Clinical trial NCT05138484: Clinical Evaluation of a Mouthwash Containing Malva Sylvestris Extract.
Official title: Clinical Evaluation of a Mouthwash Containing Malva Sylvestris Extract and Its Role in Reducing Oral Biofilm and Gingival Inflammation. A Randomized, Triple Masked Clinical Trial.
Study type: INTERVENTIONAL
Overall status: COMPLETED
Phase: PHASE3
Conditions: Periodontal Diseases
Keywords: Periodontal diseases
Inflammation
Malva sylvestris
Mouthwashes
Lead sponsor: Aravena, Pedro, DDS, PhD
Sponsor class: OTHER
Collaborators: name: Universidad Austral de Chile
First posted: 2021-12-01
Enrollment: 54
Sex: ALL
Minimum age (years): 18
Maximum age (years): 75
Standard ages: ADULT
OLDER_ADULT
Accepts healthy volunteers: no
Allocation: RANDOMIZED
Intervention model: PARALLEL
Intervention model description: Two examiners recorded the data: sex, age, hygiene habits, systemic diseases, use of drugs and smoking.

Clinical examination and registration of clinical indexes: Basic Periodontal Examination, Gingival Bleeding Index, Bacterial plaque index.

Another investigator performed, through simple randomization, the patient's assignment to one of the three parallel study groups:

G.1 (Positive control 0.12% chlorhexidine digluconate) G.2 (Negative control vehicle mouthwash) G.3 (experimental group 10% mouthwash of M. sylvestris extract). To each patient was delivering a 150 ml bottle with the corresponding mouthwash. The indications for posology were: rinse with 10 ml of mouthwash for 1 minute in the mouth, every 12 hours for 7 days. To the follow-up of the intervention, was made telephone calls at least once a day to remember the rinse.

The re-evaluation was performed 7 days after the first appointment.
Primary purpose: TREATMENT

Brief summary: Background: For centuries, plants (and / or their products) were the only resource available for the prevention and treatment of many diseases. However, its indiscriminate use without phytochemical, pharmacological and toxicological knowledge is a concern for health. The Malva sylvestris (family Malvaceae and popularly known as Malva) is mentioned in the literature as an ethnopharmacological medicine with anti-inflammatory, antimicrobial, wound healing and other properties. For this reason, M. sylvestris presents empirical indications in dentistry, mainly in the treatment of periodontal diseases (gingivitis and periodontitis), which are highly prevalent worldwide. However, scientific evidence is scarce in information that supports the biological properties and clinical benefits attributed to it.

Objective: The objective of this study was to evaluate the effect of a mouthwash based on Malva sylvestris in the control of gingival inflammation and dental biofilm.

Methods: A randomized, three-group, triple-masked clinical trial was designed. Patients from the Center of Dental Clinics of the Austral University of Chile participated with a diagnosis of gingivitis and chronic periodontitis. They were distributed randomly in three study groups: 1. Chlorhexidine mouthwash 0.12%; 2. Mouthwash with extract of M. sylvestris and 3. Mouthwash control group. The indications and dosage were identical for all groups: rinse with 10 ml, for 1 minute, every 12 hours for 7 days. The gingival index and plaque control record were recorded at the beginning and end of the follow-up period (7 days). The results obtained between the groups were compared through normality test and group analysis (ANOVA/Mann-Whitney/Dunnet p \<0.05).

Results: The pharmacological potential of M. sylvestris was determined in the reduction of the plaque control record and gingival index.

Detailed description: Design:

It´s designed a randomized clinical trial of three parallel groups, triple masking. The study and informed consent document were reviewed and approved by the Comité Ético Científico of Servicio de Salud Valdivia (ORD 181/2017) and written according to the CONSORT (EQUATOR Network) writing guidelines.

Population:

The participants were selected from the Department of Oral and Periodontal Surgery of the Dental Clinic Center of the Universidad Austral de Chile (UACh) (Valdivia, southern Chile) during the months of October 2017 to December 2018.

Sample size:

For the calculation of the sample size, the effectiveness of the use of CHX mouthwash was considered over placebo, a standardized mean in the reduction of the gingival index according to Loe \& Silness (1963) parameters of 0.21 (95% CI: 0.1-0.31) (15). Probability of error of 5%, statistical power of 80% and margin of loss of follow-up or absence of data of 20%. The participation of 18 subjects was determined by study group (calculation algorithm: "power two means 1.21 1, sd1 (0.2) sd2 (0.2) knowns", STATA V.14.0, STATASp, Texas, USA).

Preparation of M. sylvestris extract and mouthwash:

For the preparation of the mouthwash of M. sylvestris, the leaves registered in the herbarium of the University of Sao Paulo (USP) were used. Identification number ESA voucher # 121403. The leaves were dried in a forced circulation oven for 24 h, at 40 °C, and then crushed. The extraction was carried out with absolute ethanol under climatic control (25 °C) and subjected to exhaustive extraction (7 days) in a rotating shaker protected against light. In the extraction sequence, the samples were filtered, concentrated by evaporation and lyophilized for 72 h. The EEM was stored at -20 °C. The chemical monitoring was conducted by HPLC MS/MS technique (33). The mouthwash based on M. sylvestris was prepared at a standard concentration of 10% using a 1% ethanol vehicle for preservation of compounds at a concentration compatible with morphofunctional stability (32,33). The control mouthwash presents the same vehicle and composition as the previous one but is exempted from the active compound and the CHX mouthwash was prepared at a concentration of 0.12% with the same vehicle as an accentuated at a concentration of 0.12%.

