LOS BAÑOS NATIONAL HIGH SCHOOL Umali Subd., Brgy.
Batong Malake, Los Baños, Laguna
THE EFFICACY OF KANTUTAY SHOOT EXTRACT AS ANTIBACTERIAL AGENT AGAINST ESCHERICHIA COLI, AND STAPHYLOCOCCUS AUREUS
CLUSTER 1 Life Science (TEAM CATEGORY)
ABSTRACT THE EFFICACY OF KANTUTAY SHOOT EXTRACT AS ANTIBACTERIAL AGENT AGAINST ESCHERICHIACOLI AND STAPHYLOCOCCUS AUREUS
The research study is all about the Efficacy of Kantutay Shoot Extract as Antibacterial Agent against Escherichia coli and Staphylococcus Aureus. It was conducted in the Institute of Biological Sciences. The two bacteria mentioned were tested to determine the efficacy of the Kantutay Extract. Likewise, a positive control, two Kantutay shoot extracts (Pure & Boiled) and a negative control were used to determine the effects of Kantutay shoot extracts on each bacteria. The study was laid out in Kirby- Bauer method with two replications. The experiment showed that the test result was positive on E. coli, but was proven to be negative on the Staphylococcus Aureus. The results suggest that Kantutay shoot extract was an Effective Antibacterial Agent against E. coli only.
This research projects has been a challenge for us. Since we have different schedules and we experienced time conflict, we had time on doing this research project. We were just taking our time on doing the project knowing that we still have more time and it will all be fine. Until our research teacher told us the deadline of the project. It was earlier than what we expected. So, we crammed.
We were on the edge of giving up having the entire obstacle that we had but still, we were to pass our work. And we couldn’t do this without the help of a lot of people. First and foremost, we would like to praise and express our faith to the Almighty for giving us the patience and hope whenever we felt we lost it all. Thank you Lord God for everything. Thank you for blessing us with knowledge and giving us with instruments of Your love. We all love You.
And of course, we would like to thank our research teacher, Mrs. Karla Mae M. Lopez, for giving us guidelines, tips and encouragements on doing this project.
We also would like to express our gratitude to Mr. Patrick M. Rocamora. Thank you for helping us as our resource person.
Our gratitude to Rocamora family unselfishly letting us stay in their house in doing our experiments and papers.
Next is to Mr. Noel Sabino our researcher consultant, mentor and friend. Thank you of encouragement. You instilled in us the skill and the ways of a researcher.
To our loving parents, many thanks! You are our inspiration on doing this researcher project. We could not have done this without you. Thank you very much for allowing and understanding us when we are going home late going on weekends when we are conducting our experiments.
Our classmates in IV- Diamond on encouraging us that we can finish this project on time and for tour support. Thank you and good luck to us.
To our subject teachers and especially our adviser, thank you for letting us be excused on your classes especially on the day we needed the most.
To all who have been part of this research project, words can’t express our gratitude and happiness. Thanks a lot for being there for us. iii
TABLE OF CONTENTS
Title Abstract Acknowledgement Table of contents i ii-iii iv
I. The Problem and Its Background Introduction Background of the Study Statement of the Problem General Objective Specific Objectives Hypotheses Significance of the Study Scope and Delimitations Definition of Terms II. Methodology III. Results and Discussion IV. Summary of the Findings, Conclusion and Recommendations V. Bibliography
1 1 2-13 14 14 14 14 15 15 16 17-21 22-23 24 25
THE PROBLEM AND ITS BACKGROUND
Introduction The pharmaceutical industry uses about 120 different compounds derived from plants in the drugs it manufactures, and it discovered nearly three-quarters of these compounds by studying folk remedies. Medicinal Plants are sources of enormous quantities of chemical substances which are able to initiate different biological activities including those useful in the treatment of human diseases. Scientific investigations of medicinal plants used in folklore remedies have attracted increased attention in the “medical world,” especially in a bid to finding solutions to the problems of multiple resistances to the existing synthetic antimicrobials. Most of the synthetic antibiotics now available in the market have major setbacks due to the accompanying side effects on patients and the multiple resistances developed by pathogenic organisms to them. Hence, there is a justifiable need to explore for new and more potent antimicrobial compounds of natural origin to combat these pathogens. A pathogen is a biological agent that causes disease or illness to its host. The essential oil composition of the Kantutay shoots contains the parts that are needed for the application of this experiment. It contains cinecol, sabinene, and caryophylene. Basically, in testing Gram-positive bacteria, there is a need for the Gram-negative bacteria. The pathogenic capability of E. coli is usually associated with certain components of Gram-negative cell walls, in particular the lipopolysaccharide (also know n as LPS or endotoxin) layer. Unlike E. coli has a Gram-positive spherical cell.
