1 ANTI-MICROBIAL BIOACTIVITY OF ETHANOLIC EXTRACT OF DUHAT (Syzygium cumingi) BARK AGAINST Vibrio cholerae

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In partial fulfillment of the requirements in Zoology for the second quarter

by: ELLA VICTORIA AUBREY T. CASTRO JOSEPH ROEL J. MABAJEN CARL ADRIAN D. SORIANO III-CURIE

2 CHAPTER I INTRODUCTION

Today, the world is facing the advancement of technology. With that is the advancement of cure for diseases. The prominent antidote today, even for tropical diseases or chronic was drugs. Drug chemotherapy remains still one of the major options for the people worldwide. Currently, attention is being given to the use of herbal products and therapy for the treatment of most ailments whether bacterial, viral or of parasitic origin. Extracting a plants part and testing it to a certain bacteria were the preliminary steps in proving a new alternative to use for the said disease (Sofowora, 1989). The World Health Organization (WHO) reported that about 80% of the worlds population depends mainly on traditional medicine. It involves mainly the use of plant extracts. Herbal prescriptions and natural remedies were commonly employed in developing countries for the treatment of various diseases, usually tropical and common diseases, this practice being an alternative way to compensate for some perceived deficiencies in orthodox pharmacotherapy. Unfortunately, there is limited scientific evidence regarding safety and efficacy to back up the continued therapeutic application of these remedies. The rationale for the utilization of these remedies rested largely on long term clinical experience. But now, with the upsurge in the use of herbal medicine, a thorough scientific investigation of the plants will go a long way in validating their folkloric usage (Sofowora, 1989). Syzygium cumini or commonly called in the Philippines as Duhat or Lomboi, belongs to the family, Myrtaceae. The species is an evergreen tropical tree, native to

3 India, Pakistan and Indonesia. (Burkill, 1997) Syzygium cumini is native to the subtropical Himalayas, India, Sri Lanka, Malaysia and Australia, where it is also widely cultivated. The tree was introduced from India and tropical Asia to southern Africa for its edible and attractive purple-red fruits. Now grown throughout the tropics and subtropics, the best forms are frequently cultivated in Java and Florida. A duhat tree is about 8 to 14 meters high with white branchlets and reddish young shoots. Leaves are broad-tipped, opposite, shiny and leathery, ellipitic, 6 to 15 cm long. Flowers, small, pinkish, in clusters, petal arranged to form a cup. Fruit is oval, 1 to 2 cm long, dark purple to black, fleshy and one-seeded, with a sweet-astringent taste. Duhat is a fruit tree found mostly wild throughout the Philippines. Duhat seeds are known for the treatment of diabetes, as are the leaves and the juice from the fruit. The bark is astringent and in decoction is used as a mouthwash and as a gargle for ulcerations of the mouth. Analyses of the fruit show that it is a good source of calcium and fair source of iron. Burkill also states that vinegar is made from the juice of the unripe fruit in Malaya. It can also be processed to popular products such as jam and juices. Another was, bark contain 19 percent tannin and 1.67 percent garlic acid. Tannin can be useful in protecting kidneys. It also has antiviral, antibacterial and antiparasitic effects, though intake in large quantities is toxic. Duhat seed was recently suggested in reducing blood sugars of diabetic individuals. It is indeed used in India. The information needs further confirmation. There is no harm in consuming duhat seeds however. Its taste is bland. A boiled bark is a cure for dysentery, diarrhea, enema and ulcers. It can also be used as gurgle. Dried and powdered roots have the same curative effect. The fruit is known to be

4 a remedy for diabetes mellitus (the same as number Leaf and bark juice is also effective alternative. Leaf extract is an effective poultice a medical dressing consisting of a soft heated mass of meal or clay that is spread on a cloth and applied to the skin to treat inflamed areas or improve circulation etc. (www.foodrecap.net/health/duhat-benefits/, June 28, 2012) Since the researcher will determine how the Ethanolic extract of Syzygium cuminis bark will affect the bioactivity of the Vibrio cholerae, well define and study what is Vibrio cholerae. Vibrio cholerae is a type of bacteria that cause cholera (an acute, diarrheal illness that can result in severe dehydration and even death within a matter of hours). These are Gram-negative rods that are facultative anaerobic, which means they can survive either with or without oxygen. There are two general types of Vibrio cholera, the Vibrio cholerae Serogroup O1 and the Vibrio cholerae Serogroup non-O1. Vibrio cholerae Serogroup O1 is the bacteria that is most often the cause of cholera. Vibrio cholerae Serogroup O139, a Vibrio cholera Serogroup non-O1 bacterium, is the other cause of cholera. There are about 70 other species of Vibrio cholerae Serogroup non-O1; these other species rarely cause diarrhea.

