Anticandidal and Antidermatophytic Activities of Caulerpa Species from the Gulf of Mannar Coast, Mandapam Tamilnadu India

The present investigation was carried out to evaluate the antifungal activity of hexane, chloroform, ethyl acetate, acetone and methanol extracts of Caulerpa chemnitzia, (Epser) J.V. Lamououx C. racemosa (Forsk), and C. scalpelliformis (R.Br.) Weber-van-Bosse, (Chlorophyceae) against Candida albicans, C. krusei, C. guilliermondii, C. parapsilosis, C. tropicalis, C. glabrata, four dermatophytes viz., Trichophyton rubrum, T. mentagrophytes, Microsporum gypseum and Epidermophyton flocossum. The antifungal activity was evaluated by agar disc diffusion method, determination of MIC and MFC. The mean zones of inhibition produced by the extracts in disc diffusion assay against the tested fungal strains were ranged from 7.1 to 15.1 mm. The lowest MIC (250 μg/ml) and MFC (500 μg/ml) values were observed in the ethyl acetate extract of C. racemosa against C. parapsilosis, C. albicans, C. krusei, C. glabrata, C. guilliermondii, T. rubrum, M. gypseum and T. mentagrophytes. The finding suggests that ethyl acetate extract of C. racemosa showed the higher antifungal activity against C. parapsilosis and C. albicans.


Introduction
Fungal infections are an increasing threat to human health. In the developed world, these infections predominantly occur in the context of increasingly aggressive immunosuppressive therapies. The overall mortality for invasive diseases caused by Candida spp. and Aspergillus spp. is 30-50%, despite the advent of new diagnostic and therapeutic strategies. In the developing world, there are 1 million cases of cryptococcal disease per year, results in 675 000 deaths [1]. Allergic fungal syndromes are increasingly recognised. Continued efforts are required to improve the often suboptimal therapeutic outcomes associated with fungal infections.
Dermal mycoses or superficial fungal infections are among the most common infections worldwide [2]. They are believed to affect 20-25% of the world's population and the incidence of these infections have been increased significantly over last 15-20 years which may be ascribed to changing patterns of migration, growth in tourism, and changes in socioeconomic conditions. These infections are mainly caused by dermatophytes belonging to genera Epidermophyton, Microsporum and Trichophyton; and the causative species vary with geographic regions. Skin, hair, nail and subcutaneous tissues in human and animal are subjected to infection by several organisms, mainly fungi named dermatophytes and cause dermatophytoses [3]. The most common strains of pathogenic fungus in humans is C. albicans, filamentous fungi that is found in normal human skin flora. C. albicans is best known among the general populace as the fungus that cases thrush and topical Candidiasis. C. albicans is the most common causative agent of oral candidiasis, but non-albicans Candida spp., such as C. tropicalis, C. glabrata, C. parapsilosis, C. krusei and C. guilliermondii, have also emerged as significant pathogens [4].
The difficulty in diagnosis of fungal infections and delays in initiation of treatments are important factors, drugs for effective treatment of these emerging infections are in short supply, thereby contributing to a high mortality rate. Available drugs are essentially limited to the polyene natural product Amphotericin-B and various newer lipid formulations [5], the azole compounds such as fluconazole, itraconazole and flucytosine (5-fluorocytosine). Controlled release systems can address both issues. Consequently, there has been increasing interest, in both research and clinical standard of 0.5-2.5 × 10 3 cells/mL. The filamentous fungal strains were subcultured on SDA and incubated at 30°C for 4-7 days for dermatophytes. The growth was scraped aseptically, crushed and macerated thoroughly in sterile distilled water and inoculum of fungal strains were obtained according to reported procedures and adjusted to 0.4-5 × 10 4 cells/mL.

Disc-Diffusion Assay
The in vitro antifungal activities of different algal crude extracts were screened using agar diffusion method [10]. For that assay, petriplates were prepared by pouring 20 ml of sabouraud dextrose agar and allowed to solidify for 20 minutes. The standardized inoculum suspension were swabbed on the top of the solidified media and allowed to dry for 10 minutes. Discs with different concentrations of extracts (1000, 500 and 250µg/disc) were prepared and aseptically applied on the surface of the petriplates. Amphotercin-B (100 units/disc) for Yeast and Ketoconazole (5µg/disc) for dermatophytes were used as positive controls and 10 per cent DMSO was used as blind controls in all the assays. After that, the plates were incubated at 28 °C for 24 hours for yeast and 30 °C for 3-5 days with dermatophytes. The zone of the inhibitions was measured in millimeter. The experiments were carried out in triplicates.

