Impatiens Balsamina Linn. (Kamantigue) Flower Extract a Potential Antifungal Agent for Candida Albicans and Trichophyton Mentagrophytes free essay sample

way Analysis of Variance (ANOVA) decision table ‘ CHAPTER I THE PROBLEM AND ITS BACKGROUND Introduction The Impatiens balsamina Linnaeus, or commonly known as kamantigue, is a medical herb also known as garden balsam. This plant is native to South Asia and Southeast Asia belonging to the family Balsaminaceae (Touch-me-not Family). This plant can easily be seen anywhere in the Philippines. It is an annual (yearly) plant growing to 20 to 75  cm tall, with a thick branch but soft stem. The leaves are spirally-arranged, 2. 5 to 10  cm long and 1 to 2. 5  cm broad, with a deeply toothed margin. The most common flowers are pink, purple, white, or red and it is approximately 2. 5 to 5  cm diameter (http://www. plantoftheweek. org/week337. shtml). Different parts of the plant has many uses, the leaves, seeds and stem are used to treat skin afflictions and the juice extract of kamantigue was used to treat warts and even snakebite, while the flower can be applied to burns to cold the skin. The seeds of  Impatiens Balsamina contain four cysteine rich compounds with marked antimicrobial properties (http://stuartxchange. com/Kamantigi. html). The phytochemical content of Impatiens balsamina Linn. are Impatienol, pelagonidine, delphinide, cyanidine, balsaminones A and B, hosennkosides F-O and others are found in kamantigue which has antimicrobial and antifungal properties. ( A Guide to Medicinal Plants 2010 by Koh Hwee Ling, Chua Tung Kian, Tan Chay Hoon). Several species of genera Impatiens are currently still tested for their antifungal properties. 2-methoxy-1,4-napthoquinone that is used as an active ingredient of some formulation of preparation H; brand of medication manufactured by pfizer) Candida albicans is the most common pathogen within the genus Candida (Wingard, 1995; Pfaller, 2004). It is a diploid fungus that grows both as yeast and filamentous cells. Systemic fungal infections (fungemias) including those by Candida albicans have emerged as important causes of morbidity and mortality in immunocompromised patients like those who are suffering from AIDS, cancer chemotherapy, organ or bone marrow transplantation. Candida albicans biofilms may form on the surface of implantable medical devices. In addition, hospital-acquired infections by Candida albicans have become a cause of major health concerns. (Pfaller, 2002) Candida albicans is commensal and a constituent of the normal gut flora comprising microorganisms that live in the human mouth and gastrointestinal tract. Candida albicans lives in 80% of the human population without causing harmful effects, although overgrowth of the fungus results in candidiasis (candidosis). Candidiasis is often observed in immunocompromised individuals such as HIV-infected patients. A common form of candidiasis restricted to the mucosal membranes in mouth or vagina is thrush, which is usually easily cured in people who are not immunocompromised. For example, higher prevalence of colonization of Candida albicans was reported in young individuals with tongue piercing, in comparison to non-tongue-pierced matched individuals. To infect host tissue, the usual unicellular yeast-like form of Candida albicans reacts to environmental cues and switches into an invasive, multicellular filamentous form, a phenomenon called dimorphism. Trichophyton mentagrophytes  is the most widespread among the  anthropophilic dermatophytes. Anthropophilic dermatophyte is found in association with humans. It  is the most common agent of Tinea on the feet, hands, nails, groin, and the glabrous skin, however, the scalp is rarely infected. Animals are very infrequently infected as well. (Ryan KJ, Ray CG,2004). The growth rate is slow to moderately rapid, the texture of colonies ranges from downy to powdery while the surface colony color is white to pale pink while reverse is usually wine red, however at times, the color ranges from red, brown, violet, yellow or even uncolored. Presence of  microconidia  is numerous to rare, club shaped to pyriform, may be found solitary along the hyphae or sometimes in clusters, and are unicellular; and Microconidia  are frequently absent; pencil to cigar shaped, and are multi septate. Resistance to imidazole antibiotics such as ketoconazole, fluconazole, or itraconazole is unfortunately developing in isolates of Candida species. Candida glabarata and Candida krusei, in particular, are intrinsically more resistant to this class of antifungal agent than is Candida albicans (Pfaller, 2002, 2003b). The in vitro susceptibility of three clinical Trichophyton mentagrophytes  isolates to griseofulvin and tioconazole, determined by the minimal inhibitory concentration (MIC), was 2 and 0. 5 to 1. 0? g/ml, respectively. One mutant obtained after mutagenic treatment of one of these isolates was selected and showed simultaneous resistance to griseofulvin (MIC gt; 2000? g/ml) and tioconazole (MIC=1. 0? g/ml) (http://www. springerlink. com/content/q683722642467224/) Statement of the Problem This study determined the use and effectivity of Impatiens balsamina Linn. lower extract as a potential antifungal agent for Candida albicans and Trichophyton mentagrophytes; therefore the following questions had been drawn: 1. What is the zone of inhibition of Candida albicans and Trichophyton mentagrophytes. 