-emodin [physcion =chrysopha-
nol. Considering the antibacterial activity and content of
emodin, emodin was chosen as a candidate anthraquinone
derivatives for study of the antibacterial mechanism.
Bacterial membrane permeability
The effect of emodin on the membrane permeability of
A. hydrophila was investigated by measuring the amount
of potassium ions released from emodin-treated cells.
When the bacterial membrane is damaged, to a certain
extent, small ions such as potassium and phosphate tend to
leach out, and cytoplasmic constituents released from the
cell were monitored. Figure 3shows that a significant
potassium efflux from bacteria cells was induced after
incubation, and the K
?
efflux increased with increasing
incubation time from 0.5 to 4 h; when time was increased
further, only slight changes was observed. The increase in
the amount of K
?
released from A. hydrophila after treat-
ment provides evidence that emodin probably acted on the
plasma membrane by increasing permeabilization, causing
ion leakage from the cell.
Transmission electron microscopy
Transmission electron microscopy was used to observe the
morphological changes of bacterial cells treated with
emodin. The electron micrographs are displayed in Fig. 4.
Control cells of nontreated bacteria remained intact and
showed a smooth surface (Fig. 4a). However, after 4 h of
treatment, the cells showed important morphological
changes such as breakage of cell wall and membrane, and
leakage of cellular cytoplasmic contents was also observed
(Fig. 4b, c), which was similar to in previous studies [25].
Flow cytometric analysis
To investigate whether the antibacterial effect of emodin
was induced by damage to the plasma membrane, the cells
were incubated with emodin and PI. PI is a fluorochrome
that intercalates into nucleic acid as a viability marker,
which is supposed to penetrate cells and stain them only
when membrane integrity is lost [26]. Detection of internal
PI in single cells was analyzed via flow cytometry. As
shown in Fig. 5, in the absence of emodin, 97.15% of
untreated control cells showed no PI fluorescence signal
(Fig. 5a), indicating viable cells excluding the PI dye.
However, when treated with emodin and penicillin, 71.65%
and 81.5% of A. hydrophila cells were labeled fluorescently
Table 1 The relationship between antibacterial activity and anthraquinone content (mg/g) of rhubarb from different cultivation areas
Sample no. Cultivation areas Physcion Chrysophanol Emodin Rhein Aloe-emodin Total MIC (mg/ml)
1 Gansu Prov. 1.54 ±0.04 12.44 ±0.42 4.18 ±0.09 3.81 ±0.015 2.23 ±0.08 23.57 ±0.79 0.78 ±0
2 Sichuan Prov. 0.63 ±0.01 5.63 ±0.22 0.44 ±0.02 0.028 ±0.001 0.22 ±0.01 6.95 ±0.23 3.12 ±0
3 Gansu Prov. 1.86 ±0.07 13.93 ±0.64 3.17 ±0.09 2.99 ±0.12 2.91 ±0.13 24.86 ±0.81 0.78 ±0
4 Shanxi Prov. 1.26 ±0.05 9.55 ±0.56 1.38 ±0.05 0.23 ±0.004 1.40 ±0.08 12.82 ±0.64 1.56 ±0
5 Yunnan Prov. 1.70 ±0.06 8.52 ±0.41 1.57 ±0.06 0.11 ±0.004 1.58 ±0.07 13.48 ±0.63 1.56 ±0
6 Neimeng Prov. 1.33 ±0.04 8.01 ±0.46 1.41 ±0.07 0.16 ±0.003 1.44 ±0.06 12.35 ±0.54 1.56 ±0
7 Gansu Prov. 1.80 ±0.05 12.11 ±0.49 4.12 ±0.15 3.55 ±0.13 2.18 ±0.10 23.76 ±0.96 0.78 ±0
8 Guangxi Prov. 0.10 ±0.002 0.78 ±0.04 1.27 ±0.06 2.71 ±0.11 1.01 ±0.04 5.87 ±0.30 3.12 ±0
Results are expressed as mean ±SD (n=3). Strain, A. hydrophila TPS-30
MIC minimum inhibitory concentration
378 Fish Sci (2011) 77:375–384
123
after 4 h of incubation, respectively (Fig. 5b, c), thereby
indicating that emodin induced PI influx into the cells.
