SUPPLEMENTARY MATERIAL
The antibacterial activity of phytochemically characterized fractions
from Syringae Folium
Zhengyuan Zhou 1, Na Han 1, Zhihui Liu 1, Jinling Wang 1, Huanzhang Xia 2, Dezu Miao 3, Wei Li 3,
Jun Yin *1
Affiliation
1
Department of Pharmacognosy and Utilization Key Laboratory of Northeast Plant Materials, School of Traditional Chinese
Medicine, Shenyang Pharmaceutical University, Shenyang, 110016, China
2
Department of Microbiology, School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University,
Shenyang, 110016, China
3
Shandong Reyoung Pharmaceutical Co., Ltd., Yiyuan, 256100, China
Abstract
To identify the most active antimicrobial fraction of Syringae Folium (FS), four common pathogens
were used in an in vitro screening. The results showed that the combination of the 30% and 60%
ethanol fraction (FSC) obtained from the water extraction was the most active fraction with a minimal
inhibitory concentration (MIC) of 0.65 mg·ml-1. FSC was also found to be able to protect mice from a
lethal infection of Staphylococcus aureus at the clinical dosage (0.2 g·kg-1) with a survival rate of
83.3%. The antibacterial activity of FSC was then tested using the serum pharmacology method which
revealed that FSC exhibits a more long-lasting activity than the positive control (levofloxacin
hydrochloride, LH). The main components were confirmed to be iridoid glycosides and flavones by
HPLC-MS analysis.
Animals
Equal numbers of female and male Kunming mice weighing 20-25 g were used. Male SD rats weighing
200-220 g were also used. All of the experimental animals were purchased from the Pharmacology
Experimental Center of Shenyang Pharmaceutical University. All of the mice and rats were
acclimatized for one week in a light- and temperature-controlled room with a 12-h dark/12-h light cycle.
Rodent laboratory chow pellets and tap water were supplied ad libitum. The animal care and protocols
used in this study were approved by the Institutional Animal Ethics Committee of Shenyang
Pharmaceutical University. (Number: SLXK (BJ) 2012-0604).
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Chemicals and reagents
The levofloxacin hydrochloride and sodium chloride injections were purchased from Guang Dong P.D.
Pharmaceutical Co., Ltd. (0.3 g of LH and 0.9 g of NaCl in 100 ml). The plant materials used in this
study were purchased from Baishan Medicinal Materials Company in Jilin Province, China and were
identified by Professor J. Yin as dry leaves of Syringa oblate. A voucher specimen (No. ZDX-10) was
deposited in the laboratory of the Department of Pharmacognosy of Shenyang Pharmaceutical
University.
Syringopicroside,
keampferol,
luteolin
standards
were
prepared
by ourselves
(purity>95.0%).
Preparation of plant extracts and fractions
The air-dried FS (200 g) was ground into powder and extracted twice with water (1 l, reflux, 2 h). The
extract was evaporated to dryness under reduced pressure at 55°C, and 59.7 g of the water extract (WE)
was obtained. The WE (20 g) was dissolved in water and added to 95% ethanol-water (v/v) to obtain a
final EtOH concentration of 70% (v/v). The supernatant was evaporated under reduced pressure at
55°C to obtain 12.1 g of crudely purified extract, denoted purified extract (PE). 10g of PE were
dissolved in water at a concentration of 1.06 g/ml and subjected to chromatographic separation on an
D101 macroporous adsorption resin column and eluted with 15%(v/v), 30% (v/v), 60% (v/v), and 90%
(v/v) EtOH/H2O to obtain four fractions (F15, 0.4 g; F30, 1.2 g; F60, 5.2 g; F90, 0.2 g). The
combination of F30 and F60 at a mass ratio of 1.2:5.2, which was denoted FSC, was also used.
Bacterial stains and culture conditions
All of the bacteria strains used in this study, namely Staphylococcus aureus ATCC 6538, Salmonella
Typhimurium ATCC 14028, Pseudomonas aeruginosa ATCC 9027, and Shigella dysenteriae CMCC
(B) 51252, were obtained from stock cultures preserved at -75°C at the Microbiology Department. The
stains were used to the test minimum inhibitory concentrations (MICs) of the potential effective
fractions and were cultured overnight in nutrient broth (NB) or broth agar medium (NAM) at 37°C and
stored at 4°C.
Broth micro dilution assay for determination of the MIC
Minimal Inhibitory Concentrations (MICs) tests were conducted using the broth-dilutions method
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(Stalons and Thomsberry 1975). PE, F30, F60, and FSC were used as the samples in the test. Equal
volumes of each bacterial strain culture, which contained approximately 106 CFU/ml, were applied to
sterile NB supplemented with multiple proportion dilutions of PE, F30, F60, and FSC at concentrations
ranging from 66.5 mg·ml-1 to 0.002 mg·ml-1 in tubes. These serially diluted cultures were then
incubated at 37°C for 18 h. The MIC was defined as the lowest concentration of each sample that
completely suppressed colony growth.
