Phytomedicine 39 (2018) 93–99

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Phytomedicine
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Short communication

Synergism of prenylflavonoids from Morus alba root bark against clinical T

MRSA isolates
⁎ ⁎
Guo-Ying Zuoa, , Cui-Xian Yanga,b, Jun Hanb, , Yu-Qing Lib, Gen-Chun Wanga
a
Research Center for Natural Medicines, Kunming General Hospital of Chengdu Military Command, Kunming 650032, China
b
School of Basic Medical Sciences, Yunnan Traditional Chinese Medical College, Kunming 650500, China

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Clinical methicillin-resistant Staphylococcus aureus (MRSA) is a thorny problem in current anti-
Morus alba L. infective therapeutics and a challenge of new drug development. Plant prenylflavonoids possess anti-MRSA
MRSA activity, but few of the prenylflavonoids have been reported the synergistic anti-MRSA effect when they are used
Prenylflavonoid in combination with conventional antibacterial agents.
Antibacterial agents
Purpose: This study deals with anti-MRSA activity of four prenylflavonoids from the root bark of Morus alba and
Synergy
their synergism with 11 conventional antibacterial agents.
Methods: Chromatographic methods and spectral analysis were used to isolate and identify the prenylflavonoids.
The antibacterial activity and synergism were assessed by the broth microdilution method, checkerboard dilu-
tion test, and time-kill curve assay, respectively.
Results: Four prenylflavonoids, i.e., cyclocommunol (Cy, 1), morusinol (Ml, 2), morusin (Mi, 3) and kuwanon E
(Ku, 4), were isolated from Morus alba bark ethanol extract. Compounds 1, 3 and 4 showed high antimicrobial
activity on both methicillin-susceptible S. aureus (MSSA) and MRSA strains with MICs/MBCs at 4–16/32–64 and
4–32/16–128 µg/ml, respectively. Ml (2) was not active. Compound 2 showed synergy with amikacin (AK) and
streptomycin (SM) against all the ten MRSA isolates. Ml (2) and Ku (4) also showed synergy with ciprofloxacin
(CI), etimicin (EM) and vancomycin (VA) against 7–9 isolates. The fractional inhibitory concentration indices
(FICIs) ranged 0.09–1.00 and the dose reduction indices (DRIs) of these antibacterial agents ranged 2–128. Cy
(1) and Mi (3) showed synergy with the tested antibacterial agents against only 1–3 MRSA isolates except VA.
Furthermore, the MRSA resistance could be reversed in the combinations of AK with Cy, Ml, Mi and Ku; EM with
Mi and Ku; and SM with Ml by the criteria of MIC interpretive standards for Staphylococcus spp. of CLSI. All the
combinations showed only indifference in the 1 × MIC time-killing experiments. The prenylated substitutions
play an important role in the activity of the compounds used alone and combined with the tested antibacterials.
Conclusions: The study revealed for the first time the anti-MRSA synergism of prenylflavonoids 1–4 with eleven
antibacterial agents and the reversal of MRSA resistance to aminoglycosides, especially amikacin. The results
might be valuable for the development of new antibacterial drugs and synergists against MRSA infection.

Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the
most common pathogens in both hospital-acquired and community-
acquired infections (Hetem et al., 2016). Clinical MRSA strains have
developed resistance not only to β-lactams but also to aminoglycosides,
quinolones, macrolides, tetracyclines and others (Zarfel et al., 2013).
Since antibacterial drugs for MRSA are limited, it is highly necessary to
find new leading compounds that alone or in combination with anti-
bacterial drugs, can overpass the resistance issue (Li et al., 2012).
As part of our ongoing studies on the new anti-MRSA compounds
from natural sources, we have aimed to the search of compounds which
not only have anti-MRSA activity when acting alone, but also could
potentiate the anti-MRSA activity of antibacterial drugs.

Abbreviations: AK, amikacin; AM, ampicillin; CFU, colony forming unit; CI, ciprofloxacin; CLSI, Clinical and Laboratory Standards Institute; GN, gentamicin; CX, cefoxitin; Cy, cy-
clocommunol; DRI, dose reduction index; EM, etimicin; FICI, fractional inhibitory concentration index; I, intermediate; Ku, kuwanon E; LE, levofloxacin; MBC, minimal bactericidal
concentration; MH, Mueller–Hinton medium; MIC, minimal inhibitory concentration; Mi, morusin; Ml, morusinol; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-
susceptible Staphylococcus aureus; NMR, nuclear magnetic resonance; P/S, piperacillin-sulbactam; PV, penicillin; R, resistant; S, susceptible; SM, streptomycin; TP, teicoplanin; VA,
vancomycin
⁎
Corresponding authors.
E-mail addresses: zuoguoying@263.net (G.-Y. Zuo), hanzjn@126.com (J. Han).

