1809235-1274805Republic of the Philippines Department of Education National Capital Region Division of City Schools Quezon City 00Republic of the Philippines Department of Education National Capital Region Division of City Schools Quezon City 4724400-133350-19050-133350 INHIBITING CHARACTERISTICS OF IPIL-IPIL

1809235-1274805Republic of the Philippines
Department of Education
National Capital Region
Division of City Schools
Quezon City
00Republic of the Philippines
Department of Education
National Capital Region
Division of City Schools
Quezon City
4724400-133350-19050-133350
INHIBITING CHARACTERISTICS OF IPIL-IPIL (LEUCAENA LEUCOCEPHALA) SEED EXTRACT ON TRICHOPHYTON MENTAGROPHYTE, ENTEROCOCCUS FAECALIS, PROTEUS VULGARIS, SHIGELLA, AND CLOSTRIDIUM PERFRINGENS
Life Science – Team Category
(Junior High Schools)
A science investigatory project submitted in partial fulfillment of requirements in
Research II-B
Nicole R. Arias
Nicole Ann P. Fernandez
Danielle Marie M. Pua
Project Proponents
Quezon City Science High School
Division, Quezon City, NCR
Mrs. Marianne V. Jacinto
Research Adviser
This study aims to maximize the Ipil-ipil plant’s potential. The initial idea was to test the extract from the seeds as an antifungal agent against a main strand of trichophyton found in athlete’s foot Trichophyton mentagrophyte.

The Ipil-Ipil seeds were gathered in Bulacan, Philippines and were soaked in 95% ethanol for 72 hours. The seeds were discarded and the solution left behind was on the rotary evaporator at a temperature of 30°. Microbial suspension was prepared from 7-day-old culture of fungi of 0.1% peptone water.

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Pre-poured Potato Dextrose Agar (PDA) plates, about 3 mm thick, were inoculated with the microbial suspension by swabbing the agar surface. This procedure was repeated three more times, rotating the plate 90° each time to ensure even distribution of the inoculums. Three (3) equidistant wells were made on the agar plate using a cork borer (10 mm diameter). Two hundred (200) ?l of the sample was placed in each well. The PDA plates were incubated at room temperature for 3 days.
The diameter of the clearing zone gathered were 16mm, 16mm and 17 mm for Ipil-Ipil extract and 70 mm for Canesten solution. The average diameters were taken and computed. The AI was computed for Ipil-Ipil extract and Canesten solution.

Results showed that the Ipil-Ipil showed antimicrobial activity against Trichophyton mentagrophyte with an AI of 0.6.

TABLE OF CONTENTS
TOC o 2-2 “Heading, 3”
ABSTRACT2
INTRODUCTION PAGEREF _Toc1 h 4
MATERIALS AND METHODOLOGY PAGEREF _Toc2 h 5
GENERAL PROCEDURE PAGEREF _Toc3 h 7
RESULTS AND DISCUSSION PAGEREF _Toc4 h 10
CONCLUSION PAGEREF _Toc5 h 13
RECOMMENDATIONS PAGEREF _Toc6 h 14
BIBLIOGRAPHY15

INTRODUCTIONLeucaena leucocephala, or Ipil-ipil, is a small tree that can grow up to 8 meters high.Leucaena leucocephala is from kingdom plantae and the phylum classification is Tracheophyta. It from class leguminosae andorder fabales and came from a family fabaceae..

The Ipil-Ipil tree is originally from Southern Mexico and Northern Central America. It is known to have the ability to spread easily across many tropical locations. Although Ipil-Ipil is not from the Philippines, it has been spreading across the country. It has adapted to the Philippines very well and can be found easily anywhere the country because it is a type of a tropical rainforest plant.

In some provinces of the Philippines, the Ipil-Ipil seeds were used as a coffee substitute. Leaves and seeds are also used as food in Central America, Indonesia and Thailand, and eaten in processed or unprocessed forms in Java, Indonesia. Ipil-Ipil seeds are also used for agriculture and animal feed.
Enterococcus faecalis is a lactic corrosive microorganisms. Enterococci is Gram-positive cocci. Enterococcus species are facultative anaerobic living beings. E. faecalis causes around 80% of human diseases. It may cause bacteremia (the presence of bacteria in blood), abdominal and pelvic infections, urinary tract infections, oral infections (particularly with root canals), and septicemia (blood poisoning).
Proteus vulgaris, previously considered biogroup 2, has been accounted for to cause UTIs, wound contaminations, circulatory system diseases, and respiratory tract contaminations. Proteus vulgaris is known to be an aerobic, rod-shaped, Gram-negative bacterium.

