1

CHAPTER I

THE PROBLEM AND ITS SETTING

Introduction

In this modern world of technology, the price of the products has

become a challenge to everyone due to the scarcity of resources. Almost

every product at the present time has been commercialized and

modernized. In order to address such problem, people sought for products

that are more natural and cheaper. They tend to rely on using natural

resources that are renewable and non-expensive.

Natural resources are resources that exist without actions of

humankind. This includes sunlight, atmosphere, water, land (includes all

minerals) along with all vegetation, crops and animal life that can be found

within the environment. There are countless ways to use these

environmental treasures. In fact, every man-made product is composed of

natural resources. Examples of these are household equipment,

woodworks such as furniture, processed foods, and other necessities that

individuals use and consume.

However, although these natural resources are fairly sustainable,

the problem in supplying the needs of the people still remains alarming to
 2

everyone. In spite of this concern, people managed to innovate things in

various ways using materials out of these natural resources. One good

example is utilizing plants to create herbal medicines, spices for food,

essential oils, natural dyes, and even clothing. Other products derived

from plants include soaps, shampoos, perfumes, cosmetics, paint,

varnish, turpentine, rubber, and inks.

Generally, inks are known to be used in markers. Markers play an

important role in writing and in artworks in different ways. It was designed

to create writings on objects with enormous creative and practical

possibilities. In school, students and teachers extensively use marker

more commonly in making reports, homeworks, school presentations,

projects, and the like. It is also useful for permanently marking surfaces,

which is often necessary for identification purposes – putting names on

clothing tags, boxes, and tape which can be adhered to almost any item.

Typically, the inks used in most markers are made from synthetic

materials such as petroleum and chemical solvents. These kind of

substances are hazardous especially to the health of the users and to the

environment. Some markers contain methyl isobutyl ketone, a harmful

chemical that can irritate the eyes and nose, cause feelings of weakness,

headaches, nausea, vomiting, dizziness, and coordination problems

 3

through short-term use. Long-term use can cause headaches, nausea,

and enlargement of liver, burning in the eyes, intestinal pain, weakness

and insomnia. Accidentally getting marker that contains methyl isobutyl

ketone on the skin can cause redness, dry skin, and even pain.

Unfortunately, there is no remedy for such irritation.

Furthermore, the researchers of this study planned to formulate a

safer alternative ink, decreasing the possibility of acquiring diseases from

harmful chemicals brought by the commercial ones. In addition, the group

came up with the idea of making an alternative ink using natural

ingredients. Since inks are composed of binders, colorants (pigment or

dye), and other additives that affect its flow and thickness, the group have

decided on using water and ethyl alcohol (ethanol) as the binder, Achiote

and Alugbati extracts as the colorants, Gum Arabic as the thickener, and

table salt as the preservative. The group hypothesized that both plants

have the potentiality to create alternative plant-based ink.

Achiote, more commonly known as “Annatto” in the English-

speaking world, is a tall shrub to small evergreen tree 6-10 m (20-33 ft)

high. It bears clusters of 5 cm (2 in) bright white to pink flowers,

resembling single wild roses, appearing at the tips of the branches. The

fruits are in clusters: red to brown seed pods covered in soft spines. Each
 4

pod contains many seeds covered with a thin waxy blood-red aril. When

fully mature, the fruit splits open, exposing the numerous dark red seeds.

The fruit itself is not edible, however the orange-red pulp that covers the

seed is used to produce a yellow to orange food coloring. It is distributed

as well as cultivated widely in its native land in Central and South

America.

Moreover, Annatto is commonly used in Latin American and

Caribbean cuisines as both a coloring and flavoring agent. Central and

South American natives use the seeds to make body paint and lipstick.

For this reason, the Achiote is sometimes called the "lipstick-tree".

Additionally, Achiote dye (known more commonly as annatto dye) is a

widely used food colorant. It is prepared by grinding seeds or simmering

the seeds in water or oil. Owing to its fat-soluble nature, the pigment is

widely used for imparting a red to orange color to a variety of foods such

as ice cream, candy, bakery products and rice. The widest use is in the

dairy sector, where it is used to color cheese, butter and other such

products. In the Philippines the seed powder is added to stews, curries

and soups, and it is an essential ingredient in meat marinating mixes.

Fresh seed extract in water is also used for coloring rice preparations.
 5

On the other hand, Alugbati or Malabar spinach is also one of the

most popular indigenous leafy vegetables in the Philippines. It is widely

cultivated in all regions of the country particularly in the Visayas region.

The plant is a succulent branched, smooth, twining, herbaceous vine. The

stems are green or purplish. The leaves are fleshy, ovate or heart-shaped,

5-12 centimeters (cm) in length, stalked, tapering to a pointed tip and

heart shape at the base. The spikes are axillary, solitary and 5-29 cm in

length. The flowers are pink and about 4 millimeters (mm) long. The fruit is

fleshy, stalkless, ovoid or nearly spherical, 5-6 mm in length and purple

when mature.

This plant is commonly grown for its young shoots which make an

excellent succulent, slightly mucilaginous vegetable, used as a pot herb in

stews or soups, consumed boiled, fried in oil or sometimes as green

salad. Its fruits seem to have been earlier used for dyeing purposes in

China. The red fruit juice can be used as ink, cosmetic and for coloring

foods. The young leaves can be used as laxative, the pulped leaves to

poultice sores, red fruit juice as eye drops to treat conjunctivitis and the

roots as rubefacient. The red forms are commonly planted as ornamentals

and are even becoming popular in Europe as a pot plant.