https://doi.org/10.1016/j.phymed.2017.12.023
Received 4 September 2017; Received in revised form 29 November 2017; Accepted 20 December 2017
0944-7113/ © 2018 Elsevier GmbH. All rights reserved.
G.-Y. Zuo et al.
The root bark of Morus alba L. (Moraceae) (also known as white
mulberry bark and marketed in China as Sang Bai Pi (SBP)) has long
been used in Traditional Chinese Medicine (TCM) for fever asthmatic
cough, hemoptysis, edema, beriberi and diuresis (NUTCM, 2005). SBP
has showed to possess flavonoids, alkaloids and stilbenoids. Anti-
microbial, skin-whitening, cytotoxic, anti-inflammatory and anti-hy-
perlipidemic properties have been found for this species in previous
reports (Chan et al., 2016). The prenylated flavonoids from SBP and
other plant resources have shown antimicrobial properties against
methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-
resistant S. aureus (MRSA) (Sohn et al., 2004; Chen et al., 2005; Ku
et al., 2010; Mazimba et al., 2011; Zuo et al., 2012; Mun et al., 2013;
Pang et al., 2014). However, only a few of such compounds, e.g. so-
phoraflavanones B and G from Sophora species (Fabaceae) (Mun et al.,
2013; Sakagami et al., 1998) and isojacareubin from Hypericum japo-
nicum Thunb. ex Murray (Guttiferae) (Zuo et al., 2012), have been re-
ported to possess anti-MRSA synergistic properties.
In this report, we present for the first time the evaluation of anti-
MRSA synergism of the four SBP prenylflavonoids (i.e., cyclocommunol
(Cy, 1), morusinol (Ml, 2), morusin (Mi, 3) and kuwanon E (Ku, 4)) by
combining them with eleven conventional antibacterial agents, in-
cluding amikacin (AK), ampicillin (AM), ciprofloxacin (CI), gentamicin
(GN), etimicin (EM), levofloxacin (LE), piperacillin-sulbactam (P/S),
penicillin (PV), streptomycin (SM), teicoplanin (TP) and vancomycin
(VA).
Materials and methods
Plant material
M. alba root bark was bought in August 2010 from Yunnan Lv Sheng
Pharmaceutical Co., Ltd (Kunming, China). A voucher specimen (KUN
0515476) is deposited in Herbarium of Kunming Institute of Botany,
China.
Isolation and identification of compounds 1–4
The dried powder of M. alba root bark (5.0 kg) was extracted with
80% ethanol (× 3). The mixtures were filtered and the resulting fil-
trates were combined. After evaporating the solvent under reduced
pressure at 40 °C, the crude methanol extract (635 g) was suspended in
1000 ml deioned water and successively extracted with petroleum
ether, ethyl acetate (EtOAc) and butanol to give three main sub-extracts
(240 g, EtOAc; 40 g, butanol; 355 g, water). The 200 g sub-extract from
EtOAc which showed the highest activity against MRSA by disk diffu-
sion method was subjected to column chromatography with silica gel
(200–300 mesh, 1900 g; Qingdao Haiyang Chemical Co., Ltd., Qingdao,
China), gradient eluting with petroleum ether: EtOAc (P:E = 10:1–1:1)
to give 14 fractions (Mfr-1–14). The fractions were further subjected to
activity assay and the resulted active fractions were carried out re-
peated chromatography to give compounds 1–4, respectively, i.e., from
Mfr-7 with silica gel (300–400 mesh, P:E = 4:1) and Sephadex LH-20
(methanol) to give compound 3; from Mfr-9 (300–400 mesh, P:E = 4:1,
P: chloroform (C) = 30:1 and Sephadex LH-20, (CH3)2CO) to give
compound 1; from Mfr-12 (300–400 mesh, P:C = 30:1 and 20:1, re-
spectively) to give compound 4; from Mfr-13 (P:C = 30:1 and
P:E = 2:1, successively) to give compound 2. These compounds were
identified through physicochemical and spectral analyses and by com-
parison with previously reported data as Cy (1), (Lin and Shieh, 1992),
Ml (2), Mi (3) and Ku (4) (Konno et al., 1977; Kim et al., 2011). The
evidence of purity (>95%) of compounds 1–4 was assessed with 13C
NMR profile (Supplementary material, S1).
Bacterial strains
Ten MRSA strains with SCCmec III genotype and mecA gene were
94
Phytomedicine 39 (2018) 93–99
obtained and characterized from the infectious sputum samples of cri-
tically ill patients in Kunming General Hospital as previously reported
(Zhang et al., 2005; Zuo et al., 2012). The mecA-mediated oxacillin
resistance was confirmed using cefoxitin disk with inhibitory zone ≤
21 mm (CLSI, 2012). The control strain was S. aureus (ATCC25923;
MSSA) which was purchased from the Beijing Tiantan Pharmaceutical
and Biological Technology Co., Ltd., Beijing, China and were used in
this experiment.
Antibacterial agents
The eleven antibacterial agents were purchased from the manu-
facturers in China, i.e., AK (Jiangsu Wuzhong Pharmaceutical Group
Co., Ltd., Suzhou, China); AM and SM (North China Pharmaceutical
Co., Ltd., Shijiazhuang, China); CI (Sichuan Kelun Pharmaceutical Co.,
Ltd., Chengdu, China); GN and LE (Yangtze River Pharmaceutical
Group Co., Ltd., Taizhou China); EM (Wuxi Jiming Kexin Shanhe
Pharmaceutical Co., Ltd., Wuxi China); PV and P/S (Harbin
Pharmaceutical Group Co., Ltd., Harbin, China); TP (Sanofi-Aventis
(Beijing) Pharmaceutical Co., Ltd., Beijing China); VA (Eli Lilly Japan K.
K., Seishin Laboratories, Japan) was used as the positive control agent.
Cefoxitin disks were purchased from Beijing Tiantan biological pro-
ducts Co., Ltd., China. The four prenylflavonoids (1–4) were isolated
and identified from root bark of M. alba with purity >95% which was
supported by their 13C NMR spectra (S1).
Media
Standard Mueller–Hinton agar and broth (MHA and MHB, Tianhe
Microbial Agents Co., Hangzhou, China) were used as bacterial culture
media. MHB was used for all susceptibility testing and time-kill ex-
periments. Colony counts were determined using MHA plates.
Susceptibility testing
MICs/MBCs of compounds 1–4 were determined by conventional
broth microdilution technique with starting inocula of 5 × 105 CFU/ml
according to CLSI guidelines and incubated at 35 °C (CLSI, 2012).
Synergy testing
Anti-MRSA and -MSSA interactions of various antibacterial agents
in combination with compounds 1–4 were determined by checkerboard
and dynamic time-killing methods (Zuo et al., 2012). Briefly, bacteria
were subcultured on MHA plates for 24 h. Colonies were suspended in
0.9% sterile saline and adjusted to a 0.5 McFarland turbidity standard
(1.5 × 108 CFU/ml). The suspension was further diluted with MHB to
1 × 106 CFU/ml prior to testing in the 96-well-microtiter-plates. For
the checkerboard method, the uppermost row (A1) of a plate contained
an antibacterial agent (A) in a concentration of two times the expected
MIC combination. Each following row contained half the concentration
of the previous one. The same procedure was carried out along the
leftmost column (B1) with a compound (B)—but not necessarily with
the same starting concentration. So, each well contained a unique
combination of the two substances (A and B) in 100 µl MHB. At last
100 µl MHB containing 1 × 106 CFU/ml was added to the wells and
incubated at 35 °C for 24 h with a starting inoculum of 5 × 105 CFU/ml.
The concentrations of the first wells without visible growth along the
stepwise boundary between inhibition and growth were used to cal-
culate the FICI values (FICI of A combined with B) = ((MICA)Combined/
(MICA)Alone) + ((MICB)Combined/(MICB)Alone) (Iten et al., 2009); For the
time-kill testing, the wells containing 1 × MIC of the two substances
with a starting inoculum of 5 × 105 CFU/ml was also incubated at 35 °C
