Increasing awareness about chemical agents and drug risks and adverse effects fuels the demand for natural and readily available oral health care products. One of the most gained assets derived from dental practice is the potential to prevent periodontal disease by a simple but effective measure through daily dental plaque control. Although CHX has strong clinical and microbiological effects, it is reported to have many undesirable side effects [11]. On the other hand, N. Sativa may be a safer natural candidate for maintaining oral health, fostered by its antimicrobial and anti-inflammatory effects [14]. Previous studies have compared the effect of CHX and essential oils on plaque index and gingival index [19, 25]. In some cases, clinical trials assessing CHX were conducted for 2 weeks or less [19, 26], and others for longer [27, 28]. We chose a 14-day study term based on the standard prescribed duration of use for CHX. To our knowledge, this is the first study to investigate the effect of NS oil in vivo on patients with gingivitis while assessing gingival health, microbial load, and changes in the biofilm.
The NS oil used was an edible cold-pressed oil that preserves the phytochemicals to a greater degree; it is reported to have high levels of thymoquinone and other volatile and non-volatile active components [29]. Oil pulling is an ancient ayurvedic therapy for maintaining oral hygiene [16]. In the current study, a 1:1 ratio of NS oil to water was used to facilitate the mouth rinsing process and dilute the strong bitter taste.
Both NS and CHX mouth rinse significantly reduced gingival and plaque scores, although CHX showed a greater efficacy. One study showed clinical improvement of gingivitis in diabetic mice treated with N. Sativa seed oil [30]. Furthermore, in a clinical split-mouth study with 24 patients having moderatetosevere gingivitis, the efficacy of ethanolic extract of N. Sativa was tested and proved effective in treating moderate to severe gingivitis [31]. Furthermore, intracrevicular application of 0.2% thymoquinone gel in patients with periodontitis showed potential as an adjunct to SRP in treating chronic periodontitis [32]. Interestingly, the NS group showed a remarkable reduction of the gingival and plaque index scores, thus highlighting its ability to interfere with the bacterial host interactions, most likely influencing the gingival tissue via its anti-inflammatory effects. Furthermore, NS oil appeared to be equally efficacious and comparable to the gold standard chlorhexidine at reducing clinical parameters.
Although bacteria are the primary etiologic factors in periodontal disease, the patient's host response determines disease susceptibility and severity. In the current study, evaluation of IL-6 and IL-18 in the GCF was considered a useful indicator of periodontal disease and response to treatment. We found no significant difference in the efficacy of the interventions NS and CHX at reducing GCF inflammatory cytokines IL-6 or IL-18. However, there was a significant reduction in IL-6 within the NS group comparing baseline to day 15, but no effect on the IL-18 levels for both interventions. An important feature to note is the significantly higher levels of the IL-18 than the IL-6 levels found in the gingiva of the patients, which may be a sign of the degree of inflammation occurring in mild to moderate gingivitis. Other reports show that IL-6 levels are higher in chronic inflammatory diseases such as rheumatoid arthritis and periodontitis and were related to an increased risk of periodontitis and appeared to be integral in promoting periodontitis [33]. Therefore, it may be expected for IL-6 to be lower than the IL-18 in the case of gingivitis. CHX has previously been reported to have cytotoxic effects on different cell types such as gingival fibroblasts, epithelial cells, and neutrophils [34, 35] and displayed genotoxic effects [36]. CHX induced pro-inflammatory cytokines (IL-6, IL-18, and others) in odontoblast cell lines [37] and caused oral mucosal irritation [38]; thus, CHX appears to have no intrinsic anti-inflammatory effect, and its clinical anti-inflammatory effect seems to be via its strong effect as an antimicrobial agent. However, NS has cytoprotective and intrinsic anti-inflammatory effects [15]. Other studies showed that SRP effectively reduced GCF levels of IL-18 from inflamed periodontal sites in chronic periodontitis patients [39]. Therefore, SRP may be required for effective reductions of IL-18. The present study was limited to 14 days, and a more extended treatment period may be needed for NS oil to observe its anti-inflammatory effects fully.
Intervention with NS oil and CHX caused a significant reduction in the CFU, demonstrating NS's effectiveness in significantly reducing bacterial load, although CHX was better. It is well documented that CHX mouthwash is associated with a significant change in the salivary microbiome because of its high acidity and low availability of nitrates [40]. Changing the microbial environment could be the main reason for the shifting in microbial density associated with CHX. Therefore, a significant reduction in CFU was expected with the use of CHX. Furthermore, NS oil had antimicrobial activity against microbes isolated from periodontal patients (buccal surface) [4]. Other studies proved NS oil to be effective against pathogenic periodontal bacteria, P.gingivalis [18], and bacteria that make up the early biofilm [16]. Its active constituent thymoquinone was highly effective against oral biofilm formation [41]. NS and CHX interventions provided incomplete removal of pathogenic species; S. parasanguinis, S. sanguinis, S. mitis, S. oralis, and a host of non-pathogenic species initially present in the supra-gingival plaque of the patients. The presence of S. parasanguinis has always been associated with dental caries [42]. In contrast, S. mitis and S. oralis could indicate an early-stage decay[40] and periodontal diseases [43]. After treatment with NS, a 20% reduction in the number of patients with pathogenic bacteria was observed, and the pathogenic species were limited to S. mitis and S. parasanguinis. In the CHX group, there was a 50% reduction in the number of patients with the pathogenic bacteria, and the detected species after treatment were only S. sanguinis and S. parasanguinis. Although not significant, our findings show the effect of the different interventions on the qualitative alterations of the biofilm, which can be expected due to the innate differences in the properties of these interventions.
Together, our study identifies no difference in clinical efficacy between NS and CHX in primary assessed outcomes of GI and PI measurements and levels of IL-6 and IL-18. Although NS was efficacious at reducing bacterial load, CHX was significantly better. Thus, NS oil may be an alternative therapeutic option in treating mild to moderate gingivitis.
There were limitations to the study. A more extended observation period or an increase in NS active ingredient concentration could be required for more anti-bacterial therapeutic efficacy. Traditional oil pulling utilizes undiluted oils; however, since 10% NS oil was effective as an anti-microbial against P. gingivalis [18], we proposed to use a 50% oil concertation to dilute the bitter taste and reduce non-compliance by our participants. An 87% power of the study validates the number of participants used in this clinical trial, but a larger number would improve the reliability of the data overall. Since convenience sampling was conducted at the dental clinic, which had a greater number of female, registered patients, the entire sample in the current study consisted of females. However, the intention was not to limit the study to the female population. It is well known that hormonal fluctuations can influence gingival inflammation. Variations in gingival inflammation and GCF IL-6 levels occur in women at different times of the menstrual cycle [44]. Higher levels of progesterone and estradiol which occur during pregnancy may cause greater gingival inflammation [45]. Similarly, women taking hormonal contraceptives may experience greater gingival inflammation [46]. Although we had excluded pregnant females from our sample, other cyclical hormonal changes in our study participants may have impacted the findings. Therefore, our findings shed light on the effect of mouthwash in this susceptive and diverse population. The study also relies on the individual participant to mix a precise ratio of N. Sativa oil to water and then rinse for 3 min. Although not reported, it could be that inaccuracies in the ratio of NS to water may have occurred, which would lead to skewed results. The study also relied on self-reporting to identify the level of compliance. We hope we can limit the degree of bias by having a more controlled clinical trial in the future. No adverse effects were reported from any of the participants in the study, and thus, our findings indicate that 50% NS oil in a water mix may be safe for short-term use for up to 14 days.