Background of the study Lantana camara, also known as Spanish Flag, is native to tropical regions in Central and South America. Lantana camara has been introduced into other parts of the world as an ornamental plant; it is considered an invasive species in many tropical areas. It is sometimes known as "red (yellow, wild) sage", despite its classification in a separate family to sage (Lamiaceae), and a different order to sagebrush (Asterales). The essential oil of L. camara from different regions of the world has been reported by many workers (Da Silva et al., 1999: Sefidkon, 2002: Kasali et al., 2004). The oils differ in their chemical compositions according to geographic origin of the plants. Da Silva et al. (1999) reported differences in essential oil composition of L. camara collected at different places in the Amazon region of North Brazil. The essential oil obtained from leaves of Lantana camara from Cuba was analyzed by GC-FID and GC/Ms. The oil was characterized by the high percentage of sesquiterpenes. The major components were (E)-nerolidol (43.4%), lambdacadinene (7.6%), alpha-humulene (4.9%) and beta-caryphyllene (4.8%) (Journal of Essential Oil research, Cuba, 2004). The essential oil composition of Lantana camara leaves from Nigeria was reported to have 1,8-cineol (15.8%), sabinene (14.7%) and P-caryophyllene (8.9%) out of 78% of oil that were identified. E. coli is normally present in the human intestine, but the variant E. coli O157:H7 produces toxins that cause bloody diarrhea and, in some cases, far more severe problems, including kidney failure and death .A person can become infected by eating contaminated
meat (Microsoft Encarta, 2006). This pathogenic capability is usually associated with certain components of Gram-negative cell walls, in particular the lipopolysaccharide (also known as LPS or endotoxin) layer. In humans, LPS triggers an innate immune response characterized by cytokine production and immune system activation. Inflammation is a common result of cytokine production; which can also produce host toxicity. Staphylococcus aureus, literally the "golden cluster seed" or "the seed gold" and also known as golden staph) is the most common cause of staph infections. It is a spherical bacterium, frequently part of the skin flora found in the nose and on skin. S. aureus was discovered in Aberdeen, Scotland in 1880 by the surgeon Sir Alexander Ogston in pus from surgical abscesses. Each year some 500,000 patients in American hospitals contract a staphylococcal infection. In this study, the efficacy of Lantana camara, locally known as “Kantutay” against E. coli and S. aureus will be determined.
