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DETERMINATION OF MINIMUM INHIBITORY CONCENTRATION AND MINIMUM BACTERICIDAL CONCENTRATION OF ALLIUM CEPA

DETERMINATION OF MINIMUM INHIBITORY CONCENTRATION AND MINIMUM BACTERICIDAL CONCENTRATION OF ALLIUM CEPA (ONION) LEAVES EXTRACT ON ESCHERICHIA COLI ATCC # 25922
A Research Paper
Presented to the Faculty of the
College of Medicine
Cebu Doctors’ University
In Partial Fulfillment of the
Requirements for
Research in Medicine II
Block I Module 2
By:
Group 1
Askali, Naseef
Chittipolu, VinithaPriya
Estor, Maria Aubrey Psyche
Jito, LouisanVeth
Ortiz, Drazen Fernando
Raffinan, Abraham
Samson, Rhiel
Uapipatanakul, Thananon
Ypil, GraymarVen
Edwin A. Menguito, MD
Research Mentor
October 2018
ENDORSEMENT
This research proposal entitled “DETERMINATION OF MINIMUM INHIBITORY CONCENTRATION AND MINIMUM BACTERICIDAL CONCENTRATION OF ALLIUM CEPA (ONION) LEAVES EXTRACT ON ESCHERICHIA COLI ATCC # 25922” was prepared and submitted by NaseefAskali, VinithaPriyaChittipolu, Maria Aubrey Psyche Estor, LouisanVethJito, Drazen Fernando Ortiz, Abraham Raffinan, Rhiel Samson, ThananonUapipatanakul to the Proposal Hearing Committee as endorsed by:
Edwin Menguito, MD Ms. Daisy Co, RPh MSc
Research MentorResearch Coordinator
PROPOSAL HEARING COMMITTEE
Ms.LoujessaArcelo, RPh MScChair, Technical
Mr. Richard Capuyan Fidelis Quiza, MD
Panelist, MemberPanelist, Member
ACCEPTANCE OF APPROVAL
This research proposal entitled “DETERMINATION OF MINIMUM INHIBITORY CONCENTRATION AND MINIMUM BACTERICIDAL CONCENTRATION OF ALLIUM CEPA (ONION) LEAVES EXTRACT ON ESCHERICHIA COLI ATCC # 25922″upon approval of the PROPOSAL HEARING COMMITTEE with a grade of PASSED is hereby accepted for implementation in partial fulfillment of the requirements for Research in Medicine II Block I Module II.
ENRICO B. GRUET, MD
College Dean
October 11, 2018
Date of Proposal Hearing
TABLE OF CONTENTS
TITLE PAGE
ENDORSEMENT
APPROVAL SHEET
ACKNOWLEDGEMENT

LIST OF TABLES AND FIGURES
ABSTRACT
INTRODUCTION
Background of the Study
Review of Literature
Significance of the Study
Objectives of the Study
Scope and Limitations
DEFINITION OF TERMS
MATERIALS AND METHODS
Research Design
Research Locale
Research Respondents/Subjects
Research Instrument
Ethical Considerations
Data Collection
Data Processing and Analysis
RESULTS
DISCUSSION
CONCLUSION
RECOMMENDATIONS
REFERENCES

APPENDIX

Appendix A:Transmittal letters and Certifications
Appendix B:Approval from Ethics Committee
Appendix C: Phytochemical testing procedure
Appendix D:Timetable of Research Activities
Appendix E:Duties and Responsibilities of the Research Personnel
Appendix F:Research Budget
Appendix G:Documentation, Maps, Additional
Information
Appendix H:Researcher Profile
INTRODUCTION
Background of the Study
Escherichia coli, a gram negative coccobacilli, that is found in the environment, food and intestine of humans and animals. Infection caused by this bacteria otherwise known as colibacillosis, that usually cause urinary tract infection, diarrhea, respiratory illness and pneumonia 2. In the Philippines, Escherichia coli causing diarrheais the second top leading cause of morbidity and sixth leading cause of mortality for any ages 1. Disease is usually acquired by ingestion from contaminated hands, food and water. The necessity to find herbal and traditional medicine arises from the development of antibacterial resistance to common drug of choice like ampicillin, streptomycin, sulphonamides and tetracycline 9.
There is a need in making life better by introducing natural herbal supplements. For the past decade, herbal medicine has been important in its medicinal use and economical advantage. In under developed countries, as much as 80 percent of the indigenous population still depends on traditional medicine and medicinal plants. Use of herbal medicines has raised issues about their quality, safety, and efficacy. To deal about those issues, thorough examination of the country’s scientific literature of commonly used herbal medicines had been done to address the concerns 5.
Allium cepa, commonly known as onion belongs to the order Liliaceae with tunicated bulb as the underground stem modification. It is a perennial herb and has strong smelling when cut 7. Onion is proved to have antimicrobial effect and is effective against inflammation, cancer, heart diseases, diabetes, kidney problems and infectious diseases. The polyphenols present in the onion protects brain from depression and anxiety. It has also been proved that it has a protective effect against the pathogenic Escherichia coli and in fungi like Candida in dose dependent manner 10.
Onion is one of the most common known and consumed vegetable crops in the world. In addition to its culinary benefit, Onion (Allium cepa) is also used for its medicinal virtues 8. They are used medicinally mainly by utilizing its fleshy bulb that grows under the ground, but other parts of the plant like the leaves also has its traditional medicines 6.

