1 Chapter 1 Introduction

Background of the study Fungi exist in populations that are adaptable. Under the selection imposed by antifungal drugs, drug-sensitive fungal pathogens frequently evolve resistance. Although the molecular mechanisms of resistance are well-characterized, there are few measurements of the impact of these mechanisms on pathogen fitness in different environments. To predict resistance before a new drug is prescribed in the clinic, the full spectrum of potential resistance mutations and the interactions among combinations of divergent mechanisms can be determined in evolution experiments. In the search for new strategies to manage drug resistance, measuring the limits of adaptation might reveal methods for trapping fungal pathogens in evolutionary dead ends (Anderson, 2005). The cost of medicines in the Philippines is among the highest in the world. A study shows that it would take a full six days of wages for an average worker to purchase basic medicines in the country. There are more than 17,000 registered drugs in the local market, but a majority of the population can barely afford these expensive essential drugs (Defensor, 2008). To answer the problems regarding the high cost of medicine, analysis of plants for medicinal purposes rapidly increased. This includes the discovery of antibacterial, antiviral, and antifungal compounds that are present in the plants as secondary metabolites, which can exhibit a big help in treating diseases caused by pathogenic

2 microbes. As a result of this, scientists keep on exploring new compounds to treat the resistant strains (Rai, Charka, and Wadegaonkar, 2003). Bita tree A medium-sized tree, the leaves in whorls of three, oblong-obovate, 10 to 30 cm long, 5 to 7 cm wide, pointede at both ends, and shot-stalked. Flowers are small, yellowish-white, on short, terminal cymes. Calyx is small, the corolla tubular, 1 to 1.5 cm long, lobed towards the top. Fruit is a double follicle, pendant, long and slender, 20 to 40 cm long. Seeds are small and flat, with deep-brown hairs. Bita tree has been test for antidiarrheal property, Anti-malarial, Immunostimulatory, Anti-diabetic / Hypoglycemic, Antiprotozoal . It is used by locals in treating diseases and is available all throughout the year (Stuart, 2010). In order to answer the problem in the high cost of medicine and the availability of bita in our native land, the researchers screened the extract of Bita bark against Candida albicans and Aspergillus niger, by conducting a Phytochemical screening for flavonoids and tannins and an antifungal assay using Paper Disc Diffusion method, to determine its potential in becoming an antifungal agent.

Objectives of the Study This study aimed to determine the antifungal activity of Bita (Alstonia scholaris ) bark ethanolic extract.

This study specifically aimed to: 1. Determine if the following antifungal secondary metabolites are present in the Bita bark ethanolic extract: a. Flavonoids b. Tannins 2. Determine if the Bita extract has antifungal activity towards: a. Candida albicans b. Aspergillus niger 3. Determine if there is a significant difference in the antifungal activity of Bita and the positive and negative control towards: ` a. Aspergillus niger b. Candida allbicans Hypothesis There is no significant difference between the antifungal activity of the Bita ( Alstonia scholaris) bark ethanolic extract compared to the control.

Significance of the Study The results of the study may be beneficial to the following:

4 The Researchers The antifungal compounds that are present in the Bita bark which exhibited a zone of inhibition against the test organism will provide information to future researches giving them an idea that the Bita bark needs to be studied further in order to validate its antifungal property. Alternative Medicine Since alternative medicine is the trend these days considering the exorbitant cost of medicine, the information provided by this study will be invaluable in providing additional species in the list of those with medical antifungal property. Rural People The study will be of help to the rural people particularly those who have less access to medical services and medicines. Knowledge of the fungicidal property of local or indigenous materials such as Bita will provide temporary assurance for prophylactic treatment of fungi-caused disorders.

Definition of terms The following terms are given their conceptual and operational definitions for easy understanding of how they are used in the study: Antifungal Activity - is a process wherein an antifungal agent kills or inhibits fungi or its activity. It also means the activity of a compound specifically inhibiting either a dermatomycosis like ringworm or athlete's foot, or one that inhibits Candida albicans either externally as a douche or internally as a systemic fungicide (Moraleta,2003).

5 In this study, it refers to the process in which the Bita bark ethanolic extract affected the growth of the test fungi as indicated by the presence of zone of inhibition. Fungi - are eukaryotic, achlorophyllus (lacking the property of photosynthesis) organisms which are mostly aerobic and inhabit the water, soil and decaying organisms debris. They are the members of the plant kingdom that lack roots and stems and are referred to as Thallophytes (Moraleta, 2003). In this study, it refers to and limited to Candida albicans and Aspergillus niger in which the Bita bark ethanolic extract were tested for its antifungal activity. Sabouraud Glucose Agar - means a general purpose medium used in the mycology laboratory for the isolation, growth, and maintenance of fungi. Most mold fungi do well on it, but it is especially recommended for growing dermatophytes (skin, hair, and nail fungi), yeasts, and other species found on animals and humans (McIntosh, 2010). In this study, it refers to the medium where the test organisms (Aspergillus niger and Candida albicans) were inoculated to test the antifungal activity of Bita bark ethanolic extract. Disc Diffusion Assay means a method which uses antibiotic-impregnated wafers to test the reaction of a particular microorganism to the test plants and specific antibiotic. A known quantity of fungi will be grown on agar plates in the presence of thin wafers containing the test substance (Raphael, 1983). In this study, it refers to the method used to test the antifungal activity of the ethanolic extract of bita bark.

6 Scope and Limitations This study aimed to determine the antifungal activity of Bita (Alstonia scholaris) bark ethanolic extract against Aspergillus niger and Candida albicans by conducting a phytochemical screening and an antifungal assay. In Phytochemical screening Bate Smith and Metcalf and Wilstatter Test were used to detect the presence of flavonoids while Ferric chloride test was used to detect the presence of Tannins. In the Antifungal Assay, Paper Disc Diffusion Method was used as the screening method to determine if the plant extract has an antifungal activity.