Review of Related Literature Lantana camara is a low erect or subscandent, vigorous shrub with stout recurved prickles and a strong odour of black currents; it grows to 1.2-2.4 meters (or even more); its root system is very strong, and it gives out a new flush of shoots even after repeated cuttings; Leaf ovate or ovate-oblong, acute or subacute, crenate-serrate, rugose above, scabrid on both sides; Flower small, usually orange, sometimes varying from white to red in various shades and having a yellow throat, in auxiliary heads, almost throughout the year; Fruit small, greenish-blue black, blackish, drupaceous, shining, with two nutlets, almost throughout the year, dispersed by birds. Seeds germinate very easily. (Sastri and Kavathekar, 1990). The diverse and broad geographic distribution of lantana is a reflection of its wide ecological tolerances. It occurs in diverse habitats and on a variety of soil types. It generally grows best in open unshaded situations such as wastelands, rainforest edges, beachfronts, and forests recovering from fire or logging. Disturbed areas such as beside roads, railway tracks and canals are also favorable for the species (Thaman 1974; Winder and Harley 1983; Thakur et al. 1992, Munir 1996, in Day et al. 2003). Lantana does not invade intact rainforests, but is found on its margins (Diatloff 1975; Humphries and Stanton 1992, in Day et al. 2003). Where wet sclerophyll forests and rainforests have been disturbed through logging, gaps are created; this allows lantana to encroach on the forests. Further logging aggravates the condition and allows the lantana to spread or become thicker (Waterhouse 1970, in Day et al. 2003).
Kantutay has several uses, mainly as herbal medicine and in some areas as firewood and mulch. The leaves are used to reduce itching. Other used are against flu, colds coughs, fever, yellow fever, dysentery and jaundice. The roots are used for gonorrhea. Lantana oil is sometimes used for the treatment of skin itches, as an antiseptic for wounds and externally for leprosy and scabies. Lantana repels other plants and other groups of organisms such as insects. Lantana oil is used externally for leprosy and scabies. Plant extracts are used as medicine for the treatment of cancers, chicken pox, measles, asthma, ulcers, swellings, eczema, tumors, high blood pressure, bilious fevers, catarrhal infections, tetanus, rheumatism, malaria and atoxy of abdominal viscera. The toxic and repellent effects of two tropical plants, Lantana camara L. and Tephrosa vogelli Hook were Evaluated against Sitophilus zeamais Motschulsky in stored maize grain.s Five rates (1.0, 2.5, 5.0, 7.5 and 10.0% w/w) of each powdered plant material, an untreated control and a synthetic insecticide (Actellic Super 2% dust) were used to investigate treatment efficacy on mortality of the adult insect (five to eight days old), F1 progeny emergence and repellence against zeamais adults. In many regions, lantana has become dominant component of natural and agricultural ecosystems. The rapid removal of natural forests without replacement by structurally similar native vegetation may be partially replaced with tickets of lantana. Consequently, the amount of available habit for native animals may decrease. In some areas, weeds such as lantana may provide shelter and vital winter food for many native birds. A number of endangered birds species utilize lantana thickets when their natural habitat is unavailable. In Australia, the vulnerable black-breasted buttonquail, Turnix
melanogaster, feeds and roots in lantana thickets adjacent to its more favored habitat, vine forest. (Smith et al. 1998, in Day et al. 2003.). While buttonquails prefer intact vine forests, lantana provides an important temporary refuge for them between forest remnants. In Central Kenya, where natural riverine thickets have been almost completely cleared, the endangered Hinde’s babbler, Turdoides hindei, has become dependent on lantana thickets, and unless sufficient suitable natural habitat can be restored the survival of this species depends on the retention of lantana infestations (Njoroge et al. 1998). Apart from benefiting some bird species, lantana is a major nectar source for many species of butterflies and moths. Lantana camara is a highly variable species. It has been cultivated for over 300 years and now has a hundred of cultivars and hybrids. These belong mostly to the L. camara complex. Cultivars can be distinguished morphologically (variation in: flower size, shape and color, leaf size, hairiness and color; stem thorniness), physiologically (variation in: growth rates, toxicity to livestock) and by their chromosome number and DNA content (Pierre Binggeli, 1999). The key to good management of lantana is constant vigilance (Day et al. 2003.). Repeated control of regrowth is critical to success. Control of new infestations should be a priority because the species is able to expand its range during good seasons. Mechanical: Mechanical clearing and hand pulling are suitable for small areas and fire can be used over large areas. Biological: Biocontrol agents have decreased the volume of individual plants making other control methods considerably easier. None of the over 40
