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172
doi: 10.4103/2221-1691.280294 Impact Factor: 1.59
Anti-Acinetobacter baumannii activity of Rumex crispus L. and Rumex sanguineus L.
extracts
Verica Aleksic Sabo1, Emilija Svircev2, Neda Mimica-Dukic2, Dejan Orcic2, Jelena Narancic1, Petar Knezevic1
1Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 3, 21 000 Novi Sad, Vojvodina, Serbia
2Department of Chemistry, Biochemistry and environmental protection, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 3, 21 000
Novi Sad, Vojvodina, Serbia
ABSTRACT
Objective: To examine the effect of Rumex crispus (R. crispus) and
Rumex sanguineus (R. sanguineus) plant extracts against isolates of
Acinetobacter baumannii (A. baumannii) from wounds, including
multidrug-resistant strains.
Methods: Six prepared Rumex extracts were subjected to liquid
chromatography-tandem mass spectrometry. Antimicrobial activity
of extracts and pure compounds (catechin, quercetin, isoquercitrin,
emodin, and gallic acid) was examined by a microtiter plate
method, while for determination of compound binary combinations
activity a checkerboard method was applied. Active fractions of
extracts were detected by agar-overlay high-performance thin-
layer chromatography-bioautography assay followed by liquid
chromatography - diode array detection - mass spectrometry
analysis.
Results: A total of 28 compounds were detected in two extracts of
R. crispus and 26 compounds in four different R. sanguineus extracts,
with catechin as a dominant component. Anti-A. baumannii activity
was confirmed for all six R. sanguineus and R. crispus extracts at the
concentration range from 1 to 4 mg/mL. Neither examined single
compounds nor their binary combinations exhibited an anti-A.
baumannii activity (MIC>256 μg/mL). The bioautography showed
that fractions with the most prominent anti-A. baumannii activity
tended to contain more polar compounds, predominantly flavonol
(quercetin and kaempherol) glycosides; but also fractions containing
flavanone (eriodictyol) glycosides and anthraquinone (emodin)
glycosides; and less polar eriodictyol aglycone.
Conclusions: The results justify and elucidate the traditional
application of R. sanguineus and R. crispus extracts for wound
healing, indicating the necessity for their further examination in
combat against multidrug-resistant A. baumannii isolates from
wounds.
KEYWORDS: Acinetobacter baumannii; Isolates; Wound; Multi-
drug resistance; Rumex extracts; High-performance thin-layer
chromatography-bioautography
1. Introduction
Acinetobacter baumannii (A. baumannii) is a Gram-negative
coccobacillus that colonizes the oral cavity, respiratory and
gastrointestinal tract, but is also a recognized opportunistic pathogen
that causes various severe infections. A. baumannii is a frequent cause
of wound infections, particularly war-related injuries, because of
easy wound/burn contamination. On the other hand, it is a clinically
dominant species with a pronounced tendency to induce nosocomial
infections, especially in intensive care units[1,2]. The infections with
the etiological agent as A. baumannii have a high mortality risk[3,4].
This genomic species is the most troublesome member of
Acinetobacter calcocaceticus-A. baumannii complex (Acb-complex),
showing pronounced resistance to conventional antibiotics. In
the early 1970s, infections caused by this bacterium were treated
with gentamicin, minocycline, nalidixic acid, ampicillin, and
Asian Pacific Journal of Tropical Biomedicine 2020; 10(4): 172-182
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How to cite this article: Aleksic Sabo V, Svircev E, Mimica-Dukic N, Orcic D,
Narancic J, Knezevic P. Anti-Acinetobacter baumannii activity of Rumex crispus L. and
Rumex sanguineus L. extracts. Asian Pac J Trop Biomed 2020; 10(4): 172-182.
Original Article
Article history: Received 31 October 2019; Revision 9 December 2019; Accepted 6
February 2020; Available online 16 March 2020
To whom correspondence may be addressed. E-mail: petar.knezevic@dbe.uns.ac.rs
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Verica Aleksic Sabo et al./ Asian Pacific Journal of Tropical Biomedicine 2020; 10(4): 172-182
carbenicillin. However, between 1971 and 1974, this bacterium
became resistant to the aforementioned antimicrobials. During
the early 1990s, the bacterium has exhibited resistance to beta-
lactams[5], aminoglycosides[6], chloramphenicol, tetracycline, and
fluoroquinolones[7]. At the end of the 1990s, carbapenems were
the only treatment choice[5], and soon rifampicin was introduced in
combination with carbapenems. To combat multi-drug resistant strains,
tigecycline, polymyxin B, and colistin are used nowadays. However,
resistance to these antibiotics has also been demonstrated recently,
which makes this bacterium one of the greatest threats to human health
today. The emergence of A. baumannii strains resistant to all known
antibiotics, i.e. pandrug resistant strains indicates urgent necessity to
discover novel antimicrobial agents or therapeutic strategies[8]. Natural
plant products, particularly those used in traditional medicine have
become the solutions to overcome this problem[9-11]. Many studies
have proved the effectiveness of nature-derived antimicrobial agents
in the treatment of various diseases and support their usage to a great
extent nowadays.
Besides the increasing emergence and spread of multi-drug resistant
and pandrug resistant microorganisms, environmental awareness
among the population is the reason for the current application
of natural products. The use of natural antimicrobial agents as
phytopharmaceuticals and food preservatives is an increasingly
accepted alternative to the synthetic and usually toxic, teratogenic or
mutagenic chemicals. From 2000 to 2006, about 50% of new molecules
were extracted from natural products, indicating their importance in the
development of new drugs for the treatment of infectious diseases[12].
Thus, in comparison to conventional antibiotics, natural antimicrobial
agents have many advantages, such as less harm, broad acceptance
because of their traditional use, better biodegradation, less bacterial
resistance, etc.
Rumex crispus (R. crispus) L. and Rumex sanguineus (R. sanguineus)
L. from the Polygonaceae family have been used in ethnopharmacology
for various medical purposes, and some of the applications indicate
their antimicrobial activity and/or wound healing potential[13]. For
instance, R. crispus has been used in Hungary and in Romania for
diarrhea, rashes, sores and wounds treatments[14,15], while the dried
underground plant parts have been used in traditional Turkish medicine
as a blood cleanser[16]. In other parts of the world, it has also been
used against skin diseases and against dysentery[17]. R. crispus has
been applied in India and Pakistan for treating a wide range of skin
problems (sores, ulcers, and wounds) and the underground parts have
been used in diarrhea treatment[18,19]. Similarly, R. crispus was used by
American Indian tribes for the treatment of diarrhea, dysentery and skin
problems[20], including fungal infections[21]. The infusion or decoction
of R. crispus has been commonly used in folk medicines by natives
of Africa for the treatment of helminths, wounds, internal bleeding
and vascular diseases[22]. In Italy, warm leaves of R. crispus and R.
sanguineus are applied to treat abscesses. A compress made of half-
peeled leaves along with other components has been applied to wounds
and sores as the cicatrizing agent. Leaves crushed in mortar were used
against abscesses, burns and insect bites[23]. In addition, young leaves
of R. crispus that appear in the spring have been used for consumption,
while the seeds are collected during the summer and used as an Asian
national remedy. It has been reported that Rumex extracts possess
antioxidant, antimicrobial and antifungal properties[20], but their activity
against A. baumannii has not been examined.
Taking into account the traditional application of R. crispus and R.
sanguineus plant extracts in treating wound healing and the necessity
to find alternatives to combat multiple drug resistant (MDR) bacteria,
the activity of these extracts, their pure compounds individually and
in binary combinations against isolates of A. baumannii from wounds
was examined. Also, high-performance thin-layer chromatography
(HPTLC)-bioautography assay and chemical characterization of the
potent extract were performed to elucidate compound(s) responsible for
anti-A. baumannii activity.
