er defects. Ideally, before storage stage, all pre-molar teeth were cleaned with a toothbrush under running tap water. Then, all samples were stored in artificial saliva at an air-
Figure 2. Schematic illustration of human premolar tooth.
conditioned temperature of 20˚C ± 1˚C and a relative humidity of 40% ± 5% RH until use. After that, all samples to be treated were placed in their respective solutions and rinsed with reagent water ca. 30 seconds.
2.2. Extraction of Salvadora persica “Miswak”
2.2.1. Plant Materials
Dried roots of “Miswak”, as shown in Figure 1(b), were purchased from a local market in Makkah city, Kingdom of Saudi Arabia, and these had been cut in the 2015/2016 season. All the authors, in consultation with an experienced “Arak” merchant, identified the plant known as Salvadora persica “Miswak”.
2.2.2. Preparation of Salvadora persica “Miswak” Extracts
Dried roots of “Miswak” were sliced using a sharp knife then the pieces were ground to a powder with a blending machine. Then, 100 mL of deionized distilled water (from Sigma-Aldrich Chemie GmbH, Switzerland) was added to 10 mg of powder in a sterile well-capped flask; the extract was allowed to soak for 48 hrs. in the refrigerator. After that, the combination was centrifuged at 2000 rpm for around 10 minutes. The supernatant was filtered using a 0.45 mm membrane and stored in sterile screw-capped vials in the refrigerator until used. This procedure is adequate and consistent with the following publications   .
2.2.3. Polysaccharide Isolated from Salvadora persica “Miswak” Extracts
The supernatant from water soaked “Miswak” was precipitated with three volumes of acetone and stored overnight in a 4-degree refrigerator. After that, the precipitate was collected and washed three times again with acetone in order to obtain crude polysaccharide of around 2.0 g. The crude polysaccharide was dissolved in sterile deionized distilled water and then dialyzed against distilled water. The remaining quantity was lyophilized to provide total polysaccharide. This procedure is consistent with the following   .
2.2.4. Polysaccharides Composition
Three qualitative analyses of carbohydrates were carried out to confirm the nature of the carbohydrate. Firstly, Molisch’s test in which 2 mL of carbohydrate solution and 2 drops of α-naphthol solution were added together in a test tube and using a dropper and carefully inclining the tube and pouring dropwise, concentrated H2SO4 was added. The violet color at the junction of the two liquids was observed, this is because of unstable condensation product formation of β-naphthol with furfural produced by the dehydration of the carbohydrate. Secondly, Fehling’s test in which equal volumes of the test carbohydrate, Fehling A and Fehling B solution was added together and placed in a boiling water bath for several minutes. The production of brownish-red or yellow precipitate of cuprous oxide (CuO) suggests the presence of reducing sugars in the given test solution. Lastly, the Iodine test in which 2 drops of iodine solution and 2 mL of the carbohydrate test solution were mixed and a blue-black color was spotted which indicates the presence of polysaccharides. Iodine forms colored adsorption complexes with polysaccharides.
2.2.5. Solubility Test
The polysaccharide obtained from Salvadora persica “Miswak” was assessed for solubility in water (H2O), acetone (C3H6O), chloroform (CHCl3), and ethanol (C2H6O) in accordance with the British Pharmacopoeia (BP) specification.
2.2.6. Fourier Transform Infrared (FTIR)
The FTIR spectrum of the polysaccharides was recorded using a PerkinElmer Spectrum 100 FTIR spectrometer (PerkinElmer, Norwalk, CT, USA) equipped with a MIR TGS detector. The FTIR spectra were recorded between the 4000 to 400 cm−1 region at a room temperature of 20˚C ± 1˚C and a relative humidity of greater than 40% ± 5% RH. All the data analysis was collected automatically by using appropriate software (Spectrum 100 software). Each enamel sample was scanned as three different replicates under the similar settings, all of which gave identical spectra. The mean spectra of these three replicates’ data were then used in detailed data and statistical analysis.
2.2.7. Nuclear Magnetic Resonance (NMR)
The principal component analysis of 600 MHz 1H-NMR spectra of the polysaccharides was recorded using Bruker Avance III WM 600 MHz spectrometers (Karlsruhe, Germany). The sample was dissolved in D2O at a room temperature of 20˚C ± 1˚C and a relative humidity of greater than 40% ± 5% RH. The combination of peak volumes in the 2D-HSQC spectra was completed via standard Bruker TOPSPIN 3.0 software.