7 Chapter II Review of Related Literature

Kingdom Fungi includes some of the most important organisms in terms of their ecological and economic roles. These organisms break down dead organic materials continuing the cycle of nutrients in the ecosystem. Besides its usefulness in our environment these organisms can also cause a number of diseases in humans affecting organs of the body, damaging it and endangering the life of a person. Fungi are

chemically and genetically similar to animals compared to other organisms making fungal diseases difficult to cure (Waggoner and Speer, 2006). Yeast Yeasts are unicellular fungi. One of the well known characteristics is the ability to ferment sugars for the production of ethanol. They are characterized by a wide dispersion of natural habitats, present in plant leaves and flowers, soil and salt water. Yeast is also found on the skin surfaces and in the intestinal tracts of warm-blooded animals, where they may live symbiotically or as parasites. The common "yeast infection" is typically Candidiasis which is caused by the yeast-like fungus Candida albicans. They multiply as single cells that divide by budding or direct division, or they may grow as simple irregular filaments (Yeastgenome, 2010).

Candida albicans Candida albicans is a yeast that is associated with a systemic infection which later on can become extremely tenacious. There are many medications for the treatment

8 of thrush. Many top experts suggest diet in order to get rid of the fungi. Often the best treatment is to combine active cleanse elements with food in order to get rid of yeast infection (Muran, 2010). Candida albicans is a diploid fungus (a form of yeast) and a causal agent of opportunistic oral and genital infections in humans (Ryan and Ray 2010).

Filamentous fungi Filamentous fungi are fungi that grow in the form of multicellular filaments, called hyphae. A connected network of these tubular branching hyphae has multiple, genetically identical nuclei and is considered a single organism, referred to as a colony or in more technical terms a mycelium. This fungi do not form a specific taxonomic or phylogenetic grouping, but can be found in the divisions Zygomycota, Deuteromycota and Ascomycota. Although some of them cause diseases or food spoilage, others are useful for their role in biodegradation or in the production of various foods, beverages, antibiotics and enzymes. One of the most common filamentous fungi is the Aspergillus spp. (Madigan and Martinko, 2005).

Risk to Humans and Animals Reports associating A. niger with infectious diseases in healthy individuals are uncommon, although A. niger is a recognized opportunistic pathogen. Given the relative infrequency of anecdotal reports and the frequency with which all humans are exposed to A. niger, both by ingestion and inhalation, the probability of colonization in immunocompetent individuals must be quite small. The probability of colonization in

9 immunosuppressed people, however, is relatively high. Nevertheless, given the ubiquitous presence of A. niger, the increased environmental burden of A. niger due to release from commercial facilities under conditions imposed by exemption criteria is probably negligible. Thus, it may be concluded that the use of A. niger in fermentation facilities is unlikely to increase the baseline risk of infection by A. niger. The primary hazard to humans and animals appears to be toxicity associated with the production of mycotoxins known as malformins. Concern is reduced due to available information on the relevant toxins. The higher values of toxicity for malformins A and C were determined by intraperitoneal injection, a route not considered to be environmentally relevant. Furthermore, data for toxicity via ingestion indicate that the toxicity is much lower by this route. This lower toxicity may be due to the destruction of the malformins, which are cyclic pentapeptides, in the gastrointestinal tract (Yoshizawa, 1975).

Alternative Medicine Alternative medicine is any healing practice "that does not fall within the realm of conventional medicine (Steven, 1997) or "that which has not been shown consistently to be effective. It is often opposed to evidence based medicine and encompasses therapies with a historical or cultural, rather than a scientific, basis. Commonly cited examples include naturopathy, chiropractic, herbalism, traditional Chinese medicine, Unani, Ayurveda, meditation, yoga, biofeedback, hypnosis, homeopathy, acupuncture, and nutritional-based therapies, in addition to a range of other practices(MedicineNet,2004 ). It is frequently grouped with complementary medicine, which generally refers to the

10 same interventions when used in conjunction with mainstream techniques, (White House Commission on Complementary and Alternative Medicine Policy, 2002) under the umbrella term complementary and alternative medicine, or CAM. Alternative medicine practices are as diverse in their foundations as in their methodologies. Practices may incorporate or base themselves on traditional medicine, folk knowledge, spiritual beliefs, or newly conceived approaches to healing (Anshu and Acharya, 2008). Herbalism Herbalism is a traditional or folk medicine practice based on the use of plants and plant extracts. It is also known as botanical medicine, medical herbalism, herbal medicine, herbology, and phytotherapy. The scope of herbal medicine is sometimes extended to include fungal and bee products, as well as minerals, shells and certain animal parts (Archaya and Anshu, 2008). Anthropology of Herbalism People all over the world have used hundreds of thousands of indigenous plants for treatment of ailments since prehistoric times. Medicinal herbs were found in the personal effects of an "ice man", whose body was frozen in the Swiss Alps for more than 5,300 years. These herbs appear to have been used to treat the parasites found in his intestines. Thus, anthropologists theorize that an animal tends to seek out bitter plant parts in response to illness (Huffman, 2003).

11 The use of herbs and spices in cuisine developed in part as a response to the threat of food-borne pathogens. Studies show that in tropical climates where pathogens are abundant, recipes are highly spiced. Further, the spices with the most potent antimicrobial activity tend to be selected. In all cultures vegetables are spiced less than meat, presumably, because they are more resistant to spoilage (Billing and Sherman, 1998).

