MUNTINLUPA NATIONAL HIGH SCHOOL

NBP Reservation, Poblacion, Muntinlupa City

‘’Optimization of Phytochemical Screening and Paper

Chromatography Comparative Analysis of Anthocyanin by
Bloodleaf (Iresine herbstii) and Red Onion (Allium cepa) as
Indicator of Aquarelle in pH”

Decasa, Amir Edvinlee M.

De Leon, Micaela D.
Erjas, Luzviminda B.
Estabillo, Clarenz C.
Manalo, Kc Ezikiel A.
Montenegro, Luiz Miguel S.

Submitted to:

Princess C. La Rosa
Research Adviser

Christine Anne Marla, Scientific Editor of PSHS - Main

Research Consultant

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ACKNOWLEDGEMENT

The making of this project would not be possible without the help of a few
individuals. We would like to express our sincerest gratitude to the following
individuals who in a one way or another make this study possible:

To God, for providing us guidance throughout the creation of this study.

Our research advisor, Mrs. Princess Caballero La Rosa of Muntinlupa National High
School. The one who sparked the idea for this study and steered us in a right
direction.

Our research consultant, Mrs. Christine Anne Marla, licensed scientific paper editor
of Philippine Science High School-MAIN who validated the possibility of conducting
our ideas on making of aquarelle. Without her passionate participation and input, this
study would not have been successfully conducted.

To Mr. Balistoy, for supervising our test and validated in paper chromatography
respectively in Biology.

To Mr. Gatdula, Ms.Vallejo, Mr. Valdez or supervising and validated our paper.
Without their participation this would not be possibly conducted.

The Arts and Design Students, for giving their spoonfield participation every survey
and agreement know that it was all worth it for resulting an excellent result of this
project.

Especially to Mr. Peñafuerte, ADAS Coordinator, for precious approving us to survey

the random selected arts and design students. Without his participation, the excellent
important result of this project will not be possible.

Our Family especially our parents, who provided us with unfailing support financially
and emotionally. They always checked on our well-being when we, ourselves, forget
to.

To Mr. Andrei Michael P. Andes, Mr. Lorence E. Baldesco, Mr. Angelo R. Varde, and
Ms. Alexandria E. Lorica who were always ready to aid us to conduct a survey on
ADAS when possible.

Our fellow student-researchers who were always ready to aid us when possible.

To all who were involved in the making of this project, know that it was all worth it.

Thank you very much!

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 DEDICATION

This research paper is dedicated to the teachers who guided the researchers to successfully

complete this study. They were the ones who guided us through the experimentation

process. It is also dedicated to the researchers' parents who have continuously provided

emotional and financial support, met all of our demands while we developed the study, and

never stopped encouraging us. The researchers would also like to dedicate this to the school

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that provided them with an opportunity to put their skills to the test and work together to

complete the study. We dedicate this research to our All-Powerful God, who gave the

courage, intellect, security, and inspiration needed to complete it.

TABLE OF CONTENTS

INTRODUCTION………………………………………………………………………

A. Background of the Study…………………………………………………

B. Objectives…………………………………………………………………..

C. Statement of the Problem………………………………………………..

D. Hypothesis………………………………………………………………….

E. Significance of the Study………………………………………………...

F. Scope and Limitation………………………………………………………

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 G. Definition of Key Terms……………………………………………………

H. Conceptual Framework…………………………………………………….

I. Review of Related Literature……………………………………………….

METHODOLOGY……………………………………………………………………..

A. Research Design……………………………………………………………..

B. Research Locale………………………………………………………………

C. Methodology Flowchart……………………………………………………..

D. Materials and Instrumentation………………………………………...……

E. Methods and Procedures……………………………………………………

RESULTS AND DISCUSSION……………………………………………………….

CONCLUSION………………………………………………………………………….

RECOMMENDATION…………………………………………………………………

REFERENCES…………………………………………………………………………

APPENDIX…………………………………………………………………………….

STUDENT PROFILE…………………………………………………………………

PLAGIARISM REPORT………………………………………………………………

GRAMMAR CHECKER………………………………………………………………

LIST OF FIGURES

Figure 1. Conceptual Framework..............................................................................16

Figure 2. Bloodleaf (Iresine herbstii)..........................................................................17

Figure 3. Color Changes of Bloodleaf (Iresine herbstii).............................................18

Figure 4. Red Onion (Allium cepa)............................................................................19

Figure 5. Color Changes of the Red Onion (Allium cepa).........................................20

Figure 6. Acrylic Paints..............................................................................................22

Figure 7. Chemical Structure of Anthocyanin…………………………………………...25

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Figure 8. Chemical Structure of Sterols……………………………………………...….31

Figure 9. The Chemical Structure of Steroid Saponin...............................................34

Figure 10. Flow Chart…………………………………………………………….………..38

Figure 11. Paper Chromatography………………………………………..……………..50

LIST OF TABLES

Table 1. Color change of the Bloodleaf (Iresine herbstii)...........................................18

ABSTRACT

This study aims to evaluate the potential of using Bloodleaf (Iresine Herbstii) and

Red Onion (Allium Cepa) as a biodegradable alternative for aquarelle lithography by

using a pH indicator instead of using toxins like cadmium, cobalt, and gum arabic.

The study seeks to determine the phytochemical components present in the two

plants, the importance of using a pH indicator, and the difference in color intensity,

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odor acceptability, and stick consistency between the aquarelle made from the two

plants and commercial aquarelle. Additionally, the study aims to determine the

difference in the 25% concentration per pH solution between Bloodleaf and Red

Onion to create aquarelle. The study will use an experimental method and purposive

sampling to invite ten respondents from the population of Grade 12 AD students at

Muntinlupa National High-School Main Senior High School Building to test the

general acceptability of the study. The study will be conducted within two to three

weeks in the chemistry laboratory of Muntinlupa National High-School Main. The

researchers will use five pH levels, ranging from pH 2.0 to pH 13.0, to create the

safest painting medium with the smallest impact on the natural environment. The

researchers will collect data through surveys after the experiment. The study seeks

to make people understand why basic ink components are environmentally

unfriendly and how biodegradable ink can offer more using Bloodleaf and Red

Onion.

INTRODUCTION

Plant extracts tend to generate colors that are softer, more subdued, and more

realistic-looking than synthetic colorants. The University of Florida claims that it is

simpler to find plants that produce warm hues like reds, oranges, and yellows than it

is to find ones that produce cold hues like blue and purple. This is due to the fact that

warm hues are more prevalent in nature. Before harvesting, do your research on the

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plants, and make sure not to choose types that are endangered or protected. Plants

should only be harvested from your own property or with the owner's permission.

A. BACKGROUND OF THE STUDY

Ink is a common source of color on paper in the contemporary era and

is utilized widely across cultural boundaries. According to Cornell University, inks are

made up of dyes or pigments that are suspended in liquids like water, oils, or

alcohols. Although many inks are fairly easy to make, it may take some time and

chemistry knowledge to find the ideal formulation for a specific ink use Chen L. (n,d).

Inks contain compounds like tannic, gallic, and iron sulfate, according to Cofield E.

(2018). As an alternate ingredient for calligraphy ink, other researchers have already

tested extracts from Mayana leaves, Albugati fruit, Mango, and other plants. But no

one did an experiment with Red onion (Allium cepa) and Blood Leaf (Iresine herbstii)

as an aquarelle, that is why the researchers will test if Red onion (Allium cepa) and

Blood Leaf (Iresine herbstii) are an effective alternative aquarelles in lithography

using the pH scale. Typically, the inks used in most aquarelle are made from

synthetic materials like petroleum and chemical solvents. These kinds of materials

pose risks, particularly to the users' and the environment's health.

Because of this, a lot of people are now leaning towards using natural

inks as they are less toxic, the chemical makeup of onions just doesn’t support

bacteria growth McCoy (2018). They are not the best environments for the growth of

bacteria, viruses, or other pathogens due to their low pH (i.e., acidic nature) and low

protein content. In the Philippines, vegetables like Onion (Allium cepa) have been

found out to be potential ink for biological staining as they both contain anthocyanins.