 6

In today’s modern world, problems regarding the needfulness of

non-hazardous writing tools like ink and marker will become highly in

demand especially in industrialized countries that even the government

encourage young scientists to discover things that can provide solution to

the needs of these social changes. The researchers of this study were

motivated to seek for the feasibility of Achiote and Alugbati extracts as

plant-based inks, knowing that it would be of great help in addressing

environmental and economic problems.

This study undergone experimental process in order to formulate

ink from the aforementioned plants. The first phase of the process is

harvesting the fruits of Achiote and Alugbati from old cultivated places

within or outside Daet, Camarines Norte. After the collection, the plants

were submitted for identification. To extract the color, the Annatto seeds

were boiled in a certain amount of water, while the Alugbati fruits were

crushed using mortar and pestle.

In order to produce a good quality of ink, extra ingredients were

added such as Gum Arabic and table salt. Table salt is useful in

preserving the ink by preventing mold formation. Gum Arabic helps the ink

flow onto paper in a controlled fashion and also to maintain its color.

Subsequently, the researchers further evaluated the output’s acceptability

 7

through testing. The testing phase is composed of two parameters, drying

time and pH test. The two inks were then evaluated and examined by

experts. And after examining the inks, a survey was conducted among

teachers and students to further evaluate the final product. Nevertheless,

other parts of the process are thoroughly discussed on the succeeding

chapters.

Driven by the passion of contributing to the body of knowledge,

these aspiring research enthusiasts aim to yield more than just plant-

based inks, but also a legacy that will enflame the hearts and mind of

other young researchers through the creation of naturally made

innovations. Upon the accomplishment of the study, new opportunities will

be unlocked for students, teachers, community members, and also for

entrepreneurs.

Statement of the Problem

This study sought to determine the effectiveness of Achiote (Bixa

orellana) and Alugbati (Basella alba) fruits to create an alternative natural

ink. Specifically, it aimed to answer the following questions:

1. How effective is the Achiote and Alugbati inks in terms of:

a. Drying Time

b. pH Testing
 8

2. Is there a significant difference between each treatment of Achiote

and Alugbati?

3. What is the acceptability of the formulated inks in terms of:

a. Odor

b. Quality of Color

c. Flow Ability

4. What is the evaluation about the formulated plant-based inks?

Significance of the Study

The results of this study could help benefit the following stakeholders

through the formulation of plant-based ink from Achiote and Alugbati

plants.

Students. The creation of plant-based inks will guarantee the safety of

students from the harmful toxins brought by the synthetic ones.

Additionally, the yielded inks will also become handy especially in writing

and in doing other school works.

Teachers. By using plant-based ink, the chances of inhaling harmful

chemicals from synthetic ones will be reduced. Also, the ink is cheaper

and affordable which will lessen the expenses of buying refills.

 9

School. The product of this research will serve as a way to introduce

the school as a hub of innovation and become known for conducting

meaningful studies.

Entrepreneur. Through this study, entrepreneurs will be benefitted in

a way that they may use the opportunity to open new businesses

regarding the production of plant-based inks.

Future Researcher. The study will serve as a reference material for

those who may have the same interest in making natural inks. This will

also serve as a guide for future researchers.

Scope and Delimitations of the Study

This study focused on finding the feasibility of Achiote (Bixa

orellana) and Alugbati (Basella alba) extracts in making plant-based ink.

Two inks were formulated from the plants with varying colors. An orange

color was extracted from the seeds of Achiote and a dark purplish color

from the fruits of Alugbati.

The researchers harvested the aforementioned plants from old

cultivated places in Daet, Camarines Norte and then undergone validation

at the Departmet of Agriculture. The Annatto seeds were boiled in water to

produce a red coloring. To extract the color from Alugbati, researchers

crushed the fruits with mortar and pestle, strained the juice into a bowl,
 10

and discarded unwanted residues until an adequate amount of extract is

obtained. After the extraction, the researchers added gum Arabic, ethyl

alcohol (ethanol) and table salt to the juices to prolong shelf life and

enhance the color.

The experiment was conducted using three (3) treatments (Achiote

and Alugbati), with three (3) replicates each. The testing was composed of

drying time and pH test. Then, the researchers brought the two inks to five

(5) experts in the field of Arts and Design in order to gain insights and

recommendations. Furthermore, ten (10) teachers and ten (10) students

from Camarines Norte Senior High School and Camarines Norte National

High School – Main Campus also assessed the inks via survey. The

respondents were chosen purposively.

This study is delimited to the production of a safe and eco-friendly

plant-based ink. The inks were then named as Basella (Alugbati) and

Annatto (Achiote) ink. Also, the yielded inks were adhered on paper

surfaces only.
 11

Definition of Terms

The following terms are defined conceptually and operationally for

better understanding.

Ink. It refers to a liquid containing various pigments or dyes used

for coloring a surface to render an image or text. In this study, it is

defined as a colored fluid made up from the extracts of Achiote and

Alugbati.

Synthetic. It is an artificial material produced by organic chemical

synthesis. Synthetic in this study refers to commercial inks made from

harmful substances like petroleum and other chemical solvents.

Formulation. It refers to the action of devising or creating

something. In this study, formulation is defined as the process of

producing plant-based ink with the use of Achiote and Alugbati fruits.

Natural dyes. These are dyes or colorants derived from plants,

invertebrates, or minerals that are usually used in dyeing textiles and

making ink. Natural dyes in this study refer to the colorants obtained

from Achiote and Alugbati fruits via extraction.