Importance of Plants in Medicine All plants produce chemical compounds as part of their normal metabolic activities. These are divided into primary metabolites, such as sugars and fats which are found in all plants and secondary metabolites, or compounds not essential for the plants basic function, and are found in smaller range compared to its primary metabolites. However, even if these metabolites are present in small amount, they can exhibit a big help to humans (Stepp and Moerman, 2001). The functions of a secondary metabolite are varied. Some secondary metabolites are toxins, others are pheromones used to attract insects for pollination, and phytoalexins, which protects against bacterial and fungal attacks. Many plants synthesize substances that are useful in the maintenance of health among humans and other animals. These include aromatic substances, most of which are phenols or their oxygen-substituted derivatives such as tannins. Many are secondary metabolites, of which, at least 12,000 have been isolated a number estimated to be less than 10% of the total. In many cases, substances such as alkaloids serve as plant defense mechanisms against predation by microorganisms, insects, and herbivores. Many of the

12 herbs and spices used by humans for food preparation contain useful medicinal compounds (Lai, 2004 and Tapsell, 2006). Plants regulate and down regulate their biochemical paths in response to the local mix of herbivores, pollinators and microorganisms. The chemical profile of a single plant may vary over time as it reacts to changing conditions. Only the secondary metabolites and pigments have therapeutic actions in humans and can be refined to produce drugs. Plants synthesize a bewildering variety of phytochemicals but most are derivatives of a few biochemical motifs (Taiz and Zeiger, 2006). Recently, about 500,000 additional plant species were documented in the whole world, some of which are endangered of extinction. As part of the plants survival, they must produce potent antifungal compounds to protect themselves from fungi which exist in the soil. Of all the different plant types that exist, only a few have been examined for the presence of antifungal compounds. However, those that have been purified and studied are either protein or non protein compounds preventing fungal growth by different modes of action. Some lyses the cell while others block the protein synthesis of the susceptible fungi. Though some of these compounds are very potent, none of them have been successfully developed into commercial products (De Lucca, Cleveland, and Wedge, 2005).

Plants as an Antimicrobial agent As time goes by, analysis of plants for medicinal purposes rapidly increased. This includes the discovery of antibacterial, antiviral, and antifungal compounds that are present in the plants as secondary metabolites, which can exhibit a big help in treating

13 diseases caused by pathogenic microbes. But then, due to prolonged intake of the drug some organisms tend to develop resistance to it making the drug ineffective. As a result of this, scientists keep on exploring new compounds to treat the resistant strains (Rai, Charka, and Wadegaonkar, 2003). Bita Tree (Alstonia scholaris) Bita tree has a furrowed trunk and has capious lenticellate branchlets (Brown and Linnaeus, 2010). It can grow up to eighty feet high. The bark is almost odorless and very bitter, usually found in irregular fragments of 1/8 to inch thick with a spongy texture. The outer layer of the bark is rough, uneven and fissured brownish gray or sometimes blackish spots, while the inner layer is bright buff. Transverse section of the inner part shows a number of small medullar rays. The tree has an oblong stalked leaves which is six inches long and four inches wide, dispersed in four to six whorls around each stem. Its upper part is glossy while the bottom surface is white. Leaf veins run at right angles to the midrib (Grieve, 2010).

The Bark The bark is about inch thick, 1-2 inches wide and 3-6 inches long. Externally, it has a mottled pinkish or brownish and white color. It is smooth but marked by shallow fissures which are raised upon the edges and scarcely extended through the corky layer. The cork, a very thin layer represented by a dark edge, is brown. Internally the color of the bark is light. The texture is usually granular and brittle. Its taste is slightly bitter, free

14 from astringency, pleasant and may be compared to the aftertaste of a wild cherry bark (Felter and Lloyd, 2010).

Botanical Source The tree is usually seen in Asia- specifically in countries like China, India, Nepal, Sri Lanka, Cambodia, Myanmar, Thailand, Vietnam, Indonesia, Malaysia, Papua New Guinea and Philippines- and Australia (ARS Systematic Botanists, 1998).

Chemical Components The tree contains three alkaloids (Ditamine, Echitamine or Ditaine and Echitenines), several fatty acids and resinous substances, the second strongest base (Grieve, 2010). The chief constituents of the bark of Alstonia scholaris are the alkaloids ditamine, echitenine, and echitamine. Ditamine, are present at about 0.04 per cent concentration. It also has the composition C16H19NO2, which has been obtained as a bitter crystalline powder (melting-point, 75); echitenine, C20H27NO4 (melting-point, 120), is an amorphous bitter powder; echitamine or ditaine, C22H28N2O4, H2O, is a white powder, the crystals having the formula C22H28N2O4, 4H2O. The following constituents have also been extracted from the bark:Echicerin, a crystalline non-nitrogenous body; echicaoutchin, an amorphous substance resembling caoutchouc; echitin and echitein, both of which are crystalline, and echiretin, which is amorphous. All these constituents appear to be devoid of marked therapeutic properties (Kress, 2010).

15 Medicinal Uses The bark is used in patients with homeopathy for its tonic bitter and astringent properties. It is often useful, as treatment against diarrhea and dysentery (Grieve, 2010). Other than that, it has also been efficaciously employed in patients having malarial fever (Felter and Lloyd, 2010). Both bark and leaves are used to treat headache, influenza, bronchitis and pneumonia (Brown and Linnaeus, 2010). Though Alstonia, commonly known as Bita, is used in India and Eastern Colonies for malarial conditions, its efficacy in this respect is not to be compared with cinchona bark, though it does not produce the bad effects cinchona does. It is also employed as a bitter tonic, vermifuge, and as a cure for chronic diarrhea and bowel complaints (Kress, 2010).

Common Names Every country has a different native word to this tree. The names of the plants that is commonly used by people around the world are Blackboard tree, Devil tree, Dita Bark, Milkwood-pine, White Cheesewood, and Pulai (ARS Systematic Botanists, 1998).

Nystatin ( Mycostatin R) Nystatin (Mycostatin R) is an antibiotic that is effective against Candida spp. and Aspergillus spp. , Rhizopus and Mucor fungal infections. It is pale yellow and soluble in water. Nystatin (Mycostatin R) is an antifungal antibiotic that came from Streptomyces nouresi (Tuskegee University, 2010).

