Herbal medical specialties have been portion of the clever manner of life in the society since the earlier times. Crude work forces have been utilizing this sort of medical specialty as the primary method of medicine without undergoing different industrial procedures. Herbal medical specialties have truly come a long journey manner back to the ancient times of Herbalism. One of the considered innovators in Herbal medical field was Imhotep. a priest-physician of the ancient Egypt. doctor of Marcus Aurelius and Paracelsus. who have a broad array of cognition in Herbalism and made a record of the utilizations of these workss.
In line of herbal applications. Ayurveda. the traditional medical specialty of India which is developed more than 5000 old ages ago. is believed to be the medical specialty of Hindu Gods. Ayurveda is based on the belief that our organic structure is a balance and we get badly when this balance was disturbed. Consequently the five elements that Hindu’s believe to keep this alleged balance are viz. Smell. Taste. Sight. Touch and Hearing must continue its equilibrium. The first two books of Ayurveda ; Caraka Samhita and Susruta Samhita include a 1000 of mentions in herbal medical specialties.
Phytotherapy has been introduced by Gallic for the use of Herbs as medical specialties and is internationally recognized and used. To edifice a valid footing on Herbal effectivity. there are two facets that should be measured ; internal and external cogency. Internal cogency includes dependable and accurate consequences relationship with the hypothesis under controlled conditions which satisfy the scientific community from clip to clip while on the other manus. External cogency refers to the pertinence of the method either beyond or under the experimental conditions. Part of guaranting the societal value of research includes fashioning and implementing sound scientific discipline. Although international collaborative research on herbal medical specialty is no exclusion. discoursing scientific cogency as an ethical demand raises some specific challenges. including the significance of scientific cogency. set uping inclusion and exclusion standards. utilizing appropriate result steps. and finding appropriate survey designs.
The Neem tree ( Azadirachta excelsa ) is a tropical green tree that is common in the Philippines. Indonesia and other states in the Southeast Asia. The other species of Neem that has been reported is the Azadirachta indica which is believed to arise and is native in Indian subcontinent. Recently. studies have been tallied that approximately 72 states worldwide have this alleged “Miracle plant” . The Neem tree is a member of the Mahogany household holding the systematic places of:
Order: RutalesFamily: RutinaeSubfamily: MeliodeaeTribe: MelieaeGenus: AzadirachtaSpeciess: Indica or ExcelsaApproximately. there is an estimation of 25 million trees turning all over India. Neem has become the “India’s best kept secret” for a really long clip that Indians benefit from about all of its parts. Neem can be grown in countries which have 40cm to 150cm of rain yearly and can last at an uneven temperature of every bit high as 44oC to every bit low as 4oC.
Harmonizing to Indians. Amrita ( the elixir of life in Indian mythology ) was ascented into Eden and few beads fell on the Neem tree. Neem was foremost acknowledged for its healing capablenesss by the inhabitants of India and Southeast Asia every bit early as 4000 B. C. The Vedas called Neem “Sarva Roga Nivarini” . which means “one that cures all complaints and ills” . During the last 30 old ages of publication studies on the belongingss of Azadirachta. they overlooked another species that is normally found in Southeast Asia viz. the “Azadirachta excelsa” .
The subdivisions of Azadirachta excelsa are sidelong uplifting and distributing. In the immature trees. the bole is cylindrical from the base. but old trees show monolithic buttresses over the adult male roots. The bark colour ranges from pinkish grey to blanch grey depending on the age. The tree distribution on the Philippines is chiefly even on Palawan. Unlike Azadirachta indica. excelsa requires more rainfall.
Both Indica and Excelsa shows unaccountable capablenesss of bring arounding diseases. unwellnesss and including insecticidal consequence. many surveies about Neem tree have been conducted and used the infusions from different parts giving positive consequences. Spraies from the leaf infusion of Neem can be use as an insect repellent. For adult females. Neem can be usage for hygienic intents. It has been besides used to confabulate wellness issues like abrasions. dandruffs. dry tegument. caput lice. malaria and even psoriasis.
The Neem bark contains Nimbin. Nimbinin. Nimbidin. Nimbosterol. indispensable oil and tannic acid. Tannin is a water-soluble polyphenol normally located at the bark of the woody and herbaceous trees. Two types of tannic acids are known viz. the hydrolysable and the non-hydrolyzable or condensed tannic acids. Hydrolysable tannic acid. are derived functions of Gallic acid ( 3. 4. 5-trihydroxyl benzoic acid ) . Gallic acid is esterified to a nucleus polyol and the galloyl groups may be farther esterified or oxidatively crosslinked to give more complex hydrolysable tannic acids. The simplest hydrolysable tannic acids. the gallotannins. are simple polygalloyl esters of glucose which have many isomers.
Tannins are considered as “secondary compounds” . A secondary compound does non include in biogenesis. biodegradation or any metabolic procedures but plays a critical function in toxicity or protection. Traditional usage of tannic acids as agents for change overing carnal fells to leather ( besides known as tanning ) is one of the manifestation activities of tannic acids. Besides tannins can precipitate proteins including those that are found on tegument. Recent surveies show besides the ability of tannic acid to command the growing of different micro-organism. Another is the ability of the tannic acid in minimum concentration to be used as fungicide for dermophytes like Pityrosporum ovale.