Extract. It pertains to a preparation containing the active ingredient

of a substance in concentrated form. In this study, it refers to the juice

or extracted pigments from the fruits of Achiote and Alugbati.

 12

Notes

Colored Ink for Computer Printer out of Malabar Nightshade (Alugbati)

Extract (2012)

https://en.m.wikipedia.org/wiki/Bixa_orellana

Lehnhardt, P. (2015). Retrieved from https://www.hobbyfarms.com/5-

natural-ink-recipes-2/

https://en.m.wikipedia.org/wiki/Ink

Ravindran, P. N. (2017). The Encyclopedia of Herbs and Spices

http://www.madehow.com/Volume-3/Marker.html

https://en.m.wikipedia.org/wiki/Basella_alba

Aragones, J. A. – Alugbati Production Guide

https://www.merriam-webster.com/dictionary
 13

CHAPTER II

REVIEW OF RELATED LITERATURE AND STUDIES

This chapter presents the review about the related literature and

studies which supports and serves as foundation for the study’s conduct,

covering multiple articles, journals, dissertations, abstracts, and other

sources of references materials related to key concepts in the research.

The synthesis of the state-of-the-art emphasizes the similarities and

differences. The theoretical and conceptual framework with their

paradigms are also included which encompasses the various concepts

needed to be compiled in the research’ conduct.

Review of Related Literature

According to Enz F. (2014), Annatto or Achiote, one of the world’s

most important natural food colorant that yields yellow to red tint, is

derived from the seeds of the tropical tree “Bixa orellana Linn”. Such tree

is also known as the “lipstick plant” as its seed extract is also popularly

used in some countries as cosmetics and body paint. The tree can grow 4

to 6 meters with pointed heart-shaped leaves. It has a white to pinkish

flower and a brownish red, rounded to oval-shaped, spiny fruit. The fruit of

annatto when matured splits into two to reveal its seeds. The seeds are

numerous tiny kernel-like particles with red to deep orange pulp outer
 14

covering. These are usually powderized or steeped in oil, and the resulting

extract is used as dye or food coloring. The seed has two carotenoid

substances called bixin and norbixin that are responsible for its distinct

color. The substances work in the same way with beta-carotene, also a

carotenoid chemical compound that produces the orange pigment in

carrot. The planting of annatto trees is believed to have originated in the

Caribbean and South America, and it is now widely cultivated in tropical

and subtropical regions in many parts of America, Asia and Africa.

Currently, Brazil and Peru are the major producers and suppliers of
[1]
annatto products in the world. Mayan Aztec civilizations used the seeds

from the lipstick tree as ink to write scriptures. In Panama, the Embera

Indians use Achiote seeds to dye fronds for weaving baskets, producing a

vibrant red color. [2]

Additionally, Smith (2016) stated that Annatto is the name given to

the crude pigment extract (containing bixin, norbixin and other carotenoids

in different proportions) obtained from Achiote. While bixin is fat soluble,

norbixin is hydrosoluble. The possibility for obtaining water-soluble

colorants as well as oil-soluble colorants depending on the type of

extraction as well as the solvent and temperature used has converted

Achiote in one of the most interesting plant sources of vegetable

colorants. [3]
 15

Moreover, Salinas et al. (2005) indicated the use of artificial

colorants such as tartrazine (E102), allura red (E129) or sunset yellow

FCF (E110) in food products have been severely questioned in developed

countries, since there are reports showing that indiscriminate consumption

of these colorants is associated to the development of degenerative

illnesses such as some types of cancer. As a consequence, the use of

some artificial food colors such as carmoisine (E122) and Ponceau 4R

(E124) have been banned in the USA and Europe and the use of natural

colorants such as the dye extracted from the surface of B. orellana L. (E

[4]
160b, annatto extract) have been recommended.

According to Martinez et al. (2010), annatto extract has a great

economic importance worldwide and it is one of the natural colors more

widely used in the food, cosmetic and pharmaceutical industries, because

it does not alter flavor and it is practically not toxic. This natural dye is rich

on the carotenoids bixin (dark red color) and norbixin or orelline (yellow

color) which are mainly used to develop attractive colors in dairy products

(cheeses, margarine, and butter), meats, ice creams, cosmetics,

condiments, ceramic, paint, hair colors, soaps, nail polish, varnish,

[5]
lacquer, fabric colors, among others.
 16

In a published reading entitled “Alugbati Production Guide”, it

discussed about pharmacological activity of Alugbati (Malabar Spinach)

such as treatment for conjunctivitis, antioxidant, and with its richness in

Saponins that act as phytochemicals which can fight off cancer. Along with

these benefits are its other uses especially its red fruit juice that can be

used for inks. It also appears that Alugbati is already used for dyeing

purpose in China early years ago. [6]

Similarly, another reading entitled “A Review of the Taxonomy,

Ethnobotany, Phytochemistry and Pharmacology of Basella alba” (2014),

explained that Alugbati is also an important green leafy vegetable which is

commonly found in other tropical regions of the world. The plant is used as

a substitute for true spinach and also has great ethnomedicinal

importance. Different studies have proved that the plant is rich in vitamin A

and vitamin C along with flavonoids, saponins, carotenoids, many amino

acids and organic acids. Various in vivo and in vitro studies revealed that

the plants is enriched with active substances/principles having medicinal

potential. [7]

Furthermore, Mohammed et al. (2012) studied wound healing

capacity of Basella alba in male albino rats. They created burn wounds on

the back of rats and treated them with Basella alba leaf extract in glycerin
 17

for about 20 days. Their results concluded that, rats treated with aqueous

leaf extracts showed a maximum wound healing capacity with significant

[8]
wound closure and indicated wound healing capacity of Basella alba.

