Fathilah Abdul Razak

An in vitro study on the anti-adherence effect of Brucea javanica and Piper betle extracts towards oral Candida

OBJECTIVE The adherence of Candida to mucosal surfaces is the initial step for successful invasive process of the oral cavity. The study aimed to investigate the effect of two plant extracts on the non-specific and specific bindings of oral candida. METHODS In the former, adsorption to hexadecane was used to measure the hydrophobic interaction of the candida cells. In the later, glass beads coated with saliva represented the experimental pellicles in specific adhesion of oral candida to hard tissue surface. RESULTS Candida krusei, Candida dubliniensis and Candida tropicalis showed the highest adsorption to hexadecane at 30.23%, 26.19% and 19.70%, respectively, while the others within the range of 7-10%. All candidal species were significantly affected by the extracts (P<0.05) with Brucea javanica exhibited more than 60% reduction of CSH than Piper betle. Candida parapsilosis showed the highest affinity in specific-bindings to pellicle with 18.72±0.71×10(5)CFU/ml. Exposing to P. betle-treated pellicle has drastically reduced the adherence of C. tropicalis, Candida albicans and C. krusei by 86.01%, 61.41% and 56.34%, respectively. B. javanica exhibited similar effect on C. tropicalis (89.86%), Candida lusitaniae (88.95%), C. albicans (79.74%), Candida glabrata (76.85%) and C. krusei (67.61%). CONCLUSION The extracts demonstrated anti-adherence activities by modifying the CSH and the characteristics of the experimental pellicle.

The antimicrobial effect of 0.1 ppm ozonated water on 24-hour plaque microorganisms in situ

Ozone is a known oxidant present in the atmosphere and is commercially produced by simple ozonizer machines. It is a powerful antimicrobial agent in its gaseous and aqueous forms. Ozone readily dissolves in water and retains its antimicrobial property even in the dissolved state. In this study, the effect of 0.1 ppm ozonated water was analyzed on 24-hour supragingival plaque (SP) samples in situ. SP was collected from the two most posterior teeth in the contra-lateral quadrants before and after a 30-second rinse with either distilled water (control group) or 0.1 ppm ozonated water (test group). The plaque was used to count the number of total bacteria, total anaerobic bacteria, Streptococcus mutans, and Candida albicans on selective agar media. The statistical analysis of the number of colony forming units (CFUs) obtained demonstrated a significant antimicrobial effect of ozonated water on the total bacteria (p = 0.01) and anaerobes (p = 0.02). A reduction in the post-rinse CFU count for Streptococcus mutans was also observed, but the effect was not statistically significant (p = 0.07). The Candida species was only grown from one sample. Ozonated water at the 0.1 ppm concentration was effective in reducing the load of 24-hour plaque bacteria, but it did not eliminate them completely.

Evaluation of Wound Closure Activity of Nigella sativa, Melastoma malabathricum, Pluchea indica, and Piper sarmentosum Extracts on Scratched Monolayer of Human Gingival Fibroblasts

Nigella sativa, Melastoma malabathricum, Pluchea indica, and Piper sarmentosum are common Asian traditional medicines to treat minor wounds. This study aimed to investigate the in vitro wound healing properties of aqueous extracts of these plants using human gingival fibroblast (HGF) monolayer as study model. DPPH scavenging activity of the extracts was evaluated and effect on HGF proliferation was determined. Their effect on HGFs function to synthesize collagen was indicated by the level of hydroxyproline produced and effect on wound healing activity was assessed using an in vitro scratch assay. The influence of the extracts on expression of bFGF and TGF-β was also determined. Results revealed all four extracts to exhibit low free radical scavenging activity. The extract from N. sativa (NSSE) compared to the others showed favourable enhancement of HGF proliferation with EC50 of 22.67 ± 3.06 µg/mL (P < 0.05) with accelerated wound closure activity despite its nonsignificant effect on collagen synthesis. In addition to the elevated level of bFGF by up to 15% at 100 µg/mL of NSSE, a slightly better effect was observed on the expression of TGF-β. NSSE thus showed that promising wound healing properties and data obtained may contribute towards validation of its traditional use for the healing of oral wounds.Nigella sativa, Melastoma malabathricum, Pluchea indica, and Piper sarmentosum are common Asian traditional medicines to treat minor wounds. This study aimed to investigate the in vitro wound healing properties of aqueous extracts of these plants using human gingival fibroblast (HGF) monolayer as study model. DPPH scavenging activity of the extracts was evaluated and effect on HGF proliferation was determined. Their effect on HGF’s function to synthesize collagen was indicated by the level of hydroxyproline produced and effect on wound healing activity was assessed using an in vitro scratch assay. The influence of the extracts on expression of bFGF and TGF-β was also determined. Results revealed all four extracts to exhibit low free radical scavenging activity. The extract from N. sativa (NSSE) compared to the others showed favourable enhancement of HGF proliferation with EC50 of  µg/mL () with accelerated wound closure activity despite its nonsignificant effect on collagen synthesis. In addition to the elevated level of bFGF by up to 15% at 100 µg/mL of NSSE, a slightly better effect was observed on the expression of TGF-β. NSSE thus showed that promising wound healing properties and data obtained may contribute towards validation of its traditional use for the healing of oral wounds.