Statement of the ProblemWith these in head. the research workers would wish to determine the important effects on the bacterial capableness of the tannic acid contents of Neem tree to Escherichia coli and Staphylococcus aureus. Specifically. the research workers sought to reply the undermentioned inquiries: 1. Is the optical density of the sum extracted polyphenolic contents of Neem tree have important connexion with the sum of standard Tannic Acid? 2. Knowing that the sum extracted polyphenolics contain Tannin. make the sum of extracted polyphenolics straight impinge on the anti-bacterial capableness of Tannin? 3. To what extent of concentration will the add-on of these tannin contents of Neem tree will hold its maximum consequence? 4. Baging the cognition that most of this polyphenolic contents are surely found on the bark of the tree. will the presented isolation method of these contents provide great pureness and less cost and attempt?
Research HypothesisThe research workers aim to happen replies from the inquiries that we have found from our desire to analyze the antibacterial consequence of the Tannin contents of Neem tree. The undermentioned statements below are the hypotheses made by the research workers: 1. There is no important relationship between the optical density and the sum of tannic acid. 2. There is no important difference between the degrees of concentration of the polyphenolic contents to give its maximal anti-bacterial public presentation. 3. The presented method has no important consequence on the pureness of the stray stuff. 4. There is no important difference between the effects of the dissolvers used and it will non impact the public presentation of the stray polyphenolics in the growing of the trial topic.
Significance of the StudyThe consequences of this survey would be good to the followers: * INDIGENOUS Peoples: Admiting the fact that Philippines still hold topographic points that are non much developed. the result of this survey will be extremely discriminatory for them since the primary beginning can be found and can be propagated about anyplace. and accounting that the bulk of unwellnesss reported to these countries are evidences to these micro-organisms. it will be a great alleviation for them to acknowledge the benefits of these workss. * RESEARCHERS AND IN MEDICAL FIELD: Exhaustive researches refering intervention and bar of the harmful effects of these beings have been conducted and in nowadays is still on procedure. possibly this survey might assist to decrease the attempts of making chemical interventions with torturing the possible side effects due to possible use of this Neem bark infusion as a medical specialty without upseting of possible effects for the ground that it will be classified as a herbal medical specialty. * COMMUNITY: The community will besides be benefited from this survey.
The diseases related to these enteric dwellers will perchance diminish ; fewer diseases in the community means fewer disbursals for hospitalization and more budgets for nutrient and other day-to-day life demands. Therefore. transmittal will be expected to worsen and as a position. a healthy community is thrived. * Environment: Topographically. Philippines is a warm state which favors the manner of life of Neem tree and since the trending for the Global Warming has been enormously increasing. therefore seting Neem will assist renew molecular Oxygen back to its natural rhythm. On the other manus. ingestion of the other natural beginnings will diminish every bit good and as a position. a healthy society has been prospered.
Scope and Restrictions of the surveyBy and large. the survey focuses to cognize the anti-bacterial effects of the polyphenolic contents of Azadirachta excelsa ( Philippine Neem tree ) peculiarly tannic acid. The survey will affect acetonic extraction of the phenoplasts from the bark of the Neem tree and the finding of the entire tannic acid contents will be done utilizing Folin-Ciocalteu method. The extraction of the phenoplasts from the Neem tree will be governed utilizing propanone. The isolation of the tannic acid from the phenolic infusions will be done utilizing propanone besides due to the findings from legion surveies that propanone can elute tannic acid which unfasten it from other phenoplasts present. The survey will besides concern on worsening the incident instances associating to Staphylococcus aureus and Escherichia coli for these two beings will be the trial topics of the research. The trial affair will be cultured in an agar and will be subjected for rating of the tannin’s anti-bacterial consequence. The research workers do non take to claim the possible result of this survey to hold medicative value for unwellnesss refering different strains of the trial topics for assorted experiments to transform it into medical specialty should be done.
Definition of Footings* Folin-ciocateu reagent – The Folin–Ciocalteu reagent ( FCR ) or Folin’s phenol reagent or Folin–Denis reagent. besides called the Gallic Acid Equivalence method ( GAE ) . is a mixture of phosphomolybdate andphosphotungstate used for the colorimetric check of phenolic and polyphenolic antioxidants. It works by mensurating the sum of the substance being tested needed to suppress the oxidization of the reagent. * Sephadex LH-20 – Sephadex LH-20 adsorbs tannic acid in intoxicant and releases them in aqueous propanone. Its is really utile for dividing tannic acid from non-tannin phenoplasts thru chromatography * Culture medium – A growing medium or civilization medium is a liquid or gel designed to back up the growing of micro-organisms or cells. or little workss. There are different types of media for turning different types of cells. * Nutrient stock – is a liquid preparation that does non incorporate agar. Nutrient stock are used as an enrichment for specified being * Nutrient agar – Nutrient agar is a microbiological growing medium normally used for the everyday cultivation of non-fastidious bacteriums. It is utile because it remains solid even at comparatively high temperatures. Besides. bacteriums grown in alimentary agar grows on the surface. and is clearly seeable as little settlements. In alimentary stock. the bacterium grow in the liquid. and are seen as a soupy substance. non as clearly distinguishable bunchs.