Fluorescence spectra study
The spectrophotometric titrations of emodin with A. hy-
drophila genomic DNA, in the concentration range of
0–20 lg/ml, provided information about the emodin–DNA
interaction mode. As shown in Fig. 6, emodin has strong
intrinsic fluorescence; in the absence of DNA, the wave-
length maximum of emodin was about 425 nm when
excited at 290 nm. When increasing concentrations (5.0,
10, and 20 lg/ml) of DNA were added to the emodin
solution, the fluorescence intensity of emodin gradually
decreased. This could be due to intercalation of emodin
into the base pairs of the DNA helix, resulting in electric
charge transfer and change of excited electronic states,
which would lead to lower fluorescence [19]. An emission
decrease is widely recognized as an indication of interac-
tions between drugs and DNA [27].
Fig. 2 Chromatograms of
standard solution (S) and extract
of rhubarb obtained in sample
1, 2, 3, 4, 5, 6, 7, 8: aaloe-emodin,
brhein, cemodin,
dchrysophanol, and ephyscion
Table 2 Antibacterial activities of five anthraquinones against Aeromonas hydrophila
Strain MIC (lg/ml)
Physcion Chrysophanol Emodin Rhein Aloe-emodin
A. hydrophila IB101 200 200 50 50 50
A. hydrophila JG101 200 200 50 50 50
A. hydrophila TPS-30 200 200 50 50 50
A. hydrophila BSK-10 200 200 50 50 50
A. hydrophila 4LNS301 200 200 50 50 50
A. hydrophila CCH201 200 200 50 50 50
A. hydrophila LNB101 200 200 50 50 50
A. hydrophila CG101 200 200 50 50 50
Results are expressed as mean ±SD (n=3)
MIC minimum inhibitory concentration
Fig. 3 Effect of emodin on the amount of K
?
released from
A. hydrophila TPS-30. Cells were treated with emodin for predeter-
mined times, and the relative amounts of K
?
released from the cells
were measured
Fish Sci (2011) 77:375–384 379
123
Competitive binding of emodin and EB with bacterial
DNA
To confirm the mode of interaction of DNA with emodin, a
competitive binding experiment was carried out using EB
as a probe. EB is one of the most sensitive fluorescent
probes that can bind with DNA. Fluorescence of free EB is
low, but intense fluorescence is emitted after binding with
DNA, due to intercalation between adjacent base pairs
within the double helical structure of DNA. This enhanced
fluorescence can be quenched when it coexists with a
reagent molecule that undergoes a similar reaction. This
can be used to monitor the binding mode, thereby indi-
cating the ability of a compound to prevent intercalation of
EB into DNA [28]. Accordingly, the experiment was car-
ried out by titrating the EB–DNA system with emodin.
When the concentration of emodin was increased, a
remarkable fluorescence decrease of the EB–DNA system
was observed at the maximum of 590 nm (Fig. 7). This
phenomenon indicated that EB was partially replaced by
emodin in the EB–DNA system, and EB was released from
a hydrophobic environment into the water solution. The
result suggested that emodin binds to DNA in the inter-
calating mode [21,29].
KI fluorescence quenching
The I
-
ion is a dynamic quenching agent, and the mode of
action between small molecules and DNA can be deter-
mined by evaluating the effect of the I
-
ion on the
quenching of fluorescence of small molecules. When a
small molecule inserts into the bases of DNA, these bases
(in the DNA double helix structure) together with the
negatively charged phosphor-diester skeleton, inhibit the
action of the anion quencher located close to small mole-
cules, resulting in a weakening of the quenching effect of
the I
-
ion [30].
