RESEARCH PLAN

Research Title: Characterization and Synthesis of Flavanoids as Biofilm Inhibition in Ashoka leaves
(Saraca asoca ) on Staphylococcus aureus
Proponents:
Chua, Twinkle G.
Gabriel, Queen Herzlein S.
Nabong, Rhyzza Khate B.

Research Adviser: Mr. Alexis S. Rodelas

School/Address: Cayetano Arellano High School T. Alonzo Sta. Cruz, Manila
A. Rationale

Food and waterborne diseases usually take place within communities lacking
in proper sanitation. These are conditions caused by eating or drinking food or water
that is contaminated by microorganisms or the toxins they produce.

Food Poisoning is an illness caused by eating contaminated food. People

usually takes get better within a few days. In most cases, the food is contaminated by
bacteria. One of the most frequent pathogen causing this illnesses is the
Staphylococcus aureus. Present this bacteria it is sufficient to contaminate the food
enough resulting to this illness. It usually takes 2 or 3 days to recover from S. aureus
food poisoning, but some require more time to recover. To prevent this from
happening we should to employ proper food sanitation and preparation.

According to researches, Ashoka leaves (Saraca asoca) has its antibacterial

properties that could possibly eliminate bacteria due to the presence of flavonoids
(well-known antibacterial agents) and primary and secondary
metabolites. It is in this reason that the researcher thought of evaluating the
inhibitory effect of Ashoka Leaves extract against S. aureus.

This study seeks to verify the active component that in Ashoka leaves (Saraca
asoca) that can eliminate the Staphylococcus aureus that causes Food Poisoning. It
aims the characterization and Synthesis of Flavanoids as Biofilm Inhibition in Ashoka
leaves (Saraca asoca) on Staphylococcus aureus

B. Research
Questions: 1. Will the Ashoka leaves be strong enough to kill Staphylococcus aureus?
2. What is the active component in Ashoka leaves (Saraca asoca) that can inhibit
the growth of Staphylococcus aureus?
 3. Is there a harmful effect in using Ashoka leaves as a killing component for
Staphylococcus aureus?

Hypothesis: Alternative:
The Ashoka leaves will inhibit the growth of Staphylococcus aureus.
Null:
The Ashoka leaves will not be able to inhibit the growth of Staphylococcus aureus.

Engineering
Goals: Ashoka leaves as a main component in killing and reducing the amount of
Staphylococcus aureus.

Expected
outcomes: The Ashoka leaves are expected to successfully kill the Staphylococcus aureus

C. Procedures:
Procurement of Materials

The researcher will gather Ashoka leaves at Cayetano Arellano School. This
will be done in three batches as for (a) bringing to Bureau of Plant Industry for Plant
Identification and (b) for Phytochemical screening and (c) for Methicillin Resistance
Staphylococcus aureus (MRSA). The laboratory tools to be used such as Erlenmeyer
flask (500mL), electronic balance balance, graduated cylinder (1L) and Ethanol (95%)
will be obtained from Cayetano Arellano High School Science Learning Resources and
Equipment Center.

Plant Identification of Sweetsop (Annona squamosa) Leaves

Fresh Ashoka leaves will be gathered and will be brought to Bureau of Plant
Industry at San Andres St., Malate, Manila for verification of species.

Extraction of Sweetsop leaves

Five hundred (500) grams of the gathered Ashoka leaves will be washed to
remove impurities and will be air dried for 60 minutes. Using a blender, the dried
leaves will be cut and ground to smaller pieces. Ground leaves will be soaked in
 500mL 95% technical grade Ethyl alcohol and the Erlenmeyer flask will be put with a
stopper and will be kept in a cool and dark place for 48h. After soaking, the solution
will be filtered with sterilized cheese cloth and the flask with the plant material will be
rinsed with fresh portions of alcohol. The washings will be combined with the first
filtrate and the plant residue will be discarded. The filtrate will be concentrated over
water bath at 40 to 60°C to syrupy consistency or concentrated extract in the
desiccator.

Phytochemical Analysis of the Sweetsop Leaf Extract

For the phytochemical analysis of the leaf extract, a methods will be used for
this test parameters: flavonoids
To test for Flavonoids, Magnesium-Turning Test will be conducted.

Methicillin-resistant Staphylococcus aureus (MRSA)

To test for the antibiotic sensitivity of bacteria, MRSA will be conducted in the
Industrial Technology Development Institute (ITDI) Standards and Testing Division
of the Department of Science and Technology, Bicutan, Taguig City. This will show
the inhibitory activity of the leaf extract (in three replicates) against the test organism.
This will be compared to the inhibitory activity of a positive and negative control.

Risk and Safety

The researcher will conduct all the procedures and experiments of the study in a clean,
disinfected area. All of the materials used in the experiment will be disposed properly.
Testing and other procedures pertinent to the study will be conducted at the Industrial
Technology Development Institute (ITDI) Standards and Testing Division of the
Department of Science and Technology, Bicutan, Taguig City such as the Rotary
evaporation and Phytochemical screening Since Staphylococcus aureus. is a highly
pathogenic test organism, the MRSA will also be done by ITDI-STD of Department of
Science and Technology.
D. Data Analysis
Results of the presence of active components in the Ashoka leaf extract will be
interpreted based on how the extract reacted to some chemicals depending upon the
method conducted and parameter tested. For the microbial assay via disc-diffusion
method, will be reported as follows:

Reactivity Rating:
0 — None (No detectable zone around or under specimen)
1 — Slight (Some malformed or degenerated cells under the specimen)
2 — Mild (zone limited under the specimen)
3 — Moderate (zone extends 5 to 10 mm beyond specimen)
4 — Severe (zone extends greater than 10 mm beyond specimen)

Inhibitory Activity Rating: (+++) complete; (++) partial; (+) slight, and (-)
negative.

E. Bibliography https://www.nhsinform.scot/illnesses-and-conditions/infections-and-poisoning/food-
poisoning
http://www.cdc.gov/ncidod/dhqp/ar_mrsa.html

http://www.bacteriamuseum.org/species/staphaureus.shtml

http://www.dermnetnz.org/index.html

https://apic.org/monthly_alerts/staphylococcus-aureus/

http://tropical.theferns.info/viewtropical.php?id=Saraca+asoca

https://www.ijcmas.com/5-3-2016/Ch.%20Mohan,%20et%20al.pdf

https://www.healthline.com/health/what-are-flavonoids-everything-you-need-to-
know#health-benefits

https://academic.oup.com/cid/article-abstract/21/6/1417/274727