Food agar typically contains * Beef infusion – Beef Extract is derived from extract of beef and provides an vague beginning of foods. Beef Extract is non exposed to the rough intervention used for protein hydrolysis. so it can supply some of the foods lost during peptone industry. 1 Beef Extract is a mixture of peptides and aminic acids. nucleotide fractions. organic acids. minerals and some vitamins. “Its map can hence be described as complementing the alimentary belongingss of peptone by lending minerals. phosphates. energy beginnings and those indispensable factors losing from peptone. ” * Peptone – are short polymers of amino acid monomers linked by peptide bonds. They are distinguished from proteins on the footing of size. typically incorporating fewer than 50 monomer units. The shortest peptides are dipeptides. dwelling of two aminic acids joined by a individual peptide bond. * Pure civilization – A pure civilization is normally derived from a assorted civilization ( incorporating many species ) by methods that separate the single cells so that. when they multiply. each will organize an separately distinguishable settlement. which may so be used to set up new civilizations with the confidence that merely one type of being will be present. Pure civilizations may be more easy isolated if the growing medium of the original assorted civilization favours the growing of one being to the exclusion of others.
Chapter 2REVIEW OF RELATED LITERATURES AND STUDIES
Neem TreeNeem 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 ( household Meliaceae ) with A. indica to be the best-known species. Azadirachta. since non monotypic. has other two congenerous 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 parts. A. excelsa is a works of lowland monsoon woods in Southeast Asia and tolerates greater rainfall than A. indica. ” The former usually inhabits countries with an one-year 400-1000mm-precipitation.
The latter. on the other manus. requires more ; and with the Philippine’s one-year rainfall that ranges to every bit much as 5000 millimeter. A. excelsa thrives good particularly in Palawan. Basilan. and Masbate that has found sightings of the tree in copiousness. Neem Tree. harmonizing to Girish and Bhat. is the most researched trees in the universe and is said to be the “most promising tree of 21st century” . This has been concluded by other research workers and has been proven by many surveies. Elly Velez Lao Pamatong. Ph. D. of the Filipino Daily Inquirer even described neem as “The Tree of All Trees” . Neem to be associated with these rubrics has proven itself for a long clip now. It has been used on ancient medicine particularly in India. Its benefits were listed in ancient paperss ‘Charak-Samhita’ and ‘Susruta-Samhita’ . which form the foundation of the Indian system of natural intervention. Ayurveda ( used in Ayurvedic medical specialty for 4000 old ages ) .
The tree’s every portion ( wood. foliages. 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. Colak. et. Al. 2010 ) . antiallergenic. antidermatic. antidermatophytes ( Natarajan. et. Al. 2003 ) . antifeedent. fungicide ( 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 utilizations of neem tree have been cited in The Neem Tree that is produced by HDRA. an organic organisation of United Kingdom. In this brochure. appropriate organic techniques and proficient information about neem tree planting and use has been provided. Among the cited utilizations of neem tree are mosquito repellent. better dirt construction. do dirt less acidic. wood is strong and is immune to termite harm. good for shadiness and parka. and is used to handle many wellness jobs. Neem has high rate of photosynthesis and liberates more O than many other tree species. therefore sublimating the ambiance ( Nigam. Mishra. and Sharma. 1994 ; Randhawa and Parmar. 1993 ) .
Harmonizing to a survey conducted on 2006 entitled “Neem” . the temperature under the nim tree has been found to be ~10°C less than the encompassing temperature. during hot summer months in the northern parts of India. Neem is called ‘Sarvaroga nivarini’ intending ‘the curer of all ailments’ because of its repute in medicative use. In India. several viral diseases are treated with nim tree. Neem leaf paste has been used to handle little syphiliss and warts ( Girish and Bhat. 2008 ) . Neem’s surveies on consequence of disposal of neem solutions on malignant neoplastic disease. diabetes. bosom disease and AIDS are being carried out. Its Neem bark component was even considered to hold the ability to stamp down the growing of carcinogenic bacteria. streptococci sobrinus which is besides 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 nim tree and are classified into two major groups — isoprenoids and the non-isoprenoids.
The former include diterpenoids and triterpenoids incorporating protomeliacins. liminoids. azadirone. and its derived functions. genudin. and its derived functions. vilarin type of compounds and c-secomeliacins such as nimbin ( the foremost compound to be studied ) . salannin. and azadirachtin. The former ( non-isoprenoids ) include proteins and saccharides. sulphorous compounds. polyphenolics ( such as flavonoids and their glycosides. dihydrochalcone. coumarin and tannic acids ) . aliphatic compounds. phenolic acids. etc. ( Girish and Bhat. 2008 ) . Tannin
Within the range of this survey. tannic acid will be isolated from the other constituents of the neem bark infusion and will be assayed in the bacterium Staphylococcus aureus ( gram positive ) and Escherichia coli ( gram negative ) to prove its antibacterial consequence. Tannins are general descriptive name for a group of polymeric phenolic substances capable of tanning leather ( Banso and Adeyemo. 2007 ) . Haslam has more late substituted the term “polyphenol” for “tannin” . in an effort to stress the multiplicity of phenolic groups of feature of these compounds. He notes that molecular weights every bit high as 20. 000 have been reported. and that tannic acids complex non merely with proteins and alkaloids but besides with certain polyoses.
