Chapter 1

THE PROBLEM AND ITS BACKGROUND

Introduction

There is growing interest in exploiting plants for medicinal purposes especially in

Asia. Medicinal plants have assumed greater importance in recent days, due to the

tremendous potential they offer in formulating new drugs against many diseases and

illness that affect the human kind.

For many millions of people, especially to those living in rural areas within

developing countries, herbal medicines, traditional treatments and traditional

practitioners are often seen as more accessible, more affordable, more acceptable, and

is the main—and sometimes the only—source of health care. 1 Every known group in the

Philippines today has its own local "medical" specialist. This specialist is called by

various names- herbalists (arbularyo), masseurs, midwives, diviners, sorcerers, and

general practitioners. Their practice and methods of curing ailments involves

superstitions, recitation of prayers and religious rituals accompanied by the mediation of

the Holy Spirit, herbology, hydrotherapy, massage therapy, and divination.

In the Philippines, poverty and religious beliefs have fueled the development of

alternative forms of medicine. Instead of going to medical doctors, some people go to

folk healers who use herbs, massage, oils, or prayers as treatments. Although often

found active in rural communities, traditional Filipino healers can also be found in small

1
The American Association for the Advancement of Science, The Art and Science of Traditional Medicine Part 1: TCM Today – A
Case for Integration , retrieved from
http://www.sciencemag.org/sites/default/files/custompublishing/documents/TCM_Dec_19_issue_high_resolution.pdf in July
22, 2017
urban and suburban neighborhoods. The City of Puerto Princesa is also a home of

different ethnic groups that uses traditional healing.

Countless plants have been reported to have medicinal properties. These plants

are not only ornamental, they also exhibit antiviral, antifungal, antibacterial, antioxidant

and anti-inflammatory functions.2 They are sources of drugs, are used in herbal

medicine as remedy, and cure to various types of diseases. Undeniably, medicinal

plants have a big contribution on healthcare practices. For this reason, laboratory

investigations have been employed on medicinal plants for their active components and

potential.

Although many plants have valuable properties, some of them are known to carry

toxicological properties as well. Recent studies indicate that although numerous plants

are used as food sources, some of them may have mutagenic or genotoxic potential.

Numerous research studies have recently focused on both pharmacology and toxicity of

medicinal plants used by humans. This is of high importance in order to achieve a safe

treatment with plant products.

The researchers had observed that traditional medicine and therapeutic practices

are a popular culture among the residents of Purok Sandiwa, Barangay Tiniguiban.

There are herbalists in Purok Sandiwa, Barangay Tiniguiban that uses Anonang (Cordia

dichotoma) in curing poison, sprain, and body and/or muscle pain. They prepared

through the process of tincture and is applied through poultice and ingestion with a

dosage depending on the extent of the damage. With this way of application, the

2
Okwu D., et.al , “Exotic multifaceted medicinal plants of drugs and Pharmaceutical industries”, African Journal of
Biotechnology, p.8-25
researchers are encouraged wherein, the researchers will find out the preliminary

assessment of toxicity of the plant extracts.

The researchers will investigate the toxicity level of the leaves and stem bark of

the Anonang (Cordia dichotoma) which is commonly used for curing poison, sprain,

body and/or muscle pain.by the medicine men living in Purok Sandiwa, Barangay

Tiniguiban.

Conceptual Framework

Research shows that Anonang (Cordia dichotoma) offer some distinct health

benefits. Various parts of the plant are used medicinally. Some research has been

carried out and several active compounds have been isolated. The leaves contain

flavonol glycosides and phenolics.3 They are known antioxidants, for instance, which

means that they can help reduce the incidence of harmful free radicals in the body, and

they may also have the ability to improve capillary strength and naturally lower blood

pressure.4 The bark is mildly astringent and tonic. A decoction of the stem bark is taken

for treating dyspepsia, diarrhea, dysentery fever, headache, stomachache, as a tonic

and it is also beneficial after parturition. 5

This study will investigate the toxicity level of the leaves and stem bark of the

Anonang (Cordia dichotoma) which is commonly used for curing poison, sprain, body

3
Fern Ken, “Useful Tropical Plants: General Information about Cordia dichotoma” retrieved from
http://tropical.theferns.info/viewtropical.php?id=Cordia+dichotoma in July 28, 2017
4
Thomas Hollie, “What Are Flavonoid Glycosides?” retrieved from http://www.wisegeek.com/what-are-flavonoid-
glycosides.htm in July 28, 2017
5
BardeSonal, “Plant profile, phytochemistry and pharmacology of Cordia dichotoma” retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3805104/ in July 28, 2017
and/or muscle pain by the medicine men living in Purok Sandiwa, Barangay Tiniguiban

and they will determine if this is safe for the residents to ingest the tincture.

