Talisay (Terminalia catappa) Leaf Extract and Caimito (Chrysophyllum cainito) Seed

Extract: An Organic Alternative to Antimicrobial Soaps

Chapter 3
RESEARCH DESIGN

This chapter includes the research design, the data and its sources, the research

instruments, the procedure, the data gathering procedure, and the plan for data analysis.

Research Method

The objective of this research is to identify the potency of Terminalia Catappa and

Chrysophyllum cainito combined together as antibacterial agents in an antimicrobial soap to

lessen the possibility of being infected with bacteria that can be present on the surface of our

body and the environment. In the research of Borgstede and Scholz (2021), it is stated that

quantitative research is used when looking for logical mathematical results rather than theoretical

ones. Additionally, the mathematical results are obtained from connections between a set of

variables. In this research, the type of quantitative research that will be used is a true

experimental research design. According to Pubrica (2022), true experimental research has

maximum accuracy as it depends on biostatistical analysis to prove or refute a hypothesis. This

research design will be adopted since the researchers will analyze data from the antimicrobial

susceptibility tests of the different concentrations of Tainito soap using statistical method.

Research Locale

The prototype-making will be done by the researchers themselves at Barangay Karuhatan

in Valenzuela City. Under the supervision of adults, the selected place is ensured to be secured,
Talisay (Terminalia catappa) Leaf Extract and Caimito (Chrysophyllum cainito) Seed

Extract: An Organic Alternative to Antimicrobial Soaps

safe, and hazard-free so that the researchers will not be harmed during the product-making

process. The extraction of the Terminalia catappa leaf extract and the Chyrsophyllum cainito

seed extract will also be done at Barangay Karuhatan, Valenzuela City, as it is spacious and

accessible to the researchers. The antimicrobial susceptibility testing (AST) will be done at

Vetpro AMC due to the fact that its facilities, especially the laboratory and equipment that the

researchers will need, is available at the said clinic. Additionally, the clinic has been

recommended by the research advisers as it accomodates student-researchers. The place is also

well-sanitized and well-ventilated, making it an area suitable for research.

Source: Google Maps

Figure 2. Map of Vetpro AMC Clinic
Procedure

The flowchart below shows the data gathering procedure done by the researchers.
Talisay (Terminalia catappa) Leaf Extract and Caimito (Chrysophyllum cainito) Seed

Extract: An Organic Alternative to Antimicrobial Soaps

Figure 3. Flowchart of Data-Gathering Procedure

Materials

The first step in making the prototype was to gather the Terminalia catappa leaves and

Chrysophyllum cainito seeds. Each member was given a designation on which should be their

main focus.

Terminalia catappa Leaves and Chrysophyllum cainito Seeds

The leaves were gathered on Shorthorn Street located in Paradise Village, Project

8 in Quezon City. The majority of the leaves gathered were dried since according to the

study of Ally et al. (2018), the dryer the leaf, the higher the concentration of the dry

ethanolic extract. Next, the Chrysophyllum cainito fruits were gathered in Bulacan and

Project 8, Quezon City. The researchers extracted the seeds by removing the soft fleshy

pulp of the fruit since according to the study of Oranusi et al. (2015), the seed exhibits
Talisay (Terminalia catappa) Leaf Extract and Caimito (Chrysophyllum cainito) Seed

Extract: An Organic Alternative to Antimicrobial Soaps

higher nutritional and antimicrobial properties than the pulp. The devices used in

measuring the leaves and plants, such as the kitchen scale and gloves, were already

provided by the researchers.

Prototype Making and Set-ups

After gathering all the materials, the extraction of the Terminalia catappa leaves and

Chrysophyllum cainito seeds and prototype making will be done. The researchers will be guided

by professionals in order to maintain the credibility of the research.

Procedures for Extracting the Terminalia catappa leaf extract

The researchers will be guided by the study of Ally et al. (2018) in extracting the

Terminalia catappa leaf extract. First, 1 kg of dried Terminalia catappa leaves will be

crushed in order to get the ideal extract to support the solution. Grinding using a mortar

will be used to release and easily extract the leaves. Second, the process of submerging,

wherein a solute will be mixed with a solvent to form a new solution, will be utilized. 1

kg of crushed Terminalia catappa leaves will be submerged in 1 L of 95% ethanol for 5

days while being shaken periodically. After 5 days, the solution will be filtered with

Whatman No. 11 paper filter and evaporated using a rotary evaporator.