On the other hand, Oyewole and Kalejaiye (2012) used agar cup

method for the determination of antimicrobial effects of Basella alba

ethanolic extracts against Staphylococcus aureus, Pseudomonas

aeruginosa, Escherichia coli and Candida albicans. Their findings reported

that Basella alba ethanol extracts showed inhibitory activities against all
[9]
the above bacteria except Candida albicans. Likewise, Sen et al. (2010)

also examined the antibacterial activity of the Basella rubra leaves by cup

plate diffusion method and reported that aqueous, ethanol and petroleum

ether extracts of the Basella rubra leaves exhibited antibacterial activity

against E. coli, Vibrio cholera, Staphylococcus aureus and

Staphylococcus typhi. [10]

In addition, according to the article found in the Student’s

Encyclopedia, “The ink should make a clear permanent mark that when

dry does not fade from exposure to light or run from exposure to moisture.

It should flow freely and dry quickly when written in a surface and it must
[11]
contain nothing that might damage either the pen or the paper.”
 18

Neumann’s and Schluttig’s definition of ink as cited in Lindquist,

clearly stated the properties of ink that would be helpful in having a good

quality ink. The ink must be clear, filterable solution but not a suspension,

should flow easily from the pen and should not spread on paper. Since the

ink is natural, no mold must be seen on the solution and has no

pronounced unpleasant odor. Likewise, it must have an intense color

which does not become paler nor bleach out entirely as it is used in

writing. Furthermore, Lindquist added that every good ink, whether writing,

or combined writing and copying, should have qualities that give writing

that, after drying for eight days, is not removed by water or alcohol -- even
[12]
by treatment for days -- to such an extent that it becomes illegible.

Review of Related Studies

According to the study of Rajesh et al. (2014), it reveals that in

India, there are more than 450 plants that can yield dyes. In addition to

their dye-yielding characteristics, some of these plants also possess

medicinal value. Dyes are substances that can be used to impart color to

other materials, such as textiles, foodstuffs, and paper. A dye can

generally be described as a colored substance that has an affinity to the

substrate to which it is being applied. A dye that does not fade when the

material it was applied to be exposed to conditions associated with its

 19

intended use is called a fast dye. Contrariwise, a dye that loses its coloring

during proper usage is referred to as a fugitive dye. The dye is usually

used as an aqueous solution and may require a mordant to improve the

fastness of the dye on the fiber.

Archaeological evidence shows that, particularly in India and the

Middle East, dyeing has been carried out for over 5000 years. The dyes

were obtained from animal, vegetable or mineral origin with no or very little

processing. By far the greatest source of dyes has been from the plant

kingdom, notably roots, berries, bark, leaves and wood, but only a few

have ever been used on a commercial scale. The process of dyeing is

carried out in a variety of ways depending on the specific dye utilized as

well as the properties of the material.

Growing demand for natural dyes in place of the synthetic ones is

justified by low toxicity of the former ones due to the fact that the latter

ones are associated with causing cancer and other skin disorders as well

as when released in the environment takes a long time to degrade and the

intermediates could be still more toxic. Recently, in the ecology era, the

use of natural dyes has been the object for applied research in the skin

and textile industries.

 20

Natural dyes are widely used in many textile industries,

pharmaceutical industries, cosmetic industries, confectioneries, food

industries, paper industries as a diagnostic agent, as antimicrobial and

household purposes on festivals etc. Colors are used in various dosage

forms to give an attractive appearance and look.

More importantly, natural dyes are less toxic, less polluting, less

health hazardous, non-carcinogenic and non-poisonous. Added to this,

they are harmonizing colors, gentle, soft and subtle, and create a restful

effect. Above all, they are also environmental friendly and are good
[13]
colorants to make plant-based ink.

As shown in the study conducted by P. V. Powar et al. (2014), there

has been a rising interest in herbal dyes and ink as consumers have

become conscious of ecological and environmental problems. It is

observed that only synthetic inks are usually used which may be harmful

on one’s health. Because of this, it is encouraged to use inks that are safe

and can be easily found on our surrounding. Inks have so many variations

that some of them contain different kinds of element that may harm not

only the environment but also humans. On the other hand, natural ink may

offer the same functions as synthetic inks do while keeping the

environment and humans unharmed. [14]

 21

Moreover, the study of M. Ram Kumar et al. (2012) objectifies to

promote and develop the usage of natural dyes in the process of making

an edible ink. Today, the vast majority of dyes and pigments that are used

in making inks are produced synthetically. Colors produced from synthetic

dyes are more consistent from batch to batch than colors produced by

natural colorants. However, natural dyes are an eco-friendly way to impart

almost any color to textile product with the recent interest in environmental

concerns; natural dyes might be a good way to produce unique products

with a green slant.

Though all natural dyes are not 100% safe, they are less toxic than

their synthetic counterparts. Many of the natural dyes like turmeric,

annatto and saffron are permitted as food additives. They are obtained

from renewable sources. Natural dyes cause no disposal problems, as

they are biodegradable. Practically no or mild reactions are involved in

their preparation. It is possible to obtain a full range of colors using various

mordant. Added to this, edible ink overcomes some of the problems lead

by synthetic ink via less expensive and not tedious process, nontoxic, safe

to use in foods and confectionaries. At the completion of their study, all

[15]
inks were found to be edible, safe, easy to prepare and stable.
 22

Furthermore, one of the great concern of the government and the

people of every country is to have a clean and safe election. Silver nitrate

in making indelible ink is one of the major problems because of its toxicity

and corrosiveness. D. R. Co et al. conducted a study to find out the

feasibility of natural plant dyes from the market and cartridge ink as an

alternative indelible ink. The researchers have utilized cartridge ink and

natural dyes to produce a safe, low-cost and reliable indelible ink as

alternative for the commercial ones.