Apoptosis-Inducing Effect of Three Medicinal Plants on Oral Cancer Cells KB and ORL-48

Brucea javanica, Azadirachta indica, and Typhonium flagelliforme are medicinal plants commonly used to treat conditions associated with tumour formation. This study aimed to determine the antiproliferative activity of these plants extracts on KB and ORL-48 oral cancer cell lines and to suggest their mode of cell death. The concentration producing 50% cell inhibition (IC50) was determined and the activity was examined under an inverted microscope. Immunohistochemistry fluorescent staining method (TUNEL) was performed to indicate the mechanism of cell death and the fragmented DNA band pattern produced was obtained for verification. Compared to Azadirachta sp. and Typhonium sp., the antiproliferative activity of Brucea sp. extract was the most potent on both KB and ORL-48 cells with IC50 of 24.37 ± 1.75 and 6.67 ± 1.15 µg/mL, respectively. Signs of cell attrition were observed 24 hr after treatment. Green fluorescent spots indicating cell death by apoptosis were observed in images of both cells following treatment with all the three extracts. DNA fragments harvested from Brucea-treated cells produced bands in a ladder pattern suggesting the apoptotic effect of the extract. It is thus concluded that Brucea sp. extract exhibited cytotoxic activity on ORL-48 cells and their action mechanism is via apoptosis.

GAINING MORE INSIGHT INTO THE DETERMINANTS OF CANDIDA SPECIES PATHOGENICITY IN THE ORAL CAVITY

Candida infection (candidiasis) is potentially life threatening and can occur in almost all anatomical sites, including the mouth. Candida species are in fact the most common fungal pathogens isolated from the oral cavity and frequently cause superficial infections such as oral candidiasis and denture-associated erythematous stomatitis. Whilst systemic dissemination of Candida from intraoral foci is rare and largely due to severe deficits of the host immune defenses, the development of localized oral candidiasis is most commonly related to a variety of non-immune determinants such as Candida virulence factors and permissive oral microenvironment. In particular, phenotypic switching and dental biofilm have emerged as major determinants for the pathogenicity of Candida and are currently the subject of intense research. An understanding of the molecular aspects underlying the biological behavior of Candida will be the key to the development of effective preventive as well as therapeutic measures for invasive and oral candidiasis.

Removal of the apical one-third of the root improves the fixation process of the dental pulp in teeth

The fixation process in histology is an important process that will eventually determine the quality of the histology slides. Fixation of the dental pulp involves the entry of the fixative agent into the dental pulp through the root canals that are found at the apical part of the root. The objective of this study was to evaluate the effectiveness of four methods of tooth preparation (teeth cut longitudinally or cervically, removal of apical third of root and whole uncut teeth) on the fixation process of the dental pulp for producing high quality histology slides. Among the four methods of tooth preparation, removal of the apical one-third of the root produced high quality histology slides as fixation of the dental pulp was improved and hence, preservation of the pulp’s architecture and contents were achieved. It is concluded that removal of the apical one-third of the root improves the fixation process of the dental pulp in teeth.

Assessment of Antifungal Activity of Bakuchiol on Oral-Associated Candida spp.

Bakuchiol is an active component of Psoralea glandulosa and Psoralea corylifolia, used in traditional Chinese medicine. The study aimed at investigating the antifungal activity of bakuchiol on planktonic and biofilm forms of orally associated Candida species. The antifungal susceptibility testing was determined by the broth micro dilution technique. Growth kinetics and cell surface hydrophobicity (CSH) of Candida were measured to assess the inhibitory effect of bakuchiol on Candida planktonic cells. Biofilm biomass and cellular metabolic activity were quantitatively estimated by the crystal violet (CV) and the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assays. All Candida strains have been shown to be susceptible to bakuchiol with the MIC ranges from 12.5 to 100 μg/mL. Significant decrease in specific growth rates and viable counts demonstrates the inhibitory effect of bakuchiol on Candida planktonic cells. A brief exposure to bakuchiol also reduced CSH of Candida (P < 0.05), indicating altered surface properties of yeast cells towards hydrophobic interfaces. Biofilm biomass and cell metabolic activity were mostly decreased, except for C. glabrata (P = 0.29). The antifungal properties of bakuchiol on Candida species in this in vitro study may give insights into the application in therapeutic strategy against Candida infections.

