IJCRR

IJCRR - 14(11), June, 2022

Pages: 47-52

Date of Publication: 03-Jun-2022

Effect of an Indigenous Cleanser on the Microbial Biofilm on Acrylic Denture Base - A Pilot Study

Author: Vidya S. Bhat, Mrinali Maria Viegas, Sanath Kumar Shetty, Vipin C

Category: Healthcare

Abstract:Introduction: Edentulous patients wearing dentures may develop Candidiasis or denture sore mouth due to improper cleaning of the dentures. Denture cleansers remove not only the biofilm, but also stains and other forms of debris of food on dentures. Since people prefer natural substances for daily use, a natural indigenous cleanser was prepared to replace the chemical denture cleansing solution. Aim: The aim of this study was to check the effect of soapnut on Candida and Streptococcus mutans biofilm formed on dentures compared with control and commercially available Clanden tablet. Materials and Methods: 18 heat cure acrylic blocks were fabricated and (DPI heat cure) and divided into two groups for biofilm formation of C.albicans and S.mutans with 9 blocks in each group. Three solutions: distilled water(control), commercial cleanser (Clanden tablet- Global Dent Aids Pvt Ltd) and indigenous solution(soapnut) were used to check biofilm cleansing efficiency by crystal violet staining method and optical density (OD) analysis using spectrophotometry. 24 hours bacterial culture was carried out to check for zone of inhibition of the test solution. The biofilm cleansing ability tests was done thrice in triplicate. The statistical analysis was performed by Student T-test Results: The highest OD values were obtained in the S.mutans group tested with control (Water). Least values were obtained in C.albicansgroup tested with Clandon tablet. But, similar result was seen with the indigenous solution as well. No significant difference was seen between the effects of the two test specimens on either group of microorganisms compared with that of the control group. A zone of inhibition was seen around the indigenous solution. Conclusion: Indigenous denture cleanser is an effective solution for daily denture hygiene maintenance.

Keywords: Acrylic denture base, Denture cleansers, Indigenous solution, Soapnut (Sapindusmukorossi), Candida, S.Mutansbiofilm

Full Text:

INTRODUCTION

Improper cleaning of dentures may lead to denture sore mouth or candidiasis. Denture stomatitis is an oral inflammation related to denture wearing and poor oral hygiene, which occurs in 70% of denture wearers^{. 1}Associations of denture stomatitis have been reported with mucosal trauma due to poor denture fit, prolonged duration of denture use,  bacterial and fungal (primarily Candida) infection, and poor denture hygiene.² Candida cells in the oral cavity are associated with biofilms, which differ substantially from planktonic cells due to their higher antifungal resistance^.3

In the case of removable partial dentures, poor oral and denture hygiene can lead to colonization of Streptococci and that may cause inflammation of tissues and caries in abutment teeth whereas in implant-supported removable it can lead to peri-implantitis and implant failure.

Microorganisms like Candida albicans are commensals of the oral cavity which get attached to the denture in the same way that dental plaque grows on natural teeth. As time progresses, the biofilm that develops on dentures may harden and become difficult to remove. Various methods like mechanical aids, chemical methods, and microwave irradiation have been used to maintain denture hygiene. The common mechanical methods used are brushing, sonic vibrators, and ultrasonic cleaners^.4

Brushing dentures is a challenging task for old individuals.⁵Manyelderly patients suffering from dementia and poor dexterity cannot adequately brush their dentures because of disease. Various forms such as tablets, pastes and powders are available. Their relative efficacies have varied with different manufacturers, not only in removing the biofilm but also in staining food and other debris on dentures. The primary cleaning agent used is denture cleaners is sodium hypochlorite. Commercially available denture cleansers usually contain many chemical substances which could be harmful/allergic to the patients^.6

 Since nowadays people prefer natural products for day-to-day use, naturally available substances and traditionally used cleansers can be tested for cleaning ability. Soap nut (Sapindusmukorossi) also known as soapberry, ritha, aritha is an indigenous fruit found in upper Indo-Gangetic, Shivaliks and Sub Himalayan tracts. The fruit contains saponin which it is widely used as a cleanser for washing hair, and removing freckles and psoriasis.⁷

Powdered seeds had been used for arthritis, the common cold, nausea etc. The soapnut has anti-fungal and antibacterial properties^.8 Saponin from the saopnuts has been extracted by using solvents such as ethanol and water.⁹ Traditionally it was used as a detergent for clothes and also to polish gold ornaments^.10 However there has been no study carried out to check the efficiency of soapnut on denture biofilm. The aim of this study was to check the effect of soapnut on Candida and Streptococcus mutans biofilm formed on dentures compared with a control and commercially available tablet.