(http://bacteria.emedtv.com/vibrio-cholerae/vibrio-cholerae.html, June 28, 2012) The bacteria are native to the Ganges delta, which is in India and extends into Bangladesh. Since 1817, there have been seven worldwide pandemics. There is an ongoing global pandemic in Asia, Africa, and Latin America that has lasted more than four decades. Since 1995, over 80 percent of reported cases have occurred in Africa. In 2003, 111,575 cases from 45 countries were reported to the World Health Organization.

5 Cholera is a severe diarrheal disease caused by the bacterium Vibrio cholerae. Transmission to humans is by water or food. The natural reservoir of the organism is not known. It was long assumed to be humans, but some evidence suggests that it is the aquatic environment. Vibrio cholerae infections have been rare in industrialized nations for the last 100 years. In the United States, there is zero to five cases per year. Vibrio cholerae produces cholera toxin, the model for enterotoxins, whose action on the mucosal epithelium is responsible for the characteristic diarrhea of the disease cholera. In its extreme manifestation, cholera is one of the most rapidly fatal illnesses known. A healthy person may become hypotensive within an hour of the onset of symptoms and may die within 2-3 hours if no treatment is provided. More commonly, the disease progresses from the first liquid stool to shock in 4-12 hours, with death following in 18 hours to several days. The clinical description of cholera begins with sudden onset of massive diarrhea. The patient may lose gallons of protein-free fluid and associated electrolytes, bicarbonates and ions within a day or two. This results from the activity of the cholera enterotoxin which activates the adenylate cyclase enzyme in the intestinal cells, converting them into pumps which extract water and electrolytes from blood and tissues and pump it into the lumen of the intestine. This loss of fluid leads to dehydration, anuria, acidosis and shock. The watery diarrhea is speckled with flakes of mucus and epithelial cells ("rice-water stool") and contains enormous numbers of vibrios. The loss of potassium ions may result in cardiac complications and circulatory failure. Untreated

6 cholera frequently results in high (50-60%) mortality rates.

(http://textbookofbacteriology.net/cholera.html, June 28, 2012) Last April 2011, Health officials in the Philippines are struggling to contain a cholera outbreak in a remote area on the western island of Palawan. "There was no potable water, no toilets. The tribesmen defecated everywhere, and used contaminated water to both wash and as a source of drinking water," Manuel Mapue, who headed a government medical mission dispatched to the area, told reporters. At least 20 people have died and hundreds more have become sick, the country's National Disaster Risk Reduction and Management Council (NDRRMC).

(http://www.trust.org/alertnet/news/philippines-cholera-strikes-remote-ethnic-group/,June 28, 2012) Treatment of cholera involves the rapid intravenous replacement of the lost fluid and ions. Following this replacement, administration of isotonic maintenance solution should continue until the diarrhea ceases. If glucose is added to the maintenance solution it may be administered orally, thereby eliminating the need for sterility and IV administration. By this simple treatment regimen, patients on the brink of death seem to be miraculously cured and the mortality rate of cholera can be reduced more than tenfold. Most antibiotics and chemotherapeutic agents have no value in cholera therapy, although a few (e.g. tetracyclines) may shorten the duration of diarrhea and reduce fluid loss. (http://textbookofbacteriology.net/cholera.html, June 28, 2012) The aim of the present study is to determine if the Ethanolic extract of the Duhat bark will decrease or increase the microbial bioactivity of the bacteria given. Also, this

7 research will determine if the Duhat bark would be better than the commercially available drugs for Vibrio cholerae. Statement of the Problem This research will answer a general problem: What is the effect of the Ethanolic extract of Syzygium cumini against Vibrio cholerae? Specifically, this research sought to answer the following questions: 1. Will the Ethanolic extract of Syzygium cuminis bark increase or decrease the microbial bioactivity of the Vibrio cholerae? 2. What are the components of Syzygium cuminis extract that could exterminate Vibrio cholerae? 3. How will the Ethanolic extract of Syzygium cuminis bark affect Vibrio choleraes microbial bioactivity? 4. Is there a significant difference on the variuos concentrations of Syzygium cuminis Ethanolic extract and commercially available drugs on Vibrio cholerae? Significance of the Study Academe. This research enables the people to learn new things about bacteria, diseases and treatment for those. As they read and perform this study, they will know how to handle pathogenic bacteria, especially the Vibrio cholerae which is our bacteria on study. They will also know that the cure for tropical diseases is everywhere in the community. They can change the variables if they want further study about this research. Research. This research opens the room for other students whose studying the same variable for a new alternative in Cholera. If the result of this research turns into

