Revisiting and Refining Research Proposal

A research proposal serves as guide and preliminary blueprint in the conduct of

research.
The following are the parts of the scientific research proposal (adopted from
Regional Scientific Review Committee- Review and Recommendation Report,
2019):
1.Rationale. Includes a brief synopsis of background that supports the
research problem and explains why the research is important scientifically
and if applicable. It must explain the societal impact of the research.
2.Research Questions, Hypothesis (es), Engineering Goal (s), Expected
outcomes. Must be based on rationale.
3.Significance of the Study. This part establishes the societal impact of the
project.
4.Scope and Limitation. This enumerates the specific variables to be
studied, the procedures/assays or tests to be performed and where to
perform it.
5.Materials and Procedures. It includes all the materials to be used,
procedures to undertake, experimental designs, and methods for data
collection.
6.Waste Disposal. Describes how the used and unused materials,
chemicals, solutions and organisms must be disposed or handled after
experimentation.
7.Risk and Safety Precautions. Identifies all potential risks and safety
precautions needed.
8.Data Analysis. Describes the procedures you will use to analyze the data/
results.
9.Bibliography. It is a list of at least five major references from your
literature review. For example if you plan to use vertebrate animals, one of
these references must be an animal care reference. Follow consistently the
prescribed referencing style ( e.g. APA, MLA, Chicago, etc.).
10. Budget. This part includes information on the budgetary expenses
required to implement the project. Budgetary expenses include materials to
be used (if not available in the school laboratory), tests, training, honoraria
to

Research proposal must be reviewed properly by the adults (parents,

research adviser, (if any) the qualified scientist and/or designated supervisor.
Improvements, simplifications, and corrections can then be made by the
students upon the advice of the adults ( especially in cases when adults think that
the students cannot perform certain parts or is very risky for the student’s safety).
If the students and all the adults agree and approved the research plan, then the
research process proceeds.
 The school’s Scientific Review Committee (SRC) reviews and approves some
projects before experimentation using the SRC form (adopted from
Regional Scientific Review Committee- Review and Recommendation Report).

Does the research proposal include the following?

Rationale
• Does it include a brief synopsis of background that supports the research problem
and explains why the research is important scientifically?
• If applicable, does it explain the societal impact of the research?
Hypothesis (es), Research Question (s), Engineering Goal (S), Expected
Outcome/s.
• Is this based on RATIONALE?
Research Methods
Procedures
• Does it show all procedures and experimental designs, including methods for data
collection.
• There should be NO inclusion of work of mentor or others.
• Parameters should NOT be too strict to allow possible changes
Risk and Safety
• Does it identify all potential risks and safety precautions needed?
Data Analysis
• Does it describe all procedures for data analysis?
• Parameters should NOT be too strict to allow possible changes.
Bibliography
• Does it have at least 5 major references?
7

READINGS

2
Read thoroughly the sample research proposal below, then answer the
succeeding questions. Write your answer on the space provided.

Bioactivity profiling of naturally occurring Philippine strain of

Xylaria hongkongensis mycelia

John Andrei A. delos Santos, Bernadette E. Hipolito,

and Rich Milton R. Dulay

San Miguel National High School, San Miguel, Bulacan

Center for Tropical Mushroom and Development (CTMRD) Central Luzon State
University (CLSU), Science City of Muñoz, Nueva Ecija

In the development of drugs, scientist eyed fungi as notable source

because of their abundance and low cost-production. Among the many,
Penicillium species like Penicillium chrysogenum also known as Penicillium
notatum is the source of an antibiotic called penicillin. Lovastatin isolated
from an oyster mushroom, Pleurotus ostreatus and red yeast rice
Aspergillus terreus is use for lowering cholesterol and prevention of
cardiovascular disease. Cyclosporine, produced by an aerobic fungi
Tolypocladium inflatum is for rheumatoid arthritis, psoriasis, nephrotic A
syndrome and as immunosuppressant in organ transplant. Significant
mushrooms like reishi, shiitake, lingzhi, and chaga are included in Chinese
cuisine and are considered medicinal. Search and discovery for new
species of fungi and its family member is apical in the field of medicine as
they can be a source of effective substances for the treatments of different
diseases (Crampton, 2019).
 Just recently, in Hong Kong, China, a new species of Xylaria was
discovered. It is distinguished by its conical stromata, small spores and having
teleomorphic characters. It was named Xylaria hongkongnesis from the place of
its discovery (Tang et al, 2014) . However, in the Philippines, similar species
was discovered and isolated in the laboratory at Central Luzon State