Vitamin E (α-Tocopherol) Exhibits Antitumour Activity on Oral Squamous Carcinoma Cells ORL-48

Cancers involving the oral cavity, head, and neck regions are often treated with cisplatin. In cancer therapy, the main target is to eliminate unwanted cancerous cells. However, reports on the nonselective nature of this drug have raised few concerns. Incorrect nutritional habits and lifestyle practices have been directly linked to cancer incidence. Nutrients with antioxidant activity inhibit cancer cells development, destroying them through oxidative stress and apoptosis. α-tocopherol, the potent antioxidant form of vitamin E is a known scavenger of free radicals. In vitro study exhibited effective antitumor activity of α-tocopherol on ORL-48 at 2.5 ± 0.42 µg/mL. Cisplatin exhibited stronger activity at 1.0 ± 0.15 µg/mL, but unlike α-tocopherol it exhibited cytotoxicity on normal human epidermal keratinocytes at very low concentration (<0.1 µg/mL). Despite the lower potency of α-tocopherol, signs of apoptosis such as the shrinkage of cells and appearance of apoptotic bodies were observed much earlier than cisplatin in time lapse microscopy. No apoptotic vesicles were formed with cisplatin, instead an increased population of cells in the holoclone form which may suggest different induction mechanisms between both agents. High accumulation of cells in the G0/G1 phase were observed through TUNEL and annexin V-biotin assays, while the exhibition of ultrastructural changes of the cellular structures verified the apoptotic mode of cell death by both agents. Both cisplatin and α-tocopherol displayed cell cycle arrest at the Sub G0 phase. α-tocopherol thus, showed potential as an antitumour agent for the treatment of oral cancer and merits further research.

Correlation between numbers of cells in human dental pulp and age: Implications for age estimation

OBJECTIVE The aim of this study was to investigate correlations between dental pulp cell count of odontoblasts, subodontoblasts and fibroblasts and age, within different age groups. Formulation of regression equations using the dental pulp cell count for predicting age was attempted. DESIGN Eighty-one extracted teeth were grouped into two age groups (6-25 years, 26-80 years). The teeth were demineralized and histological sections were prepared for cell count. Regression equations were generated from regression analysis of cell count and tested for age estimation. RESULTS The number of dental pulp cells were found to increase until around the third decade of life and following this, the odontoblasts and subodontoblasts cell numbers began to decline while the fibroblasts seemed to remain almost stationary. The Pearson correlation test revealed a significant positive correlation between the cell number for all type of cells and age in the 6-25 years group (r=+0.791 for odontoblasts, r=+0.600 for subodontoblasts and r=+0.680 for fibroblasts). In the 26-80 years age group, a significant negative correlation of the odontoblasts (r=-0.777) and subodontoblasts (r=-0.715) with age was observed but for fibroblasts, the correlation value was negligible (r=-0.165). Regression equations generated using odontoblasts and subodontoblasts cell number were applicable for age estimation. The standard error of estimates (SEEs) were around±5years for 6-25 years and±8years for 26-80 years age groups. The mean values of the estimated and chronological ages were not significantly different. CONCLUSIONS A significant correlation between the cell count of odontoblasts and subodontoblasts with age was demonstrated. Regression equations using odontoblasts and subodontoblasts cell number can be used to predict age with some limitations.

Alternative sweeteners influence the biomass of oral biofilm

OBJECTIVE Compact-structured oral biofilm accumulates acids that upon prolonged exposure to tooth surface, causes demineralisation of enamel. This study aimed to assess the effect of alternative sweeteners Equal Stevia®, Tropicana Slim®, Pal Sweet® and xylitol on the matrix-forming activity of plaque biofilm at both the early and established stages of formation. METHODS Saliva-coated glass beads (sGB) were used as substratum for the adhesion of a mixed-bacterial suspension of Streptococcus mutans, Streptococcus sanguinis and Streptococcus mitis. Biofilms formed on sGB at 3h and 24h represented the early and established-plaque models. The biofilms were exposed to three doses of the sweeteners (10%), introduced at three intervals to simulate the exposure of dental plaque to sugar during three consecutive food intakes. The treated sGB were (i) examined under the SEM and (ii) collected for turbidity reading. The absorbance indicated the amount of plaque mass produced. Analysis was performed comparative to sucrose as control. RESULTS Higher rate of bacterial adherence was determined during the early compared to established phases of formation. Comparative to the sweeteners, sucrose showed a 40% increase in bacterial adherence and produced 70% more plaque-mass. Bacterial counts and SEM micrographs exhibited absence of matrix in all the sweetener-treated biofilms at the early phase of formation. At the established phase, presence of matrix was detected but at significantly lower degree compared to sucrose (p<0.05). CONCLUSION Alternatives sweeteners promoted the formation of oral biofilm with lighter mass and lower bacterial adherence. Hence, suggesting alternative sweeteners as potential antiplaque agents.