SUPPLEMENTARY MATERIAL
Anti-infective potential of caffeic acid and epicatechin 3-gallate isolated
from methanol extract of Euphorbia hirta (L.) against Pseudomonas
aeruginosa
S. Perumal a*, R. Mahmuda and S. Ramanathanb
a
School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia.
b
Centre for Drug Research, Universiti Sains Malaysia, 11800 Penang, Malaysia.
*
Corresponding author. Email: angelpriya30@hotmail.com
Euphorbia hirta (L.) plant is traditionally used in Malaysia for the treatment of
gastrointestinal, bronchial and respiratory ailments caused by nosocomial infectious
agents. Bioactivity-guided fractionation of methanol extract of E. hirta aerial part and
analysis using High Performance Liquid Chromatography (HPLC) has led to the
isolation of two antibacterial compounds. These compounds were identified as caffeic
acid (CA) and (-)-epicatechin 3-gallate (ECG) based on spectroscopic analyses and
comparison with previously published data. Using broth microdilution method, both
ECG and CA had demonstrated significant minimum inhibitory concentration (MIC)
of 16 µg/ml and 31 µg/ml respectively, against P. aeruginosa. Time-kill assessment
of ECG and CA displayed bactericidal effect on P. aeruginosa cells.
Keywords: Euphorbia hirta (L.); Pseudomonas aeruginosa; antibacterial activity;
epicatechin 3-gallate; caffeic acid
1. Experimental
1.1. Plant collection and authentication
The fresh aerial parts of Euphorbia hirta (L.) were obtained from Relau, Penang City,
Malaysia. The plant was collected during the period of March to April 2013. No specific
permissions were required for the field studies described. The location of the field study is
not privately owned and did not involve endangered or protected species. The plant was
authenticated by Mr. Shunmugam, the botanist of the School of Biological Sciences,
Universiti Sains Malaysia, where a voucher specimen (No.11254) was deposited in the
Herbarium Unit of the school.
1.2. Extraction of Euphorbia hirta (L.)
The air-dried aerial part of E. hirta (250 g) were ground to mesh size No.40 and macerated
solely with methanol by ratio of 1 g of ground plant material in 10 ml of methanol. The
extraction was done for three days and this process was repeated three times on the marc. The
filtered extracts obtained were evaporated under vacuum and later freeze-dried to afford
crude methanol extract (29.7 g). Subsequently the crude methanol extract was subjected to
solid- phase extraction (SPE) to remove chlorophyll. SPE cartridges (Supelco, Bellefonte,
USA) containing C18 silica gel (50 µm particle size) were pre-conditioned with methanol and
water. Cartridges were eluted with methanol: water to retain chlorophyll compounds in the
C18 silica gel whilst the eluate were collected, filtered, evaporated and eventually freeze dried
to remove any residual water. Approximately 19.6 g of chlorophyll free crude methanol
extract was obtained and stored at 4 oC prior to further analysis.
1.3. Bacterial strain
Isolates of Pseudomonas aeruginosa were obtained from clinical specimens submitted to the
Department of Medical Microbiology and Parasitology (JTMP), School of Medical Sciences,
Universiti Sains Malaysia. Species identification was confirmed by biochemical test using
API (analytical profile index) system. Isolates were stored in tryptic soy broth containing 20
% glycerol and frozen at -80 oC. Isolates were subcultured a minimum of three times prior to
experimental bioassays and mechanism studies.
1.4. Inoculum preparation
P. aeruginosa culture was recovered on a fresh tryptic soy agar (Difco, USA) plate, 24 h
prior to antimicrobial test. To prepare the standardized inoculum, four to five well isolated
colonies of microorganism with similar morphological type were selected from an agar plate
culture. Each colony was transferred with sterile swab into a tube containing 5 mL of sterile
cation-adjusted Mueller Hinton broth (CAMHB) (HiMedia, India) to obtain a liquid
suspension that matches to McFarland 0.5 turbidity standard. For the microdilution test, the
prepared standardized inoculum was immediately diluted 1:100 using CAMHB to yield 1 x
106 CFU/mL. The subsequent 1:2 dilution in tetrazolium microplate assay will bring the final
inoculum or test concentration of bacteria to 5 x 105 CFU/ml (1 x 105 CFU/well). The final
cells concentration was confirmed by viable counts.