University. The Philippine strain of A

Xylaria hongkongnesis is not yet explored compared to other species.
Hence, this study aims to find out the bioactivities of this fungus’
mycochemicals using different standard and scientifically established
preliminary procedures.

This study aims to identify the mycochemicals present

in Xylaria hongkongensis crude extract using thin layer
chromatography, antioxidant property using DPPH
B radical scavenging assay,
anti-bacterial property againt E.coli and S. aureus using
disc diffusion assay, cytotoxity on brine shrimps,
teratogenic effects on the embryonic development of
zebra fish (Danio rerio), and on Allium cepa root’s
meristematic cells.

By conducting this study, the researcher might discover the potential

medical and therapeutic uses of Xylaria hongkongensis. By knowing
these potential uses of Xylaria hongkongensis, new drugs might be
C
developed in the near future.

4
 This project is limited to the use of Xylaria hongkongensis
Philippine strain mycelia isolated and characterized from the
Center for Tropical Mushroom and Development (CTMRD)
Central Luzon State University (CLSU), Science City of Muñoz,
Nueva Ecija. The researcher will focus on evaluating the
potential of Xylaria hongkongensis ethanolic extract by
identifying the present bioactive compounds; quantifying the
anti-oxidant and phenolic content; antibacterial property against
E. coli and S. aureus; cytotoxic (Brine Shrimp Lethality Assay);
teratogenic and embryotoxic (Zebra fish (D. rerio) Assay); and
genotoxic (Onion Root Chromosomal Aberration Assay)
properties.

D
Mass production of fungus will be done at Bioassay Center,
CTMRD, CLSU, Nueva Ecija. Dried. Milled sample will be sent to the
Chemistry Laboratory Center for Natural Sciences at St. Mary’s
University in Bayombong, Nueva Vizcaya for ethanol extraction,
mycochemical screening, antioxidant and phenolic content
analysis. Antibacterial, cytotoxicity, teratogenic and
embryotoxic property screenings will be done in CLSU while the
evaluation of genotoxic and chromosomal and aberrations assay will be
conducted at Biolaboratory,
San Miguel National High School, San Miguel, Bulacan.
Research-established models will be used and considered because of their
affordability and reliability in terms of preliminary medicinal evaluation.

Independent Assessment 1

A. What part of the research proposal does each labeled parts manifest?
How did you know?
Labelled Parts of the Research
Reason of Identification
Parts Proposal

D
B. Do you think the proponents were able to establish the need for
conducting the research? Why or why not?

______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________

5
______________________________________________________________________________
______________________________________________________________________________
____________________________________________________________________________.

Independent Assessment 2
Read each statement below. Write C if the statement is correct and I if it is not.
Write your answer on the space provided.

_________1. The proponents were able to include the significance of the study.
_________2. The researchers were able to identify and coordinated with
regulated institution to where they will conduct their study.
_________3. The scope and limitation is not clearly stated.
_________4. The statement of the problem is anchored in the rationale.
_________5. The researchers will use simplified techniques within their
capability.

Independent Activity 2

6
 Bioactivity profiling of naturally occurring Philippine strain of Xylaria
hongkongensis mycelia

John Andrei A. delos Santos, Bernadette E. Hipolito, and Rich Milton R. Dulay

San Miguel National High School, San Miguel, Bulacan

Center for Tropical Mushroom and Development (CTMRD) Central Luzon State
University (CLSU), Science City of Muñoz, Nueva Ecija

Mass Production of Xylaria hongkongensis

Forty (40) sterile bottles will be filled with 40 ml aseptically filtered

coconut water. The bottles will be covered with cotton plugs, autoclaved at 15psi,
121°C for 20 minutes and allowed to cool down. One square centimetre block of
pure culture of Xylaria hongkongensis will be cut and carefully impregnated to the
liquid coconut media. The fungi will be incubated at 37 ° C for 15 days.

