Antifungal Effect of Some Medicinal Plant Extracts On Candida Albicans Adherence on Acrylic Resin Denture Base Material . An In Vitro Study

Aims: Evaluate the antifungal effect of some medicinal plant extracts (peppermint, rue, pomegranate and garlic) and their anti–adherent effect on C. albicans cells that attached on the fitting surface of acrylic resin denture base. Materials and Methods: This study evaluated the antifungal effect of plant extracts, using broth micro dilution method at two concentrations for each one. The visualization, inspection and enumeration of adherent C. albicans cells and the detection of the anti–adherent effect of these plant extracts was achieved by using fluorescent microscope. For each plant extract, two concentrations in addition to the time of immersion at three different intervals (1 h, 24 hrs and 48 hrs) were evaluated for the anti–adherent effect. Result: Antifungal effect of plant extracts on candida albicans cells, was measured by spectrophotometer which showed that both rue and pomegranate extracts had the best antifungal effect at first concentration, while in the second concentration, pomegranate and garlic extracts had the best antifungal effect . The mean number of remaining C. albicans cells which adhered on acrylic resin samples after treatment by immersion in the first concentration of plant extracts for one hour was (3.1), (3.7), (3.7) and (4.7)] cells /mm respectively; while after treatment with second concentration of plant extracts for one hour was [ (4), (4.4), (4.4) and (4)] cells / mm respectively for the above mentioned plant Conclusion: All examined medicinal plant extracts were significantly effective in dislodging C. albicans cells from acrylic resin samples at their different concentrations and statistically there was no significant difference among the different times of immersion.

,mostly in patients who wear complete dentures and have less than ideal mouth care. (2) Recently, there has been an increased interest in antimicrobial agents from medicinal plants which have been used in folk medicine . (3,4) Antibiotics are produced either synthetically or through microbial fermentation, plants, however, may provide additional source for antimicrobial substances. (5,6) Much of work has been performed to screen the antimicrobial effect of many plants (7) and for the isolation of their active ingredients (8) : 1-Peppermint:The antimicrobial activity of volatile oil isolated from peppermint has an inhibitory effect on the growth of large numbers of bacteria, yeasts including Candida (9) . In addition to anticaries effect of its watery extrac.t (10) 2-Rue:The active ingredient is the volatile oil which contain rutin. It has antispasmic and antirheumatoid effects which can be used in the treatment of gingivitis and tonsillitis (11) in addition to antibacterial, anticaries effects of rue leaves extract when used as a mouth rinse. (12) 3-Pomegranate: its water extract has an inhibitory effect on C. albicans . (13) and pathogenic bacteria. (14,15) 4-Garlic(Allium sativum):Garlic has antibacterial, antifungal, antiviral properties (12,16) ,its inhibitory effect against C. albicans by inhibiting its adhesion to buccal epithelial cells and reduces germ tube formation. For this reason, it is effective in the treatment of chronic atrophic candidiasis. (17) The aim of this study is to evaluate the antifungal anti-adherent effectof medicinal plant extracts (peppermint,rue, pomegranate and garlic) on the colonization of C. albicans on acrylic resin denture base surface ( An in vitro study).