2. Materials and methods
2.1. Standards and reagents
Reference standards of the phenolic compounds were obtained
from Sigma–Aldrich Chem (Steinheim, Germany), Fluka Chemie
Gmbh (Buchs, Switzerland), Chromadex (Santa Ana, USA), or from
Extrasythese (Genay Cedex, France). HPLC gradient grade methanol
was purchased from J. T. Baker (Deventer, The Netherlands), and
p.a. formic acid and DMSO from Merck (Darmstadt, Germany).
Naturstoff reagent A (diphenylboric acid 2-amino-ethyl ester) was
purchased from Roth (Carl Roth Cmbh + Co Karlsruhe, Germany),
polyethylene glycol 4000 from Sigma–Aldrich (Germany), HPLC
grade ethanol from J. T. Baker (Deventer, The Netherlands), toluene,
analytical grade from Centrohem (Stara Pazova, Serbia) and ethyl-
acetate, analytical grade, from Fischer Company (Fisher Scientific
UK Ltd).
2.2. Plant extract preparation
Extracts were prepared using R. crispus (voucher number 2-1721)
and R. sanguineus (voucher numbers 2-1735 and 2-1736) from
family Polygonaceae. Taxonomic determination of plant material,
voucher specimens preparation, and plants samples deposition (at
BUNS Herbarium) were done by Goran Anačkov Ph.D., University
of Novi Sad Faculty of Sciences, Department of Biology and
Ecology. Six extracts in total were prepared for testing the anti-A.
baumanni activity (Supplementary Table 1). All the extracts were
prepared by maceration of dry, grounded plant material (above
ground plant parts – for the herb extracts; and the underground parts
– for the rhizome extracts) with 80% ethanol at constant shaking for
48 h. Filtered extracts were evaporated in vacuum and re-dissolved
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174 Verica Aleksic Sabo et al./ Asian Pacific Journal of Tropical Biomedicine 2020; 10(4): 172-182
in 70% ethanol (for microbiological analysis) or dimethyl sulfoxide
(DMSO) (for chemical analysis), giving a final concentration of 200
mg/mL or 300 mg/mL. Herb extracts of Rumex species were washed
(liquid-liquid extraction) with petroleum ether to remove lipids and
pigments. Washed extracts were concentrated in vacuum and re-
dissolved in DMSO or 70% ethanol giving a final concentration of
around 200 mg/mL or 300 mg/mL.
2.3. Bacterial strains
A total of 25 bacterial strains were used in the study. Three
reference strains of A. baumannii were used, two from American
Type Culture Collection (ATCC 19606 and ATCC BAA747,
Rockville, MD, USA) and one from the National Collection of Type
Cultures (NCTC 13420, Public Health England, UK). In addition,
two reference strains, Escherichia coli (E. coli) ATCC 25922 and
Staphylococcus aureus (S. aureus) ATCC 25923, were used as quality
controls. The twenty remaining strains were MDR A. baumannii
isolates from outpatient and clinical wounds, which have been
characterized previously[16]. All the bacterial strains were stored
in Luria Bertani broth supplemented with glycerol (10% v/v) at
–70 曟. All antibacterial tests were performed using Mueller Hinton
agar (MHA) and Mueller Hinton broth (MHB).
2.4. Antibacterial activity determination
2.4.1. Effect of plant extracts
In order to find the alternative solution(s) for the eradication of A.
baumannii, the minimal inhibitory concentrations (MICs) of extracts
were examined by a slightly modified microtitre plate method[24].
In the 96-well microtiter plates, double dilutions of extracts were
prepared in sterile distilled water and the final concentrations
of each extract in the microtiter plate ranged from 0.25 to 8 mg/
mL. The final concentrations of the plant extract solvents did not
exceed 1.9% for ethanol extracts and 1% for the extracts prepared
in DMSO. All experiments included the control of the maximum
solvent concentration in the final volume to confirm the absence of
inhibition. The final bacterial count in the test was approximate 1
伊106 CFU/mL. Reference strains E. coli ATCC 25922, S. aureus
ATCC 25923 and gentamicin were used as method quality controls.
Microtiter plates were incubated overnight at 37 曟, after which 10
μL of 1% triphenyl-tetrazolium chloride (TTC) solution was added
to each well and the microtiter plates were additionally incubated for
2 h at 37 曟 until red color of formazan appeared. This modification
was made in order to make MIC values determination more
precise. The minimum concentration of extracts that prevented the
appearance of red color, i.e. formation of formazan was considered
as a MIC value.
Minimal bactericidal concentration (MBC) was determined by
spreading 10 μL of the suspension from wells without obvious
bacterial growth onto MHA, in order to determine if the type of
the bacterial inhibition was permanent or reversible. Plates were
incubated for 24 h at 37 曟. After the incubation, the presence or
absence of growth was recorded, where the lowest plant extract
concentration at which the bacterial cell count was reduced by ≥
99.9% compared to the initial number, was considered as MBC.
2.4.2. Effect of selected extract compounds
In order to determine anti-A. baumannii activity of components
from plant extracts, some dominant compounds from extracts
and those detected in the R. crispus herb extract after chemical
characterization were tested. The following five phenolic compounds
were tested: quercetin, quercetin-3-O-glucoside, catechin, emodin
and gallic acid.
Anti-A. baumannii effect of standard compounds was tested using
the same method as described for herbal extracts. Compounds were
diluted depending on their solubility in water, methanol or DMSO
(not exceeding 1%) to the final tested concentrations ranging from
0.125 to 256 μg/mL.
2.4.3. Effect of extract compounds in binary combinations
The anti-A. baumanni effect of selected compounds in the
examined extracts was tested after the preparation of different
binary combinations (1:1, v/v). This method was used due to high
MIC values (>256 μg/mL) and poor antibacterial activity of a single
compound. For each combination, double dilutions were prepared
so that the concentrations of bioactive compounds varied from
16 to 128 μg/mL. The incubation period, data interpretation and
presentation of obtained results were performed as described for
herbal extracts and individual bioactive components.
2.5. Agar-overlay HPTLC-bioautography assay
The method that combines microbiological assay with the thin-
layer chromatography[14,15] was used for further analysis of anti-A.
baumannii active substances. In this test, the focus went towards
Rumex species which showed the best activity (i.e. lowest MIC and
MBC values).
Selected extract separation was performed on HPTLC silica gel
60 F254 aluminum plates (Merck, Germany), measuring 10 cm伊20
cm. R. crispus 171_H extract was applied to the plate in the form of a
narrow band (5 μL, i.e. 1 mg/mL). Four such bands were applied on
one piece of plate. After drying the samples, the plate was developed
with the previously optimised mobile phase (ethyl acetate-toluene-
formic acid-water 80:10:5:5, v/v/v/v). The development mode was
ascendant, in a saturated chamber. After chromatographic separation,
the adsorbent layers were dried in an oven at 90 曟 for 5 min to
remove the solvent completely. Methanolic Naturstoff reagent A, NA
(1.0%) and ethanolic polyethylene glycol 4000 solution (5.0%) were
used to visualize the separated compounds. The sprayed plates were
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Verica Aleksic Sabo et al./ Asian Pacific Journal of Tropical Biomedicine 2020; 10(4): 172-182
observed under VIS and UV light (366 nm) and documented.
Separation of the extract components was done in quadruplicate,
with two bands used for the bioautographic analysis of plant extract,
one band used for visualization of separated components, and one
for the extraction of spotted fractions (zones) and further liquid
chromatography with diode array detection and mass spectrometry
(LC/DAD/MS) analysis (as described below). Fractions on the
HPTLC plates were spotted in accordance with the results of the
bioautography test.