2. Is there a significant difference in the zone of inhibition of Candida albicans and Trichophyton mentagrophytes using the flower extract of Impatiens balsamina Linn.. compared with the contr ol groups? 3. Is the Impatiens balsamina Linn. effective as antifungal agent for Candida albicans and Trichophyton mentagrophytes? Hypothesis The following hypothesis was formulated for this study: There was no significant difference in the zone of inhibition of Candida albicans and Trichophyton mentagrophytes using the flower extract of Impatiens balsamina Linn. compared with the control group. Significance of the Study This study would benefit the following: Students. This study will benefit the students specially those who were studying Mycology which will help applying their knowledge into practical uses and developing critical thinking. Enabling them to be interested in medicinal plants. Future researchers. This study will provide additional information in conducting an experiment on Impatiens balsamina Linn.. It may serve as a background for their study. Community. This study will benefit the people that have skin infection. The uses and importance of Impatiens balsamina Linn.. will provide them alternative way in treating the infection. Scope and Limitation The study was limited to the determination of the effectivity of flower extract of Impatiens balsamina Linn. as antifungal agent for  Candida albicans and Trichophyton mentagrophytes. Determination of antifungal property was limited to the quantitative sensitivity of the test organism to the extract in the antifungal bioassay performed using the Sabouraud Dextrose Agar. The plates were incubated for eighteen to twenty four hours and observations were made within this length of time. Definition of Terms Impatiens balsamina Linn. It is an annual, erect, succulent, branched herb, 1 meter high or less. Flowers are axillary, showy, 2 to 3 centimeters long, usually pink, but forms with white, red, purple, or variegated petals are also found in cultivation. Candida albicans. A yeast-like fungal organism found in small amounts in the normal human intestinal tract. Normally kept in check by the bodys own helpful bacteria, Candida albicans can increase in numbers when this balance is disturbed to cause candidiasis of the intestinal tract, or yeast infections of other parts of the body. Candida albicans causes thrush. Also called Monilia albicans. Trichophyton mentagrophytes. It is a fungi which cause inflammatory skin or scalp lesions in humans, particularly in rural workers. It also causes athletes foot, ringworm, jock itch, and similar infections of the nail, beard, skin and scalp. Candidiasis. It is a primary or secondary mycotic infection caused by members of the genus  Candida. The clinical manifestations may be acute, subacute or chronic to episodic. Involvement may be localized to the mouth, throat, skin, scalp, vagina, fingers, nails, bronchi, lungs, or the gastrointestinal tract, or become systemic as in septicemia, endocarditis and meningitis. Antifungal Agents. It is a drug that selectively eliminates fungal pathogens from a host with minimal toxicity to the host. Millimeter. is a  unit  of  length  in the  metric system, equal to one thousandth of a  metre, which is the  SI  base unit  of length. Zone of Inhibition. The clear region is an indication of the  absence, or the effective  inhibition, of  microbial  growth  by the antimicrobial  agent. CHAPTER II REVIEW OF RELATED LITERATURE This chapter indicates the ideas relevant to the present study relating to the other studies and was briefly discussed to provide the foundation of the research investigation. In order to develop an alternative medications specifically for fungal infection, careful review of related literature and studies must be done for the development of the study. Koh Hwee Ling, Chua Tung Kian, Tan Chay Hoon. (2010) stated in their book that a lotion of fresh leaves was used to treat eczema, itches and insect bites. In Vietnam, decoctions of leaves are used to stimulate growth and to wash hair. The juice was also used for warts, cancer treatment and expectorant. A decoction of flowers was taken for infections, vomiting, urine retention and as a tonic, In india, flowers are regarded as cooling, tonic and used for lumbago and intercostals neuralgia, snakebite, improves circulation and relieves stasis. In Japan, juice squeezed from the white flower petals are applied on the skin to alleviate dermatitis. In China, the seeds are prescribed for difficult labour, puerpal pains, difficult menstruation, cough, hiccups and poisonings. The seeds are mixed with arsenious acid for removing teeth. In Malaysia, the seeds are taken for gastrointestinal tract cancer,and to dislodge fish or chicken bones in throat. Richard A. McPherson and Matthew R. Pincus 2009 stated in their book that Candida albicans is the most common pathogen in all the genus of Candida. Cutaneous disease is the most frequent infection caused by the Candida species, typically presenting as erythematous lesions of the skin, sometimes accompanied by a creamy, white exudates or scaling. Richard A. McPherson and Matthew R. Pincus 2009 stated in their book that for many years, few antifungal chemotherapeutic agents were available with Nystatin virtually the only agent effective against moist of systemic pathogens. Although this potent anti fungal agent was frequently