for the predetermined time points 0, 4, 8, 12, 16, 20, and 24 h. Fifty µl
aliquots were removed at each time point from the test solution, diluted
and streaked on a MHA plate for colony count determination (Klepser
G.-Y. Zuo et al.
et al., 1997, 1998).
The bacteriostatic synergism was evaluated by the fractional in-
hibitory concentration indices (FICIs) as the following: FICI ≤ 0.5, sy-
nergy; 0.5 < FICI ≤ 4, indifference (or no interaction) and FICI > 4,
antagonism (Odds, 2003). The potentiation effects were evaluated by
the dose reduction indices (DRIs), i.e. DRI = MICalone/MICcombined
(Chou, 2006). The bactericidal synergism was evaluated by the time-
killing experiments judged by the log10CFU/ml increase in killing at
24 h (ΔLC24) in comparison with the killing by the most active single
drug as the following: ΔLC24 ≥ 2 log10CFU/ml, synergy; ΔLC24 = 1–2
log10CFU/ml, additivity; ΔLC24 = ± 1 log10CFU/ml, indifference;
ΔLC24 > − 1 log10CFU/ml, antagonism (i.e. the increased bacterial
growth > 1 log10CFU/ml in comparison with the least active single
agent) (Chin et al., 2008). The data from time-kill assays are presented
as the means ± standard deviations.
Cytotoxicity assay
The tested compounds 1 and 2 were assayed against normal human
liver HL-7702 and human lung cancer A549 cell lines for their cytotoxic
effect, by using the MTS cytotoxicity assay (a variant of the widely used
MTT assay) (Sohn et al., 2004). Briefly, HL-7702 and A549 cells were
cultured in high glucose Dulbecco's modified Eagle's medium (DMEM,
Genview, USA) in a humidified atmosphere consisting of 5% CO2 and
95% air to maintain continuous logarithmic growth. 2 × 104 cells per
well were seeded in 96-well microplates and incubated 24 h to adhere.
Then the compounds with concentrations ranging from 8 × MIC to 1/
8 × MIC (in DMSO, final concentration of DMSO ≤ 0.5% v/v) were
added and incubated for another 24 h. The MTS/PMS solution (20 µl)
was added to each well and further incubated for 3 h. The absorbance
was measured at 490 nm on a microplate reader (Sunrise, Tecan Co.,
Männedorf, Switzerland). The IC50 values were calculated from viabi-
lity curves. All data are presented as the mean values of triplicates.
Results
Antibacterial activity of the prenylflavonoids against MSSA and MRSA
Four prenylflavonoids (1–4) were isolated from the EtOAc sub-ex-
tract of M. alba root bark. Their structures were identified as Cy (1), Ml
(2), Mi (3) and Ku (4) (Fig. 1) by physicochemical and spectral analyses
(S1) and compared with previously reported data (Lin and Shieh, 1992
(1); Konno et al., 1977 (2); Kim et al., 2011 (2–4)). Compound 1 was
firstly characterized in Artocarpus communis Forster (Moraceae)
(Lin and Shieh, 1992), and 1–4 were also isolated previously from M.
3'
4' OH 5''
2'
8 6''
HO 9 O 2 5'
1'
7 6'
6
10 3 11
O potentiating effect in all the combinations despite its lowest activity
5 4
OH O
12 14
13
15 1 12
HO OH
2''
HO O 3
1'' 3''
9''
OH O 10''
The structures of compounds 1–4
Fig. 1. Structures of cyclocommunol (Cy, 1), morusinol (Ml, 2), morusin (Mi, 3) and
kuwanon E (Ku, 4).
Phytomedicine 39 (2018) 93–99
alba root bark (Geng et al., 2010; Konno et al., 1977; Nomura and
Fukai, 1978).
Compounds 1–4 were tested for antibacterial activities alone against
MSSA and MRSA (Table 1). The results of 1, 3 and 4 showed promising
effects on both MSSA and MRSA strains with MICs/MBCs (µg/ml) =
4–16/32–64 and 4–32/16–128, respectively. The 50% values (i.e.
MIC50/MBC50) against MRSA (n = 10) were determined as 4–16/
32–128 µg/ml (Table 1). Compound 2 was not active >100 µg/ml
(Table 1). The MICs of the four compounds follows the order of 4
(Ku) < 1 (Cy) < 3 (Mi) < < 2 (Ml) against both MSSA and MRSA.
Synergism of antibacterial agents with the prenylflavonoids
The results of anti-MSSA and -MRSA synergism in a total of sixteen
combinations of the 11 selected conventional antibacterial agents
(Table 1) with the four prenylflavonoids (1–4) are collected in Tables
2–3 and Fig. 2, respectively. Compounds 1(Cy) and 2 (Ml) showed the
widest spectrum of synergistic action with all the 11 agents, including
four antibacterial categories, i.e., aminoglycosides (AK, GN, EM and
SM), β-lactams (AM, P/S and PV), floroquinolone (CI, LE) and glyco-
peptides (TP and VA). For the anti-MSSA synergism in Table 2, the
bacteriostatic interactions exhibit that ten combinations showed sy-
nergy and the remaining ones were indifference. Although the activity
of compounds 2 (Ml) used alone was very weak, it showed the best
synergy with AK, CI, VA and SM, with FICIs of 0.250–0.375 and DRIs of
4–64. Next to 2, compound 1 (Cy) also showed synergy with AK, AM, P/
S and PV, together with the indifference with LE, VA, and GN. Com-
pound 3 showed only indifference with both EM and AK. Compound 4
showed synergy with EM and indifference with AK, respectively.
For the anti-MRSA synergism in Table 3, the bacteriostatic inter-
actions were also demonstrated by the sixteen combinations of the 11
antibacterial agents with compounds 1–4, which showed different ex-
tent of potentiation on their anti-MRSA activities. Compound 2 (Ml)
showed the three best combinations of synergy with AK, SM and CI
against nearly all the ten MRSA isolates, with the FICIs of 0.094–0.516
and the DRIs of 2–64, while 1 (Cy) showed the least number (≤1) of
isolates which showed synergy in combination with LE and VA
(Table 3). The remaining two combinations of VA and TP with Ml (2)
also showed seven and five isolates of synergy, with FICIs of
0.094–0.750 and DRIs of 2–64 (Table 3). The potency for the five
combinations follows the order: AK(Ml) > SM(Ml) > CI
(Ml) > VA(Ml) > TP(Ml). Other combinations also showed varied (i.e.
2–8) isolates of synergy against MRSA. Compound 4 (Ku) in the two
combinations (i.e. AK(Ku) and EM(Ku)) mainly showed synergy by 7–8
isolates of MRSA (n = 10). Meanwhile, the seven combinations with Cy
(1) showed varied potency order of AK(Cy) > AM(Cy) > GN(Cy) > LE
(Cy) > P/S(Cy) > PV(Cy) > VA(Cy). Moreover, Cy (1) showed the
3''
2''
4''
HO OH least potentiating effects among the four compounds, with sporadic
isolates of synergism, i.e., 0–4 isolates of synergy and 6–10 isolates of
O O indifference (Table 3). It is interesting that Ml (2) also showed the best
R used alone, which is similar to the case for the anti-MSSA synergism in
Table 2. While the two combinations of Mi (3) with AK and EM showed
OH O
11 only 2–3 isolates of synergy despite its activity used alone being far
more potent than that of Ml (2). In general, the anti-MRSA potentiating
R= 2
OH order (i.e. number of isolates) of the sixteen combinations follows: AK
11 (Ml) = SM(Ml) > CI(Ml) >EM(Ku) > VA(Ml) = AK(Ku) > TP(Ml) >
4'' 12
AK(Cy) > EM(Mi) = P/S(Cy) = PV(Cy) > GN(Cy) > AK(Mi) = AM
R
(Cy) > LE(Cy) > VA(Cy) (Table 3).
R= 6''
8'' Contrary to the potent bacteriostatic anti-MRSA synergistic effects,
5'' 7'' the bactericidal interactions of AK and GN in combination with all
4 tested antibacterial agents at each concentration of 1 × MIC showed
only indifference against MRSA in the dynamic time-killing experiment
judged by the log10CFU/ml increase in killing at 24 h being < 1 (i.e.
△LC24 < 1) (Fig. 2). For example, the △LC24 for AK(Cy) and GN(Cy)
were 0.38 and 0.72, respectively (Chin et al., 2008). Nevertheless, the
95
G.-Y. Zuo et al. Phytomedicine 39 (2018) 93–99

