## 3 Results
### Comparative Analysis of Biochemical and Microbiological Parameters
These bacteria are killed continuously by various innate immunological cells like PMNs, which account for more than 90% of GCF cells. The cPMNs are considered to bethe frontiers for this battle, as they form a barrier between the junctional epithelium and sub-gingival biofilm [18; 19; 30]. It is believed that any defect and/or delay in PMN recruitment and its microbicidal action will compromise periodontal health [31]. Periodontal disease becomeseven more compromised in the presence of adverse habits. It is well documented that tobacco consumption is a risk factor for developing periodontal disease [32], while the influence of area nut is still not apparent. Thus, the aim of our study was to explore area nut as a probable risk factor that can alter the bactericidal activity of neutrophils in healthy and CP patients. In the present study, the cellular viability of cPMNs was found to be inthe range of 90-92% in both groups, with no significant differencesbetween intergroup and intragroup comparisons. This implies that the concentration of area nut and reagents did not affect the cellular viability of cPMNs. The cellular viability of oral PMNs without any additiveswas found to be approximately 95% [33]. However, 84-85% of cellular viability of cPMNs was reported when treated with ANE [23]. Studies with peripheral blood PMNs reported a cellular viability of 85-95% when treated with ANE and arecoline [20]. An association hasbeen reportedbetween area nut chewing andclinically active periodontal disease sites showing bleeding on probing (BOP) [34], as well as clinical attachment loss (CAL) [8]. The subgingival plaques atthese sites have beenrecorded to harbour periodontal pathogens such as Aa and Pg [35]. Nevertheless, Aa is also found in the buccal mucosa of 36% of the healthy population [36]. The serotype c of Aa (AATCC 33384) is prevalent in the Indian population of healthy (16.7%) and CP patients (41.2%) [37]. Thus, in the current study, this strain was employedto check the bactericidal activity of cPMNs in health and CP. Most of the methods employed to measure bactericidal activity were indirect, as they evaluated the metabolic burst. [PERSON] et al. 1964 [38] reported adirect way of measuring bactericidal activity. This was achieved by plating the bacteria recovered from neutrophils after phagocytosis, and was measured in CFU. The same technique was employed in our study to assess bactericidal activity. Within individual groups, HBSS-treated cPMNs showed the least impact on CFU. Meanwhile, CFU were less numerous in the CP group than in the healthy group treated with HBSS. This observation could be because of the hyperactivity of the bactericidal activity of PMNs observed in the CP group [39; 40]. In both groups, the rANE-treated cPMNs showed a higher number of CFU on plating compared to those treated with tANE. The probable reason for this is the difference in the concentration of alkaloid/phenolic compounds. It was found that the concentration of catechin was 16 times higher in rANE than that in tANE, but investigations into the concentrations of other phenolic compounds in rANE and tANE are still underway [22]. Thus, it cannot be said affirmatively that these constituents of area nut act synergistically or have an independent function in retarding the bactericidal activity. Nevertheless, similar results were obtained with peripheral blood neutrophils [20]. A comparable reduction in CFU, although non-significant, was seen with the different extracts of area nut (rANE, tANE) and catechin between the groups (Figure 3). This suggests that the area nut and catechin altered the activity of cPMNs, thereby impairing their ability to kill microorganisms. PMNs may kill bacteria through several mechanisms, including the release of toxic oxygen metabolites (superoxide anions, H\({}_{2}\)O\({}_{2}\), etc.) and the extracellular release of antimicrobial substances [41]. We decided to undertake H\({}_{2}\)O\({}_{2}\) production for the assessment of the oxidative killing of microorganisms.