toxic, resistance to therapy was rare. The recent introduction of new antifungal gents and the subsequent development of resistance among mould and yeast pathogens has added new impetus to the development and standardization of test for laboratory guidance of antifungal chemotherapy. Alex Villafania in September 2007 stated that A Study from the University of Southeastern Philippines in Davao City has found that a compound from garden balsam (Impatiens balsamina), otherwise known as kamantigue, can control a disease striking the durian. Durian is a major export product from southern Mindanao but is constantly being attacked by serious diseases by the fungi  Phytophthora palmivora. Durian is constantly attacked by stem canker, root rot, leaf blight, die-back of seedlings and mature trees and the rotting of fruits. The study, headed by USEP Professor Belly Dionio, said that production losses in the durian industry across Southeast Asia are estimated to be around 20 percent to 25 percent annually. Dionio focused on garden balsam as an alternative to the otherwise hazardous use of expensive synthetic fungicides. An extract from red and violet garden balsam showed that it is toxic to  P. almivora  and is as effective as the commercially available fungicide Fosetyl-al. Dionio’s research team found that by using the garden balsam extract, they were able to reduce lesions on durian plants as much as 86 percent when the extract was sprayed an hour before plants are introduced to the  P. palmivora  strain. Likewise, the plants show 78 percent reduction in lesions when these are sprayed with the garden balsam extract an hour after these are exposed to  P . palmivora. Defoliation (death of leaves) due to the fungus was also lower in durian plants sprayed with the extract. They also found out that the extract can remain effective even if it was stored in room temperature for 13 weeks, making it viable as a cheap and safe long-term preventive measure against durian diseases (http://blogs. inquirer. net/insidescience/2007/09/10/study-reveals-fungicidal-benefits-of-kamantigue-on-durian/) Richard Seah in 1990 stated in their article that Garlic is one food that has powerful anti-bacterial and anti-fungal properties and some scientific studies have found it to be at least as effective as the popular anti-fungal drug, Nystatin, in destroying candida albicans. One important difference between taking drugs such as antibiotics and anti-fungals, and taking garlic, was that bacteria and fungi are not likely to develop a resistance to garlic. This was because garlic contains many different substances with anti-bacterial and anti-fungal properties, including allicin, alliin, alliinase and S-allylcysteine. To get the most of garlics ability to destroy bacteria and fungi like the candida yeast, it was best to take garlic raw (http://www. natural-cancer-cures. com/cures-for-candida. html). Lim, Jeffrey, Maamo, Marlo and Monte Grande, Claro 1993 stated in their article that the effectivity of Kampanyero (Thevetia peruviana) leaf extract as an inhibitory agent was tested on Candida albicans in vitro in extract concentration s of 100%, 75%, and 50%. In the serial dilution test, the result of five trials showed that the extract exerted significantly greater fungicidal activity in Candida albicans compared to 80 % ethanol as control. Meanwhile, results of the paper disc sensitivity test in five trials showed that there was no significant difference in the degree of effectivity among the three extract concentrations. After statistical treatment using Student’s Test, the Kampanyero leaf extract was found to posses inhibitory properties which was equally effective at a 50% concentration 75% and 100% concentration on Candida albicans in vitro. Richard Seah in 1990 stated in His article that the oil of  Oregano Vulgare,  a species of the oregano herb that is commonly used in Italian and Greek cooking, is also known to have powerful anti-fungal properties. Interestingly, Oregano is used in the Philippines primarily as a herbal medicine – for various common illnesses like coughs and the flu – rather than a herb for cooking. Oregano oil is normally sold as a pure oil. It is very potent. So all you need is a few drops mixed into a glass of water. Taking too much of oregano oil might cause irritation to the mucous membranes (http://www. natural-cancer-cures. com/cures-for-candida. html). According to Barros, Clarita T. (Ph. D. Sci. Ed. , March 2000) and Clara C. Sibolboro, Ph. D. The studily determine the antifungal activity of the crude leaf extract of three medicinal plants, namely Ocimum sanctum Linn (Bidai), Impatiens balsamina Linn (kamantigue), and Heliotropium indicum Linn (Penga-penga). The study utilize the experimental research method. The Completely Randomized Design (CRD) was the research design used. The two way ANOVA and Scheffe Test were the statistics used. Finding revealed that the ethanol extract Penga-penga, Kamantigue and Bidai were active to T. mentagrophytes and E. floccosum. The chloroform extract of kamantigue was active to C. albicans and T. metagrophytes while those of Penga-penga and Bidai were active to T. mentagrophytes. All the plant extract except the chloroform extract of kamantigue were inactive to C. lbicans due to the absence of zone of inhibition. The comparative study among Penga-penga crude extract, Penga-penga ointmentand Ketokonazole revealed that the