2.3. Test Solution
To prepare the citric acid solutions 50% (w/w), the citric acid was added to alter the pH and controlled by using a pH meter (Model: Knick pH meter 765 Calimatic, Knick Elektronische Messgerate GmbH & Co. KG, Berlin, Germany).
2.4. Enamel Erosion Samples Treatment
The enamel samples were treated at a room temperature of 20˚C ± 1˚C and a relative humidity of greater than 40% ± 5% RH with CAS for 120 seconds, to replicate the common intake times of soft drinks. For the erosion studies, three samples (n = 3) were prepared for every citric acid (CAS). For each sample, 30 mL of CAS was used. After treatment, the samples were immediately rinsed with deionized water for 30 seconds and dried with compressed air. Then, samples were stored in the same environment that would be used for testing by the Taly-surf® test-rig. For the SEM study, immediately after treatment with the CAS, one-half of the surface of the enamel sample was exposed to AESP to obtain a treated area.
2.5. Testing Procedures
The surface roughness, Ra, is defined as an average roughness of the profile around the mean line (generated by a standard filter or least squares) and it is possibly the most frequently used parameter. If z = f(x) is the profile measured from the reference mean line and L are the profile lengths measured from the reference mean line, L is the profile length being evaluated, then the average surface roughness, Ra, value is defined in Equation (2) by      :
The 3D average surface roughness can be represented by a function of z(xi, yi) with xi = i∆x and yj = j∆y, where i = 1, 2, 3, ∙∙∙, M and j = 1, 2, 3, ∙∙∙, N. In addition, ∆x and ∆y are sampling intervals. Likewise, M and N symbolize the number of sampling data points in the x-direction and y-direction. The 3D average surface roughness parameters are passed on the residual surface η(x, y), which is the difference between the original surface z(xi, yi) and the reference datum f(xi, yi). Then, the average value of the surface roughness, Sa, is defined in Equation (3) as      :
Bear in mind, Ra and/or Sa is an arithmetic mean parameter of the roughness profile. The sampling interval is insensitive to changes. Hence, the surface roughness profile of enamel teeth was analyzed quantitatively to determine the standard statistical parameter of average roughness, Ra, using a commercial precision test-rig Taly-surf® (from Taylor Hobson Precision, Inc.). Table 1 shows the specification of the device, and it conforms to British Standards (BS). Also, Figure 3 shows the image of Taly-surf® with conisphere tip r = 2 µm and pre-molar teeth with the surface profile. The errors of the surface roughness were calculated from the standard deviation (±SD) of the absolute values of height deviation and the data traces were auto-levelled to a linear least-squares straight line and then filtered with a typical cut-off.
During the course of the investigation, every single test was repeated at least three times at different and new locations on the enamel surface in order to ensure the repeatability and reproducibility of the results. The new location was at least ±300 µm from the previous test. This method should avoid any data variation of the counterbody
Table 1. Test-rig specifications.
Figure 3. Image of Taly-surf® with conisphere tip r = 2 µm (from Taylor Hobson) and pre-molar teeth with surface profile.
surface, e.g., due to micro-tribological behavior, which may appear during the test performance and indeed will affect the measurements in the following tests. All experiments were performed with a typical arrangement of sphere-on-flat applying a linear sliding contact at constant velocity over a specific distance. Tests were achieved by using a forward scan mode motion at scan length of 5 mm. For more details, see publication     .
2.6. Calibration Method
A standard ball of 22.0161 mm diameter is used to calibrate the precision instrument (from Taylor Hobson Precision, Ltd.), as shown in Figure 4. A series of ten calibration trials have been performed. This is an acceptable series of experiments as these calibration trials are predominantly about design interpretation and relative behavior to other precision systems is always vulnerable to variations in materials and dimensions. Calibration results showed that the cantilever beam system at only one end was a linear mass-spring system (R2 > 0.99), under operational and environmental conditions, with an absolute uncertainties value of <1% and measurement resolution down to at worst 50 nm. For more details, see publication  .
2.7. Scanning Electron Microscopy (SEM)
The surface topography of all pre-molar samples was analyzed by SEM of at least ten scan areas (scan size: 10 µm × 10 µm) measured at an arbitrarily located area on the surface of the sample. The SEM was performed with an LEO 440i (LEO Elektronen- mikroskopie GmbH, Oberkochen, Germany) operated at 15 kV. To study the surface visualization and to improve the reflectivity for a higher fidelity in optical profiling, all samples were fully coated with gold (25 nm thickness in 60 seconds) using Bio-Red SEM Coating System.
