Clinical Evaluation of a Mouthwash Containing Malva Sylvestris Extract.

Periodontal Diseases Clinical Trial

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.

Clinical Trial Details — Status: Completed

Administrative data

• NCT number: NCT05138484
• Other study ID #: ORD 181
• Status: Completed
• Phase: Phase 3
• Start date: October 20, 2017
• Est. completion date: January 14, 2019

Study information

• Verified date: May 2019
• Source: Aravena, Pedro, DDS, PhD
• Contact: n/a
• Is FDA regulated: No
• Study type: Interventional

Clinical Trial 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.

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

Recruitment information / eligibility

• Status: Completed
• Enrollment: 54
• Est. completion date: January 14, 2019
• Est. primary completion date: December 17, 2018
• Accepts healthy volunteers: No
• Gender: All
• Age group: 18 Years to 75 Years

Eligibility

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

Related Conditions & MeSH terms

• Periodontal Diseases

Intervention

Drug:
• 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.

Locations

Country	Name	City	State
Chile	Universidad Austral de Chile	Valdivia

Sponsors (2)

Lead Sponsor	Collaborator
Aravena, Pedro, DDS, PhD	Universidad Austral de Chile

Country where clinical trial is conducted

Chile, 

References & Publications (36)

Ainamo J, Bay I. Problems and proposals for recording gingivitis and plaque. Int Dent J. 1975 Dec;25(4):229-35. — View Citation

Araujo MWB, Charles CA, Weinstein RB, McGuire JA, Parikh-Das AM, Du Q, Zhang J, Berlin JA, Gunsolley JC. Meta-analysis of the effect of an essential oil-containing mouthrinse on gingivitis and plaque. J Am Dent Assoc. 2015 Aug;146(8):610-622. doi: 10.1016/j.adaj.2015.02.011. Review. — View Citation

Baker K. Mouthrinses in the prevention and treatment of periodontal disease. Curr Opin Periodontol. 1993:89-96. Review. — View Citation

Barros L, Carvalho AM, Ferreira IC. Leaves, flowers, immature fruits and leafy flowered stems of Malva sylvestris: a comparative study of the nutraceutical potential and composition. Food Chem Toxicol. 2010 Jun;48(6):1466-72. doi: 10.1016/j.fct.2010.03.012. Epub 2010 Mar 15. — View Citation

Benso B, Franchin M, Massarioli AP, Paschoal JA, Alencar SM, Franco GC, Rosalen PL. Anti-Inflammatory, Anti-Osteoclastogenic and Antioxidant Effects of Malva sylvestris Extract and Fractions: In Vitro and In Vivo Studies. PLoS One. 2016 Sep 19;11(9):e0162728. doi: 10.1371/journal.pone.0162728. eCollection 2016. — View Citation

Benso B, Rosalen PL, Alencar SM, Murata RM. Malva sylvestris Inhibits Inflammatory Response in Oral Human Cells. An In Vitro Infection Model. PLoS One. 2015 Oct 19;10(10):e0140331. doi: 10.1371/journal.pone.0140331. eCollection 2015. — View Citation

Collins JR, Olsen J, Cuesta A, Silva-Vetri M, Hernández M, Romanos G, Rajendra Santosh AB, Palma P. In vitro microbiological analysis on antibacterial, anti-inflammatory, and inhibitory action on matrix metalloproteinases-8 of commercially available chlorhexidine digluconate mouth rinses. Indian J Dent Res. 2018 Nov-Dec;29(6):799-807. doi: 10.4103/ijdr.IJDR_406_17. — View Citation

DellaGreca M, Cutillo F, D'Abrosca B, Fiorentino A, Pacifico S, Zarrelli A. Antioxidant and radical scavenging properties of Malva sylvestris. Nat Prod Commun. 2009 Jul;4(7):893-6. — View Citation

DRIPPS RD, LAMONT A, ECKENHOFF JE. The role of anesthesia in surgical mortality. JAMA. 1961 Oct 21;178:261-6. — View Citation