For the agar-overlay bioautography assay, two unsprayed HPTLC
plate bands were cut out after extract separation. One strip was
used as a whole, but the other was chopped into squares based on
the difference in colour obtained on the third strip. The prepared
parts of the HPTLC plates were positioned on the surface of the
MHA and then topped with an inoculated semisolid medium (a
bacterial suspension approximate 1.5伊108 CFU/mL) supplied
with 1% TTC solution (final ratio 30:1:1, v/v/v). The plates were
incubated for 24 h at 37 曟. After incubation, the absence of bacterial
growth indicated the zones with antimicrobial activities. The TTC
was used to facilitate the visualization of the presence/absence of
bacterial growth[25]. Inhibition zones are visible as transparent zones
against red-colored bacterial growth. The HPTLC plate strips were
photographed using the Canon EOS 100D camera.
2.6. Extracts' chemical composition analysis
All the measurements were done using Agilent Technologies 1200
Series High-performance liquid chromatography coupled with
Agilent Technologies 6410A Triple Quad tandem mass spectrometer
with electrospray ion source and controlled by Agilent Technologies
MassHunter Workstation software (ver. B.03.01).
2.6.1. Liquid chromatography-tandem mass spectrometry
(LC-MS/MS) analysis of plant extracts
For the quantitative LC-MS/MS analysis of the selected 45
compounds, extracts were diluted with 0.05% aqueous formic acid
and methanol (1:1) to a final concentration of 2 mg/mL. The method
used in this study for the Rumex extracts analysis was previously
developed, validated and published by Orčiċ et al[26].
2.6.2. LC/DAD/MS analysis of R. crispus herb extract
HPTLC-fractions
Following the results of the bioautography test, eight different
zones were spotted on the HPTLC plate. Every zone was scraped
from aluminum sheet, and compounds were extracted from the
silica gel with 80% methanol (400 μL), and filtered through syringe
filters, regenerated cellulose, 0.45 μm into vials. A total of 5 μL of
these samples were injected into the system, with Zorbax Eclipse
XDB-C18 (50 mm伊4.6 mm伊1.8 μm) rapid resolution column held
at 50 曟. The mobile phase was delivered at a flow rate of 0.8 mL/
min in a gradient mode (0 min 20% B, 6.67 min 60% B, 8.33 min
100% B, 12.5 min 100% B, re-equilibration time 4 min). Eluted
components were firstly recorded on diode array detector, full
spectra in 190-700 nm range, but also detected by MS, using the
ion source parameters as follows: nebulization gas (N2) pressure 40
psi, drying gas (N2) flow 9 L/min and temperature 350 曟, capillary
voltage 4 kV; and using MS2Scan run mode (negative and positive
ionization, NI/PI, m/z range of 120–1 000 and fragmentor voltage of
80 V). For the one dominant compound in fraction 7 (F7), a product
ion scan experiment was conducted using collisionally induced
dissociation (high-purity N2 as the collision gas, collision energies
ranging 10–40 V in 10-V increments).
2.7. Statistical analysis
The MICs were logarithmically transformed and data were
tested for normality of distribution. Because of the lack of normal
distribution, differences in extract activity and differences in MIC
and MBC among different plant species and plant parts were
estimated by the Wilcoxon signed-rank test. The level of significance
for all analyses was set as α=0.05.
All the experiments were performed in triplicates and on three
independent occasions and the results are represented as geometric
means of replications.
3. Results
3.1. Anti-A. baumannii effect of R. sanguineus and R.
crispus
The extracts of R. sanguineus and R. crispus showed significant
bacteriostatic and bactericidal activity against MDR A. baumannii
isolates from wounds (Table 1).
The extracts of R. sanguineus from the Zmajevac on Fruška Gora
Mountain (4NZ_H_p and 4NZ_R) exhibited bacteriostatic activity
with the MIC ranging 1.0-2.8 mg/mL for the extract of plant aerial
parts (herb) and 1.4-4.0 mg/mL for the extract of the underground
plant parts (rhizome). The bactericidal effect of these extracts
showed the same MBC values (2.0-5.7 mg/mL) regardless of the
part of the plant. Extracts of the same plant species originating
from the Iriški venac on Fruška Gora Mountain (4Z_H_p and 4Z_
R) also exhibited a bacteriostatic and bactericidal effect, with MIC
values of 1.0-2.0 mg/mL for herb extract, or 1.4-2.8 mg/mL for the
extract of rhizomes, while MBC values varied in the range 1.0-4.0
mg/mL for herb extract, or 1.4-5.7 mg/mL for rhizomes extract. The
differences in MIC and MBC values among different R. sanguineus
extracts were significant (P<0.001). Considering the different parts
of R. sanguineus, herb extracts exhibited a better antibacterial effect,
with lower MICs compared to rhizome extracts (P=0.002 for 4NZ_
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176 Verica Aleksic Sabo et al./ Asian Pacific Journal of Tropical Biomedicine 2020; 10(4): 172-182
Table 1. MICs and MBCs of Rumex sanguineus, Rumex crispus extracts, and compounds alone and in binary combinations against Acinetobacter baumannii wound isolates (mg/mL).
Acinetobacter baumannii strain ResistotypeaRumex sanguineus Rumex crispus Compoundsb and binary
combinationsc
4NZ_H_p 4NZ_R 4Z_H_p 4Z_R 171_H 179_R
MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC
ATTC 19606 CRO-CHL-GEN 1.4 2.0 1.4 2.8 1.0 2.0 1.4 2.0 1.0 2.0 1.0 2.0 >0.256 >0.256
ATCC BAA747 CRO-CHL-TET 1.4 2.0 1.4 2.8 1.0 2.0 2.0 2.8 1.4 2.0 1.4 2.0 >0.256 >0.256
NCTC 13423 N.A. 2.0 2.0 4.0 5.7 2.0 2.0 2.0 4.0 1.4 2.0 1.4 2.0 >0.256 >0.256
Aba-2572 CRO-CHL-GEN-KAN-PMB-TET 2.0 2.8 2.0 4.0 2.0 2.8 2.8 5.7 1.4 2.8 2.0 4.0 >0.256 >0.256
Aba-2793 CRO-CHL-GEN-KAN-TET 2.0 2.8 2.0 4.0 1.0 2.0 2.0 2.8 1.4 2.8 1.4 2.8 >0.256 >0.256
Aba-4156 AMK-CRO-CIP-CHL-GEN-KAN-PMB-
TET
2.0 5.7 2.0 4.0 2.0 4.0 2.0 4.0 1.4 2.8 1.4 2.8 >0.256 >0.256
Aba-4727 CRO-CIP-CHL-GEN-KAN-TET 1.4 2.0 2.0 2.8 1.0 1.4 2.0 2.8 1.4 2.8 1.4 2.8 >0.256 >0.256
Aba-4779 CRO-CIP-CHL-KAN-TET 1.4 2.8 2.0 2.8 1.0 1.4 1.4 2.8 1.0 1.4 1.4 2.0 >0.256 >0.256
