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Antibacterial properties of anthraquinones extracted from rhubarb against Aeromonas hydrophila


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Antibacterial properties of crude extract from rhubarb and its major bioactive compounds against Aeromonas hydrophila were assayed. Major bioactive compounds (anthraquinone derivatives) in rhubarb collected from different cultivation areas were determined by ultra-performance liquid chromatography (UPLC); the antibacterial 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 50–200μg/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. KeywordsRhubarb– Aeromonas hydrophila –UPLC–Antimicrobial property
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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
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
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
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
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
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
=1.83). DNA con-
centration was determined from the absorbance at 260 nm
=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
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
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
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
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
where F
and Fare the fluorescence intensities in the
absence and the presence of the DNA, respectively. K
the Stern–Volmer quenching constant, and [Q] is the con-
centration of quencher.
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
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
Competitive binding of emodin and EB with bacterial
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)
380 Fish Sci (2011) 77:375–384
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
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
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 (bd) 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
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).
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... In this study, we found 1,6-dihydro 8-propylanthraquinone to be active against E. coli ∆tolC, B. subtilis 168, S. aureus DSM 20231, and S. aureus Mu50 in a microtiter plate assay with MIC values of 10 µg/mL for E. coli ∆tolC, 8 µg/mL for S. aureus Mu50, 10 µg/mL for S. aureus DSM 20231, and B. subtilis 168 (Figure 4). Antimicrobial activity of natural anthraquinones, like emodin, aloe-emodin and rhein, was previously reported [37,44]. We found that 1,6-dihydro 8-propylanthraquinone exhibited MICs comparable to those of alnumycin against all tested bacterial strains, except for E. coli ∆tolC, against which it was approximately 10-fold more active (Table 1). ...
... For emodin, it was previously reported that the proteomic response of Staphylococcus aureus MRSA to treatment indicated a disturbance of metabolic processes due to an imbalance in the pyruvate metabolism, as well as inhibition of protein and DNA synthesis inhibition [44]. In addition, in vitro assays demonstrated that emodin binds to DNA of Aeromonas hydrophila, and it was shown to increase membrane permeability in vivo [37]. Comparative analyses of the alnumycin derivatives pre-alnumycin, as well as alnumycins b, c, and d, revealed efficacy against S. aureus biofilms [22]. ...
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Bacteria of the genus Streptomyces produce various specialized metabolites. Single biosynthetic gene clusters (BGCs) can give rise to different products that can vary in terms of their biological activities. For example, for alnumycin and the shunt product K115, antimicrobial activity was described, while no antimicrobial activity was detected for the shunt product 1,6-dihydro 8-propylanthraquinone. To investigate the antibacterial activity of 1,6-dihydro 8-propylanthraquinone, we produced alnumycin and 1,6-dihydro 8-propylanthraquinone from a Streptomyces isolate containing the alnumycin BGC. The strain was cultivated in liquid glycerol–nitrate–casein medium (GN), and both compounds were isolated using an activity and mass spectrometry-guided purification. The structures were validated via nuclear magnetic resonance (NMR) spectroscopy. A minimal inhibitory concentration (MIC) test revealed that 1,6-dihydro 8-propylanthraquinone exhibits antimicrobial activity against E. coli ΔtolC, B. subtilis, an S. aureus type strain, and a vancomycin intermediate-resistance S. aureus strain (VISA). Activity of 1,6-dihydro 8-propylanthraquinone against E. coli ΔtolC was approximately 10-fold higher than that of alnumycin. We were unable to confirm gyrase inhibition for either compound and believe that the modes of action of both compounds are worth reinvestigating.
... The MIC values against Aeromonas hydrophila were calculated using two fold microdilution broth method and found to be in the range of 50-200 µg/mL. For aloe-emodin, it was found to be 50 µg/mL [55]. ...
... 231,232 Anthraquinones inhibited cell function by penetrating the cell membrane binding with DNA, leading to cell death. 233 This was supported by Ankita's study 234,235 that anthraquinones extracted from aloe could inhibit nucleic acid synthesis of Bacillus subtilis, affect DNA replication and transcription, and block the protein expression. It has also been found that 236 rhein can inhibit some oxygen respiration and fermentation genes of S. aureus and genes of the ribonucleic acid reductase system, achieving its bacteriostasis. ...
