# Study of the Antimicrobial Effect of an Ethanolic Extract of Propolis in Periodantal Disease
## 2 Materials and Methods
### Participants and Enrolment  
This is a randomized, controlled, parallel, double-blind study conducted at the Faculty of Dentistry, University of Granada. The study was approved by the Ethics Committee of the University of Granada (reference 819). Forty patients were included in the study (Figure 1). Written consent was obtained from all the subjects and the treatment procedure was explained to the patient before treatment was given. The inclusion criteria were: Patients have explicit consent, need tooth extraction with advanced periodontal disease, and have not received active periodontal treatment in the last 3 months, bleeding of two or more teeth (bleeding on probing positive, BOP+), depth of the periodontal pocket (probing pocket depth, PPD \(\geq\) 4 mm), significant tooth extraction, highly mobile teeth, and age between 50-60 years. Exclusion criteria were: lack of informed consent, systemic illness, allergy to one of the products tested, pregnancy or lactation, use of antibiotics or anti-inflammatory drugs. After enrolment, patients are randomly assigned to test or control groups using a computer-generated sequence. All clinical measurements were performed by the same researcher. This study included two groups. In the control group (\(n\) = 20), the site was treated scaling and root planning only, and in the experimental group (\(n\) = 20), the site was treated by scaling and root planing followed by subgingival placement of ethanolic extract of Spanish propolis (EEP) (Figure 2). At the first visit, all patients had clinical measurements, bleeding on probe positive (BOP+), Periodontal pocket Deep (PPD) and Clinical attachment level (CAL). We selected the 5 deepest locations, removed the plaque on the surface with a cotton swab, and dried the area. Subsequently, a glass cylinder was inserted into the gum line for 1 min to avoid contact with the remaining oral tissue.  
The cylinder was then removed and placed in a test tube, air-dried for a few minutes with the cap open, and the sample was used for qualitative and quantitative microbiological studies by PCR of the following microorganisms: _A. actinomycetemcomitans, T. forysythemsis, P. gingivalis, P. intermedia_ and _T. denticola._ Periodantal ultrasound treatment was performed on each surface for 30 s. The device used was a Piezon(r) Master 400 from EMS Swiss Instrument Perio Slim, the power was adjusted to 75%. Polishing was done with Perio-Set(r) and rubber cups with low abrasive paste (Kerr Hawe(r)). The EEP gingival sulcus was then washed using a 3 mL UEETEK(r) graduated pipette in the Vitulia 0.9% physiological test and serum as a placebo in the control group. There was no anaesthesia or antibiotic treatment during the surgery.  
Figure 1: Flowchart diagram showing the process of the study.  
Figure 2: Study design. The registered participants were 40 chronic periodontitis patients who had signed the informed consent. They needed periodontal maintenance therapy and had at least two teeth with bleeding on probing positive (BOP+), probing depths equal or greater than 4 mm (PPD \(\geq\) 4 mm) at baseline (BL). Subgingival plaque samples were obtained with sterile vials from test and control sites at baseline and 1-month follow-up.