MATERIALS AND METHODS
The methodology in this study consists of two parts; prosthetic and mycological methods. In the first part, a total of (75) specimens from heat cured acrylic resin( Major Prodotti Dentari S.P.A Italy ) were fabricated in dimensions (10X10X3) (0.5) mm (18) ,the acrylic resin samples were conditioned and disinfected before adhesion assay. (19) The mycological methods were involved isolation of C. albicans by taking swabs from of inner surface of up-per complete denture for (8) patients selected randomly, those were wearing their dentures for more than one year (20) and they were attending prosthodontic department/College of Dentistry/Mosul University. These swabs were cultured on Sabouraud's dextrose agar medium (21) and identified according to culture characteristic, microscopic appearance and germ tube formation. The unstimulated whole saliva was collected from(30) volunteers, (healthy students about 22 years old) using spitting method , each student was instructed to rinse his / her mouth, gargling with distilled water several times for seconds to remove any remnant of food particles and debris between teeth, then, a stop watch was started. The oral cavity was closed by lips; saliva was collected in the oral cavity expelled into a graduated tube which was immediately closed with a plastic stopper at the end of collection to minimize the contact of saliva, then saliva pooled, clarified using refrigerated centrifuge. (22) The medicinal plants extracts were used in this study includes: garlic, peppermint, rue and pomegranate. The fresh leaves of rue and peppermint were collected, washed gently with water, then mixed with distilled water, ground or milled with blender. The extracts were filtered with clean gauze to separate the solid particles from liquid extracts. The same procedure was used with bulb of garlic and fruit pericarp of pomegranate. The extract powder was prepared from aqueous extract of leaves, bulb and fruit pericarp of previously mentioned plants using the freeze-dried method (Lyophilization) by using lyophilizer machine. (23) From this powder of fine particles, 1 g from each plant extract was used to prepare dilution at 1:10, 1:100 according to Abdul-Rahman (2001). All the prepared M.P.E.s were kept at (25 ± 1) ºC in a clean, dry containers. (24) Antifungal assay was carried out according to Mustafa (1995 immersion (1 hr, 24 hrs and 48 hrs) respectively. For this experiment the total number of acrylic resin disks used were (75).A standardized C. albicans suspension (2ml) was added to each specimen , incubated for one hour at 37 ºC (19,25) , then washed with phosphate buffer saline to remove the loosely adherent cells. Fixation and staining of the remaining adherent cells to the surface was achieved. (26) The number of adherent cells was examined using fluorescent microscope (Olympus, Japan) for at least three replicates for each variable was taken. (19) Statistical analysis was carried out using One Way Analysis of Variance (ANOVA) and Duncan's Multiple Range Test at levels of significance 0.01 and 0.05. to determine the difference between the studied factors.

RESULTS
In this study, the absorbance values in (nm) of replicates were measured and compared with control group by D.N.M.R. for determining the antifungal effect of the first and second conc.s of different plant extracts against C. albicans isolate. The results were listed in (Table1).   The results indicated that all the different times of immersion for 1st and 2nd conc.s of plant extracts were significantly effective in reducing the number of C. albicans cells / mm 2 adhered to acrylic resin disks, although there was no significant difference among the different time of immersion for each conc.

DISCUSSION
The fungicidal activity of plant extracts in this project was evaluated using broth microdilution method. This is because it gives more scientific result in comparison with the disk diffusion method which has low credibility for samples of plants that are difficult to diffuse in the media and also there is no relationship between diffusion power and antimicrobial activity. (27) The inhibitory effect of watery extract of peppermint against C. albicans was due to their content of volatile oils which had a significant inhibitory effect against large numbers of yeasts including C. albicans (27 ) and the inhibitory effect of the two conc.s of watery extract of rue leaves against C. albicans were due to the presence of rutin (active ingredient) in volatile oils of rue. (28) A high antifungal activity of pomegranate was evaluated which was agreed with (Mustafa 1995)result who reported that the fungicidal effect of pomegranate was significantly higher than CHX 0.2%. Lastly the antifungal activity of watery extract of garlic is agreed with (Marrino etal 1999,Ngane etal 2000 (29,30) results which reported that watery extract of garlic contain active substance in the volatile oils which had an inhibitory effect against C. albicans and also resulted from the conversion of naturally occurring allium to allicin in the presence of allinase enzyme.All the extracts of (peppermint, rue, pomegranate, and garlic) had a significant effect on the adherence of C. albicans on acrylic resin surface. This ability in removing the attached yeast cells indicated the effectiveness of these extracts as antiadherent agents. This result was obtained with even (high and low) conc.s of these extracts and at different times of immersion. The mechanism of antiadherent effect of watery extract of garlic on the adhesion of C. albicans on buccal epithelial cells, and the reduction of germ tube formation might be due to the interference of garlic with the synthesis of adhesins involved in the adhesion process or it may cause mechanical distortion of the adhesins already present on the outer envelope, thus blocking the adherence (17,31). It was possible that garlic inhibits thiol enzyme involved in the synthesis of adhesins. Our results also agreed with ( Leven etal 1979) (32) result who suggested that it would be useful to use garlic extracts as an effective measure in the treatment of (denture stomatitis) whether as a denture cleanser or mouth rinse.

CONCLUSION
All plant extracts used in this study had antifungal and antiadherent effect on candidal cells on acrylic resin denture base material. For this reason can be used as disinfectants, mouth wash or denture soaking solutions and as an alternative to drug solutions in the treatment of denture stomatitis . The study of anti-adherent effect of medicinal plant extract on C. albicans colonization on acrylic resin denture base surface using fluorescent microscope represents the first one at the Prosthodontic Department / College of Dentistry in that approach.