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With the increasing prevalence of untreatable infections caused by antibiotic-resistant bacteria, the discovery of new drugs from natural products has become a hot research topic. The antibacterial activity of anthraquinones widely distributed in traditional Chinese medicine has attracted much attention. Herein, the structure and activity relationships (SARs) of anthraquinones as bacteriostatic agents are reviewed and elucidated. The substituents of anthraquinone and its derivatives are closely related to their antibacterial activities. The stronger the polarity of anthraquinone substituents is, the more potent the antibacterial effects appear. The presence of hydroxyl groups is not necessary for the antibacterial activity of hydroxyanthraquinone derivatives. Substitution of di-isopentenyl groups can improve the antibacterial activity of anthraquinone derivatives. The rigid plane structure of anthraquinone lowers its water solubility and results in the reduced activity. Meanwhile, the antibacterial mechanisms of anthraquinone and its analogs are explored, mainly including biofilm formation inhibition, destruction of the cell wall, endotoxin inhibition, inhibition of nucleic acid and protein synthesis, and blockage of energy metabolism and other substances.
... According to the reference of Lu et al. [35], A. hydrophila NJ-35 was incubated at 28°C for the log phase, and then, the bacterial cells were collected and fnally resuspended with 1 mL sterile deionized water. Cinnamaldehyde was added into the bacterial suspension (with the fnal concentration of 1/2 MIC and 1 MIC cinnamaldehyde) and cultured in a shaking table at 28°C and 180 /min for diferent times, and ultrapure water was in the control group. ...
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Antibacterial properties of cinnamaldehyde against Aeromonas hydrophila were assayed in this study. To investigate the action mechanisms of cinnamaldehyde against A. hydrophila, we examined the antibacterial activity, bacterial membrane permeability, and ultrastructure of A. hydrophila cells treated with cinnamaldehyde. The results showed that the minimum inhibitory concentration (MIC) value of cinnamaldehyde against A. hydrophila NJ-35 was found to be 0.039 mg/mL. The trends of the growth curve of A. hydrophila treated with different concentrations of cinnamaldehyde (from 1/4 MIC to 2 MIC) were different, and there was a significant difference in the growth curve of different groups of treatment. There were significant differences in the K+ concentration among all treatment groups from 1 h to 5 h after incubation compared with that of the control. The highest K+ concentration was observed in the 1 MIC group of cinnamaldehyde. The ultrastructure of A. hydrophila cells treated with cinnamaldehyde was destroyed, and the morphology changed. These results indicated that cinnamaldehyde could inhibit the growth of A. hydrophila, increase bacterial membrane permeability, and damage cell membrane integrity, resulting in leakage of the A. hydrophila cell contents.
... Analysis of the antibacterial activity of S. alata extracts revealed that the rhein-rich extract exhibited excellent antibacterial activity against S. aureus and that the antibacterial activity (MIC) of S. alata extracts was positively correlated with the rhein content, indicating that rhein was a significant antibacterial component in S. alata extract against the growth of S. aureus. According to a previous study [68], the antibacterial activity of rhein, an anthraquinone derivative, might be related to the hydroxyanthraquinone nucleus consisting of two ketone groups at positions 9 and 10 and two hydroxyl groups at positions 1 and 8. In addition, rhein has a substituted polar carboxylic group at position 3 (Figure 1), which can enhance its antibacterial activity. ...
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Senna alata leaves display various biological activities as a result of their rhein and phenolic composition. The objective of this study was to develop bioactive de-chlorophyll rhein-rich S. alata extracts. The rhein content was quantified using a validated high-performance liquid chromatography–diode array detection (HPLC–DAD) method. The best process parameters for maximizing rhein were established using ultrasound-assisted extraction (UAE). The optimal conditions for the parameters were determined using the Box–Behnken design (BBD); 95% v/v ethanol was used as the extraction solvent at 59.52 °C for 18.4 min with a solvent-to-solid ratio of 25.48:1 (mL/g) to obtain the predicted value of rhein at 10.44 mg/g extract. However, the color of the rhein-rich extract remained dark brown. For the removal of chlorophyll, liquid–liquid extraction with vegetable oils and adsorption with bleaching agents were employed. The bleaching agents were significantly more effective at removing chlorophyll and had less of an effect on the reduction in rhein content than vegetable oils. The presence of rhein and phenolics in the de-chlorophyll extracts might be responsible for their antioxidant, anti-inflammatory, and antibacterial activities. These findings indicate that rhein-rich extract and its de-chlorophyll extracts possess sufficient biological activities for the further development of cosmeceuticals and pharmaceuticals.