16 A polyene antifungal drug to which many molds and yeast infections are sensitive, including Candida spp. Due to its toxicity profile, there are currently no injectable formulations of this drug on the market (Hahn, 2008). Mechanism of Nystatin Nystatin (MycostaticR), like amphotericin B and natamycin, binds to ergosterol, a major component of the fungal cell membrane. When present in sufficient concentrations, it forms pores in the membrane that lead to K+ leakage and death of the fungus. Ergosterol is fairly unique to fungi, so the drug does not have such catastrophic effects on animals (Espinel, 2003). Related Studies This part presents some of several studies about the barks exhibiting an antifungal activity against selected yeast and filamentous fungi. Also, results of Phytochemical analysis showing secondary metabolites having antifungal properties. A study was carried out with an objective to investigate the antifungal potential of Bark of Bombax malabaricum. The antifungal activity of the ex-tracts was evaluated on two common pathogenic fungi Aspergillus niger and Candida albicans. The testing was done by the agar diffusion method. Zones of inhibition of extracts were compared with that of standard Ketoconazole for antifungal activity. The extracts showed antifungal activities comparable with that of standard against the organisms tested. The results showed that the Petroleum ether and chloroform extracts showed no activity while the alcoholic extract showed more activity than the acetone and aqueous extracts (Girija et al, 2010).

17 The antifungal properties of aqueous and ethanol extracts of Funtumia elastica and Mallotus oppositifolius were carried out using the disc diffusion agar assay. The crude extracts exhibited definite significant antifungal activity on most of the fungi. Preliminary phytochemical studies of F. elastica and M. oppositifolius extracts revealed that they contain flavonoids and tannins (Adekunle, 2006). Studies showed antifungal activity of Ficus sycomorus Hexane, Petroleum Ether and Chloroform bark extract against Microsporum gypseum, Aspergillus niger, Aspergillus flavus and Candida albicans. Fractions of the extract revealed presence of condensed tannins, Steroids and Saponin (Hassan, 2007). Investigation of the Phytochemical constituents and the antifungal activity of the stem bark extracts Leptadenia lancifolia were carried out using various solvents (water, acetone and methanol). Phytochemical analysis showed the presence of tannins. The extracts were effective against Aspergillus flavus, Aspergillus fumgatus, Cryptococcus neoformans and Candida albicans (Doughari et. al.,2008). Phytochemical screening results showed that the different solvent extracts showed the presence of alkaloids, phenolics, saponins and tannins in Alstonia scholaris. Studies have shown that tannins that are present in the bark showed antifungal activity against a wide variety of fungi (Mahmud et al., 2009).

18 Summary of Review of Related Literature Fungi are one of the most important organisms in terms of their ecological and economic roles. However, it can also cause a number of diseases in humans affecting organs of the body, damaging it and endangering the life of a person. On the other hand, alternative medicine is any healing practice that does not fall within the realm of conventional medicine. Therefore practices done under this field needs to be confirmed by science to prove and explain the reasons behind the effect of the plant used as treatment. Since fungal infections are present, scientist tends to analyze more plants inorder to detect if it has a fungicidal effect against pathogenic fungi. Supporting their study regarding the antifungal activity and phytochemical screening, scientists tend to choose the bark because of the studies showing that most of the secondary metabolites are stored in the bark of trees. Bita is a tree with a firmed trunk and a copious lenticellate brachlets. It can grow up to 80 feet high. The bark is odorless and bitter because of its potential treatment, herbalist use it as treatment against diarrhea, dysentery, headache, influenza, bronchitis, infection and malaria.

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Chapter III Materials and Methods Research design This study used an experimental research method, specifically the Completely Randomized Design (CRD). This experimental research method followed the standard protocols and was conducted under strict laboratory conditions. The researchers used a standard protocol for yeast and filamentous fungi. Nystatin was used as positive control. The antifungal assay was performed using Paper Disc Diffusion.

Data Gathering Procedure This section includes the methods for the experimental research study. The standard protocol was taken from A Guidebook for Plant Screening by UST-RCNS, 2004. The experimental research study covered:

Plant Material Collection and Verification The plant material was gathered at Barangay San Miguel, San Miguel Iloilo. The plant was pressed using a standard press as described by the University of Santo TomasResearch Center for the Natural Science (2004). The pressed plant was left in the shade for two weeks and presented as herbarium specimen for identification by an expert from the University of the Philippines in the Visayas, Miagao Iloilo.

20

Test Organism Identification The test organisms, which are A. niger and C. albicans, were verified by a mycologist from the University of the Philippines in the Visayas, Miagao, Iloilo. Plant Extraction One hundred grams of ground fresh plant material was placed in an Erlenmeyer flask and treated with sufficient 95% ethyl alcohol to completely submerge the material. The volume of the alcohol used was noted. The flask was stoppered and the material was kept soaked for 48 hours. The mixture was filtered with the use of a glass funnel. Plant residue was discarded. The filtrate was then concentrated under vacuo (Rotary evaporator) at temperatures below 50o C. (UST-RCNS, 2004). Phytochemical Screening Test for presence of Flavonoids Bates-Smith and Metcalf method: Test for Leucoanthocyanins ( UST- RCNS, 2004) Three ml of the alcohol filtrate was treated with three drops of 12M hydrochloric acid and observed for any color change. The treated filtrate was warmed using a water bath. Further color change was observed within an hour. For positive result, a strong red or violet color indicated the presence of leucoanthocyanidins. Wilstatter cyanidin test: Test for -benzopyrone nucleus ( UST- RCNS, 2004)

21 Three ml of the alcohol filtrate was treated with three drops of 12M hydrochloric acid. Three magnesium turnings were added. Any color change was observed within 10 mins. The mixture was heated with the aid of a water bath. For positive results, colors ranging from orange to red, to crimson to magenta, and occasionally to green or blue were observed.

Test for the Presence of Tannins Ferric Chloride Test (UST- RCNS, 2004) One ml of the alcoholic filtrate was treated with three drops of 5 % ferric chloride reagent. The appearance of green to black precipitate indicates the presence of tannins.

