In the current study, TEM scanning revealed that the average particles size of the three prepared nano-herbal extracts ranged from 40- to 50-nm, which lies within the nanoscale.
MI Paste Plus™ contains Recaldent™ (CPP-ACP) in addition to 900 ppm of fluoride in form of NaF, according to its manufacturer. Such amount of fluoride is almost equivalent to the fluoride amount found in over the counter different toothpastes.
The present study evaluated a 970-nm diode laser efficacy accompanied by application of CPP-ACP-fluoride and 5% nano-extracts of clove, turmeric and garlic on surface roughness and microhardness of demineralized dentin and their bactericidal effect against S. mutans and S. sobrinus cariogenic bacteria. The results displayed a significant difference in surface roughness, microhardness, and the viable bacterial counts of the tested groups. Therefore, the null hypothesis was rejected.
In the current study, surface roughness of the demineralized dentin group (control) showed the highest significant mean values. This could be owed to removal of the constituent minerals by the action of the demineralizing solution, in which the prepared dentin specimens were kept for 72-h. Moreover, the least significant surface roughness values were recorded for MI paste Plus groups. This finding could be related to its elevated levels of Ca, P, and F ions which might have precipitated upon the demineralized dentin surface forming a CaF2 layer that could partially block the demineralized surface porosities and the patent dentinal tubules leading to decreased surface roughness. Yamazaki and Margolis (2008) concluded that a CaF2 layer was formed at the enamel surface following topical fluoride application which has reacted with Ca and P ions leading to inhibition of the demineralization and enhancement of the remineralization of the crystals. Moreover, CPP-ACP has a unique nature as an amorphous electroneutral nanocomplexes. This nano-scale complex could facilitate the diffusion of the Ca and P ions released from MI paste Plus into the porosities of the demineralized tooth structure. These released ions were found to have a significant binding-affinity to the apatite of the tooth (Reynolds et al. 2008).
On the other hand, the nano-extract of clove recorded the least significant surface roughness compared to those of turmeric and garlic, respectively. These findings could be attributed to the amount of minerals in the investigated nano-herbal extracts. According to the agricultural research services of the US department of agriculture, clove was reported to have the highest Ca and P content, followed by turmeric then garlic (Food Central Data, Agricultural Research Services, US Department of Agriculture USDA, USA). Thus, Ca and P ions might have precipitated on the demineralized dentin surface blocking the demineralized surface porosities and patent dentinal tubules and decreasing the surface roughness. Nonetheless, the influence of the nano-herbal extracts on the surface topography of the dental hard tissues is less well investigated and requires further research.
Our results disagreed with Gungor and Donmez (2020). This could be related to the difference in the control groups used in the studies. They used sound dentin specimens as control in contrast to demineralized dentin specimens that were used as control in our study. Moreover, they have equipped AFM to assess the surface roughness instead of the stereomicroscope that was employed in our study.
Data obtained from the present study showed that diode laser irradiation had significantly increased the surface roughness of the tested specimens. This could be owed to the thermal effect of the laser energy, which might lead to dentin structure alterations (Alfredo et al. 2009). These findings agreed with those of Viapiana et al. (2012), and they concluded that near-infrared wavelength range of 980-nm diode laser allowed the dentin to uptake some of the emitted laser energy by its carbonate and phosphate mineral content leading to crystalline arrangement variation and melting of the irradiated dentin surface.
Vickers microhardness results revealed that MI paste Plus showed the highest significant mean values which could be accredited to CPP, which was proven to stabilizes the levels of Ca and P ions and develops a state of supersaturation of these ions around the teeth resulting in enhancement of teeth remineralization due to increased surrounding pH and ions levels (Jayarajan et al. 2011). This finding was approved by Behrouzi et al. (2020), and they concluded that hydroxyapatite crystals density was increased by F, Ca and P ions penetration into the crystals enhancing the remineralization process.
The significance of interaction between laser irradiation and the hard tooth structures is not fully documented, and it can differ in relation to the designated parameters. Therefore, dentin inherent properties can be influenced by laser irradiation.