The researchers identified the need to find an alternative treatment against Escherichia coli due to the development of strains that are resistant to antimicrobials and its prevalence in the Philippines causing morbidity and mortality of different diseases.

Review of Literature
In the Philippines, Escherichia coli causing diarrhea is the second top leading cause of morbidity and sixth leading cause of mortality for any ages. Among Vibrio cholera and other organism, Enterotoxigenic E. coli (ETEC) is one of the most common etiologic agents of diarrhea for all ages. These infectious agents are usually spread via fecal-oral route specifically by ingestion of contaminated food or water and contact with contaminated hands 1.

Moreover, as of January to March 2018, DOH 3 identified Top 5 organisms in Acute Blood Diarrhea Cases which includes Escherichia coli as top 3 next to Entamoebahistolytica and trophozoites. The most affected age groups were from 1-4 years of age. The reported regions having most cases are from Region VII, Region X, Region IX, CARAGA and CAR.
In a study conducted by Seenivasan P., J Manickkam and I Savarimuthu4, according to the World Health Organization (WHO), majority of population depends on traditional medicine as their primary healthcare due to its major source of natural organic compound. Plants have been a beneficial source of natural products for preserving human health. The benefit of using plant extracts and phytochemicals is that, it is of great significance for therapeutic treatments with its known antimicrobial properties.

Herbal plants are popular worldwide and in the Philippines due to the clinical proofs that validates many alternative medicines used by Filipino folks that has been passed on. The knowledge on the usage of the different medicinal plants have been passed down to generations. Medicinal plants are found anywhere that plant are able to grow. Several factors affect the effectivity of medicinal plants; factors like the environment which the plant grew in will affect the components, as well as how and when it was harvested and also the processing of the plant 5.

Allium cepa, commonly known as onion, is thought as a vegetable. It is known as “sibuyastapul” which belong to a member of Lilliaceae. They are used medicinally mainly by utilizing its fleshy bulb that grows under the ground, but other parts of the plant like the leaves also has its traditional medicines. There are many varieties of onion bulbs which can be white, yellow or red. The leaves and green stems of onion are usually hollow that reaches to a height of 3 ft (1m) 6. Onion has tunicated bulb as the underground stem modification. It is a perennial herb and has strong smelling when cut 7.

Moreover, it is one of the most common known and consumed vegetable crops in the world. In addition to its culinary benefit, Onion is also used for its medicinal virtues. Many studies showed that this plant was found to have various bioactive compound with many pharmacological properties such as antioxidant, analgesic, anti-inflammatory, anti-diabetic, hypolipidemic, anti-hypertensive, immunoprotective effects and lastly antimicrobial effect 8.

Plant products especially spices and plant part extracts have used as natural antimicrobial and antioxidants agents. Results of the study showed that the onion, ginger and garlic had an antifungal effect on Cladosporium herbarum after 2 days of incubation. The growth inhibitory effect of onion extract on Aspergillus flavus, Aspergillus niger was significantly high 9. Onion is proved to have antimicrobial effect, is effective against inflammation, cancer, heart diseases, diabetes, kidney problems and infectious diseases. The polyphenols present in the onion protects brain from depression and anxiety. It has also been proved that it has a protective effect against the pathogenic Escherichia coli and in fungi like Candida in dose dependent manner 10.

A worrying issue about Escherichia coli increasingly being multidrug resistance is increasingly observed in human and also in veterinary medicine worldwide. A study by Poirel, et al 11., mentioned that E. coli samples from animals were resistant to drugs such as tetracycline, sulfonamides, phenicols, and fosfomycin. This was due to the activity of plasmids as well as other mobile genetic elements. Another study by Kahlmeter, et al 12., found out that E. coli isolated from females that had acute, uncomplicated urinary tract infections have a particular increasing resistance to the drugs, ciprofloxacin and trimethoprim.

Based on the results of the study “Antibacterial effects of Onion” by Kabrah 13, among the three types of onion, green, red and white, white onion in onion pieces method produced inhibitory effect on the growth of Pseudomonas, Escherichia coli, Salmonella, MRSA, MSSA and Klebsiella but at various bactericidal effects depending on its used concentration and type of tested bacteria. In the study Antibacterial Activity of Allium Cepa (Onion) On Some Pathogenic Bacteria Associated with Ocular Infections, the fresh onion extract was observed to inhibit bacteria isolated from ocular infection compared to cold water onion extract. In addition, Escherichia coli were found to be sensitive to fresh onion extracts as well as Staphylococcus aureus and Streptococcus pyogenes14.
In order to assess bactericidal and bacteriostatic activity of new emerging classes of antimicrobial agent, an established laboratory method is essential to assess its effectivity against certain microorganisms. Determination of bactericidal activity utilizes microdilution method. The minimal concentration of antimicrobial agent needed to prevent the growth (>99.9%) of the viable bacteria after incubation for almost 24 hours is termed as Minimium bactericidal concentration (MBC) 18. Meanwhile, minimum inhibitory concentration (MIC) is considered to be the gold standard, popular and practical for determining antimicrobial susceptibility against certain microorganisms. The advantage of this procedure includes: its reproducibility and convenience of having preprepared panels, and the availability of reagents and space that occurs due to the miniaturization of the test. It is defined as the lowest concentration of the antimicrobial agents that inhibit the growth of a viable microorganism after 24 hours of incubation 15,16,19.