Antibiotic Preparation A prepared Nystatin tablet was used in the study, then the outer shell was removed, and inner portion was powderized with the used of mortar and pestle. 500 mg of powderized Nystatin was mixed with 5 ml sterile distilled water and was poured to a sterile amber bottle and refrigerated. The concentration of the antibiotic used was 100mg/ml.

22

Antifungal assay A. Paper Disc Diffusion Preparation of the Inoculum Yeast A loopful of organism from culture slants of Candida albicans was inoculated in 5 ml of Sabouraud glucose broth medium followed by incubation for 18 hours at room temperature. After incubation, adjustment of yeast cell suspension is followed using 0.5 McFarland standard. This serves as the inoculum for swabbing (UST-RCNS, 2004). Filamentous fungi A colony of Aspergillus niger was inoculated to Potato Dextrose Agar plate. The plate was incubated at room temperature, allowing it to grow well. Once the conidia have luxuriantly developed, scrapping off of five loopfuls of conidia was done. Then the conidia were immersed in a 5ml sterile isotonic saline-Tween 80 solution contained in a screw capped tube. The cap was replaced and the contents were shaken for 1 minute. After shaking, the concentration of the spore suspension was compared to 0.5 McFarland standard, adjustments was done in order to achieve an identical turbidity of 0.5ml of the spore suspension. This serves as the inoculum for swabbing (UST-RCNS, 2004). Preparation of the assay plates Twenty ml of melted Potato Dextrose Agar was poured in a dry and sterile petri dish followed by solidification of the agar. The surface of the agar was streaked three times, rotating the plate approximately 60 degrees after each application to ensure an

23 even distribution of the inoculum, with the use of a sterile cotton swab dipped in the test organism suspension. The plate was allowed to stand for five minutes (UST-RCNS, 2004). Placing of paper Disc Following the aseptic techniques, the sterile forceps was used to pick and lay on the agar surface the paper disc that has been immersed in plant extracts, Nystatin (positive control) and distilled water (negative control) (UST-RCNS, 2004). Reading the Assay Plates and Documenting the Result A halo or clearing, called zone of inhibition was observed around each disc. This zone was measured, in millimetres, using the ruler. The results was expressed as inactive, partially active, active, and very active when the mean zone of inhibition that was observed is <10mm, 10-13 mm, 14-19mm and >19mm, respectively (UST-RCNS, 2004). Interpretation of the results For the Paper Disc Diffusion Method, the zone of inhibition exhibited around each of the antibiotic discs (positive control disc), negative control disc and the plant extract solution was measured using a ruler. Any zone of inhibition with a diameter exceeding >19 mm means that the fungi are highly sensitive to the treatment. All statistical data were analyzed using the Statistical Package for Social Science Software using the One Way Analysis of Variance or the ANOVA for CRD. Least Significant Difference (LSD) was used to determine significant differences between treatment and control.

24

Ethical Consideration All the used agar and broth cultures were disposed in the to be autoclaved bin. Then cultures were decontaminated immediately by autoclaving before cleaning the glassware and disposing immediately the spent agar and broth media (UST-RCNS, 2004). All the apparatus used, were autoclaved to assure that all microorganisms are killed. The culture was handled with caution. Wearing of the personal protective (clothing) or gear was imposed during the experiment proper to avoid infection. Data Analysis Procedure Mean Mean zone of inhibition was computed for each treatment based on the values for the three trials. Analysis of Variance (One-Way ANOVA) for CRD This was used to determine if there is a significant difference in the antifungal activity of Bita bark compared to Nystatin and was validated by the negative control. Least Significant Difference The least significant difference (LSD) was used to determine the significant difference between the antifungal activity of the plant extract against the positive and negative control.

25 Chapter IV Results and Discussion

Phytochemicals Present in the Plant Extracts of Bita Bark Phytochemical screening was conducted to determine the presence of secondary metabolites having fungicidal component. Specifically, the phytochemical screening was intended to detect the presence of flavonoids and tannins. This was done using standard procedures. Phytochemical screening of the plant extract showed presence of tannins and absence of flavonoids in the plant extracts. This implies that Bita, which is positive for tannins, has the potential as a fungicide because it contains the tannin found among plants that exhibit antifungal activity.

Figure 1. Results of Flavonoid Screening (A) Bate-Smith and Metcalf Method and (B) Wilstatter cyanidin test Since flavonoids are absent in Bita bark extract this indicates that the plant extracts may not be useful for therapeutic actions.

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Figure 2. Result of Tannin Screening (Ferric Chloride Test)

Antifungal Activity of the Plant Extract . The results showed that the Bita bark ethanolic extract have an antifungal activity,

specifically on Aspergillus niger but not on Candida albicans. The control groups in this study were Nystatin (positive control) and distilled water (negative control).

Table 1 Effect of Bita Bark Ethanolic Extract to Test Organisms Based on the Mean Zone of Inhibition Test Organism Aspergillus niger Candida albicans Mean Zone of Inhibition (mm) Nystatin (+) Distilled water (-) 23.9 0 26.9 0 Bita 13.0 5.1

Table 1 shows a high sensitivity of Candida albicans and Aspergillus niger to Nystatin which is the positive (+) control used in this study. On the other hand Bita bark

27 extract was partially reactive against Aspergillus niger but inactive for Candida albicans.

Figure 4 Results of Antifungal Screening (Ethanolic extract on Aspergillus niger)

Table 2. Analysis of Variance for A. niger Zone of Inhibition with Bita Bark Extract and Control Treatments Source of Variance Sum of Squares Between groups 856.702 Within groups 10.107 Total 866.809 *significant at p<.05 Df 6 8 Mean Square 428.351 1.684 F 254.298 Sig. .000

Table 3. LSD Comparison of Zone of Inhibition for Aspergillus niger

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(J) Controls Positive Negative *significant at the 0.05 level (I) Treatment Bita

Mean Difference (I-J) -10.86667* 13.00000*

Std. Error 1.05970 1.05970

Sig. .000 .000

Analysis of variance for CRD showed significant differences in the antifungal activity of different treatments against Aspergillus niger (Table 2). The F value was 254.298 at .000 level of significance. Comparison of the zone of inhibition of A. niger between Bita bark ethanolic extract and Nystatin (+control) showed significantly wider zone of inhibition for the + control compared with Bita bark extract. On the other hand, the zone of inhibition of A. niger was significantly wider for Bita ethanolic extract

compared with the negative control. Therefore, Bita can be used as treatment against diseases cause by A. niger -in the absence of Nystatin - compared to no treatment at all. The data is given in Table 3.