Additionally, the investigation involved the creation of indelible ink

from Achiote and kasubha extracts. The objective is to determine the

feasibility of the alternative ink compared to the commercially ones sold in

the market. It was also the purpose of the study to create a safe and

cheap indelible ink. At the end of the study, results confirmed that Bixa

orellana L. can be a powerful source of an effective adherence in making

an indelible ink.

In this case, if the study proves that Achiote has an effective quality

as ink, it can be concluded that this plant is a good source of extract not

just in making indelible ink but also for other purposes such as the ones

used in markers. [16]

 23

Nevertheless, inks are not closed for the purpose of writing only. In

an interesting research study of N. B. Uma et al. (2014), pigments were

extracted from plants to create “Tri-color Printer Ink”. It was conducted to

study an economical, simple, as well as bio-friendly method in producing

ink for printers. The result showed that the ink produced was as

outstanding as commercial ink as it is acidic, possess high boiling point at

75°C, and it was able to operate successfully by commercial Hewlett-

Packard printer. It dries as fast, produce not just three main color but as

well as other color that can be chosen from printing options in computer
[17]
and as an added value, it is unexceptionally bio-friendly to nature.

Currently, there are several ways for extracting the colorant from

Achiote (Bixa orellana). One of the oldest and most traditional methods is

the extraction with water. The efficiency of the pigment extraction depends

on the method. In this sense, the general statistical analysis showed that

the highest percentage of pigment extraction yields were obtained using

KOH (4.84%, in average) as opposed to extractions using ethanol or water

(3.46% and 3.37% respectively) according to the Tukey-Kramer Multiple-

Comparison Test. Besides, no significant differences (P > 0:05) in the

pigment extraction yield were observed between annatto extracts where

[18]
ethanol or water had been used.
 24

One study from International Research Journal of Pharmacy states

that the usage of natural dyes obtained from Achiote in textile finishing has

good light fastness and may help in keeping the skin healthy by preventing

from allergy and reduce the risk of skin cancer. The dye obtained is

biodegradable and non-toxic. The natural dye obtained from Achiote also

has medicinal properties and finds its application traditionally in the

preparation of sindhoors. Natural dye from Achiote was evaluated by the

study and showed better result in staining of cotton cloth, silk fiber and

cotton fiber. The dye is cheap, soothing, long lasting and has anti-

microbial property. Because of its anti-microbial property, its usage in

dyeing Band-Aid cloth will be helpful for fast wound healing. The

application of annatto was found to be easy, more practical and reliable.

Lastly, the natural dye serves as eco-friendly agent for the clean and

green environment. [19]

Asish Mitra and Sanat Kumar Das (2015) carried out an

investigation to extract natural pigment from the fruits of Alugbati (Basella

alba) plant. The dye was extracted with methanol and then centrifuged at

10000 rpm and the supernatant was taken for analysis. The Basella alba

fruit dye was used for dyeing the scoured cotton cloth as such and also

using mordants like alum, FeSO 4 and CuSO4. The dye component was

extracted and dyeing of cotton was also studied. It is found that pigments
 25

from Basella alba fruits was moderately applicable in dyeing of cotton fiber

as well as other fibers. The findings also suggest that Basella alba is a rich

source of betalains and could have potentiality for use in the development

of food colorants, cosmetics, paper coloring, use as acid-base indicator

and nutraceuticals in future. [20]

In the study of M. F. L. Amon and L. P. Pladio, the purplish red

colored extract from the fruits of spinach vine or “Alugbati” (Basella rubra

L.) was investigated. This plant thrives in tropical Asia, Africa, Malaya, and

is found cultivated throughout the Philippines. Among the three solvent

solutions used, solvent 1 which is 1% HCl in ethanol (1:100 mL solvent

ratio) solution exhibited the best extraction property as indicated by the

high color density, polymeric color, and degradation index of the purplish

red extract. UV-Vis spectral analysis of the extract showed two peak

detections at 272 nm and 500 nm indicative of anthocyanin presence in

the extract. The absence of a third absorption band in the 310 - 360 nm

range means that the anthocyanin in the crude extract is not acylated.

TLC screening revealed that the extract have radical scavenging property

(antioxidant) against DPPH radical using ascorbic acid as positive control.

Result of the Brine Shrimp toxicity test showed the extract is nontoxic with

an LC50 value calculated at 1,995.26 mg/L in 24 hrs. Initial results

 26

revealed that Alugbati extract imparts a reddish color when mixed with
[21]
food samples. This shows that it can be used as a food coloring.

M.Singh et al. (2016) reviewed the pharmacological activities and

other properties of Alugbati. In the review article, Alugbati was said to be

used to treat large number of human ailments as mentioned in Charaka

Samhita, and Sushruta Samhita. It has been found to be a good source of

calcium, iron, vitamin A and vitamin C. In Ayurveda, the plant has shown

immense potential in androgenic, antiulcer, antioxidant, cytotoxic,

antibacterial activity, anti-inflammatory, central nervous system (CNS)

depressant activity, nephroprotective and wound healing properties etc.