Periodontal indices record:

Prior to the clinical trial, two examiners (E.A and A.N) underwent a calibration process for the registration of clinical indices (BPE, gingival index and plaque control record) with a specialist in periodontics. To this end, 10 patients from the Center of Dental Clinics of the UACh were evaluated, diagnosed by the specialist with gingival index (GI) over 90% and moderate chronic periodontitis in one of their dental sextants. Inter-examiner and intra-examiner kappa index of 0.89 and 0.80, respectively.

The objective and methodology of the study were explained to the patients who fulfilled the selection criteria and who had already been previously diagnosed. Those who agreed to participate, signed an informed consent document.

On the day of the appointment, the examiners, confirmed the fulfillment of the selection criteria and recorded the data: sex, age, hygiene habits, systemic diseases, use of drugs and smoking.

Then we proceeded to the clinical examination and the following indices were evaluated:

* Basic Periodontal Examination (BPE): The mouth is divided into sextants and using a WHO-standardized periodontal probe, the probing of each tooth is performed in 3 points (mesial, medial and distal) by vestibular and lingual-palatal of each tooth, registering the highest value per sextant, according to the following coding: 0 (healthy tissue), 1 (positive bleeding, absence of periodontal pocket), 2 (presence of tartar, defective fillings, black area of the probe completely visible), 3 (periodontal pocket greater than 5.5 mm) (35).
* Gingival Index (GI): With a WHO-standardized periodontal probe, the buccal, lingual and interdental papillae marginal gingival are probed, using a four-point scale from 0 (absence of inflammation) to 3 (severe inflammation) (37,38).
* Plaque control record (PC): 5 drops of Plaque Revealing Solution are applied, and the patient is instructed to make movements with the tongue in such a way that it touches all dental surfaces in a time of 15 seconds. The vestibular, lingual and interproximal surfaces of all the teeth are then examined. To calculate the index, add the total number of dental surfaces with plaque, divide this number by the total number of surfaces present in the mouth and multiply by 100 (39).

Randomization and use of mouthwash:

The randomization and masked identification of the mouthwashes was done by a single researcher (B.B.). All mouthwashes were dispensed in a 150 mL white vial and each content was identified by a different colored marker known only to the investigator in charge.

To determine the randomization of each patient within the three groups, the page www.random.org was used, with the numbers 1,2 or 3 corresponding to the groups G1, G2 and G3, respectively, being returned as a result.

For this, the patients were randomized into 3 parallel study groups:

* G1 (Green): Positive Control (0.12% CHX)
* G2 (Orange): Negative control (alcohol vehicle mouthwash 0.1%)
* G3 (Yellow): Experimental group (10% mouthwash of M. sylvestris extract). After the clinical examination, another researcher (V.Ll.) recorded in each clinical record the patient's coding assigned by number, group and color. He delivered the bottle and posology to each patient corresponding to the randomized group, having to rinse with 10 ml of mouthwash for 1 minute in the mouth, every 12 h for 7 days. To confirm the follow-up of the intervention, the same researcher made telephone calls at least once a day to remember the daily rinse. The instructions were delivered verbally and in writing.

The re-evaluation of the patients was carried out 7 days after the first appointment, by the same examiner registering the PBE, GI and PC indices.

Control of risk of bias:

The masking mechanisms through coding by number, group and color, ensured that the patients did not know the specific mouthwash they received; examiners E.A and A.N were limited only to assessing clinical aspects and data analysis was performed with the database using numerical coding. Only researcher B.B was aware of the real assignment by color, with its corresponding study group and number.

To control the risk of random bias in the changes of hygiene habits of the participants on the registration of the periodontal indices, the frequency of brushing, type of toothpaste and use of oral antiseptic mouthwashes was registered; indicating to each participant to maintain these habits without needing to change the oral hygiene frequency.

Statistics:

Mouthwash study groups were defined as an independent variable. The PC and GI analyzed at the beginning and 7 days after the intervention of the study were defined as dependent variable. An analysis of descriptive statistics of the following data was performed: sex (male/female), age (in years), frequency of brushing/day, smoking (smokes over 10 cigarettes/day), presence of chronic disease (Yes/No) and medication consumption (Yes/No). In addition, the values of PC and GI. The continuous variables were presented as average values (Standard Deviation - SD).

In the analytical statistics, the normality of the data was analyzed by the Shapiro Wilk test (p\> 0.05). To compare the indices between the 3 groups before and after the intervention, the data were analyzed by repeated measures ANOVA (MR). The nonparametric data were presented as scatter plot of the samples, comparison with T-test to detect differences.

Considering the variability of the initial data of the patients belonging to each study group that can generate a result bias, an analysis of covariance (ANCOVA) of the baseline data was performed. To check the study hypotheses, the mean values were compared, with a 95% confidence interval of the results between study groups, establishing a level of significance of p \<0.05

Eligibility criteria: Inclusion Criteria:

* Code equal of higher than 2 (Basic Periodontal Examination-BPE).
* I or II ASA classification.
* Without periodontal treatment on past three months.

Exclusion Criteria:

* Acute infection
* Dental trauma
* Alveolar osteitis (Dry socket)
* Elderly dependent patients
* Orthodontic appliances
* Antibiotic therapy
* Analgesic therapy
* Heavy smokers
* Menstrual period
* Pregnants
* Lactating woman

Primary outcomes:
measure: Basic periodontal examination
timeFrame: 7 days
measure: Gingival index
timeFrame: 7 days
measure: Plaque control record
timeFrame: 7 days
Secondary outcomes:
Other outcomes:
Locations:
city: Valdivia
country: Chile
facility: Universidad Austral de Chile
Study officials:
affiliation: Faculty of Medicine, Pontificia Universidad Católica de Chile
name: Bruna Benso, PhD
role: PRINCIPAL_INVESTIGATOR
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