3. Results and Discussions
Every test was repeated at least three times, and the results were presented as mean ±SD value. All citric acid solution of the current investigation was characterized by pH values. The pH value of the CAS was 3.3. As has been previously mentioned, the present work aims to study the effect of aqueous extracts of Salvadora persica on human dental enamel acid erosion. Surface roughness, Ra, has been extensively used to determine the total average roughness and the coverage net (before and after the treatment). Figure 5 illustrates an example of a human dental enamel surface roughness before and after the treatment of AESP.
3.1. Stylus-Base Data Analysis
The mean surface roughness, Ra, of the pre-molar teeth was measured using “ball-on- flat” arrangement and “single scan mode”. For group I: pre-molar protective, under normal conditions (where there is no inhibitor, and the tooth has been washed with deionized water for 30 seconds), the average surface roughness of the pre-molar tooth was 3.38 µm (±SD = 0.02 µm). Indeed, after the premolar was exposed to the AESP (for approximately 2 minutes), the performance of the human dental enamel was improved, and the average roughness of the premolar tooth was Ra ≈ 2.56 µm (±SD = 0.035 µm), which means that the surface net was Ra ≈ 0.82 µm. However, after the sample was immersed again in CAS, the surface was damaged by 0.63 µm as the surface roughness of the sample was Ra ≈ 3.19 µm (±SD = 0.027 µm). In addition, the remaining layer of the
Figure 4. Measurement calibration on a sphere of known radius.
Figure 5. A typical example of a human dental enamel surface roughness before and after treatment.
protective zone was around Ra ≈ 0.19 µm with a 95% level of confidence, as shown in Figure 6.
On the other hand, for group II: pre-molar restoring, under the normal conditions (where there is no inhibitor, and the tooth has been washed with deionized water for 30 seconds), the average surface roughness of the pre-molar tooth was 3.58 µm (±SD = 0.084 µm). After the pre-molar was exposed to the CAS (for around 2 minutes), the performance of the human dental enamel was degraded, and the average roughness of the pre-molar tooth was Ra ≈ 3.92 µm (±SD = 0.075 µm), which means that the surface net was Ra ≈ 0.34 µm. AESP was introduced to the human dental enamel after CAS (for around 2 minutes), the surface roughness was measured, and it gives us the Ra ≈ 2.97 µm (±SD = 0.058 µm), indicating that the surface net was Ra ≈ 0.95 µm (a few layers of the pre-molar after exposure to AESP was improved). Finally, the sample was exposed to CAS again for around 2 minutes, and the surface roughness value was Ra ≈ 3.30 µm (±SD = 0.17 µm), indicating that the surface net was Ra ≈ 0.33 µm. Surprisingly, the net value between two different mediums (normal-CAS-AESP) or (CAS-AESP-CAS) were Ra ≈ 0.61 µm and Ra ≈ 0.62 µm, respectively. Besides, the remaining layer of the restoration zone was around Ra ≈ 0.01 µm after treatment for two minutes with CAS, as shown in Figure 7, with a 95% level of confidence.
3.2. FTIR Data Analysis
The IR spectrum of polysaccharide isolated from Salvadora persica “Miswak” is presented in Figure 8. Peaks from 900 to 1400 are due to the fingerprint areas for carbohydrates, the absorption peaks at 1500 cm−1 and 1600 cm−1 represents the acetyl group.
Figure 6. Effect of different medium on human dental enamel at different test conditions (normal, AESP and CAS).
Figure 7. Effect of different medium on human dental enamel at different test conditions (normal, CAS, AESP and CAS).
Figure 8. IR of polysaccharide isolated from Salvadora persica “Miswak”.
The sharp band at 2900 cm−1 is characteristic of methyl C-H stretching associated with aromatic rings. The very obvious absorption at 3000 cm−1 is due to the stretching vibration and angular vibration of O-H linkage. The intensity of bands of about 3300 cm−1 in the IR spectrum was due to the hydroxyl stretching vibration of the polysaccharide, and as predicted these were broad, which make up the gross structure of carbohydrates. This is all in line with the polysaccharide structure that is neither starch nor cellulose, see      .
3.3. NMR Data Analysis
The 1H-NMR of polysaccharide isolated from Salvadora persica “Miswak” is illustrated in Figure 9. There is spectral evidence of the presence of polysaccharide. The peaks in the anomeric region and more peaks in the region of 3.3 ppm and 4.9 ppm, are indicative of the presence of polysaccharide in the extraction. This is an encouraging result and is indeed consistent with the results obtained in    .