The paper also includes the evidence-based overview of pharmacological

and phytochemical properties of the Basella rubra, which may be helpful

to establish a standard natural drug for further research. Evidently,

Alugbati was concluded as a good source of plant extract that is safe,

[22]
which can be potentially used in formulating natural inks.

Synthesis of the State-of-the-Art

 27

In the study of D. R. Co et al., they have utilized cartridge ink and

natural dyes from Achiote and kasubha to produce a safe, low-cost and

reliable indelible ink as alternative for the commercial ones. Results of the

study proved that Achiote can be a powerful source of an effective

adherence in making indelible ink. Their study is somewhat similar to the

current study since they have used Achiote to create natural indelible ink.

In the contrary, they differ in terms of the purpose because the

researchers of the current study will use Achiote and Alugbati to create

plant-based ink for markers instead of indelible ink.

Similarly, the study conducted by N. B. Uma et al. proved that

pigments extracted from plants can be used to create tri-color printer ink.

The results showed that the ink produced from Turmeric, Roselle, and

Asian pigeon wings flower were as outstanding as commercial ink and bio-

friendly to nature. This study is slightly similar to the current study because

they both use plant pigments to make ink and they only differ in terms of

purpose and ingredients used.

Moreover, one study from International Research Journal of

Pharmacy states that the usage of natural dyes obtained from Achiote in

textile finishing has good light fastness and may help in keeping the skin

healthy. The natural dye from Achiote was evaluated and showed better
 28

result in staining of cotton cloth, silk fiber and cotton fiber. This study is

similar to the current study in such a way that they both chose Achiote as

the test subject. In the contrary, the current study will be experimenting

Achiote and Alugbati fruits to produce plant-based ink.

In another investigation by Asish Mitra and Sanat Kumar Das found

that the pigments from Alugbati fruits was moderately applicable in dyeing

of cotton fiber as well as other fibers. The findings also suggest that

Basella alba is a rich source of betalains and could have potentiality

foruse in the development of food colorants, cosmetics, paper coloring,

use as acid-base indicator and nutraceuticalsin future. This study became

different with the current study because of its purpose wherein the extract

was applied in cotton fiber. It is somewhat similar since the dyeing of

cotton fiber used Alugbati fruits as colorant.

Parallel to that, M. F. L. Amon and L. P. Pladio also investigated the

extracts from the fruits of Alugbati. Initial results revealed that Alugbati

extract imparts a reddish color when mixed with food samples. This shows

that it can be used as a food coloring. For this reason, the investigation is

somewhat related to the current study because they both use Alugbati

fruits. However, the current study will use the fruits to formulate ink rather

than food coloring.

 29

Theoretical Framework

This study is supported by the Theory of Multiple Chemical

Sensitivity (MCS) proposed by biochemist Martin L. Pall. Multiple

chemical sensitivity is a chronic condition in which a person develops

adverse symptoms from exposure to chemicals in the environment. In

other words, it is a disease initiated by toxic chemical exposure, leading to

brain injury that produces high level of sensitivity to the same set of

chemicals that are implicated in initiation of the disease. Although healthy

people often react to toxic substances, the difference is that a person with

MCS has much more severe, debilitating, prolonged symptoms, and also

reacts to much smaller amount of chemicals. Their reactions are repetitive

and reproducible, and symptoms are generally relieved after the toxic
[21]
chemicals are removed or within a consistent amount of time thereafter.

In this study, the theory helped the researchers to understand that

there is such a disease that can be acquired due to the long term

exposure and contact with synthetic chemicals. Therefore, in order to

prevent this disease, people need to lessen the use of products that

contain harmful substances.

Another theory that supports this study is the Total Load Theory.

The idea is that the body has relatively finite space to work with and the
 30

detoxification pathways, specifically, have a certain capacity of how much

toxic material they can remove and how quickly. Since people are

exposed to and absorb such an overwhelming amount of toxins, the body

cannot effectively remove it all and trace amounts are left behind. Even

healthy person cannot effectively process and remove everything. The

toxic material left behind builds up and is stored in fatty tissues and other

areas of the body. The more build up there is, the less efficient the body

becomes at detoxing from new exposures. Eventually a tipping point is

reached and the body is so severely hampered in its detoxification

[22]
process that symptoms or illness develop.

In connection with the MCS Theory, people’s daily exposure to

everyday toxins can be enough to actually cause Chemical Sensitivities

and direct damage to the body. With these supporting theories, the

researchers were be able to conclude that the emergence of such health

conditions leads to a possible correlation with the increased prevalence of

cancer and many other illnesses. In order to reduce the risk of acquiring

diseases from chemicals, people have to rely on using natural products

that are safe and nontoxic. Thus, formulating plant-based ink as an

alternative to commercial ones will be of great help in protecting one’s

health as well as the environment.

 31

EORY OF MULTIPLE CHEMICAL SENSITIVITIES

TOTAL LOAD THEORY
 32

ACHIOTE AND ALUGBATI INKS

Figure 1

Theoretical Paradigm

Conceptual Framework

The study hypothesized that Achiote and Alugbati fruits can be a

good source of colorants that can be used in making natural ink. It is also
 33

focused on determining the effectiveness of Achiote and Alugbati as

alternative plant-based inks.

The Achiote seeds and Alugbati fruits were harvested and were

identified by the Department of Agriculture. The colors from Annatto seeds

were extracted through boiling while the Alugbati fruits were crushed using

mortar and pestle. The extracts collected were mixed with gum Arabic to

thicken the mixture, along with table salt that served as preservatives. The

experiment was comprised of three (3) treatments with three (3) replicates

for each ink.