8 positive, they can use this research and results as a basis for future experiments. The other researchers can change the variables if they want further investigation about the topic. They can make other treatments for Cholera and they can make the disease easier to control than the past. Medicine. As said, it will open room for other cure for the same disease, Cholera. This research can widen up the scientific peoples mind about this and they can yield up with new medicines that will improve the present status of antidotes and the present medical level about the cure against Cholera. It will also help people to buy cheaper medicines than the ones in drugstores or to get plants in their own trees to cure their own diseases. Economic Value and the Society. Duhat barks were very affordable and for a tropical disease, it will turn out as a better cure. As said, boiled Duhat bark can cure diarrhea. It is very cheap and everyone can afford it. It is easier for the cholera victims to find a cure on their illness. Also, it decreases the labor of the people finding medicines for their patients. Fewer expenses for transportation, for food and for the price of the medicine will be opened up by this research. It is very essential to the people. Duhat barks can be easily seen in our community. It will help our world lessen the cases of this disease. Scopes and Limitations This research covers the study of the antimicrobial bioactivity effects of Syzygium cumini barks Ethanolic extract against Vibrio cholerae. It also talks about the effectivity of the Ethanolic extract of Duhat bark against the pathogenic bacteria Vibrio cholerae

9 and it will determine how ill the Duhat bark affect the microbial bioactivity of Vibrio cholerae. This research does not talk about the factors that involve the factors that involve the comparison of the commercially available drugs to the Duhat bark extract. It does not tackle the chemicals that were present in each group (the treatment and the control group) that affects the microbial activity of the Vibrio cholerae. It also does not cover the comparison of the Duhat bark to the other parts of the Duhat tree and the other plants. Lastly, it does not tackle about the chemicals present in the Syzygium cuminis Ethanolic extract that reacted by the Vibrio cholerae and vice versa. DEFINITION OF TERMS: Ethanol. Ethanol is an alcohol obtained from the fermentation of sugars and starches or by chemical synthesis. (http://yahoo.answers.com, July 12, 2012) Duhat. A fruit that was about 8 to 14 meters high with white branchlets and reddish young shoots. (www.foodrecap.net/health/duhat-benefits/, June 28, 2012) Vibrio cholerae. It is a bacterium that mainly causes cholera, rarely diarrhea. (http://bacteria.emedtv.com/vibrio-cholerae/vibrio-cholerae.html, June 28, 2012) Cholera. It is a severe diarrheal disease caused by the bacterium Vibrio cholerae. Transmission to humans is by water or food.

(http://textbookofbacteriology.net/cholera.html, June 28, 2012) Bioactivity. The effect of a given agent, such as a vaccine, upon a living organism or on living tissue. (http://yahoo.answers.com, July 12, 2012)

10 Percolation. To cause (liquid, for example) to pass through a porous substance or small holes; filter. (http://www.thefreedictionary.com/Percolation, August 12, 2012) Rotary Evaporator. Rotavap; Rotary evaporators (also called "rotavaps") are used to remove solvents from reaction mixtures and can accommodate volumes as large as 3 liters. (http://www.chem.ucla.edu/~bacher/Specialtopics/rotavap.html, August 12, 2012) Autoclave. An autoclave is a piece of equipment which uses pressurized steam to kill microorganisms. The machine can be used to sterilize most glassware, but a hot air oven operating a temperature of 160C for one hour should be used instead for equipment which will be damaged by the wet heat of the autoclave. (http://water.me.vccs.edu/courses/env211/lesson14_2.htm, September 3, 2012)

11 CHAPTER II REVIEW OF RELATED LITERATURE

Here are some of the related studies and literatures that tackle the variables that were involved in the study of the antimicrobial bioactivity of Ethanolic extract of Duhat bark against Vibrio cholerae. Vibrio cholerae Eventually, the Italian scientist, Filippo Pacini, would gain prominence for his discovery of Vibrio cholera, but not until 82 years after his death, when the international committee on nomenclature in 1965 adopted Vibrio cholerae Pacini 1854 as the correct name of the cholera-causing organism. Until then, many credited Robert Koch (18431910) with this seminal discovery. Cholera came to Florence in 1854 during the Asiatic Cholera Pandemic of 184663. Pacini became very interested in the disease. Immediately following the death of cholera patients, he performed an autopsy and with his microscope, conducted histological examinations of the intestinal mucosa. During such studies, Pacini first discovered a comma-shaped bacillus which he described as a Vibrio. He published a paper in 1854 entitled, "Microscopical observations and pathological deductions on cholera" in which he described the organism and its relation to the disease. His microscopic slides of the organism were clearly labeled, identifying the date and nature of his investigations. While later found to be important, Pacini's work was completely ignored by the scientific community. It is unlikely that John Snow knew of this earlier publication,

