PhiliPPine Journal of otolaryngology-head and neck Surgery Vol. 23 no. 1 January – June 2008 ORIGINAL ARTICLES PhiliPPine Journal of otolaryngology-head and neck Surgery 5 Philipp J Otolaryngol Head Neck Surg 2008; 23 (1): 5-8 c Philippine Society of Otolaryngology – Head and Neck Surgery, Inc. ABSTRACT Objective: To determine the antimycotic activity of the four medicinal plant extracts, kalachuchi bark (Plumeria acuminata Ait.), atsuete bark (Bixa orellana Linn.), akapulko leaves (Cassia alata Linn.), and neem leaves (Azadirachta indica Adr. Juss), when compared to the standard clotrimazole in the treatment of otomycosis. Study Design: Experimental Study Methods: Taxonomically identified plants, kalachuchi, atsuete, akapulko, and neem tree were collected and deposited in an herbarium. Extracts of these plants and the standard clotrimazole were tested against isolates of Aspergillus flavus, Aspergillus niger, and Candida albicans taken from patients with otomycosis. Three trials were made for each extract using different solvents and results subjected to statistical analysis. Result: Of the four medicinal plant extracts studied, only kalachuchi bark extract exhibited antifungal activity against Aspergillus flavus and Aspergillus niger using methylethylketone as solvent when compared to the standard clotrimazole. It was equally effective in inhibiting the growth of A. flavus and A. niger. However, all plant extracts using all types of solvents were equally ineffective in inhibiting the growth of Candida albicans. Conclusion: This in vitro study suggested that kalachuchi (Plumeria acuminata Linn.) bark extract inhibits the growth of Aspergillus species and was comparable to the standard clotrimazole. Following appropriate further studies and clinical trials, it may be a potential alternative treatment option for otomycosis caused by Aspergillus species. Key words: otomycosis; kalachuchi (Plumeria acuminata Linn.) bark; antimycotic; Aspergillus flavus; Aspergillus niger In Vitro Antimycotic Activity of Four Medicinal Plants Versus Clotrimazole in the Treatment of Otomycosis: A Preliminary Study Joebert M.Villanueva, MD Marida Arend V. Arugay, MD Rachel Zita H. Ramos, MD Department of Otorhinolaryngology Head & Neck Surgery Western Visayas Medical Center Correspondence: Joebert M. Villanueva, MD Western Visayas Medical Center ENT Office Q. Abeto St, Mandurriao, Iloilo City 5000 Phone: (6333) 509 0077 Fax: (6333) 321 1797 Email: joebert_md@yahoo.com.ph Reprints will not be available from the author. Funding support for this study was received from the West Visayas Medical Center Research Committee. The authors signed a disclosure that they have no proprietary or financial interest with any organization that may have a direct interest in the subject matter of this manuscript, or in any product used or cited in this study. Presented at Analytical Research Contest (1st Place), Philippine Society of Otolaryngology Head and Neck Surgery 50th Annual Convention, EDSA ShangriLa, Mandaluyong City, December 1, 2006. PhiliPPine Journal of otolaryngology-head and neck Surgery Vol. 23 no. 1 January – June 2008 ORIGINAL ARTICLES 6 PhiliPPine Journal of otolaryngology-head and neck Surgery Otomycosis, a fungal infection of the external auditory canal, is found throughout the world. Its prevalence is greatest in hot, humid, and dusty areas of the tropics and subtropics. Although a wide spectrum of fungi are involved, Aspergillus is the most common.1 Studies by Geaney2 and by Lakshmipathi and Murti3 attributed all observed cases to either Aspergillus or Candida species. Several studies have cited the antifungal properties of certain medicinal plants. Of the 10 medicinal plants recommended by the Department of Health (DOH) of the Republic of the Philippines, akapulko (Cassia alata Linn.) showed antifungal activity4 . A study of 10 medicinal plants in the priority list of the Philippine Council for Health Research Development (PCHRD) and Plant Resource of South East Asia (PROSEA) by Penecilla et al5 concluded that kalachuchi (Plumeria acuminata Ait.) and atsuete (Bixa orellana Linn.) had the highest activity in the assays against Candida species. Biswas et al observed that the extracts of neem leaf and neem seed oil kernels are effective against certain fungi including Trichophyton, Epidermophyton, Microspor, Trichosporon, Geotricum and Candida.6 Interested in finding out which among these medicinal plants—kalachuchi (Plumeria acuminata Ait.) bark, atsuete (Bixa orellana Linn.) bark, akapulko (Cassia alata Linn.) leaves, and neem (Azadirachta indica Linn.) leaves, had fungicidal properties against the common fungal pathogens causing otomycosis, our study aimed to determine the antimycotic activity of these four medicinal plant extracts when compared to the standard Clotrimazole in the treatment of otomycosis. METHODOLOGY A. Collection and Identification of Plants Collection and taxonomical identification of the following