CHAPTER III

METHODOLOGY

This chapter shows the research design, research locale, participants of the study,

research instruments, and the data gathering procedure.

Research Design

The researchers will be using completely randomized design in short CRD

because CRD is used when there are at most 10 treatments present in a study, and it is

used in experimental methods This will make their research flexible when a problem

comes up that is unsolvable or the researchers are incapable of doing. The mice that

the researchers procure on a licensed breeder will be subjected to four treatments and

each of those treatments will be different from each other with their composition. If

everything is successful in the mice testing, the next thing the researchers will then

test it on human subjects that volunteer.

Research Locale

The study will be conducted at #174 Wawa Lingayen, Pangasinan. The

independent variable, Bamboo shoots, will be gathered there as well. The said

variable will be subjected to Phytochemical Analysis at Virgen Milagros University

Foundation at San Carlos City, Pangasinan. The laboratory testing will be conducted

at A.G.S. Diagnostic and Drug Testing Laboratory at San Carlos City, Pangasinan.

Blood Cholesterol Tests will also be conducred on the experimental mice to find out

which concentration of the bamboo shoot treatments is best in reducing the

cholesterol level. The study will be conducted from September 2020 to April 2022.
Participants of the Study

The researchers in this study will find and select people who will stand as

respondents. The selected respondents of the researchers are fifty people of any

gender who are 20 years old and above, and the researchers are currently looking in

their own baranggay. The aforementioned age range was the selected respondents

because they are one of the people with the biggest health problems in today’s era in

having high cholesterol levels. They will be invited to participate in the study. Their

respective voices are essential to the study’s evolution and findings. Respondents in

this study would choose what they would prefer to use between the commercial

product or the labong as tea in lowering cholesterol level.

Also, Swiss Albino Mice will be respondents in this study. The researchers chose

Swiss Albino mice as their respondents because they are commonly used in lab

experiments and that they are less expensive than other lab animals. The cholesterol-

lowering effect of Bamboo shoots will be tested to Swiss Albino mice. Using the

mice, researchers will determine the difference between experimental group, mice

treated with the Bamboo shoot treatment and commercial group, mice treated with the

simvastatin drug treatment in the study.

Research Instruments

A. Gathering of Materials

The first material to be gathered would be the bamboo shoot. It is the main

ingredient in this research and the sole purpose this research was started. The bamboo

shoots would be gathered in 174 Wawa Lingayen, Pangasinan because the resources

are abundant there and has no risk of endangering the species. The next material will

be the mice. It will be bought and also tested in A.G.S. Diagnostic and Drug Testing
Laboratory. The minor materials can be bought in the supermarket, this material are

the honey and tea bag, while the blender, knife, oven, and sifter will be coming from

the researchers.

B. Procurement of Animals

The researchers will get the majority of their animals from commercial vendors

who deliver SPF-quality animals to A.G.S. Diagnostic and Drug Testing Laboratory..

The Facility Manager or AV has assessed and authorized a list of "preferred vendors"

at A.G.S. Diagnostic and Drug Testing Laboratory. The Facility Manager or AV will

review vendor-supplied health status data on a regular basis, and animals ordered

from these favored vendors will not require pre-approval in terms of health status.

The Facility Manager must get information regarding the genetic and pathogen

status of the animals in issue, as well as the pathogen status of all animals kept in the

unapproved vendor facility, when acquiring animals from unapproved vendors or

other academic institutions. Before an animal is shipped, the institutions must give

info such as pathogen antibody serology data and parasite screens.

Before accepting animal exports, the Facility Manager or AV will review all

animal health records. Depending on the animal's health history, additional testing,

longer quarantine, or rederivation may be requested, or the import may be denied.

Animals from a non-preferred vendor will be quarantined in isolation cubicles

and tested for ecto- and endoparasites 48 hours after arrival, as well as a PCR swab

for fur mite DNA identification.

 C. Ethical Considerations ‘

Ethical assessments related to the use of animals in research are wide-ranging.

The three Rs (Replace, Reduce, Refine) are established principles that are also

enshrined in legislation. Assessments of harm and benefit associated with experiments

on animals are particularly demanding, because the future benefits are often uncertain.