agents trialed have resulted in control but some have been partially successful including Teleonemia scrupulosa Stal (Hemiptera), Octotoma scabripennis (Coleoptera), Uroplata girardi Pic (Coleoptera) and Ophiomyia lantanae (Froggatt) (Diptera) (Dap et al. 2003) L. camara was the first weed ever targeted for classical biological control at the turn of the century, and since then 36 insect species have been released in 33 countries throughout the exotic range. Despite these efforts, control of the weed has generally been disappointing (Thomas and Ellison 2000). Many reasons have been suggested for this failure: the great genetic diversity of the plant, its ability to hybridize, and that fact that its origin as a hybrid ornamental plant complicates the search for its center of origin and thus for potential agents (Thomas and Ellison 1999, Day et al. 2003). Twenty nine biotypes exist in Australia alone (Smith and Smith 1982, in Thomas and Ellison 1999). No insect agent released to date has caused significant damage to the very important Common Pink biotype (Thomas and Ellison 1999). In general, the insect agents released have a restricted host range within this complex, and, in addition, the weed is able to tolerate wider climatic and geographical areas (Thomas and Ellison 1999). Searchers have been made in Mexico, Central America, the West Indies, and Brazil, and insects have been collected from several different lantana species. These insects have been host-tested and released in Hawaii, South Africa, Australia, several countries in East Africa, South and East Asia and the Pacific (Day. 300). A strain of the rust Prospodium tuberculatum from Brazil was screened as a biocontrol agent against 40 Australian L. camara forms and 52 closely related, nontarget plant species. Result under glasshouse conditions showed that the Brazilian rust strain is pathogenic to only two flower color forms: pink ad pink-edged red. Macro- and
microsymptoms were recorded using 11 assessment categories and 4 susceptibility ratings. No macrosymptoms were observed on any of the non-target plans (Thomas et al. 2006). Lantana flowers in most places all year round if adequate moisture and light are available (Gujral and Vasudevan 983, Graaff 1986, in Day et al. 2003), with flowering peaking duringthe wet summer months. In cooler or drier regions, flowering occurs inly in the warmer or wetter months, due to frost or drought damage (Winder 1980, Swarbrick et al. 1998 in Day et al. 2003). Plants can flower as early as the second growing season. Initially, lepidopteron species were thought to be primary pollinators of lantana (Dronamraju 1958, Schemske 1976, Kugler 1980, Hilje 1985, in Day et al. 2003)
Statement of the Problem The project aims to determine the effectiveness of the extract from Kantutay shoots as an antibacterial agent against E. coli and Staphylococcus aureus. General Objective To determine the effectiveness of the extract from kantutay shoots as an antibacterial agent against E. coli and Staphylococcus aureus. Specific objectives 1. Is the extract from kantutay shoots an effective antibacterial agent against E. coli and Staphylococcus aureus? 2. Is the pure extract from kantutay more effective than the boiled extract? 3. Is the antibacterial affordable?
Hypotheses 1. Kantutay shoots extract is an effective antibacterial agent against E. coli and Staphylococcus aureus. 2. The pure extract is more effective than the boiled extract. 3. The antibacterial if affordable.
Significance of the study This study was conducted due to the large spread of diseases, namely diarrhea, cramps, meningitis, arthritis, sore throat, fever, etc. which is usually caused by the bacteria E. coli and Staphylococcus aureus. This experiment/research study was also conducted to prevent the spread of communicable diseases. Kantutay shoots are commercially produced and easy to grow. They are accessible and have a high antibacterial content. This study also provides information to the public on the causes of diseases, its spread and prevention.
Scope and delimitation This study focused on the use of kantutay shoot extract as antibacterial against E. coli and Staphylococcus aureus. Other bacteria were not considered, hence the study is focused upon those two strains of bacteria. The kantutay shoots were fresh and newly picked. The researchers were not able to consider other plant sources due to time and resource constraints.