After 15 days of culturing, the fungi will be harvested, dried for five days and
powdered using a domestic blender. Milled Xylaria hongkongensis will be sent to the
Chemistry Laboratory Center for Natural Sciences at St. Mary’s University in
Bayombong, Nueva Vizcaya for ethanol extraction, mycochemical screening,
antioxidant and phenolic content analysis.

Mycochemical Screening of Xylaria hongkongensis

Mycochemical screening will be carried out to detect the secondary

metabolites present. The plant extract will be spotted on marked and labeled TLC
(thin layer chromatography) 7 x 4 cm, in the acetate-methanol (7.3) mixture
developing chamber. The spots for a certain metabolite will be visualized on the TLC
plates and will be exposed under UV light and hot plate to check the separation of
different compounds.

For typical visualization of secondary metabolites, vanillin-sulfuric acid

reagents will be utilized. This solution can determine the presence of Phenols,
Sterols, triterpenes and essential oils. Methanolic potassium hydroxide will be used
to test anthraquinones, coumarians, and anthrones while phenolic compounds and
tannins will be detected through the use of Potassium ferricyanide-ferric chloride
reagent. Dragendorff’s reagent will be used to spot alkaloids and Antimony (III)
chloride will be used to detect the presence of flavonoids.

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Quantification of Anti-oxidant (DPPH Radical Scavenging Capacity) and Total
Phenolic Content

Antioxidants are defined as man-made or natural substances that may inhibit

or delay some types of cellular damage from potentially harmful molecules called free
radicals (NCCIH, 2013). Our body use some free radicals to fight bacteria and
viruses. In the other hand, free radicals can damage the cells and might contribute to
health problems like heart disease, some types of cancer and cataract (Health Direct,
2018).

DPPH Radical Scavenging Capacity. Xylaria hongkongensis concentrated

extract will be used to make a stock solution. An aliquot will be taken to make
1000ppm dilution and 1000ppm of Catechin as control (1mg/ml). One (1) ml of
prepared stock solution will be mixed with four (4) ml of 0.1mM DPPH solution in
separate plastic cuvette. Reactions will be done in triplicate. The prepared mixtures
will be incubated in the dark at 37°C for 30 minutes. The reading will be monitored at
517 nm using a UV VIS spectrophotometer. A lower absorbance of the reaction
mixture indicated higher free radical scavenging activity. The radical scavenging
activities will be compared to the activity of the control Catechin. The ability to
scavenge the DPPH radical will be calculated using the formula:

% of Radical Scavenging Effect = [(Acontrol – Asample) / Acontrol] x 100

Where Acontrol is the absorbance of the control which is the DPPH without the test
sample. Asample is the absorbance of the test sample containing the mixture of DPPH
and the sample. Catechin will be used as the positive control.

Total Phenolic Content. The total phenolic content in Xylaria hongkongensis

extract will be detected with the Folin-Ciocalteu reagent. Gallic acid will be used as a standard and
the total phenolic content will be expressed as mg/g Gallic Acid Equivalents (GAE). Concentrations of
31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49 and 1mg/ml of Gallic acid will be prepared in methanol.
Concentration of 1mg/ml of plant extract will be prepared in methanol and 0.5 ml of each sample will
be introduced into test tubes and mixed with 2.5 ml of a 10 fold diluted Folin-Ciocalteu
reagent and 2ml of 7.5% Sodium carbonate. The tubes will be covered with parafilm and allowed to
stand for 30 minutes at room temperature before the absorbance read at 760nm
spectrophotometrically. All determination will be performed in triplicates.

Anti- bacterial Activity of Xylaria hongkongensis

Escherichia coli are bacterium that is normally found in humans and

animal’s intestines. However, some types of E.coli can cause intestinal infections like
diarrhoea, abdominal pain, and fever which can lead to other complications if not
treated (Seladi-Schulman, 2017) .