Figure 8shows Stern–Volmer plots of the KI quenching
effect in the absence and presence of DNA. Quenched
fluorescence yielded Y=1.0151 ?0.069X,r=0.998
and Y=1.0015 ?0.045X,r=0.998, respectively. The
quenching constant of I
-
to emodin was 0.069 L/mmol, but
the quenching constant of the KI–emodin system in the
presence of DNA was 0.045 L/mmol. From Fig. 8, it can
be seen that, in the absence DNA, increasing the KI con-
centration caused efficient quenching of the fluorescence
of emodin in a concentration-dependent manner. In the
presence of DNA, however, KI showed less effective
Fig. 4 Transmission electron microscopy observations of A. hydro-
phila TPS-30 treated with emodin (b) or penicillin (c, positive
control), and untreated (a)
c
380 Fish Sci (2011) 77:375–384
123
quenching of emodin fluorescence than that observed in the
absence of DNA. This phenomenon suggests that emodin
binds to DNA, possibly in the intercalating mode. The
intercalation leads to a decrease in the collision frequency
of quenching molecules, so DNA plays a protective role
[22].
Discussion
Analysis of the antibacterial activity of rhubarb showed
that the crude extract exhibited excellent antibacterial
activity against A. hydrophila and the antibacterial activity
(MIC) of rhubarb was positively related to the anthraqui-
none content (r=0.9306, P\0.01), which indicated that
anthraquinones was a major antibacterial component in
rhubarb against the growth of A. hydrophila. However,
based on their average MICs against A. hydrophila
(50–200 lg/ml) (Table 2), five anthraquinones showed
different antibacterial activities. Comparisons of the
activities of the five anthraquinones revealed that the
effects of emodin, rhein, and aloe-emodin against all bac-
terial strains were higher than those of physcion and
chrysophanol. This was consistent with the results of
Fig. 5 Flow cytometric
measurement of the effects of
emodin. The increments of the
log fluorescence signal
represent uptake of PI by the
bacteria cells. Cells not treated
with emodin (a), and cells
treated with emodin (b)or
penicillin (c)
Fish Sci (2011) 77:375–384 381
123
previous reports [8,24], which suggested that the anti-
bacterial activity of these anthraquinone derivatives might
be related to the type of substituent groups on the molec-
ular structure. All of these anthraquinone derivatives have
the same hydroxyanthraquinone nucleus composed of two
ketone groups at C9 and C10 and two hydroxyl groups at
C1 and C8, while different groups are substituted at C3 and
C6 of the phenyl ring (Fig. 1). Three anthraquinones
(rhein, emodin, and aloe-emodin) have polar substituent
carboxyl, hydroxyl, and hydroxymethyl groups at C3, C6,
and C3, respectively. It was reported that the presence of
polar functional group (carboxyl, hydroxyl, and hydroxy-
methyl) can increase antibacterial activity [8,24].
Although physcion and chrysophanol also have hydroxyl
groups at C1 and C8 (Fig. 1), the apolar methyl and weakly
polar methoxyl in chrysophanol and physcion might
weaken their antibacterial activity.
Emodin, one of the important bioactive compounds in
rhubarb, has shown a wide variety of pharmacological
activities, such as anti-inflammatory [31], antioxidant [7],
antimicrobial [8], and antitumor activities [32]. Among
their wide biological activity, only in a few cases has their
molecular mechanism been elucidated. In particular, the
antibacterial activity and mechanisms of action of emodin
against A. hydrophila have been little reported. To learn
about the possible mechanism of antibacterial activity
against A. hydrophila, here we investigated the morphol-
ogy of treated cells and the molecular mechanism of
emodin–DNA interactions. Several possible mechanisms of
action were proposed.
Damage to the bacterial cell wall and cytoplasmic
membrane might indicate loss of structural integrity and of
the membrane’s ability to act as a permeable barrier. In our
experiments, FACScan analysis showed that emodin
increased the plasma membrane permeability for influx of
ONPG into cells (Fig. 5), and caused large leakage of
potassium ions from treated cells (Fig. 3). Moreover,
morphological changes and leakage of cytoplasmic con-
tents were also demonstrated by electron micrographs of
A. hydrophila cells treated with emodin (Fig. 4). All results
elucidated that emodin increased membrane permeabili-
zation and caused leakage of intracellular contents. Cell
death might be the result of cell contents leakage or the
initiation of autolytic processes [33].