Table 1
MICs/MBCs of compounds 1–4 and related antibacterial agents alone against MSSA and MRSA (µg/ml)a.

Strainsb 1 2 3 4 AK AM CI GN EM LE P/S PV SM TP VA

MSSA 4/32 1024/nsc 16/64 4/32 16/32 4/32 128/512 32/128 4/16 4/16 4/16 8/32 32/256 0.5/1 0.5/2
MRSA01 16/64 512/ns 16/128 16/128 16/64 64/512 128/512 16/64 4/8 64/128 25/6512 128/512 128/512 0.5/1 1/1
MRSA02 16/128 512/ns 32/128 8/32 64/256 32/256 32/256 32/64 8/32 32/64 256/512 32/128 64/512 4/8 0. 25/1
MRSA03 16/64 512/ns 8/32 4/32 32/128 64/256 64/256 16/64 4/32 32/64 128/256 32/128 32/256 0.5/8 1/1
MRSA04 8/64 1024/ns 16/128 4/32 32/128 64/256 256/512 32/256 8/32 32/64 128/256 64/256 64/256 1/2 0.5/2
MRSA05 16/64 1024/ns 32/64 8/64 16/64 64/512 64/256 32/128 4/16 32/128 256/512 128/512 64/256 1/4 2/2
MRSA06 16/64 1024/ns 16/64 8/32 32/128 128/512 64/512 32/256 8/32 32/128 128/512 128/512 64/512 1/4 1/2
MRSA07 8/32 1024/ns 16/128 4/16 32/256 128/512 128/256 32/128 8/32 16/64 128/512 64/256 128/512 2/4 1/2
MRSA08 8/128 1024/ns 16/128 4/16 32/256 32/256 128/512 32/256 8/64 8/32 128/256 64/128 32/128 1/2 1/2
MRSA09 16/64 1024/ns 8/32 4/32 32/128 32/256 128/512 32/128 4/16 16/128 128/256 128/512 64/256 1/2 1/2
MRSA10 16/64 1024/ns 32/128 8/64 32/128 32/64 64/256 32/128 4/16 32/64 256/512 128/256 64/256 4/8 1/2
MRSA range 8–16/ 512–1024/ 8–32/ 4–16/ 16–64/ 32–128/ 32–256/ 16–32/ 4–8/ 8–64/ 128–256/ 32–128/ 32–128/ 0.5–4/ 0.25–2/
32–128 ns 32–128 16–128 64–256 64–512 256–512 64–256 8–64 32–128 256–512 128–512 128–512 1–8 1–2
MRSA (50%) 16/64 1024/ns 16/128 4/32 32/128 64/256 64/256 32/128 4/32 32/64 128/512 64/256 64/256 1/4 1/2
MRSA (90%) 16/128 1024/ns 32/128 8/64 32/256 128/512 128/512 32/256 8/32 32/128 256/512 128/512 128/512 4/8 1/2

a
1: cyclocommunol (Cy), 2: morusinol (Ml), 3: morusin (Mi), 4: kuwanon E (Ku), AK: amikacin, AM: ampicillin, CI: ciprofloxacin, GN: gentamicin, EM: etimicin, LE: levofloxacin, PV:
penicillin, P/S: piperacillin-sulbactam, SM: streptomycin, TP: teicoplanin, VA: vancomycin.
b
50% and 90%: values of inhibition against 50% and 90% of the 10 MRSA strains, respectively.
c
ns > 1024.