To determine whether an organisms is said to be sensitive or resistant against antimicrobial agents, an Kirby – bauer disc diffusion method are generally used and recommended by the CLSI guidelines. The advantages of this method are its test simplicity which doesn’t require any special equipment, the results that are easily interpreted and its flexibility in selection of disks for testing. Among all the susceptibility methods, this is the least costly. This requires incubation for certain time and the mean diameter of the zone of inhibition is measured to determine the sensitivity or resistance of a certain microorganism to several antimicrobial agent 17,19.

Significance of the study
The result of the study provided information on the antimicrobial and optimal effective concentration of Allium cepa(onion) leaves extract on Escherichia coli.

To the Community, it will help them find a cheaper, affordable and readily available antimicrobial that is adjunct to the treatment of common bacterial diseases caused by Escherichia coli.

To the Manufacturers, it will be a great breakthrough in producing and developing a cheap but effective antimicrobial drug made out ofonion leaves extract, because onion are cheaper one than the ones currently used in commercially made antimicrobial drugs. They may also utilize the results of the study to deepen the understanding and widen the scope concerning its antimicrobial activity.

To the future researchers, that they may use the result of this study as a basis for developing low-cost and effective treatment for diseases caused by Escherichia coli. Further research can be done with other extracts with properties similar to onion.

Objectives of the study
General Objective
To determine the antimicrobial effect of Allium cepa (onion) leaves extract against Escherichia coli.

Specific Objectives
To determine the diameter of zone of inhibition (in millimiter) against Escherichia coli, exhibited by:
Experimental Group (onion leaves extract)
Positive Control Group (Ampicillin)
Negative Control Group (Normal Saline Solution)
To compare the mean diameter of zone of inhibition whether there is a significant difference among the experimental treatment onion leaves extract from the positive control (Ampicillin) and negative control (Normal Saline solution) against Escherichia coli.

To determine minimum inhibitory concentration and minimum bactericidal concentration of experiment treatment onion leaves extract from the positive control (Ampicillin) and negative control (Normal Saline solution) on Escherichia coli.

To determine the optimum concentration of onion leaves extract with the greatest antimicrobial effect against Escherichia coli.

Statement of Null Hypothesis
There was no significant difference in the mean diameter of zones of inhibition between experimental treatment onion leaves extract from the positive control (Ampicillin) and negative control (Normal Saline solution) against Escherichia coli.

Statement of Alternative Hypothesis
There was a significant difference in the mean diameter of zone of inhibition of onion leaves extract from the positive control (Ampicillin) and negative control (Normal Saline solution) against E.coli.

Onion leaves extract in different concentration have significant antimicrobial effect against Escherichia coli.

Scope and Limitations
This study focused on the determination of the antimicrobial effect and the zone of inhibition around the antimicrobial discs of the various concentration of onion leaf extract on plates against the organism Escherichia coli. This study will utilize only the onion leaves through the manual pounding of its leaves by using mortar and pestle.
In the Mueller – Hinton agar, the application of the antimicrobial disc to the plate was another limitation. The disc may be placed deep or too superficial on the agar. To minimized errors like these, the researcher who are skilled and Registered Medical Technologist applied the antimicrobial disc into the Mueller – Hinton agar plate. To measure the diameter of zone of inhibition, a vernier calliper was used in order to average all the results which were read also by Registered Medical Technologist that help minimize reading error.

CONSORT FLOW CHARTEscherichia coli ATCC # 25922

Excluded
Other strains of E.coli
Preparation of Filter paper disc
Experimental Group
Leaf extract of Allium cepa (onion)
n = 8
Measuring its effectiveness
Determination of minimum inhibitory concentration (MIC)
Analysis and Interpretation of results
Positive Control
Ampicillin Antimicrobial disc
n = 8
Negative Control
Normal Saline Solution
Antimicrobial disc
n = 8
Determination of minimum bactericidal concentration (MBC)
Measurement of zone of inhibition
2552700571500255270057150041910023368004191002336800571500378460057150037846006223004597400622300459740062230058674006223005867400406400234950040640023495002438400234950024384002349500430530023495004305300234950024384003784600243840037846006096004597400609600459740026162004597400261620045974004508500462280045085004622800245110020447002451100204470024511003568700245110035687005588003568700558800356870044577003568700445770035687002628900441960026289004419600622300565150062230056515002692400565150026924005651500452120056515004521200565150026924005880100269240058801002565400102870025654001028700Definition of terms:
Antimicrobial effect – refers to the action of the leaf extract of onion that will inhibit and prevent the growth of bacteria through microdilution method and disc susceptibility testing .

Allium cepa (onion) leaf extract – belongs to the Lilliaciae family that will be used to produce the juices through mechanically pounding of the onion leaves.
Escherichia coli ATCC # 25922 – a gram positive pure culture that will be purchased from MaCare in Cebu City.

