Effects of Brucea javanica and Piper betle extracts on oral Candida SPP / Mohd-Al-Faisal Nordin

Candida species is an opportunistic microorganism residing in the human oral cavity. There is increasing prevalence of candidal infections in the oral cavity largely because of the increasing size of the population at risk. The rise in diagnosed cases of oral candidiasis is also due to the immunosuppressive effect of prescribed antifungal agents on resistant hosts. The ability of Candida species to adhere on mucosal tissues and/or denture surfaces, and the production of hydrolytic enzymes along with defective host immunity are among the known key factors of invasions and pathogenesis of oral candida. Although Candida albicans remains as the most pathogenic organism, the emergence of non-Candida albicans Candida (NCAC) species with reduced susceptibility to prescribed antifungal agents has prompted efforts to study antifungal agents from natural sources. Plants have long been known to possess medicinal values and are rich in chemical constituents that can be used in the development of antifungal products. Initially, seven local plants were systematically screened for their antifungal activity and two of them - Brucea javanica L. and Piper betle L. were selected based on their positive antifungal activities on seven Candida species tested. The specific objectives of this study were; (i) to determine the minimum inhibition concentration (MIC) and minimum fungicidal concentration (MFC) values of the extracts, (ii) to investigate the growth inhibitory effect of the extracts based on changes in the pattern of growth profile of each Candida species, (iii) to investigate the influence of extracts on adherence mechanisms which include the non-specific and specific bindings, (iv) to investigate any morphological changes of the cells following treatment with the extracts, and (v) to explore the differential expressions of multigene family of secreted aspartyl proteinases (SAPs) and hyphal cell wall protein 1 (HWP1). Candida species purchased from the American Types Culture Collection (ATCC) were Candida albicans ATCC 14053, Candida dubliniensis ATCC MYA-2975, Candida glabrata ATCC 90030, Candida krusei ATCC 14243, Candida lusitaniae ATCC 64125, Candida parapsilosis ATCC 22019 and Candida tropicalis ATCC 13803. Growth inhibitory responses of the Candida species to the extracts include determination of the MIC and MFC, while effect on the growth curve was determined based on spectrophotometric assay. Deviations in the doubling time (g) and specific growth rates (μ) were computed as percentage to extract-treated cells relative to that of the total cells in the absence of extracts. In addition, the anti-adherence effect of the B. javanica and P. betle extracts which included study on the cell-surface hydrophobicity (CSH) and specific bindings on pellicles, ultrastructure and the regulations of SAP1-10 and HWP1 were also analysed. 0.12% w/v chlorhexidine gluconate-containing mouthrinse was used as a reference. In preliminary screening, the diameter of inhibition zone (DIZ) values showed that B. javanica and P. betle aqueous extracts exhibited a wide range of antifungal activities over the seven Candida species with C. dubliniensis identified as the most sensitive. Of the seven Candida species, C. tropicalis showed the highest growth rates (0.319 ± 0.002 h-1) while the others were in the range of 0.141 ± 0.001 to 0.265 ± 0.005 h-1. This indicated that different species of candida reproduce at different rate. In the presence of extracts, the lag and log phases were extended and shifted to the right. This resulted in the deviations of the g- and μ-values, indicating that the extracts may have exerted fungistatic activity towards the candidal cells. Growth kinetics of the candidal species was also elucidated based on colony forming unit (CFU) enumeration. Different Candida species have shown different CSH values and adhering capacity to the pellicle. In view of the CSH, C. krusei, C. dubliniensis and C. tropicalis showed the highest adsorption to hexadecane at 30.23%, 26.19% and 19.70%, respectively, while the others were much lower within the range of 7% to 10%. The CSH of all Candida species were significantly affected by these two extracts (P < 0.05), with B. javanica exhibiting more than 60% reduction of CSH than P. betle. Specific bindings of the candidal cells on the pellicles were also shown to be affected by the treatment of extracts. Exposure to P. betle-treated pellicle drastically reduced the adhering capacity of three out of seven candidal species by more than 50% (C. tropicalis 86.02%, C. albicans 61.41% and C. krusei 56.34%). Pellicles treated with B. javanica exhibited similar effect on C. tropicalis (89.86%), C. lusitaniae (89.66%), C. albicans (79.74%), C. glabrata (76.85%) and C. krusei (67.61%). Comparatively, the adherence interference effect of B. javanica towards the candidal cells was slightly higher than P. betle. In addition to the growth inhibitory and anti-adherence effects, physical changes in the cell walls of Candida species were also demonstrated following treatment of the candidal cells with the extracts. The expressions of SAP1-10 and HWP1 were affected by the extracts treatment, suggesting that the extracts have successfully penetrated and disrupted the intrinsic environment of the cells. The genes seemed to be suppressed and this may then revoke the pathogenesis of oral Candida. As a conclusion, B. javanica and P. betle exhibited antifungal activities towards the seven oral Candida species tested. Data from this study strongly suggest the fungistatic and growth inhibitory effects of the extracts. Thus, B. javanica and P. betle extracts may be considered as promising adjuncts in oral health products