Aba-4803 AMK-CRO-CIP-CHL-GEN-KAN-TET 1.4 2.0 2.0 2.0 1.0 1.0 1.4 1.4 1.0 2.0 1.4 2.0 >0.256 >0.256
Aba-4804 AMK-CRO-CIP-CHL-GEN-KAN-PMB-
TET
2.8 2.8 2.8 2.8 1.4 1.4 2.0 2.0 2.0 2.0 2.0 2.8 >0.256 >0.256
Aba-4890 AMK-CRO-CIP-CHL-GEN-KAN-TET 1.4 2.0 2.8 4.0 1.0 1.4 2.0 2.8 1.0 2.0 1.4 2.8 >0.256 >0.256
Aba-4914 AMK-CRO-CIP-CHL-GEN-KAN-PMB-
TET
2.0 2.8 2.0 4.0 1.0 2.8 2.0 4.0 1.4 2.8 1.4 2.8 >0.256 >0.256
Aba-5055 CRO-CIP-CHL-GEN-KAN-PMB-TET 1.0 2.8 1.4 2.0 1.0 2.0 1.4 2.8 1.0 2.0 1.4 2.8 >0.256 >0.256
Aba-5074 AMK-CRO-CIP-CHL-GEN-KAN-TET 2.0 5.7 2.8 5.7 1.4 2.8 2.0 4.0 1.4 4.0 1.4 2.8 >0.256 >0.256
Aba-5081 CRO-CIP-CHL-GEN-KAN-TET 2.0 2.8 2.8 4.0 1.0 2.0 2.0 4.0 1.0 2.8 1.0 2.0 >0.256 >0.256
Aba-5372 CRO-CHL-KAN-TET 2.0 4.0 2.8 4.0 1.4 2.8 2.8 5.7 1.4 2.8 1.4 2.8 >0.256 >0.256
Aba-6673 CRO-CIP-CHL-GEN-KAN-TET 1.4 2.0 2.8 4.0 1.0 2.0 2.8 5.7 1.4 2.8 1.4 2.8 >0.256 >0.256
Aba-7860 AMK-CRO-CIP-CHL-GEN-KAN-PMB-
TET
2.0 2.8 4.0 4.0 1.0 1.4 2.8 4.0 2.0 2.8 2.0 4.0 >0.256 >0.256
Aba-8255 CRO-CHL-KAN-TET 2.8 4.0 4.0 5.7 2.0 2.8 2.8 4.0 1.4 4.0 2.0 5.6 >0.256 >0.256
Aba-8781 CRO-CHL-KAN 2.0 4.0 2.0 4.0 1.4 2.8 2.0 4.0 1.4 2.8 2.0 4.0 >0.256 >0.256
Aba-8833 CRO-CHL-KAN 2.0 2.8 2.0 2.8 1.4 2.0 2.0 2.8 1.4 4.0 2.0 4.0 >0.256 >0.256
Aba-34963 CRO-CIP-CHL-GEN-KAN-TET 2.0 4.0 2.0 4.0 1.0 2.8 2.0 4.0 1.0 2.0 1.4 2.8 >0.256 >0.256
Aba-40100 CRO-CIP-CHL-GEN-KAN-TET 1.4 2.0 1.4 2.8 1.0 2.0 1.4 2.0 1.0 2.0 1.0 2.0 >0.256 >0.256
Escherichia coli ATCC 25922d- 1.0 1.4 2.0 2.0 1.0 2.0 1.0 1.4 1.0 2.0 1.0 2.0 >0.256 >0.256
Staphylococcus aureus ATC C
25923d
- 1.0 1.4 2.0 2.0 1.0 1.4 1.0 2.0 0.5 1.0 0.5 1.0 >0.256 >0.256
MIC: Minimal inhibitory concentration; MBC: Minimum bactericidal concentration and they were expressed as geometrical mean of the values obtained in three independent repetitions; a Based on MIC values from
ref. 11; N.A. not available; b Quercetin, isoquercitrin, catechin, emodin, gallic acid; c binary combinations were: quercetin-isoquercitrin, quercetin-catechin, quercetin-emodin, quercetin-gallic acid, isoquercitrin-
catechin, isoquercitrin-emodin, isoquercetin-gallic acid, catechin-emodin, catechin-gallic acid, and emodin-gallic acid; d MIC/MBC for a control antibiotic, i.e. gentamicin was 0.5/1.0 μg/mL for Escherichia coli and
0.5/2.0 μg/mL for Staphylococcus aureus. H: plant aerial parts (herb) R: underground plant parts (rhizome).
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H and P<0.001 for 4NZ). Similarly, the plant origin, i.e. locality,
also influenced antimicrobial activity, since both rhizome and herb
extracts from Iriški venac showed better anti-A. baumannii activity
than those obtained from Fruška Gora (P<0.001 and P=0.027,
respectively).
The extracts of R. crispus also exhibited significant antimicrobial
activity against A. baumannii isolates from wounds. Bacteriostatic
activity against MDR A. baumannii was recorded with MIC values
as 1.0-2.0 mg/mL for both extracts (Table 1), while the bactericidal
effect was observed at higher concentrations of 1.4-4.0 mg/mL for
extract 171_H and 2.0-5.6 mg/mL for extract 179_R, with significant
difference in MICs and MBCs (P<0.001). Herb extracts showed
lower MICs in comparison to rhizome extracts (P=0.041).
3.2. Compounds of Rumex plant extracts by LC-MS/MS
quantitative analysis
The preliminary results of LC-MS/MS analysis showed that
R. sanguineus and R. crispus plant extracts consisted of various
secondary metabolites which indicate that these plants are a
potentially rich source of the biologically active phenolic compounds
(Table 2). A total of 30 compounds were detected in the extracts
of both Rumex species and the presence of 26 compounds in four
different R. sanguineus extracts was confirmed. For extracts of
R. sanguineus plants, the present phenolic compounds can be
classified in different groups as follows: phenyl-carboxylic acids
(p-hydroxybenzoic acid, protocatechuic acid, p-coumaric acid, gallic
acid, caffeic acid, ferulic acid, syringic acid, and 5-O-caffeoylquinic
acid); flavones (apigenin); flavonols and their glycosides
(kaempferol, kaempferol-3-O-glucoside, quercetin, quercitrin,
quercetin-3-O-glucoside, rutin); flavanones (naringenin); flavane-3-
ols (catechin and epicatechin); and quinic acid. Of the compounds
in the R. sanguineus extracts, catechin was dominant in amounts
12.31-16.63 mg/g of dry extract in the herb, and 3.49-3.60 mg/g of
dry extract in the rhizome. In the herb extracts, it was followed by
naringenin (0.63-0.68 mg/g of dry extract) and gallic acid (0.57-0.66
mg/g of dry extract in the herb extracts), and followed by epicatechin
in the rhizome extracts (0.66-0.77 mg/g of dry extract),
In R. crispus extracts, 28 compounds was confirmed (Table 2),
and can be classified in different groups as follows: flavonoid
aglycones (apigenin, epicatechin, catechin, luteolin, myricetin,
quercetin, naringenin) and methylated derivatives (isorhamnetin and
chrysoephanol); flavonoid glycosides: 3-O-glycoside (quercetin-3-O-
glucoside, kaempferol-3-O-glucoside, and quercitrin), 7-O-glucoside
Table 2. Results of quantitative (LC-MS/MS) analysis of the phenolic compounds in Rumex plant extracts (mg of compound/g of dry extract).
No. LQa[M-H]-Compounds Rumex sanguineus extracts Rumex crispus extracts
4NZ_H_p 4NZ_R 4Z_H_p 4Z_R 171_H 179_R
1 0.007 191- Quinic acid 0.231 0.026 0.437 0.056 0.710 0.057
2 0.010 169- Gallic acid 0.573 0.316 0.661 0.229 0.949 0.251
3 0.002 153- Protocatechuic acid 0.089 0.015 0.141 0.012 0.145 0.019
4 0.023 289- Catechin 12.310 3.604 16.631 3.487 41.957 13.248
5 0.004 353- 5-O-Caffeoylquinic acid 0.019 <LQ 0.037 <LQ <LQ n.d.
6 0.049 457- Epigallocatechin gallate <LQ <LQ <LQ <LQ <LQ <LQ
7 0.004 137- p-Hydroxybenzoic acid 0.021 0.005 0.010 0.007 0.015 0.009
8 0.031 289- Epicatechin 0.328 0.766 0.384 0.658 n.d. 1.688
9 0.003 179- Caffeic acid 0.041 0.012 0.022 0.021 0.032 0.020
10 0.020 197- Syringic acid 0.037 0.054 0.024 0.024 0.041 0.092
11 0.002 163- p-Coumaric acid 0.019 0.008 0.014 0.009 0.038 0.027
12 0.007 193- Ferulic acid 0.022 0.019 0.010 0.030 0.054 0.024
13 0.002 431- Vitexin (apigenin-8-C-glucoside) <LQ <LQ <LQ <LQ <LQ n.d.