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 The onion, also known as Allium cepa, is a herbaceous biennial plant

that is grown for its edible bulb and is a member of the Amaryllidaceae family.

Despite having few nutrients, onions are valued for their flavor and are frequently

used in cooking. They are also used as a cooked vegetable and provide flavor to

foods like stews, roasts, soups, and salads (The Editors of Encyclopedia Britannica,

1998). According to Murray & Nowicki (2020), various clinical investigations have

demonstrated that onion extracts can lower blood pressure, promote fibrinolysis,

decrease platelet aggregation, and lower blood lipid levels.

Blood leaf or Iresine herbstii, it is part of the Amaranthaceae or Amarant

family, it is an uncommon plant that has around 30 species in its genus, all of which

are native to South America, especially Brazil. It can grow to a height of 2 feet and is

a herbaceous subshrub. It mostly has pink or crimson variegation but it can also be

green and yellow Khale (2008). It is mostly used as a decoration in a front yard, as it

is one gorgeous plant. Most of the species of Iresine plants are perennial; they range

in size from small to medium-sized shrubs. These plants are also notable for their

common names, such as the chicken gizzard plant, the beefsteak plant, and other

descriptive names. These plants are novelties in the northern hemisphere, as they

are more common in the southern hemisphere Vanzile (2021).

The researchers will check if there is anything that needs to be

adjusted, or changed, so that the experiment will be a success. Students, artists,

calligraphers, and the general public have a desire for ink made with alternative

substances. The researchers think that the Onion (Allium cepa) and Blood Leaf

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(Iresine herbstii) has great potential in having an alternative aquarelle ink due to its

advantages. The effectiveness of it as a component of aquarelle was decided upon

by the researchers. To create aquarelle inks from plants (vegetables), researchers

must balance the amount of water and source, so it can produce a natural odor and

color, so that spreading of ink on the paper will be avoided and can maintain a good

quality of aquarelle.

B. OBJECTIVES

These research objectives aim to address the knowledge gap on the potential

of red onion and bloodleaf extracts as alternative sources of natural aquarelle inks.

Through this study, the phytochemical compounds present in the extracts will be

identified, and the optimization process for their maximum yield of anthocyanin as

aquarelle inks will be determined. Paper chromatography analysis will also be

conducted to compare the effectiveness of the extracted anthocyanin from red onion

and bloodleaf extracts as aquarelle inks. The study also aims to evaluate the pH

sensitivity, color stability, and durability of the aquarelle inks obtained from the

extracts. Lastly, the toxicity and environmental impact of the aquarelle inks will be

assessed to provide recommendations for further optimization and utilization of red

onion and bloodleaf extracts as aquarelle inks.

C. STATEMENT OF THE PROBLEM

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 This study is aims to test, analyze & compare the general

acceptability of the phenomena of Bloodleaf (Iresine Herbstii) and Red Onion

(Allium Cepa) to make an aquarelle using the potential hydrogen or pH

indicator that will substitute the aquarelle lithography instead of using toxins

ingredients like cadmium, cobalt, and gum arabic. Specifically, it attempts to

answer the following questions:

1. What are the phytochemical components that can be found in

Bloodleaf (Iresine Herbstii) and Red Onion (Allium Cepa)?

2. Is there a significant difference in the 25% concentration per pH solution

between the a Bloodleaf (Iresine Herbstii) and Red Onion (Allium Cepa) that

will be mixed to make an aquarelle in terms of:

● Color Intensity

● Environmental Friendly

● Stick Consistency

D.HYPOTHESES.

1. Ho - There are no significant components of Red Onion (Allium Cepa) that are

essential to make an aquarelle.

Ha - There are significant components of Red Onion (Allium Cepa) that are

essential to make an aquarelle.

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 2. Ho - There are no significant components of Bloodleaf (Iresine Herbstii) that

are essential to make an aquarelle.

Ha - There are significant components of Bloodleaf (Iresine Herbstii) that are

essential to make an aquarelle.

3. Ho - There are no significant difference in the 25% concentration per solution

between the aquarelle made of Bloodleaf (Iresine Herbstii) and Red Onion

(Allium Cepa) in terms of; color intensity, environmental friendliness, and stick

consistency.

Ha - There are significant difference in the 25% concentration per solution

between the aquarelle made of Bloodleaf (Iresine Herbstii) and Red Onion

(Allium Cepa) in terms of; color intensity, environmental friendliness, and stick

consistency.

E. SIGNIFICANCE OF THE STUDY

This study aims to provide valuable information on “Optimization of

Phytochemical Screening and Soxhlet Extraction Comparative Analysis of

Anthocyanin by Bloodleaf (Iresine herbstii) and Red Onion (Allium cepa) as

Indicator of Aquarelle in pH” as an alternative for synthetic ink. This study

would contribute to the following:

● Architecture- Architects were trained to make drawings and paintings

from their schematic plans so that clients could visualize how their

completed buildings would look. Most synthetic water colors or

aquarelles are expensive and may affect the environment so natural

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 ink made from Bloodleaf and Red Onion will help them not just to save

but also make a good quality painting made of natural ink and save the

environment because of its eco-friendliness.

● Arts and Design Students- Natural pigments can help AD students in a

lot of ways but in specifically helping replenishable, easy to use,

environment-friendly and most importantly; being cheap and easy to

find.

● Environment - Due to its combination of quality, affordability, and eco-

friendliness. Water-based ink is much more gentle on the environment

than synthetic ink because of its non-carcinogenic properties.

● Future Researchers - As this study promotes itself to explore the

chemical properties of the Bloodleaf (Iresine Herbstii) and Red Onion

(Allium Cepa), the results will contribute to the knowledge of the field of

science and serve as a basis or reference for future studies.

● Painters- Synthetic aquarelles are now expensive, causing many

painters to make some effort to make their own ink to be able to paint.

This natural aquarelle made from Bloodleaf and Red Onion will give

them ideas and opportunities to save money and make some natural

ink with assurance of its effectiveness and good quality.

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F. SCOPE AND LIMITATIONS

The purpose of this study is to make people understand why the basic

components of an ink are environmentally unfriendly and why a biodegradable

ink could offer more using Bloodleaf and Red Onion. The scope includes all

equipment, personnel, and procedures used to carry out the specified

research. The study will primarily focus on the comparative analysis between

Bloodleaf (Iresine herbstii) and Red Onion (Allium cepa) while using pH-level

such as pH 2.0- Calamansi Extract, pH 3.0- Vinegar, pH 7.0 - Pure water, pH

9.0- Baking Soda, pH 13.0- Bleach to make a safest painting mediums for

making a small impact on the natural environment, water in aquarelle paint

makes it economically friendly and environmentally preferable. The

experimentation will be conducted in the premises of Muntinlupa National

High-School Main, specifically in the chemistry laboratory. For the research

equipment during the experimentation, the researchers will be given the lab

apparatus from the chemistry laboratory by their research teacher that they

will require. The researcher will invite 10 respondents from the population of

the Grade 12 AD Students at Muntinlupa National High-School Main Senior

High School Building 3 trials per pH-level experiment to test the general

acceptability of the study. As for the collection of data, the researchers will

conduct a survey after the experiment since the independent variables are

considered as quantitative data.

Due to the unavailability of a specific date for conducting our study, this

study aims to complete the experiment within two (2) to three (3) weeks,

keeping in mind that the research instructor may give only a short period of

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 time for the researchers. Other pH-level solutions are beyond the scope of the

study; therefore, the researchers chose 5 pH levels that can be used for the

Bloodleaf and Red Onion indicator solution. Due to time and budget limits, the

chemical composition will not be checked in this research study since it is

beyond the capabilities of researchers to do that without the use of

appropriate materials.

G. DEFINITION OF KEY TERMS

Aquarelle ink – it is the substance that is placed in a commercial aquarelle ink. In

this study we will be conducting an experiment to create a natural source of ink that

commercial aquarelle have.