Procedures for Extracting the Chrysophyllum cainito Seed Extract

The researchers will be guided by the study of Oputah et al. (2016) in extracting

the Chrysophyllum cainito seed extract. First, each C. cainito seed will be broken up to

yield cotyledon and the seed coat, which will be both used in the extraction. Second, the
Talisay (Terminalia catappa) Leaf Extract and Caimito (Chrysophyllum cainito) Seed

Extract: An Organic Alternative to Antimicrobial Soaps

ground powder (200 g) of the seeds will be extracted using 1000 mL of ethanol in a

Soxhlet extractor for 72 hours. Lastly, the ethanol extract of the Chrysophyllum cainito

seeds will be concentrated using a rotary evaporator.

Procedures for Making the Organic Tainito Extract Soaps

In making natural soaps, only three main ingredients (water, oils, and lye) are

utilized. However, in this research, the addition of the Terminalia catappa and

Chrysophyllum cainito will be used. The components of the soap are as follows: 500 mL

of olive oil, 500 mL of vegetable oil, 30 mL of castor oil, 60 mL of coconut oil, 140 g of

lye, and 140 g of water. The hot process will be used as it is favorable to the researchers

because it does not require to be cured, unlike the cold process (Centre for Affordable

Water and Sanitation Technology, 2014). First, the lye will be dissolved using distilled

water. During this process, the lye will create and produce its own heat. Then, in a

separate pot, the oils must be heated at a low temperature until they are consistently

liquid. After this, mix the oil and lye solution until it has reached the desired consistency.

Next, the soap mixture must be heated at a temperature not higher than 82 ℃ for 1.5 to 2

hours. Carefully stir the mixture occasionally and do not allow the soap to boil over.

Afterward, the soap mixture must be divided into three in order to add the different

concentrations of Terminalia catappa leaf extract and Chrysophyllum cainito seed

extract. Each batch will be poured into molds and labeled to avoid confusion. After

cooling, the soap can be removed from the mold and cut into bars. There will be three

Tainito extract soap bars created for each concentration.

Talisay (Terminalia catappa) Leaf Extract and Caimito (Chrysophyllum cainito) Seed

Extract: An Organic Alternative to Antimicrobial Soaps

Table 1

Prototype Soaps of the Experiment

Prototype Description

70 T.C., 30 C.C. Tainito Soap

● 167mL Olive Oil

● 167 mL Vegetable Oil

● 10 mL Castor Oil

● 20 mL Coconut Oil

● 47 g Caustic Soda

● 47 g Distilled Water

● 12 mL 70 T.C., 30 C.C. Extract

● 2 g Rosemary

50 T.C., 50 C.C. Tainito Soap

● 167mL Olive Oil

● 167 mL Vegetable Oil

● 10 mL Castor Oil

● 20 mL Coconut Oil

● 47 g Caustic Soda

● 47 g Distilled Water

● 12 mL 50 T.C., 50 C.C. Extract

Talisay (Terminalia catappa) Leaf Extract and Caimito (Chrysophyllum cainito) Seed

Extract: An Organic Alternative to Antimicrobial Soaps

Prototype Description

● 2 g Rosemary

30 T.C., 70 C.C. Tainito Soap

● 167mL Olive Oil

● 167 mL Vegetable Oil

● 10 mL Castor Oil

● 20 mL Coconut Oil

● 47 g Caustic Soda

● 47 g Distilled Water

● 12 mL 30 T.C., 70 C.C. Extract

● 2 g Rosemary

Parameters

To test the effectiveness of the created prototype, it will be inspected using the

Antimicrobial Susceptibility Test (AST) which is responsible for finding and identifying the

different effective amounts of concentrations that are recommended to which amount of

concentrations has the ability to inhibit bacteria. The prototype will be examined through the

zone of inhibition.
Talisay (Terminalia catappa) Leaf Extract and Caimito (Chrysophyllum cainito) Seed

Extract: An Organic Alternative to Antimicrobial Soaps

Inhibition Zone Diameter

After creating the different concentrations of Tainito extract, the inhibition zone

diameter of each concentration will be examined by professionals of Vetpro AMC using

the standard procedure. In measuring the diameter of the zone of inhibition, the lid of the

plate must be kept in place while using rule or caliper that measures in millimeters and

place “0” in the center of the antibiotic disk. It must be measured from the center of the

disk to the edge of the area with zero growth to get the radius. In order to get the

diameter, the diameter must be multiplied by 2. The following table will be used to

evaluate the bacterial response to each concentration of Tainito extract:

Table 2

Interpretation of Inhibition Zone Diameter

Diameter of Zone of Inhibition (mm)

Resistant 10 or less

Intermediate 11-15

Susceptible 16 or more

Safety and Precaution

Handling of Lye

In handling Lye, the equipment that is needed should be appropriate for working

inside the laboratory - plastic or rubber gloves, eye protection such as goggles, clothing
Talisay (Terminalia catappa) Leaf Extract and Caimito (Chrysophyllum cainito) Seed

Extract: An Organic Alternative to Antimicrobial Soaps

that protects your skin from splashes or spills, and closed-toed shoes. Always add the lye

to water (not water to lye). This is required in laboratories and will be done by a person

with knowledge of handling chemicals inside Adamson University. In the disposal of the

lye, It can be mixed by combining with distilled water and pouring it down the drain.

