Background
Dental caries is an irreversible localized infection that results in progressive tooth decay [
1]. It’s a common type of dental disease associated with microorganisms present on the tooth surface in dental plaque. One of the main etiologic factors of dental caries is considered to be
Streptococcus sobrinus, which belong to the gram-positive mutans streptococci group. The presence of
S. sobrinus is relatively higher on the molars compared to the anterior teeth [
1,
2]. Several epidemiological studies have shown that the prevalence of
S. sobrinus is more closely associated with high caries activities [
3].
S. sobrinus can colonize the tooth surface and initiate plaque formation by synthesizing water-insoluble glucan from sucrose by glucosyltransferases (GTF), resulting in a firm attachment to the tooth surface and form lactic and other organic acids by fermentation of various sugars in foods [
4]. Different strategies for preventing dental caries caused by cariogenic bacteria, such as depression of the growth of streptococcus, inhibition of GTF activity, and hydrolysis of glucans by enzymes, have been developed [
5]. Further accumulation of plaque around the gingival margin and subgingival region may lead to shifts in the balance of the microflora from mainly gram-positive bacteria to gram-negative bacteria, and an increased number of gram-negative anaerobic bacteria will cause the development of periodontal diseases [
6].
Periodontal diseases are chronic inflammatory disorders of bacterial origin that affect tooth-supporting tissues [
7]. Over 700 bacterial species that have been identified in the oral cavity [
8], only a few are associated with periodontitis, including
Porphyromonas gingivalis [
9].
P. gingivalis is a gram-negative bacterium closely associated with chronic periodontitis. High numbers of
P. gingivalis, together with other periodontopathogens, induce a host immune response, which in turn leads to a destructive inflammatory process.
Oxidative stress observed in a diseased periodontium could result directly from excess reactive oxygen species (ROS) activity or antioxidant deficiency or indirectly by creating a pro-inflammatory state. Some studies reported that the excess production of ROS resulted in damages of gingival tissues, periodontal ligament, and alveolar bone [
10‐
12]. Therefore, the search for an antioxidant that could be used to control these diseases as polyphenolic compounds are likely candidates [
13].
Acacia nilotica (L.) Delile sub
nilotica (Leguminosae) is a tree found in the central and northern parts of Sudan and is known in Sudanese folk medicine by the common name ‘Garad or Sunt.’ The fruit and the stem bark are regarded as a tonic and astringent and are used internally to treat colds, bronchitis, pneumonia, diarrhea, and dysentery [
14,
15]. Fractionation of
A. nilotica leaves and bark showed the presence of phenols condensed tannin [
16], gallic acid, (+) catechin, (−) epigallocatechin-7-g’allate, catechin derivatives [
17,
18], ellagic acid, kaempferol, and quercetin [
19]. Parallelly many researches demonstrated its wide array of pharmacological activities, such as anti- HIV-1 protease [
20], antibacterial [
21], antioxidant, anticarcinogenic [
22,
23] and anti-inflammatory activities [
24]. In Sudanese and Indian traditional medicine, gargle the decoction of the
A. nilotica bark is used to strengthens teeth and eliminates toothache [
25‐
27]. Even though there is a little investigation on the potential beneficial effects of
A. nilotica bark on oral health. Thus, we evaluated the effects of
A. nilotica bark methanolic extract and its fractions on the growth of two oral bacteria included
S. sobrinus and
P. gingivalis. We also investigated their GTF inhibitory activity and antioxidant functions for oral hygiene purposes.
Methods
Reagents
All materials purchased from (Wako-Japan) except p-iodonitrotetrazolium violet from (Sigma-Aldrich-Japan).
Plant materials and extraction
A. nilotica bark was collected from Sennar State, Sudan in May 2013 and then authenticated by the University of Khartoum, Faculty of Forest. Voucher specimens (SD-SS-01) are deposited in the Horticultural Laboratory, Department of Horticulture, Faculty of Agriculture, University of Khartoum. A. nilotica bark was shade dried and powdered before being extracted with methanol for 12 h three times. The extracts were filtered through Whatman No. 2 filter paper, and the solvent was removed under vacuum using a rotary evaporator.
