Adjuvant Treatment in Elderly Patients With Periodontitis Through the Administration of a Supplement Rich in Oleuropein From the Olive Leaf

To calculate the sample size, the GRANMO tool (https://www.imim.es/ofertadeserveis/software-public/granmo/) has been used, considering an alpha risk of 0.05, a bilateral contrast, a beta risk of 0.20, a standard deviation of differences of 2.5, a minimum difference of 1 mm reduction in probing depth to be detected between baseline and end of study, and a predicted proportion of loss of 20% tracking.

Treatments All patients will receive a complete oral examination at baseline and after 4 months following the treatment. Initial therapy will be performed in all patients and will consist on full-mouth scaling and root planning by hand and with ultrasonic instrumentation as necessary. This treatment will be completed with oral hygiene instructions. All initial therapy procedures will be performed by the same periodontist.

After periodontal treatment, enrolled volunteers will be randomly assigned to one of two groups by a second periodontist. Control group will receive hard gelatin capsules containing placebo (only the excipients), whereas the experimental group will receive the same hard gelatin capsules but containing 200 mg of an olive leaf extract. The randomization will be done by the use of random number charts (by coin-toss), which will be performed at the time of the first subject's enrollment, and the next subject will be assigned to the other group based on the first subject's assignment. Both placebo and capsules will be dispensed by the same person, and subjects will be given verbal and written instructions on its use. As volunteers will be enrolled they will be allocated sequential study numbers and provided with their capsules, sufficient for one month. Subjects will come to the Faculty every 4 weeks during the course of the trial to replenish their capsules. The capsules intake will last for a total of 4 months. Remaining medications will be checked for compliance. Capsules will be prepared at the CIDAF (Centro de Investigación Sobre Alimentos Funcionales) at the Technological Health Park in Granada. The olive extract will be obtained from Natac company (Madrid, España).

Sample collection and analyses All data collection and determinations will be performed at baseline and after 4 months.

1. Oral examinations Periodontal clinical measurements will include the presence or absence of supragingival dental plaque and gingival bleeding on probing (BOP), as well as periodontal probing depth (PPD) and recession of the gingival margin (GM). Parameters will be measured at six sites (mesiobuccal, buccal, distobuccal, distolingual, lingual, and mesiolingual) on every tooth by means of a calibrated periodontal probe (Hu-Friedy®, Chicago, IL, USA), 15 mm in length and 0.35 mm in diameter. Clinical attachment level (CAL) will be calculated by adding recession to PDD. The number of remaining teeth will also be recorded. O´Leary's plaque index, Van der Velden's bleeding index will be also calculated. Patients with BOP site <10%, PPD ≤3 mm, erythema and edema absence will be considered periodontal-healthy subjects.
2. Sociodemographic data collection
3. Basic sociodemographic data: these will include age, gender, years of education.
4. Financial status: participants will be asked to report their mean income during the previous three years using a four-point scale (low, inadequate to cover daily expenses = 1, medium, trying hard to cover daily expenses = 2, good, adequate to cover daily expenses = 3, very good, very adequate to cover daily expenses = 4).
5. Smoking status: current smokers will be defined as smokers at the time of the interview. Former smokers will be defined as those who had previously smoked, but had not done so for a year or more. The remaining participants will be defined as occasional or non-current smokers.
6. Physical activity level: it will be evaluated in metabolic equivalent (MET)-minutes per week, using the self-reported International Physical Activity Questionnaire (IPAQ). Those who reported at least 3 MET-minutes per week will be classified as physically active (minimally active - or "health enhancing physical activity (HEPA) active"). All others will be defined as physically inactive. Furthermore, the weekly frequency of physical activity will be recorded.
7. Last month's depression symptoms: they will be assessed using a validated shortened, self-report Geriatric Depression Scale (GDS) (range 0-20). A GDS score between 0 and 4 will be used to indicate no depression, while scores of 5-10 and N 10, will indicate mild and severe depression, respectively.
8. Healthcare use: the presence of private physicians' offices, public or private health care centers or hospitals in the area of residence, as well as the annual number of visits for regular check-up of the health status, will be recorded.
9. Adult's social participation: weekly frequency of their social activities with their family, their friends as well as their yearly frequency of excursions will be recorded for this purpose.
10. Anthropometric characteristics and blood pressure
11. Weight, height and waist circumference will be measured. BMI and waist-to-hip ratio will be calculated. Overweight will be defined as BMI between 25.0 and 29.9 kg/m2, while obesity will be defined as BMI >29.9 kg/m2.
12. Blood pressure: patients with blood pressure levels>140/90 mmHg or using antihypertensive medications will be classified as hypertensive.
13. Dietary habits assessment The evaluation of dietary habits will be based on a semi-quantitative food-frequency questionnaire (FFQ). FFQ will be used to evaluate the level of adherence to the Mediterranean diet, the MedDietScore (theoretical range 0-55) was used.
14. Blood sampling and analysis Blood samples (16 mL) will be collected by forearm venipuncture after 12 h over-night fasting, without stasis in the sitting position. Whole-blood samples will be used for hematological evaluations. Most part of blood will be drawn into EDTA-containing tubes (1 mg/ml) and plasma will be isolated by centrifugation at 1000 xg, for 10 min and divided into aliquots of 250 μl for immediate analysis or for storage at -80 °C. Blood also will be used for obtaining isolated blood mononuclear cells (BMCs) for other additional analyses. Aliquots containing both, plasma or BMCs, will be preserved at -80º C until analyses.