MATERIALS AND METHODS

The experimental invitro study was conducted after obtaining ethical clearance. The bacterial and fungal strains were procured from the Yenepoya Research Centre microbial repository. The sample size was calculated based on triplicate values i.e three samples for each group of microorganisms and cleanser solution.

METHODOLOGY

Fabrication of acrylic blocks

A 2mm thickness of modelling wax sheet was flasked and dewaxed. The mould space obtained was packed with heat-cure clear acrylic (DPI heat cure resin) and cured in a short curing cycle. The sheet of heat cure acrylic was polished to obtain smooth surfaces devoid of any roughness and irregularities and cut into blocks of 10mm x 10mm x 2mm each (Fig.1).

These were disinfected with surgical spirit and stored in distilled water. A total of 18 blocks were obtained and divided into group A (C. albicans) and group B (S.mutans) with 9 blocks in each group.

Preparation of cleansing solutions:

Ripe soap nuts collected from a tree in Mangalore were dried at room temperature for 10 days (fig 2). 10- 12 dry Soap nuts were boiled in 60 ml of water for 30 minutes at 270ºC. The nuts were strained from the liquid and the concentrated soap nut solution was stored in a sterilized container and refrigerated. (Fig 3) The solution was sterilized under germicidal Ultra Violet light prior to use.

One Clandon tablet (Global Dent Aids Pvt Ltd) was dissolved in 50 ml of water and this solution was used for testing.

Antimicrobial activity:

The antimicrobial assay was done by disc diffusion method. The 4hour microbial culture was uniformly spread on Muller-Hinton agar plates. The microorganism strains used were ATCC 90028 and ATCC 25175 for C. albicans and S.mutans respectively. A known concentration of test compounds were placed on the inoculated plates. The zone of inhibition was measured after incubating for 24hours at 37º C.A ruler was used to measure the diameter of the disk plus the surrounding clear area in millimetres (mm). 

Biofilm assay by crystal violet stain method

Overnight bacterial culture was inoculated to sterile conical flasks containing potato dextrose AGAR growth media. After 8 hours of shaking incubation, the acrylic denture resin squares were immersed in the culture and allowed to form biofilm under static conditions in multiwall culture plates. (Fig 4)

After 48 hours the acrylic blocks were taken out and immersed in distilled water, soapnut and commercial denture cleanser for 8 hours. (Fig 5). They were washed and allowed to dry and the biofilm was fixed using 95 % methanol for ten minutes. The dried and fixed biofilms were stained with crystal violet (0.1%). Acrylic blocks were rinsed with PBS and air-dried again. The stained biofilm was solubilised in an acetic acid solution (33%) and the optical density (OD) of each sample was recorded at a wavelength of 580 nm using a spectrophotometer.

Analyses:

The experiments were performed thrice in triplicate. Statistical analysis was performed by student’s t test using SPSS Version 22 software (SPSS Inc., Chicago, USA). p values <0.05 were regarded as significant to compare the optical density values of 3 cl.24-hour inoculation on a culture plate was obtained. (Fig. 6)

The mean values of zone of inhibition were obtained and it was found to be highest around the commercial tablet(table1).

The control group showed no zone of inhibition indicating no antimicrobial activity. While the tablet group and soapnut group showed a mean zone of 17mm and 14 mm respectively against C.albicans. A mean of 13mm and 15mm was found against S.mutans for soapnut and tablet respectively.

Optical density after crystal violet staining:

The highest OD values were obtained in the B group (S.mutans) tested with the control (Water). Least values were obtained in the A group (C.albicans) tested with Clanden tablet. No significant difference was seen between the effect of the two test specimens on either group of microorganisms compared with that of the control group. (graph.1)

Graph 1: Effectiveness of cleasing solution in terms of optical density. (x axis- Optical Density values, y axis – microorganisms).
* indicates a significant difference between control and test groups.  p values < 0.05

# Indicates no significant difference between cleanser and soapnut groups. p value > 0.05

DISCUSSION

Denture hygiene maintenance is the most important step to be followed by the patient. Denture cleaning methods used on daily basis may be mechanical or chemical. Studies show a positive effect of mechanical and chemical cleaning and of the combination of both on denture cleanliness^.12

According to a study by Peracini et al. mechanical cleaning method was the most prevalent among various denture users.¹³ Various pastes used to clean dentures contain abrasive agents that may lead to surface roughness of the denture. This surface roughness would increase the adherence of plaque and microorganisms on the abraded denture surface. Also, mechanical cleaning with pastes requires patients to manually clean the dentures, which becomes cumbersome in old age due to loss of dexterity and increased dependence.

Commercial denture cleaning products are based on chemicals like sodium hypochlorite, peroxides, neutral peroxides with enzymes, or acids.