Alternative – it is used to have another material or as another possibility. This will be

used to create an alternative for the aquarelle ink.

Color – it is the human visual perception. This will be used to determine if the

hibiscus with pH solution will have efficient colors.

Indicator - presence or absence of a threshold concentration of a chemical species,

such as an acid or an alkali in a solution, generally by a colour change.

Ink – a colored fluid used for artwork and painting. This will be used as a basis for

our study.

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Odor – it is perceiving odors or scents by nose. This will be used to see if the

extract has a good scent or odor for the aquarelle ink.

pH - quantitative measure of the acidity or basicity of aqueous or other liquid

solutions.

Phytochemical - variety of nonnutritive chemical compounds found in plant foods,

such as flavonoids.

H. CONCEPTUAL FRAMEWORK

FIgure 1. Conceptual Framework

Figure 1. Conceptual Framework

Figure 1 depicts the framework and conceptual structure of the study. The

color intensity and stick consistency of the aquarelle ink will be tested through

survey, the recipients will be using the product to judge its qualities. It will also be

tested for how environmentally friendly the aquarelle is. The study is a comparative

analysis of Red Onion and Bloodleaf as an aquarelle ink. The researcher will

compare the results of 2 agents and choose what’s better.

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I. REVIEW OF RELATED LITERATURE

Bloodleaf (Iresine herbstii)

Figure 2. Bloodleaf (Iresine herbstii)

In temperate regions, bloodleaf, a herbaceous subshrub that grows up to 2

feet tall, is grown in pots. The plant can be classified into two groups based on

the shape of its leaves: ovate plants and obcordate (inversely heart-shaped)

plants. The plant contains green and yellow variegation, but it also frequently

exhibits pink or scarlet. The majority of popular names come from the dark

variegated obcordate leaves. On top of its foliage, the erect flower spikes

bloom. The blooms aren't particularly striking when compared to the leaves,

though. Aarti Khale, n.d. The majority of popular names are derived from the

obcordate leaves' dark variegation. The upright flower spikes are in bloom,

rising above the leaves. Compared to other flowers, the blooms are not

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exceptionally showy. The blooms are not particularly showy when compared

to the foliage. Khale Aarti (n.d.).

Color change of the Bloodleaf (Iresine herbstii)

Figure 3. Color change of the Bloodleaf (Iresine herbstii)

Table 1. Color change of the Bloodleaf (Iresine herbstii)

The PH scale determines whether aqueous or other liquid solutions

are basic or acidic. A pH of greater than 7 denotes a base, whereas a pH of

less than 7 suggests acidity. The range is 0 to 14, with 7 being neutral. PH

gauges how much free hydrogen and hydroxyl ions are present in the water

relative to other ions. The PH of water is a sign of chemical alterations that

may be caused by contaminants in the water (OpenStax College, 2019).

A solution that contains a pH level of 2.0 mixed with Bloodleaf ( Iresine

herbstii) extract will have a Very Light Pink color, A solution that contains pH

level of 3.0 will have a Light Pink color, A solution that contains pH level of 7.0

will have a Dark Pink color, A solution that contains pH level of 9.0 will have a

Pink color and lastly, a solution that contains pH level of 13.0 will have a Light

Green color.(Cababat & Rubang, n.d.)

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Red Onion (Allium cepa)

Figure 4. Red Onion (Allium cepa)

Red onions, commonly referred to as purple onions, have skin that is

purplish-red and white meat that has a reddish tint. The skin has also been

used as a dye, though they are most frequently employed in cooking. In

comparison to white or yellow onions, red onions are typically medium to large

in size and have a sweeter flavor. They are frequently eaten raw, grilled, or

very lightly cooked with other foods. They can also be added to salads for

color and flavor. Compared to white and yellow onions, red onions are readily

available all year long and are higher in flavonoids and fiber. To lessen the

"bite" and pungency of a red onion after it has been chopped, immerse it in

cool water for a time and then drain the water (Wikipedia, 2022).

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Color change of the Red Onion (Allium cepa)

Figure 5. Color change of the Red Onion (Allium cepa)

Most onions have a pH rating of 5 – 5.8 depending on the type and

whether they're raw or cooked. That's why they are acidic, the good news is

that they still lean closer to a neutral rating compared with lemons or limes.

The pH of onions may change slightly as you cook them, but they'll still be

acidic in nature. (Mira, 2022, para. 2)

A solution that contains a pH level of 2.0 mixed with Red Onion (Allium

cepa) extract will have a Light Red color, A solution that contains pH level of

2.0 will have a Faintly Red color, A solution that contains pH level of 7.0 will

have a White color, A solution that contains pH level of 9.0 will have a Light

Green color and lastly, a solution that contains pH level of 13.0 will have a

Green color. (Jadhav, n.d)

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Phytochemical Analysis

Phytochemicals are biologically active substances that are found in

plants. Since different solvents are used at various extraction circumstances,

such as duration and temperature, extraction from the plant is an empirical

endeavor. It is crucial to keep bioactive components separate from co-

extracting substances as the plants' bioactive components are extracted

further. Compounds that were isolated were further fractionated according to

their acidity, polarity, or molecular size. Solvent extraction has lately employed

the Universal Extraction System (Buchi). For extraction using various

solvents, the dried powder of different plant parts is put in a glass thimble.

Each extract undergoes the operation ten times, adjusting the temperature to

be just below the boiling point of the appropriate solvents. The extracted

solvent is then purified, condensed in a vacuum concentrator, and tested for

phytoconstituents.

The term "phytochemical screening" is another name for this method. In this

procedure, plant samples such as leaves, stems, roots, or bark that are the

reservoir of secondary metabolites are processed into aqueous and organic

extracts. The existence of secondary metabolites in the plant extracts,

including terpenes, flavonoids, and alkaloids, is then investigated. For each

class of substances to be examined, standard tests are listed in the literature.

Following this, the presence and nature of the mixture's components are often

examined using a straightforward separation technique like thin-layer

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chromatography (TLC). In TLC, the extracts are put into a glass dish covered

in silica gel or another adsorbent and stored in a chromatographic chamber

with an appropriate flowing solvent. The essential components of this method

are a mobile phase and a stationary phase, where the compounds are divided

according to their polarity. After the plate has been removed from the

chromatographic chamber, a developing solvent may occasionally be used to

identify the compounds. In developing nations, this strategy has historically

been employed and is still being used. This method allows for the early

detection of known metabolites in the extracts and is therefore economically

practical because the isolation of pure bioactive components is a time-

consuming and laborious operation. The tests are straightforward to run, but

because they have low selectivity and sensitivity of detection and are not ideal

for the efficient separation of metabolites, it is challenging to find minute

amounts of components in the sample.

Aquarelle

Figure 6. Acrylic Paints

It is a method of painting using transparent watercolors as opposed to

opaque watercolors. Painting using the aquarelle technique has been

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practiced for millennia, but due to the popularity of other popular techniques at

the time, it did not catch on right away. A water-based solution known as the

aquarelle technique is either transparent or has a low level of opacity. The

method has its roots in ancient Egypt, where artists of the time were familiar

with the method and its visual outcome. Among watercolor paintings is

Aquarelle. It is a printed image that has been colored with watercolors applied

through stencils, with a new stencil used for each color. In other words, an

aquarelle is a design or painting that uses thin, translucent watercolors as well

as the technique for doing so.

