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Total Phenolic Contents of Neem Tree

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Herbal medicines have been part of the ingenious way of living in the society since the earlier times. Primitive men have been using this kind of medicine as the primary method of medication without undergoing different industrial processes. Herbal medicines have really come a long journey way back to the ancient times of Herbalism. One of the considered pioneers in Herbal medical field was Imhotep, a priest-physician of the ancient Egypt, physician of Marcus Aurelius and Paracelsus, who have a wide array of knowledge in Herbalism and made a record of the uses of these plants.

In line of herbal applications, Ayurveda, the traditional medicine of India which is developed more than 5000 years ago, is believed to be the medicine of Hindu Gods. Ayurveda is based on the belief that our body is a balance and we get ill when this balance was disturbed. Consequently the five elements that Hindu’s believe to maintain this so-called balance are namely Smell, Taste, Sight, Touch and Hearing must preserve its equilibrium. The first two books of Ayurveda; Caraka Samhita and Susruta Samhita include a thousand of references in herbal medicines.

Phytotherapy has been introduced by French for the usage of Herbs as medicines and is internationally accepted and used. To edifice a valid basis on Herbal effectiveness, there are two aspects that should be measured; internal and external validity. Internal validity includes reliable and accurate results relationship with the hypothesis under controlled conditions which satisfy the scientific community from time to time while on the other hand, External validity refers to the applicability of the method either beyond or under the experimental conditions. Part of ensuring the social value of research includes devising and implementing sound science. Although international collaborative research on herbal medicine is no exception, discussing scientific validity as an ethical requirement raises some specific challenges, including the meaning of scientific validity, establishing inclusion and exclusion criteria, using appropriate outcome measures, and determining appropriate study designs.

The Neem tree (Azadirachta excelsa) is a tropical green tree that is common in the Philippines, Indonesia and other countries in the Southeast Asia. The other species of Neem that has been reported is the Azadirachta indica which is believed to originate and is native in Indian subcontinent. Recently, reports have been tallied that about 72 countries worldwide have this so-called “Miracle plant”. The Neem tree is a member of the Mahogany family having the taxonomic positions of:

Order: Rutales
Family: Rutinae
Subfamily: Meliodeae
Tribe: Melieae
Genus: Azadirachta
Species: Indica or Excelsa
Approximately, there is an estimate of 25 million trees growing all over India. Neem has become the “India’s best kept secret” for a very long time that Indians benefit from almost all of its parts. Neem can be grown in areas which have 40cm to 150cm of rain annually and can survive at an uneven temperature of as high as 44oC to as low as 4oC.

According to Indians, Amrita (the elixir of life in Indian mythology) was ascented into heaven and few drops fell on the Neem tree. Neem was first acknowledged for its healing capabilities by the dwellers of India and Southeast Asia as early as 4000 B.C. The Vedas called Neem “Sarva Roga Nivarini”, which means “one that cures all ailments and ills”. During the last 30 years of publishing reports on the properties of Azadirachta, they overlooked another species that is commonly found in Southeast asia namely the “Azadirachta excelsa”.

The branches of Azadirachta excelsa are obliquely uplifting and spreading. In the young trees, the trunk is cylindrical from the base, but old trees show massive buttresses over the man roots. The bark color ranges from pinkish gray to pale gray depending on the age. The tree distribution on the Philippines is mainly even on Palawan. Unlike Azadirachta indica, excelsa requires more rainfall.

Both Indica and Excelsa shows unexplainable capabilities of curing diseases, illnesses and including insecticidal effect, many studies about Neem tree have been conducted and used the extracts from different parts yielding positive results. Sprays from the leaf extract of Neem can be use as an insect repellant. For women, Neem can be use for hygienic purposes. It has been also used to confer health issues like scratches, dandruffs, dry skin, head lice, malaria and even psoriasis.

The Neem bark contains Nimbin, Nimbinin, Nimbidin, Nimbosterol, essential oil and tannin. Tannin is a water-soluble polyphenol commonly located at the bark of the woody and herbaceous trees. Two types of tannins are known namely the hydrolysable and the non-hydrolyzable or condensed tannins. Hydrolysable tannin, are derivatives of gallic acid (3,4,5-trihydroxyl benzoic acid). Gallic acid is esterified to a core polyol and the galloyl groups may be further esterified or oxidatively crosslinked to yield more complex hydrolysable tannins. The simplest hydrolysable tannins, the gallotannins, are simple polygalloyl esters of glucose which have many isomers.

Tannins are considered as “secondary compounds”. A secondary compound does not include in biosynthesis, biodegradation or any metabolic processes but plays a vital role in toxicity or protection. Traditional use of tannins as agents for converting animal hides to leather (also known as tanning) is one of the manifestation activities of tannins. Also tannins can precipitate proteins including those that are found on skin. Recent studies show also the ability of tannin to control the growth of different microorganism. Another is the ability of the tannin in minimal concentration to be used as antifungal for dermophytes like Pityrosporum ovale.