After finalizing the plant-based inks, they were visually observed

and studied using parameters. The inks were evaluated by teachers and

students via survey, and a rubrics guide for the experts. The outputs were

improved with the help of the feedbacks from the respondents and

evaluators.

Annatto Seeds Alugbati Fruits

Extraction of Colorants

Ink from Annatto seed extracts Ink from Alugbati fruit extracts
Treatments 1, 2 and 3
34

Observations and Tests

Survey and Evaluation

Plant-based Inks

Figure 2

Conceptual Paradigm

Notes
 35

Enz F. (2014). Retrieved from

http://www.pinoykusinero.com/2014/04/homemade-atsuete-achiote-or-

annatto-oil.html?m=1

Retrieved from http://www.canopytower.com/achiote

Smith (2016). Achiote (Bixa orellana L.): a natural source of pigment and

vitamin E

Salinas et al. (2005)

Martinez et al. (2010)

J. A. Aragones. Alugbati Production Guide

A review of the taxonomy, ethnobotany, phytochemistry and

pharmacology of Basella alba (2014)

Mohammed et al. (2012)

Oyewole and Kalejaiye (2012)

Sen et al. (2010)

Antioquia, John Paul C. (2012) Alternative Ink.

Lindquist, Evan. Old Ink. n. d.

http://www.evanlindquist.com/oldink.html
 36

A Review: Dye Yielding Sources and their Importance (2014)

Herbal Ink: Safe, Easy and Ecofriendly Alternative (2014)

Formulation and Standardization of Herbal Based Edible Ink (2012)

Substitute Indelible Ink Using Bixa orellana L. (Annatto) and Corcussativus

L. (Kasubha) extracts

Production of Tri-Color Printer Ink by Natural Pigment Extraction (2014)

Chemical Characterization and Quantification of the Pigment Extraction

Yield of Seven Mexican Accessions of Bixa orellana (2016)

Annatto: Eco-friendly and Potential Source of Natural Dye (2013)

Fabric dyeing with natural dye extract from Basella alba fruit and

spectroscopic analysis of the extract at different conditions (2015)

Potential Food Colorant from the Extracts of Alugbati (Basella rubra L.)

Basella rubra Linn – A Review (2016)

Retrieved from https://www.prohealth.com/library/dr-palls-theory-of-

multiple-chemical-sensitivity-disease-mechanism-confirmed-by-roman-

study-of-mcs-patients-27433

Retrieved from http://thinkbeforeyoustink.com/site/mobile?url=http%3A

%2F%2Fthinkbeforeyoustink.com%2FwhatisCS.html#2507
 37

CHAPTER III

RESEARCH DESIGN AND METHODOLOGY

 38

This chapter provides the discussion with regards to the design and

methods of research to be used for the conduct of the study. Likewise, this

part also comprises the sources of data, research instruments, data

gathering procedures, and the statistical tool.

Research Design

This study utilized experimental research design, which is the

primary approach used to study the relationships between one variable

and another. This is a traditional type of research that is quantitative in

nature. In short, researchers used experimental research to compare two

or more groups on one or more measures.

In this design, one variable is manipulated to see if it has an effect

on the other variable. Experimental designs are used in this way to answer

hypotheses. The researchers designed an experimental study which will

then support or disprove the hypothesis.

Methodology

Preparation of Materials and Ingredients

 39

The researchers gathered all the needed ingredients and prepared

the materials that was used in the creation of ink. Before the final

experiment, the group conducted a preliminary experiment to test different

concentration of ingredients. The best concentrations were chosen for the

final experiment. The extraction process was done in a laboratory at

Camarines Norte Senior High School, Daet, Camarines Norte.

Extraction of Colorants from Annatto Seeds

The method of extraction for Achiote was through boiling the seeds. All

of the replicates was done with similar methods.

1. Collect Annatto seeds and remove any dirt before extraction.

2. Weigh the needed

amount of Plate 1. Annatto seeds seeds then

put them in a beaker with water.

 40

Plate 2. Weighing the seeds in a digit

balance
3. Boil the seeds with water into a hot plate at a temperature of 300 oC

for 8 minutes or until an orange to reddish color is obtained.

Plate 3. Boiling the annatto

seeds

4. Wait until the extract is cooled. Then, discard the remaining plant

materials using a strainer into a bowl.

Plate 4. Straining the seeds

 41

5. To remove any unwanted residues from the extract, filter it using a

clean cloth.

Plate 5. Filtering the extract

using Chinese cotton

6. After the filtration, add Gum Arabic and table salt to the extract.

7. Stir the mixture until it reaches the desired consistency.

8. Lastly, transfer the product to a container/jar.

Extraction of Colorants from Alugbati fruits

 42

The method of extraction for Alugbati is by crushing the fruits. All of the

replicates was done with similar methods.

1. Collect ripe Alugbati fruits. Wash the fruits and remove any dirt

before extraction.

Plate 6. Removal of dirt from the

fruits

2. Weigh an adequate amount of fruits into a digital balance.

Plate 7. Weighing Alugbati fruits

3. Crush the fruits using mortar and pestle to extract the juice.
 43

4. After pounding, strain the extracts into a bowl.

Plate 9. Straining the extract

5. Remove any remaining residues by filtering the extracts using a

clean cloth.

6. When the extract is ready, add ethyl alcohol (ethanol), Gum Arabic

and table salt.