Aquarelle comes in two varieties: natural and synthetic ingredients,

both of which can have positive and negative effects on users. There are two

types of aquarelle: natural and manufactured compounds, both of which can

have both favorable and unfavorable effects on their users. The pigment

known as synthetic aquarelle is made by mixing several chemicals and may

be detrimental to those with sensitive skin. It goes without saying that the

ingredients used to make this synthetic pigment as an aquarelle will cost

more. Due to this, we considered creating a natural aquarelle using the

anthocyanins taken from Bloodleaf (Iresine Herbstii) and Red Onion (Allium

Cepa). Natural aquarelle is risk-free, healthful, and eco-friendly because it

contains only safe natural ingredients. Natural aquarelle is more affordable for

most people than synthetic aquarelle, which can be harmful to others due to

the various chemicals mixed in it. In the aquarelle technique, there are two

techniques you can use, it is called "Wet on wet" and "Wet on dry"

technique.Any watercolorist's painting vocabulary should include both wet on

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wet and wet on dry techniques. Each technique has unique qualities that

produce results that are highly distinctive and lovely. "Wet on wet" refers to

the application of new, wet paint over wet paper or another surface. "Wet on

dry" simply means that you're applying wet paint onto dry paper, or wet paint

onto an area of dry paint (Anthony Roebuck, 2018).

Paper Chromatography

Specialized paper is often used for separation in paper

chromatography. In order to distinguish the components present in plant

extracts, Porter Martin and Richard Synge utilized paper chromatography in

1943. To ascertain the purity of medicinal items is where it is most frequently

utilized. In addition to being utilized for separation in businesses, it is used to

find contamination in food and drinks. According to Kumari et al.,(2022), In

chemistry labs, paper chromatography is used to examine reaction mixtures.

Because it can successfully separate a complicated mixture with identical

polarity, this approach has the advantage of being versatile (such as amino

acids). This method is advantageous for evaluating materials with limited

quantities since the purity of the constituents is given priority. The foundation

of the paper chromatography principle is the variable degree of contact

between the elements and the stationary phase. Paper adsorption and paper

partition chromatography are the two categories.

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Anthocyanin

Figure 7. Chemical Structure of Anthocyanin

Anthocyanin Content Variations with Plant Development. Leaf color is

generally correlated with pigment metabolism. While chlorophyll is involved in

the pigmentation of green leaves, anthocyanin is typically responsible for the

colors of red, blue, pink, and purple leaves (January 24). The flavonoid

pathway in the cytoplasm of a pigmented plant cell produces the water-

soluble pigments known as anthocyanins. Sugar molecules bond together,

making it extremely soluble in vacuolar sap. Once released, these molecules

are kept in vacuoles. The majority of flower petals, red fruits (like apples), and

nearly all of the red leaves that appear in the autumn are all pink-red in hue

because of anthocyanin. The red wavelengths are scattered by the plant

tissues and are visible to us as red due to anthocyanins' absorption of light in

the blue-green range (Harvard Forest, n.d.). Since the very early days of plant

domestication, anthocyanin have been a target trait for selection. There is a

renewed interest in biotechnologies for the anthocyanin biosynthetic pathway

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because of the new discoveries of the benefits it contributes to human health.

From the start of the experimental genetics, they have been modeling a

research system of choice through current advances on the molecular

genetics of plant speciation. In the last 20 years the number of publications

regarding anthocyanin has grown dramatically. For at least the next decade, it

is expected that scientific publication will be the prime focus of research (K.M

Davies et al,. 2017).

Safety of Anthocyanin

Anthocyanin is one of the plant compounds that can benefit your

health. Anthocyanin is an antioxidant of the flavonoid family that is purportedly

able to reduce inflammation and protect people from conditions like cancer,

Alzheimer’s disease, heart disease, and type 2 diabetes. Foods that are

anthocyanin rich are generally considered safe. But in case of anthocyanin

supplements cannot be described as the same, since anthocyanin

supplements may produce more polyphenols. High doses of polyphenol

supplements may damage your kidneys, cause tumors or unbalance the

thyroid hormones according to animal studies. It can also interact with

medication and lower the absorption of certain nutrients. Therefore, as long as

the antioxidant comes from fresh, ripe plant foods then it will only bring good

benefit to the human body (A. Petre et al,. 2022).

Color Intensity

The intensity of a color (sometimes referred to as saturation or chroma)

determines how pure it is. A color with high intensity is vivid, whereas a color

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with low intensity is more subdued or neutral. When colors are taken directly

from the tube and are not combined with another hue, they are at their purest.

When you add another color to the mix, the intensity of both colors decreases

(Understanding Color : Intensity, 2019). Primary colors are believed to be the

most "pure" in terms of intensity. Intensity can also be defined as a color's

brightness or dullness. It is altered by implementing new colors into the pure

hue. By adding gray to a color, it can be made less intense. In some ways, the

amount of gray in the hue can be used to measure intensity (Fussell, n.d.).

The color that our eyes perceive is actually the complimentary color

that has already dissolved into the substance or solution. The more absorbent

the color, the more intense it will be. More absorbance happens as the

concentration of the solution through which the color is absorbed increases.

Furthermore, Beer's law states that the concentration of a solution is directly

proportional to its absorption of light, and it is widely known that absorbance is

proportional to intensity. As an outcome, the higher the concentration, the

higher the absorbance, and therefore the higher the intensity. Beer's law is a

formula that expresses the relationship between light intensity and the

concentration of a substance. Explore the history of light transmittance and

absorbance, and in addition to how the equation of Beer's law is used in

science (Study.com, n.d.).

Environmentally Friendly

27
 The researchers will observe each of the Bloodleaf(Iresine herbstii)

and Red onion(Allium cepa) ph mixture if it causes any harm to the

environment and to the operator.

Stick Consistency

The Water Sensitivity Test enables us to determine how well the paper will

absorb fountain solution at the printing surface. This test involves concurrently

applying ink and fountain solution to the paper. Using a densitometer, the

differences between the "dry" and the "wet" prints are measured and

compared. In this test, the test sample paper is simultaneously covered in

fountain solution and ink. The results are expressed as "% Transfer," which

measures the amount of ink that can be printed in the wet area in comparison

to how much can be transferred in the dry area. This test determines if the

paper's surface can absorb enough fountain solution to print without

snowflaking or smudged ink. The percentage of ink transfer in the "water

area" is calculated using a densitometer, which also records the percentage of

wet coating pick, if any, on the sample. Since differences in wet pick can be

caused by drying conditions (the amount and degree of the binder's cure) or

the coating of the paper being tested, this test is especially helpful when

testing coated papers. This test is especially helpful for assessing coated

papers since variations in wet pick may be caused by drying circumstances

(the amount and degree of the binder's cure) or the starch content of the

binder system (Plowman, n.d.).

Flavonoids

28
 Flavonoid, also called flavone, is a class of non-nitrogenous biological

pigments that is represented in plants. It is mostly found in the vacuoles of

plant cells, it is a water-soluble phenolic compound(having a -OH group

attached to an aromatic ring). Flavonoids have been described to have more

than 3,000 different members.

A major group, anthoxanthins, often impart yellow colours to the petals

of flowers. A second major second major group, the anthocyanins, are

responsible for the purple and purple-red colours of autumn leaves, as well as

the red colouring of buds and young shoots. Flavonoids and flavonols,

pigments that are typically yellow or ivory-coloured. Flavonoids may provide

protection against damage from ultraviolet radiation and may serve as

antioxidants. Attracting bees, butterflies and other pollen-transporting animals

that implement fertilization in plants is a important role that the flavonoids

need to do. They impart colour to flowers in order to do the job. How plants

interact with nitrogen-fixing bacteria in their roots is also an effect of

flavonoids present (Petruzzello, n.d.)

Glycosides

When a monosaccharide and an alcohol interact with an acid catalyst,

glycosides are produced as the acetal derivatives. In biological systems,

glycosides are ubiquitous. The solubility and other characteristics of the

molecule can be significantly altered by adding a sugar moiety to a lipid

benzenoid structure. Numerous glycosidases enzyme systems have

developed for the attachment and removal of sugars from alcohols, phenols,

29
and amines as a result of the significant modifying influence that such

derivatization has. (S. Farmer et al., 2023.)

Any of the many naturally occurring compounds known as glycosides

have a carbohydrate part made up of one or more sugars or uronic acids and

are joined by a hydroxy molecule. The hydrophilic molecule, also known as an

aglycon, is typically a non-sugar substance but might potentially be another

carbohydrate, such as cellulose, glycogen, or starch, which contains large

amounts of glucose. One glycoside that is frequently seen as the pigment in

flowers and fruits is anthocyanin. (Britannica, n.d.)

