## 4 Discussion
While it has been reported that the immediate bond strength of the etch-and-rinse adhesive material post chlorhexidine application remains unaffected [64; 65; 66], a study by [PERSON] et al. [67] highlighted that the combined application of 2% chlorhexidine and etch-and-rinse adhesive material leads to statistically significant decreases in bond strength values in the short term. Subsequent to a 37% phosphoric acid application, attempts were made to remove chlorhexidine from the surface of chlorhexidine-treated dentin tissue using an absorbent cotton pellet, yet the hydroxyapatite within the dentin tissue continued to harbor chlorhexidine remnants. Through X-ray photoelectron spectroscopy, a study by [PERSON] et al. [68] determined that chlorhexidine residues persisted on acid-etched hydroxyapatite surfaces, possibly undermining the subsequent primer's binding capacity. Moreover, [PERSON] et al. [69] demonstrated through SEM analysis that the detrimental effects of chlorhexidine applied after acid treatment, such as in plug formation and debris accumulation in the dentin canals, impeded the formation of resin extensions in the desired dimensions. In present study, it was considered that the low bond strength values observed in the short term in the groups where chlorhexidine was applied may be attributed to these reasons. Research has demonstrated that a-tomatine, a representative glycoalkaloid present in the roots, stems, and leaves of tomatoes, plays a crucial role in countering potential pathogens and is universally found across tomato genotypes [70]. It comprises a tomatoidine (aglycone moiety), 8-licotetraose (tetrasaccharide moiety) containing two glucose molecules, a xylose, and a galactose [71; 72]. a-tomatine has been found to exhibit the suppression of certain cancer cell lines [73; 22; 74]. Notably, it significantly inhibits MMP-2 and -9 activation compared to untreated positive controls [9; 75]. Additionally, a-tomatine, known for its antiproliferative activity, can restrain the growth of cancer cells, creating cytotoxicity and suppressing lymphoma and lung cancer cell proliferation [76; 77]. When combined with anti-cancer drugs, tomatine has been reported to be able to hinder cancer cells' spread via curbing MMP activation [9]. Some studies have also indicated its antibiotic activity against diverse microorganisms [78; 79; 80]. Although, in the medical field, a-tomatine has demonstrated the capacity to inhibit MMP-2 and -9 release [9; 76; 77], it has yet to be utilized in dentistry. This study marks the inaugural application of a-tomatine as an MMP inhibitor in dentin tissue. A pilot study was required to determine the concentration and duration of application of a-tomatine on the dentin surfaces before starting the study. Regarding the application duration, when examining studies related to CHX in the literature, it was known that the CHX application time is a crucial factor for bond strength. Therefore, durations of 20 and 30 s, based on the application time of CHX, which are among two of the most commonly used values, were selected for a-tomatine [81]. A preliminary investigation involving the dentin surfaces of six sound teeth was conducted to ascertain the appropriate molarity and application duration for a-tomatine. 0.75-1 and 1.5 uM of tomatine solutions, previously reported as effective against MMP enzymes, were prepared following suitable conditions and formulations [9; 10; 82]. Subsequent to applying the a-tomatine solutions at three distinct uM levels for durations of 20 and 30 s, microdensile bond strength values were assessed and subjected to statistical analysis. The findings led to the selection of 1.5 uM of a-tomatine, with a determined application time of 20 s to dentin surfaces. a-Tomatine impedes the transport and intracellular signaling of specific DNA cues facilitating MMP-2 and -9 enzyme production [76], and it hinders the formation of these enzymes by obstructing mRNA structures [10].