Clostridium perfringens is a gram-positive bacillus that is known produce extracellular toxins C. perfringens type A produces an alpha toxin that can cause acute foodborne illness in human that can result to diarrhea and abdominal discomfort. C. perfringens type A is the cause of necrotizing enteritis in chickens. C. perfringens can be found on raw meat and poultry. It also prefers to grow in conditions with very little or no oxygen, and under ideal conditions can multiply very rapidly
MATERIALS AND METHODOLOGY
Location and duration of the study
The methodology started on August 23, 2018, collecting the Ipil-Ipil and separating the seeds from its pods. The pods, after soaked in 95% ethanol for 3 days, were brought to UP Institute of Chemistry to undergo the process of rotary evaporation. The antimicrobial testing of the of Ipil-Ipil seed extract on Trichophyton mentagrophyte was performed by the scientists of Natural Sciences Research Institute in UP Diliman while the cultivation of Enterococcus faecalis, Proteus vulgaris, Bacillus cereus, and Clostridium perfringens was performed by the researchers in BGR Hydrolab and Testing Center. The reading of the results was done on October 24, 2018 that marks the last day of the whole study. The solution was brought to UP Institute of Chemistry, UP Diliman, Metro Manila on August 29, 2018.
Research Design
Dependent variables/Resulting variables
Inhibition zone of Trichophyton mentagrophyte
Independent Variables
Inhibition zone of Trichophyton mentagrophyte, Enterococcus faecalis, Proteus vulgaris, Shigella, and Clostridium perfringens
In this study, the researchers tested the antimicrobial property of the extraction from Ipil-Ipil seeds (with a concentration of 100%) on different types of microorganisms.

Materials
The materials and the equipments needed for the experiment were as follows: solution, Alcohol lamp, BaSO4 standard equivalent to McFarland Standard No. 2, 10 mm bore size stainless cork borer, sterile cotton swabs, 95% Ethanol in a wide-mouthed jar, inoculating loop, matches, McFarland Standard No. 2, mortar and pestle, permanent marker, 10 ml and 1.0 ml sterile pipettes, plant material, test tube rack, tissue paper, and a white board with 2-4 heavy black lines for comparison of turbidity of the microbial suspense.-118110105537054073517220980043331027249300-3124202759247Extracted solution using rotary evaporation
00Extracted solution using rotary evaporation
19806852740626Filtration of the seeds from the ethanol
4000020000Filtration of the seeds from the ethanol
-443247-451863Collection of Ipil-Ipil fruit
00Collection of Ipil-Ipil fruit
2172937-519448Separation of the seeds from the Ipil-Ipil fruit
4000020000Separation of the seeds from the Ipil-Ipil fruit
47614702133600Soaking the Ipil-Ipil seeds in 95% of ethanol
00Soaking the Ipil-Ipil seeds in 95% of ethanol
1753235866774004757420188595-3215513059670
2075403355836800-174660239511700
435464128348515040062859568240586326779Culturing of bacteria/fungi
00Culturing of bacteria/fungi

301434527432000-17299526659600
39490656301105Anti-microbial testing
400000Anti-microbial testing

10287006575425
39236656782435
-172720173355-445897111366500
31248357302500
-174660239511700GENERAL PROCEDURECollection of Ipil-Ipil seeds
A kilogram of Leucaena leucocephala seeds were hand-picked from San Jose Del Monte, Bulacan and have been authenticated at the UP Institute of Biology. Trichophyton mentagrophyte will be provided and authenticated at the Natural Sciences Research Institute of UP Diliman. The Enterococcus faecalis, Proteus vulgaris, Shigella, and Clostridium perfringens will be provided by BGR Hydrolab and Testing Center.

Preparation of Ipil-Ipil seeds
The Ipil-Ipil seeds were placed in a glass jar. A measure of 1,000mL of 95% ethanol was poured into the glass jar and sat for 72 hours. After 72 hours, the solution and the seeds were separated.

Extraction of the solution
The solution was extracted using rotary evaporator.

Antimicrobial assay of the extract
Preparation of suspension for each test organism using 9 ml 0.1 % peptone water.

Preparation of the seeded agar was done by inoculating appropriate agar with the microbial suspension (i.e. NA for bacteria, GYP for yeast, PDA for molds) and swabbing the agar surface.

A cork borer was flame-sterilized with a 10 mm diameter hole. Three equidistant wells were cut out on each seeded agar plate using the cork borer on the agar plate. Wells were not bored at the center of the seeded plate. Ensure that there is sufficient space between the wells. Transfer the cut out agar discs in a Petri dish. The cork borer was flame-sterilized after usage.
Each test substance was aseptically pipetted out 200µl of each well.

For the antibiotic (positive) control in solution, a single well was cut at the center of the seeded agar plate using a frame-sterilized cork borer. Dispense 100 to 200 µl of the solution. For the antibiotic discs, a disc was placed at the center of a seeded agar plate.