Most drain cleaners really contain lye. If concerned about the drain, the local waste

management services can be contacted to learn about ways to get rid of dangerous things.

It is also possible to neutralize the solution, which is still a reliable method of getting rid

of lye. When neutralizing, it is essential to use full protective equipment to prevent injury,

obtain the right acid (Low PH Acid), which will effectively neutralize the lye, and

combine the lye and acid in small amounts in a sizable stainless steel or pyrex container.

Moreover, Lye is a very caustic and reactive substance, it should only be disposed of with

extreme caution because improper disposal can be harmful to both human health and the

environment.

Handling of Ethanol

In handling Ethanol, the equipment that is needed should be appropriate for

working inside a laboratory are the lab coat, Safety glasses, nitrile gloves, and face mask.

This is to ensure the safety of oneself in handling Ethanol. Another parameter in handling

ethanol is to avoid inhaling gas, mist, or fumes, make sure there is enough airflow,

remove all potential ignition sources, personnel should be moved to safe areas, and watch

out for vapor concentrations that can explode. This is required in laboratories and will be

done by persons with knowledge of handling chemicals inside Adamson University.

Talisay (Terminalia catappa) Leaf Extract and Caimito (Chrysophyllum cainito) Seed

Extract: An Organic Alternative to Antimicrobial Soaps

Disposing of Ethanol can go down the drain as long as its concentration is less than 24%

for it to be considered non-hazardous. Water is used in lessening the concentration of

Ethanol.

Handling of Bacteria

Handling microorganisms and bacteria is always sensitive and needs proper

handling. The following handling protocols should be observed and practiced by the

health professionals of Adamson University. The bacteria should be greatly observed and

treated as a potential pathogen. Next, cleanse and sterilize all the equipment and tools –

pipettes, tubes, needles, and other materials for culturing, and disinfect the working area.

Second, always wear safety gear such as a lab coat, safety glasses, gloves, and face mask,

always observe laboratory protocol, and label all the information in a certain

microorganism. Third, use an autoclave to dispose of materials that have the potential to

spread dangerous microorganisms in 30 to 40 minutes at a pressure of 20 pounds and

121°C. Lastly, in an event of a spill or broken culture tube, use a solution of 70% ethanol

and 10% bleach.

Data Analysis

The data collected in the study was arranged and classified in line with the research

design and problems drawn out by the researchers. The data analysis that will be used will vary

depending on the statement of the problems given in this study. Percent analysis will be used to

specify the most effective concentration among the three different concentrations of Tainito
Talisay (Terminalia catappa) Leaf Extract and Caimito (Chrysophyllum cainito) Seed

Extract: An Organic Alternative to Antimicrobial Soaps

extract soaps. It is defined by Unacademy (2023) as “the process of inspecting, narrowing,

transforming, and modeling data from the huge amounts of information or data.” It will also be

used to determine whether there will be a difference between the most effective Tainito

concentration and penicillin in terms of their inhibition zone diameter.

For the first statement of the problem, the researchers will identify and give the results

and means of the different concentrations of the Tainito extracts that will be produced in terms of

their inhibition zone diameter. The researchers will create tables wherein the gathered data will

be listed and compiled.

In analyzing and interpreting the gathered data for the second statement of the problem

the percent analysis as a data analysis method will be used. This method will be utilized as the

researchers will compare the inhibition zone diameter of each of the three different

concentrations of Tainito extracts against each other in order to determine if there would be a

percentage difference between groups. The formulation for this data analysis method is;

𝑁1+𝑁2
𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝐷𝑖𝑓𝑓𝑒𝑟𝑒𝑛𝑐𝑒 = (𝑁1+𝑁2 × 100
⎡ ⎤
⎢ 2 ⎥
⎣ ⎦

Moreover, this formulation will be used on Microsoft Excel in order to create accurate results.