 44

7. Stir the mixture until it reaches the desired consistency.

8. Lastly, transfer the product to a container/jar.

Observation and Testing

After the extraction and mixing process, the researchers began the

observation of treatments in a laboratory at the prior school. The inks

undergone testing using two (2) parameters which includes drying time,

and pH testing. Also, the group carefully recorded the drying time and pH

value of the inks using an observation guide.

Evaluation and Acceptability Testing

Once the observation is done, the inks were subjected for evaluation.

The evaluators were limited to five (5) experts. The experts assessed the

plant-based inks using their own professional background. To evaluate the

 45

two inks, the researchers provided a rubrics guide. An agreement of

schedule was made between the evaluators and the researchers.

Also, to test the acceptability, a survey was conducted to 10 teachers

and 10 students from Camarines Norte National High School – Main

Campus and Camarines Norte Senior High School. The group gave

sample product to the respondents and rated its quality based on their

own personal perspectives.

 46

Preparation of Materials and Ingredients

Extraction of Annatto seeds and Alugbati fruits

Combining of additional ingredients

Observation and Testing of Treatments

Evaluation of Experts

Acceptability Testing via Survey

Plant-based Inks from Achiote and Alubati Extracts

Figure 3

Experimental Flowchart
 47

Sources of Data

To be able to create the plant-based inks, reliable data that are

essential to the study were gathered from the cited related readings. Since

the study is focused on identifying the various properties of Achiote and

Alugbati extracts as inks, data were attained through observations and

testing. Additionally, a rubrics was used to objectively gather evaluation

and perspectives from experts. Also, a survey was conducted to generate

feedbacks of students and teachers within the school premises.

Related Readings. Various articles, journals, and studies with similar

ideas have contributed a lot to the conduct of this study.

Observation. The application of an observation guide used by the

researchers determined the effectiveness and quality of Achiote and

Alugbati inks based on parameters. Results from the observation provided

valuable information regarding the performance of the outputs.

Evaluation of Experts. The experts from the field of Arts and Design

evaluated the formulated inks using rubrics to objectively evaluate the

product.
 48

Survey Results. Students and teachers were selected purposively to

answer the survey questionnaire regarding the acceptability of the

products.

Research Instruments

The research instruments used in conducting this study are limited to

Observation Guide, Rubrics, and Survey Questionnaire.

Observation Guide is one of the instruments in gathering data. The

researchers were able to obtain meaningful information through

observations and testing which happened after the creation of inks.

Results from the observation helped define the effectiveness of the study

and determine the different properties and features of Achiote and

Alugbati inks.

Rubrics was also included as a tool for data gathering. The rubrics

was composed of indicators along with their corresponding adjectival

descriptions to measure the acceptability of the inks. The results from the

evaluation brought credible opinions and suggestions which further

improved the study.

Survey Questionnaire was also used to collect general amount of

acceptability from teachers and students at Camarines Norte Senior High

 49

School. In order to establish good point as to why Achiote and Alugbati

inks could potentially serve as alternatives to the synthetic one,

respondents used the products and gave their own insights as one of the

important beneficiaries of this study.

Data Gathering Procedure

In gathering such information for the research process, the

researchers conducted observation, evaluation and survey for the purpose

of obtaining the needed data throughout the whole study. First, the

observation and testing phase was conducted in a laboratory at

Camarines Norte Senior High School. An observation guide was prepared

by the researchers to sequentially assess and examine the properties and

qualities of the inks for each treatment.

Second, the yielded plant-based inks were submitted for evaluation

by five (5) experts in the field of Arts and Design. An agreement of

schedule for the evaluation was made between the researchers and the

experts. Also, a rubrics was given to the experts to rate the study’s output.

The feedbacks and suggestions of the evaluators were of great help in

enhancing the quality of the inks.

Lastly, survey questionnaire were distributed to 20 selected

respondents, composing of ten (10) teachers and ten (10) students from
 50

Camarines Norte National High School – Main Campus and Camarines

Norte Senior High School. Information and suggestions from the

respondents were essential to further develop the acceptability of the inks.

The whole data gathering procedure was recorded and

documented. All the information from the rubrics and survey guide were

treated and quantified statistically.

Statistical Tool

For the numerical analysis, the researchers used One-Way ANOVA

(Analysis of Variance). It compares the mean differences between the

factors in an independent variable. In this study, the factor that was

compared between the Achiote and Alugbati ink was the drying time. An

online calculator specifically designed for One-Way ANOVA on

Vassarstats.net, a website for statistical computation, was used. On

setting up the website, the button for independent samples was clicked

first. Next, the number of samples was inputted. The data gathered from

the observation was inputted on the designated textboxes with for each

factor. By clicking to the calculate button, the website did the proper

calculations. After that, it immediately shows the ANOVA Summary which

contains all the significant values for interpretation, most importantly,

having P-value as well.

 51

Furthermore, in order to determine the differences and relationships

of variables, the data gathered by the researchers will be subjected to

some statistical treatment. One of the statistical methods is finding the

weighted mean according to frequency. Weighted mean is used as a

summarizing value for responses that vary in terms of degree of

importance such as responses with rating scale, grades with different

units, etc. The results from the survey will be analyzed by computing the

weighted mean.

Formula:

Σ wx
Weighted mean =
Σw

Where:

Σ (Sigma) means “Sum Up”

w = weight

x = value
 52

Notes

Mixed Methods Research. Retrieved

fromhttp://resourcecentre.foodrisc.org/mixed-methods-research_185.html

Weighted Mean. Retrieved from

https://www.mathsisfun.com/data/weighted-mean.html