12 presented in far-away Italy four years before Snow's death. Nor 30 years later would Robert Koch come across Pacini's report. As a supporter of the germ theory, Pacini insisted that cholera was contagious. Yet similar to debate throughout Europe, his ideas were contradicted by influential Italian physicians who believed in the miasmatic theory. Until one year after his death in 1883, Pacini's cholera data were ignored by the scientific community. During 1883, cholera was epidemic in Egypt. Koch traveled with a group of German colleagues from Berlin to Alexandria, Egypt in August, 1883. Following necropsies, they found a bacillus in the intestinal mucosa in persons who died of cholera, but not of other diseases. He reasoned that the bacillus was related to the cholera process, but was not sure if it was causal or consequential. He stipulated that the time sequence could only be resolved by isolating the organism, growing it in pure culture, and reproducing a similar disease in animals. He was not able to obtain such a pure culture, but did try to infect animals with choleraic material. None became infected. His thoughts and early findings were sent in a dispatch to the German government and shared with the German press. Despite the earlier work of Dr. John Snow, many still believed that cholera was caused by miasmata. Just 10 years earlier at a major 1874 international sanitary conference, representatives of 21 governments voted unanimously that "ambient air is the principal vehicle of the generative agent of cholera." With Koch findings, however, the tide of scientific and public opinion began to increasingly to change, although slowly. Scientists were divided in Germany, almost entirely negative to Koch's theory in France, and nearly so in England. In the international sanitary conference of 1885 attended by

13 Robert Koch along with representatives of 28 countries, the British delegation successfully blocked any "theoretical discussion on the etiology of cholera," thereby denying evidence that British John Snow had so carefully described in his 1855 book, Italian Filippo Pacini had witnessed in his microscopic studies, and German Robert Koch had cultured in his field and laboratory studies.

(http://www.ph.ucla.edu/epi/snow/firstdiscoveredcholera.html , July 12, 2012) Studies were conducted about Vibrio cholerae and one of these studies was conducted about Vibrio cholerae by P. M. Desmarchelier, F. Y. Wong, and K. Mallard. It was entitled as An epidemiological study of Vibrio cholerae O1 in the Australian environment based on rRNA gene polymorphisms. (Epidemiol Infect. 1995 December; 115(3): 435446.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2271589/) This aims to study the relationships between clinical isolates and environmental isolates from rivers and aquatic life, widely distributed throughout the country, a wide range of molecular typing methods were employed. In the paper, they report the analysis of the 180 Australian isolates (10 clinical and 170 environmental) using ribotyping. Seven ribotype patterns were observed among the Australian inaba isolates. The ogawa isolates were more diverse. Ribotype patterns were not shared between the serotypes with the exception of one ogawa isolate which could be distinguished using PFGE. Ribotyping has been useful in confirming an association between epidemiologically related clinical isolates and the aquatic environment and the persistence of several clones of the O1 serovar in the Australian environment during an 8-year period.

14 Another study was conducted by T. Popovic, C. Bopp, O. Olsvik and K. Wachsmut. It was entitled as Epidemiologic application of a standardized ribotype scheme for Vibrio cholerae O1. (J. Clin. Microbiol. 1993, 31(9):2474.

http://jcm.asm.org/content/31/9/2474) The ribotype patterns obtained are reproducible and stable over time. Seven different but very similar ribotypes (la to lg) were observed among 16 strains of the classical biotype. Twenty ribotypes and subtypes were identified among 198 Vibrio cholerae 01 strains of the El Tor biotype. Six different patterns were found among the strains causing the current seventh pandemic. The most widely distributed strains were those of ribotype 6, which was subdivided into three very similar but still distinguishable subtypes. Strains associated with two documented environmental reservoirs exhibited three distinct ribotype patterns; those isolated from patients who ate food from the U.S. Gulf waters were all of ribotype 2, while the strains related to the northeast Australian rivers were of ribotypes 9 and 10. Nontoxigenic Vibrio cholerae 01 strains originating in Latin America and the U.S. Gulf Coast did not form a specific cluster of ribotypes. Ribotyping in combination with other well-defined methods can assist in epidemiologic investigations, helping to trace the movement of strains and to identify their geographic origins. The last study for the Vibrio cholerae was conducted by Mohammed R. Pourshafie, Francine Grimont, Mahnez Saifi and Patrick A. D. Grimont. It was entitled as Molecular epidemiological study of Vibrio cholerae isolates from infected patients in Teheran, Iran. (Department of Bacteriology, Pasteur Institute of Iran, Teheran, Iran and Institut Pasteur, Unite des Enterobacteries, Unite INSERM 389, 75724 Paris Cedex 15, France, Received 1 Nov. 1999; revised version accepted 3 April 2000.) A