Diode laser irradiation prior to MI paste Plus application showed a significant increase in the present study. This could be explained by increased dental structure uptake of F, Ca and P ions when combined with laser application. The combined treatment of laser irradiation and fluoride application was proved to increase the acid resistance of hard tooth structures than either of the previously mentioned treatments alone (Kumar et al. 2016).
Our findings were in accordance with Wiegand et al. (2010), and they disclosed that a proper remineralization of the dental structures necessitates the availability of materials that could successfully deliver Ca and P ions to the tooth structure with the aid of laser irradiation.
The investigated nano-herbal extracts showed a comparable microhardness values to MI paste Plus. Clove nano-extract showed the highest significant Vickers microhardness, followed by turmeric then garlic. This might be owed to the discrepancies in their Ca and P content, which might have been directly related to their remineralization potential. As Ca and P ions are the main keys in balance between de- and re-mineralization processes and they could alter teeth vulnerability to caries development (Hara and Zero 2010).
Furthermore, it was determined that the decreased pH values in the demineralized dentin will activate the MMPs (Matrix-Metallo-Proteinases) types 2, 8, and 9 causing hydrolysis of the dentin organic matrix (collagen), which can be prevented by using MMPs inhibitors (Chaussain-Miller et al. 2006). MMPs inhibitors include many natural herbs which usually comprise active biological compounds with powerful antioxidant properties such as phenolic and polyphenolic compounds.
Our results agreed with Al-lami and Al-Alousi (2011), who concluded that clove extract could efficiently rise the demineralized enamel surface microhardness. They owed their findings to the Ca and P ions content of clove extract. Moreover, Gungor and Domenz 2020 investigated different herbal teas for their effect on erosive dentin microhardness and found that the highest values were recorded for clove due to its high Ca and P ions content. Additionally, tannin is one of the constituent polyphenolics compounds of clove and an in vitro study demonstrated its ability to increase the enamel acid resistance, thereby increasing the remineralization capacity of clove extract (Yu et al. 1995).
On the other hand, Basir et al. (2018) investigated the anti-caries effect and microhardness of different curcumin concentrations on enamel. They declared that curcumin showed an anti-caries effect and increased enamel microhardness with all tested concentrations. Furthermore, Prabhakar et al. (2013) compared the effect of 2% chlorhexidine and turmeric extract on root dentin microhardness, and they found out that turmeric extract had significantly increased dentin microhardness. Nevertheless, curcumin (an antioxidant polyphenolic principal compound of turmeric) could have impeded the action of MMP-9 responsible for the degradation of the collagen matrix of the demineralized dentin. As curcumin can chelate the catalytic Zinc ions that are vital for the activity of MMPs, thus inhibiting their action (Zhang et al. 2012).Furthermore, Słowianek and Leszczyńska (2016) showed that clove had the highest antioxidant activity by far followed by turmeric then garlic and this was owed to the differences in their phenolic compounds types which have variable antioxidant properties with variable effect on MMPs inhibition, which agrees with our results.
On the other hand, our results showed that diode laser irradiation combined with MI paste Plus and nano-extracts had an increased significant antibacterial effect against S. mutans and S. sobrinus than the tested materials alone. This could be attributed to combining the antibacterial efficiency of the diode laser on one hand with that of the tested materials on the other hand which might have an intense significant effect on the overall viable bacterial count for both strains. These results were in accordance with Lee et al. (2006), who reported that the antibacterial efficacy of diode laser against S. mutans is obviously elevated in relation to the antibacterial mouthwash used in their study.
The antibacterial efficiency of diode laser irradiation could be explained through its photo-disruptive and thermal effects. It is not necessary for an immediate bacterial cell death to happen during lasing, but a sub-lethal cell damage repressing its growth, is more likely to occur. Such damage might possibly accumulate denatured proteins and destroy cell wall integrity. Thus, terminating the cell growth and causing cell lysis. Furthermore, thermal changes greatly affect cellular protein. Laser irradiation might lead to denaturation of cell proteins inducing the cell to produce new proteins. Cells will prevent building-up of debris from denatured protein causing cell death (Lee et al. 2006).