In analyzing and interpreting the gathered data for the third statement of the problem, the

researchers will also use percentage analysis. This data analysis method will be used by the

researchers to compare the inhibition zone diameter of the best variation of the Tainito extract

soap against Penicillin to determine if there would be a difference between the two groups.
Talisay (Terminalia catappa) Leaf Extract and Caimito (Chrysophyllum cainito) Seed

Extract: An Organic Alternative to Antimicrobial Soaps

Resources

Allyn, O. Q., Kusumawati, E., & Nugroho, R. A. (2018). Antimicrobial activity of Terminalia

catappa brown leaf extracts against Staphylococcus aureus ATCC 25923 and

Pseudomonas aeruginosa ATCC 27853. F1000Research, 7, 1406.

https://doi.org/10.12688/f1000research.15998.1

Borgstede, M., & Scholz, M. (2021). Quantitative and Qualitative Approaches to Generalization

and Replication–A Representationalist View. Frontiers in Psychology, 12.

https://doi.org/10.3389/fpsyg.2021.605191

Center for Affordable Water and Sanitation Technology. (2014, August ).Soap Fact Sheet : Soap

Making.

https://globalhandwashing.org/wp-content/uploads/2020/04/Soap-Making_Fact-Sheet_20

14-08-22_en.pdf

Cholkar, S. S., Gawade, A. R., & Kuchekar, A. B. (2022). The Use of Medicinal Plant Extract in

Hand Sanitizer and Spray to Combat Against Covid-19. Biosciences Biotechnology

Research Asia, 19(1), 183–189. https://doi.org/10.13005/bbra/2977

Kaur, P., Stoltzfus, J., & Yellapu, V. (2018). Biostatistics: Desriptive Statistics. International

Journal of Academic Medicine, 4(1), 60–63.

http://www.ijam-web.org/text.asp?2018/4/1/60/230853

Kent State University. (n.d.). SPSS TUTORIALS: ONE-WAY ANOVA. Retrieved April 2, 2023,

from
Talisay (Terminalia catappa) Leaf Extract and Caimito (Chrysophyllum cainito) Seed

Extract: An Organic Alternative to Antimicrobial Soaps

https://libguides.library.kent.edu/spss/onewayanova#:~:text=One%2DWay%20ANOVA

%20

Kim, T. K. (2015). T test as a parametric statistic. Korean Journal of Anesthesiology, 68(6),

540–546. https://doi.org/10.4097/kjae.2015.68.6.540

Guidelines Review Committee. (2009, January 15). WHO guidelines on hand hygiene in health

care. https://www.who.int/publications/i/item/9789241597906

Hoover, L. (2021, May 20). What Is Qualitative vs. Quantitative Study? GCU.

https://www.gcu.edu/blog/doctoral-journey/what-qualitative-vs-quantitative-study#:~:text

=Quantitative%20studies%20rely%20on%20numerical,think%20or%20respond%20with

in%20society

Ngouana, T. K., Mbouna, C. D. J., Kuipou, R., Tchuenmogne, M. a. T., Zeuko’o, E. M.,

Ngouana, V., Mallié, M., Bertout, S., & Boyom, F. F. (2015). Potent and Synergistic

Extract Combinations from Terminalia Catappa, Terminalia Mantaly and Monodora

tenuifolia Against Pathogenic Yeasts. Medicines, 2(3), 220–235.

https://doi.org/10.3390/medicines2030220

Oputah, S. L., Ajanaku, K. O., Mordi, R. C., Olugbuyiro, J., Olorunshola, S. J., & Azuh, D. E.

(2016). Phytochemical and Antibacterial Properties of Ethanolic Seed Extracts of

Chrysophyllum albidum (African Star Apple). Oriental Journal of Physical Sciences,

1(1–2), 05–09. https://doi.org/10.13005/ojps01.0102.02

Oranusi, S. U., Braide, W., & Umeze, R. U. (2015). Antimicrobial activities and chemical

compositions of Chrysophyllum cainito (star apple) fruit. Microbiology Research

International, 3(3), 41–50. http://eprints.covenantuniversity.edu.ng/5773/

Talisay (Terminalia catappa) Leaf Extract and Caimito (Chrysophyllum cainito) Seed

Extract: An Organic Alternative to Antimicrobial Soaps

Pubrica. (2022). Experimental Study Design: Research, Types of Design, Methods and

Advantages. Retrieved April 1, 2023, from

https://pubrica.com/academy/experimental-design/experimental-study-design-research-ty

pes-of-design-methods-and-advantages/

Thomas, L. (2022, December 5). Quasi-Experimental Design | Definition, Types & Examples.

Scribbr. https://www.scribbr.com/methodology/quasi-experimental-design/