15 total of 110 clinical isolates of Vibrio cholerae O1 biotype El Tor serotype Ogawa isolated in a recent outbreak from different districts of Teheran, Iran, was subjected to 99 carbon source utilisation tests, ribotyping and toxinogenotyping. PCR showed that the genes encoding cholera toxin ( ctxA), toxin co-regulated pilus (tcp), accessory cholera enterotoxin (ace) and zonula occludens toxin ( zot) were present in 100%, 100%, 97.3% and 99.1% of the isolates, respectively. Restriction fragment length polymorphism (RFLP) study of the BglI-digested DNA probed with five oligonucleotides targeting the conserved regions of 16S and 23S rRNA genes revealed a similar ribotype pattern for 109 isolates. All but one isolate showed ribotype pattern B21a, containing seven bands with molecular sizes ranging from 11 to 3.9 kb. The toxin gene restriction pattern (toxinogenotype) showed that the isolates carried either three or two copies of the toxin genes (ace, zot, ctx) which were recognized as TB41a and TB69 patterns, respectively. Overall, the ribotyping data showed that, despite biochemical differences in 12 of the 99 carbon sources, most of the isolates studied belonged to a single clone.

(http://jmm.sgmjournals.org/content/49/12/1085.abstract, August 12, 2012) Duhat Bark Syzygium cumini or commonly called in the Philippines as Duhat or Lomboi, belongs to the family, Myrtaceae. The species is an evergreen tropical tree, native to India, Pakistan and Indonesia. (Burkill, 1997) Syzygium cumini is native to the subtropical Himalayas, India, Sri Lanka, Malaysia and Australia, where it is also widely cultivated. The tree was introduced from India and tropical Asia to southern Africa for its

16 edible and attractive purple-red fruits. Now grown throughout the tropics and subtropics, the best forms are frequently cultivated in Java and Florida. A duhat tree is about 8 to 14 meters high with white branchlets and reddish young shoots. Leaves are broad-tipped, opposite, shiny and leathery, ellipitic, 6 to 15 cm long. Flowers, small, pinkish, in clusters, petal arranged to form a cup. Fruit is oval, 1 to 2 cm long, dark purple to black, fleshy and one-seeded, with a sweet-astringent taste. Duhat is a fruit tree found mostly wild throughout the Philippines. Duhat seeds are known for the treatment of diabetes, as are the leaves and the juice from the fruit. The bark is astringent and in decoction is used as a mouthwash and as a gargle for ulcerations of the mouth. (www.foodrecap.net/health/duhat-benefits/, June 28, 2012) Studies were also conducted about Syzygium cumini and one of these studies was conducted by Ugbabe, G.E, Ezeunala, M.N., Edmond, I.N., Apev, J. and Salawu, O.A. It was entitled as Preliminary Phytochemical, Antimicrobial and Acute Toxicity Studies of the Stem, bark and the Leaves of a cultivated Syzygium cumini in Nigeria. (African Journal of Biotechnology Vol. 9(41), pp. 6943-6747, 11 October, 2010,

http://www.academicjournals.org/AJB). The phytochemical screening revealed the presence of carbohydrates, saponins, tannins, terpenes, volatile oil, sterols, resins balsam, phlobatanins and flavonoids in the leaves and stem bark of the species studied. Alkaloids and anthraquinones were absent in all the plant parts studied. 70% methanol extracts were used for antimicrobial and acute toxicity studies. The six test organisms used for antimicrobial studies were: Pseudomonas auriginosa, Escherichia coli, Staphylococcus aureus, Candida albicans, Bacillus subtilis and Salmonella typhii. The oil from the leaves showed activity on all the test organisms; the leaf extract showed activity on all test