Minimum inhibitory concentration – the lowest concentration of
onion leaf extract that will inhibit the evidence of growth of bacteria after 24 hour of incubation on the tubes or microdilution wells as detected by the unaided eye.

Minimum bactericidal concentration – the lowest concentration of onion leaf extract that will prevent the growth of bacteria on freshly inoculated agar plates which will be assessed by the naked eye.

Zone of inhibition – the clear area surrounding the antibiotic disc produced by the onion leaf extract.
MATERIALS AND METHODS
Research Design
This study will utilize the experimental method.
Research Locale
The study will be conducted at Cebu Doctors’ University research laboratory and Microbiology – Parasitology Laboratory at College of Medicine.

Research Subjects
The research subjects of the study include Escherichia coli (ATCC # 25922), which will be purchased from MaCare Cebu City, Philippines. It will be divided into three groups by sub-culturing it into twenty four (24) plates.

Inclusion Criteria
Only Escherichia coli ATCC # 25922 will be used in this study.

Exclusion Criteria
The researchers will not include other strains of Escherichia coliz
Research Instrument
This study will make use of the Vernier calliper to measure the zone of inhibition exhibited by onion leaves extract discs, the positive control (Ampicillin) and negative control (NSS) disc within the Mueller – Hinton agar plate.

Data Collection Procedures
Preliminary Procedure
After the title will be approved by the CDU research council, the research paper will be evaluated for any duplication in the main and medicine library. It will be then submitted to the research office for scheduling of statistician to evaluate the title. The researchers will be submitting a transmittal letter to the Dean of College of Medicine, to allow the study to be conducted and to the head of the research laboratory, to allow the researchers to use the necessary laboratory apparatus.
Collection and Identification of Organism
A stock culture of Escherichia coli ATCC # 25922 will be purchased from MaCare Cebu city, Philippines.

Collection of onion leaves
A fresh greeny onion leaves will be selected for this study. It will be purchased from a vegetable garden in Naga city barangay Na-alad and will be authenticated by the Department of Agriculture. It will be then washed using distilled water to remove any unwanted dirt and allowed to air dry for almost an hour. The researcher will collect all leaves of onion by wearing plastic gloves to maintain its sterility.
Preparation of onion leaf extract
A weighing scale will be used to weigh a hundred grams (g) of onion leaves. It will be sliced and chopped into smaller pieces to allow easy way of pounding manually using mortar and pestle. The extracts that will be obtained from manually pounding the onion leaves will be strained in a sterile filter paper and will be collected into a petri dish.
Preparation of Filter Paper Discs
Using an office puncher, holes of approximately 6 mm diameter were punched. It will be made sure to avoid overlapping of holes. The discs will then be autoclaved at 15 lbs pressure for 30 minutes.

Preparation of Disc Impregnation with the leaf extract of onion
The sterile discs were placed approximately 5 mm apart inside a petri dish. Using an automated mechanical pipette, a fixed volume of 20?l was impregnated on each disc.

Preparation of Disc Impregnation with Negative Control (NSS).

In another sterile petri dish that contains 5 ml of Normal Saline Solution, another eight (8) pieces of circular filter paper disc will be submerge for an hour to allow enough time to be absorbed by the filter paper disc.

Preparation of Positive control (Ampicillin) is a commercially prepared antibiotic disc.

Experimental Procedure
Preparation of Bacterial Suspension
From the stock culture, a loopful colony will be isolated and suspended into a 5 ml normal saline solution using a sterile tube. This procedure will further be repeated until the suspension of Normal saline solution turbidity matches that of the McFarland standard. It will be then incubated for 15 minutes at 370C.

Inoculum Preparation
A sterile cotton swab will be used to dip into the standardized suspension and will be inoculated into the Mueller Hinton Infusion agar using streak method.

Disc Application on the Culture
A total of (24) twenty four plates with Mueller Hinton Infusion agar will be used. It will be divided into three groups wherein each groups have (8) petri dishes comprising the experimental, positive and negative group. A sterile forceps will be used to place the antibiotic disc (Ampicillin), designated as positive group in eight (8) plates; other eight (8) plates will contain the Normal saline solution, designated as negative group, and the last eight (8) plates will be impregnated with leaf extract of onion as the experimental group. The incubated Mueller Hinton Infusion agar will then be incubated at 370C for 24 hours in an upside down to prevent the droplets of moisture collecting in the surface of the agar to cause separate colonies to join together. After incubation for 24 hours, the mean diameter of zone of inhibition of the disc will be measured and read.

Determination of Minimum Inhibitory Concentration:
Leaf extracts of Allium cepa (onion) which exhibit a strong antibacterial activity will be manipulated to determine its minimum inhibitory concentration (MIC) microdilution method. Through the use of sterilize plastic microdilution trays that is described to have conical and round wells, microdilution method will be perform. In each microdilution trays, a broth of 0.1 mL of final volume is added. In each row, the researchers will add to each well of column 2 through 10 a volume of 0.05 mL of broth CAMHB. The same broth which were used in wells 2 through 10 is used to prepare the antibiotic-containing solution. The researchers will then add to each of the first two wells a 0.05 mL of this solution. After that, wells from column 2 through 9 will be use to make a serial twofold dilutions of the antibiotic using semiautomatic microdilution device that employs a 0.05-mL microdiluters. then moving and transferring 0.05 mL to each well. In the ninth well, discard 0.05 mL. The tenth well which contains 0.1 mL of broth being used with no antibiotic will serve as a positive growth control.