1.5. Determination of minimum inhibitory concentration (MIC) and minimum
bactericidal concentration (MBC)
The minimum inhibitory concentrations (MIC) of E.hirta were determined by using
tetrazolium microplate assay which were slightly modified from serial broth microdilution
method as described by Eloff (2004). This assay was performed using flat-bottomed
polystyrene 96-well clear microtitre plates (Greiner Bio-one, Germany). In short, the varying
polarity extracts of E. hirta were dissolved in Tween 40 (Sigma-Aldrich, Germany) and an
identical two-fold serial dilution using MH broth (Difco, USA) were made to form 2.0 0.031 mg/mL. One hundred microlitres of bacterial inoculum was added and mixed
thoroughly in all the wells (1.0 - 0.015 mg/mL as the highest in-test concentration). The
microtitre plates were incubated overnight at 37 °C. An appropriate mixture of solvent Tween
40, medium and inoculum were included as drug-free control and the final concentration of
Tween 40 in the well was ensured to be less than 1 % (v/v). Clinically established antibiotic,
cefepime (2.0-125 µg/mL) was used in parallel experiment as a positive drug control. An
additional non-infected medium was included as growth control. The MIC of E. hirta extract
was detected following addition of 50 μL of INT (2-4-Iodophenyl-3-4-nitrophenyl-5-phenyl2H-tetrazolium chloride) at a final concentration 0.2 mg/mL in all the wells and incubated for
further 30 min at 37 °C. Bacterial growth was determined by observing the color change of
INT in the microplate wells. MIC was defined as the lowest extract concentration that
completely inhibits the growth of microorganisms. For the determination of minimum
bactericidal concentration (MBC), 20 μL of culture medium from the microtitre plate wells
that showed no changes in color will be re-inoculated on MH agar plates (Difco, USA). After
24 h of incubation at 37 oC, MBCs were determined as the lowest concentration that yielded
nil bacterial growth on MH agar plates. The MIC and MBC determination were performed
three times in duplicate.
1.6. Bioactivity guided fractionation and isolation
The fractionation of the active crude methanol extract was carried out by gravity column
chromatography (CC). A sintered glass column measuring exact height of 60 cm and width of
6 cm attached to the reservoir with a capacity of 1 L was used to pack the stationary phase.
The column chromatography fractionation was performed on silica gel (70-230 mesh). The
column was packed by using 30 g of silica gel per 1 g of the extract to be separated. The
methanol extract (19.6 g) which was found to be active against P. aeruginosa, with an MIC
value of 63.0 µg/mL was subjected to fractionation over a silica gel column (silica gel 60;
70-230 mesh), eluted with stepwise gradient mixture of n- hexane and acetone of increasing
polarity (9:1 to 4:5, then pure acetone). A total of 240 fractions (100 mL each) were
collected. The fractions were analyzed by thin layer chromatography (TLC) and identical
fractions with similar Rf values were merged to yield 7 fractions, denoted as M1-M7. All the
7 fractions were evaluated against P. aeruginosa, and M3 was observed to be the most active,
with MIC value of 63.0 µg/mL followed by M6 with MIC value of 125 µg/mL. Fraction M3
(2.73 g) was further chromatographed over silica gel column (height 60 cm, diameter 3 cm,
silica gel 60; 70-230 mesh) eluting with gradient mixture of dichloromethane and acetone
(9.9:0.1 to 9:1) to afford four subfractions (M3 – M304). The subfraction M302 (750 mg)
which showed potent antibacterial activity was subjected to passage over a Sephadex LH-20
column, eluted with dichloromethane and acetone (9.8 : 0.2) to yield 3 subfractions
(M30201–M30203). Subfraction M30202 (150 mg) was finally purified by preparative TLC
to afford one pure compound 1 (78 mg). Fraction M6 (980 mg) was repeatedly
chromatographed using silica gel column (height 60 cm, diameter 3 cm, 70-230 mesh) with a
gradient solvent systems of dichloromethane and acetone (9.9:0.1 to 9:1) to obtain 5
subfractions (M601 – M605). Subfraction M602 (210 mg) was applied to a Sephadex LH-20
column eluted with dichloromethane and acetone (9.6: 0.4) to yield subfraction M60201 (100
mg). Subfraction M60201 was further submitted to purification by preparative TLC to afford
pure compound 2 (83 mg).The purity of the two isolated antibacterial compounds verified by
HPLC- photodiode array was more than 95%.