17 organisms except Pseudomonas auriginosa while the stem bark extract showed no activity on any of the test organisms used. The oil had a saponification value of 363 and an acid value of 4.21. Another study about Syzygium cumini that was conducted was entitled as Influence of Samac bark, Samac fruit, Samac leaf, and Duhat bark on growth of microorganisms. by Mura, K.; Tanimura, W. 1986, Hakkokogaku Kaishi (01 May 1987. 64 (1) 1-7, http://www.academicjournals.org/AJB) This paper describes the influence on the growth of lactic acid bacteria (LAB), acetic acid bacteria (AAB) and yeast (Y), of samac (Macaranga grandifolia) bark, samac fruit, samac leaf, and duhat (Syzygium cumini) bark, which are added to basi (Philippine sugarcane wine). An aqueous extract of samac bark greatly inhibited growth of LAB and AAB, but Y growth was greatly increased. It is therefore presumed that samac bark prevents acidification of basi, and accelerates alcoholic fermentation. An aqueous extract of samac fruit not only inhibited growth of LAB but also of Y. It is therefore presumed that samac fruit prevents acidification and inhibits alcoholic fermentation. An aqueous extract of samac leaf promoted growth of Y, but caused little growth inhibition of LAB or AAB. It is therefore presumed that samac leaf has little effect on acidification of basi. An aqueous extract of duhat bark greatly inhibited growth of AAB so it is presumed that it prevents acidification of basi; it also inhibited Y growth, so it is presumed that it inhibits alcoholic fermentation. All samples contained a great deal of polyphenol, which probably inhibits microbial growth. Aggregated cells of LAB, AAB and Y grown in a medium containing various aqueous extracts were examined; the surface of these cells was covered with granular

18 matter. This adhered granular matter is presumably related to microbial growth inhibition. Also, In vitro study of antioxidant activity of Syzygium cumini fruit was conducted. (Pharmacognosy Research Laboratory, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Kolkata 700019, India. Received 9 February 2004. Revised 23 April 2004. Accepted 23 April 2004. Available online 17 July 2004. http://dx.doi.org/10.1016/j.foodchem.2004.04.033) Food rich in antioxidants plays an essential role in the prevention of diseases. The fruits of wild Indian Syzygium cumini (L.) Skeels (Myrtaceae), also known as black plum, are edible. Traditionally they are also used to cure a number of ailments. In this paper, the antioxidant activity of the fruit skin has been analyzed using different assays, such as hydroxyl radical-scavenging assay, based on the benzoic acid hydroxylation method, superoxide radical-scavenging assay, based on photochemical reduction of nitroblue tetrazolium (NBT) in the presence of a riboflavin-light-NBT system, DPPH radical-scavenging assay, and lipid peroxidation assay, using egg yolk as the lipid-rich source. Total antioxidant capacity was determined by the assay based on the reduction of Mo(VI)Mo(V) by the extract and subsequent formation of a green phosphate/Mo(V) complex. In all the systems, a significant correlation existed between concentration of the extract and percentage inhibition of free radicals or percentage inhibition of lipid peroxidation. The antioxidant property of the fruit skin may come in part from the antioxidant vitamins, phenolics or tannins and anthocyanins present in the fruit. (http://findmeacure.org, August 12, 2012) Another was Evaluation of the Antioxidant Activity of Syzygium cumini Leaves by Zhi Ping Ruan Liang Liang Zhang and Yi Ming Lin; (Key Lab of Ministry of

19 Education for Coast and Wetland Ecosystems, Xiamen 361005; P.R. China; Department of Biology, School of Life Sciences, Xiamen University, Xiamen 361005; P.R. China; Received: 22 August 2008; in revised form: 10 October 2008 / Accepted: 13 October 2008 / Published: 16 October 2008) The antioxidant activity of Syzygium cumini leaf extracts was investigated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicalscavenging and ferric-reducing antioxidant power (FRAP) assays. The methanolic extract and its four water, ethyl acetate, chloroform, and n-hexane fractions were prepared and subjected to antioxidant evaluation. The results showed that the ethyl acetate fraction had stronger antioxidant activity than the other ones. HPLC data indicated that S. cumini leaf extracts contained phenolic compounds, such as ferulic acid and catechin, responsible for their antioxidant activity. A significant linear relationship between antioxidant potency, free radical-scavenging ability and the content of phenolic compounds of leaf extracts supported this observation. (www.mdpi.org/molecules, August 12, 2012) Effects of Syzygium cumini Bark on Blood Glucose, Plasma Insulin and Cpeptide in Streptozotocin induced Diabetic rats was conducted. (January 2006, G. Saravanan, Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar-608002, Tamil Nadu, India P Leelavinothan Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar-608002, Tamil Nadu, India) In recent years, several plant extracts have been examined for their anti-diabetic properties in an effort to identify alternative treatment strategies that pose less of a risk for diabetics. The present study was undertaken to investigate the anti-diabetic effects of Syzygium cumini bark in experimental diabetes mellitus. Materials and Methods: Diabetes was induced in male