Sterols

Figure 8. Chemical Structure of Sterols

Sterol is an organic substance with the chemical formula C17H28O. Its

structure differs from that of gonane by having a hydroxyl group in position 3

instead of a hydrogen atom. Thereby, it is a gonane alcohol. Steroids are a

broad term for any substances that have changed ring systems, extra

functional groups, or the gonane structure. Sterols are a subclass of steroids

as a result. Most eukaryotes, such as plants, animals, and fungi, make them

naturally. Some bacteria can as well (however likely with different functions).

30
 Sterols, also known as phytosterols, zoosterols, and mycosterols, are found

in the membranes of plants, animals, and microbes, respectively. Cholesterol

is the primary zoosterol, while phytosterols such -sitosterol, campesterol, and

stigmasterol are three of the most important phytosterols. (Gordon, 2003)

Triterpenes

One of the classes of higher terpenes is the triterpenes. It appears to

be extremely common in nature and is present in practically all terrestrial and

aquatic animals. Nevertheless, higher plants have been found to contain the

majority of bioactive triterpenes. It is included in the broad and structurally

diverse group of triterpenoids that are naturally occurring alkenes, which also

include natural degradation products, oxidation, and hydrogenation products.

It can be divided into linear and cyclic ones, mainly including tetracyclic and

pentacyclic carbocycles, after taking into considerations the architecture of its

carbon skeleton.

Triterpenoids frequently shows a wide range of biological activities,

such as cytotoxic, anti-cancer, anti-flammatory, anti-oxidative, anti-fungal, and

wound healing activity because of its complex and diverse structures.

Triterpenes are regarded as a potential family of molecules for the creation of

novel medications, even with our existing knowledge. (Roussis & Ioannou,

2020)

31
Alkaloids

A large class of naturally occurring chemical substances known as

alkaloids have nitrogen atoms, sometimes amino or amido atoms, in their

structures. These nitrogen atoms cause the alkalinity of these compounds,

and are usually situated in a ring system. Alkaloids can be divided into

classes, such as indoles, quinolines, isoquinolines, pyrrolidines, pyridines,

pyrrolidines, tropanes, and terpenoids, and steroids. There are other

classification systems that are connected to the family of plants they occur in,

opium alkaloid, is one of the examples of classification systems which occurs

in opium poppy(Papaver somnife rum). Due to the lack of a clear relationship

between the two separate classification systems, these lead to confusion

between the biological distribution of alkaloids and their chemical kinds. The

name alkaloids comes from "alkali-like". just like inorganic alkalis, it can react

with acids and salts. Alkaloids are usually colorless, odorless crystalline solids

in their pure form, they can be yellowish liquid sometimes. They usually have

a bitter taste. There are more than 3000 of alkaloids that are known in over

4000 plant species. (Kurek, 2019)

Saponins

32
 Figure 9. The Chemical Structure of Steroid Saponins

Saponins isolated from medicinal plants are naturally occurring

bioorganic molecules of high molecular weight, containing at least 6 or 12

carbon atoms, respectively. and their relationship to their medicinal efficacy or

pharmacological behavior.Recently, an increasing demand for saponin

applications has been observed due to various biological, medical and

pharmaceutical effects. As a result, the chemistry of saponins, particularly

triterpenoid saponins, is well covered in this review paper. All that was

included with respect to saponins was their classification, chemical makeup,

potential traditional isolation pathways, and qualitative and quantitative

measurements. Hederagenin and oleanolic acid mono- and bidesmoside

structural examples are also shown. According to their atoms, rings, and

functional groups, the structural variations of triterpenoid, steroid, and alkaloid

glycosides have been compiled (MMA El Aziz et al., 2019).

Tannins

Tannins are chemical compounds that belongs to a larger group called

polyphenols, it is a type of chemical compound that is bitter and astringent.

33
Occurring mostly in nature, they occur abundantly namely in the bark of trees

and variety of leaves, legumes, and fruits including grapes. They possess a

special capacity to quickly combine with other molecules, particularly proteins,

leading to precipitation. Unlike other kinds of polyphenols, tannins often have

bigger molecules. They are responsible for dissuading animals from eating

unripe fruits and seed by making them unpalatable. (Salpico, 2021)

Liebermann-Burchard Test

Shinoda Test

A test for flavones' presence is the Shinoda test. Under the conditions

of the Shinoda reaction, they are converted to anthocyanins if they are

present in the test sample (Shinoda test for flavonoids fundamental chemistry,

2014) Alcoholic solutions of flavone or flavonol that have been exposed to

metallic Hydrochloric acid with magnesium (or zinc) produce an orange, red,

or color violet. (Flavonoids, 2013)

Mayer’s Test

Froth Test

34
 Fehling’s Test

Ferric Chloride Test

METHODOLOGY

A. RESEARCH DESIGN

This study uses an Experimental method. According to Gaille (2017), it is

easy to duplicate settings that could take a long time when conducting research in a

lab setting, allowing the variables to be tested adequately. As each variable is

carefully examined, this enables researchers to exert more control over any

additional variables that may exist, thereby reducing the unpredictable nature of

nature.

The type of sampling method that this study will be using is Purposive

sampling method. It is also known as judgmental, selective, or subjective

sampling. It is a type of non-probability sampling where researchers choose

individuals from the population to take part in their surveys using their own

discretion. The researchers will thoroughly think through how they will establish a

sample population, even if it is not statistically representative of the greater

population at hand. As the name suggests, researchers will go to this community

35
on purpose because they think that these individuals fit the profile of the people

that they need to reach. (Purposive sampling 101, 2021)

B. VARIABLES AND MEASURES

C. RESEARCH LOCALE

The researchers requested that the plant i.d. verification be done by

the Jose Vera Santos Memorial Herbarium of the Institute of Biology, College

of Science, University of the Philippines - Diliman for use in any legal

proceedings for the research project pertaining and related to this

authentication.

The Muntinlupa National High-School Main-Senior High Building,

specifically the research and engineering laboratory, is where the Paper

Chromatography and Application of Indicator and Solution will take place. The

chemical lab apparatus that the researchers will need will be provided to them

by their research teacher for use as their research equipment during the

experiment. The DOST Industrial Technology Development Institute is where

the phytochemical screening will take place. Students majoring in arts and

design are the intended target group for the survey exam, for which

questionnaires will be distributed.

Color Intensity- On the unique paper that the researchers gave each

respondent, they are free to paint whatever they like. The respondents will

36
 assess whether they are pleased with the result or whether they find it

appealing.

Odor Acceptability- The researchers will drop a pinch of the ink

on the special paper. After dropping a pinch of the aquarelle, the

respondents will sniff the aroma of the aquarelle. The respondents

will also observe if the odor is pleasant to their nose, or the odor is too strong

for them.

Stick Consistency- Each respondent will expose the ink to water to see if

there are any changes with the consistency of the aquarelle. The clarity of

strokes and color must be checked to know if the consistency of the aquarelle

is good enough.

D. METHODOLOGY FLOWCHART

Gathered the Ingredients

37
Manual Separation Food Processor

(Mortar & Pestle)

Paper Chromatography

Specimens mixed with Different pH Levels

38
 Bloodleaf Red Onion

Conduct survey & Test the Results

E. MATERIALS AND INSTRUMENTATION

This equipments will be used for the extraction of Anthocyanin in

Bloodleaf (Iresine herbstii) and Red Onion (Allium cepa) to determine which

specimen is more effective as a substitute for commercial aquarelle.

Materials Purpose

150 grams BLOODLEAF (Iresine Serve as one of the main indicator for

herbstii) the samples

75% extract

150 grams RED ONION (Allium cepa) Serve as one of the main indicator for
75% extract
the samples

DISTILLED WATER Used to affect pH of the solutions.