As defined by Bate-Smith. tannic acids are “water-soluble phenolic compound holding molecular weights between 500 and 3000… [ giving ] the usual phenolic reactions… [ and holding ] particular belongingss such as the ability to precipitate alkaloids. gelatin and other proteins” . They are secondary metabolites of workss and although non portion of the primary metamorphosis such as biogenesis. biodegradation. and other transitions of intermediary metamorphosis. they do hold diverse biological activities runing from toxicity to hormonal apery. and may play a function in protecting workss from herbivory and disease ( Hagermann. 2002 ) . Phenolic substance can be obtained from assorted parts ( in tree bark. wood. fruit. fruitpod. foliages and roots and in works saddle sore ) of different workss belonging to multiple species ; concentration of present tannic acids varies. though. In one survey. it was found out that tannic acid is more concentrated in the cambium bed ( interior bark ) of the tree. In add-on. an older tree has more tannic acid than a younger one and there is more tannic acid in the lower portion than the upper parts. The de-barking of the tree with the desire of pull outing tannin depends on the tree of beginning ; and for this survey. the neem tree. There had non yet any research conducted to how old a neem tree possesses the highest concentration of tannic acid content. Other surveies suggested to purchase chopped barks saw at proverb factory if one is interested in obtaining tree barks for tannic acid.
In purchasing chopped bark proverb. it must be ascertained that the purchase must non come from logs that has been left unfastened to be soaked in the rain. Rain-soaked chopped bark is non of much usage as it does non incorporate high tannic acid. Tannin is soluble in H2O and if left in the rain will run out out with other sap. For best consequences. barks are sundried as suggested by legion surveies and powderized to be stored for farther use. But harmonizing to the advisers of FAO/IAEA. sundried barks may do oxidization of tannic acid to quinones taking to polymerisation since the compound is light-sensitive. As a recommendation. they suggest to force-heat the barks inside the oven. There are two wide categorizations of tannic acids: the hydrolysable and the non-hydrolysable tannic acids. Hydrolysable tannic acids are esters of sugars. chiefly 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 tannic acids are readily degraded under hydrolytic conditions into these cardinal constituents. Early work on hydrolysable tannic acids included Haslam’s important elucidations of the constructions of the simple gallotannins ( the simplest hydrolyzable tannic acid ) . More late. Okuda et Al. have been peculiarly active in word picture and categorization of complex hydrolysable tannic acids.
Feldman’s man-made work has provided utile penetrations into likely biosynthetic paths for the complex hydrolysable tannic acids. Condensed tannic acids or proanthocyanidins or besides known as non-hydrolysable tannic acids are polymeric flavanoids ( Hagermann. 2010 ) . They are much more immune to decomposition and simply yield polymers or formless precipitates under the influence of acids. The basic monomer of condensed tannic acids is ( Eysenck Personality Inventory ) cathechin. which is extended by the consecutive add-on of similar units to organize 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 tannic acids are considered as officinal in Europe and North America. ” It was even been included to older editions of many pharmacopoeias. Hydrolysable tannic acids are specifically referred to as “acidum tannicum” or tannic acid ( British Pharmacopoeia. 1932 ; Deutsches Arzneibuch. 1926 ; Pharmacopee Francaise. 1937 ; Pharmacopoeia of the United States of America. 1926 ) .
It is said that hydrolysable tannic acids when heated releases pyrogallic acid which is known to be hepatoxic and has antiseptic every bit good as acerb belongingss. In 1938. Wilson. MacGregor. and Stewart were the first to describe on the happening of liver lesions in tannic acid-treated burn patients. Post-mortem scrutiny in a series of 33 severly burned. fatal instances revealed a characteristic devolution and mortification of liver cell. which was much more intense than that seen in other variety meats. In the mildest signifier. it appeared as a fatty devolution of the epithelial cells environing the venas in the cardinal zone of the hepatic lobules but could. in more advanced illustrations. advancement to a entire devastation of the cardinal zones in which merely a narrow strip environing each portal piece of land showed lasting liver cells. Several other communications were published depicting liver harm in burn patients who had some signifier of tannic acid therapy. Buis and Hartmann confirmed the findings by Wilson. et Al. . and in add-on. noted that the lesions in worlds were indistinguishable to those found in by experimentation burned animate beings treated with tannic acid jelly. Though none of the said writers associated the phenomena seen in burn patients with the application of tannic acid. Wells. Humphrey. and Coll in 1942. straight related the happening of liver harm to the tannic acid therapy. In their article in the New England Journal of Medicine. these writers described four patients who died three to five yearss after the hurt. in the period which was by and large associated with toxaemia of pregnancy.