Antibacterial test of Kantutay shoot extract
Research design The researchers used Kirby-Bauer Technique for the bioassay of the study. Two bacteria: E. coli and S. aureus were used for the study. Streptomycin was used for the positive control and distilled water for the negative control. The assay uses 3 agar plates with 2 replications on each culture. Research materials The materials used in the media preparation of the activity were: 6 Petri dishes, 4 indicator strains, blender, chopping board, knife, agar plate and other necessary glassware. Conduction of the experiment 1. • • culturing the test organism E. Coli Staphylococcus aureus The bacterial strains were placed in vials (10ml) and were inoculated for 16 to 18 hours. 2. Solvent extraction of Kantutay shoots
Solvent: water Formulation of solution: The weight of the solvent is 100ml (each) and the weight of the kantutay shoots 200g.
The Kantutay shoots and each kind of solvent were mixed using a blender. The extracts undergo decantation to produce a debris-free solution. 3. Bioassay (Kirby-Bauer Method) The sensitivity-disc method of determining which of a wide variety of antibiotics is effective against an organism is a rapid, accurate, and inexpensive diagnostic tool. Broad spectrum antibiotics affect a wide variety of both gram-negative and gram-positive organisms. The use of the carefully standardized techniques reduces the influence of variables in the preparation of the test. This illustrates the differences in the gram-negative bacteria to several antibiotics. This one bacterium.
sensitivity of gram-positive and
will measure the antibiotic sensitivity of only Procedure 1.
The sterile swab was dipped into the one bacteria broth culture of standard
density: then rotated several times with firm pressure on the inside wall of the tube above the fluid level to remove excess inoculums. 2. The swab was streaked over the entire Muller-Hinton agar plate surface and
repeat two more times rotating the plate about 60 degrees to insure even distribution of inoculums. Replace plate top and allow 3-15 minutes for excess surface moisture to absorb. 3. The discs were applied to the surface using sterile forceps and dispenser.
Distribute the discs evenly. Gently press down each disc to insure complete contact with the agar surface.
Within 15 minutes, after the discs were applied, the plates were incubated at 37
degrees Celsius. 5. After 16-18 hours of incubation, each plate was examined and the diameters were
measured of the zones of complete inhibition using a ruler or template. 6. The zones of inhibition were interpreted referring to the tables provided and
reported the organisms tested to be susceptible, intermediate, or resistant to the various agents used. 7. The plate of the second bacterium was obtained and the zones of inhibition were
measured to determine the susceptibility of these organisms to the various antibiotics.
RESULTS, FINDINGS, AND DISCUSSIONS
Antibacterial test of Kantutay extract To test which culture was greatly affected by the crude extracts, the sensitivity discs were put in agar plate. Data were gathered after 18 to 24 hours ofinhibitionand was carefully analyzed by zone of interpretation as the statistical tool.
1st crude extract
2nd crude extract
Table 1: The results of Antibacterial test of Kantutay shoots extract
R (positive control)
I (positive control)
S (positive control)
N(none) (positive control)
Table 2: Zone Size Interpretation
SUMMARY OF FINDINGS, CONCLUSION AND RECOMMENDATION
Summary of findings The results of the antibacterial test are as follows: for the positive control, both E. coli and S. aureus were detected in both trials: for pure extract, for both trials, E. coli was detected, but S. aureus, it was detected in only one trial. Results are the same for the boiled extract and both bacteria were not detected in the negative control. For the zone of inhibition, E. coli was detected up to the intermediate zone, while there was no inhibition for S. aureus. Conclusion The study conducted was proven to be effective as an antibacterial agent against E.coli only and not against Staphylococcus Aureus. The results testified this conclusion made.
Recommendation The researchers recommend having further experimentation using more bacteria. The researchers suggest that the antibacterial agent should be used against pathogenic bacteria.
BIBLIOGRAPHY http://www.motherherbs.com/lantana.html http://