Figure 4. Results of Antifungal Screening (Ethanolic extract on Candida albicans) Table 4. Analysis of Variance for Candida albicans Zone of Inhibition with Bita Bark Extract and Control Treatments

29 Source of Sum of Variance Squares Df Between groups 1224.860 2 Within groups 28.900 6 Total 1253.760 8 *significant at 0.05 level of significance

Mean Square 612.430 4.817

F 127.148

Sig. .000

Table 4 is the result of the analysis of variance on the zone of inhibition of Candida albicans treated with Bita bark ethanolic extract. There were significant

differences in the zone of inhibition of C. albicans when grown in Nystatin (+ control) containing medium, Bita bark ethanolic extract, and distilled water (- control). value was 127.148 at .000 level of significance . The F

Table 5. LSD Comparison of Zone of Inhibition for Candida albicans Mean (J) Controls Difference (I-J) Positive -21.80000* Negative 5.10000* *significant at 0.05 level of significance (I) Treatment Bita Std. Error 1.79196 1.79196 Sig. .000 .029

The comparison of zone of inhibition of C. albicans using the LSD is given in Table 5. The results showed that zone of inhibition was significantly wider when

Nystatin was used compared with Bita bark ethanolic extract. Furthermore, zone of inhibition of C. albicans treated with Bita bark ethanolic extract was significantly bigger over the negative control. The results presented indicate that Nystatin is the best treatment for both Aspergillus niger and Candida albicans. In the absence of Nystatin, bita bark ethanolic

30 extract could serve the purpose particularly for treating infections caused by Aspergillus niger . The result of the antifungal activity of the bark extract showed an effect on the organisms tested. This is due to the presence of tannins that are found in the bark. Also, it has been demonstrated that tannins have a powerful anti-fungal and astringent action in a multitude of clinical studies (Zahourk, 2007). Studies have shown that tannins that are present in the bark showed antifungal activity against a wide variety of fungi (Mahmud et al., 2009).

Chapter V Summary, Conclusions and Recommendations

31 Summary This study was done to determine the antifungal activity of bita bark extract against Candida albicans and Aspergillus niger by conducting a phytochemical screening and antifungal assay. Standard procedures for phytochemical screening were used to screen for the presence of secondary metabolites flavonoids and tannins. The specific procedures used were the Bate Smith and Metcalf Method and Wilstatter cyaniding test for the presence of flavonoids. Ferric chloride test was used to determine the presence of tannins. Antifungal assay was conducted to determine the antifungal activity of the Bita bark ethanolic extract. Filamentous fungi ( Aspergillus niger) and yeast (Candida

albicans) were used as test organisms for the study. Paper Disc Diffusion Method was used for the antifungal assay. Nystatin was used as the positive control for both A. niger and C. albicans while distilled water was used as the negative control. The mean, analysis of variance for CRD, and LSD were the statistical tools used. The findings disclosed the absence of flavonoids on Bita bark extract while tannins were positively detected. Apparently, tannins are responsible for the fungicidal property of the bita bark extract as indicated in various literatues. The zone of inhibition of both Candida

albicans and Aspergillus niger was significantly smaller compared to the positive control (Nystatin), indicating that Nystatin is very active compared with the Bita bark ethanolic extract which is slightly active. Both the Analysis of Variance in CRD and the LSD showed that there is a significant difference on the antifungal activity of the plant extract compared to the positive control (Nystatin).Therefore, the plant extract is not as effective as the positive control in terms of treatment of fungal infections.

32

Conclusions From the findings presented, the researchers conclude that Bita bark has an antifungal property, based on the zone of inhibition noted for Aspergillus niger. Therefore, it can be a potential antifungal agent in treating diseases caused by Aspergillus niger . Recommendations Based on the findings presented, the following are recommended: 1. Establish the tannin level in various parts of Bita plant to identify the plant parts with the highest concentration of the substance apparently responsible for the fungicidal property. 2. In areas where synthetic drugs are not available to treat external mold infection, continuous or\ repeated and frequent application of Bita bark extract could serve the purpose. 3. Other pathogenic filamentous fungi may be tested as to their sensitivity to extracts from various parts of Bita plant. 4. Establish the Minimum Inhibitory Concentration of the Bita bark ethanolic extract against the test organisms used. 5. Other Antifungal Assay may be used like the Agar-Well-Diffusion Method.