Table 2 only two isolates of AK(Mi) and one isolate of AK(Cy) showed inter-
MICs of antibiotics alone and in combination with four prenylflavonoids (1–4) against the mediate activity (Table 4). Other combinations also showed resistance
MSSA (ATCC25923) strain.
reversal effect, such as three isolates reversed in the combination of SM
Combinationa MIC (µg/ml) DRIb FICIc Itnd (Ml). Furthermore, the combinations of EM with Mi and Ku also showed
6–7 isolates to be changed as the same potency as the MSSA strain
Alone Combined (ATCC25923) (Table 4).
AK(Ml) 16(1024) 2 (128) 8(8) 0.25 s
CI(Ml) 128(1024) 32(16) 4(64) 0.27 s Cytotoxicity of Cy (1) and Ml (2)
VA(Ml) 0.5(1024) 0.125(64) 4(16) 0.31 s
SM(Ml) 32(1024) 8 (128) 4(8) 0.38 s The in vitro cytotoxicity of Cy (1) and Ml (2) was measured in HL-
EM(Ku) 4(4) 1(0.5) 4(8) 0.38 s
7702 and A549 cell lines. The IC50 values were 27.4 and 170.2 µg/ml
AK(Cy) 8(4) 2(0.5) 4(8) 0.38 s
AM(Cy) 64(4) 16(1) 4(4) 0.50 s against HL-7702, and 31.6 and 260 µg/ml against A549, respectively.
P/S(Cy) 32(4) 16(1) 2(4) 0.50 s Considering their relatively low cytotoxicity and strong anti-MRSA sy-
PV(Cy) 8(4) 2(1) 4(4) 0.50 s nergy activity, compounds 1 and 2 could possibly be applied to treat
AK(Ku) 16(4) 4(1) 4(4) 0.50 s MRSA or MSSA infections. However, their combined cytotoxicity re-
TP(Ml) 0.5(1024) 0.25(64) 2(16) 0.56 i
mains to be investigated.
LE(Cy) 32(4) 8(2) 4(2) 0.75 i
VA(Cy) 0.5(4) 0.25(1) 2(4) 0.75 i
EM(Mi) 4(16) 1(8) 4(2) 0.75 i Discussion
GN(Cy) 16(4) 8(2) 2(2) 1.00 i
AK(Mi) 16(16) 8(8) 2(2) 1.00 i
The prenylated flavonoids possess an isoprenyl, a geranyl, a 1,1-
a
AK: amikacin, AM: ampicillin, CI: ciprofloxacin, GN: gentamicin, EM: etimicin, LE: dimethylallyl, and/or a lavandulyl substituent in their chemical struc-
levofloxacin, PV: penicillin, P/S: piperacillin-sulbactam, SM: streptomycin, TP: teico- tures. They have previously demonstrated to be the active antimicrobial
planin, VA: vancomycin, Cy: cyclocommunol (1), Ml: morusinol (2), Mi: morusin (3), Ku: entities in phytochemicals when they were used alone (Sohn et al.,
kuwanon E (4). “AK(Ml)” means amikacin was used in combination with morusinol, and 2004). In this report, we further revealed the effective synergism of the
the same below. All data in the parentheses are designated to those of the prenyl-
prenylated flavonoids 1–4 from M. alba when they were used in com-
flavonoids which were used in combination with the antibacterial agents.
b bination with conventional antibacterial agents against MRSA (Tables 2
DRI: dose reduction index = MICAlone/MICCombined.
c
FICI (of A combined with B )= ((MICA)combined/(MICA)alone) + ((MICB)combined/ and 3), including the effects when used alone (Table 1).
(MICB)alone); FICI ≤ 0.5, synergy (s); 0.5 < FICI ≤ 4, indifference (i). Sohn et al. (2004) reported the antimicrobial activity of 18 pre-
d
Itn: interaction. nylated flavonoids isolated from M. alba and other medicinal plants,
including Mi (3) which was effective only against the Gram (+) bac-
bactericidal interactions always resulted in potentiating effects in all teria S. aureus KCTC 1621 and S. epidermis ATCC 12228 with MICs of 25
the combinations (Fig. 2). In the combination of AK(Cy) (A), for ex- and 20 µg/ml, respectively. Mi (3) also showed to be suppressor of
ample, the killing value by log10CFU/ml at 24 h (LC24) in the curve of iNOS, antinociceptive, platelet aggregation activities, etc.
the combination is smaller than those of AK and Cy used alone. The (J) Mazimba et al. (2011) reported Mi against S. aureus (NCTC 4163) with
in Fig. 2 shows the dose-dependent bactericidal effects from the con- MIC of 7.81 µg/ml, together with MICs of 3.91–62.5 against Bacillus
centrations of 8 × MIC to 1/4 × MIC of Cy used alone. subtilis, Micrococcus flavus, Streptococcus faecalis, Salmonella abony and
Pseudomonas aeruginosa. In a patent, Ku et al. (2010) determined Mi 3
Reversal effects of MRSA resistance against aminoglycosides with MICs of 10 µg/ml against MSSA (Smith) and 100 µg/ml against
MRSA (ATCC 33591). Pang et al. (2014) also reported MICs of 10
The bacteriostatic synergy and potentiating effects of the four compounds from M. alba against 4 pathogenic bacteria, in which
compounds on the antibacterial agents made some of the MRSA isolates compounds 2–4 showed 50.0 (114), 6.3 (14.9) and 25.0 (58.9) µg/ml
change from resistant (R) to susceptible (S) according to the CLSI cri- (µM) against MSSA (ATCC6538), respectively. We determined their
teria (CLSI, 2012). Significantly, AK combined with Cy, Ml, Mi and Ku MICs/MBCs of 1024/ > 1024, 16/64 and 4/32 µg/ml against ATCC
resulted in 8–10 of the ten isolates being changed to susceptible, with 25923 and MIC50/MBC50 of 512–1024/ > 1024, 8–32/32–128 and

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Table 3
Potentiation effects of various antibacterial agents in combination with the four prenylflavonoids (1–4) against the ten MRSA strains.