Fractionation of A. nilotica bark
The crude extract (400 mg /2 ml of 50% methanol) was applied to a Sephadex LH-20 column. The column was eluted with methanol (250 ml), methanol-water (80:20, v/v; 150 ml, Fr1), methanol-water (50:50, v/v; 250 ml, Fr2), methanol-water (5:95, v/v; 100 ml, Fr3) and finally acetone-water (70:30, v/v; 250 ml, Fr4). The fractions (Fr1–4) were concentrated in vacuo (38 °C) and freeze-dried to give four powders with approximate weights of 34.2 mg, 35.7 mg, 44.8 mg, and 244.5 mg for Fr1, Fr2, Fr3, and Fr4 respectively. Furthermore, the crude extract and fractions were subjected to high performance liquid chromatography (HPLC) with reversed-phase column C18 (Sunniest 4.5 mm i.d. X 250 mm). The solvent system used was as follows: a gradient program for 65 min from 5 to 100% methanol in water with 0.05% TFA (Trifluoroacetic acid) at a flow rate of 1 ml/min, monitored at 280 nm.
Antibacterial activity assay
Minimum inhibitory concentration
(MIC) was determined by the broth dilution method [
28].
S. sobrinus 6715 and
P. gingivalis ATCC 33277 were cultured in brain heart infusion broth. For
P. gingivalis broth supplemented with 0.5 μg/ml vitamin K
3 and 5 μg/ml hemin. The samples were tested for antibacterial activity in sterile 96-well plates. The inoculums were prepared by diluting the broth culture to 10
6cell/ml for
S. sobrinus and 10
8cell/ml for
P. gingivalis approximately. The experiments were performed in triplicate. Chlorhexidine was included in the assay as a positive control. Then cultures were incubated 24 h for
S. sobrinus and 72 h for
P. gingivalis at 37 °C under anaerobic condition. Microbial growth was indicated by adding 50 μl of (0.2 mg/ml) p-iodonitrotetrazolium violet (INT) to culture and incubated at 37 °C for 2 h. The MIC was defined as the lowest concentration that inhibited the color change of INT [
29]. For minimum bactericidal concentration (MBC), 10 μl from wells that showed no color change were transferred to 100 μl of fresh media and then incubated for 24 h under the anaerobic condition at 37 °C. Microbial growth was also indicated by adding INT to culture. The MBC was defined as the lowest concentration that inhibited the color change of INT.
Preparation of glucosyltransferase (GTF)
S. sobrinus 6715 was grown for 20 h at 37 °C in 4 L of Todd Hewitt broth. After centrifugation of the culture at 1300
g for 10 min at 4 °C, the cells were collected and then extracted with 8 M urea for 1 h with stirring. The crude enzyme solution containing urea was dialyzed against 10 mM sodium phosphate buffer (pH 6) until the urea was removed entirely. One milliliter of the crude enzyme solution was pipetted into a microtube and stored in a freezer at − 80 °C [
30].
GTF inhibitory activity assay
Insoluble glucan synthesized by GTF was measured turbidimetrically. GTF was incubated in 300 μl of 0.1 M phosphate buffer (pH 6.0) containing 1% sucrose, 0.5% dextran T-10, and in the presence or absence of samples at 37 °C for 3 h. The volume of the crude GTF solution used in the assay was determined by absorbance of around 1.0 at 590 nm. Chlorhexidine was used as a positive control [
30]. The inhibition rate is expressed by the following equation: Inhibition (%) = 100 × (Ac – As)/Ac.
Where.
Ac: Absorbance of the control.
As: Absorbance of the sample.
ABTS radical scavenging activity assay
2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) was dissolved in water to make a concentration of 7 mM. ABTS
+ was produced by reacting to the ABTS stock solution with 2.45 mM potassium persulfate and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. For the study of samples, the ABTS
+ was diluted with phosphate-buffered saline 5 mM, pH 7.4 to obtain an absorbance of 0.70 at 734 nm. After the addition of 980 μl of diluted ABTS to 20 μl of samples, the absorbance reading was taken five minutes after the initial mixing [
31]. Trolox was used as a positive control. The activity was measured as follows:
% ABTS scavenging activity =.
[(control absorbance – sample absorbance)/ (control absorbance)] × 100.
Statistical analysis
The inhibitory activity of GTFs and antioxidant activities were expressed as the mean (mean ± standard deviation) value. The significant differences between samples were assessed by one-way analysis of variance (ANOVA) followed by pairwise comparison of the mean using Tukey’s multiple comparison test. Values were determined to be significant when p was less than 0.05 (p<0.05).
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