Freires IA, Denny C, Benso B, de Alencar SM, Rosalen PL. Antibacterial Activity of Essential Oils and Their Isolated Constituents against Cariogenic Bacteria: A Systematic Review. Molecules. 2015 Apr 22;20(4):7329-58. doi: 10.3390/molecules20047329. Review. — View Citation

Gao L, Xu T, Huang G, Jiang S, Gu Y, Chen F. Oral microbiomes: more and more importance in oral cavity and whole body. Protein Cell. 2018 May;9(5):488-500. doi: 10.1007/s13238-018-0548-1. Epub 2018 May 7. Review. — View Citation

Gasparetto JC, Martins CA, Hayashi SS, Otuky MF, Pontarolo R. Ethnobotanical and scientific aspects of Malva sylvestris L.: a millennial herbal medicine. J Pharm Pharmacol. 2012 Feb;64(2):172-89. doi: 10.1111/j.2042-7158.2011.01383.x. Epub 2011 Nov 4. Review. — View Citation

Guarrera PM. Traditional phytotherapy in Central Italy (Marche, Abruzzo, and Latium). Fitoterapia. 2005 Jan;76(1):1-25. Review. — View Citation

Gunsolley JC. Clinical efficacy of antimicrobial mouthrinses. J Dent. 2010 Jun;38 Suppl 1:S6-10. doi: 10.1016/S0300-5712(10)70004-X. Review. — View Citation

Gürgan CA, Zaim E, Bakirsoy I, Soykan E. Short-term side effects of 0.2% alcohol-free chlorhexidine mouthrinse used as an adjunct to non-surgical periodontal treatment: a double-blind clinical study. J Periodontol. 2006 Mar;77(3):370-84. — View Citation

Hajishengallis G. Periodontitis: from microbial immune subversion to systemic inflammation. Nat Rev Immunol. 2015 Jan;15(1):30-44. doi: 10.1038/nri3785. Review. — View Citation

Hooks KB, O'Malley MA. Dysbiosis and Its Discontents. mBio. 2017 Oct 10;8(5). pii: e01492-17. doi: 10.1128/mBio.01492-17. — View Citation

James P, Worthington HV, Parnell C, Harding M, Lamont T, Cheung A, Whelton H, Riley P. Chlorhexidine mouthrinse as an adjunctive treatment for gingival health. Cochrane Database Syst Rev. 2017 Mar 31;3:CD008676. doi: 10.1002/14651858.CD008676.pub2. Review. — View Citation

Jeffcoat MK, McGuire M, Newman MG. Evidence-based periodontal treatment. Highlights from the 1996 World Workshop in Periodontics. J Am Dent Assoc. 1997 Jun;128(6):713-24. Review. — View Citation

Larsen T, Fiehn NE. Dental biofilm infections - an update. APMIS. 2017 Apr;125(4):376-384. doi: 10.1111/apm.12688. Review. — View Citation

Lazar V, Saviuc CM, Chifiriuc MC. Periodontitis and Periodontal Disease - Innovative Strategies for Reversing the Chronic Infectious and Inflammatory Condition by Natural Products. Curr Pharm Des. 2016;22(2):230-7. Review. — View Citation

Löe H, Schiött CR, Karring G, Karring T. Two years oral use of chlorhexidine in man. I. General design and clinical effects. J Periodontal Res. 1976 Jun;11(3):135-44. — View Citation

Löe H. The Gingival Index, the Plaque Index and the Retention Index Systems. J Periodontol. 1967 Nov-Dec;38(6):Suppl:610-6. — View Citation

Murakami S, Mealey BL, Mariotti A, Chapple ILC. Dental plaque-induced gingival conditions. J Clin Periodontol. 2018 Jun;45 Suppl 20:S17-S27. doi: 10.1111/jcpe.12937. Review. — View Citation

Newman DJ, Cragg GM. Natural Products as Sources of New Drugs from 1981 to 2014. J Nat Prod. 2016 Mar 25;79(3):629-61. doi: 10.1021/acs.jnatprod.5b01055. Epub 2016 Feb 7. Review. — View Citation