25% CALAMANSI EXTRACT - 20 Used for making pH solutions around

39
 grams Solution 2.0

25% VINEGAR - 20 grams Solution Used for making pH solution around 2.5

25% PURE WATER - 20 grams Solution Used for making pH solution around 7.0

25% BAKING SODA - 20 grams Used for making pH solution around 8.3

Solution

25% BLEACH - 20 grams Solution Used for making pH solution around

12.0

ALL PURPOSE FLOUR - 200 grams Used to make the aquarelle more

thicker and have a vibrant color.

WHITE SUGAR - 100 grams Used to extend the drying time and

prevent hard edges and irregularities in

underpaintings, flat washes, and

gradation of aquarelle.

10PCS SPECIAL PAPER Used for the survey.

GLOVES Used to protect against skin absorption

of chemicals, chemical burns, thermal

burns, lacerations, and cryogenic liquid

exposure.

ETHANOL 70% Solvent used for Soxhlet method

extraction.

40
 Instruments Purpose

MORTAR AND PESTLE Used to prepare reactants for a solid-

state synthesis (the ceramic method).

BEAKER Used for holding the distilled water.

STIRRING ROD Used for mixing solutions and

indicators.

STRAINER Used to strain liquids away from other

ingredients but also to occasionally sift

fine ingredients away from larger

ingredients.

MEASURING SPOONS Used to measure the proper amount of

liquid precisely.

CONICAL FLASK Used for making indicators for mixing.

BALANCE Used for measuring in grams.

TEST-TUBES Used for storing mixed solutions and

indicators.

TEST-TUBES RACK Used to hold the test tubes.

WEIGH BOAT Used for weighing the baking soda,

sugar and all purpose flour.

GRADUATED CYLINDER Used to measure the volume of

liquids..A

41
 ERLENMEYER’S FLASK Used to store the sample extracts.

CORK STOPPER Used with the erlenmeyer's flask to

reduce or eliminate evaporation on

extracts or contamination of airborne.

F. METHODS AND PROCEDURES

F.1 Paper chromatography

Paper chromatography is an analytical technique for separating

dissolved chemical substances based on their different migration rates across

sheets of paper. It is a cheap but effective analytical tool that needs little raw

material. Masood et. al., (2021)

Extract preparation for bloodleaf

The extract of the bloodleaf is prepared by using mortar and pestle.

The stem of the bloodleaf will be grinded and compressed using a fish net.

The extracted plant material will be placed in the conical flask. A measure of

14 cm long strip of chromatography paper will be used, one end is sharp,

whereas the other is blunt. A 2 cm line will be drawn using a pencil from the

pointed end of the paper. Using a capillary tube, microdroplets of stem extract

will be applied at the center of the pencil line in the chromatography paper

allowing it to air dry and repeat the application for 5 to 10 times. Then the

solvent will be poured into the beaker and the paper strip will be put into the

42
beaker attached to a stick by bending it at the blunt end. The solvent (70%

ethanol) must come into contact with the paper's pointed end but shouldn't

grasp the spot where the extract is placed. The beaker is left alone so it can

stand watch the band separation and the solvent movement. When the bands

of pigment have formed after the solvent has gone roughly half as far as the

paper, then the cover would be lifted with the paper from the beaker attached.

The solvent front's edge will be marked with a pencil and allow it to dry

completely.

Extract preparation for red onion

The extract of the red onion is prepared by using a food processor.

The red onion will be grinded and compressed using a fish net. The extracted

plant material will be placed in a beaker. A measure of 14 cm long strip of

chromatography paper will be used, one end is sharp, whereas the other is

blunt. A 2 cm line will be drawn using a pencil from the pointed end of the

paper. Using a capillary tube, microdroplets of plant extract will be applied at

the center of the pencil line in the chromatography paper allowing it to air dry

and repeat the application for 5 to 10 times. Then the solvent will be poured

into the beaker and the paper strip will be put into the beaker attached to the

stick by bending it at the blunt end. The solvent (70% ethanol) must come into

contact with the paper's pointed end but shouldn't grasp the spot where the

extract is placed. The beaker is left alone so it can stand watch the band

separation and the solvent movement. When the bands of pigment have

formed after the solvent has gone roughly half as far as the paper, then the

43
cover would be lifted with the paper from the beaker attached. The solvent

front's edge will be marked with a pencil and allow it to dry completely.

F.2 Phytochemical analysis

The extraction, screening, and identification of medicinally active

substances found in plants is referred to as phytochemical analysis, also

known as phytochemical screening. Plants can produce bioactive substances

such as flavonoids, alkaloids, carotenoids, tannin, antioxidants, and phenolic

compounds..Phytochemical Screening (n.d).

Plant Material for bloodleaf

In the present study, the stem of bloodleaf will be used as plant material.

The material has just been picked from a plant. The stems are cut, cleaned,

washed under running tap water, drained, and boiled for 10 minutes at 90

degrees Celsius. The samples are placed in polyethylene bags and kept at 4

degrees Celsius for laboratory analysis.

Drugs and Chemicals

Solvents and chemical used are distilled water and trifluoroacetic acid 0.1

% (v/v),

Extract Preparation for bloodleaf

The extract was prepared as stated by the method of Kouakou et. al (2013).

One hundred fifty grams (150 g) of bloodleaf leaves will be extracted for 24

hours at 4 °C in 200 mL of acidified methanol with trifluoroacetic acid 0.1%

44
(v/v). The macerate is strained on cotton wool and Whatman paper in

succession. We will obtain a dry extract after low-pressure vacuum

evaporation of the methanol in the BÜCHI Rotavapor R-114 at 38 °C. In order

to get rid of the sugars and chlorophyll pigments, the dried extract was

blended with 200 mL of distilled water. The combined aqueous solution was

then filtered using gel XAD-7. The XAD-7 gel was covered with 100 mL of

acidified methanol that contained 0.1% trifluoroacetic acid (v/v), and the

resulting methanolic filtrate was presumably evaporated under low pressure in

a BÜCHI Rotavapor R-114 at 38 °C. The obtained dry extract is dissolved in

100 mL of distilled water. The aqueous solution was lyophilized using the

CHRIST ALPHA 1-2 freeze dryer. The dried extract obtained represented the

bloodleaf leaves extract whose polyphenol content and compounds had

previously been determined by Nuez-Estevez et al (2021).

Plant Material for Red Onion

In the current study, red onions will be used as plant material. The material

was purchased at a supermarket. The onions are cut, cleaned, washed under

running tap water, drained, and boiled for 10 minutes at 90 °C. The samples

are placed in polyethylene bags and kept at 4 degrees Celsius for laboratory

analysis.

Drugs and Chemicals

Solvents and chemicals used are distilled water and trifluoroacetic acid 0.1

% (v/v).

45
Extract Preparation for Red Onion

The extract was prepared as stated by the method of Kouakou et al (2013).

One hundred fifty grams (150 g) of bloodleaf leaves will be extracted for 24

hours at 4 °C in 200 mL of acidified methanol with trifluoroacetic acid 0.1%

(v/v). The macerate is strained on cotton wool and Whatman paper in

succession. We will obtain a dry extract after low-pressure vacuum

evaporation of the methanol in the BÜCHI Rotavapor R-114 at 38 °C. The

dried extract was mixed with 200 mL of distilled water, and the resulting

aqueous solution was filtered through gel XAD-7 to remove any sugars or

chlorophyll pigments. The gel XAD-7 was covered with 100 mL of acidified

methanol that contained 0.1% trifluoroacetic acid (v/v). The filtrate from this

process, which would likely be a methanolic one, was then likely treated to

low-pressure vacuum evaporation in a BÜCHI Rotavapor R-114 at 38 °C. A

100 mL solution of distilled water is made from the obtained dry extract. The

aqueous solution was lyophilized using the CHRIST ALPHA 1-2 freeze dryer.

The dried extract obtained represented the bloodleaf leaves extract whose

polyphenol content and compounds had previously been determined by Ren

& Zhou (2021).

