CHAPTER I
THE PROBLEM AND ITS SETTING
This chapter informs the reader about the problem under the researchers’ study. Introduction In these times, many are having and suffering from skin diseases by bacteria called, Staphylococcus aureus bacteria. Bacterial and fungal infections are widespread throughout the world. The situation is more critical especially in the third world countries were in most cases lack of adequate sanitation and primary health care programs make it difficult and expensive to combat diseases. A number of higher plants have been used for centuries as remedies for human diseases. People having bacterial and fungal infections are taking chemical medications that may harm their bodies and may show some side-effects. In this case, it is better to take natural medicines instead. One of the examples for natural medicines is Moringa oleifera (Malunggay). Staphylococcus aureus, often referred to simply as “Staph”, are bacteria commonly found on the skin and in the noses of healthy people.
Sometimes, S. aureus can cause infection and is one of the most common causes of skin infections in the United States. Most of these infections are minor (such as pimples, boils, and other skin conditions). The S. aureus bacteria can also cause serious, and sometimes, fatal infections such as bloodstream infections, surgical wound infections, and pneumonia. Staphlococcus aureus bacteria are a break in a continuity of a tissue of the body. These bacteria are primarily caused by external forces. People easily get infections through open wounds if not treated well. When bacteria enter through these open wounds, they may grow and cause infection. To avoid these infections, commercial antiseptic is immediately applied on the wounded area until the injury heals. But these commercial antiseptics can cause irritation on skin and allergic reaction which may worsen the wounded surface. Moreover, some of these antiseptics are costly, so most of the people cannot afford. For this reason, traditional healers depend much on plant resources primarily for herbal medicines and antiseptics. But some of these plants weren’t proven by science to contain curies properties. In fact, some of it has side effects. This means that wider information about the chemical constituents of these plants should be known.
Statement of the Problem
This study aims to determine the effect of Moringa oleifera leaf extract to Staphylococcus aureus bacteria. This study aims to answer the following questions:
1.) Does Moringa oleifera extract have antiseptic property?
2.) Does Penicillin have antiseptic property?
3.) Does Amoxicillin have antiseptic property?
Scope and Limitation
This study is limited only with the antiseptic effect of Moringa oleifera, Penicillin and Amoxicillin to the Staphylococcus aureus bacteria. The bacteria will be cultured in the General Santos Doctors’ Medical Foundation, Inc. Laboratory this summer of 2013. This study aims to prove that the Moringa oleifera is effective and better than the other antiseptics in fighting diseases such as the Staphylococcus aureus bacteria.
Significance of the study
Medicine can be a great help to those people who have illnesses but only to those who can afford to buy. Poor people remain using herbal medicine in which some are not therapeutically approved. This is hazardous because it is left treated improperly, it may lead to death. Through this study, we will be able to provide better opportunity for medication, purposely to treat wounds. The study does not dwell on alternative medicine but also in possibility of creating preventive supplements for ailments.
CHAPTER II
REVIEW OF RELATED STUDIES AND CONCEPTUAL FRAMEWORKS
This chapter contains the different bodies of knowledge synthesized together to fit into the study’s needs.
Related Literature
Moringa Oleifera
Moringa is a healer, food magician, beauty and beautician, a plant with surprising water purification capabilities, a best friend and humanitarian who works for so little. It is one of the most useful trees on earth. Moringa makes a major contribution to human and animal health; in many cases, it can mean the difference between life and death. Absolutely no negative effects to daily consumption of Moringa leaves have ever been reported. As a healing plant, Moringa is even more amazing. There is much evidence from around the world, from various traditions and cultures that have used this Miracle Tree for so many ailments and troubles. The leaves are believed to have a stabilizing effect on blood pressure and control glucose levels. They are also used to treat anxiety, diarrhea and inflammation of the colon, skin infections, scurvy, intestinal parasites, and many other complications.
Amoxicillin
Amoxicillin, formerly amoxycillin in some markets, and abbreviated amox, is a moderate-spectrum, bacteriolytic, β-lactam antibiotic used to treat bacterial infections caused by susceptible microorganisms. It is usually the drug of choice within the class because it is better absorbed, following oral administration, than other β-lactam antibiotics. Amoxicillin is one of the most common antibiotics prescribed for children.