... Anthraquinones belong to anthracyclines, a class of antibiotics that act as an inhibitor of topoisomerase II, preventing the relaxing of supercoiled DNA and thus blocking DNA transcription and replication [37,38]. They have also been reported to act against bacteria through other mechanisms, including their ability to disorder bacterial cytoplasmic membrane, increasing their permeability, thus resulting in the loss of the cytoplasmic content leading to the death of bacteria [36,39]. In another mode of action, they produce hydroxyl radicals which act by destroying the cell wall of microorganisms and the nitrogen base pairs of their DNA [40]. ...
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Rumex abyssinicus Jacq. is a perennial medicinal herb widely used in traditional medicine to treat many diseases. Phytochemi-cals of the plant were isolated using column chromatography and thin layer chromatography techniques. Extract, fractions and pure compounds were screened for antimicrobial activity against sensitive and multi-drug resistant microbes and their cytotoxicity was performed on different cancer cell lines. The mechanism of action of purified helminthosporin as well as the potent fraction containing a mixture of two compounds was assessed. Fraction R7C3 was the most potent antibacterial with the lowest MIC value of 0.12 µg/mL. Helminthosporin was the most potent compound with the lowest MIC value of 1.95 µg/mL. The compound was more potent than the antibiotic chloramphenicol against multi-drug resistant (MDR) bacteria with MIC equal to 16 µg/mL. The fraction and helminthosporin were shown to destroy the cell wall of the yeast and bacteria, and DNA fragmentation effect on the genome of Candida albicans and Bacillus cereus. Helminthosporin was the most cytotoxic compound with IC 50 ˂ 10 µM. Fraction R7C3 showed the most potent cytotoxic effects on all cancer cell lines, with IC 50 ranging from ˂1 to 4.35 ng/mL. Our study is the first report on the mechanism of action of helminthosporin, a potent candidate in the development of new drugs against multi-resistant bacteria and cancer cells. In addition, this study uncovered Rumex abyssinicus as a new source of syringic acid and bis(2-ethyloctyl) phthalate.
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Relevance. Despite the variety of antibacterial drugs, including multicomponent ones, chlorhexidine-containing antiseptics are nowadays the gold standard of antiseptic products relevant for periodontal therapy. At the same time, adverse side effects and new data about unfavourable chlorhexidine effects on the microbiome determine it necessary to search for a new optimal chlorhexidine-containing product that combines high effectiveness and relative safety. Purpose. The study aimed to compare and analyze the effectiveness of chlorhexidine-containing antiseptics used in dental practice. Material and Methods. The study analyzed the results of clinical studies published from 2018 to 2023 and devoted to the investigation of chlorhexidine effectiveness in the treatment of periodontal diseases. The publications were searched in the Pub Med database by the keywords 'chlorhexidine', 'periodontal disease', and 'periodontitis'. A total of eighty-four publications corresponded to the search criteria. After the primary analysis of all available publications corresponding to the inclusion and exclusion criteria, we studied and analyzed 32 publications. Results . The analysis of the publications for the past five years extracted a trend for a more frequent increase of chlorhexidine concentrations to 0.12% and 0.2% in periodontal therapy medications. Conclusion . Prescribing chlorhexidine-containing medications should be reasonable and respond to the characteristics of the clinical picture. At that, a thorough selection of chlorhexidine active agent concentration, a form of presentation and duration of use should correspond to the clinical situation and goal.