Genetically modifying laboratory animals gives rise to a special responsibility as this

involves a double intervention: first, intervention in the animal's genetic material and

second, use of the animal as a research object.

1. Responsibility for considering options (Replace)

Researchers are responsible for studying whether there are alternatives to

experiments on animals. If the same knowledge can be obtained without using

laboratory animals, alternative methods must be preferred. If no viable alternatives

exist, researchers should assess whether the study may be postponed until alternative

methodologies are developed. When justifying experiments on animals, researchers

therefore must be able to account for the absence of options and the need to acquire

knowledge immediately.

2. The principle of proportionality: responsibility for considering and balancing

suffering and benefit

Researchers must consider the risk that laboratory animals experience pain and

other sufferings (see guideline 5) and assess them about the value of the research for

animals, people, or the environment. Researchers are responsible for determining if

the experiment will benefit animals, people, or the environment. The study's potential

benefits must be considered, validated, and described in both the short and long term.

Researchers must also assess the scientific quality of the studies as well as whether
they will provide important scientific advantages. Suffering can only be caused to

animals if it is counterbalanced by a substantial and probable benefit for animals,

people, or the environment. Research institutions should provide training on how to

balance these factors when planning experiments on animals. There are many

different methods for analyzing harm and benefit.

3. Responsibility for considering reducing the number of animals (Reduce)

Researchers are responsible for considering whether it is possible to reduce the

number of animals the experiment plans to use and must only include the number

necessary to maintain the scientific quality of the

experiments and the relevance of the results. This means, among other things, that

researchers must conduct literature studies, consider alternative experiment designs

and perform design calculations before beginning experiments.

4. Responsibility for minimizing the risk of suffering and improving animal welfare

(Refine)

Researchers are responsible for assessing the expected effect on laboratory

animals. Researchers must minimize the risk of suffering and provide good animal

welfare. Suffering includes pain, hunger, thirst, malnutrition, abnormal cold or heat,

fear, stress, injury, illness, and restrictions on the ability to behave normally/naturally.

Researchers must take into account both the direct and indirect suffering that may

be endured during an animal experiment, as well as the risk of suffering before and

after the experiment. This means that researchers must also take account of the need

for periods of adaptation between the experiments and the animals themselves.
5. Responsibility for maintaining biological diversity

Researchers are responsible for ensuring that the use of laboratory animals does

not endanger biological diversity. This means that they must consider the

consequences to the stock and the ecosystem as a whole. The use of endangered and

vulnerable species must be reduced to an absolute minimum. When it is credible but

uncertain, researchers must observe the precautionary principle - the knowledge that

the inclusion of animals in research or certain methods may have ethically

unacceptable consequences for the stock.

6. Responsibility when intervening in a habitat

Researchers are responsible for reducing disruption and any impact on the natural

behavior of individual animals, including those that are not direct subjects of research,

as well as of populations and their surroundings. Some research and technology-

related projects may impact animals and their living conditions, for example as a

result of installing radar masts, antennas, or other measurement instruments. In such

cases, researchers must seek to observe the principle of proportionality (see guideline

3) and minimize the possible negative impact.

7. Responsibility for openness and sharing of data and material

Researchers are responsible for ensuring that there is transparency about research

findings and facilitating the sharing of data and material from experiments on

animals. Transparency is important to avoid unnecessary repetition of experiments

and to ensure that the public is informed. Disclosing negative results may give other

researchers information about which experiments are not worth pursuing and help

reduce the use of animals in research.

8. Requirement of expertise on animals

People who handle live animals must have updated and documented expertise on

animals - this includes specific knowledge about the biology of the animal species in

question, and a willingness and ability to take care of animals properly. Researchers

and other parties who handle animals must also have adequately updated and

documentation on animals.

9. Requirement of due care

There are national laws and rules in place regarding the use of animals in

research, and researchers and managers must comply with these. Any person who

plans to use animals in experiments must familiarise themselves with the current

rules.Respect for animals' dignity

Researchers must have respect for animals' worth, regardless of their utility value,

and for animals' interests as living, sentient creatures. Researchers must be respectful

when choosing their topic and methods, and when disseminating their research.

Researchers must provide care that is adapted to the needs of each laboratory animal.