Statement of the Problem
With these in mind, the researchers would like to ascertain the significant effects on the bacterial capability of the tannin contents of Neem tree to Escherichia coli and Staphylococcus aureus. Specifically, the researchers sought to answer the following questions: 1. Is the absorbance of the total extracted polyphenolic contents of Neem tree have significant connection with the amount of standard Tannic Acid? 2. Knowing that the total extracted polyphenolics contain Tannin, do the amount of extracted polyphenolics directly impinge on the anti-bacterial capability of Tannin? 3. To what extent of concentration will the addition of these tannin contents of Neem tree will have its maximal effect? 4. Bagging the knowledge that most of this polyphenolic contents are certainly found on the bark of the tree, will the presented isolation method of these contents provide great purity and less cost and effort?

Research Hypothesis
The researchers aim to find answers from the questions that we have found from our desire to examine the antibacterial effect of the Tannin contents of Neem tree. The following statements below are the hypotheses made by the researchers: 1. There is no significant relationship between the absorbance and the amount of tannic acid. 2. There is no significant difference between the levels of concentration of the polyphenolic contents to yield its maximum anti-bacterial performance. 3. The presented method has no significant effect on the purity of the isolated material. 4. There is no significant difference between the effects of the solvents used and it will not affect the performance of the isolated polyphenolics in the growth of the test subject.

Significance of the Study
The results of this study would be beneficial to the following: * INDIGENOUS PEOPLE: Acknowledging the fact that Philippines still have places that are not much developed, the outcome of this study will be highly preferential for them since the primary source can be found and can be propagated almost anywhere, and accounting that the majority of illnesses reported to these areas are grounds to these microorganisms, it will be a great relief for them to recognize the benefits of these plants. * RESEARCHERS AND IN MEDICAL FIELD: Exhaustive researches concerning treatment and prevention of the harmful effects of these organisms have been conducted and in present is still on process, perhaps this study might help to lessen the efforts of doing chemical treatments with tormenting the possible side effects due to possible usage of this Neem bark extract as a medicine without disturbing of possible effects for the reason that it will be classified as a herbal medicine. * COMMUNITY: The community will also be benefited from this study.

The diseases related to these intestinal inhabitants will possibly decrease; fewer diseases in the community means fewer expenses for hospitalization and more budgets for food and other daily living needs. Thus, transmission will be expected to decline and as a perspective, a healthy community is thrived. * ENVIRONMENT: Topographically, Philippines is a warm country which favors the way of living of Neem tree and since the trending for the Global Warming has been tremendously increasing, thus planting Neem will help regenerate molecular Oxygen back to its natural cycle. On the other hand, consumption of the other natural sources will decrease as well and as a perspective, a healthy society has been prospered.

Scope and Limitations of the study
Generally, the study focuses to know the anti-bacterial effects of the polyphenolic contents of Azadirachta excelsa (Philippine Neem tree) particularly tannin. The study will involve acetonic extraction of the phenolics from the bark of the Neem tree and the determination of the total tannin contents will be done using Folin-Ciocalteu method. The extraction of the phenolics from the Neem tree will be governed using acetone. The isolation of the tannin from the phenolic extracts will be done using acetone also due to the findings from numerous studies that acetone can elute tannin which unfasten it from other phenolics present. The study will also concern on declining the incident cases relating to Staphylococcus aureus and Escherichia coli for these two organisms will be the test subjects of the research. The test matter will be cultured in an agar and will be subjected for evaluation of the tannin’s anti-bacterial effect. The researchers do not aim to claim the possible outcome of this study to have medicinal value for illnesses concerning different strains of the test subjects for various experiments to transform it into medicine should be done.

Definition of Terms
* Folin-ciocateu reagent – The Folin–Ciocalteu reagent (FCR) or Folin’s phenol reagent or Folin–Denis reagent, also called the Gallic Acid Equivalence method (GAE), is a mixture of phosphomolybdate andphosphotungstate used for the colorimetric assay of phenolic and polyphenolic antioxidants. It works by measuring the amount of the substance being tested needed to inhibit the oxidation of the reagent. * Sephadex LH-20 – Sephadex LH-20 adsorbs tannin in alcohol and releases them in aqueous acetone. Its is very useful for separating tannin from non-tannin phenolics thru chromatography * Culture medium – A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells, or small plants. There are different types of media for growing different types of cells. * Nutrient broth – is a liquid formulation that does not contain agar. Nutrient broth are used as an enrichment for specified organism * Nutrient agar – Nutrient agar is a microbiological growth medium commonly used for the routine cultivation of non-fastidious bacteria. It is useful because it remains solid even at relatively high temperatures. Also, bacteria grown in nutrient agar grows on the surface, and is clearly visible as small colonies. In nutrient broth, the bacteria grow in the liquid, and are seen as a soupy substance, not as clearly distinguishable clumps.

Nutrient agar typically contains * Beef extract – Beef Extract is derived from infusion of beef and provides an undefined source of nutrients. Beef Extract is not exposed to the harsh treatment used for protein hydrolysis, so it can provide some of the nutrients lost during peptone manufacture.1 Beef Extract is a mixture of peptides and amino acids, nucleotide fractions, organic acids, minerals and some vitamins. “Its function can therefore be described as complementing the nutritive properties of peptone by contributing minerals, phosphates, energy sources and those essential factors missing from peptone.” * Peptone – are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the basis of size, typically containing fewer than 50 monomer units. The shortest peptides are dipeptides, consisting of two amino acids joined by a single peptide bond. * Pure culture – A pure culture is usually derived from a mixed culture (containing many species) by methods that separate the individual cells so that, when they multiply, each will form an individually distinct colony, which may then be used to establish new cultures with the assurance that only one type of organism will be present. Pure cultures may be more easily isolated if the growth medium of the original mixed culture favours the growth of one organism to the exclusion of others.