In understanding with old observations. on autopsy these patients exhibited cardinal lobular liver mortification as the outstanding characteristic or as the exclusive cause of decease. However. the false hepatotoxicity of tannic acerb poisoning were questioned when those early publications were analyzed in retrospect and with the cognition acquired in the consecutive old ages. From the collected information. it was concluded that the grounds yet was inconclusive. From the past surveies. tannic acerb readyings of sick definition and hapless quality were used. frequently in highly high concentration. These harmonizing to some surveies were believed to hold affected the result of those surveies. Furthermore. liver harm and damage of liver map besides occurred in patients who did non have tannic acid intervention at all. and these phenomena are now considered portion of the burn syndrome. Tannin’s Anti-bacterial Properties
The antimicrobic mechanism of tannic acids can be summarized as follows: ( i. ) The acerb belongings of the tannic acid may bring on complexation with enzymes or substrates. Many microbic enzymes in natural civilization filtrates or in purified civilization are inhibited when assorted with tannic acid. ( two ) A tannin’s toxicity may be related to its action on the membranes of the micro-organism. ( three ) Complexation of metal ions by tannic acids may account for tannin toxicity. Chung et Al. reported that the repressive consequence of tannic acid on the growing of enteric bacteriums may be caused by its strong iron-binding capacity. Chung et Al. besides reported that tannic acid inhibited the growing of all 15 of the bacterium tested. but Gallic acid and ellagic acid did non suppress any of them. They concluded that the ester linkage between Gallic acid and and glucose ( to organize tannic acid ) was of import to the antimicrobic potency of these compounds. Tannic acid was found to be repressive to the growing of of enteric bacteriums such as Bacteroides fragilis. Clostridium perfringes. Eschirichia coli. and Enterobacter cloacae. Staphylococcus aureus
S. aureus is a Gram-positive. spherical bacteria ( cocci ) with a diameter of 1 – 1. 3 m. When viewed microscopically. S. aureus appears in bunchs. like Bunches of grapes. Turning in nutrient. some strains can bring forth 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 proceedingss. The chief reservoirs of S. aureus are worlds and animate beings. Healthy people carry the being in their olfactory organ and pharynx ( 50 % ) . on their custodies ( 5-30 % ) . and in lesions. S. aureus can besides colonize nutrient contact surfaces. and it can go a relentless being in abattoirs. S. aureus can pollute nutrients through contact with contaminated custodies. stuffs and surfaces. but besides via the air ( coughing ) . S. aureus is a Gram-positive. enterotoxin bring forthing being. 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 merely does it bring forth enterotoxin. which causes nutrient poisoning when ingested. the being besides causes a figure of other diseases. e. g. lesion infections and blood toxic condition ( sepsis ) . toxic daze etc. On a rich medium. S. aureus signifiers reasonably big. xanthous settlements. The being can turn both with and without O ( 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 grocery. In order to bring forth noticeable degrees of toxin. the figure of beings must be over 105-6 per gm of merchandise. The clip between consumption of the toxin and the symptoms is merely two to five hours and depends on the sum and type of nutrient and the province of wellness of the individual. With respects to the survey of effects of tannic acid in this sort of bacteriums. several have already been conducted. Akiyama. Fujii. Yamasaki. Oono. and Iwatsuki examined the antibacterial activity of several tannic acids on the curdling of plasma by Staphylococcus aureus and the consequence 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. bacillar bacteria that lives in homo and animate being bowels where it is present in big Numberss. There are 100s of E. coli strains. most of which are comparatively harmless. some are even good to worlds. While most E. coli strains are harmless. one exclusion is the E. coli strain O157: H7. This peculiar strain is a pathogen which produces a powerful toxin frequently referred to as Shiga toxin or verotoxin. which can do terrible unwellness. E. coli thrive in warm. moisture. dark topographic points that are rich in foods. such as human and carnal enteric piece of lands. Once excreted. the bacteriums can non last the rough conditions of the outside universe ; nevertheless. some manage to happen their manner into lakes and watercourses. or another host. Once in H2O or deposit. E. coli can prevail for several hebdomads. Current research suggests that E. coli can last. turn. and persist in damp beach sand. Some common beginnings of E. coli are runoff from developments. direct release of untreated sewerage. escape from sewerage pipes. and dungs from sea chumps. water bird. and pets.
Storm cloacas can transport Canis familiaris and cat fecal matters off pavements or streets straight into watercourses. Improperly maintained infected systems may let go of pathogens into groundwater. Cattle. domesticated animate beings. wild animate beings. and birds straight let go of fecal matters into watercourses and lakes. Escherichia coli is one of the most intensively studied life species ( Elena et al. 2005 ) . While it has long served as a theoretical account being for biochemistry. genetic sciences. and molecular biological science. more late. it has been widely used in experimental surveies of development. E. coli is a normal portion of the microbiota of the lower GI piece of land of mammals. including worlds. and normally exists as a harmless commensal. However. there besides exist many infective strains of E. coli that can do a assortment of diarrheal and other diseases in worlds and animate beings. These infective strains express virulency factors that are involved in pathogenesis. but which are normally accessary to normal metabolic maps.
Chapter 3Methodology
The chemical methods used in this research were gathered from three beginnings: foremost 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 Bettering the Safety and Efficiency of Feeding Ruminants on Tanniniferous Tree Foliage” has been done. In order to beef up this research undertaking. advisers were called to specify the analytical methods to be used. The nucleus intent of this beginning was to supply the research workers with the chemical method to be used in quantifying the tannic acids present in the sample. The 2nd beginning was the Tannin Handbook prepared by Dr. Ann Hagerman ; this enchiridion was originally published for the usage in the Hagerman research lab.