D. Summary of Phytochemical Analysis

Antioxidant, antibacterial potential and the phytochemical constituents of

Bambusa blumeana and Bambusa vulgaris shoots extracts were explored. As

eradicant against E. coli, at 12 and 24 hours of incubation the widest zone of

inhibition was observed in paper discs treated with B. blumeana ethanol extract with

10.05 mm while in S. aureus the widest zone of inhibition was observed in paper

discs treated with B. vulgaris with 14.63 mm and 13.76 mm, respectively.

Moreover, protectant test showed that the smallest zone of bacterial colonization
of E. coli and S. aureus, were observed in plates treated with B. vulgaris ethanol

extract with 7.54 mm, 6 mm, 14.25 mm and 8.89 mm respectively, at 12 and 24 hours

of incubation. DPPH radical scavenging assay and total phenolic content revealed the

antioxidant potential of bamboo shoots. Among the tested bamboo shoot, B.

Blumeana ethanol extract had the highest radical scavenging activity while B. vulgaris

had the highest total phenolic content. These pharmacological activities can be due to

the presence of phytochemicals such as cardiac glycosides, saponins, tannins, steroids

and terpenoids.

Results of the phytochemical screeningshowed that tannins, steroids, saponins,

terpenoids and cardiac glysosides are present in all acetone and ethanol leaf extracts

of bamboo while alkaloids are absent in all bamboo leaf extracts. On the other hand,

hot water extracts of B. blumeana lack tannins, steroids, cardiac glycosides and

terpenoids. Also, no traces of tannins and steroids in B. blumeana var Luzonensis hot

water extracts were observed. Bioassay of the antimicrobial activity of the bamboo

leaf extracts showed their potentials as protectant against the bacterial and fungal test

pathogens. For the bactericidal activity of the bamboo leaf extracts, small zones of

bacterial colonization were recorded ranging from 7.0mm to 32.1mm for E.coli and

6.0mm to 16.9mm against S. aureus at 12 hrs and 24 hrs of incubation. Whereas,

eradicant test showed their inability to inhibit the growth of the bacterial pathogens.

For the fungicidal activity, results also revealed their ability as protectant against A.

niger and F. semitectum preventing fungal growth at 24 hrs of incubation. However,

an observable decreased in their efficacy as protectant was observed after 48 hrs of

incubation which resulted to the colonization of the fungalpathogens in all plates

treated with different leaf extracts. Based on the results of the study, it can be

concluded that ethanol, acetone and hot water extracts of three species of bamboo
contain phytochemicals which can be attributed to their pharmacological activities.

Also, their antimicrobial potentials as protectant for both bacteria and fungi were

revealed suppressing the growth of the test pathogens.

E. General Procedure

Phytochemical Screening

The bamboo shoots will be collected and cut into small pieces until it reaches the

weight of 100 grams. The 100-gram bamboo shoots will be put inside an Erlenmeyer

Flask. 300 milliliters of Ethyl alcohol will be added to the Erlenmeyer flask, and then

it will be covered using a cork.

Care and Management of the mice

The care and management of the mice will be done by considering the settlement

of the mice. The mice will undergo the process of acclimatization for seven days

while the mice are being fed. The management of the mice will be done by means of

cleaning their cages and by getting an official consultation from a professional

veterinarian, before and after the experiment.

Increasing the Cholesterol of the Mice

The mice will be fed foods with high cholesterol to increase the rate of their

cholesterol level after the mice undergo the process of acclimatization.

Gathering of Bamboo Shoots

The bamboo shoots will be gathered in the backyard of one of the researchers’

house at San Fabian, Pangasinan.

Peeling of Bamboo Shoots

The researchers will peel the Bamboo shoots to remove the outer part. This

will remove the unwanted impurities in the bamboo shoot.

Washing the Bamboo Shoots

The bamboo shoots will be washed after it was gathered and peeled to remove

excess dirt and in preparation for oven-drying.

Oven-Drying

The bamboo shoots will be oven-dried for 4-5 minutes to make it easier to

powder. It will no longer undergo sun-drying.

Blending

The oven-dried bamboo shoots will be powdered using a blender. It will be

blended repeatedly until powdered.

Sifting
 The powdered bamboo shoots will be sifted to remove parts that are not

powdered. It will be done repeatedly until the powdered bamboo shoots no longer

contains visible impurities.