CHAPTER 2
REVIEW OF RELATED LITERATURES AND STUDIES

Neem Tree
Neem Tree is a tropical evergreen tree that is native and widely spread in Indian subcontinent. This tree grows in much West Africa and Southeast Asia (Valenzuela, 2007) including the Philippines. Neem tree belongs to the genus Azadirachta (family Meliaceae) with A. indica to be the best-known species. Azadirachta, since not monotypic, has other two congeneric species: the A. siamensis (Thai neem tree) and A. excelsa (Philippine Neem tree). Schmutterer and Doll’s The Marrango or Philippine Neem Tree, Azadirachta excelsa (=A. integrifoliala): A New Source of Insecticides with Growth-Regulating Properties has pointed out the differences between A. indica and A. excelsa. It was stated that “Whereas A. indica thrives in hot dry regions, A. excelsa is a plant of lowland monsoon forests in Southeast Asia and tolerates greater rainfall than A. indica.” The former normally inhabits areas with an annual 400-1000mm-precipitation.

The latter, on the other hand, requires more; and with the Philippine’s annual rainfall that ranges to as much as 5000 mm, A. excelsa thrives well especially in Palawan, Basilan, and Masbate that has found sightings of the tree in abundance. Neem Tree, according to Girish and Bhat, is the most researched trees in the world and is said to be the “most promising tree of 21st century”. This has been concluded by other researchers and has been proven by many studies. Elly Velez Lao Pamatong, Ph. D. of the Philippine Daily Inquirer even described neem as “The Tree of All Trees”. Neem to be associated with these titles has proven itself for a long time now. It has been used on ancient medication especially in India. Its benefits were listed in ancient documents ‘Charak-Samhita’ and ‘Susruta-Samhita’, which form the foundation of the Indian system of natural treatment, Ayurveda (used in Ayurvedic medicine for 4000 years).

The tree’s every part (wood, leaves, seeds, flower, fruit, roots, bark, roots, and stems) has been studied to possess biological activities such as antibacterial (Bhuiyan, et. al, 1997; Akiyama, et.al, 2001; Banso and Adeyemo, 2007; Funatogawa, et.al, Çolak, et.al, 2010), antiallergenic, antidermatic, antidermatophytes (Natarajan, et. al, 2003), antifeedent, antifungal (Niharika et. al, 2010; Mondali et al, 2008), anti-inflammatory, antipyorrhoeic, antiscabic, cardiac, diuretic, insecticidal (Ahmed and Grainge, 1985; ), larvicidal (Mustafa and Al-Khazraji, 2008), nematicidal, spermicidal, etc. Different uses of neem tree have been cited in The Neem Tree that is produced by HDRA, an organic organization of United Kingdom. In this booklet, appropriate organic techniques and technical information about neem tree planting and usage has been provided. Among the cited uses of neem tree are mosquito repellant, improve soil structure, make soil less acidic, wood is strong and is resistant to termite damage, good for shade and windbreaker, and is used to treat many health problems. Neem has high rate of photosynthesis and liberates more oxygen than many other tree species, thus purifying the atmosphere (Nigam, Mishra, and Sharma, 1994; Randhawa and Parmar, 1993).

According to a study conducted on 2006 entitled “Neem”, the temperature under the neem has been found to be ~10°C less than the surrounding temperature, during hot summer months in the northern parts of India. Neem is called ‘Sarvaroga nivarini’ meaning ‘the curer of all ailments’ because of its reputation in medicinal usage. In India, several viral diseases are treated with neem. Neem leaf paste has been used to treat small pox and warts (Girish and Bhat, 2008). Neem’s studies on effect of administration of neem solutions on cancer, diabetes, heart disease and AIDS are being carried out. Its Neem bark constituent was even considered to have the ability to suppress the growth of carcinogenic bacterium, streptococcus sobrinus which is also involved in dental plaque formation, based on the research conducted by Bhuiyan, Nishimura, Matsumura and Shimono of Okayama University Dental School. More than 135 compounds have been isolated from the different parts of neem and are classified into two major groups — isoprenoids and the non-isoprenoids.

The former include diterpenoids and triterpenoids containing protomeliacins, liminoids, azadirone, and its derivatives, genudin, and its derivatives, vilarin type of compounds and c-secomeliacins such as nimbin (the first compound to be studied), salannin, and azadirachtin. The former (non-isoprenoids) include proteins and carbohydrates, sulphorous compounds, polyphenolics (such as flavonoids and their glycosides, dihydrochalcone, coumarin and tannins), aliphatic compounds, phenolic acids, etc. (Girish and Bhat, 2008). Tannin

Within the scope of this study, tannin will be isolated from the other components of the neem bark extract and will be assayed in the bacterias Staphylococcus aureus (gram positive) and Escherichia coli (gram negative) to test its antibacterial effect. Tannins are general descriptive name for a group of polymeric phenolic substances capable of tanning leather (Banso and Adeyemo, 2007). Haslam has more recently substituted the term “polyphenol” for “tannin”, in an attempt to emphasize the multiplicity of phenolic groups of characteristic of these compounds. He notes that molecular weights as high as 20,000 have been reported, and that tannins complex not only with proteins and alkaloids but also with certain polysaccharides.