Within 15 minutes after standardized of the actively growing inoculum, it will then be added to each tube an adjusted 0.1 mL of inoculum in a dilution series using a pipettor to expel beneath the surface of antimicrobial-containing broth the inocula. It will then be shake, mix through vortex at low speed or by using hands.
The microdilution wells will be inoculated by first adding 0.05 mL of broth to each well after the dilutions have been made and then add 0.01 mL of inoculum.

Through the use of a well calibrated pipetor. In this case, the number of organisms in the seed tray should be 5 x 106 CFU/mL and the actual inoculum in a growth control well must be determined immediately after inoculation. During incubation, to prevent evaporation, each tray in a plastic bag should be sealed properly with the use of a plastic tape, or with a tightly fitted plastic cover before incubation.

Determination of Minimun Bactericidal Concentration:
Minimum bactericidal concentration (MBC) will be determined using microdilution by using a logarithmic-phase culture for the final inoculum that is 5 x 10^5 CFU/mL.The final inoculum will then be added to the microdilution tubes in 0.1 mLsmall volume below the surface with no shaking. It will be then incubated at 35 ºC. Gently mix by vortex at a low speed or by hand after 20 hours and again at 24 hours before sampling. Through the use of a calibrated pipettor, subculture 0.01mL from each clear test tube; duplicate subcultures must be done. On an agar plate, streak the broth subcultured, then incubate it for 24 hours. Use the number of colonies subsequently grown to determine the bactericidal endpoint based on the final inoculum and the rejection values.

Phytochemical screening
The leaf extract of onion will be further analyze to determine its phytochemical contents by undergoing preliminary phytochemical test using standard methods. Saponins will be determined through Froth test. The dragendroff’s test were carried out for the presence of alkaloid. Presence of increase amount of brick red precipitate denotes for glycosides. The Ferric chloride test will be used to determine presence of tannins. Dissappearance of yellow coloration through dilution of ammonia solution followed by addition of concentrated sulphuric acid indicates the presence of flavonoids. Salkowski’swill be used for detection of steroids and Triterpenoids (see appendix C).

Actual Data Gathering
Vernier calliper will be used in order to measure the diameter of the zone of inhibition exhibited by all twenty – four disc. The diameter of zone of inhibition will be compared if Escherichia coli is resistant, intermediate or susceptible. sMinimum inhibitory concentration is the lowest concentration of antimicrobial agent that completely inhibits growth of the microorganisms in the tubes or microdilution wells as detected by the unaided eye.Minimum bactericidal concentration will be determined by no growth on subculture on agar plate which will be assessed by the naked eye.

Data Processing and Analysis
Descriptive statistics using mean and standard deviation will be computed from the data obtained in the experiment
Reference
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Center for disease control and prevention.E.coli (Escherichia coli). https://www.cdc.gov/ecoli/. (accessed 22 September 2018)
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Seenivasan P, J Manickkam, Savarimuthu I. In vitro antibacterial activity of some plant essential oils. BMC Complem. Altern. M. 2016; 6(1): 39 – 46. (accessed 19 September 2018).

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Teshika JD, Zakariyyah AM, Toorabally Z, Zengin G, Rengasamy KR, Pandian SK, et al. Traditional and modern uses of onion bulb (Allium cepa L.): A systematic review. Critical Reviews in Food Science and Nutrition. 2018;1–75. (accessed 20 September 2018)
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APPENDIX A
TRANSMITTAL LETTER AND CERTIFICATION

October 2, 2018
Dr. Enrico B. Gruet
Dean, College of Medicine
Cebu Doctors’ University
Mandaue City, Cebu, Philippines
Dear. Dr.Gruet,
Greetings of peace
We are members of Level II Group 1 medicine students. As a requirement for medical students from the Cebu Doctors’ University College of Medicine, we would like to conduct a research entitled “Determination of minimum inhibitory concentration and minimum bactericidal concentration of Allium cepa(onion) leaves extract on Escherichia coli ATCC # 25922.

We are hoping for your kind consideration and approval. Thank you very much and God bless!
Respectfully,
Naseef S. Askali
Research Group Leader
Endorsed by:
Edwin Menguito, MDDaisy Co, RPh MSc
Research MentorResearch Coordinator, Level 2
Endorsed by:
Teresita Chiu, MD MHPEdMaria Fe F. Abejar, MA MAT RPsyc
Head, Medical Educational Unit Head, Research Office
Approved by:
Enrico B. Gruet, MD
Vice President for Academic Affairs

October 2, 2018
Dr.Roserie T. Quisumbing
Head Microbiology Department
Cebu Doctors’ University
Mandaue City, Cebu, Philippines
Dear. Dr.Quisumbing,
We are members of Level II Group 1 medicine students at Cebu Doctors’ University. We would like to humbly ask your permission to allow us to conduct our research entitled “Determination of minimum inhibitory concentration and minimum bactericidal concentration of Allium cepa(onion) leaves extract on Escherichia coli ATCC # 25922”.We hope to make use of the material and instruments in the microbiology laboratory, most especially the incubator, autoclave, biosafety cabinet, micropipette, test tube racks, vernier calliper, wire loop, forceps, alcohol lamps and stove once the bacteria arrives.