Incubate the seed agar with the test substance and antibiotic standard at 35ºC for 24 hours for bacterial and yeast test organisms and at 30ºC for 3 days for filamentous fungal (mold) test organisms.

After incubation, the clearing zone or zone of inhibition around the well or the disc was observed. Clearing zones were measured in millimeters. The average of the diameters of the clearing zones for the three wells for each extract and compute the antimicrobial index AI using the following formula:
AI = Diameterofclearingzone-DiameterofwellDiameterofwellData Analysis
The diameter of the zone of inhibition for the ethanoic extract was measured using a ruler.
SCOPE AND LIMITATION OF THE STUDY
This study is to determine the effectiveness of the Ipil-Ipil extract against the stated microorganisms in the BGA hydrolab. The measurement of inhibition zones and antimicrobial indexes of each sample will be recorded within a week. The usage of bacteria was considered due to insufficient time. 100% extract was used for the same reason.

Risk and Safety
Biological hazards can be a threat to the health of living organisms, primarily that of humans. This can include samples of a microorganism, virus or toxin (from a biological source) that can affect human health. Doing anything related to microbiology poses a safety hazard. Some examples are skin infection and inhalation of airborne chemicals. In this research project, glassware, bacterium, and flammable materials were utilized. Biosafety level two was considered in dealing with the microorganisms.
Proper Disposal
Since a fungus was used, safety precautions were observed when handling it. All the equipment and materials used were sterilized after work. Work areas were disinfected.

Statistical Analysis
The statistical T-Test (Student’s T-Test) was used to determine the significant differences between the inhibition zones of the fungi as well as the fitness of the study.

RESULTS AND DISCUSSION180975021463000
.
Figure 14.

Figure 14 shows the diameter of the clearing zone (mm) in T. Menta, Enterococcus faecalis, Shigella, Clostridium perfringens, Proteus vulgaris with Ipil-Ipil extract
Figure15.

Test Organism Sample Clearing zone, mm AI
1 2 3 T. mentagrophyte Ipil- Ipil extract 16 16 17 0.6
Canesten solutiona, 20?lPDA 70 6.0
Enterococcus faecalis Ipil- Ipil extract
(MHA) Mueller Hinton Agar 1 1 .75 -0.83
, Distilled water 1 -0.83
Shigella
Ipil- Ipil extract
(MHA) Mueller Hinton Agar .1 .1 .1 -.98
Proteus vulgaris
Ipil- Ipil extract
(MHA) Mueller Hinton Agar .1 .1 .1 -.98
Clostrodium perfringens Ipil- Ipil extract
(MHA) Mueller Hinton Agar 2 .1 -0.66
Distilled water 0 0
Figure 15 shows the antimicrobial index of Ipil-Ipil extract with 100% concentration against the microorganisms. The Ipil-Ipil seed extract is most effective against trichophyton mentagrophyte. The antimicrobial Index of the extract against the fungi was 0.6
CONCLUSIONThe larger the diameter of the clearing zone, the more effective the extract is against Trichophyton mentagrophyte which shows that the Ipil-Ipil seed extract can be used as an alternative ingredient in antifungal creams and ointments against Trichophyton mentagrophyte which is one of the most common fungi found in athletes foot.

This study proves that Ipil-Ipil seeds have an antifungal property to inhibit the Trichophyton mentagrophyte in Athlete’s foot.
RECOMMENDATIONS Since this study proved that the extract from Ipil-Ipil (Leucaena leucocephala) seeds are effective against the fungi (Trichophyton mentagrophyte), the following recommendations could be considered for future studies:
Since the study proves that Ipil-Ipil seed extract can inhibit the Trichophyton mentagrophyte, it can also be tested on other kinds of fungi.
The researchers can test if the Ipil-Ipil plant could have other properties other than antifungal property.

BIBLIOGRAPHY
BIBLIOGRAPHY n.d. Bode science center. Accessed October 25, 2018.

n.d. Center for disease control and Prevention. Accessed October 25, 2018.

n.d. Enterococcus Faecalis. Accessed October 21, 2018.

n.d. Health line. Accessed October 21, 2018.

2004-2015. Ipil-Ipil scientific name: Leucaena Leucocephala. Accessed January 18, 2018.

Peter M. Rabinowitz, Lisa A. Conti. 2010. Clostridium perfringens. Accessed October 21, 2018.

BIBLIOGRAPHY m Jay18 l 1033 Jaya Sureshbabu, Russell W Steele. n.d. Medscape. Accessed October 25, 2018.

Stuart, Godofredo. 2016. Philippine Medical Plants. July. Accessed january 18, 2018