Penicillin
Penicillin (sometimes abbreviated PCN or pen) is a group of antibiotics derived from Penicillium fungi. They include penicillin G, procaine penicillin, benzathine penicillin, and penicillin V. Penicillin antibiotics are historically significant because they are the first drugs that were effective against many previously serious diseases, such as syphilis, and infections caused by staphylococci and streptococci. Penicillins are still widely used today, though many types of bacteria are now resistant. All penicillins are β-lactam antibiotics and are used in the treatment of bacterial infections caused by susceptible, usually Gram-positive, organisms.
Staphylococcus Aureus
Staphylococcus aureus is a bacterium that is a member of the Firmicutes, and is frequently found in the human respiratory tract and on the skin. Although S. aureus is not always pathogenic, it is a common cause of skin infections (e.g. boils), respiratory disease (e.g. sinusitis), and food poisoning. Disease-associated strains often promote infections by producing potent protein toxins, and expressing cell-surface proteins that bind and inactivate antibodies. The emergence of antibiotic-resistant forms of pathogenic S. aureus (e.g. MRSA) is a worldwide problem in clinical medicine. Staphylococcus was first identified in Aberdeen, Scotland (1880) by the surgeon Sir Alexander Ogston in pus from a surgical abscess in a knee joint. This name was later appended to Staphylococcus aureus by Rosenbach who was credited by the official system of nomenclature at the time.
It is estimated that 20% of the human population are long-term carriers of S. aureus which can be found as part of the normal skin flora and in anterior nares of the nasal passages. S. aureus is the most common species of staphylococcus to cause Staph infections and is a successful pathogen due to a combination of nasal carriage and bacterial immuno-evasive strategies. S. aureus can cause a range of illnesses, from minor skin infections, such as pimples, impetigo, boils (furuncles), cellulitis folliculitis, carbuncles, scalded skin syndrome, and abscesses, to life-threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome (TSS), bacteremia, and sepsis. Its incidence ranges from skin, soft tissue, respiratory, bone, joint, endovascular to wound infections. It is still one of the five most common causes of nosocomial infections and is often the cause of postsurgical wound infections. Each year, some 500,000 patients in American hospitals contract a staphylococcal infection.
Related Studies
The antimicrobial activities of Malunggay (Moringa oleifera) leaves, roots, bark and seeds were investigated in vitro against bacteria, yeast, dermatophytes and helminths pathogenic to man. By a disk-diffusion method, it was demonstrated that the fresh leaf juice and aqueous extracts from the seeds inhibit the growth of Pseudomonas aeruginosa and Staphylococcus aureus and that extraction temperatures above 56°C inhibited this activity. No activity was demonstrated against four other pathogenic Gram-positive and Gram-negative bacteria and Candida albicans. By a dilution method, no activity was demonstrated against six pathogenic dermatophytes. A method was standardized for studying the effect of aqueous extracts on Ascaris lumbricoides eggs, but no activity was exhibited by any part of the tree in contrast to Chenopodium ambrosioides leaf extracts. Antibacterial effects of aqueous and ethanolic extracts of seeds of moringa (Moringa oleifera) in the concentration of 1:5 and 1:10 in volumes 50, 100, 150 and 200 µL were examined against Staphylococcus aureus, Vibrio cholerae, Escherichia coli (isolated from the organism and the aquatic environment) and Salmonella Enteritidis.
Antibacterial activity against S. aureus, V. cholerae and E. coli isolated from the whiteleg shrimp, Litopenaeus vannmaei, was detected in aqueous and ethanolic extracts of moringa. E. coli isolated from tilapiafish, Oreochromis niloticus, was sensitive to the ethanolic extract of moringa. The aqueous extracts of soursop showed an antibacterial effect against S. aureus, but the antibacterial activity by the ethanol extracts of this plant was not demonstrated. The extracts were tested for their antimicrobial potency against Escherichia coli, Staphylococcus aureus, and Candida albicans using the filter paper disk method. Analyses of the results revealed that solution A exhibited antimicrobial potency against Escherichia coli and Staphylococcus aureaus. Solution B, C and D showed antibacterial bioactivity against Escherichia coli.