20 albino Wistar rats by a single intraperitoneal injection of streptozotocin (45 mg/kg body weight), after which, the animals were randomly allocated into five experimental groups as follows: Group 1: normal rats, Group 2: normal rats received Syzygium cumini bark extract (SBEt; 300mg/kg body weight), Group 3: diabetic control rats, Group 4: diabetic rats receiving SBEt (300mg/kg), Group 5: diabetic rats received glibenclamide (600g/kg body weight). The effects of 45 days treatment of SBEt on blood glu-cose, plasma insulin, C-peptide, urine sugar and body weight were studied in comparison to those of glibenclamide. Results: Blood glucose levels (268.1019.25 mg/dL) and urine sugar increased significantly whereas the levels of plasma insulin (5.01 0.29 U/L) and Cpeptide (167.68 8.50 pmol/L) decreased significantly in diabetic rats as compared to normal rats. Oral administration of SBEt exhibited anti-diabetic activity by significantly (p<0.05) lowering blood glucose (84.304.25) and urine sugar levels in diabetic rats. Additionally, diabetic rats treated with SBEt had significantly (p<0.05) elevated levels of plasma insulin (10.290.59) and C-peptide (236.5011.87). During OGTT, long-term administration of SBEt was able to significantly (p<0.05) decrease blood glucose concentrations (93.94 3.17; 120min) at various time intervals when compared to the OGTT pattern of diabetic rats (316.03 18.03). As com-pared to glibenclamide, SBEt has better anti-diabetic effects. Conclusions: The findings of this study indicate that the anti-diabetic activity of SBEt, and both the pancreatic and the extra-pancreatic mechanisms might be involved such apparent dual actions of SBEt would be more advantageous to the existing oral anti-diabetic monotherapy.

(http://www.academicjournals.org/JMPR, August 12, 2012)

21 Lastly, In vitro study on the antimicrobial property of duhat ( Syzygium cumini) was conducted by E.P.Famatiga. (Journal of the Philippine Dental Association, 57(1), 65, 2002) Herb-based medicines are slowly gaining acceptance from the Filipino market due to the high cost of commercially available drugs. However, their efficacy is still in question. Scientists are continuously searching for bases for herbal medication. Syzygium cumini, is one of the herbal plants used in traditional medicine. The objective of this study was to determine the antimicrobial properties of Syzgium cuminii (duhat) against Staphylococcus aureus UPCC 1143, Escherichia coli UPCC 1195, and Candida albicans UPCC 2168 in vitro. The decoction of duhat leaves and bark, at 40 percent, 80 percent, and 160 percent concentrations were tested for antimicrobial activity against the test organisms using the Agar Cup Methods. Results of the antimicrobial test were gathered by ocular observation and the diameters of zone of inhibition were measured using a ruler under reflected light. The results of the antimicrobial test showed that duhat extracts have no antimicrobial effect against Escherichia coli and Candida albicans. Only Staphylococcus aureus exhibited susceptibility to the duhat extracts. Different concentrations of duhat samples produced varying diameters of clearing zone that increased with increasing concentration.

(http://scinet.dost.gov.ph/union/ShowSearchResult.php? s=2&f&p&x&page&sid=1&id=An+in+vitro+study+on+the+antimicrobial+property+of+ duhat+%28Syzgium+cuminii%29&Mtype=ANALYTICS, August 12, 2012) Antimicrobial Bioactivities. The history of antimicrobials begins with the observations of Pasteur and Joubert, who discovered that one type of bacteria could prevent the growth of another. They did not know at that time that the reason one

22 bacterium failed to grow was that the other bacterium was producing an antibiotic. Technically, antibiotics are only those substances that are produced by one microorganism that kill, or prevent the growth, of another microorganism. Of course, in today's common usage, the term antibiotic is used to refer to almost any drug that attempts to rid your body of a bacterial infection. Antimicrobials include not just antibiotics, but synthetically formed compounds as well. The discovery of antimicrobials like penicillin and tetracycline paved the way for better health for millions around the world. Before penicillin became a viable medical treatment in the early 1940s, no true cure for gonorrhea, strep throat,

or pneumonia existed. Patients with infected wounds often had to have a wounded limb removed, or face death from infection. Now, most of these infections can be cured easily with a short course of antimicrobials. However, with the development of antimicrobials, microorganisms have adapted and become resistant to previous antimicrobial agents. The old antimicrobial technology was based either on poisons or heavy metals, which may not have killed the microbe completely, allowing the microbe to survive, change, and become resistant to the poisons and/or heavy metals. Antimicrobial nanotechnology is a recent addition to the fight against disease causing organisms, replacing heavy metals and toxins and may someday be a viable alternative. Infections that are acquired during a hospital visit are called "hospital acquired infections" or nosocomial infections. Similarly, when the infectious disease is picked up

23 in the non-hospital setting it is considered "community acquired".

(http://yahoo.answers.com, July 12, 2012)

CHAPTER III METHODOLOGY

This chapter will discuss what will the researchers need for their experiment, how will they do their experiment and the safety measures that they should follow in doing the experiment.