References

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35 Kress, Henriette. (1995-2010). Alstonia, I.C.A. Alstonia. Henriettes Herbal Homepage. Retrieved April 23, 2010 from http://www.henriettesherbal.com/ eclectic/bpc1911/alstonia.html. Labay, P.M. (2003). Ethnobotanical and phytochemical studies on beneficial flora of Marinduque . PCCARD Commodities - DOST . Retrieved: April 22, 2010 from http://maidon.pcarrd.d ost.gov.ph/joomla/index.php Lai PK (June 2004). "Antimicrobial and chemopreventive properties of herbs and spices". Curr Med Chem: 145160. PMID 15180577. Lanting Jr. , Maximo V. and Palaypayon, Concepcion M. (2002) . Forest Tree Species with Medicinal Uses . Retrieved: from http://erdb.denr.gov.ph/pu blications/den r/den r_v11.pdf Lincoln Taiz and Eduardo Zeiger.(2006). Unraveling the Function of Secondary Metabolites. A Companion to Plant Physiology.4th Edition. Essay 13.2 . Retrived: April 14, 2010 from http://4e.plantphys.net/article.php? ch=13&id=313 Madigan M and Martinko J . (2005). Brock Biology of Microorganisms (11th ed.). Prentice Hall. ISBN 0131443291. OCLC 57001814. Retrived:April 5, 2010 . Mahmud et al. (2009). ANTIFUNGAL ACTIVITIES OF VITEX NEGUNDO LINN. Pak. J. Bot., 41(4): 1941-1943. Retrieved July 14 ,2010. MedicineNet.(2004). Definition of Complementary medicine. Retrieved: April 14, 2010 from http://www.medterms.com/script/main/art.asp?articlekey=31077 Mondofocto .(2009).Mondofocto. United Kingdom. Retrieved March 25, 2010 from http;www.mondofoc.com Moraleta, Rolando .(2003). Review of Microbiology. Manila Philippines. pg 164.Retrieved March 25,2010. McIntosh, Phillip. (2010).Retrrieved March 25, 2010 from http://mycology.suite101.com /article.cfm/sabouraud_dextrose_agar Muran, Paula .(2010). Parasitic Yeast Infection. U.S. Retrieved March 25, 2010 from http://healing.about.com/od/candida/a/candida_muran.html Rai, M.K,Deepak Acharya and P.Wadegaonkar. (2003). Plant derived-antimycotics: Potential of Asteracios Plant, In. Plant derived-antimycotics:Current Trends and Future prospects, Haworth press, N-york, London, Oxford, pp.165-185.

36 Raphael, Stanley S. (1983). Lynchs Medical Laboratory Technology. Fourth Edition. W.B. Saunders Company . pg.433-434. Reddy, Vinay R., M.D.(2010). Antibiotics, Bacteria and (usually not) Viruses. Dr. Reddy's Pediatric Office on the Web. Retrived: April 17, 2010 from http://www.drreddy. com/antibx.html. Ryan KJ, Ray CG (2004). Sherris Medical Microbiology (4th ed.). McGraw Hill. ISBN 0-8385-8529-9. Retrived : April 5, 2010. Spring, Manda.(2008).What is Aspergillus niger?. An overview of Aspergillus niger.Retrived: April 17, 2010 from http://www.brighthub.com/health/technology/articles/9543.aspx. Stepp, John R. and Moerman ,Daniel E..(2001).The importance of weeds in ethnopharmacology. Journal of Ethnopharmacology. Retrived: April 14, 2010 from http://www.sciencedirect.com/science.doi:10.1016/S03788741(00)00385-8 Tapsell LC (2006). "Health benefits of herbs and spices: the past, the present, the future". Med J Aust 1. PMID 17022438 Tarloff, Joan B., PhD. (2007). Allergies to Drugs. Adverse Drug Reactions.The Merck Manuals Online Medical Library. Retrived: April 17, 2010 from http://www.merck.com/m mhe /sec02/ch015/ch015f.html. Tuskegee University. (2010). Nystatin(MycrostaticR). Antifungal drugs. Retrived: April 12, 2010 from http://compepid.tuskegee.edu/syllabi/biomedical/Physiol ogy/physiolo gy352/chapter 12.html University of Santo Thomas-Research Center for the Natural Sciences (UST-RCNS). (2004). Guidebook to Plant Screening: Phytochemical and Biological. University of Santo Thomas. Espaa. Manila. Philippines Wagoner, Ben and Speer Brain. (2006). Universitry of California. Introduction to Fungi. Retrieved March 25,2010 from http://www.ucmp.berkeley.edu/fungi. WHCCAMP,(2002).White House Commission on Complementary and Alternative Medicine Policy. Retrieved: April 14, 2010 from http://whccamp.hhs.gov/fr2.html YeastGenome. (2010). What are yeast? . Retrived: April 5, 2010 from http://www.yeast genome.org /VL-what_are_yeast.html

37 Yoshizawa, T., Y. Tsuchiya, N. Morooka and Y. Sawada.(1975). Malformin Al as a mammalian toxicant from Aspergillus niger. Agric. Biol. Chem. 39:13251326.Retrived: April 18, 2010 from http://www.epa.gov/biotech_rule/pubs/fra/fra006.htm. Zahoruk, Cynthia (2007). Autism Canada Foundation. Medication- Antifungal. Retrieved July 15,2010 from http://www.autismcanada.org/antifungalmed.html

38

Appendices

Appendix A Communications

39

College of Pharmacy and Medical Technology University of San Agustin Iloilo City May 7, 2010

40

DR. RESURRECION B. SADABA Division of Biological Sciences College of Arts and Sciences University of the Philippines in the Visayas Miagao, Iloilo Dear Dr. Sadaba, Greetings! We, the third year Medical Technology students are required by the school to conduct a thesis related to our field. In relation to this, our group will be conducting a study titled, Screening and Antifungal Activity of Bita (Alstonia scholaris) Bark Ethanolic Extract. With this, we humbly ask for your help to verify the plant that we are about to use in our proposal. Your verification will help reassure the output of our experiment. We are hoping for your kind support and consideration. Thank you very much and God Bless!

Sincerely yours, Ace John Felix S. de la Cruz

Group Leader

NOTED: Mrs. Christine A. Villanueva Research Adviser

College of Pharmacy and Medical Technology University of San Agustin Iloilo City July 1, 2010

41 Dr. Gerard L. Penecilla Associate Director WVSU, Research and Development Center Dear Dr. Penecilla; Greetings! We, the fourth year Medical Technology students are required by the school to conduct a thesis related to our field. In relation to this, our group will be conducting a study titled, Screening and Antifungal Activity of Bita (Alstonia scholaris) Bark Ethanolic Extract. With this, we respectfully ask for your help to assist us during our phytochemical screening. As part it, we are going to test for the presence of Flavonoids and Tannins. Rather than that, we would like to ask permission if we could use your laboratory and reagents in order to run these test. In return, we are going to pay a fair amount for the usage of the reagents, apparatus, and equipments. Your assistance and cooperation would surely be a big help. We are hoping for your positive response. Thank you very much and God Bless! Sincerely yours, Ace John Felix S. de la Cruz
Group Leader

NOTED Mrs. Christine A. Villanueva Research Adviser

Appendix B Standard Protocol Plant Material Collection and Identification

42 Plant Extraction

Through experts in plant taxonomy

Extraction by the use of 95% Ethanol

Phytochemical Screening Determination of secondary metabolites (Flavonoids and Tannins).