We used 3 different solutions to check the efficiency on microbial biofilm growing on the denture surface. Since in recent years people have adapted to naturally available substances for their daily use, a cleansing solution was extracted from an indigenous fruit and compared with that of a chemical cleansing tablet. The control solution used was water and it showed to have the least cleaning efficiency on biofilm formed on the denture surface. This proved that use of water alone would not lead to any cleaning of dentures.

The denture cleansing tablet used was Clandon which is an effervescent tablet. Effervescent denture cleansing tablets contain alkaline peroxides; the tablet used in this study contains sodium perborate monohydrate. The tablets have an effervescent action when they come in contact with water by producing an alkaline solution of hydrogen peroxide containing active oxygen. This effervescence has a mechanical action of removing debris, and oxygen has antimicrobial and stain-removing effects.¹⁴

The main disadvantage of conventional chemical cleansers, such as sodium hypochlorite, is their whitening properties on dentures when used for prolonged periods.^15,16 The increased resistance of pathogenic organisms to synthetic antifungal and antibacterial agents is drawing attention to plant extracts having antifungal efficiency. For geriatric patients cost and availability may be a factor while selecting a denture cleanser.¹⁷

Nowadays various plant materials having antifungal properties are being tested in the research field. The main advantage of opting for naturally occurring substances over commercial chemical solutions is minimal chances of harmful effects as well as allergic reactions.

A study was conducted to check the efficiency of lemongrass extract on the C. Albicans denture biofilm and found that there was a reduction in cell counts^{. 18 Another} study to check the effect of Triphala as a denture cleanser revealed that it had superior cleansing ability compared to chlorhexidine gluconate.¹⁹

In our study, an indigenous naturally occurring cleanser was tested for antimicrobial activity and cleaning efficiency on acrylic denture material. Sapindustrifoliatus Linn., a small deciduous tree belongs to the family Sapindaceae which is known as soapnut in English, Ritha in Bengali and Ponnangottai in Tamil^.20 The major constituents of the fruits are saponins (10%-11.5%), sugars (10%) and mucilage^.21 In a study conducted to evaluate the antimicrobial activity of soapnut against S.mutans and C.albicansfound that soapnut had higher antifungal activity against C.albicans.²²

But in our study, the antibacterial action of soapnut was higher than antifungal activity according to the biofilm assay test. The action of soapnut is mainly by saponins. Plant-derived saponins are secondary metabolites and are traditionally used as detergent. It has amphiphilic nature due to the presence of lipid-soluble aglycone and water-soluble chains in its structure.²³ Saponins are divided into two groups which are triterpenoid and steroid glycosides. Glycoside structure characterizations are varied by the number of sugar units attached at different positions.²⁴

Various methods for extraction of saponins have been used, but according to a study by Merve et al., Ethanol water solution (50 % v/v) with 1:10 solid- liquid ratio was the best. Bioassay directed isolation and characterization of a new acetylated triterpene saponin,  named hederagenin 3-O-(2,4-O-diacetyl-α-L--arabinopyranoside)-(1-3)-α-L-rhamnopyranosyl- (1-2)- α-L-arabinopyranoside (1), together with six known triterpenoidal hederagenin saponin (2-7) from S. Mukorossi^.25

 The indigenous soapnut solution had a significant effect against the denture biofilm but slightly lesser compared to the commercially available tablet cleanser. The antimicrobial activity test by disc diffusion method also showed an inhibitory zone formed around the well-containing soapnut solution, thus proving it has antimicrobial action.

Conclusion: The indigenous soapnut solution had a significant effect against the denture biofilm but slightly lesser compared to the commercially available tablet cleanser. The antimicrobial activity test by disc diffusion method also showed an inhibitory zone formed around the well-containing soapnut solution, thus proving it has antimicrobial action Thus naturally available sources could be an economic and chemical-free alternative.

Limitations:

Effects of the indigenous solution on the Physical and esthetic properties of the acrylic material were not evaluated in the present study.

Biofilm formation was not carried out in an environment simulating the oral cavity

Acknowledgement: We acknowledge Yenepoya Research Center for the facilities provided. We acknowledge the immense help received from the scholars whose articles are cited and included in the references of this manuscript. The authors are also grateful to authors/editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.

Conflict of Interest: Nil

Source of funding: Self-funded

Authors’ Contribution:  Dr.Vidya Bhat –concept, guide and overall conduct of study, Dr.Mrinali- methodology and conduct of study. Mr.Vipin- Microbial assay

Dr. Sanath Shetty-approval of study design

References:

1. Zissis A, Yannikakis S, Harrison A. Comparison of denture stomatitis prevalence in 2 population groups. Int J Prosthodont. 2006 Nov-Dec;19(6):621-5.

2. Gendreau L, Loewy ZG. Epidemiology and etiology of denture stomatitis. Journal of Prosthodontics: Implant, Esthetic and Reconstructive Dentistry. 2011 Jun;20(4):251-60.

