Clinical Evaluation of a Mouthwash Containing Malva Sylvestris Extract.
Condition: Periodontal Diseases
MeSH terms: Periodontal Diseases
Study type: Interventional
Phase: Phase 3

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
    

Intervention name(s): experimental group 10% mouthwash of M. sylvestris extract
Intervention type(s): Drug
Intervention description(s): 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.
Other intervention name(s): Positive control 0.12% chlorhexidine digluconate; Negative control vehicle mouthwash
Intervention MeSH terms: Chlorhexidine; Chlorhexidine gluconate
Allocation: Randomized
Intervention model: Parallel Assignment
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.
Masking: Triple (Participant, Care Provider, Investigator)
Primary purpose: Treatment
Keywords: Periodontal diseases; Inflammation; Malva sylvestris; Mouthwashes