CLINICAL AND BIOCHEMICAL EVALUATION OF THYMOQUINONE GEL IN THE TREATMENT OF CHRONIC PERIODONTITIS

Ahmed Abdallah Khalil 1 and Eman Alaaeldin 2,3 . 1. Lecturer of Oral Medicine, Oral Diagnosis and Periodontology, Faculty of Dentistry, Minia University, Egypt. 2. Department of Pharmaceutics, Faculty of Pharmacy, Minia University, Egypt. 3. Department of Clinical pharmacy, Faculty of Pharmacy, Deraya University, Minia, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 03 February 2019 Final Accepted: 05 March 2019 Published: April 2019

Bacterial pathogens stimulate host cells to release pro-inflammatory cytokines (e.g., interleukin-1 and tumor necrosis factor-α) as part of the immune responses (Sculley DV, Langley-Evans SC 2003). These cytokines recruit polymorphonuclear leukocytes to the site of infection (Lamont RJ, Jenkinson LRJ, 1998). Inflammatory mediators are important to the pathogenesis of periodontal diseases and may be used as diagnostic markers. Interleukin (IL)-1 is present in two active forms, IL-1α and IL-1β. Both are potent Pro-inflammatory molecules and are the main components of osteoclast activating factor (M Mogi, 1999).Interleukin (IL)-1 is produced by macrophage, lymphocytes, monocyte, dendritic cells, lymphocytes, neutrophils, endothelial cells and fibroblasts (Stashenko P et al., 1991). Interleukin-1β (IL-1 β) is a potent Proinflammatory cytokine which plays a major role in inflammation and bone resorption, therefore, it becomes an important parameter in periodontal research (Bergmann A, Deinzer R., 2008).
Reactive oxygen species (ROS) have been implicated in the pathogenesis of many diseases, including rheumatoid arthritis (Mapp PI et al., 1995), chronic obstructive pulmonary disease (Macnee W, Rahman I, 1999), atherosclerosis (Halliwell B, 1993) and periodontal disease (Teng YT., 2003). Upon stimulation by bacterial antigens (e.g., lipopolysaccharide), polymorphonuclear leukocytes produce reactive oxygen species (ROS) via the oxidative burst and released into the extracellular environment. As the released ROS was not target-specific, damage to host tissue also occurs. (Sculley DV, Langley-Evans SC 2003).
When ROS attacks proteins and lipids, a series of nonenzymatic reactions occur to produce a large variety of intermediate and end products. Oxidative stress (OS) may be evaluated either by observing by-products of the interaction of ROS with lipids, proteins, and DNA or by examining alterations in total antioxidant capacity (TAOC) (Chevion M et al., 2000).
The total antioxidant capacity (TAOC) has been widely used to investigate OS in periodontal disease. It is an integrated parameter that reflects the cumulative activity of nonenzymatic antioxidants present in the plasma and body fluids (Baser U et al., 2015).
Many treatment modalities are available to achieve the goal of periodontal therapy. This includes nonsurgical periodontal therapy, such as scaling and root planing (SRP) alone or SRP plus systemic or local antimicrobial/antiinflammatory agents, and surgical periodontal therapy (Alam S et al., 2009).
Nigella sativa (NS), commonly known as black seed or black cumin, is an annual plant belonging to the Ranunculaceae family. Nigella sativa seeds contain diverse but well-characterized chemical components, many of which are capable of inducing beneficial pharmacological effects in humans (M. A. Randhawa and M. S. Al-Ghamdi, 2002).
Thymoquinone (TQ) is the major biologically active compound of N. sativa, is known as the bioactive constituent of the volatile black seed oil. TQ exhibits many pharmacological properties, which include antibacterial, antihistaminic, anti-inflammatory and hypoglycemic actions (Kara MI et al., 2012& Taka E. et al., 2015. TQ also displays an antioxidant response through its potent superoxide-scavenging ability (Nader MA et al., 2010). The purpose of this research was to investigate the effect of TQ gel on the clinical periodontal parameters as well as the level of IL-1β and total anti-oxidant capacity levels in gingival crevicular fluid of chronic periodontitis patients.