14 0.002 447- Luteolin-7-O-glucoside <LQ <LQ <LQ <LQ <LQ n.d.
15 0.003 463- Hyperoside n.d. n.d. n.d. n.d. n.d. <LQ
16 0.002 609- Rutin 0.048 0.009 0.034 0.010 0.114 0.021
17 0.002 463- Quercetin-3-O-glucoside 0.237 0.019 0.243 0.018 1.681 0.091
18 0.003 431- Apigenin-7-O-glucoside <LQ <LQ <LQ <LQ n.d. n.d.
19 0.049 317- Myricetin <LQ <LQ <LQ <LQ n.d. n.d.
20 0.001 447- Quercitrin (quercetin-3-O-rhamnoside) 0.044 0.035 0.041 0.030 0.074 0.190
21 0.002 447- Kaempferol-3-O-glucoside 0.293 <LQ 0.203 0.001 0.972 <LQ
22 0.049 301- Quercetin <LQ 0.064 <LQ <LQ 0.247 <LQ
23 0.003 271- Naringenin 0.683 0.082 0.634 0.075 0.442 0.203
24 0.002 285- Luteolin <LQ <LQ <LQ <LQ 0.443 0.004
25 0.005 269- Apigenin <LQ <LQ 0.006 <LQ 0.064 0.010
26 0.003 285- Kaempferol 0.098 0.001 0.131 0.001 0.071 n.d.
27 0.013 315- Isoramnetin <LQ <LQ <LQ <LQ 0.006 n.d.
28 269- Aloe-emodin n.a. n.a. n.a. n.a. 0.006 0.061
29 283- Rein n.a. n.a. n.a. n.a. n.d. n.d.
30 269- Emodin n.a. n.a. n.a. n.a. 2.601 2.271
31 253- Chrysophanol n.a. n.a. n.a. n.a. n.d. 1.440
LQ – limit of quantification; n.d. – not detected; n.a. not analyzed. LC-MS/MS: liquid chromatography-tandem mass spectrometry.
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178 Verica Aleksic Sabo et al./ Asian Pacific Journal of Tropical Biomedicine 2020; 10(4): 172-182
(luteolin-7-O-glucoside and apigenin-7-O-glucoside), 8-C-glycoside
(vitexin) and 3-O-ester (epigallocatechin gallate); hydroxybenzoic
acids (gallic acid, p-hydroxybenzoic acid, protocatechuic acid, and
syringic acid); quinic acid, phenylpropene acids (ferulic acid, caffeic
acid, p-coumaric acid) and 5-O-caffeoylquinic acid and anthraquinones
(aloe-emodin, emodin, chrysophanol). The most dominant compounds
in both R. crispus extracts were catechin (41.96 mg/g of dry herb extract
and 13.25 mg/g of dry rhizome extract) and emodin (2.60 mg/g of dry
herb extract and 2.27 mg/g of dry rhizome extract).
3.3. Effect of extract compounds alone and in binary
combinations
In order to identify the biologically active components responsible
for the antimicrobial activity of plant extracts, five standard bioactive
components: catechin, quercetin, quercitrin, gallic acid, and emodin
were tested. These components were selected according to the
criteria for their presence in plant extracts in which antimicrobial
activity was detected against the MDR A. baumannii, as well as on
the basis of data previously published in the literature.
In all R. sanguineus extracts, catechin was the most dominant
component with the amount of 3.487 to 16.631 mg per gram of
dry extract (Table 2). The extract of R. sanguineus (4Z_H_p) with
the highest catechin content (16.631 mg per gram of dry extract)
exhibited the best anti-A. baumannii activity. However, catechin
alone did not exhibit antibacterial activity against MDR A. baumannii
isolates from wounds, even at the highest tested concentration (256
μg/mL) (Table 1).
Table 3. Results of LC/DAD/MS analysis of 8 different HPTLC-fractions of Rumex crispus herb extract.
Fractions Dominant Rt (min) Detected compounds Molecular weight [M-H]-/+/ NI/PI Predicted amount (μg)
F1 Dark brown 0.70 Disaccharide 377-, 365+
0.71 Quinic acid 190 191- 0.708
5.53 n.i. 511-
9.39 n.i. 250-
F2 Dark orange 0.75 n.i. 133-, 135+
4.47 Quercetin-3-O-glucuronide 478 477-, 479+
4.63 Quercetin-3-O-glucoside 464 463- 1.680
F3 Pale blue 4.65 Quercetin-3-O-glucoside 464 463- 1.680
4.70 Quercetin-3-O-derivative
5.45 Quercitrin (Q-3-O-rhamnoside) 448 447- 0.074
F4 Orange-brown 5.12 Eriodictyol-hexoside 450 449-
5.31 n.i. 462 461-, 463+
5.41 Kaempferol-3-O-glucoside 448 447- 0.969
7.56 Emodin-8-O-glucoside 432 431-
7.88 Emodin-8-O-malonyl-hexoside 518 517-
F5 Yellow 3.27 Galloyl-catechin 442 441-, 443+
5.87 Eriodictyol-rhamnoside 434 433-
6.28 n.i. 433-, 469-, 491-, 547-, 736-
7.62 Emodin-8-O-glucoside 432 431-
F6 Blue-violet-blue 0.95 Gallic acid 170 169- 0.946
1.66 Catechin 290 289- 41.800
1.48 Protocatechuic acid 154 153- 0.145
3.63 Ferulic acid 194 193- 0.054
F7 Light brown 1.49 Protocatechuic acid 154 153- 0.145
2.44 n.i. 172-, 174+
3.84 n.i. 187 254-, 186-, 188+, 170+, 210+
5.79 Eriodictyol 288 287-
6.5 Quercetin 302 301- 0.246
6.89 Luteolin 286 285- 0.442
7.58 Kaempherol 286 285- 0.071
9.27 Emodin 270 269- 2.590
1.74 n.i. 251-, 265-, 239+, 261+
F8 Yellow 2.4 n.i. 236-, 238+, 295-
2.42 282-, 419+
3.85 n.i. 187 186-, 244-, 188+, 210+,170+,
415+, 432+, 438+
4.42 n.i. 138-
6.76 Naringenin 272 271- 0.441
7.31 Isorhamnetin 316 315- 0.006
9.28 Emodin 270 269- 2.590
n.i. - not identified. The compounds are listed in the order of occurrence in the fractions (zones) of the high-performance thin-layer chromatography (HPTLC)
and the amount of the compound in the zone represents the approximate values (based on quantitative analysis of total extract). NI/PI negative /positive
ionisation mode. LC-DAD-ESI-MS: liquid chromatography coupled with diode array detector and electrospray ionization mass spectrometry.
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Verica Aleksic Sabo et al./ Asian Pacific Journal of Tropical Biomedicine 2020; 10(4): 172-182
The antibacterial activities of quercetin and its derivative quercetin-
3-O-glucoside (isoquercitrin) as the bioactive components of the
flavonoid class were also tested. Quercetin was 0.064 mg/g in R.
sanguineus (4NZ_R) dry extract (Table 2). Methanol and DMSO
solutions of quercetin and water solution of isoquercitrin did not
exhibit considerable antibacterial activity against MDR A. baumannii
isolates since MIC values were greater than 256 μg/mL (Table 1).