As defined by Bate-Smith, tannins are “water-soluble phenolic compound having molecular weights between 500 and 3000… [giving] the usual phenolic reactions… [and having] special properties such as the ability to precipitate alkaloids, gelatin and other proteins”. They are secondary metabolites of plants and although not part of the primary metabolism such as biosynthesis, biodegradation, and other conversions of intermediary metabolism, they do have diverse biological activities ranging from toxicity to hormonal mimicry, and may play a role in protecting plants from herbivory and disease (Hagermann, 2002). Phenolic substance can be obtained from various parts (in tree bark, wood, fruit, fruitpod, leaves and roots and in plant gall) of different plants belonging to multiple species; concentration of present tannins varies, though. In one study, it was found out that tannin is more concentrated in the cambium layer (inner bark) of the tree. In addition, an older tree has more tannin than a younger one and there is more tannin in the lower part than the upper parts. The de-barking of the tree with the desire of extracting tannin depends on the tree of source; and for this study, the neem tree. There had not yet any research conducted to how old a neem tree possesses the highest concentration of tannin content. Other studies suggested to buy shredded barks saw at saw mill if one is interested in obtaining tree barks for tannin.

In buying shredded bark saw, it must be ascertained that the purchase must not come from logs that has been left open to be soaked in the rain. Rain-soaked shredded bark is not of much use as it does not contain high tannin. Tannin is soluble in water and if left in the rain will drain out with other sap. For best results, barks are sun-dried as suggested by numerous studies and powderized to be stored for further usage. But according to the consultants of FAO/IAEA, sun-dried barks may cause oxidation of tannin to quinones leading to polymerization since the compound is photosensitive. As a recommendation, they suggest to force-heat the barks inside the oven. There are two broad classifications of tannins: the hydrolysable and the non-hydrolysable tannins. Hydrolysable tannins are esters of sugars, mainly glucose, and phenol carboxylic acids, such as gallic acid (3,4,5-trihydroxyl benzoic acid) (Hagermann, 2010), hexahydroxydiphenic acid, or its stble dilactone ellagic acid. As their name infers, hydrolysable tannins are readily degraded under hydrolytic conditions into these fundamental components. Early work on hydrolysable tannins included Haslam’s significant elucidations of the structures of the simple gallotannins (the simplest hydrolyzable tannin). More recently, Okuda et al. have been particularly active in characterization and classification of complex hydrolysable tannins.

Feldman’s synthetic work has provided useful insights into likely biosynthetic routes for the complex hydrolysable tannins. Condensed tannins or proanthocyanidins or also known as non-hydrolysable tannins are polymeric flavanoids (Hagermann, 2010). They are much more resistant to decomposition and merely yield polymers or amorphous precipitates under the influence of acids. The basic monomer of condensed tannins is (epi)cathechin, which is extended by the successive addition of similar units to form oligomers and polymers. An article from Medscape entitled “The Use of Tannic Acid in the Local Treatment of Burn Wounds: Intriguing Old and New Perspectives” statted that “the hydrolysable tannins are considered as officinal in Europe and North America.” It was even been included to older editions of many pharmacopoeias. Hydrolysable tannins are specifically referred to as “acidum tannicum” or tannic acid (British Pharmacopoeia, 1932; Deutsches Arzneibuch, 1926; Pharmacopeé Française, 1937; Pharmacopoeia of the United States of America, 1926).

It is said that hydrolysable tannins when heated releases pyrogallol which is known to be hepatoxic and has antiseptic as well as caustic properties. In 1938, Wilson, MacGregor, and Stewart were the first to report on the occurrence of liver lesions in tannic acid-treated burn patients. Post-mortem examination in a series of 33 severly burned, fatal cases revealed a characteristic degeneration and necrosis of liver cell, which was much more intense than that seen in other organs. In the mildest form, it appeared as a fatty degeneration of the epithelial cells surrounding the veins in the central zone of the hepatic lobules but could, in more advanced examples, progress to a total destruction of the central zones in which only a narrow strip surrounding each portal tract showed surviving liver cells. Several other communications were published describing liver damage in burn patients who had some form of tannic acid therapy. Buis and Hartmann confirmed the findings by Wilson, et al., and in addition, noted that the lesions in humans were identical to those found in experimentally burned animals treated with tannic acid jelly. Though none of the said authors associated the phenomena seen in burn patients with the application of tannic acid, Wells, Humphrey, and Coll in 1942, directly related the occurrence of liver damage to the tannic acid therapy. In their article in the New England Journal of Medicine, these authors described four patients who died three to five days after the injury, in the period which was generally associated with toxemia.