Minimum Inhibitory Concentration (MIC) for Fungi
The MIC of the crude extracts of Caulerpa species were determined by using broth micro dilution technique as recommended by CLSI [11] and [12] for yeast and filamentous fungi respectively. The MIC values were determined in RPMI-1640 (Himedia, Mumbai) with L-glutamine without sodium bicarbonate, pH 7.0 with morpholine propane sulfonic acid (MOPS). Fifty milligram of crude extracts were dissolved in 1 mL of 10% DMSO and stock solution was obtained for the determination of MIC. For crude extracts, 20 µL of each plant extracts, were dissolved with 980 µL of RPMI-1640 medium (2 mg/mL). From that, two fold serial dilutions in the range from 1000 to 15.7 µg/mL were prepared. 200 µL of solution was poured into first well of 96 well microtitre plates and then, 100 µL were transformed to the next well containing 100 µL of RPMI-1640. The same procedure was performed for all wells. 10 µL of fungal standardized inoculum suspensions containing 0.5-2.5×10 3 cfu/mL for yeast 0.4-5×10 4 cfu/mL for dermatophytes were transfered to each well. The control well contained only sterile water and devoid of inoculum. The microtitre tray plates were incubated without agitation at 28°C for 24 h for yeast and 30°C for 4-7 days for dermatophytes. The MIC of the extracts were recorded as the lowest concentration of extracts were inhibited the growth of the Candida and dermatophytic strains when compared to that of control.

Minimum Fungicidal Concentration (MFC)
MFC of the extracts were determined by plating 100 µl of samples from each MIC assay well with growth inhibition. The wells were transferred to freshly prepared sabouraud dextrose agar plates and incubated in incubator at 28 °C for 24 hours for yeasts and 30 °C for 3-5 days for dermatophytes. The MFC was recorded as the lowest concentration of the extracts that did not permit any visible fungal growth after the period of incubation.

Statistical Analysis
The results are expressed as the mean  SD. All statistical analyses were performed using SPSS version 16.0 statistical software (SPSS Inc., Chicago, IL, USA). Student's t-test was performed to determine any significant difference between different extracts for in vitro antifungal assays. Comparison of means for in vitro antifungal assessment was carried out using one-way analysis of variance (ANOVA) and Duncan test. P value < 0.05 was considered statistically significant.

Results
The different solvents of viz., hexane, chloroform, ethyl acetate, acetone and methanol extracts of C. chemnitzia, C. racemosa and C. scalpelliformis were screened against fungal strains. Among the tested extracts, the ethyl acetate and chloroform extracts showed the antifungal activity against fungal strains tested. The mean zones of inhibition of the extracts, assayed against the test organisms ranged between 7.1 and 15.1 mm. The ethylacetate extract of C. racemosa showed promising activity against C. parapsilosis (15.1 mm), followed by C. albicans (14.3 mm) and T. rubrum (14.1 mm). The chloroform extracts showed activity against C. parapsilosis (13.1 mm), followed by T. rubrum (13.3 mm), C. albicans (12.6 mm) and T. rubrum (12.3 mm) are presented in Tables 1, 2 & 3. The Amphotericin-B (100 units/disc), anticandidal positive control produced zones of inhibition were from 9.0 to14.5 mm. Ketoconazole (10 µg/disc), antidermatophytic positive control produced zones of inhibition ranged from 14 to 19 mm. The negative control (10% DMSO) did not produce any zone of inhibition for all the fungal strains tested. The result of MIC values of the different extracts of C. chemnitzia, C. racemosa and C. scalpelliformis ranged between 250 and 500 µg/ml. While the MFC values were between 500 and 1000 µg/ml.       Table 3. Antifungal activity of different extracts of Caulerpa scapelliformis.
In the present study we have used two control drugs namely Amphotericin B and ketoconazole. Amphotericin B (Amp B) and the azoles are mainly used in common clinical situations. Furthermore, Amp B is considered as the drug of choice for the treatment of fungal infections [25]. However, dose-limiting nephrotoxicity associated with amphotericin B, rapid development of resistance with flucytosine, drug-drug interactions, fungistatic mode of action and resistance development with the azoles. In case of Amp B, due to its poor permeability across the membrane an increased amount of Amp B must be administered to patients, which can result in severe side effects such as renal damage [26]. Ketoconazole is one of the commonly used antifungal drugs administered orally for the treatment of both superficial and deep infections caused by Trichophyton. However, the unpleasant side effects of this drug include nausea, abdominal pain, and itching and its toxicity limits its therapeutic use in many cases [27]. The utilization of various latex in combination with an antifungal drug such as amphotericin-B or ketoconazole may involve a reduction of the dose of drugs used in treatment of mycoses and therefore in a reduction of their side-effects.
Since natural products have been proven to be an excellent source of novel chemical entities, we have employed screening of microbial extracts in our search for new novel compounds.

Conclusion
Finally, it can be concluded that the ethyl acetate extracts of C. chemnitzia, C. racemosa and C. scalpelliformis showed the best antifungal activity against C. parapsilosis and C. albicans.
Among the types of fungi tested, the Candida species were more susceptible than dermatophytes. Further, the isolation of antifungal compound from the ethyl acetate extract of C. racemosa is in progress.