Subject selection
This study was conducted on 68 subjects of both sexes with age ranged 25-58 years old. Forty eight patients (19 males and 29 females) were clinically diagnosed with moderate to severe chronic periodontitis with at least 10 periodontal pockets with minimum depth 5mm, and twenty healthy subjects were chosen as volunteers for negative control group. The patients were selected from the out patients clinic of the Oral Medicine, Oral Diagnosis, and Periodontology Department. Faculty of Dentistry, Minia University.
All subjects were free of systemic disease and had not received any type of periodontal therapy, prescribed antibiotics or anti-inflammatory medication within the preceding 6 months. Pregnant, lactating females and smokers were excluded from the study.

Ethical regulations:
The complete treatment plan was explained to all patients including detailed steps, risks, and expected results and their full signed consent was obtained prior to entry into the study. The study was complied with the rules set by the International Conference on Harmonization Good Clinical Practice Guidelines, and the Declaration of Helsinki and the research ethics committee of the Faculty of Dentistry, Mini University.

Preparation of Thymoquinone gel:
Thymoquinone gel was prepared in the department of pharmaceutics, faculty of pharmacy, Minia University. Chitosan gel of thymoquinone was prepared as follows: half of water required to form the gel was added to aqueous acetic acid (1 % v/v). The pre-weighed amount of chitosan was added gradually to the prepared acetic solution and was left to swell. The remaining amount of water was added to the swollen gel and mixed to form a final concentration of 1.8 % (Mostafa, M et al., 2018). The formed gel was allowed to stand for 24 hour at room temperature before further treatment. Finally, about 5.5 ml of freshly prepared aqueous thymoquinone suspension was dispersed into 10 gm of chitosan gel to give a final TQ concentration of 0.1 % (w/w). 86 9. The strips were individually placed in plastic Eppendorf and stored at -20C until be used for the laboratory analysis. 10. GCF from filter paper strips was diluted 1:2 -1:20 into Reaction Buffer ultrafiltered through a 10,000 MWCO filter and used directly in the assay. The buffer used is deionized or distilled water. Samples were incubated and centrifuged at 4°C. 11. Commercially available Enzyme linked immunosorbent assay (ELISA) kits were utilized, and assays were applied according to the manufacturers' instructions to examine the level of IL-1β and TAOC (Fig. 2). 12. The GCF sample was also collected from group III (control group) to determine the level of IL-1β and TAOC at baseline only.

Results:-
This study was conducted on 68 subjects of both sexes with age ranged 25-58 years old. Forty eight patients (19 males and 29 females) were clinically diagnosed with moderate to severe chronic periodontitis. The patients were selected from the out patients clinic of the Oral Medicine, Oral Diagnosis, and Periodontology Department. Faculty of Dentistry, Minia University. The 68 subjects were grouped into three groups; Group I (TQ group): received nonsurgical periodontal therapy and TQ gel, Group II: only non-surgical periodontal therapy and Group III: 20 healthy subjects.

The results of Clinical parameters:
The clinical parameters including plaque index (PI), gingival index (GI), probing depth (PD) and clinical attachment level (CAL) were recorded at baseline (prior to treatment) and at weeks 4 and 12 after treatment in group I and II.
Both groups showed significant improvement regarding all clinical parameters from baseline to 4 and 12 weeks after treatment.
The changes in the clinical parameters of the two studied patients groups are summarized in tables (1, 2).      , which was statistically significant (p = <0.0001). Additionally, after 12 weeks IL-1 β level had been reduced to 106.4 ± 9.50 pg/µl L, which was statistically significantly different to baseline and 4-weeks after treatment.

Comparison between level of IL-1β between group I and group II:
Comparison between level of IL-1β between group I and group II at different intervals was shown in table (7). Comparison of IL-1 β level between the three groups was shown in table 8 & 9.

Results of TAOC in group I:
Descriptive changes in the mean values and stranded deviation of TAOC at baseline and after periodontal therapy at different intervals in group І were shown in table (10). Comparison of the mean difference of TAOC levels at different intervals were shown in table (11)

Results of TAOC in group II:
Descriptive changes in the mean values and stranded deviation of TAOC at baseline and after periodontal therapy at different intervals in group І were shown in table (12). Comparison of the mean difference of TAOC levels at different intervals were shown in table (13)  12.62 (µm/l), respectively), which was statistically significant (p = <0.0001). Additionally, after 12 weeks TAOC level had been increased to 762.9683± 12.84 µm/l, which was statistically significantly different to baseline and 4weeks after treatment.