The Malunggay root and root bark extracts were effective in inhibiting the growth of Escherichia coli whereas only the root bark extract with 75:25 concentration was effective in inhibiting the growth of Staphylococcus aureaus. The extracts, however, had no effect on yeast. The group recommends further testing to be done to other microorganisms to determine the antimicrobial potency against other bacterial species. Moringa oleifera is the most widely cultivated species of the monogeneric family Moringaceae (order Brassicales). This family includes 13 species of trees and shrubs distributed in sub-Himalayan ranges of Arabia, India, Madagascar, North Eastern and South Western Africa, and Sri Lanka. [8,9] It contains several phytochemicals, some of which are of high interest because of their medicinal value, as described in the next section. In particular, this plant family is rich in a fairly unique group of glycoside compounds called glucosinolates and isothiocyanates.
Antibacterial Effects
Moringa plant compounds have significant antibacterial properties. A 2010 study published in the journal, “Bayero Journal of Pure and Applied Sciences,” found that Moringa seed and leaf extracts killed Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Enterobacter aerogenes bacteria. Moringa extracts were also effective against Salmonella typhimurium, a germ that causes dysentery and inflammation of the stomach and intestines. In addition, the researchers found that Moringa has significant anti-fungal activity. Another 2010 study published in the Brazilian journal, “Revista do Instituto de Medicina Tropical de São Paulo,” confirmed the germ-fighting ability of water-based extracts of Moringa.
Seed extracts of Moringa oleifera were evaluated for their antimicrobial activity against four types of bacteria namely Staphylococcus aureus, Bacillus subtilis, Eschreiashia coli and Pseudomonas aeruginosa. Two species of fungi i.e. Aspergillus niger and Candida albicans were also bioassayed for their response when the seed extracts were used. All of the seed extracts irrespective of their types, in different concentrations inhibited the growth of all microbes to varying degrees. Aqueous extract showed strong and superior antibacterial activity against all bacterial strains especially with regard to gram positive bacteria (Staphylococcus aureus and Bacillus subtilis) as compared to methanol or petroleum ether. Less or no activity was observed against Aspergillus niger and Candida albicans. These findings support the traditional use of the plant in the treatment of different infections in the area.
The Action of Penicillin on Staphylococcus
Bacteria exposed to one of the active members of the sulfonamide group of drugs continue for a time to grow at a normal rate; growth is then interrupted and bacterial death begins (Hirsch, 1944). Some of the earlier work with penicillin indicated that, in contrast to this, penicillin causes the death of a certain proportion of the dividing cells of a growing culture without any appreciable lag period (Hobby et al., 1942; Lee et al., 1944). Rantz and Kirby (1944), however, have shown that at limiting dilutions of penicillin there may be a definite interval between the addition of penicillin and the onset of growth inhibition, as evidenced by turbidimetric determinations. From this, as well as from other studies of the action of penicillin, it has been concluded that the substance acts only on dividing cells, leaving nondividing cells unaffected. Hobby and Dawson (1944) likewise demonstrated that bacteria continued to multiply for a time after being planted in broth containing small concentrations of penicillin. According to these experiments, however, a similar lag in the onset of effective action of penicillin was also apparent when higher concentrations were used.
The present studies were undertaken in order to gain more precise knowledge on this point and to determine how long the influence of penicillin on bacteria might last after its complete removal from the culture medium. A commercial preparation of penicillin was used, the manufacturer’s assay of its potency being accepted. The Staphylococcus employed was designated as strain Mx by Julianelle and Wieghard (1935) and is their type B. It is inhibited by 0.06, but not by 0.03, units of penicillin per ml and is thus somewhat less sensitive to penicillin than the Oxford strain. Tryptose phosphate broth and tryptose phosphate agar, manufactured by the Digestive Ferments Company, were used as culture media. Viable bacteria were determined by plate count after 24 hours’ incubation.
When samples were taken from the growing culture, they were chilled to 0 C in an ice bath after being withdrawn and held there until dilutions could be prepared or plating. The extracts from plants contain not only minerals and primary metabolites but also a diverse variety of secondary metabolites. The search for biologically active compounds has been vigorous in recent years due to the growing cases of microbial resistance to the time honored antibiotics8. In recent years numbers of studies have been reported dealing with antimicrobial screening of extracts of medicinal plants. Antibacterial properties of various plants parts like leaves, seeds and fruits have been well documented for some of the medicinal plants for the past two decades10. A variety of compounds are accumulated in plant parts accounting for their constitutive antimicrobial activities11.