In agreement with our study, Patel et al. (2017) reported that fluoride-containing CPP-ACP varnish was effective in decreasing S. mutans count in saliva which could be owed to CPP-ACP anticariogenic effect in addition to its F ions, which might lead to ACP-F localization at the tooth surface by the action of casein protein. It was determined that CPP-ACP binds successfully to S. mutans bacteria which was mediated by cell moieties of surface phosphate through cross-linking of calcium and by the hydrophobic and hydrogen bond-mediated cellular interactions (Memarpour et al. 2015).
The chief component of clove essential oil is eugenol, which has a powerful antibacterial effect on different oral bacteria. Its mechanism of action is related to interaction with cell membrane of bacteria (Oyedemi et al. 2009). Our results agreed with Mirpour et al. (2015), who concluded that clove showed an antibacterial activity against S. mutans and S. aures. They owed their results to the antibacterial effect of saponins and flavonoids components of the investigated plant extract. Furthermore, Lapinska et al. (2020) concluded that clove essential oil had distinct antibacterial activity on S. mutans even when it was incorporated in a flowable composite.
On the other hand, “curcumin”, the exclusive bioactive agent of turmeric, was proven to have an adequate antibacterial influence. Javid et al. (2017) suggested that curcumin could enhance prevention of caries through decreasing the amounts of produced acids from S. mutans, proteins, and polysaccharides.
It was demonstrated that turmeric induced membrane permeabilization of bacterial cells, which causes cell damage (Gera et al. 2017). Furthermore, when curcumin was used in different surfactants formulas, it showed a photo-sensitizing efficacy during antibacterial photo-dynamic treatments. The mechanism by which curcumin resulted in photo-induced bacterial cell death has not yet been fully recognized. However, it is believed that binding of photosensitizer to the outer membrane of microbial cells is an essential requirement for microbial photosensitization (Jori and Coppellotti 2007). This might explain the interaction between the turmeric and diode laser which has a significant bactericidal effect against both tested bacteria in our study.
Whereas garlic is a powerful antibiotic. It is a reliable antimicrobial agent, and it can inhibit gram-positive and gram-negative bacteria (Kshirsagar et al. 2018). The main antibacterial mechanism of garlic is through interaction with the enzymes responsible for bacterial metabolism and nutrition (Bakri and Douglas 2005).
Padiyar et al. (2018) demonstrated the effectiveness of garlic mouthwash against S. mutans bacteria which could be due to its antioxidant sulfur compound “allicin” which can easily diffuse inside the bacterial cells through their membranes and bind the sulfur groups to the bacterial enzymes and proteins, causing modification and inhibition of the bacterial activities.
Nonetheless, a former study inspected the antibacterial and antioxidant properties of ginger, turmeric and garlic spice extracts and it was concluded that turmeric recorded higher antioxidant and antibacterial activity than garlic (Panpatil et al. 2013). These results agreed with our study. They owed their results to the higher amounts of polyphenolic antioxidant compounds of turmeric than garlic. Moreover, Sofia et al. (2007) investigated the antibacterial effect of different Indian spices including garlic and clove. They revealed that 3% clove extract displayed a broad antibacterial activity against all investigated bacteria than garlic, as it can be concluded from our results.
It worth mentioning that the mechanism of antibacterial activity of clove, turmeric and garlic nano-herbal extracts combined with lasers irradiation is not entirely fulfilled and require further research. The therapeutic potential of different herbal formulas is now justified. Therefore, the urge of using natural plants as an alternative medicine will not only decrease clinical implications of bacterial drug resistance but also total cost and unwanted drugs side-effects. However, more studies to evaluate different clinical effects of herbal extracts are recommended for teeth remineralization and prevention of different oral diseases.