In agreement with previous observations, on autopsy these patients exhibited central lobular liver necrosis as the outstanding feature or as the sole cause of death. However, the assumed hepatotoxicity of tannic acid intoxication were questioned when those early publications were analyzed in retrospect and with the knowledge acquired in the successive years. From the collected data, it was concluded that the evidence yet was inconclusive. From the past studies, tannic acid preparations of ill definition and poor quality were used, often in extremely high concentration. These according to some studies were believed to have affected the outcome of those studies. Moreover, liver damage and impairment of liver function also occurred in patients who did not receive tannic acid treatment at all, and these phenomena are now considered part of the burn syndrome. Tannin’s Anti-bacterial Properties

The antimicrobial mechanism of tannins can be summarized as follows: (i.) The astringent property of the tannin may induce complexation with enzymes or substrates. Many microbial enzymes in raw culture filtrates or in purified culture are inhibited when mixed with tannin. (ii) A tannin’s toxicity may be related to its action on the membranes of the microorganisms. (iii) Complexation of metal ions by tannins may account for tannin toxicity. Chung et al. reported that the inhibitory effect of tannic acid on the growth of intestinal bacteria may be caused by its strong iron-binding capacity. Chung et al. also reported that tannic acid inhibited the growth of all 15 of the bacteria tested, but gallic acid and ellagic acid did not inhibit any of them. They concluded that the ester linkage between gallic acid and and glucose (to form tannic acid) was important to the antimicrobial potential of these compounds. Tannic acid was found to be inhibitory to the growth of of intestinal bacteria such as Bacteroides fragilis, Clostridium perfringes, Eschirichia coli, and Enterobacter cloacae. Staphylococcus aureus

S. aureus is a Gram-positive, spherical bacterium (coccus) with a diameter of 1 – 1.3 m. When viewed microscopically, S. aureus appears in clusters, like bunches of grapes. Growing in food, some strains can produce toxins which cause acute gastro-intestinal diseases if ingested. The enterotoxin produced by S. aureus is a heat-stable protein, which survives heating at 100 °C for 30 – 700 minutes. The main reservoirs of S. aureus are humans and animals. Healthy people carry the organism in their nose and throat (50 %), on their hands (5-30 %), and in wounds. S. aureus can also colonise food contact surfaces, and it can become a persistent organism in slaughterhouses. S. aureus can contaminate foods through contact with contaminated hands, materials and surfaces, but also via the air (coughing). S. aureus is a Gram-positive, enterotoxin producing organism. Together with other species, such as S. intermedius, S. hyicus and S. epidermidis, S. aureus belongs to the genus Staphylococcus. S. aureus can be distinguished from S. epidermidis by the production of the enzymes coagulase and thermonuclease.

Not only does it produce enterotoxin, which causes food poisoning when ingested, the organism also causes a number of other diseases, e.g. wound infections and blood poisoning (sepsis), toxic shock etc. On a rich medium, S. aureus forms fairly large, yellow colonies. The organism can grow both with and without oxygen (facultatively anaerobic), and is catalase-positive and oxidasenegative. Virtually all S. aureus strains produce the enzyme coagulase. Illness is caused by the toxin which S. aureus has produced in the foodstuff. In order to produce detectable levels of toxin, the number of organisms must be over 105-6 per gram of product. The time between ingestion of the toxin and the symptoms is only two to five hours and depends on the amount and type of food and the state of health of the person. With regards to the study of effects of tannin in this kind of bacteria, several have already been conducted. Akiyama, Fujii, Yamasaki, Oono, and Iwatsuki examined the antibacterial activity of several tannins on the coagulation of plasma by Staphylococcus aureus and the effect of incubating S. aureus for 24 hours in the combination of conventional chemotherapy and tannic acid below MIC (Minimum Inhibitory Concentration). Escherichia coli

Escherichia coli , or E. coli for short, is a common, rod-shaped bacterium that lives in human and animal intestines where it is present in large numbers. There are hundreds of E. coli strains, most of which are relatively harmless, some are even beneficial to humans. While most E. coli strains are harmless, one exception is the E. coli strain O157:H7. This particular strain is a pathogen which produces a powerful toxin often referred to as Shiga toxin or verotoxin, which can cause severe illness. E. coli thrive in warm, wet, dark places that are rich in nutrients, such as human and animal intestinal tracts. Once excreted, the bacteria cannot survive the harsh conditions of the outside world; however, some manage to find their way into lakes and streams, or another host. Once in water or sediment, E. coli can persist for several weeks. Current research suggests that E. coli can survive, grow, and persist in moist beach sand. Some common sources of E. coli are runoff from developments, direct release of untreated sewage, leakage from sewage pipes, and droppings from sea gulls, waterfowl, and pets.

Storm sewers can carry dog and cat feces off sidewalks or streets directly into streams. Improperly maintained septic systems may release pathogens into groundwater. Cattle, domesticated animals, wild animals, and birds directly release feces into streams and lakes. Escherichia coli is one of the most intensively studied living species (Elena et al, 2005). While it has long served as a model organism for biochemistry, genetics, and molecular biology, more recently, it has been widely used in experimental studies of evolution. E. coli is a normal part of the microbiota of the lower gastrointestinal tract of mammals, including humans, and usually exists as a harmless commensal. However, there also exist many pathogenic strains of E. coli that can cause a variety of diarrheal and other diseases in humans and animals. These pathogenic strains express virulence factors that are involved in pathogenesis, but which are usually accessory to normal metabolic functions.