Antibacterial effect (in vitro) of Moringa oleifera and Annona muricata against Gram positive and Gram negative bacteria Antibacterial effects of aqueous and ethanolic extracts of seeds of moringa (Moringa oleifera) and pods of soursop (Annona muricata) in the concentration of 1:5 and 1:10 in volumes 50, 100, 150 and 200 µL were examined against Staphylococcus aureus, Vibrio cholerae, Escherichia coli (isolated from the organism and the aquatic environment) and Salmonella Enteritidis. Antibacterial activity (inhibition halo > 13 mm) against S. aureus, V. cholerae and E. coli isolated from the whiteleg shrimp, Litopenaeus vannmaei, was detected in aqueous and ethanolic extracts of moringa. E. coli isolated from tilapiafish, Oreochromis niloticus, was sensitive to the ethanolic extract of moringa. The aqueous extracts of soursop showed an antibacterial effect against S. aureus and V. cholerae, but the antibacterial activity by the ethanol extracts of this plant was not demonstrated.
Moringa Medical Preparations
From the roots to the sap, every part of the Moringa plant yields some health benefit. Moringa plants are rich in phytochemicals — compounds produced by plants that have health benefits. Moringa medicinals are prepared as water-based or alcoholic extracts, decoctions (boiled in water), creams, oils, moisturizers, ointments or powders. Fresh leaves or roots are applied as poultices to treat rheumatism and other joint pains, to relieve lower back pain, to stop headaches and to treat wounds.
Conceptual Framework
Dependent Variable
Independent Variables
Staphylococcucs Aureus Bacteria
Moringa Oleifera
Amoxicillin
Penicillin
Figure 1: Conceptual Framework
Hypotheses
This study is conducted to test the following hypotheses: 1.) Malunggay (Moringa oleifera) has an effect on Staphylococcus aureus. 2.) Malunggay (Moringa oleifera) can be an alternative to penicillin and amoxicillin.
Definition of Terms
Terms used in the study are defined operationally:
Moringa oleifera – is the most widely cultivated species of the genus Moringa, which is the only genus in the family Moringaceae. Horseradish tree or Malunggay. Staphylococcus aureus bacteria – is a bacterium that is a member of the Firmicutes, and is frequently found in the human respiratory tract and on the skin. Penicillin – antibiotics are historically significant because they are the first drugs that were effective against many previously serious diseases, such as syphilis, and infections caused by staphylococci and streptococci. Amoxicillin – is a moderate-spectrum, bacteriolytic, β-lactam antibiotic used to treat bacterial infections caused by susceptible microorganisms. It is usually the drug of choice within the class because it is better absorbed, following oral administration, than other β-lactam antibiotics.
CHAPTER III
RESEARCH DESIGN AND METHODOLOGY
This chapter discusses the procedures and methods in the screening for antiseptic property of Moringa oleifera (Malunggay). Research Design
Figure 2: Research Design
Research Materials
The materials used in this study were 200 grams of Moringa oleifera leaves, 6 petri-dishes (sterilized), 30 mL of nutrient agar, staphylococcus aureus bacteria, 18 punched filter paper, 3 beakers, 3 forceps, inoculation loop (sterilized), alcohol lamp and incubator. Independent variables: Amoxicillin, penicillin and Moringa oleifera (malunggay). Dependent variable: Area of innovation of the S. aureus bacteria. Controlled variables: The volume or quantity of nutrient agar, the number of punched filter paper dipped in each of the three: amoxicillin suspension, penicillin suspension and Moringa oleifera extract, the temperature (35° C) of the incubator in which the bacteria shall be kept for 24 hours.