plant species was performed: kalachuchi (Plumeria acuminata Ait.) bark, atsuete (Bixa orellana Linn.) bark, akapulko (Cassia alata Linn.) leaves, and neem (Azadirachta indica Linn.) leaves. A minimum of 500 grams of each species were collected and air dried for three to five days at the West Visayas State University herbarium. B. Extraction of Plant Material About 500 grams of each dried plant material (kalachuchi, atchuete, akapulko, and neem) were crushed using mortar and pestle. The solvents hexane, methylethylketone and ethanol were used to serially extract the organic constituents from the plants.7 A 30-gram sample of each was taken for extraction using hexane, methylethylketone, and ethanol respectively. The sample which was dissolved using hexane was macerated for 48 hours with constant shaking using a mechanical shaking bath BT25 (Yamato, Japan). The mixture was then filtered using No. 33 filter paper (Whatman, U.S.A.) and evaporated to dryness using a Heidolph vv 2000 rotavap (Heidolph, Germany) machine. The solid marc was dissolved in another solvent, methylethylketone, and the same process of shaking, filtering, and evaporation to dryness was done. This process was repeated using ethanol. Extracts were stored in 10 ml amber bottles and labeled properly. C. Laboratory Testing 1. Gathering of Fungal Strains With informed consent, specimens were taken from patients diagnosed with otomycosis at the Out-Patient Department of a government tertiary hospital by a single otolaryngology resident using sterile cotton swabs and sterile saboraud’s dextrose broth tubes. Sample specimens were incubated in complete darkness at room temperature for four days to one week. Tubes were then examined for presence of surface growth or the appearance of mycelial growth structures and spores. 2. Identification of Fungal Species Only two species of fungi were isolated and identified – Aspergillus flavus and Aspergillus niger. Pure isolates of Candida albicans taken from the microbiology laboratory were obtained to represent Candida species in order to measure the efficacy of the plant extracts against Candida causing otomycosis. 3. Preparation of Pure Culture and Agar Disks Pure cultures were prepared using the agar-blocked method.8 4. Bioassay Proper The agar disc method was performed against pure isolates of Aspergillus flavus, Aspergillus niger and Candida albicans. The agar disc size was 14.5 mm in diameter. Three trials were made for the control group and for each extract using different solvents. A 100% concentration of the positive control (clotrimazole) was used. Examination of all plates for any zone of inhibition formation was performed. The diameters of the zones of inhibition were PhiliPPine Journal of otolaryngology-head and neck Surgery Vol. 23 no. 1 January – June 2008 ORIGINAL ARTICLES PhiliPPine Journal of otolaryngology-head and neck Surgery 7 Table 1. Mean Zones of Inhibition Between the Different Treatment Groups against Fungal Pathogens FUNGAL PATHOGEN EXTRACTS SOLVENT MEAN ZONES OF INHIBITION (in mm.) INITIAL (n=3) FINAL (n=3) Aspergillus flavus Aspergillus niger Candida albicans Kalachuchi bark Atsuete bark Akapulko leaves Neem leaves Control (Clotrimazole) Kalachuchi bark Atsuete bark Akapulko leaves Neem leaves Control (Clotrimazole) Kalachuchi bark Atsuete bark Akapulko leaves Neem leaves Control (Clotrimazole) Hexane MEK Ethanol Hexane MEK Ethanol Hexane MEK Ethanol Hexane MEK Ethanol Hexane MEK Ethanol Hexane MEK Ethanol Hexane MEK Ethanol Hexane MEK Ethanol Hexane MEK Ethanol Hexane MEK Ethanol Hexane MEK Ethanol Hexane MEK Ethanol 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 38.5 46.17 34.83 14.5 14.5 35.33 14.5 14.5 14.5 14.5 14.5 14.5 44.17 24.83 41.17 33 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 43 14.5 17.33 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5 22.33 then measured by getting the average of the zone measuring lengthwise and clockwise.8 D. Statistical Analysis The mean diameter of zones of inhibition of the different treatment groups were compared using analysis of variance in completely randomized design. Pairs of treatment means were compared using the Duncan’s Multiple Range Test. Absence of a statistically significant difference compared to the positive control (clotrimazole) was considered a significant finding. RESULTS The positive control (clotrimazole) had a mean zone of inhibition diameter against Aspergillus flavus of 44.17 mm. Among the medicinal plants, kalachuchi (Plumeria acuminata Ait.) using methylethylketone (MEK) as solvent had the highest mean zone of inhibition diameter against Aspergillus flavus of 46.17 mm. Other extracts showed no increase in the mean zone of inhibition diameters against Aspergillus flavus. (Table 1) The zones of inhibition using the control, clotrimazole for Aspergillus niger were 43.00 mm mean diameter. Kalachuchi extracts using MEK had mean zone of inhibition diameter at 41.17 mm. Other extracts showed