three samples were equally active against T. mentagrophyte. Similarly, the Kamantigue crude extract and kamantigue ointment were comparable in effect, but Ketokonazole (control drug) was significantly different. On the nature of inhibitory response of the three samples, the inhibition wa s fungicidal for both Penga-penga and Kamantigue extracts. Charles W. Hagen Jr. In 1996 Stated in his article that Chromatographic analysis of stems, sepals and petals of inbred Impatiens balsamina of the red-flowered genotype llHHPrPr has revealed a characteristic assemblage of flavonoid pigments in each organ. The more conspicuous compounds have been identified or partially characterized. The stems possess 3-monoglucosides of kaempferol, quercetin, pelargonidin, cyanidin and, presumably, delphinidin. The variety of pigments is less in flower parts than in stems, and less in etals than in sepals, but the flower parts exhibit a greater elaboration of substituents on the aromatic nuclei. The paired petals of mature flowers are pigmented by p-coumaroyl and feruloyl esters of pelargonidin-3, 5-diglucoside supplemented by more highly substituted derivatives of pelargonidin and by large amounts of kaempferol as the aglycone and two glucosides. The distribution of pigments has significance in the biology of the plant as well as providing an approach to studi es of factors which control flower differentiation ( file:///E:/flavonoids. htm). Richard L. Mansell,  Joyce A. Seder in September 1971 stated in their article that Petals from stage 3 flower buds of the purple (LLhhPrPr) genotype of  Impatiens balsamina  were examined for O-methyltransferase activity. Acetone powders of the tissue gave active preparations which would methylate caffeic acid to ferulic acid in the presence of  S-adenosyl-l-methionin (  file:///E:/S0031942200971942. htm). Masoomeh Shams Ghahfarokhi, Masoud Goodarzi,  Mehdi Razzaghi Abyanehb,  Taghi Al-Tiraihi,  Gholamhosein Seyedipourd in December 2004 stated in their article that the antifungal activity of onion (Allium cepa  L. on two important dermatophytes,  Trichophyton rubrum   and Trichophyton mentagrophytes, with special reference to morphological aspects was studied. Growth of both fungi was found to be strongly inhibited by aqueous onion extract (AOE) as a dose-dependent manner. The extract showed fungicidal effect for both fungi at concentrations gt;3. 12% (v/v). The fungus  Trichophyton mentagro phytes  was more affected by the onion as compared to  Trichophyton rubrum  at all concentrations used. Morphological effects of onion exposure were examined in correlation with fungal growth. Corresponding to the growth inhibition, light and electron microscopy observations revealed morphological anomalies in hyphal compartments. The results demonstrated that AOE targets the cell membrane of the fungi as breaking down of both inner and outer membranes with consequent extrution of materials into the surrounding medium. Cytoplasmic membranes and other membranous structures of organelles, such as nuclei and mitochondria, were also disrupted. In correlation to the fungal growth, morphological alterations occurred to a less content for  Trichophyton rubrum  compared with  Trichophyton mentagrophytes. The hyphae of  Trichophyton rubrum  were found to be mainly affected by converting to resistant forms, i. e. , chlamidospores as a consequence of phenotype switching response to AOE. Plasmolysis accompanied by an almost complete depletion and disorganization of cytoplasmic structures were found to be the final event which led to cell death. Ultrastructural evidences obtained from this study strongly support that morphological changes of  Trichophyton rubrum and  Trichophyton mentagrophytes  caused by AOE are associated with its fungistatic and fungicidal activities. With respect to the morphological results and the preliminary data on fungal biochemistry, a mechanism of action by interacting of AOE with thiol (–SH) groups present in essential compartments of the fungal cells was postulated. (http://www. sciencedirect. com/science/article/pii/S0367326X04001510) According to Philippine Council for Agriculture, Forestry and Natural Resources Research and Development, Los Banos, Laguna (Philippines)-Department of Science and Technology (2007)that the pathogen, Phytophthora palmivora Butler infects all parts of the durian tree in all stages of its development causing patch or stem canker, root rot, leaf blight, die-back of seedlings, and mature trees, and rotting of mature and immature fruits. Current disease management strategies involve the use of fungicides and proper cultural practices. However, due to high cost of fungicides and the risk to health and environment, there is a need to search for alternative method that is safe, effective and economical. Thus, Dionio et al. (USeP) conducted a 1-year study to determine the effectiveness of Kamantigue or garden balsam (Impatiens balsamina) in controlling the Phytophthora disease of durian. Findings of the study revealed the following: Extracts from mature seeds of red and violet Kamantigue had greater toxic activity than pink and white Kamantigue. Extracts prepared under sterile condition and stored earlier under ambient room condition (28 °C to 30  °C ) or in air-conditioned room (26  °C to 27 °C ) completely inhibited growth of P. palmivora in vitro up to 13 weeks of storage. The time of extract applicated affected the number of lesions induced by P. palmivora on 6-month-old durian seedlings of the Monthong variety. Spraying of extract 1 hour before and 1 hour after spray inoculation of P. palmivora reduced lesion number by 86% and 78%, respectively. Spraying of extract 24 hours before inoculation of P. almivora gave comparable result of 60% reduction in lesion number. Compared with seedlings inoculated with P. palmivora alone, seedlings which were also sprayed with the Kamantigue extract had significantly lower percentage defoliation. Seedlings sprayed with the extract 1 hour before inoculation had 13. 