3. Ramage G, Robertson SN, Williams C. Strength in numbers: antifungal strategies against fungal biofilms. Int J Antimicrob Agents. 2014 Feb;43(2):114-20

4. Sharma H, Patil R, Nagmoti JM. Comparative evaluation of the effect of commercially available two different forms of denture cleansers on denture biofilm in diabetic and nondiabetic individuals: An in vivo study. Indian J Health Sci Biomed Res 2017;10:196-202

5. Kulak?Ozkan Y, Kazazoglu E, Arikan A. Oral hygiene habits, denture cleanliness, presence of yeasts and stomatitis in elderly people. J. Oral Rehabil. 2002 Mar;29(3):300-4.

6. Gajwani-Jain S, Magdum D, Karagir A, Pharane P. Denture cleansers: A review. IOSR J. Med. Dent. Sci. 2015;1(14):94-6.

7. Kirtikar KR, Basu B.D. (1991) Indian Medicinal Plants Vol. II, p. 1017, 2220. Lalit Mohan Basu, Allahabad, India.

8. Meena VN, Rajakohila M, Syndia LA, Prasad PN, Ariharan VN. Multifacetious uses of Soapnut Tree–A mini-review. Res. J. Pharm. Biol. Chem. Sci. 2012;3:420-4.

9. Dhawan D, Gupta J. Research Article Comparison of Different Solvents for Phytochemical Extraction Potential from Datura metel Plant Leaves. Int. J. Biol. Chem. 2017;11:17-22.

10. Templeton RH. Reetha and shikakai as natural surfactants for cleaning of historic textiles. Int J Res Analyt Rev. 2018;5:2348- 50.

11. Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Pathol. 1966 Apr 1;45(4):493-6.

12. Duyck J, Vandamme K, Krausch-Hofmann S, Boon L, De Keersmaecker K, Jalon E et al. Impact of denture cleaning method and overnight storage condition on denture biofilm mass and composition: a cross-over randomized clinical trial. PloS one. 2016 Jan 5;11(1):e0145837.

13. Peracini A, Andrade IM, Paranhos HD, Silva CH, Souza RF. Bebehaviors and hygiene habits of complete denture wearers. Brazilian dental journal. 2010;21(3):247-52.

14. de Lucena-Ferreira SC, Ricomini-Filho AP, Da Silva WJ, Cury JA, Del Bel Cury AA. Influence of daily immersion in denture cleanser on multispecies biofilm. Clinical oral investigations. 2014 Dec;18(9):2179-85.

15. Moffa EB, Giampaolo ET, Izumida FE, Pavarina AC, Machado AL, Vergani CE. Colour stability of relined dentures after chemical disinfection. A randomized clinical trial. Journal of dentistry. 2011 Dec 1;39:65-71.

16. Moon A, Powers JM, Kiat?Amnuay S. Color stability of denture teeth and acrylic base resin subjected daily to various consumer cleansers. J Esthet Restor Dent.2014 Aug;26(4):247-55.

17. Haloci E, Manfredini S, Toska V, Vertuani S, Ziosi P, Topi I et al. Antibacterial and antifungal activity assesment of nigella sativa essential oils. Int. J. Pharm. Pharm. Sci. 2012 Jun 26;6(6):270-2.

18. Madeira PL, Carvalho LT, Paschoal MAB, de Sousa EM, Moffa EB, dos Santos da silva MA et al. In vitro effects of lemongrass extract on Candida albicans biofilms, human cells viability, and denture surface. Frontiers in cellular and infection microbiology. 2016 Jun 28;6:71.

19. Sushma R, Sathe TT, Farias A, Sanyal PK, Kiran S. “Nature cures:” An alternative herbal formulation as a denture cleanser. Annals of African medicine. 2017 Jan;16(1):6.

20. Manning SA. Systematic Guide to Flowering plants of the world. Museum Press, 1965.

21. Kokate CK. Practical Pharmacognosy. 4th ed. New Delhi: Vallabh Prakashan; 1993. pp. 107–111.pp. 178–181.

22. Aneja KR, Joshi R, Sharma C. In vitro antimicrobial activity of Sapindusmukorossi and Emblica Officinalis against dental caries pathogens. Ethnobotanical Leaflets. 2010;2010(4):3.

23. Güçlü-Ustünda? O, Mazza G. Saponins: properties, applications and processing. Crit Rev Food Sci Nutr. 2007;47(3):231-58

24. Hostettmann K, Marston A. Chemistry and pharmacology of natural products. Cambridge, UK: Cambridge University Press; 1995.

25. Kose MD, Bayraktar O. Extraction of saponins from soapnut (SapindusMukorossi) and their antimicrobial properties. World Journal of Research and Review (WJRR). 2016;2(5):262952.