Benzyl isothiocyanate, a major component from the roots of Salvadora persica is highly active against Gram-negative bacteria

Abier Sofrata, Ellen M Santangelo, Muhammad Azeem, Anna-Karin Borg-Karlson, Anders Gustafsson, Katrin Pütsep, Abier Sofrata, Ellen M Santangelo, Muhammad Azeem, Anna-Karin Borg-Karlson, Anders Gustafsson, Katrin Pütsep

Abstract

Plants produce a number of antimicrobial substances and the roots of the shrub Salvadora persica have been demonstrated to possess antimicrobial activity. Sticks from the roots of S. persica, Miswak sticks, have been used for centuries as a traditional method of cleaning teeth. Diverging reports on the chemical nature and antimicrobial repertoire of the chewing sticks from S. persica led us to explore its antibacterial properties against a panel of pathogenic or commensal bacteria and to identify the antibacterial component/s by methodical chemical characterization. S. persica root essential oil was prepared by steam distillation and solid-phase microextraction was used to sample volatiles released from fresh root. The active compound was identified by gas chromatography-mass spectrometry and antibacterial assays. The antibacterial compound was isolated using medium-pressure liquid chromatography. Transmission electron microscopy was used to visualize the effect on bacterial cells. The main antibacterial component of both S. persica root extracts and volatiles was benzyl isothiocyanate. Root extracts as well as commercial synthetic benzyl isothiocyanate exhibited rapid and strong bactericidal effect against oral pathogens involved in periodontal disease as well as against other Gram-negative bacteria, while Gram-positive bacteria mainly displayed growth inhibition or remained unaffected. The short exposure needed to obtain bactericidal effect implies that the chewing sticks and the essential oil may have a specific role in treatment of periodontal disease in reducing Gram-negative periodontal pathogens. Our results indicate the need for further investigation into the mechanism of the specific killing of Gram-negative bacteria by S. persica root stick extracts and its active component benzyl isothiocyanate.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Antibacterial activity of S. persica…
Figure 1. Antibacterial activity of S. persica essential oil determined by colony forming units (CFU) assay.
The individual bars show the statistical mean and standard deviation of the number of surviving bacteria from three experiments. The essential oil concentration is in percentage of final assay volume. Control is with DMSO only. (A) Gram-negative bacteria (B) Gram-positive bacteria (C) Kinetics of antibacterial activity against E. coli MC4100 (Ec) and A. actinomycetemcomitans (Aa) Samples were withdrawn for CFU determination at time-points indicated. Oil dilution was 0.1%. The CFU values displayed are the statistical mean of three experiments.
Figure 2. Gas chromatography-mass spectrometry (GC-MS) analysis…
Figure 2. Gas chromatography-mass spectrometry (GC-MS) analysis of S. persica root volatiles and essential oil.
(A) Chromatogram of Miswak volatiles (B) Chromatogram of Miswak essential oil. (C) Mass spectrometry (MS) analysis and identification of compound a as benzyl isothiocyanate MW = 148.6 (theoretical mass 149.21) and compound b as benzyl nitrile, MW = 116.5 (theoretical mass 117.15). The chemical structures are presented as insets in the figure.
Figure 3. Antibacterial activity of commercial synthetic…
Figure 3. Antibacterial activity of commercial synthetic BITC against two Gram-negative bacteria.
The individual bars show the statistical mean and standard deviation of the number of surviving bacteria from three experiments. The amount of BITC applied corresponds to the BITC content in the different oil dilutions of figure 1; 28 µmole (1% oil), 2.8 µmole (0.1% oil), 1.4 µmole (0.05% oil), 0.56 µmole (0.02% oil), 0.28 µmole (0.01% oil), 0.14 µmole (0.005% oil) and 0.028 µmole (0.001% oil) pure BITC, respectively. Control is with DMSO only.
Figure 4. Analysis of S persica root…
Figure 4. Analysis of S persica root components by thin layer chromatography, antibacterial activity and GC-MS.
(A) TLC analysis of MPLC extracted-fractionated samples 1–6. Samples 7 and 8 are not shown since their components were too hydrophilic to be mobile in the ethyl acetate-hexane solvent. (B) Antibacterial activity of MPLC samples against two Gram-negative and two Gram-positive bacteria. The individual bars show the statistical mean and standard deviation of the number of surviving bacteria from two experiments. Pooled, denotes samples 1–8 mixed together. Control is with DMSO only. (C) Chromatogram of MPLC-samples with antibacterial activity. Sample 2 (left) and sample 3 (middle) displayed one major compound (a) = benzyl isothiocyanate. Sample 5 (right) displayed both compound (a) and compound (b) = Benzyl nitrile.
Figure 5. Transmission electron micrographs of A.…
Figure 5. Transmission electron micrographs of A. actinomycetemcomitans treated with essential oil, BITC or the antibiotic ampicillin.
Samples were withdrawn at time points indicated. Concentration of essential oil was 0.1%, which corresponds to a content of 2.8 µmole BITC. The amount added synthetic BITC was 2.8 µmole and ampicillin concentration was 1 mg/ml. Control for essential oil and BITC contained 1% DMSO. For ampicillin, the control was with 1% water. The control bacteria were withdrawn after 40 min of incubation.

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