4% defoliation. Those sprayed 24 hours before inoculation had 12. 6% defoliation while those sprayed with extract o ne hour after inoculation had 12. 2% defoliation. Scraped canker lesions sprayed with 1:1 (wt/r) extract and the pure crude extract were generally dry 3 weeks after extract application. All treated lesions were dry up to the 4th week of application while untreated lesions remained wet. Results of the study showed that matured fruit/seed of red and violet Kamantigue or garden balsam can be used as protectant botanical fungicide for the control of Phytophthora leaf blight of durian. However, for patch canker treatment, the affected portion must be well scraped before application of the extract in order to get better results. The researchers recommended validation trials for both leaf blight and patch cankers treatments. Evaluation of the effectiveness of the extract to control major diseases of other crops should also be done. Information generated by the study is important in reducing production cost. Kamantigue is available all year round. Furthermore, the environmental and health hazards caused by synthetic pesticides can be reduced. (http://agris. fao. org/agrissearch/search/display. do? f=2009%2FPH%2FPH0901. xml%3BPH2008001319). According to Ma. Victoria M. dela Cruz, M. D. , Julius A. Lecciones, M. D. , Indira Gandhilenet. Jundam, M. D. Joann Gomez, M. D. , Vicente Dennis Dio, M. D. , Luz V. Samulde, M. D. on Pediatric division stated that The specie most frequently cultured from clinical specimens is Candida albicans- a member of the normal flora of the mucous membranes in the respiratory, gastrointestinal, and female genital tracts which if in such locations they gain dominance, may also be associated with pathological condition. Sometimes, the condition may even severe enough to produce sys temic disease, especially if cell-mediated immunity is impaired. Although, antifungal drugs are currently in use, only a limited number is available, most have to seek alternative means of combating these diseases, which is evidently increasing in incidence and occurrence. Setiawati, Erna  stated in 2006 stated that candidiasis is infection which cause of Candida. Ketoconazole is an effective antifungal. Bougainvillea glabra Choicy. is an traditional drug to treatment of leucorrhea. Objective: To compare the effectiveness of 100% Bougainvillea glabra Choicy. versus 2% ketoconazole in tratment of vaginal candidiasis. Method: This study was done by a laboratory experimental. As samples were the result of (+)Candida albicans inoculations at SDA medias. These were made the solution equal to 0,5 Mc Farland. 0,1 cc of solution was cultivated on the SDA media supplemented with 100% Bougainvillea glabra Choicy. and 2% ketoconazole. Incubated at 370 C for 2 days and looked those growth. If grow yeast on SDA media thus the result is (+) Candida albicans, and if don’t grow yeast on SDA media thus the result is (-) Candida albicans. The difference proportion of growth was analyzed by chi-square test with degree of signifance of plt;0,05. Result: 30 medias of SDA which contained 100% Bougainvillea glabra Choicy. ,30 medias were found (+)/positive for growth Candida albicans. Meanwhile, 30 medias which contained 2% ketoconazole, 15 medias were found positive and 15 medias were found negative. The result of the chi-square test is significant ( p= 0,000 ). Conclusion: 100% Bougainvillea glabra Choicy. is uneffective in inhibiting the growth of Candida albicans in kandidiasis vaginalis whereas 2% ketoconazole is effective. Key words: Vaginal candidiasis, Candida albicans, 100% Bougainvillea glabra choicy 2 % ketokonazole (file:///C://UJI/BANDING/EFEKTIVITA/BUGENFIL. ebarchive) According to A. Ownagh, R. Majdani, N. Yaghobzadeh, and Z. Nemati, spoilage and poisoning of foods by fungi is a major problem, especially in developing countries. Aspergillus is the most important fungi causing spoilage of foodstuffs. Candida albicans as a yeast is the most important problem in local and mucosal opportunistic infections. The effect s of growth inhibition of Thyme oil on Candida albicans and Aspergillus fumigates were studied in vitro. The disc diffusion method was used to elevate the zone of fungal growth inhibiton. Minimal Inhibitory Concentration(MIC) of the oil was used as a broth dilution method and minimal Fungicidal Concentration (MFC) of the oil was determined by agarsurface culture method and compared with each other. The formaline was used as a positive control and normal saline was used as negative control. Zone of inhibition of thyme on Aspergillus fumigates was 34mm and on Candida albicans was 35mm. Minimal Inhibition Concentration (MIC) of the oil on Aspergillus furmigatus was 7 lit/ml and Minimal Inhibition Concentration (MIC) of the oil on Candida albicans was 250 ? it/ml. The thyme oil was found to be strongly fungistatical agent against Candida albicans and Aspergillus fumigatus, werestudies in vitro (file:///C:/AntifungaleffectsofThyme0oilon. Ownagh. RMajdani. webarchive). According to Rajeshkumar R,  Sundararaman M in 2001, The opportunistic yeast pathogen Candida albicans and the emerging non-albicans Candida spp. cause life-threatening infections in immuno-compromised pati ents, leading to an increase in mortality rate. At present, the emergence of non-albicans Candida spp. auses serious infections that are difficult to treat the human populations worldwide. The available, synthetic antifungal drugs show high toxicity to host tissues causing adverse effects. Many metabolites of terrestrial and marine plants, microbes, algae, etc. , contain a rich source of unexplored novel leads of different types, which are under use to treat various diseases. Such natural drugs are less expensive and have lower toxicity to host tissues. The patent search on identified and potential anticandidal-lead molecules, from various patent databases, has been described in this review. Furthermore, this article consolidates the trends in the development of anticandidal drug discovery worldwide. Most of the investigations on natural, bioactive molecules against candidiasis are in various phases of clinical trials, of which, two drugs Caspofungin acetate and Micafungin sodium were approved by the U. S. FDA. In conclusion, the exploration of drugs from natural resources serves as a better alternative source in anticandidal therapeutics, having great scope for drug discovery in the future. file:///C://EmergenceofCandidaspp. andexplorationofnatural.. 5BMycoses2011PubMed-NCBI. webarchive) According to Dong Gun Lee,  Song Yub Shin,  Dae-Hee Kim,  Moo Yeol Seo,  Joo Hyun Kang,  Younghoon Lee,  Kil Lyong Kim  and  Kyung-Soo Hahm the antifungal mechanism of a 20-mer peptide, Ib-AMP1, derived from  Impatiens balsamina  was investigated. The oxidized (disulfide bridged) Ib-AMP1 showed a 4-fold increase in antifungal activity against  Aspergillus flavus  and  Candida albicans  than reduced (non-disulfide bridged) Ib-AMP1. Ib-AMP1 had very low activity for phospholipid disruption when compared with cecropin A(1-8)-magainin 2(1-12), ? -helical amphiphatic, antimicrobial peptide. Confocal microscopy showed that Ib-AMP1 binds on cell surface or penetrates into cell membranes. These results suggested that Ib-AMP1 may manifest its antifungal activity against  Candida albicans  by inhibiting a distinct cellular process rather than ion channel or pore formation in cell membrane. Antifungal mechanism  Ã‚  Ã‚  Candida albicans  Ã‚  Ã‚  confocal microscopy  Ã‚  Ib-AMP1 (http://www. springerlink. om/content/v1503k6062649346/) Justification Of The Study The above study shows that the following plants specifically Garlic, Oregano, Kampanyero, Penga-penga and others are used as alternative medicine for fungal infection in humans. Other study reveals that Impatiens balsamina Linn. was used as a fungicide for the prevention of fungal growth in fruit such as Durian. The present study wanted to prove that Impa tiens balsamina Linn. can inhibit the growth of Candida albicans and Trichophyton mentagrophytes. Not only fungal growth in plants but also fungal infection in humans. This will determine the effectivity of the flower extract of Impatiens balsamina Linn. as potential antifungal agent. CHAPTER III METHODOLOGY Research Design The study Impatiens balsamina Linn. Flower Extract: A Potential Antifungal Agent for Candida albicans and Trichophyton mentagrophytes was an experimental research design. The research design used was Quantitative Analysis that examined the antifungal properties of Impatiens balsamina Linn. flower extract that attempts to prove the Effectivity of Impatiens balsamina Linn. In inhibiting the growth of Candida albicans and Trichophyton mentagrophytes. Test Organism The Culture Organism which was Candida albicans and Trichophyton mentagrophytes were gathered from the Department Of Science and Technology in Bicutan, Taguig City. Gathering Of Data The researchers applied their knowledge in the preparation of the media that was used in the experimental procedure and in performing the inoculation of the test organism which was Candida albicans and Trichophyton mentagrophytes. Other procedures like flower extraction was done with the help of Medical Technology in Unciano Colleges Inc. -Antipolo Collection Of Data The experiment was divided into three groups, the positive control group was Nystatin, an antibiotic which was commonly used for fungal infection; the negative control group was 80% ethyl alcohol while the experimental group contains Impatiens balsamina Linn. flower extract. The zones of inhibition of both control groups and the experimental group were compared then analyzed. The zone of inhibition was measured by the use of vernier caliper. The unit of measurement was reported in millimeter. Collection Of The Plant Sample The plant sample was collected in Barangay Caniogan, Morong Rizal. Only the red flowers of Impatiens balsamina Linn. was used in this study. Preparation Of The Plant Sample: The researchers collected fresh and mature flowers of the plant sample, then removed any extraneous matter by washing the flower petals with running water, then the flower petals were wiped with a dry and clean cloth, and followed by reducing the