Gallic acid, as a representative of hydroxybenzoic acid derivates,
was present in Rumex extracts, especially in R. sanguineus 4Z_H_
p (0.661 mg per gram of dry extract) (Table 2). The results show no
activity against MDR A. baumannii (Table 1). The lack of activity
was also recorded for emodin in the herb and rhizome extracts in the
amount of 2.3-2.6 mg per gram of dry extract (Table 2).
In addition, similar to the results of the individual bioactive
components of herbal extracts, their binary combinations did not
exhibit an antibacterial effect (Table 1).
3.4. Agar-overlay HPTLC-bioautography detection coupled
with LC/DAD/MS analysis
After the HPTLC separation of R. crispus herb extract (sample
171_H), eight different zones of compounds similar in colour
were detected (Figure 1B). Some of these fractions (F1-F5 and
F7) showed considerable anti-A. baumannii activity according to
bioautography (Figure 1A). The quantitative LC-MS/MS indicated
that this plant is a potentially rich source of biologically active
phenolic compounds (Table 3). Using LC/DAD/MS analysis of
different fractions, additional compounds were detected: quercetin-
3-O-glucuronide (F2), eriodictyol (F7) and its derivatives -hexoside
(F4) and -rhamnoside (F5), emodin derivatives -8-O-glucoside (F4
and F5), and -8-O-malonyl hexoside (F4); but also some of the
compounds confirmed by LC-MS/MS analysis (e.g. rutin, 5-O-
caffeoylquinic acid) were not detected in the fractions due to their
small amount present on HPTLC or wastage during the process of
compounds extraction from the silica gel.
According to the LC/DAD/MS analysis of each fraction, a total of
36 different compounds was detected in R. crispus 171_H extract,
among which 25 were identified (Table 3). The obtained spectral
data were not sufficient for more detailed identification of the
remaining 11 compounds. The most abundant compounds were
catechin (~41.800 μg) in the zone F6, emodin (~2.590 μg) in F7, and
quercetin-3-O-glucoside (~1.680 μg) in F2.
The strips were cut according to eight fractions spotted under
the UV illumination and bioautography was carried out (Figure
1). The bacterial growth inhibition was obvious on the start of the
strip, i.e. in the zone with fractions F1-F5 (Rf<0.4) on the HPTLC
strip (Figure 1A). In these zones mostly the flavonoid glycosides
were detected while their aglycones had higher Rf values (less polar
compounds), located in the upper fractions, near the front of the
chromatogram (Figure 1B and C). According to the bioautography
assay coupled with LC/MS analysis, the detected compounds with
anti-A. baumannii activity were mostly in the fractions of quercetin
derivatives: quercetin-3-O-glucuronide (F2), isoquercitrin (quercetin-
3-O-glucoside) (F2 and F3), quercetin-3-O-derivative (F3), quercitrin
(quercetin-3-O-rhamnoside) (F3) and quercetin-3-O-glucoside (F2
and F3) (Figure 1 and Table 3). The other polyphenols glycosides
Figure 1. Bioautogram (A), high-performance thin-layer chromatography chromatogram,detection with NP/PEG reagent under 366 nm (B), and liquid
chromatography-diode array-mass spectrometry detection of the compounds in each zone (fraction) (C) of Rumex crispus herb extract agaist Acinetobacter
baumannii ATCC 19606 strain.
F8
F7
F6
F5
F4
F3
F2
F1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
伊106
A B C
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5
Counts vs. acquisition time (min)
Eriodictyol
Naringenin
Isorhamnetin
Emodin
Gallic acid
Catechin
Emodin-8-O-glucoside
Quercetin-3-O-glucuronide
Emodin-8-O-malonyl-hexoside
Eriodictyol-rhamnoside
Eriodictyol-hexoside
Galloyl-catechin
Kaempferol-3-O-glucoside
Quercitrin
Quercetin-3-O-glucoside
Quinic acid 33
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180 Verica Aleksic Sabo et al./ Asian Pacific Journal of Tropical Biomedicine 2020; 10(4): 172-182
were present in A. baumannii inhibiting fractions: kaempferol-3-
O-glucoside (F4), emodin-8-O-glucoside (F4 and F5), emodin-8-
O-malonyl-hexoside and eriodictyol-hexoside in F4, eriodictyol-
rhamnoside and galloyl-catechin in F5. However, the water
solution of pure quercetin-3-O-glucoside tested alone did not
show antibacterial activity. The predicted amount of quercetin-3-
O-glucoside on the HPTLC was 1.681 μg (F3 and F4) (Table 3).
Other phenolic glycosides present were not as affordable as the
pure compounds for further testing in this study. Similarly, in the
fraction F7, which showed bacterial inhibition in bioautography,
the quercetin (0.247 μg) and emodin (2.590 μg) were detected and
they did not show anti-A. baumannii activity when tested alone. The
approximate amount of gallic acid, emodin, and catechin in F6 or F8
was 0.946 μg (F6), 2.590 μg (F8), and 41.800 μg (F6), respectively.
4. Discussion
Extracts of R. sanguineus and R. crispus plants have not been
examined previously against A. baumannii, despite their application as
traditional wound remedies. In the present study, we confirmed anti-A.
baumannii activity of the ethanol extracts. Yildirim et al.[27] also proved
that ether and ethanol extracts of R. crispus leaves and seeds showed
antibacterial activity in contrast to aqueous extracts. However, they
have demonstrated antibacterial activity by disc diffusion method only
against bacterial strains S. aureus and Bacillus subtilis with an inhibition
zone of 0.8-1.1 cm, whereas strains of Pseudomonas aeruginosa, E.
coli, and Candida albicans were resistant to these extracts. In our study,
the antibacterial activity of R. crispus extracts was detected against
Gram-negative MDR isolates by the microdilution method. It seems
that the antibacterial activity of ethanol extracts of herb and rhizomes
is better compared with the activity of ether and aqueous extracts tested
by Yildirim et al.[27]. Ethanol extracts of R. crispus rhizome showed
similar activity against Pseudomonas aeruginosa and E. coli and it was
superior to water, acetone, and methanol extracts[28].
The variation in the composition of extracts had an impact on anti-A.
baumannii activity, depending on species, plant part, and geographical
origin. The effect of geographical variations on the composition of the
extract can be overcome by plant growth in a greenhouse under strictly
controlled conditions.
All examined extracts showed significant anti-A. baumannii activity
against MDR isolates from wounds. These MDR strains could
be eradicated by tested Rumex extracts avoiding last line defense
antibiotics, such as polymyxins. For these reasons, the extracts should
be further examined for its potential topical application in the treatment
of A. baumannii infected wounds in vivo.
The selected components of extracts did not show considerable
activity against A. baumannii when they were applied as single agents.
Catechins are generally considered as efficient antimicrobial agents
that exhibit antibacterial activity by inhibiting the N-terminal fragment
of DNA gyrase or interacting with its ATP binding site[28]. Catechin is
the dominant component of the genus Rumex plants extracts, and its
level was significantly higher in herb than in rhizome extracts, which
indicates that this component may be responsible for antibacterial
activity. However, this activity is not confirmed by the study with
catechin activity alone. The antibacterial activity of catechin derivatives,
that were not examined in the present study, seems to be more active
against Gram-negative bacteria, with MICs in range of 32-512 μg/
mL, although some are inactive (MIC>800 μg/mL)[29-31]. Quercetin
and quercetin-3-O-glucoside were also inactive against A. baumanni,
which is in accordance with the previous report, which found that
quercetin-3-O-glucoside was inactive against various bacteria even
in extremely high concentration (100 mg/mL)[32,33]. Gallic acid was
inactive against A. baumannii at higher concentrations, although it was
reported that gallic acid showed MICs against A. baumannii transposon
mutant strains as 128-256 μg/mL[34]. The discrepancy is probably due
to the difference in the degree of sensitivity/resistance, compared to
MDR isolates from wounds in the present study. Similarly, after 24
h of treatment with 200 μg/mL catechin and gallic acid, the number
of clinical isolates of MDR A. baumannii belonging to European
clones 栺and 栻 was generally low with 1.2%-9.7% and 4.3%-8.7% of
reduction, respectively[35]. When emodin was administered alone, it did
not exhibit considerable anti-A. baumannii activity neither, which is in
accordance with the previous report, which showed no activity against
Gram-negative bacteria Klebsiella pneumoniae and E. coli in emodin
(MIC>500 μg/mL)[36].