Unfortunately. the original printed Tannin Handbook was no longer available but all its contents were posted at the site nowadays in the mentions of this survey. The intent of this beginning for the research workers was to indue them with cognition on how to insulate and sublimate their tannic acids being studied. The last beginning 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 research workers with cognition on how to civilization and inoculate different types of bacterium which has a large portion in their survey.
Research DesignBy and large. the method used by the research workers in this survey is the Experimental method. Travers ( 1978 ) says of the scientific experiment that it has become the most esteemed method of progressing scientific cognition. Gay ( 1976 ) thinks that this method is the lone method of research which can truly test hypotheses refering cause-and-effect relationship. He says further that the experimental method represents the most valid attack to the solution of jobs. both practical and theoretical. Ary. et Al. ( 1972 ) added that an experiment is by and large regarded as the most sophisticated research method for proving hypothesis. The experimental method is defined as the one which represents Mill’s rule of research known as the method of difference. This lone means that the consequence of a individual variable applied to one state of affairs can be assessed and the difference is determined ( Mill. 1872 in Good. et Al. 1935 ) . In this survey. the experiments contain merely two groups. an experimental group and a control group. The experimental group receives the intervention under probe while the control group receives a different intervention or the usual method it was utilizing before. Data Gathering Procedure
1. Preparation of Plant ExtractThe general job in the survey of natural works merchandises is that their nature and sum are dependent on assorted factors. which must be controlled every bit far as possible. One of these factors is stress ; the metabolic province of the works may alter when it is stressed in any mode. This can be a job before every bit good as after reaping a works portion for analysis. As cells die. the cellular unity is lost and as a consequence the enzymes come in contact with substrates to which they are non usually exposed in life cells. In add-on. it besides increases the oxidization procedure. which is a job with phenoplasts. most particularly in tannic acids since these are prone to oxidization. If a works is cut and dried under near ambient conditions. which by and large requires a big clip to dry. the nature and content of phenolic compounds can alter. In order to avoid these alterations. the metabolic activities of the cells need to be curbed instantly. 1. 1 Collection. drying and storage of works stuff
Bark age and its phase of development affect degrees and nature of phenoplasts. When the aggregation site is near to the research lab. the stuff can be transported to the research lab in fresh province. The fresh stuff should be kept on ice and transported under dark conditions. Transportation system of big sum of barks in plastic bags should be avoided. since temperature in the bag could lift taking to sudating and wilting which can alter the nature and degree of phenoplasts. If liquid N is available. the better option is to stop dead the sample and so freeze-dry the stuff without dissolving it. Dissolving can tear cell membranes taking to alterations in phenoplasts. If the stuff is frozen utilizing a deep-freeze. do certain that the stuff is non thawed during conveyance. Solid C dioxide should be used to transport such stuff.
Once the stuff is dried. it should be kept in a dry topographic point. sooner in a desiccator in the dark. The lyophilized stuff by and large is hygroscopic. Light is besides known to alter the nature of phenoplasts. After freeze-drying. the cell construction is broken and the enzymes are in the native province. With the soaking up of H2O. enzymes and phenoplasts can respond. which can bring forth drastic alterations in phenoplasts. The freeze-drying. though considered to be one of the safest method for saving of phenoplasts. can take to drastic alterations if the storage conditions are non appropriate. If a lyophilizer is non available. the works stuff has to be dried under far from ideal conditions. The sample can be dried at approximately 50–52°C utilizing a forced air oven. This will rush the procedure of drying. and the enzymes present in the works sample will non hold much clip to respond with phenoplasts. Drying at temperatures higher than 55°C should be avoided. since it can take to inactivation of phenoplasts or could diminish their extractability in dissolvers and impact the quantification. 1. 2 Crunching of sample
Fresh or frozen stuffs are hard to work with. Crunching could be a job utilizing these stuffs. Fresh stuff. when frozen utilizing liquid N. can be ground utilizing ‘Polytron’ homogenizers. One has to be cautious that the temperature does non lift during homogenisation ; addition in temperature can take to enzymatic alterations in phenoplasts. Phenolic resins are by and large extracted in aqueous organic dissolvers. The wet nowadays in the fresh stuff demands to be taken into history while fixing organic dissolvers for extraction. It is suggested to crunch the sample after drying it. About 500 g of the works stuff should be land foremost to go through a 2 millimeter screen. If the land samples in a desiccator are kept in a icebox. the desiccator must be opened after the contents has reached the ambient temperature. otherwise wet will distill on the sample which will take to alterations in the province of phenoplasts during storage.
1. 3 Extraction of Phenolic resinsThe purpose is to quantitatively spread phenoplasts present in the works stuff to liquid stage. For the extraction procedure. a suited dissolver is required. Generally. aqueous propanone ( 70 % ) is a popular pick. It has been reported by assorted workers to be better in pull outing phenoplasts from works stuffs. Dried ( finely land ) works stuff ( 20 g ) is taken in a glass flasks of about 250 ml capacity. 160 milliliter of aqueous propanone ( 70 % ) is added and the flask is suspended in an supersonic H2O bath and subjected to supersonic intervention for 20 min at room temperature. The contents of the flasks is so transferred to centrifugate tubings and subjected to centrifugation for 10 min at about 3000g at 4°C ( if refrigerated extractor is non available. cool the contents by maintaining the extractor tubing on ice and so centrifugate at 3000g utilizing an ordinary clinical extractor ) . Roll up the supernatant and maintain it on ice. Transfer the pellet left in the extractor tubing to the beaker utilizing two parts of 5 milliliters each of 70 % aqueous propanone and once more subjects the contents to supersonic intervention for 20 min.