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Anthraquinones having an anthracene core structure with two carbonyl groups are essential compounds in therapeutic applications. One of the growing concerns within the medical community is antimicrobial resistance. Multiple drug‐resistant microbes can worsen a disease that can be easily treated with normal drugs. Microbes also produce biofilms that further enhance their resistance to antimicrobials, rendering biofilm‐associated infections challenging to eradicate. This review provides an update on the antibacterial and antifungal activities of natural and synthetic anthraquinone derivatives. The mechanism of antibacterial and antifungal action of anthraquinones are summarised, which will help in designing new therapeutically effective anthraquinone compounds. Anthraquinones are chemical compounds produced by plants, bacteria and fungi and possess important therapeutic properties. They show excellent activity against various microbial pathogens including multidrug resistant organisms. Both the natural and synthetic anthraquinones have been shown to inhibit biofilm formation and exhibit synergistic action with antibiotics making them highly efficacious.
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Background Rheum tanguticum Maxim . ex Balf is a traditional Chinese medicinal plant that is commonly used to treat many ailments. It belongs to the Polygonacae family and grows in northwest and southwest China. At high elevations, the color of the plant’s young leaves is purple, which gradually changes to green during the growth cycle. Anthraquinone, which is known for various biological activities, is the main bioactive compound in R. tanguticum . Although a significant amount of research has been done on R. tanguticum in the past, the lack of transcriptome data limits our knowledge of the gene regulatory networks involved in pigmentation and in the metabolism of bioactive compounds in Rheum species. Methods To fill this knowledge gap, we generated high-quality RNA-seq data and performed multi-tissue transcriptomic analyses of R. tanguticum . Results We found that three chlorophyll degradation enzymes ( RtPPH, RtPao and RtRCCR ) were highly expressed in purple samples, which suggests that the purple pigmentation is mainly due to the effects of chlorophyll degradation. Overall, these data may aid in drafting the transcriptional network in the regulation and biosynthesis of medicinally active compounds in the future.
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Sequence selective energy transfer from adenine–thymine bases of DNA to the anthracene chromophore has been observed.
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(9-Anthrylmethyl)ammonium chloride (AMAC, 1) binds to natural and synthetic DNA sequences with a high affinity, as deduced from the absorption and fluorescence spectral data. Scatchard plots constructed from these data gave binding constants in the range (2-8) x 10(4) M-1 of base pairs. Extensive hypochromism, broadening, and red shifts in the absorption spectra were observed when AMAC binds to various sequences of synthetic and natural DNA. Upon binding to DNA, the fluorescence from the anthryl chromophore was efficiently quenched by the DNA bases and the fluorescence spectra at high concentrations of CT DNA show significant broadening of the vibronic bands. Stern-Volmer quenching constants obtained from the linear quenching plots strongly depended on the DNA sequence. A high quenching constant of 1.4 x 10(4) M-1 for dI-dC sequences and a low value of 2.1 x 10(3) M-1 for homo AT sequences were estimated from this data. Time-resolved fluorescence measurements clearly show a biexponential decay behavior (lifetimes 8.2 and 30.6 ns) for AMAC bound to CT DNA. The fluorescence spectra obtained 50 ns after the excitation showed considerable red shift when compared to the spectra at early times. The red-shifted, long-lived emission spectra were consistent with the intercalative binding of 1. Triplet-triplet absorption spectra of AMAC in the presence of CT DNA show the complete quenching of the anthryl triplet by the DNA bases. Fluorescence polarization measurements with AMAC and various DNA sequences suggest that the bound chromophore is rigid on nanosecond time scales. The melting temperatures of CT DNA and poly(dA-dT) samples were increased by AMAC binding, from 78 and 56.6-degrees-C to 83 and 63-degrees-C, respectively. Excitation into the absorption bands of the DNA in the 260-300-nm region, where the anthryl absorption was negligible, resulted in an intense, red-shifted, and broad fluorescence spectrum from the anthryl chromophore. The sensitized fluorescence spectra were assigned to the anthryl chromophore on the basis of the excitation spectra as well as its resemblance to the emission spectrum of the long lived component detected in the time-resolved studies. Energy transfer from DNA bases depended on the temperature. For example, when the helix was melted, energy transfer could no longer be observed. Thus, the double-helical structure of the DNA polymer was essential for the energy transfer, consistent with the intercalative mode of binding of AMAC. It is noteworthy that binding of AMAC to DNA results in induced CD bands with distinct peaks that correspond to the absorption spectrum of the bound probe. Several pieces of strong evidence for the intercalative binding of the anthryl probe to the DNA helix are presented. The anthryl excited state serves as a sensitive probe to distinguish between homo and hetero AT sites as well as between AT and GC sites.