size of the flower petals through cutting and then autoclaved at 121 ° C for 15 minutes. Extraction Procedure: The researchers weighed about 100g of the ground plant material, placed in an Erlenmeyer flask, soaked in sufficient amount of 80% ethyl alcohol and stand for 24 to 48 hours. The volume of alcohol used was noted, and the mixture was filtered through a funnel with gentle suction, then the plant material with fresh portions of alcohol was rinsed by the flask and then the first filtrate was combined with the plant residue being discarded. The flower extract is placed in an evaporating dish on top of the casserole with boiling water to become more concentrated, until the scent of the alcohol was lessened. Then the flower extract was stored in a tightly stopped amber bottle and stored, preferably in cold condition, or if kept at room temperature, treat the extract with a trace of chloroform or toluene. Standard Ingredients In Sabouraud Dextrose Agar Water. The dried powdered ingredients are added to water. A typical batch uses 1 liter (1000 ml) of water although any amount can be made. Tap water was used unless a more defined formulation was required. Tap water usually contains minerals that are beneficial for fungal growth. If local tap water has high concentrations of undesirable minerals, deionized water should be sed. Dextrose. The carbon source in Saboraud Dextrose was dextrose (glucose), usually provided at a concentration of 40 to 70 grams per liter. This was notably more sugar than was included in most media. Saboraud Dextrose also has a low (acidic) pH. Both of these properties tend to facilitate the isolation of fungi, which are generally more tolerant of these conditions tha n bacteria. The high sugar concentration results in a high osmotic pressure, which tends to inhibit the growth of bacteria more than it does to fungi. Agar. Agar is a gelling agent, extracted from certain seaweeds and provided in powdered form. When mixed with water and heated, it cools to form a semi-solid gel that must be poured into a test tubes or Petri dishes before it cools. The agar concentration in Saboraud Dextrose can be from 10 to 25 grams per liter, with more agar producing a stiffer gel. Peptone. All organisms need nitrogen to grow. Organic nitrogen is provided by peptone or neopeptone at about 10 grams per liter. Peptone contains amino acids and other organic molecules obtained by the enzymatic digestion of animal proteins. Yeast extract is sometimes substituted for peptone. Preparing  Sabouraud Dextrose Agar The premix Sabouraud Dextrose Agarwas used, the proper amount of agar was around 65 grams and was mixed with one liter of water in an Erlenmeyer Flask and heated in the stove until the agar dissolved. The medium was then autoclaved or sterilized at 121? C for about 15 to 20 minutes. And was cooled, after cooling the medium was poured into sterile Petri plate and allow it to solidify. When the medium was solidified and cooled to room temperature it was ready for inoculation. Preparation of Peptone Water Dissolve a proper amount of 15g of the medium in 1 liter of distilled water. Mix thoroughly until it dissolves and become clear. Autoclave at 121? C for 15 minutes. Then stand it at room temperature until it becomes tolerable. Preparation of Broth Pour a sufficient amount of peptone water on two test tubes, the first tube was for Candida albicans and the second tube was for Trichophyton mentagrophytes. Let the prepared peptone water cooled at room temperature until it becomes tolerable. Using a wire loop get the organism and mix it with the peptone water in both test tube, observe until it becomes hazy but still visible to its background. More over when you put the test tube over a news paper the letters must still be visible. Then it was ready for inoculation. Preparation of Antibiotic Disc Make a filter paper disc using a punching machine. Autoclave the filter paper disc at 121 ° Celcius for 30 mins. Then soaked it in a sufficient amount of Nystatin. Inoculation The sterile petri plates containing solidified Sabouraud Dextrose Agar medium were used as a media in performing inoculation of the organism using sterile cotton swab. The entire surface of the agar were streaked evenly in all directions. And the plates were incubated at 37? C for 24 hours for the yeast and 27? C for the moulds. Disc Diffusion Method For Antifungal Activity Whatmann no. 1 filter paper were used to cut 7mm sized paper disc by a punching machine. This disc were sterilized at 121? C for 30 minutes at 15 lbs. At the centre of the petriplate a disc were positioned and 20 µl of each essential extract were soaked on the disc. Then fungal broth were streaked one by one over the agar plate radially. The petriplates were incubated at 37? C for 24hours. The zone of inhibition were measured after 24 hours incubation. Then the diameter of the zone of inhibition were observed and recorded. The following interpretative range of standard zone was adopted from Ontengco (2000). Zone of Inhibition| Inhibitory activity| ?17mm| +++, strong| 15-16mm| ++, moderate| 7-12mm| +, weak| 6 or 0mm| -, negative| Statistical Analysis The following statistical was utilized in this research. Analysis of variance (ANOVA) is a technique for analyzing the way in which the mean of a variable was affected by different types and combinations of factors. One way analysis of variable is the simplest form. It is