Contributed equally
Natural products have been optimized to interact with biological systems through a long natural selection process (
The air-dried, powdered fruit of sumac plant (25 g) was packed in an Erlenmeyer flask and extracted with 250 ml methanol (MeOH), sonicated for 2 h at 45°C, then left in a dark glass bottle at room temperature for 24 h for complete extraction. The methanolic extract was filtered and evaporated to dryness with a rotary vacuum evaporator.
A Waters Alliance e2695 separations module, 2998 photo diode array (PDA) and Empower version 3 software was used (Waters, Eschborn, Germany). The preparative HPLC system consisted of a 3535-quaternary gradient module and a 996 PDA detector.
1H-NMR and 13C-NMR measurements of isolated MG from sumac was carried out on a Bruker Avance II 500 spectrometer, which was equipped with a 5 mm indirect detection probe with Z gradient.
The UHPLC system (Accela; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was equipped with an XBridge ODS-column (150×2.1 mm i.d.), 3.5-µm particle size (Waters, Milford, MA, USA) and a mobile phase containing 0.1% formic acid (FA) as (eluent A) and MeOH containing 0.1% FA as (eluent B). Linear gradient elution was used starting with 95% A and continuously increased to 100% B in 20 min. The UHPLC system was coupled to LTQ Orbitrap XL system (Thermo Fisher Scientific, Inc.) equipped with an electrospray ionization (ESI) source. The positive ionization mode was used at a scan range of m/z 100–1000.
MG quantitation was run on a Waters HPLC ODS Column (XBridge, 4.6 ID ×150 mm, 5 µm) with a guard column (Xbridge ODS, 20×4.6 mm ID, 5 µm). The mobile phase consisted of water (labeled A) and acetonitrile (labeled B) solvent mixture. The gradient was as follows: 95% A and 5% B at 0 min, held there for 2 min, then raised to 50% A and 50% B over 13 min, then to 10% A and 90% B over 1 min, held there for 3 min, and finally returned to 95% A, 5% B over 1 min. All of the samples were filtered with a 0.45 µm micro-porous filter. The PDA wavelengths ranged from 210 to 500 nm, and the monitoring wavelength of MG was 272 nm. The flow rate was 1 ml/min. The injection volume was 10 µl, and the column temperature was at room temperature.
The UHPLC was equipped with an XBridge ODS-column (150×2.1 mm i.d.), 3.5 µm particle size (Waters) and a mobile phase containing 0.1% formic acid (FA) as (eluent A) and MeOH containing 0.1% FA as (eluent B). Linear gradient elution was used starting with 95% A and continuously increased to 100% B in 20 min.
The prep-HPLC experiments were run on an ODS column (Agilent PrepHT C18, 22.2×250 mm, 10 µm). The linear gradient started at 98% A, where it stayed for 3 min; it was then raised to 5% A over 15 min, where it remained for another 4 min and was then returned to 98% A over 2 min. The flow rate used was 15 ml/min, the injection volume was 1,000 µl.
Five different concentration levels of standards for MG were prepared: 10, 50, 100, 250 and 500 ppm in methanol diluents. These standards were used to construct a calibration curve to quantitate MG in sumac.
Crude sumac was prepared by dissolving ~3,000 mg in 15 ml of methanol, sonicated for 3 min and then filtered via a 0.45 µm membrane filter before injection. The concentration of the final red solution was 200 mg/ml. This solution (1 ml) was injected into the preparative HPLC Chromatograph and six fractions were collected.
To evaluate the effect of treatments,
After 24 h of incubation, THB was discarded from plates, wells were rinsed with distilled water to remove loosely bound bacterial cells, and then adherent bacteria were fixed with 95% ethanol. For quantification of biofilm biomass, the fixed and air-dried
Following 24 h of incubation, glass slides with adherent
Where
Where
Data were analyzed using SPSS version 23.0 (IBM Corp., Armonk, NY, USA). Differences between the control (untreated) and treatment groups were evaluated applying one-way analysis of variance followed by a post hoc least significant difference test for multiple comparisons. Data are presented as the mean ± standard error. P<0.05 was considered to indicate a statistically significant difference.