Centrifuge and roll up supernatant as described above. 2. Measurement of Entire Phenolics and Tannins utilizing the Folin-Ciocalteu Method Harmonizing to Makkar et Al. the method for entire phenol is utile in order to cognize the efficiency of extraction of phenoplasts in dissolvers. The consequences can be expressed as tannic acid equivalent. The nature of tannic acid varies from one commercial beginning 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 H2O. Transfer it in a brown bottle and shop in a icebox ( 4°C ) . It should be aureate in colour. Do non utilize it if it turns olive viridity. * Sodium carbonate ( 20 % ) : Weigh 40 g Na carbonate. fade out it in approximately 150 milliliters distilled H2O and do up to 200 milliliter with distilled H2O. * Standard tannic acid solution ( 0. 1 mg/ml ) : Dissolve 25 milligram tannic acid ( TA ) obtained from Merck in 25 milliliters distilled H2O and so thin 1:10 in distilled H2O ( ever use a newly prepared solution ) .
2. 2 Preparation of Calibration CurveTake different aliquots of the standard Tannic Acid solution ( 0. 00. 0. 02. 0. 04. 0. 06. 0. 08. 0. 10 milliliter ) in trial tubing. do up the volume to 0. 5 milliliter with distilled H2O. Add 0. 25 milliliter of Folin-Ciocalteu reagent and so 1. 25 milliliter of Sodium Carbonate solution. Vortex the tubings for 40 proceedingss so enter the optical density at 725 nanometer. Plot the sum of Tannic Acid in µg as a map of the optical density at 725 nanometer. Calculate the incline and the intercept and obtain the equation of the line. 2. 3 Analysis of Entire Phenolic resins
Take suited aliquots of tannin-containing infusion. ab initio 0. 02. 0. 05 and 0. 1 milliliter in trial tubing. do up the volume to 0. 5 milliliter with distilled H2O so add 0. 25 milliliter of the Folin-Ciocalteu reagent. Add 1. 25 milliliter of Sodium Carbonate solution. Vortex the tubings for 40 proceedingss and step its soaking up at 725 nanometer. Calculate the sum of entire phenoplasts as Tannic Acid equivalent from the standardization curve. 3. Isolation of Tannin
Sephadex LH-20 is a liquid chromatography medium designed for molecular size of natural merchandises ; it is a beaded cross-linked dextran that has been hydroxypropylated to give a chromatography medium with both hydrophilic and lipotropic character. Tannins adsorb to Sephadex LH-20 in intoxicant while little phenoplasts elute from the stuff. Tannins can so be eluted with aqueous propanone. Chromatography on Sephadex LH-20 is really utile for dividing Tannin from non-tannin phenoplasts. 3. 1 Reagents
* 95 % Ethanol* 70 % Acetone* Sephadex LH-20* Column ( 5 x 40 centimeters )* Fraction Collector* Continuous UV proctor ( utile for supervising the eluate. but non indispensable ) 3. 2 ProcedureEquilibrate the Sephadex LH-20 harmonizing the manufacturer’s way in ethyl alcohol. Since the isolation requires a column. rinse it with several bed volumes of ethyl alcohol after packing the column. Plant extracts of different volumes are applied to the column after taking all propanone from the sample through rotary vaporization. Elute with ethyl alcohol at a rate of 1 mL/min. if UV proctor is available. elute until the optical density at 280 nanometer is no longer altering and is near the baseline. If no UV proctor is available. usage 1 litre of Ethanol to elute the sample. Then elute the column with 70 % propanone. tannic acids are normally seen as brown set of pigments. This eluate is non monitored in the UV because of the strong soaking up of propanone. Continue rinsing until the beads return to their original colour. Combine all tannin fractions and usage rotary evaporator to take all dissolvers. The tannic acid collected should be placed in phials and stored in dark deep-freezes. 4. Preparation of Culture Media
Generalized and specialized media are required for the growing of bacteriums. Culture media is an unreal dirt that contains nutritionary and environmental demands for the nutriment and reproduction of micro-organisms. The media that are used in research labs to civilization bacteriums are referred to as unreal media or man-made media. because they do non happen of course. instead. they are prepared in the research lab. 4. 1 Reagents
* Agar: a complex saccharide extracted from Marine algae that solidifies below the temperature of 45oC. * Beef Extract* Peptone
4. 2 Preparation of Culture Media4. 2. 1 Nutrient BrothDissolve 3. 0 gms of beef infusion and 5. 0 gms of peptone in 1000 milliliter of distilled H2O and mix exhaustively. Dispense 10 milliliter of the prepared Nutrient Broth in clean trial tubings and set a cotton stopper right off. 4. 2. 2 Alimentary Agar
Dissolve 3. 0 gms of beef infusion. 5. 0 gms of peptone and 15. 0 gms of agar pulverization in 1000 milliliter of distilled H2O. Mixing should be done over an ordinary H2O bath. Heat the solution over a fire beginning and furuncle until all solids are dissolved. Transfer the solution to smaller containers. such as Erlenmeyer flasks and cover it with cotton stoppers. 4. 2. 3 Sterilization