Combinationa Effectb MIC (µg/ml, n = 10) DRIc FICId Interaction (n′)e

Alone Combined s i

AK(Ml) Range 16–64(512–1024) 2–16(32–256) 16–4(32–4) 0.09–0.50 10 0

50% 32(1024) 8(64) 4(16) 0.31
90% 32(1024) 8(128) 4(4) 0.38
AK(Ku) Range 16–64(4–16) 0.125–16(1–4) 128–2(8–2) 0.26–1.00 7 3
50% 32(4) 4(2) 4(4) 0.50
90% 32(8) 8(2) 4(2) 0.63
AK(Cy) Range 16–64(8–16) 2–32(2–8) 8–2(8–2) 0.50–1.00 4 6
50% 32 (16) 8(4) 4(4) 0.63
90% 64(16) 16(8) 2(2) 1.00
AK(Mi) Range 16–64(8–32) 2–32(2–8) 16–2(8–2) 0.19–1.00 2 8
50% 32(16) 8(4) 2(4) 0.75
90% 32(32) 16(8) 2(2) 0.75
AM(Cy) Range 32–64(8–16) 4–16(2–16) 16–2(4–2) 0.50–1.50 2 8
50% 64(16) 16(4) 4(2) 0.75
90% 128(16) 32(8) 2(2) 1.00
CI(Ml) Range 32–256(512–1024) 8–32(16–128) 8–2(64–4) 0.14–0.52 9 1
50% 64(1024) 16(32) 8(32) 0.25
90% 128(1024) 32(128) 4(8) 0.38
EM(Ku) Range 4–8(4–16) 0.125–2(1–2) 64–4(8–2) 0.37–0.75 8 2
50% 4(4) 1(1) 4(4) 0.50
90% 8(8) 2(2) 4(2) 0.52
EM(Mi) Range 4–8(8–32) 0.5–4(1–8) 8–2(16–2) 0.25–0.75 3 7
50% 4(16) 1(4) 4(8) 0.56
90% 8(32) 4(8) 2(2) 0.75
GN(Cy) Range 16–32(8–16) 4–16(1–4) 4–2(8–4) 0.37–0.75 2 8
50% 16(16) 8(2) 2(8) 0.63
90% 32(16) 8(4) 2(4) 0.75
LE(Cy) Range 8–64(8–16) 2–16(0.5–16) 32–2(16–1) 0.37–1.50 1 9
50% 32(16) 4(8) 4(2) 0.75
90% 32(16) 8(16) 2(1) 1.06
P/S(Cy) Range 128–256(8–16) 32–128(1–16) 4–2(8–1) 0.500–1.500 3 7
50% 128(16) 64(4) 2(4) 0.63
90% 256(16) 128(16) 2(1) 0.75
PV(Cy) Range 32–128(8–16) 2–64(1–16) 64–2(16–1) 0.50–1.02 3 7
50% 64(16) 16(2) 4(4) 0.63
90% 128(16) 64(16) 2(1) 1.02
SM(Ml) Range 32–128(512–1024) 8–32(16–256) 8–4(32–4) 0.19–0.50 10 0
50% 64(1024) 16(32) 4(16) 0.28
90% 128(1024) 16(256) 4(4) 0.50
TP(Ml) Range 0.5–4(512–1024) 0.125–2(16–256) 16–2(64–4) 0.12–0.75 5 5
50% 1(1024) 0.25(64) 4(16) 0.50
90% 4(1024) 2(128) 2(4) 0.63
VA(Ml) Range 0.25–2(512–1024) 0.063–0.5(8–64) 16–2(64–16) 0.12–0.56 7 3
50% 1(1024) 0.25(32) 4(16) 0.31
90% 1(1024) 0.5(64) 2(16) 0.56
VA(Cy) Range 0.25–2(8–16) 0.25–1(0.5–8) 2–1(32–2) 0.56–1.06 0 10
50% 0.5(16) 0.5(1) 2(16) 0.75
90% 1(16) 1(8) 1(2) 1.00

a
AK: amikacin, AM: ampicillin, CI: ciprofloxacin, EM: etimicin, GN: gentamicin, LE: levofloxacin, PV: penicillin, P/S: piperacillin-sulbactam, SM: streptomycin, TP: teicoplanin, VA:
vancomycin; Cy: cyclocommunol (1); Ml: morusinol (2); Mi: morusin (3); Ku: kuwanon E (4). “AK(Cy)” means amikacin was used in combination with cyclocommunol, and the same
below. All data in the parentheses are designated to the compounds, and the data outside are designated to the antibacterial agents, respectively.
b
50% and 90%: values of inhibition against 50% and 90% of the 10 MRSA strains, respectively.
c
DRI: dose reduction index = MICAlone/MICCombined, ranged from the maximal to the minimal.
d
FICI (of A combined with B )= ((MICA)Combined/(MICA)Alone) + ((MICB)Combined/(MICB)Alone); FICI ≤ 0.5, synergy (s); 0.5 < FICI ≤ 4, indifference (i).
e
n′: Number of MRSA strains showing the interactions.

4–16/16–128 µg/ml against MRSA (n = 10) (Table 1). Compounds 1–3
are all prenylated and 4 is geranylated (or dimeric prenylated). How-
ever, the modified C12]C13 double bond (i.e. hydroxylated at C13 and
broken double bond) in 2 led to a great decrease of its activity com-
pared to that of compound 3 used alone (Table 1), which is similar to
the case in the previous report (Ngoupayo et al., 2009).
Before evaluation the synergistic actions, an initial combinatory
trial of potential conventional antibacterial agents was carried out
using disk diffusion test on a MHA plate. We finally selected the 11
agents to investigate their synergism. However, limited by the lack of
sufficient amount of compound 4, we could not have yet an in-depth
study of its potential effects on GN, TP, trimethoprim/sulfamethoxazole
and nitrofurantoin. Similarly, the potential synergism of 1 with mino-
cycline and netilmicin, and 3 with nitrofurantoin is also remained to be
ascertained.
We provide in this report the novel results of the synergy of anti-
bacterial agents of different groups of conventionally used in clinic with
plant compounds through the values of FICI ≤ 0.5. Odds (2003)
pointed out that it is not rational for authors to make fine-scale inter-
pretations of data from FICI experiments, such as additivity by 0.5 <
FICI ≤ 1, and indifference by 1 < FICI < 2 (Zuo et al., 2012), con-
sidering the possible reproducibility errors by three-dilution range
(mode ± 1 dilution) in a MIC chequerboard experiment. Thus the FICI
interpretation was suggested as ‘synergy’ when (FICI ≤ 0.5),

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Fig. 2. Time-kill curves of the synergistic effect of the nine combinations at 1 × MIC (alone) concentration of AK(Cy) (A), GN(Cy) (B), AK(Ml) (C), SM(Ml) (D), CI(Ml) (E), VA(Ml) (F), TP
(Ml) (G), AK(Mi) (H) and EM(Mi) (I) against a clinical MRSA strains of SCCmec III type. All the combinations showed only additive effect, for the △LC24 each was determined as <2
log10CFU/ml. The last line chart in (J) presents the killing effects of (8–1/4) × MIC of Cy used alone, respectively. Results are mean of triplicate samples.