The HPLC analysis had confirmed the presence of isolated compounds in the
methanol extract of E. hirta. The HPLC profile of crude methanol extract of E.hirta aerial
part exhibited numerous minor peaks and few abundant peaks (Figure S1). The presence of
the isolated antimicrobial compounds in the crude methanol extract was identified by
comparing their retention times. The retention times of compound 1 and 2 are as follows;
6.925 min and 10.613 min, respectively (Figure S2). These retention times were
corresponding with the retention time of 7.088 min and 10.653 min on HPLC profile of crude
methanol extract of E. hirta.
1.7. HPLC-DAD analysis
The analysis was performed using an Agilent 1260 Infinity HPLC instrument (Agilent
Technologies, Santa Clara, CA, USA). Separation was carried out on a reversed phase
column (Agilent Zorbax Eclipse Plus C18, 4.6 x 250 mm, 5 µm), guarded by a Agilent
Zorbax Reliance analytical cartridge guard column (4 x 80 mm, 5 µm). The HPLC mobile
phases composed of 10% (v/v) acetonitrile in water (solvent A) and and 55 % (v/v) of
acentonitrile in water (solvent B). Both solvent A and B were attuned to pH 3 with acetic
acid. The solvent gradient elution programme was as follows with total analysis time of 40
min: 0-30 min, linear gradient 15-75% B; 30-35 min, isocratic 100% B; 35-40 min, isocratic
15% B. The flow rate was at 1.0 mL/min. The sample injection volume and column
temperature were set at 20 µL and 30 oC, respectively. The DAD acquisitions were
implemented in the range 190-420 nm.
1.8. Ultra-Violet and Visible spectroscopy (UV-Vis) and Fourier Transform Infrared
Spectroscopy (FTIR)
The antibacterial active principles isolated from methanol extract of E. hirta were submitted
to UV-Vis and FTIR profiling. A Perkin-Elmer 45 UV-VIS spectrometer was operated to
collect UV-Visible spectra of the solution in the wavelength range of 200 to 700 nm against
methanol. For FTIR analysis, KBr discs produced were analyzed in the mid IR range 4000400 cm-1 using Perkin– Elmer System 2000 FTIR.
1.9. Electrospray ionisation-mass spectrometry (ESI-MS) and Nuclear Magnetic
Resonance spectroscopy (NMR) analysis
Characterization of the isolated antibacterial compounds from E. hirta was carried out by
direct inlet flow using ion trap mass spectrometer equipped with an ESI ion source, in
negative and positive ion mode (Agilent 1100 Series, Waldbronn, Germany). Direct infusion
was performed using a syringe pump of 500 µL volume at a flow rate of 10µL/min. The
following conditions were used: spray voltage 4.70 kV; capillary temperature 275 oC; sheath
gas flow 15 arbitary units (a.u.); auxiliary gas flow 21 a.u.; and sweep gas flow 0 a.u. For MS
analysis, mass spectrometer was scanned from 10 to 1000 m/z. The structures of the isolated
compounds were identified by NMR spectroscopy analysis. 1H-NMR and
13
C-NMR spectra
were recorded on a Bruker Avance III 500 and Bruker Ascend 500 MHz NMR (Fallenden,
Switzerland) spectrometer with TMS as internal standard. The identified compound
structures were confirmed by comparison with NMR spectral data with those available in the
literature.