CHAPTER 3
METHODOLOGY

The chemical methods used in this research were gathered from three sources: first is from the joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. A joint FAO/IAEA Coordinated Research Project (CRP) on the “Use of Nuclear and Related Techniques to Develop Simple Tannin Assays for Predicting and Improving the Safety and Efficiency of Feeding Ruminants on Tanniniferous Tree Foliage” has been done. In order to strengthen this research project, consultants were called to define the analytical methods to be used. The core purpose of this source was to provide the researchers with the chemical method to be used in quantifying the tannins present in the sample. The second source was the Tannin Handbook prepared by Dr. Ann Hagerman; this handbook was originally published for the use in the Hagerman laboratory.

Unfortunately, the original printed Tannin Handbook was no longer available but all its contents were posted at the site present in the references of this study. The purpose of this source for the researchers was to endow them with knowledge on how to isolate and purify their tannins being studied. The last source was the Laboratory Manual in General Microbiology authored by Faculty Members of the Department of Biology of the Polytechnic University of the Philippines. It provides the researchers with knowledge on how to culture and inoculate different types of bacteria which has a big part in their study.

Research Design
Generally, the method used by the researchers in this study is the Experimental method. Travers (1978) says of the scientific experiment that it has become the most prestigious method of advancing scientific knowledge. Gay (1976) thinks that this method is the only method of research which can truly test hypotheses concerning cause-and-effect relationship. He says further that the experimental method represents the most valid approach to the solution of problems, both practical and theoretical. Ary, et al. (1972) added that an experiment is generally regarded as the most sophisticated research method for testing hypothesis. The experimental method is defined as the one which represents Mill’s principle of research known as the method of difference. This only means that the effect of a single variable applied to one situation can be assessed and the difference is determined (Mill, 1872 in Good, et al. 1935). In this study, the experiments contain only two groups, an experimental group and a control group. The experimental group receives the treatment under investigation while the control group receives a different treatment or the usual method it was using before. Data Gathering Procedure

1. Preparation of Plant Extract
The general problem in the study of natural plant products is that their nature and amount are dependent on various factors, which must be controlled as far as possible. One of these factors is stress; the metabolic state of the plant may change when it is stressed in any manner. This can be a problem before as well as after harvesting a plant part for analysis. As cells die, the cellular integrity is lost and as a result the enzymes come in contact with substrates to which they are not normally exposed in living cells. In addition, it also increases the oxidation process, which is a problem with phenolics, most especially in tannins since these are prone to oxidation. If a plant is cut and dried under near ambient conditions, which generally requires a large time to dry, the nature and content of phenolic compounds can change. In order to avoid these changes, the metabolic activities of the cells need to be curbed immediately. 1.1 Collection, drying and storage of plant material

Bark age and its stage of development affect levels and nature of phenolics. When the collection site is close to the laboratory, the material can be transported to the laboratory in fresh state. The fresh material should be kept on ice and transported under dark conditions. Transportation of large amount of barks in plastic bags should be avoided, since temperature in the bag could rise leading to sweating and wilting which can change the nature and level of phenolics. If liquid nitrogen is available, the better option is to freeze the sample and then freeze-dry the material without thawing it. Thawing can rupture cell membranes leading to changes in phenolics. If the material is frozen using a freezer, make sure that the material is not thawed during transport. Solid carbon dioxide should be used to transport such material.

Once the material is dried, it should be kept in a dry place, preferably in a desiccator in the dark. The freeze-dried material generally is hygroscopic. Light is also known to change the nature of phenolics. After freeze-drying, the cell structure is broken and the enzymes are in the native state. With the absorption of water, enzymes and phenolics can react, which can produce drastic changes in phenolics. The freeze-drying, though considered to be one of the safest method for preservation of phenolics, can lead to drastic changes if the storage conditions are not appropriate. If a lyophilizer is not available, the plant material has to be dried under far from ideal conditions. The sample can be dried at about 50–52°C using a forced air oven. This will hasten the process of drying, and the enzymes present in the plant sample will not have much time to react with phenolics. Drying at temperatures higher than 55°C should be avoided, since it can lead to inactivation of phenolics or could decrease their extractability in solvents and affect the quantification. 1.2 Grinding of sample

Fresh or frozen materials are difficult to work with. Grinding could be a problem using these materials. Fresh material, when frozen using liquid nitrogen, can be ground using ‘Polytron’ homogenizers. One has to be cautious that the temperature does not rise during homogenization; increase in temperature can lead to enzymatic changes in phenolics. Phenolics are generally extracted in aqueous organic solvents. The moisture present in the fresh material needs to be taken into account while preparing organic solvents for extraction. It is suggested to grind the sample after drying it. About 500 g of the plant material should be ground first to pass a 2 mm screen. If the ground samples in a desiccator are kept in a refrigerator, the desiccator must be opened after the contents has reached the ambient temperature, otherwise moisture will condense on the sample which will lead to changes in the state of phenolics during storage.