Gathering/Collection of Materials
Application of Duhat bark Ethanolic extract to Vibrio cholerae Disposal of Vibrio cholerae

Ethanolic Extraction and Percolation of Duhat Bark Preparation of the Concentrations

Figure 1. This is the summary of the methodology of the experiment. This can be a simple guide for all the researchers that will perform this experiment too. Further explanations and details can be found and read below.

Gathering/Collection of Materials

24 `The researchers will gather 1 sack of Duhat Bark in Baltazar St. 7 th Ave. Caloocan City. Also, they will buy Mutacol for the positive control. For the negative control, they will buy 350 mL Natures Spring distilled water in Mercury Drug, cor. 11th Ave. Caloocan City. They will have their experiment at the Research Institute of Tropical Medicine in Department of Health Compound, FILINVEST Corporate City, Alabang, Muntinlupa City, 1781 Philippines. (http://www.ritm.gov.ph/, July 30, 2012) The researcher will be guided by a supervisor from the RITM and the RITM will give the supervisors identity in the actual day of the experiment. The bacteria will be given at the institute. Ethanolic Extraction and Percolation of Duhat Bark The researcher will need 1000 mL of Ethanol for the extraction solvent and 25 kilos of Duhat barks. The researchers will first air-dry the Duhat barks then they will put the Duhat barks in a large bowl and pour the Ethanol. After extraction the researchers will percolate the solvent. To get a pure extract of the Duhat bark, the researchers will use a rotary evaporator. Because rotary evaporators are only in premiere laboratories in Philippines, the researcher went to University of the Philippines, Diliman Campus to make the Rotavap extraction. Preparation of the Concentrations The researchers will prepare 5 set-ups for the experiment: one for the positive control (Mutacol vaccine), three with the extracts with different amounts (2 mL, 3 mL and 5 mL), and one for the negative control (distilled water).
Negative Control (-)

1 (2 mL)

2 (3 mL)

3 (5 mL)

Positive Control (+)

R1

R1

R1

R1

R1

25

R2 R3

R2 R3

R2

R2 R3

R2 R3

R3

Figure 2. The Research Design. It will be needed when the concentrations were being prepared. In each set-up, there were three sub-set-ups. The researchers will get the average of the results of the three sub-set-ups so that they can get a reliable Proper Handling of a Pathogenic Bacteria data for this study. Many laboratory exercises involving bacteria require that all equipment be sterilized before use. Sterilization is a method used to kill all bacteria on the equipment so that residual bacteria will not contaminate the sample. The two primary methods of sterilization are to use chemicals, such as alcohol, or to use high heat, usually in an autoclave. (http://water.me.vccs.edu/courses/env211/lesson14_2.htm, 2012) Before doing the experiment, the researchers must follow the proper handling of pathogenic bacteria techniques. First, clear your work area of all non-essential items such as purses, clothing, backpacks, and books you will not need during lab. Wipe off your lab table with disinfectant solution before you begin. Remove dangling jewelry and make sure long hair is pinned or tied out of the way. Wear gloves and protective eyewear when working with bacterial cultures and dispose of the gloves in a BIOHAZARD bag. Food and beverages are NOT allowed in the lab room. Never place pens, pencils, fingers, labels, or any other items in your mouth while in the lab. Avoid spilling solutions that contain bacteria. In case of an accidental spill, inform your instructor. Wipe up as much of the spilled material as you can with paper towels and dispose of them properly. Thoroughly soak the September 3,

26 area where the spill occurred with disinfectant solution. Wait 20 minutes before wiping up the disinfectant solution. When you have completed the lab activity, remove all items from your lab table and wipe it with disinfectant solution. Before you leave the lab, wash your hands thoroughly with soap and warm water.

(http://www.austincc.edu/biocr/1406/labm/ex9a/prelab_9a_1.htm, July 30, 2012) Disposal of Pathogenic Bacteria When the researchers are completely done with their experiment, they will need to decontaminate any plates theyve used. More than likely, they will not have access to an autoclave for sterilization. Another way to decontaminate their experimental materials is to use disinfectants. The best disinfectant is household bleach at 10% strength. The researchers can make a 10% bleach solution by mixing one part of regular laundry bleach (e.g. Clorox) with 9 parts of water. Other general common household cleaning reagents are also effective at decontaminating bacteria, and can be used. Decontaminate plates by carefully opening, and pouring a generous amount of disinfectant (i.e., 10% bleach) onto the agar surface. Leave the plates to soak for at least an hour. The sterilized, decontaminated plates can then be disposed of in their regular household garbage, but ONLY after sterilization, as described, is complete. (http://www.sciencebuddies.org/science-fair-projects/project_ideas/Micro_Safety.shtml, July 30, 2012)