O'Leary TJ, Drake RB, Naylor JE. The plaque control record. J Periodontol. 1972 Jan;43(1):38. — View Citation

Offenbacher S, Barros SP, Beck JD. Rethinking periodontal inflammation. J Periodontol. 2008 Aug;79(8 Suppl):1577-84. doi: 10.1902/jop.2008.080220. Review. — View Citation

Pihlstrom BL, Michalowicz BS, Johnson NW. Periodontal diseases. Lancet. 2005 Nov 19;366(9499):1809-20. Review. — View Citation

Rafiei M, Kiani F, Sayehmiri F, Sayehmiri K, Sheikhi A, Zamanian Azodi M. Study of Porphyromonas gingivalis in periodontal diseases: A systematic review and meta-analysis. Med J Islam Repub Iran. 2017 Sep 12;31:62. doi: 10.18869/mjiri.31.62. eCollection 2017. — View Citation

Razavi SM, Zarrini G, Molavi G, Ghasemi G. Bioactivity of malva sylvestris L., a medicinal plant from iran. Iran J Basic Med Sci. 2011 Nov;14(6):574-9. — View Citation

Tartaglia GM, Kumar S, Fornari CD, Corti E, Connelly ST. Mouthwashes in the 21(st) century: a narrative review about active molecules and effectiveness on the periodontal outcomes. Expert Opin Drug Deliv. 2017 Aug;14(8):973-982. doi: 10.1080/17425247.2017.1260118. Epub 2016 Nov 20. Review. — View Citation

Teles RP, Teles FR. Antimicrobial agents used in the control of periodontal biofilms: effective adjuncts to mechanical plaque control? Braz Oral Res. 2009;23 Suppl 1:39-48. Review. — View Citation

Tonetti MS, Greenwell H, Kornman KS. Staging and grading of periodontitis: Framework and proposal of a new classification and case definition. J Clin Periodontol. 2018 Jun;45 Suppl 20:S149-S161. doi: 10.1111/jcpe.12945. Review. Erratum in: J Clin Periodontol. 2019 Jul;46(7):787. — View Citation

Topcuoglu N, Kulekci G. 16S rRNA based microarray analysis of ten periodontal bacteria in patients with different forms of periodontitis. Anaerobe. 2015 Oct;35(Pt A):35-40. doi: 10.1016/j.anaerobe.2015.01.011. Epub 2015 Jan 29. — View Citation

Van Leeuwen MP, Slot DE, Van der Weijden GA. Essential oils compared to chlorhexidine with respect to plaque and parameters of gingival inflammation: a systematic review. J Periodontol. 2011 Feb;82(2):174-94. doi: 10.1902/jop.2010.100266. Epub 2010 Nov 2. Review. — View Citation

Van Strydonck DA, Slot DE, Van der Velden U, Van der Weijden F. Effect of a chlorhexidine mouthrinse on plaque, gingival inflammation and staining in gingivitis patients: a systematic review. J Clin Periodontol. 2012 Nov;39(11):1042-55. doi: 10.1111/j.1600-051X.2012.01883.x. Epub 2012 Sep 7. Review. — View Citation

* Note: There are 36 references in all — Click here to view all references

Outcome

Type	Measure	Description	Time frame	Safety issue
Primary	Basic periodontal examination	The mouth is divided into sextants and using a WHO-standardized periodontal probe (Hu-Friedy?), 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 (black area of the probe partially visible, periodontal pocket of 3.5 - 5.5 mm), 4 (black area of the probe below the gingival margin, periodontal pocket greater than 5.5 mm).	7 days
Primary	Gingival index	With a WHO-standardized periodontal probe (Hu-Friedy?), the buccal, lingual and interdental papillae marginal gingivae are probed, using a four-point scale from 0 (absence of inflammation) to 3 (severe inflammation)	7 days
Primary	Plaque control record	5 drops of Bacterial Plaque Revealing Solution (Caristop-Revelador DUAL TONE, MAVER?) 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 bacterial plaque, divide this number by the total number of surfaces present in the mouth and multiply by 100	7 days