Mixing

The researchers will put warm water and mix it together with the powdered

bamboo shoots and add a reasonable amount of honey for the enhancement of the

taste.

Administering Treatments in Cholesteric Mice

The mice will be given bamboo shoot tea twice a day. This will be done for

two weeks. The mice will take in the tea orally using syringe without a needle.

Interpreting the Gathered Data

The mice will undergo the tests to interpret their cholesterol level using the

Automated Biochemical Analyzer. The researchers will be supervised by a

professional while testing the mice.

Application of Treatments and Parameters

The treatments for high cholesteric mice are made out of bamboo shoots as tea

and compared to the commercial group which is the group of mice that will be treated

with Simvastatin drugs. The different treatments will be applied for about twice a day

for 2 weeks after the mice are fed.

The product will be consumed by 15 mice with high cholesteric level. If the

cholesterol level decreases, and the mice responded well to the experimental treatment

of Bamboo shoots as tea, the effectiveness of the shoots as a cholesterol lowering

agent is positive.
After the procedure is done and applied, the researchers will compare the group that

will treated with Bamboo shoots as tea to the commercial group. They will be

compared to determine the significant difference between the two in terms of:

a) Lowering Cholesterol Effect

b) Cost

c) Acceptability to People

Data Gathering Procedures

Through the phytochemical analysis, the researchers will find out what are the

components present in the variable, bamboo shoots. The researchers will then analyze

the collected data from the phytochemical analysis and determine if there are

components present in bamboo shoots that has the lowering cholesterol effect.

The collection of data from other laboratory test, specifically the blood

cholesterol test will be interpreted through the statistical treatment One-way ANOVA.

Since there are four treatments in the study, the use of this statistical treatment is the

most appropriate.

In interpreting the results of the one-way ANOVA, if the f-calculated is higher

than the f-tabulated data, then there is a significant difference between the treatments.

But, if the f-tabulated is higher than the f-calculated, then there is no significant

difference between the treatments.

If it was proven that there is a significant difference between the treatments, the

statistical treatment, Scheffe’s test would be used. Same as one-way ANOVA, if the f-

calculated of each treatments is higher than f-tabulated of each treatments, then there

is a significant difference. The treatment that has the highest difference of f-calculated
and f-tabulated would be interpreted as the best treatment.

To compare if there is a significant difference between the commercial variable

treatment and the best treatment, one-sample t-test would be used. If t-calculated is

higher than t-tabulated, then it can be interpreted that there is a significant difference

between the two treatments.

To compare the cost of both treatments, a table showing the prices of both

products to find out which costs more. A questionnaire will also be used to find out

which from the two treatments is better in terms of people’s acceptability to the

products.

Commercial Bamboo
Questions
Product Shoot Tea
1. Which product is easier to consume?
2. Which product is pleasing to the eyes when deciding to
buy either of the products?
3. Which product would you recommend to other people?
4. Which product would you use in lowering your
cholesterol level?
5. Which product do you think can be used by people of any
age?

The formula for one-way ANOVA that will be used:

∑x = T1 + T2 + T3 + T4

∑x2 = T12+ T22 + T32+ T32

( Ʃ x )2
CF=
N

TSS= ∑ T12+ T22 + T32+ T42 – CF

BSS = Σ¿ CF

WSS = TSS – BSS

Degrees of Freedom

df total =N-1 df BG = r df WG = d f – d f
t b

SS BG
MSBG =
df BG

724.3367
MSBG =
3

SS WG
MSWG =
df WG

MS BG
Fcalculated =
MSWG

The formula of Scheffe’s Test:

T1 vs T2

' ( X 1−X 2 )2
F=
S W 2 ( n 1+n 2 )
n 1n 2

T2 vs T3

' ( X 1−X 2 )2
F=
S W 2 ( n 1+n 2 )
n 1n 2

T1 vs T3

' ( X 1−X 2 )2
F=
S W 2 ( n 1+n 2 )
n 1n 2
The formula for T-test:

SS1 = ∑T02 - ∑ ¿ ¿

∑(T 4 )2
SS2 = ∑T42 -
N1

T 0−T 4
Tcomputed =
√[ SS 1 +SS 2
] 1 1
( + )
N 1 + N 2−2 N 1 N 2