In vivo antibacterial experiment
The mice were randomly divided into five groups with 10 mice in each group. The mice in the
medicated group received the FSC fraction twice a day. The oral administration dose of the high-dose
group was 0.2 g·kg-1 (in dry extraction), whereas the middle-dose and the low-dose groups received
half and a quarter of the high dose, i.e., 0.1 g·kg-1 and 0.05 g·kg-1, respectively. The positive control
groups were administered LH based on a clinical dose of 0.036 g·kg-1, and the blank group received
normal saline. The administration lasted for seven days. On the 3rd and 4th days, all of the mice were
intraperitoneally injected with Staphylococcus aureus (109 CFU·ml-1) at doses of 0.1 ml·10 g-1 and 0.15
ml·10 g-1, respectively. The survival of each group was then calculated on the 7th day.
Preparation of FSC metabolite-containing serum
Thirty rats were randomly divided into five groups with six rats in each group. The rats were fasted for
12 h before the experiment. The rats in the blank control group were given 0.5% sodium
carboxymethylcellulose (CMC-Na) at a dose of 10 ml/kg. The three medicine groups were orally
administered FSC in 0.5% CMC-Na at dosages of 10.9, 21.9, and 43.8 mg/kg (of the crude drug). The
rats in the positive control group were given LH at a dosage of 80 mg·kg-1. Blood samples were
collected from orbita of the rats at 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, and 10 h after administration and
centrifuged immediately at 4000 r/min. One milliliter of methanol was added to the supernatant to
precipitate the protein. After vortexing, the serum suspensions were centrifuged at 13,000 rpm. The
final supernatants were dried with sterile nitrogen gas, resolved with sterile water, and stored at 4°C.
The serum samples were also filtered through a 0.22-μm microfiltration membrane for disinfection
before the antibacterial assay.
K-B method for assaying the antibacterial effect of FS-containing serum
This assay was performed using the paper disc diffusion method (Awadh et al. 2001) The bacterial
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strains grown in NB at 37°C for 18 h were diluted with saline solution (0.85% NaCl) to a turbidity of
106 CFU·ml-1. The bacterial suspension was inoculated into NAM to obtain a bacterium mixing
medium and poured into Petri plates. A sterile paper disc (diameter = 6 mm) filled with 30 μl of the
sample per disc was placed on the surface of the agar after it was thoroughly cooled to solid. The plates
were then incubated at 37°C for 24 h. The antibacterial activities were evaluated by observing the
bacterium growth. The experiments were conducted in triplicate. The diameter of the inhibition zone in
each of the discs was measured and recorded at the end of the incubation period.
Qualitative HPLC-MS analysis of the active fraction
The qualitative analysis of the active fraction (FSC) was performed using an Agilent series 1100 HPLC
system combined with a mass selective detector and a XUnion C18 column (150 mm × 4.6 mm, 5 μm).
A mobile phase of methanol and 0.1% formic acid solution (37:63, v/v) was used at a flow rate of 1.0
ml/min throughout the run. The flow rate was set to 0.2 ml/min, and the column temperature was
maintained at 25°C. By investigating the full-scan mass spectra of FSC, we found that the signal in the
negative mode was much higher than that obtained in the positive ion mode. The mobile phase was
selected to optimize the separation, and formic acid (0.1%) was chosen to promote the ionization of the
constituents The mass spectrometer was run in the negative scan mode and used to detect a mass range
of m/z 100 to 2000. (Thermo LCQ Fleet)The ion spray voltage and capillary voltage were set to 4.0 kV
and -30 V, respectively, and the capillary temperature was maintained at 275°C. The flow rates of
auxiliary gas and sheath gas were set to 5 l·h-1 and 30 l·h-1, respectively.
Figure Captions:
Fig.S1 Survival rate of the Staphylococcus aureus-infected mice. The mice in the control group were
given normal saline (i.g.). The mice in the positive group were administered levofloxacin hydrochloride
(LH) at a dose of 0.012 g·kg-1 (i.g.). The mice in the FSC groups mice received FSC at doses of 0.05
g·kg-1, 0.1 g·kg-1, or 0.2 g·kg-1.
Fig.S2 Dose-response relationship of FSC-containing serum obtained from the blood collected 1 h after
injection. The FSC was suspended with 0.5% CMC-Na. The dosage design of the FSC suspensions was
10.9, 21.9, and 43.8 mg·kg-1 with respect to the crude drug.
Fig.S3 Time-effect relationships of the high dosages of the FSC- and LH-containing serums against
Staphylococcus aureus (a), Salmonella Typhimurium (b), and Shigella dysenteriae (c).
Fig.S4 Total ion current (TIC) chromatogram of FSC. Syringopicroside(1);Keampferol(2); Luteolin(3).
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1
Table S1. HPLC-MS data for identified compounds of FSC
Compound
tR (min)
MW
Syringopicroside
18.62
494
Structures
ESI- MS539.49 [M+COOH]493.98 [M-H]-
Keampferol
26.10
286
331.53 [M+COOH]377.03[M+HCOOH
+COOH]-
Luteolin
28.70
286
331.46 [M+COOH]377.01[M+HCOOH
+COOH]-
2
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