We are looking forward to your approval on this matter as this can greatly help in the success of our research conduct.

We hope for your kind consideration. Thank you very much and God bless!
Respectfully,
Naseef S. Askali
Research Group Leader
Endorsed by:
Edwin Menguito, MDDaisy Co, RPh MSc
Research Mentor Research Coordinator, Level 2
Endorsed by:
Teresita Chiu, MD MHPEd Maria Fe F. Abejar, MA MAT RPsyc
Head, Medical Educational Unit Head, Research Office
Recommending Approval:
Roserie T. Quisumbing, MD
Head Microbiology Department
Approved by:
Enrico B. Gruet, MD
Vice President for Academic Affairs

APPENDIX B
APPROVAL FROM ETHICS COMMITTEE
APPENDIX C
PHYTOCHEMICAL TESTING PROCEDURE
Determination of Saponins(Guevarra, 2005)
Boil around 500 mg of onion leaf extract with 5 ml of distilled water, filtered and cooled.

Froths test: To a 2.5 ml filtrate of onion leaves, add 10 ml of distilled water and shake vigorously for about 30 seconds. Let it stand for 10 minutes and observe for the presence of more than 2 cm height of “honeycomb” froth at the surface of the liquid.

Determination of Alkaloid (Iqbal et al., 2015)
Stir around 15 mg of onion leaf extract with 1% HCL (5 ml) for 5 minutes on a water bath then filtered.
Dragendorff’s test: To a 2.5 ml filtrate of onion leaves, add 1 ml of
dragendroff’s reagent (Potassium bismuth iodide solution). Presence of alkaloids will show orange red precipitate.

Determination of Glycosides (Ongtengco and Capal, 2000)
Dissolved around 500 mg of onion leaf extract with 5 ml of hot water and filtered. The filtrate will be used for the test. Prepare 2 test tubes and place a sample of 2 ml in each test tube. To sample 1, 1 ml of 10% HCL will be added. To sample 2, nothing is added. For 5 minutes, place the 2 test tube in a boiling water bath and allowed it to cool after. Both sample will be neutralized using anhydrous sodium carbonate until no detectable effervescence is produced. Fehling’s solution will be added by mixing 3 ml of Fehlings A and 3 ml of Fehlings B. Use 1 ml of Fehlings solution and heat the 2 test tubes for 2 minutes on a water bath. Observe for the amount of brick red precipitate that is formed. The increased in the amount of brick red precipitate in hydrolyzed sample (sample with dilute acid) as compared to the other sample will denote the presence of glycosides.

Determination of Tannins (Ongtengco and Capal, 2000)
a..Ferric Chloride Test: Dissolved around 500 mg of onion leaf extract in a test tube with 5 ml hot water and filter it while it’s still hot. Add 2 drops of 0.1% ferric chloride solution to the filtrate. A dark color precipitate which can be either black, blue – black, dark blue, green, blue green or brownish green coloration indicates presence of tannins.

5. Determination of Flavonoids (Ara et al., 2010)
a. Method 1: About 5 ml of diluted ammonia solution will be added to aqueous filtrate of a 500 mg leaf extract of onion that is dissolved in a 5 ml of water. An 1 ml of concentrated sulphuric acid will then be added. The presence of flavonoids will be detected upon yellow coloration that disappears on standing.

6. Determination of Steroid and Triterpenoids(Ara et al., 2010)
a. Salkowski’s Test: To around 100 mg of onion leaf extract, 2 ml of chloroform will be added and 3 ml of concentrated H2SO4 was added to each portion to form a layer. The presence of steroids is determined through a reddish brown coloration of the interface.

APPENDIX D
TIMETABLE OF RESEARCH ACTIVITIES
Task Name September October
WEEK 1 WEEK 2 WEEK 3 WEEK 4 WEEK 1 WEEK 2 WEEK 3 WEEK 4
Brainstorming Refining Title and Objectives Finish writing and
pass research plan Accomplish Title Screening Search and Write RRL First Draft (For comments and suggestions) plus signatures Process forms 4A-4D Edit and Rewrite Process Anti-Plagiarism (4E) Process Phase 2 of Form 1 Submission of Research Proposal to Panelist Research Proposal Edit Research Proposal Approval/ Signature of Panel (Form 7) Submission of Revised Research Proposal Ethical Review of Paper APPENDIX E
DUTIES AND RESPONSIBILITIES
Name of the Researchers
Role/ task of contributors
Naseef Askali In-charge of keeping all documents valid. Manages task for the group. Responsible for scheduling consultations between the group and the research coordinator and mentor. Content contributor for the RRL about Herbal and traditional medicines, methods, result and discussion.

Vinitha Priya ChIttipolu Responsible for RRL about onion taxonomy and characteristics. Content contributor for significance of the study. Assigned for proof-reading.