3.4. UV Data Analysis
The UV-vis spectra showed that the polysaccharide had an absorption peak at 190 nm, which is the UV characteristic for the polysaccharide, which is in agreement with the data published in  . Figure 10 shows the UV-vis spectra of polysaccharide isolated from Salvadora persica “Miswak”.
Figure 9. The 1H-NMR of polysaccharide isolated from Salvadora persica “Miswak”.
Figure 10. The UV-vis spectra of polysaccharide isolated from Salvadora persica “Miswak”.
3.5. SEM Data Analysis
The surface topography of all pre-molar samples was analyzed by Scanning Electronic Microscopy (SEM) after all samples were fully coated with gold (25 nm thickness in 60 seconds). Figure 11(a) shows the SEM image of the effects of a single application of CAS (pH = 3.3 and t = 120 seconds) on the dental enamel surface. Whereas, Figure 11(b) shows the SEM image of the enamel surface treated with AESP (t = 120 second). Besides, Figure 11(c) and Figure 11(d) show a close-up view of the CAS and AESP images, respectively. For all treated samples illustrated in Figure 11, surface flaws and defects such as voids or rough surfaces are observable. SEM images of treated enamel surfaces with CAS show severe erosion with voids and loss of material over the whole surface compared to the images of AESP treatment where the surfaces seem to be much smoother with very flat areas. This can be seen only for the sections treated with CAS (see Figure 8(a)), the characteristic of eroded HAP prism structure of enamel and periphery is visible on the micrograph. Thus, it can be assumed that this interaction causes
Figure 11. SEM micrograph of the sound human enamel surfaces with different condition (a, b) and close-up features (c, d).
the loss of HAP crystals at the surface and results in the erosion process. To demonstrate this effect of acid erosion on dental enamel, the etched enamel prisms core surfaces after the acid erosive treatment are illustrated in SEM images.
The AESP treated enamel surfaces seen in Figure 11(b) are fairly smooth compared to the enamel surface treated with CAS. Therefore, it is hypothesized that the polymers perhaps adsorb on the eroded enamel surface and form protective layers covering the enamel surface following AESP treatment. Therefore, when the tooth was treated with the AESP the morphology of the surface changed and the etch pattern characteristics for CAS treatment on the surface of enamel are not apparent after this process. It can thus be suggested that the protective layer, which absorbs directly onto the human enamel surface may be due to the presence of polysaccharide. To confirm the presence of polysaccharide, the polysaccharide was isolated from extraction and some tests were carried out to characterize this. The polysaccharide is a combination of a number of varied macromolecular substances, and the composition and yield of the polymer can differ according to the methods of isolation. The polysaccharide indicated negative Fehling’s reagent and iodine-potassium iodide reactions, showing that it did not contain reducing sugar and starch type polysaccharide. It is considered as soluble in water and practically insoluble in ethanol, acetone, and chloroform.
The SEM images illustrated distinctive etch morphologies that support the surface roughness data and interpretations of the current investigation. There is a strong correlation between the Ra value and the SEM morphology image for both CAS and AESP. For those CAS treated, the surface profile was strongly eroded which corresponds to the measured value of Ra. On the other hand, for those AESP treated, the surface profile was less affected. Thus, the surface roughness corresponds to the value of Ra.
In addition, the SEM images show some changes in the surface morphology of enamel such as the presence of erosion and porosity. This finding is an encouraging result, and is in agreement with the following publications     .
The scientific results obtained from this study show that the AESP derived can significantly reduce surface roughness value, Ra, few sub-nano meter level of human dental enamel affected by citric acid. It was also illustrated for the first time, measured with high precision test-rig Taly-surf® (from Taylor Hobson, Inc.), that AESP protects the human dental enamel against the effects of citric acid solutions. The protection is mainly due to the “shielding” effect of the polymers, which is self-formed directly on the surface of the human enamel during treatment with AESP. Perhaps, the modification of acidic soft drinks with such natural polymers (polysaccharide) may decrease the erosive effect considerably. Further investigation is essential to characterize the external polymer layer formed on the enamel surface. An important outcome of the studies herein is the ability to assess the effectiveness of inhibitors on enamel dissolution that could be expanded to include many types of substrate. In conclusion and in brief, the new methodology that has been used in this paper has enormous potential in the field of micro-mechanisms and nano-technology.
There is no ethical issue regarding this paper.
Conflicts of Interest
The authors have no conflicts of interest.