Fig. 6 Fluorescence spectra of emodin in the absence (a) and
presence of A. hydrophila genomic DNA (b–d) in Tris buffer (pH
7.2). Total concentration of emodin: 200 lg/ml. Cell path length:
1 cm. a0, b5.0, c10, d20 lg/ml DNA
Fig. 7 Competitive binding of emodin and EB with A. hydrophila
genomic DNA: acontrol (2 lMEB?10 lg/ml DNA), b2lM
EB ?10 lg/ml DNA ?50 lg/ml emodin, c2lMEB?10 lg/ml
DNA ?100 lg/ml emodin, and d2lMEB?10 lg/ml DNA ?
200 lg/ml emodin
Fig. 8 Stern–Volmer plots for quenching of emodin fluorescence on
sequential addition of KI in the absence (a) or presence (b), of DNA;
DNA =10.0 lg/ml; EGCG =200 lg/ml
382 Fish Sci (2011) 77:375–384
123
The assays reported herein and previous reports [25,34]
indicated that emodin can bind and insert into the cell
membrane, leading to loss of cytoplasmic membrane
integrity. What then does emodin do inside the cell? Can it
act on intracellular targets in bacteria? Previous studies
have demonstrated that anthraquinone derivatives of Chi-
nese rhubarb could inhibit macromolecular synthesis in
cells [35,36], so it was also hypothesized to target intra-
cellular processes in bacteria beyond membrane perme-
abilization. Therefore, we investigated the molecular
mechanism of the bactericidal activity of emodin on
A. hydrophila genomic DNA. Interestingly, fluorescence
spectroscopic studies showed that emodin could bind with
the phosphate group of DNA and intercalate into the base
pairs of the DNA helix, suggesting that DNA may be a
target for the antibacterial activity of this anthraquinone,
which might affect replication and transcription, repress
expression, and even lead to cell death [36].
In addition, the antimicrobial activity of emodin might
involve other modes of action. Previous studies have
demonstrated that anthraquinone derivatives could inhibit
the activities of nicotinamide adenine dinucleotide
(NADH) oxidase and succinate oxidase of mitochondria
[37]. Therefore, we propose that emodin might inhibit
electron transfer of the respiratory chain, substrate oxida-
tion, and dehydrogenation processes in the bacteria. As a
result, such inhibition could lead to uncoupling of oxidative
phosphorylation, restraining of active transport, and loss of
pool metabolites [33,36].
Acknowledgments This research work was jointly supported by the
project of the Key Open Laboratory for Genetic Breeding of Aquatic
Animals and Aquaculture Biology from the Ministry of Agriculture
(BZ2009-24) and the earmarked fund for Modern Agro-industry
Technology Research System, China (nycytx-49).
