Investigations of a Possible Chemical Effect of Salvadora persica Chewing Sticks

Abstract

Salvadora persica is commonly used chewing sticks in many parts of the world as an oral hygiene tool. This study measured the amount of benzyl isothiocyanate (BITC) released into the mouth and assessed its retention time in saliva. The study also tested if the released amount of BITC could potentially be antibacterial or cytotoxic. Twelve subjects brushed their teeth with fresh Miswak once, twice, and four times. The amount of BITC in the saliva and in the used brushes was quantified using gas chromatography-mass spectrometry. The antibacterial effect of BITC and Miswak essential oil (MEO) was tested against Haemophilus influenzae, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis. The cytotoxic effect on gingival fibroblasts and keratinocytes was tested using MTT. The highest amount of the active compounds was detected in saliva after using the Miswak tip for once and immediately. It significantly decreased when the Miswak tip was used more than once and thus after 10 min. The growth of the tested bacteria was inhibited by MEO and BITC in a dose dependent manner, P. gingivalis being the most sensitive. MTT assay showed that BITC and MEO were cytotoxic towards gingival fibroblasts while oral keratinocytes showed resistance. This study suggests that the Miswak tip should be cut before each use to ensure the maximum effect.

Figure 1

Benzyl isothiocyanate (BITC), benzyl cyanide (BC), and benzaldehyde (BA) concentration in saliva. (a) The concentration of compounds in saliva collected during fresh Miswak use (Saliva-1), saliva collected when Miswak was used for a second time (Saliva-2), and saliva collected while using Miswak for a fourth time (Saliva-4). (b) The concentration of compounds in saliva before brushing, immediately after brushing with fresh Miswak, and after 5, 10, and 30 min of Miswak brushing. In the above figure the bars denoted with ∗ and ∗∗ are significantly different when the comparison was made between the concentration of same compound detected in different samples.

Figure 2

Gingival fibroblasts cells exposed for 10 min to different concentrations of Miswak oil, benzyl isothiocyanate (BITC), benzyl cyanide, benzaldehyde, and 2.5 μg/mL of chlorhexidine. The bars denoted with ∗ (p < 0.05), ∗∗ (p < 0.01) are significantly different from negative control (DMSO) and media control.

Figure 3

Gingival fibroblasts cell exposed for 24 h to different concentrations of miswak essential oil, benzyl isothiocyanate (BITC), benzyl cyanide, and benzaldehyde. The bars denoted with ∗ (p < 0.05), ∗∗ (p < 0.01) are significantly different from negative control (DMSO) and media control.

Figure 4

Oral keratinocytes exposed for 24 hr to different concentrations of Miswak oil and benzyl isothiocyanate (BITC). The points indicate cell viability in relation to the viability using media alone. Points marked with ∗ indicate a statistical difference from media control (p < 0.05).

Figure 5

Inhibition of growth of A. actinomycetemcomitans (Aa), H. influenzae (Hi), and P. gingivalis (Pg). The individual bars represent the number of colony forming units (CFU) at various concentrations of Miswak oil or chlorhexidine (Chlx) compared to the negative control (DMSO). In the above figure ∗ stands for p < 0.01–0.001 and ∗∗ stands for p < 0.0001 when number of CFU were compared between negative control and different concentrations of Miswak essential oil or Chlx. Different letters on the bars represent significant difference (p < 0.05) between the CFU of the three bacteria tested at a given concentration of Miswak essential oil or Chlx independently, b indicate a significant difference compared to a, and c indicate a difference compared to both a and b.

Figure 6

Inhibition of growth of A. actinomycetemcomitans (Aa), H. influenzae (Hi), and P. gingivalis (Pg). The individual bars represent the number of colony forming units (CFU) at various concentrations of benzyl isothiocyanate (BITC) or chlorhexidine (Chlx) compared to the negative control (DMSO). In the above figure ∗ stands for p < 0.01–0.001 and ∗∗ stands for p < 0.0001 when number of CFU were compared between negative control and different concentrations of BITC or Chlx. Different letters on the bars represent significant difference (p < 0.05) between the CFU of the three bacteria tested at a given concentration of BITC or Chlx independently, b indicate a significant difference compared to a, and c indicate a difference compared to both a and b.

Figure 7

Inhibition of growth of A. actinomycetemcomitans (Aa), H. influenzae (Hi), and P. gingivalis (Pg). The individual bars represent the number of colony forming units (CFU) at various concentrations of benzyl cyanide or chlorhexidine (Chlx) compared to the negative control (DMSO). In the above figure ∗ stands for p < 0.01–0.001 and ∗∗ stands for p < 0.0001 when number of CFU were compared between negative control and different concentrations of benzyl cyanide or Chlx. Different letters on the bars represent significant difference (p < 0.05) between the CFU of the three bacteria tested at a given concentration of benzyl cyanide or Chlx independently, b indicate a significant difference compared to a, and c indicate a difference compared to both a and b.

Figure 8

Inhibition of growth of A. actinomycetemcomitans (Aa), H. influenzae (Hi), and P. gingivalis (Pg). The individual bars represent the number of colony forming units (CFU) at various concentrations of benzaldehyde or chlorhexidine (Chlx) compared to the negative control (DMSO), set to 100%. In the above figure ∗ stands for p < 0.01–0.001 and ∗∗ stands for p < 0.0001 when number of CFU were compared between negative control and different concentrations of benzaldehyde or Chlx. Different letters on the bars represent significant difference (p < 0.05) between the CFU of the three bacteria tested at a given concentration of benzaldehyde or Chlx independently, b indicate a significant difference compared to a, and c indicate a difference compared to both a and b.