Blood analyses will be as follows:

1. Biochemistry evaluations: fasting blood lipid levels (including high-density lipoprotein-cholesterol (HDL), low-density lipoprotein-cholesterol (LDL), direct LDL-cholesterol, very low density-cholesterol (VLDL) and triglycerides (TG), plasma glucose, high-sensitive C-reactive protein, LPa, cortisol, fibrinogen and homocysteine levels will be determined in plasma using an automatic analyzer. Patients showing fasting blood glucose > 126 mg/dL, using of antidiabetic medication or with a prior diagnosis of diabetes will be determined as Type 2 diabetes mellitus in accordance with the American Diabetes Association diagnostic criteria. Hypercholesterolemia will be defined as total serum cholesterol levels >200 mg/dL or use of lipid-lowering agents, according to the National Cholesterol Education Program Adult Treatment Panel III guidelines.
2. Hematological study: haemogram and blood cell profile in whole blood samples.
3. Circulating pro-inflammatory markers: circulating levels of multiple inflammatory cytokines including IL-1β and IL-18 will be analyzed by a commercial multiplex kit (Cytokine Panel 1B 25plex, Thermo Fisher Scientific). In addition, protein levels of active caspase-1 and gene expression of Nlrp3 (CIAS1) and caspase-1 will be determined in isolated PBMCs by Western-Blotting and RT-PCR, respectively, to evaluate inflammasome activity.
4. Autophagy markers: MAP-LC3, ATG12, p62, TFEB, Beclin-1 and B and D cathepsines levels will be determined by Western-Blotting in isolated PBMC protein isolates. Beclin-1, y Maplc3 gene expressions will be determined by RT-PCR in isolated PBMCs using beta-actin as housekeeper gene.
5. Oxidative stress markers and antioxidant capacity: damage to lipids will be analyzed through the determination in urine of 15-F2 isoprostanes, damage to proteins by determination of carbonyl proteins in plasma68 and damage to DNA through the determination in urine of 8-hydroxyguanosine (8OHdG). In addition, the determination of the in situ redox state will be performed in the subjects at the clinic by using a blood drop from a finger. It will be carried out through the Redoxsys system (Aytu BioScience, Inc., Englewood, USA), which also measures the antioxidant capacity of the sample. This is a novel system that measures with a reliability before not available the total redox charge in biological samples as well as the antioxidant capacity of them.
6. Metabolomic study: to carry out this analyses, the metabolites of the different samples are extracted first. Then the molecules present in the methanolic extract are separated by chromatography. The eluate is monitored in an ESI-Q-TOF-MS / MS system by electrospray98. The analysis of results will be carried out in collaboration with Dr. Reinald Pamplona, from the University of Lleida, with whom we have been collaborating on this issue for years and with whom we share a line of research and publications on metabolomics.
7. Periodontal microbiota study. Subgingival plaque samples will be collected from the three sites with the highest PPDs by putting on the periodontal pocket the tip of a filter paper during 30 s after isolating the zone.

Subgingival plaque samples collected in dry papers will be analyzed by PCR to evaluate the presence of known peridontopathogens: Phorphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Fusobacterium nucleatum. The microbiota studies will be carried out using 16S rDNA mass sequencing techniques. Sequencing and bioinformatic analysis will be performed by the Integrated Microbiome Resource, from the University of Dalhousie, in Canada.

1. Determinations in urine.

Fasting urine will be collected and the following determinations will be performed:

1. Oxidative damage markers: 8-hydroxy-2-deoxyguanosine and total F2-isoprostanes will be measured in urine by a competitive enzyme immunoassay. Results will be normalized to urinary creatinine.
2. Oleuropein metabolites concentration: they will be quantified by LC-ESI-MS/MS as in previous studies.
3. Successful Aging Index calculus Successful aging index (SAI), with potential scores ranging from 0 to 10, will be employed to evaluate aging for study participants. The full index encompasses education, financial status, physical activity, BMI, GDS score, participation in social activities with friends, with family, yearly excursions, CVD risk factors score and Mediterranean diet adherence level to develop the index. A cumulative variable (range 0-4) indicating the overall burden of classical cardiovascular disease risk factors (i.e., obesity and history of hypertension, diabetes and hypercholesterolemia) will be developed (participants having none of the aforementioned risk factors will receive score 0, having one factor score 1, etc.).
4. Intervention success criteria To assess the success of the intervention with oleuropein, the reduction of 1 mm or more in probing depth (PD) will be taken as the main variable. In addition, and as secondary success criteria, a reduction of 1 mm or more in the attachment level will be taken, as well as a reduction of more than 20% in gingival bleeding on probing.
5. Statistic analysis A descriptive analysis of the main variables studied will be carried out. For normal quantitative variables, the mean and standard deviation will be calculated, or the median and percentiles in the case of non-normality. Qualitative variables will be expressed using absolute and relative frequencies. The normality of the variables will be checked with the Kolmogorov-Smirnov test in order to determine the need to carry out transformations (logarithmic and others) before carrying out the comparison studies. To study the effect of treatment on the different variables in the same group, the Student's t-test for related samples will be used. To carry out comparative analyzes at the same experimental time between both experimental groups, the Student's t-test for independent samples will be used.