References
1. Larsen JL, Jensen NJ (1977) An Aeromonas species implicated in
ulcer-disease of the cod (Gadus morhua). Nordisk Vet Med
29:199–211
2. Lu CP (1992) Pathogenic Aeromonas hydrophila and the fish
diseases caused by it. J Fish China 16:282–288 (in Chinese)
3. Ardo
´L, Yin G, Xu P, Va
´radi L, Szigeti G, Jeney Z, Jeney G
(2008) Chinese herbs (Astragalus membranaceus and Lonicera
japonica) and boron enhance the non-specific immune response
of Nile tilapia (Oreochromis niloticus) and resistance against
Aeromonas hydrophila. Aquaculture 275:26–33
4. Harkrishnan R, Balasundaram C (2008) In vitro and in vivo
studies of the use of some medicinal herbals against the pathogen
Aeromonas hydrophila in goldfish. J Aquat Anim Health
20:165–176
5. Nya EJ, Austin B (2009) Use of garlic, Allium sativum, to control
Aeromonas hydrophila infection in rainbow trout, Oncorhynchus
mykiss (Walbaum). J Fish Dis 32:963–970
6. Xie J, Liu B, Zhou QL, Su YT, He YJ, Pan LK, Ge XP, Xu P
(2008) Effects of anthraquinone extract from rhubarb Rheum
officinale Bail on the crowding stress response and growth of
common carp Cyprinus carpio var Jian. Aquaculture 281:5–11
7. Iizuka A, Iijima OT, Kondo K, Itakura H, Yoshie F, Miyamoto H,
Kubo M, Higuchi M, Takeda H, Matsumiya T (2004) Evaluation
of rhubarb using antioxidative activity as an index of pharma-
cological usefulness. J Ethnopharmacol 91:89–94
8. Wang J, Zhao H, Kong W, Jin C, Zhao Y, Qu Y, Xiao X (2010)
Microcalorimetric assay on the antimicrobial property of five
hydroxyanthraquinone derivatives in rhubarb (Rheum palmatum
L.) to Bifidobacterium adolescentis. Phytomedicine 17:684–689
9. Hsiang CY, Hsieh CL, Wu SL, Lai IL, Ho TY (2001) Inhibitory
effect of anti-pyretic and anti-inflammatory herbs on herpes
simplex virus replication. Am J Chin Med 29:459–467
10. Agarwal SK, Singh SS, Verma S, Kumar S (2000) Antifungal
activity of anthraquinone derivatives from Rheum emodi.J
Ethnopharmacol 72:43–46
11. Liu YF, Yan FF, Liu Y, Zhang C, Yu HM, Zhang Y, Zhao YX
(2008) Aqueous extract of rhubarb stabilizes vulnerable athero-
sclerotic plaques due to depression of inflammation and lipid
accumulation. Phytother Res 22:935–942
12. Huang Q, Lu GD, Shen HM, Chung MCM, Ong CN (2007) Anti-
cancer properties of anthraquinones from rhubarb. Med Res Rev
27:609–630
13. Liu B, Xie J, Ge XP, Xu P, Wang AM, He YJ, Zhou QL, Pan LK,
Chen RL (2010) Effects of anthraquinone extract from Rheum
officinale Bail on the growth performance and physiological
responses of Macrobrachium rosenbergii under high temperature
stress. Fish Shellfish Immunol 29:49–57
14. Rupesh KR, Deepalatha S, Krishnaveni M, Venkatesan R, Jaya-
chandran S (2006) Synthesis, characterization and in vitro bio-
logical activity studies of Cu-M (M =Cu
2?
,Co
2?
,Ni
2?
,Mn
2?
,
Zn
2?
) bimetallic complexes. Eur J Med Chem 41:1494–1503
15. Naghmouchi K, Drider D, Khead E, Lacroix C, Prevost H, Fliss I
(2006) Multiple characterizations of Listeria monocytogenes
sensitive and insensitive variants to divergicin M35, a new ped-
iocin-like bacteriocin. J Appl Microbiol 100:29–39
16. Hao G, Shi YH, Tang YL, Le GW (2009) The membrane action
mechanism of analogs of the antimicrobial peptide Buforin 2.
Peptide 30:1421–1427
17. Friedrich CL, Rozek A, Patrzykat A, Hancock REW (2001)
Structure and mechanism of action of an indolicidin peptide
derivative with improved activity against gram-positive bacteria.
J Biol Chem 276:24015–24022
18. Jang WS, Kim HK, Lee KY, Kim SA, Han YS, Lee IH (2006)
Antifungal activity of synthetic peptide derived from halocidin,
antimicrobial peptide from the tunicate, Halocynthia aurantium.
Febs Lett 580:1490–1496
19. Guo JB, Zhang GW, Chen XX, Wang JJ (2008) Studies on the
Interaction between D-(?) catechin and DNA. J Anal Sci
24:507–511 (in Chinese)
20. Tang YL, Shi YH, Zhao W, Hao G, Le GW (2009) Interaction of
MDpep9, a novel antimicrobial peptide from Chinese traditional
edible larvae of housefly, with Escherichia coli genomic DNA.