Two new compounds isolated from rhizomes of Rheum emodi have been characterised as rheinal 1 and rhein-11-O-β-D-glucoside 2 by spectral data and chemical studies.
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To assess the mode of action of high-pressure treatment on Lactobacillus rhamnosus GG (LGG) a flow cytometric analysis was applied. This fluorescence-based approach could give additional insights on process-induced changes of cellular events, which were not explicitly assessable by culture techniques, such as cellular inactivation sites, specific metabolic activities, etc. To achieve this goal, combined staining with carboxyfluorescein diacetate (cFDA) and propidium iodide (PI) was applied. With this staining strategy pressure-induced changes of microbial esterase activity and membrane integrity were monitored. Moreover, the ability of the cells to extrude intracellular accumulated carboxyfluorescein (cF) upon energisation was ascertained as an additional vitality marker. The determination of microbial viability status with help of different physiological and metabolic parameters and their relative changes following pressure treatment is of increasing importance in evaluating sterilisation and/or pasteurisation processes. Comparison of conventional culture techniques with flow cytometric viability assessment (after pressure treatments at 100–600 MPa) revealed the occurrence of certain cell populations, which were stressed and lost their ability to grow on agar, but still showed metabolic activity. This fraction is often described as viable but not culturable cells. The presence of such injured bacteria in food might be critical in terms of their potential activity on excreting toxic or food spoiling metabolites.
Antibacterial activity, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of crude extract from Polygonum cuspidatum roots were assayed against five common foodborne bacteria (Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, and Salmonella anatum). The crude extract exhibited potent antibacterial properties. Major bioactive compounds in P. cuspidatum roots were identified as stilbenes (e.g., piceid, resveratroloside, and resveratrol) and hydroxyanthraquinones (e.g., emodin, emodin-1-O-glucoside, and physcion) by LC–ESI-MS. Both stilbenes and hydroxyanthraquinoines greatly contributed to the antibacterial properties. Additionally, scanning electron microscopy was used to observe morphological changes of the bacteria treated with the crude extract and its major antibacterial components. Possible mechanisms of the antibacterial action were also discussed. This study suggests that the roots of P. cuspidatum and its antibacterial components may have potential for use as natural preservatives.
Antimicrobial peptides from edible insects may serve as a potentially significant group of food preservatives. In the present work, the mode of action of a novel antimicrobial peptide MDpep9 from Chinese traditional edible housefly larvae was investigated. MDpep9 was shown to bind to bacterial DNA from the results of gel retardation and fluorescence quenching experiments. Further investigations confirmed that MDpep9 could bind with the phosphate group of DNA and intercalate into the base pairs in a helix of DNA or locate in hydrophobic environment of DNA. The previous and present results demonstrated that MDpep9 has dual mechanisms of bactericidal activity: disrupting bacterial cell membranes and binding to bacterial genomic DNA to inhibit cellular functions, ultimately leading to cell death. The results of DNA-binding mode may be contributive in designing new and promising antimicrobial peptides for food preservatives.
Previously, the antimicrobial peptides BF2-A and BF2-B, two analogs of Buforin 2 that was hypothesized to kill bacteria by entering cells and binding nucleic acids, had been designed based on the structure–activity analysis of Buforin 2. In the present study, BF2-A and BF2-B were chemically synthesized and their activities and lipopolysaccharide affinity were assayed. To elucidate the mechanism of action with cytoplasmic membranes, we subsequently examined the membrane permeability of both peptides in detail. Both peptides showed stronger antimicrobial activities against a broad spectrum of microorganisms than their parent peptide. Interestingly, BF2-A did not cause significant membrane permeabilization for influx of ONPG into cells, and hardly caused the leakage of intracellular macromolecules, probably BF2-A slightly disturbed cell membrane causing the K+ leakage during peptide crossing phospholipids bilayer. Electron micrographs indicated that the cell membrane treated by BF2-A was still intact within 20 min. On the contrary, BF2-B obviously increased the outer and inner membrane permeability, even induced the slight leakage of macromolecules in the cytoplasm. The leakage of cytoplasmic contents was also demonstrated by the electron micrographs. The increase of membrane permeabilization explained why BF2-B displayed better antimicrobial activity and rapid killing kinetics than BF2-A.