Test Organism Verification

Through an Expert in Mycology Antifungal Assay

Figure 5. Schematic Diagram of Biologic Waste Disposal Workflow

Paper Disc Method

Diffusion

Figure 5. Schematic Diagram of Workflow A. Preparation of the Plant Extracts (UST-RCNS,2004)

43

Collection of fresh plant

Weighing of plant part (100g plant)

Cutting of the bark into pieces and crushing of Leaves and Bark using a mortar and pestle

Soaking of plant part in 95% Ethanol solution for 48 hours at room temperature in a mechanical shaker

Solutions will be incubated for 48 hours at room temperature on a shaker and filtered.

Filtrate will then be concentrated via VACUO (rotary evaporator) at a temperature below 50 degrees

The concentrated extract will be used in the Antifungal Assay.

Figure 6. Schematic Diagram of Plant Extraction B. Phytochemical Screening (UST-RCNS,2004)

44 Flavonoids: Bate-Smith and Metcalf Method and Wilstatter cyanidin test a. Three ml of the alcoholic filtrate is added with three dorps of 12M hydrochloric acid. Observe for any color change b. Warm for 15 min in a water bath and observe for further color change with one hour. c. The other test tube, having three ml of the alcoholic filtrate, is added with three drops of 12M hydrochloric acid. Followed by addition of three pieces magnesium turnings. d. Observe for color change within 10 min.

Tannins: Ferric chloride test 1. Take one ml of alcohol filtrate and transfer it to a clean testube. 2. Treat tube with three drops of ferric chloride reagent. Observe for greenblack precipitate.

B. Antifungal Assay : Disc Diffusion Assay ( UST-RCNS,2004)

45

Preparation of the Inoculum of Aspergillus niger and Candida albicans

Preparation of the Assay Plates

Placing of paper discs impregnated with the control and the plant extract solution.

Reading of the results

Interpretation of the results

Figure 7. Schematic Diagram of Antifungal Aassay

Inoculum preparation of Filamentous Fungi (Aspergillus niger)

46 1. Inoculation of A. niger colonies on Sabouraud glucose agar plate 2. Room temperature incubation until the conidia luxuriantly develops 3. Scrapping of 5 loopfuls, of conidia. 4. Immersion of the conidia to 5ml sterile saline- Tween 80 solution in the screw capped tubes 5. Shaking of contents for 1 minute. 6. Comparison of the inoculums turbidity with the 0.5 McFarland standard. Inoculum preparation of Yeast (Candida albicans) 1. A loopful of C. albicans will be inoculated from culture slants to a 50ml Sabourauds glucose broth medium. 2. Incubate for 18 hours at room temperature. 3. Adjustment of yeast cell suspension with the use of 0.5 McFarland standard as the basis of adjustment and Tween 80 as the diluent, to adjust the turbidity. Antibiotic preparation A prepared Nystatin tablet was used in the study, then the outer shell was removed, and inner portion was powderized with the used of mortar and pestle. 500 mg powderized Nystatin was mix together with distilled water.

Preparation of the Assay Plates 1. 20 ml of the Potato Dextrose Agar will be poured into a dry and sterile Petri dish. 2. Wait until the agar is solidified 3. Surface of the agar will be streaked three times, rotating the plate

47 approximately 60 degrees after each swab, with a cotton swab dipped in the test organism suspension. 4. Allow to stand for 5 minutes.

Placing of Paper Disc and Incubation

1. Using a sterile forceps, pick and lay on the Agar surface the paper disc that has been immersed into its respective solutions. 2. Incubate for 18- 24 hours at room temperature.

Reading of results 1. The Zone of inhibition will be measured with the use of a ruler (Measurement will be millimetres).

Appendix C

48 Documentation

Figure 8 Bita (Alstonia scholaris) gathered at Barangay San Miguel, San Miguel,Iloilo.

A. Plant Extraction

49

Figure 9 Plant part used in this study

Figure 10 Preparation of plant part for Extraction

50

Figure 11 Preparation of Plant Extract

B. Phytochemical Screening

51

Figure 12 Plant extract for Phytochemical screening of Flavonoids and Tannins

Figure 13 Test for Flavonoids

Figure 14 Test for Tannins C. Anti-Fungal Assay

52

Figure 15 Preparation and Labeling of Potato Dextrose Plates

Figure 16 Standardization of Inoculum

53

Figure 17 Swabbing of inoculum to plates

Figure 18 Plant extract and control

54 Figure 19 Placing of Disc

Figure 20 Output of the Antifungal Assay

55

Appendix D Raw Data

Trial(C. albicans) Control (+) 1 1) 30 Trial (A. niger) Control (+) 2) 27 1 1) 22 3) 18 2) 24 2 1) 29 3) 30 2) 32 2 4) 19 3) 28 5) 23 3 1) 27 6) 22 2) 28 3 4) 25 3) 23 5) 23 6) 27

Control (-) 1) 0 2) 0 Control (-) 1) 0 3) 0 2) 0 1) 0 3) 0 2) 0 1) 0 3) 0 2) 0 1) 0 3) 0 2) 0 1) 0 3) 0 2) 0 3) 0

Bita 1) 0 Bita 2) 7 1) 6.5 22 3) 2) 18 1) 7.3 3) Invalid 2) 0 1) 16 3) 0 2) 11 1) 10 3) 12 2) 7.2 1) 12 3) 8 2) 13 3) 13

56