1.3 Extraction of Phenolics
The aim is to quantitatively diffuse phenolics present in the plant material to liquid phase. For the extraction process, a suitable solvent is required. Generally, aqueous acetone (70%) is a popular choice. It has been reported by various workers to be better in extracting phenolics from plant materials. Dried (finely ground) plant material (20 g) is taken in a glass flasks of approximately 250 ml capacity. 160 mL of aqueous acetone (70%) is added and the flask is suspended in an ultrasonic water bath and subjected to ultrasonic treatment for 20 min at room temperature. The contents of the flasks is then transferred to centrifuge tubes and subjected to centrifugation for 10 min at approximately 3000g at 4°C (if refrigerated centrifuge is not available, cool the contents by keeping the centrifuge tube on ice and then centrifuge at 3000g using an ordinary clinical centrifuge). Collect the supernatant and keep it on ice. Transfer the pellet left in the centrifuge tube to the beaker using two portions of 5 ml each of 70% aqueous acetone and again subjects the contents to ultrasonic treatment for 20 min.

Centrifuge and collect supernatant as described above. 2. Measurement of Total Phenolics and Tannins using the Folin-Ciocalteu Method According to Makkar et al, the method for total phenol is useful in order to know the efficiency of extraction of phenolics in solvents. The results can be expressed as tannic acid equivalent. The nature of tannic acid varies from one commercial source to the other. Tannic acid from Merck was found to be the best.

2.1 Reagents
* Folin-Ciocalteu reagent (1 N): Dilute commercially available Folin-Ciocalteu reagent (2 N) with an equal volume of distilled water. Transfer it in a brown bottle and store in a refrigerator (4°C). It should be golden in color. Do not use it if it turns olive green. * Sodium carbonate (20%): Weigh 40 g sodium carbonate, dissolve it in about 150 ml distilled water and make up to 200 ml with distilled water. * Standard tannic acid solution (0.1 mg/ml): Dissolve 25 mg tannic acid (TA) obtained from Merck in 25 ml distilled water and then dilute 1:10 in distilled water (always use a freshly prepared solution).

2.2 Preparation of Calibration Curve
Take different aliquots of the standard Tannic Acid solution (0.00, 0.02, 0.04, 0.06, 0.08, 0.10 mL) in test tubes, make up the volume to 0.5 mL with distilled water. Add 0.25 mL of Folin-Ciocalteu reagent and then 1.25 mL of Sodium Carbonate solution. Vortex the tubes for 40 minutes then record the absorbance at 725 nm. Plot the amount of Tannic Acid in µg as a function of the absorbance at 725 nm. Calculate the slope and the intercept and obtain the equation of the line. 2.3 Analysis of Total Phenolics

Take suitable aliquots of tannin-containing extract, initially 0.02, 0.05 and 0.1 mL in test tubes, make up the volume to 0.5 mL with distilled water then add 0.25 mL of the Folin-Ciocalteu reagent. Add 1.25 mL of Sodium Carbonate solution. Vortex the tubes for 40 minutes and measure its absorption at 725 nm. Calculate the amount of total phenolics as Tannic Acid equivalent from the calibration curve. 3. Isolation of Tannin

Sephadex LH-20 is a liquid chromatography medium designed for molecular sizing of natural products; it is a beaded cross-linked dextran that has been hydroxypropylated to yield a chromatography medium with both hydrophilic and lipophilic character. Tannins adsorb to Sephadex LH-20 in alcohol while small phenolics elute from the material. Tannins can then be eluted with aqueous acetone. Chromatography on Sephadex LH-20 is very useful for separating Tannin from non-tannin phenolics. 3.1 Reagents

* 95 % Ethanol
* 70% Acetone
* Sephadex LH-20
* Column (5 x 40 cm)
* Fraction Collector
* Continuous UV monitor (useful for monitoring the eluate, but not essential) 3.2 Procedure
Equilibrate the Sephadex LH-20 according the manufacturer’s direction in ethanol. Since the isolation requires a column, wash it with several bed volumes of ethanol after packing the column. Plant extracts of different volumes are applied to the column after removing all acetone from the sample through rotary evaporation. Elute with ethanol at a rate of 1 mL/min, if UV monitor is available, elute until the absorbance at 280 nm is no longer changing and is near the baseline. If no UV monitor is available, use 1 liter of Ethanol to elute the sample. Then elute the column with 70% acetone, tannins are usually seen as brown band of pigments. This eluate is not monitored in the UV because of the strong absorption of acetone. Continue washing until the beads return to their original color. Combine all tannin fractions and use rotary evaporator to remove all solvents. The tannin collected should be placed in vials and stored in dark freezers. 4. Preparation of Culture Media

Generalized and specialized media are required for the growth of bacteria. Culture media is an artificial soil that contains nutritional and environmental requirements for the nourishment and reproduction of microorganisms. The media that are used in laboratories to culture bacteria are referred to as artificial media or synthetic media, because they do not occur naturally, rather, they are prepared in the laboratory. 4.1 Reagents

* Agar: a complex carbohydrate extracted from marine algae that solidifies below the temperature of 45oC. * Beef Extract
* Peptone

4.2 Preparation of Culture Media
4.2.1 Nutrient Broth
Dissolve 3.0 grams of beef extract and 5.0 grams of peptone in 1000 mL of distilled water and mix thoroughly. Dispense 10 mL of the prepared Nutrient Broth in clean test tubes and put a cotton plug right away. 4.2.2 Nutrient Agar