Table 4 The reason why we chose these antimicrobial agents for combina-
The CLSI MIC interpretative criteria and susceptibility spectrum of MRSA strains to the tory evaluation is also that MRSA is multi-drug resistant to the anti-
antibacterial agents in combination with the four prenylflavonoids (1–4).
microbial agents in the previous reports (Zarfel et al., 2013; Zuo et al.,
Agenta MIC interpretative criteria (µg/ml)b 2012). We demonstrated that the anti-MRSA and -MSSA activities of the
four conventional groups of clinical used antibacterial agents (β-lac-
S I R tams, aminoglycosides, quinolones and glycopeptides) can be effec-
tively increased through their combination with the four prenylated
AK ≤16 32 ≥64
(Cy;Ml;Mi;Ku)c (9;10;8;10)c (1;0;2;0) (0;0;0;0)
flavonoids in different degrees. The most effective group is that of
AM ≤0.25 – ≥0.5 aminoglycosides which showed synergy with Ml and Ku against most of
(Cy) (0) (0) (10) the tested MRSA isolates though the alone used Ml was not active
CI ≤1 2 ≥4 (Tables 2–3). It is worth to take into account that Cy and Mi that
(Ml) (0) (0) (10)
showed high activity alone, had low activities when tested in combi-
GN ≤4 8 ≥16
(Cy) (1) (8) (1) nation (Tables 1 and 3), while the difference is relatively not so sig-
EMd (≤1) – (≥1) nificant in the synergism against MSSA (Table 2). The multiple sy-
(Mi;Ku) (6;7) (0;0) (4;3) nergistic effects of the four prenylated flavonoids (1–4), especially Cy
LE ≤1 2 ≥4 and Ml, on different groups of antibacterial agents must be owing to
(Cy) (0) (4) (6)
P/S (≤16) (-) (≥16)
their multiple mechanisms of action on MRSA, including the bacterial
(Cy) (0) (0) (10) cell barrier permeability, efflux pump inhibition and the inhibition of
PV ≤0.12 – ≥0.25 bacterial enzymes (Wagner and Ulrich-Merzenich, 2009).
(Cy) (0) (0) (10) Mun et al. (2014) studied the morphological changes when the
SM (≤8) – (≥8)
MRSA strains were treated with a prenylated flavonoid sophora-
(Ml) (3) (-) (7)
TP ≤8 16 ≥32 flavanone B (SPF-B). They found SPF-B could directly bind with the
(Ml) (10) (0) (0) pathogens’ PGN. Moreover, it was reported previously that cell wall
VA ≤2 4–8 ≥16 thickening was associated with adaptive resistance to AK in MRSA
(Cy;Ml) (10;10) (0;0) (0;0) clinical isolates (Yuan et al., 2013). Therefore, one aspect of the anti-
a bacterial mechanisms of the present four prenylated flavonoids (1–4)
AK: amikacin, AM: ampicillin, CI: ciprofloxacin, EM: etimicin, GN: gentamicin, LE:
levofloxacin, PV: penicillin, P/S: piperacillin-sulbactam, SM: streptomycin, TP: teico- might also be associated with interference of the PGN of S. aureus,
planin, VA: vancomycin;. Cy: cyclocommunol (1); Ml: morusinol (2); Mi: morusin (3); Ku: especially MRSA, which made the drug, such as AK, more easily pe-
kuwanon E (4). netrate through the cell wall and reach the target of action. It might as
b
S: susceptible; I: intermediate: R: resistant. well be the reason why the MRSA resistance to AK could be reversed by
c
The numbers in parentheses are the number of MRSA strains which showed S, I or R
1–4. This speculation may also partly explain the differences of anti-
to antibacterial agents in the combinations corresponding to the left column. A total of 10
MSSA/MRSA activities between Ml and Mi because of their different
MRSA strains were evaluated.
d
P/S, EM and SM have no MIC criteria in the CLSI standard and we took the MIC cells’ permeability. It has been noted that the prenylated flavonoids are
results against ATCC 25923 for the comparison. more hydrophobic than common flavonoids; they may easily penetrate
through the cell barrier, which facilitates the compounds targeting the
‘antagonism’ (FICI > 4.0) and ‘no interaction’ (FICI > 0.5–4.0). El. We active site (Sohn et al., 2004; Ngoupayo et al., 2009). Therefore, the
also calculated the DRI of each agent or compound in a combination free hydroxyl and prenyl-like substituents might be the important co-
(Tables 2–3) (Chou, 2006). factors of anti-MSSA and -MRSA of compounds 1–4 both used alone and