1.10. Bactericidal activity of the isolated compounds
1.10.1. Antibacterial agent
Laborator -grade standard powder of cefepime (lot no: 341037; International Laboratory, San
Francisco, USA) was obtained and reconstituted according to the manufacturer’s instruction.
Stock solution of cefepime was freshly prepared on the day of the experiments. Cefepime
was required to pass quality control standard (Pseudomonas aeruginosa; ATCC 27853) on
the day of the experiment (Clinical and Laboratory Standards Institute, 2012).
1.10.2. Time-kill assay
The time-kill studies of isolated antibacterial compounds against P. aeruginosa were
performed in accordance with guidelines provided by the Clinical and Laboratory Standards
Institute (2012), formerly known as NCCLS (National Committee for Clinical Laboratory
Standards, 1992). Briefly, the test compounds were dissolved into flasks containing Mueller
Hinton broth at ½ x MIC, 1 x MIC and 2 x MIC. Suspension of P. aeruginosa (5 x 105
CFU/mL) was inoculated into the entire test and control flasks. Two control flasks, one
without test compounds but with inoculum (growth control) and another without inoculum
but with test compounds (sterility control) were included in this study. The flasks were
incubated at 37 oC and at selected time intervals of 0.5, 4, 8, 12 and 24 h of incubation period,
an aliquot of 1 mL was withdrawn from culture medium and performed 10-fold serial
dilution. An amount of 100 µL from each dilution was inoculated on Mueller Hinton agar and
the plates were incubated at 37 oC for 24 h. Colonies formed on the agar plates were
enumerated and CFU/mL was determined. The experiment was performed in triplicate. The
lower limit of detection was 300 CFU/mL. The results presented were the means of three
independent assays.
References
Clinical and Laboratory Standards Institute. 2012. Performance standards for antimicrobial
susceptibility testing; twenty-second informational supplement. CLSI document
M100-S22. Clinical and Laboratory Standards Institute, Wayne, Pa.
Eloff JN. 2004. Quantification the bioactivity of plant extracts during screening and bioassay
guided fractionation. Phytomedicine. 11:370-371.
Captions
Table S1. Minimum inhibitory concentrations (MIC) and minimum bactericidal
concentrations (MBC) of crude methanol extract and isolated compounds from Euphorbia
hirta (L.) against Pseudomonas aeruginosa.
Figure S1. HPLC-DAD chromatogram of crude methanol extract of Euphorbia hirta (L.)
aerial part at 260 nm. Two isolated antibacterial compounds were identified based on their
retention times. Compound 1 (7.088 min) and compound 2 (10.653 min).
Figure S2. HPLC-DAD chromatogram of compound 1 and compound 2 isolated from crude
methanol extract of Euphorbia hirta (L.) aerial part. (A) Retention time of compound 1
(6.925 min) and (B) compound 2 (10.613 min).
.
Table S1. Minimum inhibitory concentrations (MIC) and minimum bactericidal
concentrations (MBC) of crude methanol extract and isolated compounds from Euphorbia
hirta (L.) against Pseudomonas aeruginosa.
Samples
MIC
MBC
Methanol extract
63.0a
125.0
Compound 1
31.0
63.0
Compound 2
16.0
31.0
Cefepimeb
16.0
31.0
a
Values expressed are mean of three independent experiments (n=3); values given as µg/mL.
b
Cefepime (positive control).
Compound 1
Compound 2
Figure S1. HPLC-DAD chromatogram of crude methanol extract of Euphorbia hirta (L.)
aerial part at 260 nm. Two isolated antibacterial compounds were identified based on their
retention times. Compound 1 (7.088 min) and compound 2 (10.653 min).
(A)
(B)
Figure S2. HPLC-DAD chromatogram of compound 1 and compound 2 isolated from crude
methanol extract of Euphorbia hirta (L.) aerial part. (A) Retention time of compound 1
(6.925 min) and (B) compound 2 (10.613 min).