Staphylococcus aureus is a non-moving small round shape type of

bacteria. It is one of the five most common causes of infections after injury or
surgery. This bacterium can cause many types of health problems like minor skin
infections, boils, cellulitis folliculitis, carbuncles, skin syndrome and abscesses, lung
infections or pneumonia, brain infections or meningitis, bone infections or
osteomyelitis, heart infections or endocarditis and generalized life threatening blood
infections or Toxic Shock Syndrome (TSS) (Mandal, 2018).

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 Preparation of Test Organisms. Pure E. coli and S. aureus culture will be obtained
from Department of Biological Sciences, Central Luzon State University. The bacteria will be
grown in Nutrient Agar (NA) for 48 hours and transferred in nutrient broth and incubated for
24 hours. It will be sterilized to 1.5 x 10 8 cells/ml using 0.5 McFarland standards
respectively. This will serve as the bacterial inoculants.

Media Preparation and Sterilization. Thirteen (13) grams of Mueller-Hilton agar will
be suspended in 500 ml distilled water in an Erlenmeyer flask. The suspension
will be boiled until homogenized. The homogenized media will be sterilized using an autoclave
at 121°C, 15 psi for 20 minutes. After sterilization, approximately 20ml of the media will
be transferred aseptically in sterile petri dish and allow to solidify. The plate should stand in an
inverted position for 24 hours prior to use.

Preparation of Paper disc. Whattman filter paper no. 1 will be punched to produce
discs measuring 6mm in diameter. The discs will be placed in a petri plates and sterile in
autoclave at 15 psi for 30 minutes.

Preparation of treatments. Three sterilized paper discs will be placed in three petri
dishes, impregnated and labeled with Treatment 1 (Xylaria hongkongensis),
Treatment 2 (Streptomycin) and Treatment 3 (95% Ethanol).

Disc Diffusion Assay. The disc diffusion assay is a method of testing the antibacterial
properties of substances using a channel paper disc. The paper disc is saturated with
substance to be tested and aseptically place in bacteriological media containing the test
organism to which zone of inhibition will be measured (Hudzicki,
2009).

An amount of 0.2 ml from the bacterium inoculum will be spread evenly in the assay
plates using sterile cotton swab. The filter paper discs treated with test substances will
be placed equidistantly using sterile needles. The plates will be labeled properly and be
incubated at room temperature for 24 hours in an inverted position. The zone of inhibitions will
be measured using a ruler in millimetre.

Teratogenicity and Embryotoxicity Assay

The transparency of the zebra fish embryos makes it easy for identification of cardiac and
skeletal deformities. It exhibits similarity to higher form of vertebrates which makes them a
standard model for toxicity testing (Memita, et al., 2018). According to studies, 84 percent of
the genes known to be associated with human disease have a counterpart in the zebra fish
genome and 70 percent of proteincoding human genes are related to zebra fish genes. These
findings highlight the importance of the zebra fish model in human disease research
(Muralidhar S.Talkad, 2014).

Spawning of Danio rerio. An oxygen-saturated glass aquarium will be used for

spawning matured female and male zebra fish with a ratio of 1:2. Adult zebra fish will be
isolated using a plastic mesh and covered with black plastic for 12 hours to induce spawning.
After incubation in the dark, the fishes will be exposed to light to start fertilization. After 30
minutes, the fertilized eggs will be siphoned out of the aquarium. Embryos will be rinse thrice
using embryo water and observed under a stereomicroscope to examine uniformity and normal
condition.

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 Preparation of Treatments. An amount of 0.1 ml of X. hongkongensis will be
measured and dissolved in 9.9 ml of embryo water to produce a stock solution of 10,
000 ppm and serially diluted to produce 10,000, 1000, 100, 10 and 1 ppm,
respectively. Table 1 shows the treatments prepared including the positive control
(1.5% ethanol) and negative control (embryo water).