Topographic point all the prepared Nutrient Broth and Nutrient Agar in an sterilizer. Besides put cotton swabs. forceps. Petri dishes and punched filter documents which will be used for the following experiments. Sterilize the setups at 15 pounds per square inchs and 121oC for 15 proceedingss. 4. 3 Culturing the Bacteria
Pure civilizations of both Escherichia coli and Staphylococcus aureus are purchased from the National Science Research institute ( NSRI ) which is shacking at the compound of the University of the Philippines Diliman. Perform sub-culturing by reassigning a loop full of the pure civilization of each being to different unfertile Nutrient Broth with the usage of an vaccinating cringle aseptically. Set aside the civilizations and allow them multiply for a twenty-four hours. 4. 4 Distributing the Culture Medium
Sterile Nutrient Agar is transferred to sets of unfertile Petri dishes aseptically. Wait until the home bases solidify. and fix them for vaccination. 5. Inoculation to the Culture MediaInoculation is the procedure for adding specimen to the civilization medium. Cultures from this survey are obtained from a plated medium. 5. 1 Swab Method cDip a unfertile cotton swab in a Nutrient stock incorporating Escherichia coli. Remove surplus inoculants by lightly pressing the swab against the tubing at a degree above that of the Nutrient stock. Inoculate the Agar home base by streaking with the swab incorporating the inoculants. Revolve the home base by 60o and reiterate the streaking utilizing the same swab. Repeat the rotating and streaking process five times to guarantee an even distribution of the inoculants. Let the surface of the civilization to dry for seven proceedingss to let the soaking up of the extra wet. reiterate the process for four other home bases with the usage of other sets of cotton swabs. Do the same process for Staphylococcus aureus.
6. Anti-bacterial Trial of the Isolated Tannin to the Cultured Bacteria via the Filter Paper Method. Harmonizing to Engelkirk ( 2011 ) . an antibacterial agent will be accepted if it inhibits or destroys the pathogen without destructing the host. To carry through this. the agent must aim a metabolic procedure or constructions possessed by the pathogen but non by the host. 6. 1 Reagents
* Acetonic Tannin: Dissolve equal mass of the stray Tannin with different volumes of 70 % Acetone ( space. 1. 0. 2. 0. 3. 0. 4. 0 and 5. 0 milliliter ) . * 95 % Ethyl alcohol: Used for flame sterilisation of the forceps 6. 2 Procedure
Immediately after leting the plated civilizations to absorb the wet. fix them for the anti-bacterial trial. Dip the unfertile filter paper halfway in the Acetonic Tannin of different concentrations with the aid of a fire sterilized forceps. Put the filter documents at each of the centre of the five plated civilizations. Incubate the home bases invertedly at 37oC until the following two yearss and step 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 survey can besides be determined by the civilization giving the smallest zone of clearance or none at all. Statistical Tool
Two statistical interventions were used in this survey 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 alterations of the given variables ; the relationship can be computed and shown in a spread Diagram ( Cruz. et Al. 2009 ) . Utzurrum. et Al. said that the most widely used computational expression for correlativity is the Pearson Product Moment Correlation Coefficient. In calculating for the Pearson’s r. there are two basic premises ; the presence of a additive relationship and the interval ratio degree of measuring of informations. The expression for Pearson’s R is: r=NXY-XYNX2-X2 [ NY2-Y2 ]
Where:X= informations for independent variableY= informations for dependant variableN= sample sizer= grade of relationship between X and YTo prove for the significance of r. t-test is used. The expression for this trial are: df=N-2tcalc=rN-21-r2Decision regulation ; the research workers assumed a degree of significance of 0. 05. If tcalc & gt ; ttab. reject Ho. if tcalc & lt ; ttab. accept Ho. ANOVA is a method for partitioning the fluctuation observed in experimental informations into different parts. each portion attributable to a known beginning. This statistical trial 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 divergence of a measuring from the population mean is made up of two constituents. These constituents are the divergence of a measuring from the mean of the group where it belongs and the divergence of the group mean from the population ( Sevilla. et Al. 2007 ) . The expressions used for One-way ANOVA are:
SSBT=i=AC ( XA2+XB2+XC2 ) r-XT2NSSWT=i=ACXA12+XA22+…XC32- ( XA2+XB2+XC2 ) RSST=SSBT+SSWTDFBT=t-1DFWT=tr-1DFT=DFBT+DFWTMSBT=SSBTDFBTMSWT=SSWTDFWTFcalc=MSBTMSWTWhere:SS= amount of the squaresBT= between interventionsWT= within interventionsDF= grades of freedomMS= mean squaredX= given informationsr= figure of replicatest= figure of interventionsDecision regulation ; the research workers assumed a degree of significance of 0. 05. If Fcalc & gt ; Ftab. cull Ho. if Fcalc & lt ; Ftab. accept Ho.