Test for Anthocyanins

By combining 2 mL of the plant extract with 2 mL of 2 N HCl, the

presence of anthocyanins was demonstrated. The presence of anthocyanins

is indicated by the appearance of a pink-red color that turns purplish blue after

the addition of ammonia.

46
F.3 Procedure

F.3.1 Preparing for the pH solutions

The solutions that can be found in their home such as; pH 2.0-

Calamansi Extract, pH 3.0- Vinegar, pH 7.0- Pure water, pH 9.0- Baking

Soda, pH 13.0- Bleach, to make a safest painting medium for making the

smallest impact on the natural environment, water in aquarelle paint makes it

economically friendly and environmentally preferable. Every solution of pH-

level will be used is 20 grams, after putting the solutions on each small

container, the extracted or grinded red onion indicator will be added. After

adding the indicator, observe the changes of color in each solution which is

the acid to base, then add the water in each solution. The more indicators

added in the solutions, the more vibrant the color.

F.3.2 Preparation of the Aquarelle

Needed materials will prepare first in making an aquarelle which is the

200g of All-purpose flour, 100g sugar, 5 pieces small container, water, and

the main ingredient the 5 extracted Blood Leaf (Iresine Herbstii) and Red

Onion (Allium Cepa) with solutions in pH-level of making the aquarelle. The

first step of making an aquarelle is to put the 20g all-purpose flour on each

small container, and 10g of sugar will be added. After preparing the measured

flour and sugar, mix thoroughly then add the extracted Onion with pH-level

solutions in each container. Mix thoroughly for the finest result.

47
 After the experiment, the researchers will conduct a survey to know if

the Bloodleaf (Iresine Herbstii) and Red Onion (Allium Cepa) as aquarelle

while using the pH-level experiment meet the expected results.

G. DATA GATHERING

To know if the Bloodleaf (Iresine Herbstii) and Red Onion (Allium Cepa)

as aquarelle while using the pH-level experiment meet the expected results it

should achieve the qualifications in terms of: color intensity, odor

acceptability, and stick consistency of Blood Leaf and Red Onion on 3 trials

per pH-level experiment. There will be 60 samples from the population of the

Grade 12 ADAS Students at Muntinlupa National High-School Main Senior

High School Building to test the general acceptability of the study. As for the

collection of data, the researchers will conduct a survey after the experiment

since the independent variables are considered as quantitative data.

H. PLAN FOR ANALYSIS

48
 RESULT AND DISCUSSION

The discussions of the results below were the salient findings of the study on the

comparative results of the specimens.

A. PLANNED PROCEDURE FOR CONDUCTING THE AQUARELLE

B. PHYTOCHEMICAL TEST FOR SPECIMEN CONSTITUENTS

Sample: Plant Extract (2 Samples)

Description and Identification:

OCS-2022-0826: About 150 mL of dark green liquid in glass container,
unmarked
OCS-2022-0827: About 150 mL of violet liquid in a glass container, unmarked

OCS-2022-0826 OCS-2022-0827
Test Parameters Test Method
Bloodleaf Red Onion
(Iresine herbstii) (Allium cepa)

Sterols Liebermann-Burchard

49
Triterpenes Test

Flavonoids Shinoda Test

Alkaloids Mayer’s Test

Saponins Froth Test

Glycosides Fehling’s Test

Tannins Ferric Chloride Test

C. PAPER CHROMATOGRAPHY

(figure 11)

The bloodleaf (Iresine herbstii) and red onion (Allium cepa) extracts are both

examined to determine its hue. The bloodleaf has yellow chlorophyll in its

chlorophyll B and green chlorophyll in its chlorophyll A. The bloodleaf's red

anthocyanin pigment component, when separated, releases a vibrant red-pink

50
 hue. However, the red anthocyanin pigment in red onions produces a poor

purple-red hue.

D. RESPONDENTS AGREEMENT OF COMPARATIVE ANALYSIS IN TERMS

OF COLOR INTENSITY, ENVIRONMENTAL FRIENDLINESS, AND STICK

CONSISTENCY

(Analysis of the results: 1 paragraph

Pakilagay kung may difference yung dalawang specimen naka percent

dapat per variables)

Table 2, 3, 4 show the result of respondents evaluation from the participants

who tested the alternative ink Bloodleaf (Iresine herbstii) in terms of;

Table 2. Color Intensity. The summary of the respondents' evaluation of the

alternative ink following the intensity of the color.

Percentage Rate M Descriptive Equivalent

100% 0.72 Strongly Agree

75% 0.28 Agree

50% 0 Disagree

Table 3. Environmental Friendliness. The summary of the respondents’

evaluation of the alternative ink following the environmental friendliness of the

aquarelle.

Percentage Rate M Descriptive Equivalent

51
 100% 0.88 Strongly Agree

75% 0.12 Agree

50% 0 Disagree

Table 4. Stick Consistency. The summary of the respondents’ evaluation of

the alternative ink following the stick consistency of the ink color.

Percentage Rate M Descriptive Equivalent

100% 0.6 Strongly Agree

75% 0.4 Agree

50% 0 Disagree

Table 5, 6, 7 show the result of respondents evaluation from the participants

who tested the alternative ink Red Onion (Allium Cepa) in terms of;

Table 5. Color Intensity. The summary of the respondents' evaluation of the

alternative ink following the intensity of the color.

Percentage Rate M Descriptive Equivalent

100% 0.12 Strongly Agree

75% 0.28 Agree

50% 0.6 Disagree

52
 Table 6. Environmental Friendliness. The summary of the respondents’

evaluation of the alternative ink following the environmental friendliness of the

aquarelle.

Percentage Rate M Descriptive Equivalent

100% 0.12 Strongly Agree

75% 0.88 Agree

50% 0 Disagree

Table 7. Stick Consistency. The summary of the respondents’ evaluation of

the alternative ink following the stick consistency of the ink color.

Percentage Rate M Descriptive Equivalent

100% 0.32 Strongly Agree

75% 0.28 Agree

50% 0.4 Disagree

E. SIGNIFICANCE OF DIFFERENCE IN THE AGREEMENT RATES OF

RESPONDENTS BETWEEN THE COMPARATIVE ANALYSIS OF BLOOD

LEAF (Iresine herbstii) AND RED ONION (Allium cepa)

The calculations were done to compare the acceptance rates of

bloodleaf (Iresine herbstii) and red onion (Allium cepa) inks. The t-test result

showed a statistical difference (p<0.05), indicating variations in the approval

rates of the two tested products. As for color intensity, stick consistency, and

53
environmental friendliness, the outcome was consistent across all 3 factors.

Reviewing the goods' overall means showed that the ink manufactured from

Bloodleaf (Iresine herbstii) was far more well-liked by the subjects than the ink

made from Red Onion (Allium cepa). This further supports the validity of the

prior assumption regarding the efficiency of Bloodleaf extracts as an aquarelle

ink substitute over Red Onion extracts based on the responses provided by

the student respondents. Nonetheless, thorough studies need to be

conducted to establish the reliability of the result considering other

respondents.

Table 8. Significance of Difference. Comparison of acceptability rates of

respondents between aquarelle made of Bloodleaf(Iresine herbstii) extract

and aquarelle made of Red Onion (Allium cepa).

Categories Bloodleaf Red Onion Significance

(Iresine herbstii) (Allium cepa)

Color Intensity 0.5 0.3 Significant

Environmental 0.5 0.5 Significant

Friendliness

Stick 0.5 0.3 Significant

Consistency

54
 (Figure 12)

The color intensity, Environmental Friendliness and Stick

Consistency of Bloodleaf (Iresine herbstii) and Red Onion (Allium cepa) is

tested into a testing paper. It shows the more effectiveness of Bloodleaf

(Iresine herbstii) than Red Onion (Allium cepa) as an alternative ink.

CONCLUSION

55
 RECOMMENDATION

Based on the respondents, Red onions (Allium cepa) have been found to have no

negative environmental effects, however they did exhibit poor color consistency and

intensity. The bloodleaf (Iresine hersbtii) has a good color intensity and has no

harmful impact on the environment, it appears to have a negative trend as a result of

its lack of sticky consistency.