Food Chem 115:867–872
21. Khan S, Nami SAA, Siddiqi KS, Husain E, Naseem I (2009)
Synthesis and characterization of transition metal 2,6-pyridine-
dicarboxylic acid derivatives, interactions of Cu(II) and Ni(II)
complexes with DNA in vitro. Spectrochim Acta A 72:421–428
22. Song YM, Kang JW, Wang ZH, Lu XQ, Gao JH, Wang LF
(2002) Study on the interactions between CuL
2
and morin with
DNA. J Inorg Biochem 91:470–474
23. Kumar CV, Asuncion EH (1993) DNA binding studies and site
selective fluorescence sensitization of an anthryl probe. J Am
Chem Soc 115:8547–8553
24. Chen QH, Zheng WF, Su XL, Lai WS (1962) Studies on chinese
rhubarb I. Preliminary study on the antibacterial activity of
Fish Sci (2011) 77:375–384 383
123
anthraquinone derivatives of chinese rhubarb (Rheum palmatum
L.). Acta Pharm Sin 9:757–762 (in Chinese)
25. Shan B, Cai YZ, Brooks JD, Corke H (2008) Antibacterial
properties of Polygonum cuspidatum roots and their major bio-
active constituents. Food Chem 109:530–537
26. Ananta E, Heinz V, Knorr D (2004) Assessment of high pressure
induced damage on Lactobacillus rhamnosus GG by flow
cytometry. Food Microbiol 21:567–577
27. Trommel JS, Marzilli LG (2001) Synthesis and DNA binding of
novel water-soluble cationic methylcobalt porphyrins. Inorg
Chem 40:4374–4383
28. Lepecq JB, Paoletti C (1967) A fluorescent complex between
ethidium bromide and nucleic acid. J Mol Biol 27:87–106
29. Song YM, Kang JW, Zhou J, Wang ZH, Lu XQ, Wang LF, Gao
JZ (2000) Study on the fluorescence spectra and electrochemical
behavior of ZnL
2
and morin with DNA. Spectrochim Acta A
56:2491–2497
30. Kumar CV, Asuncio EH (1992) Sequence dependent energy
transfer from DNA to a simple aromatic chromophore. J Chem
Soc Chem Commun 6:470–472
31. Kuo YC, Meng HC, Tsai WJ (2001) Regulation of cell prolif-
eration, inflammatory cytokine production and calcium mobili-
zation in primary human T lymphocytes by emodin from
Polygonum hypoleucum Ohwi. Inflamm Res 2:73–82
32. Wang CG, Yang JQ, Liu BZ, Jin DT, Wang C, Zhong L, Zhu D,
Yan Wu (2010) Anti-tumor activity of emodin against human
chronic myelocytic leukemia K562 cell lines in vitro and in vivo.
Eur J Pharmacol 627:33–41
33. Denyer SP (1990) Mechanisms of action of biocides. Int Biodeter
2–4:89–100
34. Alves DS, Pe
´rez-Fons L, Estepa A, Micol V (2004) Membrane-
related effects underlying the biological activity of the anthra-
quinones emodin and barbaloin. Biochem Pharmacol 68:549–561
35. Wang SR, Chen QH (1977) Studies of chinese rhubarb XIII.
Interaction of anthraquinone derivatives of chinese rhubarb with
DNA. Acta Biochem Biophys Sin 1:95–98 (in Chinese)
36. Li DD, Su XL, Chen QH (1964) Studies of chinese rhubarb VIII.
Mechanism of antibiotic action of anthraquinone derivatives (2)
effect on the metabolism of nitrogen containing compounds in
S. aureus. Acta Biochem Biophys Sin 2:151–160 (in Chinese)
37. Chen CL, He BF, Chen QH (1988) Biochemical study of chinese
rhubarb inhibiton of anthraquinone derivatives on NADH oxidase
and succinate oxidase of mitochondrion. J Chin Biochem 1:36–41
(in Chinese)
384 Fish Sci (2011) 77:375–384
123