no increase in the mean zone of inhibition diameters against Aspergillus niger. (Table 1) Clotrimazole had only a mean diameter zone of inhibition at 22.33 mm for Candida albicans. Kalachuchi bark extract using only methylethylketone as solvent had only a mean diameter zone of inhibition of 17.3 mm. The three remaining medicinal plant extracts (atsuete, akapulko, and neem) using all three solvents exhibited no increase in the mean zone of inhibition diameters against Candida albicans. (Table 1) Since only kalachuchi extract showed a comparable zone of inhibition diameter to the control (Clotrimazole), it was the only extract that was statistically analyzed. Statistical analysis using analysis of variance showed that the positive control (clotrimazole) was effective for Candida albicans compared to kalachuchi extract using different solvents. On the other hand, kalachuchi extract using methylethylketone (MEK) as solvent exhibited the same response as clotrimazole against PhiliPPine Journal of otolaryngology-head and neck Surgery Vol. 23 no. 1 January – June 2008 ORIGINAL ARTICLES 8 PhiliPPine Journal of otolaryngology-head and neck Surgery Aspergillus flavus and Aspergillus niger. (Figure 1) DISCUSSION Of the four plant extracts studied, only kalachuchi bark extract exhibited antifungal activity against Aspergillus flavus and Aspergillus niger using methylethylketone as solvent when compared to the standard clotrimazole. Kalachuchi (Plumeria acuminata Ait.) belongs to the family Apocynaceae. Plumeria species have formally been investigated for isolation of a variety of iridoids and triterpenoids, which exhibited algicidal, antibacterial, cytotoxic and plant growth inhibitor activity. A study by Pandey et al revealed that repeated column chromatography of the methanolic extract of the bark of Plumeria species obtained the purified iridoid-saccharide plumieride compound.9 Plumieride may be responsible for the antimycotic activity of kalachuchi in this study. This in vitro study suggested that kalachuchi bark extract inhibits the growth of Aspergillus species and was comparable to clotrimazole. Following appropriate further studies and clinical trials, it may be a potential alternative treatment option ACKNOWLEDGEMENT The authors wish to thank Dr. Jose Mari Fermin, Medical Director of Western Visayas Medical Center and its Research Committee for the financial support; Dr. Gerard Penecilla and staff, for their help and guidance during the process of plant identification and extraction; Professor Roman Sanares for the statistical analysis; Professor Celia P. Magno for her time and service during the bioassay; and the resident staff of the Department of Otorhinolaryngology Head & Neck Surgery, Western Visayas Medical Center. REFERENCES 1. Joy MJ, Agarwal MK, Samant HC, et al. Mycological and bacteriological studies in otomycosis. Indian J Otolaryngol 1980;32:72-5. 2. Geaney GP. Tropical otomycosis. J Laryngol Otol 1967;81:987-97. 3. Lakshmipati G, Murti RB. Otomycosis. J Indian Med Assoc 1960;34:439-41. 4. Philippine Council for Health Research and Development. Herbal products developed by Filipino scientists. 2003. Available from: http://www.pchrd.dost.gov.ph 5. Penecilla G, Magno C, de Castro J, et al. Production and Testing of Natural Products for Antimicrobial and Antifungal Action. West Visayas State University College of Arts and Sciences Research Journal. 2001;2(1):10-20. 6. Biswas, Kausik, Ishita C, Ranajit KB, and Uday B. Biological activities and medicinal properties of Neem (Azadirachta indica). Current Science. 2002;82(11):1336-1345. 7. Guevara BQ, Claustro AL, Aguinaldo AM, Madulid RS, Espeso EI, Nonato MG, et al. A Guidebook to plant screening: phytochemical and biological. Rev ed. Manila: Research Center for the Natural Sciences, University of Santo Tomas;2005. 8. Magno CP. Manual in Medical Microbiology. 2nd ed. Iloilo: West Visayas State University;2005. 9. Pandey R, Dobhal M, Graham A, Oseroff A. Iridoid-saccharide compound and method using same. Available from: http://www.freepatentsonline.com/EP1527783.html 10. Bojrab DI, Bruderly T, Abdulrazzak Y: Otitis externa. Otolaryngologic Clin North Am. 1996;29(5):761-781. Figure 1. Response of Control (Clotrimazole) and Kalachuchi Extract using Different solvents against Fungal Pathogens *Solvent with the same letter showed no significant difference. for otomycosis caused by Aspergillus species. However, it cannot be overemphasized that the key to successful treatment of otomycosis is gentle efficient cleaning of the ear canal, carefully removing its accumulated debris, thorough drying and application of antifungal agents. 10 Further research can validate the results of this in vitro study by ascertaining specific chemical properties through fractionation and structural elucidation of kalachuchi bark extract. Toxicity studies and in vivo trials can then determine kalachuchi bark extract’s benefit as a topical antifungal agent against otomycosis.