The primary assessment of crude MSE efficiency for the inhibition of
Sumac is very rich in phenolic and anthocyanin compounds. These classes of compounds are known to exhibit a range of biological activities. In the attempt to isolate the active ingredients from sumac, several analytical HPLC runs were performed using different mobile phases. Upon scaling up using preparative HPLC, separations were carried out by collecting six fractions, which were subjected to antibacterial assays. A typical preparative HPLC chromatogram of the crude methanol mixture and its corresponding overlaid UV-Vis spectra, in the range of 210–500 nm, are presented in
The ultraviolet-visible spectra demonstrated many phenolics that possess typical absorption maximums between 268.8 and 277.1 nm. There was also a very strong absorption maximum at 519.5 nm, which is a typical anthocyanin compound that is responsible for the red pigment in sumac (
Fraction III (
Accurate high-resolution mass spectroscopy (HR-MS) and 1H-NMR and 13C-NMR disclosed the identity of the peak at 7.88 min to be MG (
Analytical HPLC was used to construct a calibration curve of MG at 5 levels (10, 25, 50, 100, 250 and 500 ppm) with a coefficient of determination (
In an effort to evaluate anti-biofilm activity, the isolated MG (fraction III) was further tested on
The optical profilometry technique, applied in analysis of the surfaces of glass slides with
The pH measurements of the collected
The present study demonstrated that the dominant and most antibacterial active compound of
In conclusion, the present study demonstrated that
The present study was supported by the Al-Qasemi Research Foundation, the Ministry of Science, Technology and Space (Israel) and the Faculty of Medicine, Vilnius University (Lithuania).
Streptococcus mutans
methanolic sumac extract
methyl gallate
high-performance liquid chromatography
Streptococcus mutans biofilm biomass formed on the polystyrene surface after 24 h of incubation in THB containing 1% sucrose and different concentrations of MSE. Data are presented as the mean ± standard error from three independent experiments (n=4-11). *P<0.05 vs. control group. MSE, methanolic sumac extract.
A typical preparative high performance liquid chromatography chromatogram of a crude methanol mixture of sumac-1,000 µl was injected at a flow rate of 15 ml/min. The monitoring wavelength was 272 nm. The intervals of the fractions were as follows: I (1–8 min), II (8–9.1 min), III (9.1–9.9 min), IV (9.9–12.6 min), V (12.6–16 min), and VI (16–22 min). Fraction III is methyl gallate.
Overlay of ultraviolet-visible spectra of the eluted peaks of the crude methanolic extract of sumac in the range of 210–500 nm.
Chromatogram of the reinjected fraction III on analytical high performance liquid chromatography, which contained relatively pure methyl gallate at 7.88 min, with its corresponding ultraviolet-visible spectrum (210–500 nm), presented in the right corner of the figure.
Numbered carbon atoms in methyl gallate for nuclear magnetic resonance spectroscopy analysis.
Chromatogram showing the methyl gallate peak in the methanol extract, with its corresponding ultraviolet-visible spectrum (210–500 nm).
Streptococcus mutans biofilm biomass formed on the polystyrene surface after 24 h of incubation in THB containing 1% sucrose and different concentrations of MG. Data are presented as the mean ± standard error from three independent experiments (n=10-11). *P<0.05 vs. control group. MG, methyl gallate.
Optical profile of the glass slides with Streptococcus mutans culture biofilm after 24 h of incubation in the presence of different concentrations of MG. Glass slide surface of bacteria incubated (A) without MG, in the absence of sucrose, (B) without MG, in the presence of 1% sucrose, and treated with (C) 0.55, (D) 0.70, (E) 0.85 and (F) 1.00 mg/ml MG. Magnification, ×50. MG, methyl gallate.
Quantities of Streptococcus mutans biofilm formed on the glass slide surface after 24 h of incubation in THB containing 1% sucrose and different concentrations of MG. (A) the surface roughness parameter Rq of the biofilm on glass slides and (B) the biofilm thickness. Data are presented as the mean ± standard error from three independent experiments (n=18, biofilm roughness; n=15, biofilm thickness). *P<0.05 vs. control group.
pH levels of the
Experimental group | pH |
---|---|
Blank | 7.20±0.04 |
Control | 4.07±0.03 |
MSE (4 mg/ml) | 4.77±0.09 |
MSE (5 mg/ml) | 5.21±0.11 |
MSE (6 mg/ml) | 5.30±0.07 |
Data are presented as the mean ± standard error from three independent experiments (n=4-11).
P<0.05 vs. control group.
pH levels of the
Experimental group | pH |
---|---|
Blank | 7.47±0.02 |
Control | 4.28±0.02 |
MG (0.55 mg/ml) | 6.14±0.17 |
MG (0.7 mg/ml) | 7.17±0.03 |
MG (0.85 mg/ml) | 7.27±0.01 |
MG (1 mg/ml) | 7.28±0.02 |
Data are presented as the mean ± standard error from four independent experiments (n=10-11).
P<0.05 vs. control group.