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G.-Y. Zuo et al.
in combination with antibacterial agents.
Conclusion
We revealed that four groups of antibiotics including eleven con-
ventional antibacterial agents showed synergistic effects in combina-
tion with the prenylated flavonoids 1–4 against MRSA and MSSA by
varying degrees, especially the prominent synergy exhibited by mor-
usinol (2) and Kuwanon E (4). The MRSA resistance to aminoglycosides
(AK, EM and SM) could even be reversed by 1–4. The findings could be
valuable for the development of new antibacterial drugs and the sy-
nergists against MRSA infection in order to overcome the problematic
multidrug-resistance.
Conflict of interest
We wish to confirm that there are no conflicts of interest associated
with this publication and there has been no significant financial support
for this work that could have influenced its outcome.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (NSFC, 81173504) and the supporting fund of
Yunnan Province of China (2008PY001). We are also grateful to
Kunming Institute of Botany (CAS) for spectral analysis.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.phymed.2017.12.023.
References
Chan, E.W.C., Lye, P.Y., Wong, S.K., 2016. Phytochemistry, pharmacology, and clinical
trials of Morus alba. Chin. J. Nat. Med. 14, 17–30.
Chen, L., Cheng, X., Shi, W., Lu, Q., Go, V.L., Heber, D., Ma, L., 2005. Inhibition of growth
of Streptococcus mutans, methicillin-resistant Staphylococcus aureus, and vancomycin-
resistant Enterococci by kurarinone, a bioactive flavonoid isolated from Sophora fla-
vescens. J. Clin. Microbiol. 43, 3574–3575.
Chin, J.N., Jones, R.N., Sader, H.S., Savage, P.B., Rybak, M.J., 2008. Potential synergy
activity of the novel ceragenin, CAS-13, against clinical isolates of Pseudomonas
aeruginosa, including multidrug-resistant P. aeruginosa. J. Antimicrob. Chemother. 61,
365–370.
Chou, T.C., 2006. Theoretical basis, experimental design, and computerized simulation of
synergism and antagonism in drug combination studies. Pharmacol. Rev. 58,
621–681.
Clinical and Laboratory Standards Institute, 2012. Table 2C. Zone diameter and MIC
interpretive standards for Staphylococcus spp. Performance standards for anti-
microbial susceptibility testing. Twenty Second Informational Supplement. Approved
Standard (M100–S22). CLSI, Wayne, PA.
Geng, C., Yao, S., Xue, D., Zuo, A., Zhang, X., Jiang, Z., Ma, Y., Chen, J., 2010. New
isoprenylated flavonoid from Morus alba. Chin. J. Chin. Mat. Med. 35, 1560–1565.
Hetem, D.J, Derde, L.P.G., Empel, J., Mroczkowska, A., Orczykowska-Kotyna, M.,
Kozi´nska, A., Hryniewicz, W., Goossens, H., Bonten, M.J.M., on behalf of the MOSAR
WP3 Study Group, 2016. Molecular epidemiology of MRSA in 13 ICUs from eight
European countries. J. Antimicrob. Chemother. 71, 45–52.
99
Phytomedicine 39 (2018) 93–99
Iten, F., Saller, R., Abel, G., Reichling, J., 2009. Additive antimicrobial [corrected] effects
of the active components of the essential oil of Thymus vulgaris—chemotype carva-
crol. Planta Med. 75, 1231–1236.
Kim, J.Y., Lee, W.S., Kim, Y.S., Curtis-Long, M.J., Lee, B.W., Ryu, Y.B., Park, K.H., 2011.
Isolation of cholinesterase-inhibiting flavonoids from Morus ihou. J. Agric. Food
Chem. 59, 4589–4596.
Klepser, M.E., Wolfe, E.J., Jones, R.N., Nightingale, C.H., Pfaller, M.A., 1997. Antifungal
pharmacodynamic characteristics of fluconazole and amphotericin B tested against
Candida albicans. Antimicrob. Agents Chemother. 41, 1392–1395.
Klepser, M.E., Wolfe, E.J., Pfaller, M.A., 1998. Antifungal pharmacodynamic character-
istics of fluconazole and amphotericin B against Cryptococcus neoformans. J.
Antimicrob. Chemother. 41, 397–401.
Konno, C., Oshimam, Y., Hikino, H., 1977. Morusinol, isoprenoid flavone from Morus root
barks. Planta Med. 32, 118–142.
Ku, Y.L., Liang, S.T., Lin, Y.H., Chen, L.H., Kuo, Y.Y., 2010. Anti-bacterial use of extract
from Morus australis poir and compound kuwanon H. US2010/166898A1, 2010, 1.
Li, M., Du, X., Villaruz, A.E., Diep, B.A., Wang, D., Song, Y., Tian, Y., Hu, J., Yu, F., Lu, Y.,
Otto, M., 2012. MRSA epidemic linked to a quickly spreading colonization and
virulence determinant. Nat. Med. 18, 816–819.
Lin, C.N., Shieh, W.L., 1992. Pyranoflavonoids from Artocarpus communis. Phytochemistry
31, 2922–2924.
Mazimba, O., Majinda, R.R.T., Motlhanka, D., 2011. Antioxidant and antibacterial con-
stituents from Morus nigra. Afr. J. Pharm. Pharmacol. 5, 751–754.
Mun, S.H., Joung, D.K., Kim, S.B., Park, S.J., Seo, Y.S., Gong, R., Choi, J.G., Shin, D.W.,
Rho, J.R., Kang, O.H., 2014. The mechanism of antimicrobial activity of sophora-
flavanone B against methicillin-resistant Staphylococcus aureus. Foodborne Pathog.
Dis. 11, 234–239.
Mun, S.H., Kang, O.H., Joung, D.K., Kim, S.B., Seo, Y.S., Choi, J.G., Lee, Y.S., Cha, S.W.,
Ahn, Y.S., Han, S.H., 2013. Combination therapy of sophoraflavanone B against
MRSA: in vitro synergy testing. Evid. Based Compl. Alt. 2013, 823794.
Ngoupayo, J., Tabopda, T.K., Ali, M.S., 2009. Antimicrobial and immunomodulatory
properties of prenylated xanthones from twigs of Garcinia staudtii. Bioorg. Med.
Chem. 17, 5688–5695.
Nomura, T., Fukai, T., 1978. Kuwanon E, a new flavanone derivatives from the root bark
of the cultivated Mulberry tree (Morus alba L.). Heterocycles 9, 1295–1300.
NUTCM, 2005. Nanjing University of Traditional Chinese Medicine (NUTCM) (Ed.), 2005.
Dictionary of Chinese Crude Drugs, second ed. Shanghai Scientific Technologic
Publisher, Shanghai, China, pp. 2786–2787.
Odds, 2003. Synergy, antagonism, and what the chequerboard puts between them. J.
Antimicrob. Chemother. 52, 1.
Pang, D., Liu, F., Shi, Y., Liu, J., Shen, W., Zou, Y., Liao, S., Xiao, G., 2014. Antibacterial
activity of 10 phenolic compounds from mulberry. J. Chin. Pharm. Univ. 45,
221–226.
Sakagami, Y., Mimura, M., Kajimura, K., Yokoyama, H., Linuma, M., Tanaka, T., Ohyama,
M., 1998. Anti-MRSA activity of sophoraflavanone G and synergism with other an-
tibacterial agents. Lett. Appl. Microbiol. 27, 98–100.
Sohn, H.Y., Son, K.H., Kwon, C.S., Kwon, G.S., Kang, S.S., 2004. Antimicrobial and cy-
totoxic activity of 18 prenylated flavonoids isolated from medicinal plants: Morus
alba L., Morus mongolica Schneider, Broussnetia papyrifera (L.) Vent. Sophora flavescens
Ait and Echinosophora koreensis Nakai. Phytomedicine 11, 666–672.
Wagner, H., Ulrich-Merzenich, G., 2009. Synergy research: approaching a new generation
of phytopharmaceuticals. Phytomedicine 16, 97–110.
Yuan, W., Hu, Q., Cheng, H., Shang, W., Liu, N., Hua, Z., Zhu, J., Hu, Z., Yuan, J., Zhang,
X., 2013. Cell wall thickening is associated with adaptive resistance to amikacin in
methicillin-resistant Staphylococcus aureus clinical isolates. J. Antimicrob.
Chemother. 68, 1089–1096.
Zarfel, G., Krziwanek, K., Johler, S., Hoenigl, M., Leitner, E., Kittinger, C., Masoud, L.,
Feierl, G., Grisold, A.J., 2013. Virulence and antimicrobial resistance genes in human
MRSA ST398 isolates in Austria. Epidemiol. Infect. 141, 888–892.
Zhang, K., Mcclure, J.A., Elsayed, S., Louie, T., Conly, J.M., 2005. Novel multiplex PCR
assay for characterization and concomitant subtyping of staphylococcal cassette
chromosome mec types I to V in methicillin-resistant Staphylococcus aureus. J. Clin.
Microbiol. 43, 5026–5033.
Zuo, G.Y, Jing An, Y., Han, J., Zhang, Y.L., Wang, G.C., Hao, X.Y., Bian, Z.Q., 2012.
Isojacareubin from the Chinese Herb Hypericum japonicum: potent antibacterial and
synergistic effects on clinical methicillin-resistant Staphylococcus aureus (MRSA). Int.
J. Mol. Sci. 13, 8210–8218.