The activity of the components in binary combinations against
A. baumannii was not observed. The synergy between quercetin-
epigallocatechin gallate against meticillin-resistant staphylococci has
been previously proven[37], as well as between quercetin and gallic
acid, p-anisic acid and cinnamic acid against Aeromonas salmonicida,
but in all cases, MICs were very high (MIC> 256 μg/mL)[38]. It is a
lack of data in the literature on the other combinations tested in this
study (quercetin-isoquercitrin, quercetin-catechin, quercetin-emodin,
quercetin-gallic acid, isoquercitrin-catechin, isoquercitrin-emodin,
isoquercetin-gallic acid, catechin-emodin, catechin-gallic acid, and
emodin-gallic acid), and the activities of these combinations against
A. baumannii. The antibacterial activity was absent when standard
compounds were administered alone and in binary combinations,
while the activity was detected in the Rumex extracts by microdilution
methods and bioautography assay. It suggests that interactions among
these detected and other undetected compounds play a major role in
the anti-A. baumannii activity. Further study should include higher
concentrations of compounds (516 and 1 024 μg/mL) for a more precise
estimation of their activity and interactions (additive or synergistic).
The bioautography showed that only fractions F6 and F8 did not
express anti-A. baumannii activity and that active fractions/compounds
are the most polar ones. The active fractions F1-F5 contained more
polar compounds, flavonoid (quercetin-, kaempherol-, eriodictyol-)
glycosides and/or anthraquinone (emodin-) glycosides that have not
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Verica Aleksic Sabo et al./ Asian Pacific Journal of Tropical Biomedicine 2020; 10(4): 172-182
been examined in the present study as a single agent, indicating their
potential anti-A. baumannii effect. In the inactive fractions, dominant
compounds of F6 and F8 are gallic acid and catechin which had no
considerable anti-A. baumannii activity when they were tested alone.
The activity of F7 suggests that either compounds eriodictyol and/
or luteolin is potentially responsible for the antibacterial activity and/
or rather all the compounds of the fraction F7 act synergistically.
This assumption is supported by the previous report of eriodicyol
antimicrobial activity with MICs ranging from 250-800 μg/mL against
Gram-negative bacteria E. coli, Salmonella enterica subsp. enterica
serovar. Typhimurium, and Pseudomonas putida and its synergistic
activity in binary combination with hesperetin and naringenin[39].
Similarly, luteolin was identified as a responsible component for
antimicrobial activity of Rumex extracts during food preservation[40].
It is possible that, in general, synergism plays a major role in the
extract activity when the MIC of the mixture was determined, and the
individual compounds had weaker antimicrobial activity. Previously,
it was reported that when all the active compounds based on the
bioautography were isolated and characterized, they usually showed
much lower activity than the expected, indicating the presence of
the synergism[41]. Besides the better activity of extracts than single
compounds of binary combinations and potential additive/synergistic
interactions of the components, the extracts have other beneficial
properties, such as antioxidant[42] and anti-inflammatory[43], which
additionally can contribute to in vivo anti-A. baumannii effect. Thus,
further experiments should focus on in vivo study on the effect
of topical application on the healing of wounds infected with A.
baumannii.
In this study, the antibacterial activity of the Rumex extracts against
MDR A. baumannii isolates from wounds was confirmed, justifying
their traditional application in the treatment of wound healing. Therapy
options for wound infections due to MDR A. baumannii are limited
and both R. sanguineus and R. crispus extracts are potential natural
alternatives. The agar-overlay HPTLC-bioautography coupled with LC/
DAD/MS analysis of spotted fractions gave more insight into the types
of secondary biomolecules contributing to the extract activity. The
fractions of R. crispus herb extract, containing flavonol (quercetin and
kaempherol) glycosides, flavanon- (eriodictyol) and anthraquinone-
(emodin) glycosides, and eriodictyol aglycone, have higher anti-A.
baumannii activity. This study supports further in vitro and in vivo
studies on these ethnopharmacological remedies as a valuable and
promising source of antibacterial compounds against MDR A.
baumannii.
Conflict of interest statement
We declare that there is no conflict of interest.
Funding
This study was supported by the Ministry of Education, Science
and Technological Development of the Republic of Serbia, grant OI
172058.
Authors’ contributions
VAS, PK, and JN performed microbiological analyses, while ES,
DO, and NMD performed chemical analyses. VAS and PK wrote
the manuscript, and all authors discussed and analyzed the data. PK
supervised the work.
References
[1] Almasaudi SB. Acinetobacter spp. as nosocomial pathogens: Epidemiology
and resistance features. Saudi J Biol Sci 2018; 25(3): 586–596.
[2] Xie R, Zhang XD, Zhao Q, Peng B, Zheng J. Analysis of global
prevalence of antibiotic resistance in Acinetobacter baumannii infections
disclosed a faster increase in OECD countries. Emerg Microbes Infect
2018; 7(1): 31.
[3] da Silva KE, Maciel WG, Croda J, Cayô R, Ramos AC, de Sales RO, et
al. A high mortality rate associated with multidrug-resistant Acinetobacter
baumannii ST79 and ST25 carrying OXA-23 in a Brazilian intensive care
unit. PLoS One 2018; 13(12): e0209367.
[4] Zhou H, Yao Y, Zhu BQ, Ren DH, Yang Q, Fu YQ, et al. Risk factors for
acquisition and mortality of multidrug-resistant Acinetobacter baumannii
bacteremia: A retrospective study from a Chinese hospital. Medicine
(Baltimore) 2019; 98(13): e14937.
[5] Zarrilli R, Crispino M, Bagattini M, Barretta E, Di Popolo A, Triassi M,
et al. Molecular epidemiology of sequential outbreaks of Acintobacter
baumannii in an intensive care unit shows the emergence of carbapenem
resistance. J Clin Microbiol 2004; 4: 946-953.
[6] Seward RJ, Lambert T, Towner KJ. Molecular epidemiology of
aminoglycoside resistance in Acinetobacter spp. J Med Microbiol 1998; 47:
455-462.
[7] Fournier PE, Vallenet D, Barbe V, Audic S, Ogata H, Poirel L, et
al. Comparative genomics of multidrug resistance in Acinetobacter
baumannii. PLoS Genet 2006; 2: e7.
[8] Isler B, Doi Y, Bonomo RA, Paterson DL. New treatment options against
carbapenem-resistant Acinetobacter baumannii infections. Antimicrob
Agents Chemother 2018; 63(1): e01110-e01118.
[9] Intorasoot A, Chornchoem P, Sookkhee S, Intorasoot S. Bactericidal
activity of herbal volatile oil extracts against multidrug-resistant
Acinetobacter baumannii. J Intercult Ethnopharmacol 2017; 6(2): 218–222.
[10] Tiwari V, Roy R, Tiwari M. Antimicrobial active herbal compounds
against Acinetobacter baumannii and other pathogens. Front Microbiol
2015; 18(6): 618.
[11] Aleksic V, Mimica-Dukic N, Simin N, Nedeljkovic NS, Knezevic P.