Maria Aubrey Psyche A. Estor Responsible for proof-reading, RRL about health benefits of onion and prevelance of E.coli. Content contributor for the output of our studies and CV. Manage the task by reminding everyone about the status of our studies.
Louisan Veth P. Jito Responsible for review of related literature about E.coli common diseases and discussion. Content contributor of significance of the study, Background and definition of terms.

Drazen Fernando N. Ortiz Content contributor for the Scope and Limitations, background of study, materials and methods, RRL about E.coli. Responsible for proof-reading and checking for topographical errors.

Abraham P. Raffinan Responsible for proof-reading, RRL on methods. Content contributor of materials and methods and Background of the study.

Rhiel S. Samson Responsible for RRL about E.coli being resistant to some drugs. Responsible for having the documents signed when other members are not available. Content contributor for material and methods, Scope and Limitations and Gantt chart.

Thananon Uapipatanakul Content contributor for the RRL about onion leaves health benefits, results and budget expenses. Responsible for proof-reading and checking topographical errors.

Graymar Ven E. Ypil Content contributor of significance of the study, RRL for E.colo description, Ethical consideration and definition of terms.
Appendix F:
Research BudgetExpenses Cost (Php)Three reams of bond paper (8.5×13) 690 Photocopy400 Ink for printer – Black (2) 500 Snacks during the proposal hearing 300 Bookbinding (3 copies) 1,350 Antibiotic (Ampicillin) 300Onion leaves150Petri Dish with MHIA 1000Test Tubes (4) 150Stock Culture of E.coli3000Statistical Analysis Fee 500 Anti-Plagiarism Fee 1,400 Research Honorarium 1,000 TOTAL: P 10,740
Research Profile
CURRICULUM VITAE3856990215265
Personal Background:
Name: NaseefSakandalAskali
Date of Birth: May 19, 1995
City Address: Mandaue, Cebu City
Provincial Address: San Jose Gusu, Zamboanga City
Email Address: [email protected] of Parents: Abdulnajar A. Askali and Nuraina S. Askali
Address of Parents: Mambalot, Palawan
Gender: Male
Religion: Islam
Civil Status: Single
Educational Background:
Tertiary
Course: Bachelor of Science in Medical Technology
School: Universidad de ZamboangaAddress: Main Campus, Don Toribiost., Tetuan, Zamboanga CityInclusive Dates: 2012- 2016
Secondary
School: Philippine School of DohaAddress: MadinathKhalifa South; Doha, QatarInclusive Dates: 2008-2012
ElementarySchool: Notre Dame of Jolo, Sulu / Notre Dame of BongaoAddress: Astoria Street., Jolo, Sulu /BongaoTawi – TawiInclusive Dates: 2002-2008
CURRICULUM VITAE362394548895
Personal Background:
Name: Chittipolu, VinithaPriya
Date of Birth: May 08,1997
City Address: India
Provincial Address: Mandaue City
Email Address: [email protected]
Name of Parents: Mr. and Mrs.Satyanarayana
Address of Parents: India
Gender: Female
Religion: Hindu
Civil Status: Single
Educational Background:
Tertiary
Course: Bachelor of Science in Biology
School: Cebu Doctors’ UniversityAddress: NRA, Mandaue City, Cebu, 6014, Philippines.
Inclusive Dates: 2015-2016
Secondary
School:Hyderabad Public School/ Sri GayatriEdu. InstitutionsAddress:Ramanthapur, Hyderbad,Telangana, IndiaInclusive Dates: 2009-2012, 2012-2014
ElementarySchool: The Hyderabad Public School
Address: Ramanthapur, Hyderbad,Telangana, India
Inclusive Dates:2002-2009
CURRICULUM VITAE380047547625
Personal Background:
Name: Maria Aubrey Psyche A. Estor
Date of Birth: September 30, 1996
City Address: Mandaue city, Cebu
Provincial Address: Jasaan, Misamis Oriental
Email Address: [email protected]
Name of Parents: Sol and Corazon A. Estor
Address of Parents: Jasaan, Misamis Oriental
Gender: Female
Religion: Roman Catholic
Civil Status: Single
Educational Background:
Tertiary
Course: Bachelor of Science in Medical Technology
School: Cebu Doctors’ UniversityAddress: North Reclamation, Mandaue City, CebuInclusive Dates: 2013- 2017
Secondary
School: Xavier University High School- Ateneo de CagayanAddress: Pueblo de Oro, Cagayan de Oro CityInclusive Dates: 2009 -2013
ElementarySchool: Jasaan Central SchoolAddress: Jasaan, Misamis OrientalInclusive Dates: 2003-2009
CURRICULUM VITAE3814763285750
Personal Background:
Name: LouisanVeth P. Jito
Date of Birth: February 16, 1996
City Address: 187 SB Cabahug St. Ibabao,
Mandaue City.

Provincial Address: Ticad, Bantayan, Cebu
Email Address: [email protected]
Name of Parents: Velvet P. Jito and Santiago G. Jito Jr.