Table 1

Concentrations used for Teratogenicity and Embryo toxicity Assay

Treatments Concentrations (ppm)
Treatment 1 0
Treatment 2 1
Treatment 3 10
Treatment 4 100
Treatment 5 1,000
Treatment 6 10,000
Positive control 1.5% Ethanol
Negative Control Embryo water
Exposure to treatments. Three ml each of the prepared treatments of Xylaria
hongkongensis and controls will be placed into a 24-well ELISA plate. Into each well,
four embryos at segmentation phase (12 hpf) will be transferred. The plates will be
incubated at 26°C ± 1°C. At 12, 24, 36 and 42 hpt the mortality and hatchability of
the embryos will be recorded. At 36 hpt, embryos’ heart beat will be recorded.
Teratogenic effect of Xylaria hongkongensis will be examined using a stereo
microscope under 40X magnification at 60 hpt. Teratogenic (malformation of head,
tail and heart, scoliosis, deformity of yolk, and growth retardation),lethal (coagulation,
tail not detached, no somites, and no heart-beat), and normal are the parameters for
the morphological endpoint evaluation of zebra fish. Coagulated embryos and no
visual heartbeat embryos are considered dead.

Brine Shrimp Lethality Assay (BSLA)

Brine Shrimp Lethality Assay (BLSA) is used for preliminary cytotoxic assay of a
specific substance and is dependent on its capacity to kill nauplii (Dulay, et al,
2014) . It is used as guide for the detection of antitumor and pesticidal compounds
and furthermore an indicator for general toxicity (Olowa & Nuñeza, 2013). The
availability of brine shrimp eggs and the ease of performing the assay makes it a top
method (El-Fadal, 2017).
14
 Spawning of Brine shrimp cysts. Nineteen grams of Sodium chrloride
(NaCl) will be dissolved in 500 mL of distilled water to produce 38% artificial
sea water (ASW) suitable for the spawning brine shrimps (Artemia salina). Two
milligram of brine shrimp cysts will be allowed to hatch in the ASW for 48 hours
in constant aeration and illumination. After 48 hours, nauplii will be separated
from the shells using a micropipette.

Preparation of Treatments. Various concentrations of

X. hongkongensis (10,000, 1,000, 100, 10, and 1 ppm) will be prepared. Table
2 shows the treatments including the positive (Potassium dichromate) and
negative (ASW) controls.

Table 2

Treatments used for Brine Shrimp Lethality Assay

Treatments Concentrations (ppm)

Treatment 1 10000
Treatment 2 1000
Treatment 3 100
Treatment 4 10
Treatment 4 1
Positive Control (Potassium dichromate)
Negative control ( Artificial sea water)

Exposure to Treatments. Ten brine shrimps will be introduced in each

test tube containing four mL each of test solutions including the controls. The
mortality of the nauplii will be observed and recorded after 24 hours of
exposure. The procedure will be done in triplicates and in three trials.

Mortality determination on A. Salina. After 24 hours of exposure to

the different treatments, the mortality of the brine shrimp will be counted and
recorded. By dividing the number of dead nauplii by the total number of brine
shrimp and multiplying it to 100 will give the percent mortality. To ensure that
the mortality of nauplii will be due to the bioactive compounds and not by
starvation, Abbot`s formula will be used.
 Abbot`s Mortality Formula
% Mortality Due to Treatment = y-x x 100

Where: x = numbers of dead brine shrimps in the treatment

y = number of dead brine shrimps in the control
100-x

Onion Root Tip Chromosomal Aberration Assay

Allium cepa assay is a practical and economical anticancer pre-screening
system. It is used to determine the genotoxic effect of bio-active compound on
onion cells (Cabuga, Jr. et al, 2017).

Preparation of treatments. Serial dilution will be used in making 1000,

500, 250 and 125 μL/ml of Xylaria hongkongensis. Table 3 shows the treatments
prepared including the positive (5 mg/L Maleic hydrazide) and negative controls
(Distilled water with 1% DMSO).

Table 3.

Preparation of treatments for ORTCAA

Macroscopic Analysis. Macroscopic analysis will be carried out to find

out the effect of Xylaria hongkongensis on A. cepa’s root length and to compute
the EC50 half maximal lethal dose, the effective concentration to inhibit the root
lengths exposed in the negative control. Identical 18 onion bulbs will be
purchased from San Miguel Public Market. Onions’ dry scales will be peeled off.
Old roots will be removed using a scalpel to expose the root primordia. The
bulbs will be exposed to X. hongkongensis different concentrations, positive
control and negative control in 72 hours. After three days, the root lengths will be
measured using a ruler and submerged to Farmer’s fluid (three parts Ethyl
alcohol and one part Glacial acetic acid) in 24 hours for microscopic analysis.