Dissolve 3.0 grams of beef extract, 5.0 grams of peptone and 15.0 grams of agar powder in 1000 mL of distilled water. Mixing should be done over an ordinary water bath. Heat the solution over a flame source and boil until all solids are dissolved. Transfer the solution to smaller containers, such as Erlenmeyer flasks and cover it with cotton plugs. 4.2.3 Sterilization

Place all the prepared Nutrient Broth and Nutrient Agar in an autoclave. Also put cotton swabs, forceps, Petri dishes and punched filter papers which will be used for the next experiments. Sterilize the apparatuses at 15 psi and 121oC for 15 minutes. 4.3 Culturing the Bacteria

Pure cultures of both Escherichia coli and Staphylococcus aureus are purchased from the National Science Research institute (NSRI) which is residing at the compound of the University of the Philippines Diliman. Perform sub-culturing by transferring a loop full of the pure culture of each organism to different sterile Nutrient Broth with the use of an inoculating loop aseptically. Set aside the cultures and let them multiply for a day. 4.4 Dispensing the Culture Medium

Sterile Nutrient Agar is transferred to sets of sterile Petri dishes aseptically. Wait until the plates solidify, and prepare them for inoculation. 5. Inoculation to the Culture Media
Inoculation is the process for adding specimen to the culture medium. Cultures from this study are obtained from a plated medium. 5.1 Swab Method cDip a sterile cotton swab in a Nutrient broth containing Escherichia coli. Remove excess inoculums by lightly pressing the swab against the tube at a level above that of the Nutrient broth. Inoculate the Agar plate by streaking with the swab containing the inoculums. Rotate the plate by 60o and repeat the streaking using the same swab. Repeat the rotating and streaking procedure five times to ensure an even distribution of the inoculums. Allow the surface of the culture to dry for seven minutes to allow the absorption of the excess moisture, repeat the procedure for four other plates with the use of other sets of cotton swabs. Do the same procedure for Staphylococcus aureus.

6. Anti-bacterial Test of the Isolated Tannin to the Cultured Bacteria via the Filter Paper Method. According to Engelkirk (2011), an antibacterial agent will be accepted if it inhibits or destroys the pathogen without destroying the host. To accomplish this, the agent must target a metabolic process or structures possessed by the pathogen but not by the host. 6.1 Reagents

* Acetonic Tannin: Dissolve equal mass of the isolated Tannin with different volumes of 70% Acetone (blank, 1.0, 2.0, 3.0, 4.0 and 5.0 mL). * 95% Ethanol: Used for flame sterilization of the forceps 6.2 Procedure

Immediately after allowing the plated cultures to absorb the moisture, prepare them for the anti-bacterial test. Dip the sterile filter paper halfway in the Acetonic Tannin of different concentrations with the help of a flame sterilized forceps. Place the filter papers at each of the center of the five plated cultures. Incubate the plates invertedly at 37oC until the next two days and measure the diameter of the zone of clearance if there is any of it formed. The Minimum Bactericidal Concentration (MBC) of the Acetonic Tannin based on this study can also be determined by the culture giving the smallest zone of clearance or none at all. Statistical Tool

Two statistical treatments were used in this study and those were the Pearson Product Moment Correlation Coefficient and One-Way Analysis of Variance (ANOVA). Correlation Analysis is concerned with the relationship in the changes of the given variables; the relationship can be computed and shown in a scatter Diagram (Cruz, et al. 2009). Utzurrum, et al. said that the most widely used computational formula for correlation is the Pearson Product Moment Correlation Coefficient. In computing for the Pearson’s r, there are two basic assumptions; the presence of a linear relationship and the interval ratio level of measurement of data. The formula for Pearson’s r is: r=NXY-XYNX2-X2[NY2-Y2]

Where:
X= data for independent variable
Y= data for dependent variable
N= sample size
r= degree of relationship between X and Y
To test for the significance of r, t-test is used. The formulas for this test are: df=N-2
tcalc=rN-21-r2
Decision rule; the researchers assumed a level of significance of 0.05. If tcalc > ttab, reject Ho, if tcalc < ttab, accept Ho. ANOVA is a method for partitioning the variation observed in experimental data into different parts, each part attributable to a known source. This statistical test of significance is employed when three or more groups are involved and when the variable measured is of the ratio or interval type. One-way ANOVA for short is an F-test. It assumes that the deviation of a measurement from the population mean is made up of two components. These components are the deviation of a measurement from the mean of the group where it belongs and the deviation of the group mean from the population (Sevilla, et al. 2007). The formulas used for One-way ANOVA are:

SSBT=i=AC(XA2+XB2+XC2)r-XT2N
SSWT=i=ACXA12+XA22+…XC32-(XA2+XB2+XC2)r
SST=SSBT+SSWT
DFBT=t-1
DFWT=tr-1
DFT=DFBT+DFWT
MSBT=SSBTDFBT
MSWT=SSWTDFWT
Fcalc=MSBTMSWT
Where:
SS= sum of the squares
BT= between treatments
WT= within treatments
DF= degrees of freedom
MS= mean squared
X= given data
r= number of replicates
t= number of treatments
Decision rule; the researchers assumed a level of significance of 0.05. If Fcalc > Ftab, reject Ho, if Fcalc < Ftab, accept Ho.

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