Comparison between level of TAOC between group I and group II:
Comparison between level of TAOC between group I and group II at different intervals was shown in table (14).
Comparison of TAOC at baseline between the three groups was shown in table 15 &16

Discussion:-
Periodontitis is an inflammatory disease leads to local elevation in levels of pro-inflammatory cytokine which plays a vital role in the process of inflammation associated with the destruction of the periodontium (Teng YT. 2006, Reher VG. et al., 2007. IL-1β is a cell immune response mediator released as an outcome of bacterial components as lipopolysaccharides. This cytokine increases the neutrophils recruitment and the expression of adhesion molecules and it can result in destruction of periodontal tissue (Lö YJ. et al., 1999). Measurement of IL-1β, in gingival crevicular fluid (GCF) or tissues adjacent to periodontitis-affected sites in patients, has been suggested as a sensitive and remarkable method in monitoring severity of periodontal disease (Hou LT. et al, 1995). Hence, IL-1β would seem an attractive candidate molecule for monitoring periodontal disease initiation, progression, and therapeutic outcome. (McGuire & Nunn,1999) It is a well-known fact that free radicals and reactive oxygen species (ROS) are essential for many normal biologic processes. At low concentration, these free radicals stimulate the growth of fibroblasts and epithelial cells in culture, but at higher concentrations it may result in tissue injury (Battino M. et al., 1995). The measurement of total antioxidant capacity (TAOC) was developed as a cost-effective tool to assess the activity of the whole antioxidant system (Chapple et al., 1997). Assays of TAOC have the advantage of analyzing the combined effectiveness of contributing antioxidant species, which may be greater than the sum of the effects of the individual antioxidant (Maxwell SR et al., 2006) 91 The fact that the bacteria in the subgingival area are organised in a biofilm, which seriously limits the effectiveness of antimicrobials (Brown & Gilbert 1993), so, the use of local drug therapy adjuntively to scaling and root planing is better than as a monotherapy (Tonetti 1997).
Thymoquinone is considered to be the main active ingredient in nigella sativa oil (M. A. Randhawa  To evaluate the effect of TQ gel on the clinical periodontal parameters as well as the level of IL-1β and total antioxidant capacity levels in gingival crevicular fluid, two groups of patients with chronic periodontitis were either submitted to combined pharmacological and clinical treatment (Group I) or single clinical treatment (group II). Periodontal probing depth, clinical attachment level, gingival index and plaque index as well as the level of IL-1β and total anti-oxidant capacity were recorded in the two groups at baseline (prior to treatment), 4 and 8weeks.
Regarding the clinical parameters, a statistically-significant improvement, compared to base line, was found in both groups after 4 and 12 weeks of treatment. Although group I showed more clinical improvement compared to group II, it was not statistically significant.
The clinical improvement in both groups may be attributed to the non-surgical periodontal therapy, employed in the study; since scaling and root planning treatment approach in periodontal therapy is an efficient method to reduce the amount of calculus and biofilm bacteria attached to the sub-gingival root surface. It also induces beneficial changes to the periodontal tissues, as reduction of the gingival inflammation, reduction of probing pocket depth, and gain in clinical attachment level (Adriaens and Adriaens, 2004).
Regarding the biochemical improvement, the elevated base line of IL-1β in patients with periodontitis (groups I, II) compared to that of healthy volunteers (group III) was in consistence with the results of many studies (Orozco A et  Concerning TAOC level, the results of the present study have shown that there was significant increase in the TAOC level after 4 and 12 weeks of treatment compared to baseline in each individual treatment group. When comparing the level of TAOC of group I and group II at baseline with group III, significant differences were found. The data from the present study are in agreement with the results of Pavlica (Pavlica et al., 2004) who found positive relations between plasma TAOC and periodontitis in dogs. Also, our results are consistent with (Brock et al., 2004) who found that the mean total antioxidant concentrations of serum and plasma from patients with periodontal disease were lower than healthy control samples, and the results of Chapple

Conclusions:-
According to our results, non-surgical periodontal therapy improves all clinical and biochemical parameters in both groups significantly. Adjunctive use of thymoqinone gel showed significant improvement in the biochemical parameters after 4 weeks of treatment compared to single non-surgical periodontal therapy. Adjunctive therapy of 92 TQ has failed to maintain that significant improvement after 12 weeks of treatment due to the instability and pharmacokinetics of the drug. Further studies should be established to enhance the physicochemical properties and pharmacokinetics of the drug.