Address of Parents: Bantayan, Cebu
Gender: Female
Religion:Roman Catholic
Civil Status: Single
Educational Background:
Tertiary
Course: Bachelor of Science in Medical Technology
School: Velez CollegeAddress: 121 F. Ramos St., Cebu CityInclusive Dates: 2013- 2017
Secondary
School: Bantayan Science High SchoolAddress: M.Y. Village, Ticad, Bantayan, CebuInclusive Dates: 2009-2013
ElementarySchool: Bantayan Central Elementary SchoolAddress: Ticad, Bantayan, CebuInclusive Dates: 2003-2009
CURRICULUM VITAE359568876200
Personal Background:
Name:Drazen Fernando N. Ortiz
Date of Birth: September 11, 1995
City Address: Cambiohan, Consolacion, Cebu
Provincial Address: Cebu city
Email Address: [email protected]
Name of Parents: Fernando F. Ortiz and Thelma N. Ortiz
Address of Parents: Cambiohan, Consolacion, Cebu
Gender: Male
Religion: Roman Catholic
Civil Status: Single
Educational Background:
Tertiary
Course: Bachelor of Science in Biology
School: Cebu Doctors’ University
Address: Mandaueciy, Cebu
Inclusive Dates: 2012- 2017
Secondary
School: Sacred Heart School – Ateneo de Cebu
Address: Mandaue city, Cebu
Inclusive Dates: 2008-2012
Elementary
School: Maria Ernestine School
Address: BudiaanTalamban, Cebu city
Inclusive Dates: 2002-2008
CURRICULUM VITAE3754120186055
Personal Background:
Name: Abraham P. Raffiñan
Date of Birth: November 11, 1994
City Address: Mandaue City
Provincial Address: M.L. Quezon Street, Maguikay
Email Address: [email protected]
Name of Parents: Rey Juan Agapito V. Raffiñan, Giovanna P. Raffiñan
Address of Parents: Luisa Perez Cmpd, M.L. Quezon St., MaguikayMandaue City
Gender: male
Religion: Roman Catholic
Civil Status: Single
Educational Background:
Tertiary
Course: Bachelor of Science in Medical Technology
School: Cebu Doctors’ UniversityAddress: 1 Dr.P.V. Larrazabaljr. Avenue, North Reclamation area Inclusive Dates: 2012- 2016
Secondary
School: Saint Louis College-CebuAddress: Sudlon, Maguikay, Mandaue CityInclusive Dates: 2008-2012
ElementarySchool: Sotero B. Cabahug Forum for LiteracyAddress: Winfields street, PulpuganConsolacionInclusive Dates: 2002-2008
CURRICULUM VITAE3590925-3808
Personal Background:
Name: Rhiel Soon Samson
Date of Birth: October 16, 1991
City Address: Naga City
Provincial Address: Mandaue City
Email Address: [email protected]
Name of Parents: Aniceta and Odile Samson
Address of Parents: New York Harriman 10926
Gender: Male
Religion: Roman Catholic
Civil Status: Single
Educational Background:
Tertiary
Course: Bachelor of Science in Medical Technology
School: Cebu Doctors’ UniversityAddress: North Reclamation Area, Mandaue city, CebuInclusive Dates: 2012- 2016
Secondary
School: New York Military AcademyAddress: Cornwall on Hudson, New York 10926Inclusive Dates: 2008-2012
ElementarySchool: Pine Tree ElementaryAddress: Monroe Woodbury, New York 109250Inclusive Dates: 2000-2007
CURRICULUM VITAE3766283292735
Personal Background:
Name: ThananonUapipatanakul
Date of Birth: November 7, 1993
City Address: Boardwalk residence, Mandaue city
Provincial Address:Cebu
Email Address: [email protected]
Name of Parents: SupakoraneeTangchitnob
Address of Parents: Bangkok, Thailand
Gender: Male
Religion:Buddhist
Civil Status: Single
Educational Background:
Tertiary
Course: Bachelor of Science in Physical Therapy
School: Chulalongkorn UniversityAddress: Bangkok, ThailandInclusive Dates: 2012- 2015
Secondary
School: Assumption College SchoolAddress: Bangkok, ThailandInclusive Dates: 2009-2011
ElementarySchool: SarasasEktra SchoolAddress: Bangkok, ThailandInclusive Dates: 1999-2009
CURRICULUM VITAE3756163-408337576134763
Personal Background:
Name: GraymarVen E. Ypil
Date of Birth: July 09, 1993
City Address: 3 TambisUrgello St., Cebu City
Provincial Address: 3 TambisUrgello St., Cebu City
Email Address: [email protected]
Name of Parents: Grace Orma E. Ypil (Mother)
Venustiano D. Ypil (Father)
Address of Parents: 3 TambisUrgello St., Cebu City
Gender: Male
Religion: Protestant
Civil Status: Single
Educational Background:
Tertiary
Course: Bs – physical therapy
School: Cebu Doctors’ UniversityAddress: North Reclamation area, Mandaue City
Inclusive Dates: 2000- 2015
Secondary
School: Southwestern University/ USC/ M.Zed Christian schoolAddress: Aznar road, urgellost., Cebu cityInclusive Dates: 2009 – 2010, 2007 – 2009, 2006 – 2007
ElementarySchool: M.Zed Christian schoolAddress: Horsehoe hills, banawa, Cebu cityInclusive Dates: 2000 – 2006