16

13
 Microscopic analysis. Fixed onion root tips will be carefully placed in a
slide and cut by 2mm. A drop of 1N HCl (Hydrochloric acid) will be used to
hydrolyse the roots for 4-5 minutes. Aceto-carmine will be added to the treated
slide to stain the chromosomes. The slide will be set over the flame of an alcohol
lamp and covered using a cover slip. The cells will be pressed and excess stain
will be expelled. A clear nail polish will be used to seal the cover slip. The cells
will be observed under a binocular microscope with 400x magnification in the
laboratory of Natural Science Research Institute (NSRI) – UP Diliman Campus to
take a closer observation to the cells, and the stages of mitosis. The mitotic index
and mitotic phase index will be computed using the data obtained.

The mitotic index (MI) will be computed by counting the numbers of cells
under the mitotic phase in 1000 cells using the formula below:

Total number of dividing cells

Mitotic Index (MI) = X 100
1000

In addition, Mitotic Phase Index (MPI) will be computed by counting the

number of cells in the prophase, metaphase, anaphase, and telophase using the
formula below:

Total number of dividing cells (PMAT)

Mitotic Phase Index (MI) = X 100
Total number of dividing cells
Waste Disposal

After the conduct of the study, the chemicals, reagents, and solvents used
will be disposed into their organic and inorganic containers. Used and unused
Allium cepa will be put in a black plastic bag to be brought in the municipal facility
recovery area.

The microorganisms and agar used in the antimicrobial test will be

autoclaved. The brine shrimps will be bleached while zebra fish embryos will be put into
ice and disposed according to the regulated research institution’ s guidelines.

Data analysis

To determine the effect of various concentrations of X. hongkongensis extract,

Analysis on Variance (ANOVA) will be used. Trend Line Fit Regression Analysis
in Microsoft Excel 2016 will be used to determine the half-maximal
effective concentration (EC50) on Onion Root Tip Chromosomal Aberration Assay
and brine shrimp lethality assays.

15
Independent Assessment 3
Use the SRC Evaluation form for Research Methods and evaluate the text for
methodology (Independent Activity 2) by putting a check mark ( ⁄) in the complete
and incomplete column (check only one). State your reason/s on your judgment
using your own words in the remarks column. Write your answer on the space
provided.

Does the research methods

include the following: Complete Incomplete Remarks

a. Procedures

1. Does it show all procedures and

experimental designs, including
methods for data collection?
2. There should be NO inclusion of
work of mentor or others.
3. Parameters should NOT be too
strict to allow possible changes
b. Risk and Safety

1. Does it identify all potential risks

and safety precautions needed?
c. Data Analysis
1. Does it describe all procedures
for data analysis?
2. Parameters should NOT be too
strict to allow possible changes

Independent Activity 3
Revisit and assess the proposal you prepared from your Research I class. Use
and accomplish the SRC form concerning the research plan.

Independent Assessment 3
After accomplishing Independent Activity 3, use the rubric below to evaluate your
research proposal to the criteria presented in the SRC form. Put a check (/) on
the appropriate column.

Scale Level of Description

 Expectation
Exceeds
4
Expectation
3 Meets Expectation
Needs
2 incomplete. Many
Improvement
1 Inadequate
Parts of your Research Proposal
1. Rationale
2. Research Questions
3. Significance of the Study
4. Scope and Limitation
5. Materials to be used
6. Procedures
7. Waste Disposal
8. Risk and Safety Precautions
9. Data Analysis
10. Budget and Allowance
The part is present and
clearly stated. Information is
complete.
No grammatical error. No
need for revision.
The part is present and
clearly stated. Information is
complete.
No grammatical error. Needs
slight revision.
The part is present but not
clearly stated. Information is
grammatical error. Needs
minor revisions.
The part is not
present.
Information is incomplete
and not clearly stated. Many
grammatical errors.
Needs
major revision
4 3 2 1