Procedure
(The bacteria and other necessary apparatus shall be prepared and provided by the laboratory). Label the beakers: “A” for amoxicillin suspension, “B” for penicillin suspension and “C” for Moringa oleifera extract. Dip 6 punched filter paper in each beaker. Set aside the beakers. Prepare the 6 petri-dishes with the same amount of nutrient agar which was prepared 24 hrs ago. Label these petri-dishes. Two petri-dishes will be labeled “A” for amoxicillin suspension, two petri-dishes will be labeled “B” for penicillin suspension and two petri-dishes will be labeled “C” for Moringa oleifera. Transfer the S. aureus bacteria using an inoculation loop. The wire end with the loop will gently touched to a bacterial colony on a source plate. Then, wipe the end of the inoculation loop with bacteria across the surface of the petri-dish with the nutrient agar. Do this process promptly yet gently in all the petri-dishes. Using a forceps, transfer the dipped filter papers to their respective labeled petri-dishes with S. aureus bacteria. Put 3 filter papers in each petri-dish. If all procedures are done, put the three petri-dishes into the incubator (@35° C) for 24 hrs.
ANTISEPTIC SCREENING
Sterilization of Materials
All glass and punched filter paper discs will be sterilized through dry heat sterilization using an oven at 121°C for 1 hour. These will be set
aside for future use. Preparation of Culture Media
5.75 grams of nutrient agar will be dissolved in 250mL of distilled water. The dissolved agar will be placed in an Erlenmeyer flask. Then it was heated on a hot plate and will be stirred using a magnetic stirrer until the solution is thick/gelatin-like. This will be then sterilized at 121°C for 1 hour (standard sterilization time and temperature). After heating and sterilization, it will be put in a plate stirrer and then it will be transferred to an Erlenmeyer flask and will be set aside to cool down. Using the pour-plate method, 15mL of nutrient agar will be poured into each petri dish. The petri dishes will be set aside to let the agar solidify.
Inoculation of Bacteria
The cap of the culture tube will be removed and the bacteria from the swabs will be transferred to the culture media.
Preparation of Plant Extract
The Moringa oleifera (Malunggay) leaf sample will be gathered from the house garden of the researchers. The leaves will be wasted and wiped with a piece of cloth to make it dry. The Malunggay leaves will be crushed in a mortar and pestle to collect its pure extract.
Preparation of Treatments
The Moringa oleifera extract will be poured in their corresponding petri dishes and prepared into the following treatments: 100% moringa oleifera pure extract, amoxicillin powder with water, and penicillin powder with water. Each treatment has three replications.
Application of Treatments and Incubation of the Bacteria
The test disc was made by punching holes in the filter paper. The sterilized filter paper discs were placed in each respective petri dish with the extracts and were soaked for 15 minutes. The soaked filter paper discs were placed on each petri dish containing the cultured bacteria. Three filter paper discs with equal distances from each other were placed in each petri dish. The petri dishes were inverted and incubated for 24 hours at Bio35°C.
BIBLIOGRAPHY
http://www.sciencedirect.com/science/article/pii/037887419190078R http://jb.asm.org/content/51/2/181.full.pdf
http://www.moringaoleifera.org/
http://en.wikipedia.org/wiki/Penicillin
http://en.wikipedia.org/wiki/Moringa_oleifera
http://en.wikipedia.org/wiki/Staphylococcus_aureus
http://en.wikipedia.org/wiki/Amoxicillins
http://www.scielo.br/scielo.php?pid=S0036-46652010000300003&script=sci_arttext http://www.livestrong.com/article/431418-the-health-benefits-of-moringa-oleifera-plants/ http://www.researchmoringa.com/uploads/3CMU_J_Nat_Sci_Antibacterial_Activity.pdf http://www.irjponline.com/admin/php/uploads/1008_pdf.pdf
“Phytotherapy Research”; Moringa Oleifera: A Food Plant with Multiple Medicinal Uses; Farooq Anwar, et al.; November 2006 “Bayero Journal of Pure and Applied Sciences”, 3(1): Antimicrobial Profile of Moringa Oleifera Lam. Extracts Against Some Food-Borne Microoganism; Bukar, A., et al. ; April 2010 “The Nigerian Tribune”: Growing Moringa Oleifera for Money; Oluwatomi Olatoye; June 2010 “Scientific Research and Essay”; Nutritional Potential of Two Leafy Vegetables; Moringa Oleifera and Lpomoea Batatas Leaves; Ibok Oduro et al.; February 2008