used to compare the means of more than two independent groups. It was an extension of the independent sample t-test and can be used to compare any number of groups or treatments. Analysis of variance gives a single overall test of whether there are differences between groups or treatments. The Duncan’s Multiple- Range Test (DMRT) was based on the comparison of the range of a subset of the sample means with a calculated least significant range. This least significant range increases with the number of sample means in the subset. If the range of the subset exceeds the least significant range. Then the population means can be considered significantly different. Once a range was found not to be significant, no further subsets of this group are tested. The Duncan’s Multiple- Range Test was used to determine which of the population means are equal and which are significantly different. The Duncan’s Multiple-Range Test was utilized to determine which of the compared groups was significantly different. The Duncan’s Multiple-Range Test was utilized if the One-way ANOVA shows a significant difference in the population means (Adrian, 2010) CHAPTER IV RESULTS AND DISCUSSION Table 1. The total and the mean of the zones of inhibition of Nystatin (Positive Control), Impatiens balsamina Linn. Experimental) and 80% Ethanol (Negative control) against Candida albicans in Trials 1,2,3. Trial| Nystatin(Positive) | Impatiens balsamina Linn. (Experimental) | 80% Ethanol(Negative)| Trial 1| 9| 0| 8. 5| Trial 2| 11| 11| 8| Trial 3| 11| 8| 9| Total| 31| 19| 25. 5| Mean| 10. 3333| 6. 3333| 8. 5| Note: Unit is in millimetres (mm. ) Table 1 show s the zone of inhibition on Candida albicans of the experimental group (Impatiens balsaminna Linn.. flower extract) as well as the positive (Nystatin) and negative (80% Ethanol) controls in all of the three trials were shown as well as their sums and means that were calculated. On the first trial, Nystatin (Positive control) had 9 mm zone of inhibition and the 80% Ethanol (Negative Control) had 8. 5mm zone of inhibition. On the other hand, the Impatiens balsamina Linn. flower extract (Experimental) was unsusceptible since it had no zone of inhibition. For the second trial, Nystatin (Positive control) had 11 mm zone of inhibition and the 80% Ethanol (Negative Control) had 8 mm zone of inhibition. The Impatiens balsamina Linn. flower extract (Experimental) had 11 mm zone of inhibition. Lastly, for the third trial, Nystatin (Positive control) had 11 mm zone of inhibition and the 80% Ethanol (Negative Control) had 9 mm zone of inhibition. The Impatiens balsamina Linn. flower extract (Experimental) had 8 mm zone of inhibition. The total zone of inhibition of the positive control (Nystatin) on three trials was 31 mm and their mean was 10. 3333mm. On the other hand, the sum and mean of the zone of inhibition of experimental group (Impatiens balsamina Linn. ) on the three trials was 19 mm and 6. 3333 mm respectively. Lastly, the total zone of inhibition of the negative control (80% Ethanol) was 25. mm and the mean was 8. 5 mm. Table 2. One-way Analysis of Variance (ANOVA) decision table Sum of`DfMeanF Sig. SquaresSquare Between Groups24. 056212. 0281. 064. 402 Within Groups67. 833611. 306 Total 91. 8898 Table 2 shows the results of the one-way ANOVA (Analysis of Variance) computations which includes the sum of squares between groups and within groups which was 24. 056 an d 67. 833 respectively. The Degrees of Freedom (df) are 2 and 6 and the mean square are 12. 028 and 11. 306. The tabular value of F as shown in Table 5 was 1. 064 and with 95% Confidence interval, the value of significance from the experiment was . 02. Since the accepted value of significance for 95% Confidence interval must be less than or equal to 0. 05 and the value of significance calculated from the experimental data does not fall within the reference range, it was concluded that there was no significant difference between the zone of inhibition of the experimental group (Impatiens balsamina Linn. ) and the positive (Nystatin) and negative (80% Ethanol) group against Candida albicans. Therefore, the null hypothesis was accepted. Table 3. The total and the mean of the zones of inhibition of Nystatin (Positive Control), Impatiens balsamina Linn. Experimental) and 80% Ethanol (Negative control) against Trichophython mentagrophytes in Trials 1,2,3. | Nystatin(Positive) | Impatiens balsamina Linn. (Experimental) | 80% Ethanol(Negative)| Trial 1| 11| 8| 10| Trial 2| 12| 11| 10| Trial 3| 13| 12| 11| Total| 36 | 31| 31| Mean| 12| 10. 3333| 10. 3333| NOTE: Unit is in millimetres (mm. ) Table 3 shows the zone of inhibition on Trichophyton mentagrophytes of the experimental group (Impatiens balsamina Linn.. flower extract) as well as the positive (Nystatin) and negative (80% Ethanol) controls in all of the three trials were shown as well as their sums and means that were calculated. Based on the data gathered in Trial 1, the Nystatin (Positive control) had 11 mm zone of inhibition and the 80% Ethanol (Negative Control) had 10 mm zone of inhibition. On the other hand, the Impatiens balsamina Linn. flower extract (Experimental) had 8 mm zone of inhibition. For the second trial, Nystatin (Positive control) had 13 mm zone of inhibition and the 80% Ethanol (Negative Control) had 11 mm zone of inhibition. The Impatiens balsamina Linn. flower extract (Experimental) had 12 mm zone of inhibition.