[Downloaded free from http://www.apjtb.org on Tuesday, March 17, 2020, IP: 10.232.74.22]
182 Verica Aleksic Sabo et al./ Asian Pacific Journal of Tropical Biomedicine 2020; 10(4): 172-182
Synergistic effect of Myrtus communis L. essential oils and conventional
antibiotics against multi-drug resistant Acinetobacter baumannii wound
isolates. Phytomedicine 2014; 21(12): 1666-1674.
[12] Newman DJ, Cragg GM. Natural products as source of new drugs over
the last 25 years. J Nat Prod 2007; 70: 461-477.
[13] Vasas A, Orbán-Gyapai O, Hohmann J. The genus Rumex: Review of
traditional uses, phytochemistry and pharmacology. J Ethnopharmacol
2015; 175: 198-228.
[14] Denes A, Papp N, Babai D, Czúcz B, Molnár Z. Ehetö, vadon
termöőnövények és felhasználásuk a Kárpát-medencében élö magyarok
körében néprajzi és etnobotanikai kutatások alapján. In: Andrea D (ed.)
Ehetö vadnövények a Kárpát-medencében
. Janus Pannonius Múzeum,
Pécs; 2013, p. 35–76.
[15] Butura V. Romanian ethnobotany encyclopedia [in Romanian]. Bucharest,
Romania: The Scientific and Encyclopedic Publishing; 1979.
[16] Baskan S, Daut-Özdemir A, Günaydin K, Erim FB. Analysis
of anthraquinones in Rumex crispus by micellar electrokinetic
chromatography. Talanta 2007; 71: 747–750.
[17] Shiwani S, Kumar Singh N, Hyeon Wang M. Carbohydrase inhibition
and anti-cancerous and free radical scavenging properties along with
DNA and protein protection ability of methanolic root extracts of Rumex
crispus. Nutr Res Pract 2012; 6(5): 389-395.
[18] Pareek A, Kumar A. Rumex crispus L. –a plant of traditional value. Drug
Discovery 2014; 9: 20–23.
[19] Ahmed SS, Erum S, Khan SM, Nawaz M, Wahid A. Exploring the
medicinal plants wealth: A traditional medico-botanical knowledge of
local communities in Changa Manga Forest, Pakistan. Middle-East. J Sci
Res 2014; 20: 1772–1779.
[20] Moerman D. Native American ethnobotany. Timber Press; 2003.
[21] Suh HJ, Lee KS, Kim SR, Shin MH, Park S, Park S. Determination of
singlet oxygen quenching and protection of biological systems by various
extracts from seed of Rumex crispus L. J Photoch Photobiol B 2010;
102(2): 102-107.
[22] Idris A, Wintola OA, Afolayan AJ. Phytochemical and antioxidant
activities of Rumex crispus L. in treatment of gastrointestinal helminths
in Eastern Cape Province, South Africa. Asian Pac J Trop Biomed 2017;
7(12): 1071–1078.
[23] Cornara L, La Rocca A, Marsili S, Mariotti MG. Traditional uses of
plants in the Eastern Riviera (Liguria, Italy). J Ethnopharmacol 2009;
125(1): 16–30.
[24] Clinical Laboratory Standards Institute. Methods for dilution antimicrobial
susceptibility tests for bacteria that grow aerobically. 7th ed. Approved
Standard M7-A7. CLSI, Wayne, PA; 2006.
[25] Knezevic P, Aleksic V, Simin N, Svircev E, Petrovic A, Mimica-Dukic N.
Antimicrobial activity of Eucalyptus camaldulensis essential oils and their
interactions with conventional antimicrobial agents against multi-drug
resistant Acinetobacter baumannii. J Ethnopharmacol 2016; 178: 125-136.
[26] Orčiċ D, Francišković M, Bekvalac K, Svirčev E, Beara I, Lesjak M, et
al. Quantitative determination of plant phenolics in Urtica dioica extracts
by high-performance liquid chromatography coupled with tandem mass
spectrometric detection. Food Chem 2014; 143: 48–53.
[27] Yıldırım A, Mavi A, Kara AA. Determination of antioxidant and
antimicrobial activities of Rumex crispus L. extracts. J Agric Food Chem
2001; 49(8): 4083-4089.
[28] Idris OA, Wintola OA, Afolayan AJ. Evaluation of the bioactivities of
Rumex crispus L. leaves and root extracts using toxicity, antimicrobial,
and antiparasitic assays. Evid-Based Compl Alt Med 2019. ID 6825297.
[29] Gradisar H, Pristovsek P, Plaper A, Jerala R. Green tea catechins inhibit
bacterial DNA gyrase by interaction with its ATP binding site. J Med
Chem 2007; 50(2): 264–271.
[30] Mabe K, Yamada M, Oguni I, Takahashi T. In vitro and in vivo activities
of tea catechins against Helicobacter pylori. Antimicrob Agents Chemother
1999; 43(7): 1788-1791.
[31] Hamilton-Miller JMT. Chemical and biological properties of tea infusions.
Frankfurt: U&M, Germany; 1997, p. 63-75.
[32] Yoda Y, Hu ZQ, Zhao WH, Shumamura T. Different suscepttibilities of
Staphylococcus and Gram-negative rods to epigallocatechin gallate. J
Infect Chemother 2004; 10(1): 55-58.
[33] Nitiema LW, Savadogo A, Simpore J, Dianou D, Traore AS. In vitro
antimicrobial activity of some phenolic compounds (coumarin and
quercetin) against gastroenteritis bacterial strains. Int J Microbiol Res
2012; 3(3): 183-187.
[34] Razavi SM, Zahri S, Zarrini G, Nazemiyeh H, Mohammadi S. Biological
activity of quercetin-3-O-glucoside, a known plant flavonoid. Bioorg
Khim 2009; 35(3): 376–378.
[35] Ajiboye TO, Skiebe E, Wilharm G. Phenolic acids potentiate colistin-
mediated killing of Acinetobacter baumannii by inducing redox
imbalance. Biomed Pharmacother 2018; 101: 737–744.
[36]
ćurković-Perica M, Hrenović J, Kugler N, Goić-Barišić I, Tkalec M.
Antibacterial activity of Pinus pinaster bark extract and its components
against multidrug-resistant clinical isolates of Acinetobacter baumannii.
Croatia Chemica Acta 2015; 88(2): 133–137.
[37] Chukwujekwu JC, Coombes PH, Mulholland DA, van Staden J. Emodin,
an antibacterial anthraquinone from the roots of Cassia occidentalis. S Afr
J Bot 2006; 72(2): 295-297.
[38] Coopoosamy RM, Magwa ML. Antibacterial activity of aloe emodin and
aloin A isolated from Aloe excelsa. Afr J Biotech 2006; 5(11): 1092-1094.
[39] Betts JW, Hornsey M, Wareham DW. In vitro activity of epigallocatechin
gallate (EGCG) and quercetin alone and in combination versus clinical
isolates of methicillin-resistant Staphylococcus aureus. ASM 2014 Barts
and the London, School of Medicine and Dentistry, UK; 2014.
[40] Mhalla D, Bouaziz A, Ennouri K, Chawech R, Smaoui S, Jarraya B, et al.
Antimicrobial activity and bioguided fractionation of Rumex tingitanus
extracts for meat preservation. Meat Sci 2017; 125: 22-26.
[41] Eloff JN, Katerere DR, McGaw LJ. The biological activity and chemistry
of the southern African Combretaceae. J Ethnopharm 2008; 119: 689-
699.
[42] Idris AO, Wintola AO, Afolayan AA. Phytochemical and antioxidant
activities of Rumex crispus L. in treatment of gastrointestinal helminths
in Eastern Cape Province, South Africa. Asian Pac J Trop Biomed 2017;
7(12): 1071-1078.
[43] Singh M, Purohit MC. Anti-inflammatory activity of methanolic extract
of roots of Rumex obtusifolius. Int J Pharm Sci & Res 2018; 9(8): 3519-
3522.
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