Future research could investigate other pH solution to utilize in the substance, the

complementary paper recommendations given in this study can be further

considered, like as:

56
REFERENCES AND RESOURCES

57
Animal Biotechnology (2014) Herbal Medicine and Biotechnology for the Benefit of

Human Health. Retrieved from https://www.sciencedirect.com/topics/medicine-and-

dentistry/phytochemical-screening]

Andrew Wilton & Anne Lyles. The Great Age of British Watercolours (1750–1880).

Prestel, 1993. ISBN 3-7913-1254-5. Retrieved from

https://en.m.wikipedia.org/wiki/Watercolor_painting#:~:text=Watercolor%20refers%2

0to%20both%20the,to%20pass%20out%20of%20use.

Ask Difference (2019, August 28) Watercolor vs. Aquarelle. Retrieved from

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Lauren Josey n.d., Soxhlet Extractor. Retrieved from

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APPENDIX

65
 Student Profile

Manalo, Kc Ezikiel A.
Sunflower Street, Patdu Compound, South GreenHeights Village,
Putatan Muntinlupa City

_________________________________________________________________________

Personal Information

Age : 18

Gender : Female

Civil Status : Single

Birthday : January 30, 2005

Birthplace : Muntinlupa City

Religion : Roman Catholic

Citizenship : Filipino

Language : Filipino/English

Father’s Name : Venson Miranda Manalo

Mother’s Name : Dyan Abig Manalo

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_________________________________________________________________________

Educational Background

Senior High : Muntinlupa National High School (2022-2023)

Junior High : Tunasan National High School (2020-2021)

Elementary : Bayanan Elementary School Unit-1 (2016-2017)

“To provide a basis for the development of natural and sustainable aquarelle ink from
locally available plants, promoting eco-friendly practices in the art and calligraphy
industry.”

Student Profile
Erjas, Luzviminda B.
Block 3 Lot 13 D Chunghee St. Carey Homes Summitville Subdivision,
Putatan Muntinlupa City, Metro Manila 1772

_________________________________________________________________________

Personal Information

Age : 17

Gender : Female

Civil Status : Single

Birthday : June 27, 2005

Birthplace : Pasay City

Religion : Roman Catholic

Citizenship : Filipino

Language : Filipino/English

Father’s Name : Joel Erjas

Mother’s Name : Rosemary Erjas

_________________________________________________________________________

67
 Educational Background

Senior High : Muntinlupa National High School (2021-2023)

Junior High :
● Cagayan National High School (2017-2019)
● Pitogo High School (2019-2021)

Elementary :
● Ciriaco P. Tinga Elementary School (2011-2014)
● Tuguegarao North Central School (2014-2016)
● Tenement Elementary School (2016-2017)

“To evaluate the effectiveness of Red onion and Blood leaf extracts as alternative
aquarelle ink compared to synthetic materials in terms of color quality, stability, and
toxicity.”

Student Profile

De Leon, Micaela D.
Dela Cruz comp. Summitville Subd. Putatan Muntinlupa City

_________________________________________________________________________

Personal Information

Age : 18

Gender : Female

Civil Status : Single

Birthday : March 6, 2005

Birthplace : Muntinlupa City

Religion : Roman Catholic

Citizenship : Filipino

Language : Filipino/English

Father’s Name : Jorge De Leon

Mother’s Name : Monaliza De Leon

_________________________________________________________________________

68
 Educational Background

Senior High : Muntinlupa National High School (2021-2023)

Junior High : Tunasan National High School (2017-2020)

Elementary : F. de Mesa Elementary School (2010-2016)

“To provide a basis for the development of natural and sustainable aquarelle ink from
locally available plants, promoting eco-friendly practices in the art and calligraphy
industry.”

Student Profile
Estabillo, Clarenz C.
Block 7, Lot 43, Phase 3, St.Mary Street, Sto.niño Village, Tunasan, Muntinlupa City,
Metro Manila, 1773
_________________________________________________________________________

Personal Information

Age : 17

Gender : Male

Civil Status : Single

Birthday : June 4, 2005

Birthplace : Manila

Religion : Roman Catholic

Citizenship : Filipino

Language : Filipino/English

Father’s Name : Arvin Cristin V. Estabillo

Mother’s Name : Rowena C. Estabillo

_________________________________________________________________________

Educational Background

69
 Senior High : Muntinlupa National High School (2021-2023)

Junior High : Sto. Niño School of Muntinlupa (2017- 2021)

Elementary : Tunasan Elementary School (2011 - 2017)

“To optimize the phytochemical screening of Red onion (Allium cepa) and Blood leaf
(Iresine herbstii) extracts for aquarelle ink production.”

Student Profile
Montenegro, Luiz Miguel S.
Block 4 Lot 10 Amaranth St, Southview Homes 3,
Brgy. San Vicente, San Pedro City, Laguna

_________________________________________________________________________

Personal Information

Age : 18

Gender : Male

Civil Status : In a Relationship

Birthday : September 4, 2004

Birthplace : Muntinlupa City

Religion : Roman Catholic

Citizenship : Filipino

Language : Filipino/English

Father’s Name : Matthew Montenegro

Mother’s Name : Donna Montenegro

_________________________________________________________________________

70
 Educational Background

Senior High : Muntinlupa National High School (2021-2023)

Junior High : Sto. Niño School of Muntinlupa (2017-2021)

Elementary : Sto. Niño School of Muntinlupa (2011-2017)

“To provide a basis for the development of natural and sustainable aquarelle ink from
locally available plants, promoting eco-friendly practices in the art and calligraphy
industry.’’

Student Profile
Decasa, Amir Edvinlee M.
BLK 9 LOT 754, Bagong Silang, Sucat, Muntinlupa City

_________________________________________________________________________

Personal Information

Age : 17

Gender : Male

Civil Status : Single

Birthday : June 12, 2005

Birthplace : Calaca, Batangas

Religion : Roman Catholic

Citizenship : Filipino

Language : Filipino/English

Father’s Name : Louie B. Decasa

Mother’s Name : Geraldine M. Decasa

_________________________________________________________________________

71
Educational Background

Senior High : Muntinlupa National High School (2021-2023)

Junior High : Muntinlupa Business High School 2016-2021

Elementary : Bagong Silang Elementary school 2010-2016

“To provide recommendations for further optimization and utilization of red onion and
bloodleaf extracts as aquarelle inks.”

MUNTINLUPA NATIONAL HIGH SCHOOL

NBP Reservation, Poblacion, Muntinlupa City

PLAGIARISM REPORT

‘’Optimization of Phytochemical Screening and Paper

Chromatography Comparative Analysis of Anthocyanin by
Bloodleaf (Iresine herbstii) and Red Onion (Allium cepa) as
Indicator of Aquarelle in pH”

Decasa, Amir Edvinlee M.

De Leon, Micaela D.
Erjas, Luzviminda B.
Estabillo, Clarenz C.
Manalo, Kc Ezikiel A.
Montenegro, Luiz Miguel S.

72
 Submitted to:

Princess C. La Rosa
Research Adviser

Christine Anne Marla, Scientific Editor of PSHS - Main

Research Consultant

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 MUNTINLUPA NATIONAL HIGH SCHOOL

NBP Reservation, Poblacion, Muntinlupa City

GRAMMAR CHECKER

‘’Optimization of Phytochemical Screening and Paper

Chromatography Comparative Analysis of Anthocyanin by
Bloodleaf (Iresine herbstii) and Red Onion (Allium cepa) as
Indicator of Aquarelle in pH”

Decasa, Amir Edvinlee M.

De Leon, Micaela D.
Erjas, Luzviminda B.
Estabillo, Clarenz C.
Manalo, Kc Ezikiel A.
Montenegro, Luiz Miguel S.

Submitted to:

Princess C. La Rosa
Research Adviser

Christine Anne Marla, Scientific Editor of PSHS - Main

Research Consultant

118
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