In order to determine the toxicity level of the plant extract of Anonang (Cordia

dichotoma) leaves and bark, the researchers will undergo a general bioassay that

appears capable of detecting a broad spectrum of bioactivity present in plant crude

extracts which is the Brine Shrimp (Artemia sp.) Lethality Assay (BSLA). BSLA is used

as an indicator for general toxicity and also as a guide for the detection of antitumor and

pesticidal compounds.6 The low cost and ease of performing the assay and the

commercial availability of inexpensive brine shrimp eggs makes BSLA a very useful

bench top method.7 This assay has been noted as a useful tool for the isolation of

bioactive compounds from plant extracts.

In this present study, plant extract of the Anonang (Cordia dichotoma) Leaves

and Stem bark collected in Purok Sandiwa, Barangay Tiniguiban will be test in vivo for

their toxicity effect against the brine shrimp. Thus, the findings of this present work

would give baseline information on this plant that could be use as a basis for the

development of new tools of great therapeutic importance. The researchers conducted

an interview in Purok Sandiwa, Barangay Tiniguiban into the local medicine men to

gather information regarding the medicinal plants and traditional therapies used by the

medicine men in treating disease and illnesses.

To illustrate the research to be conducted, the research paradigm is presented

on the next page.

6
Meyer Bernhard, et.al, “Brine shrimp: A convenient general bioassay for active plant constituents”, Plant Med, retrieved from
https://www.ncbi.nlm.nih.gov/pubmed/17396775 in July 26, 2017
7
McLaughlin J.L., et.al, “Benchtop bioassays the discovery of bioactive natural products: an update” retrieved from
https://www.scienceopen.com/document?vid=3f7149a7-c52c-4fd2-bcc0-c14918020e7e in July 26, 2017
 Figure 1

Research Paradigm

Input Process Output

A. Anonang (Cordia Product 1:

dichotoma) found in
Purok Sandiwa,
Barangay
Tiniguiban, Puerto
Princesa City
B. Medicinal Uses
and Traditional
therapies of selected
medicine men in
Purok Sandiwa,
Barangay tiniguiban
C. Traditional Uses
of Anonang (Cordia
dichotoma) Leaves
and Stem bark
A. Interview to the residents Brochure of the
of Purok Sandiwa, Anonang (Cordia
Barangay Tiniguiban, dichotoma) containing
Puerto Princesa City its background
B. Plant Decoction of information, traditional
Anonang (Cordia uses, traditional
dichotoma) Leaves and therapies used by the
Stem bark selected medicine men
in Purok Sandiwa,
C. Lethailty Assay of the Barangay tiniguiban
plant extract of Anonang and the results of the
Leaves and Stem bark Brine Shrimp Lethality
using Brine Shrimp Assay (BSLA)
Product 2:
Decoction

Figure 1 shows the Anonang (Cordia dichotoma) found in Purok Sandiwa,

Barangay Tiniguiban, Puerto Princesa City, medicinal uses and traditional therapies of

selected medicine men in Purok Sandiwa, Barangay tiniguiban and Traditional Uses of

Anonang (Cordia dichotoma) Leaves and Stem bark as the input of the study. The

process includes the interview to the residents of Purok Sandiwa, Barangay Tiniguiban,

Puerto Princesa City, plant decoction of Anonang (Cordia dichotoma) leaves and stem

bark, and lastly is the lethailty assay of the ethanolic extract of Anonang leaves and
stem bark using brine shrimp. The first product will be the brochure of the Anonang

(Cordia dichotoma) containing its background information, traditional uses, traditional

therapies used by the selected medicine men in Purok Sandiwa, Barangay tiniguiban

and the results of the Brine Shrimp Lethality Assay (BSLA) and the second product will

be the decoction that will serve as the output of the study.

Statement of the Problem

This study aims to determine the toxicity level of the plant extract of leaves and

stem bark of the Anonang (Cordia dichotoma) found in Purok Sandiwa, Barangay

Tiniguiban, Puerto Princesa City by the use of Brine Shrimp Lethality Assay.

Specifically, this study seeks answer to the following questions:

1. How much concentration of the plant extract of the Anonang (Cordia

dichotoma) leaves and stem bark will be used to determine the toxicity?

2. What is the number of surviving brine shrimp after 24 hours in terms of the

following concentrations:

a. 25%,

b. 50%, and

c. 75%?

3. What is the percent molarity of the number of surviving brine shrimp after 24

hours in terms of the following concentrations:

a. 25%,

b. 50%, and
 c. 75%?

Assumptions

The study was anchored on the basic assumption that the result of the lethality

assay revealed cytotoxic activity against the brine shrimp. The leaf plant and stem bark

toxicity against brine shrimp is chronically toxic. This finding supports the traditional

medicinal alternative use of this plant. Based on the possible relationship between brine

shrimp lethality and plant bioactivity, this study could serve for further ethnobotanical

research, anti-inflammatory .and anti-microbial activities.

Hypothesis

The researchers hypothesized at 0.01 level of significance that:

There is no significance difference between the concentration of the

decoction of the Anonang (Cordia dichotoma) leaves and stem bark to the

degree of lethality of the brine shrimp.

Scope and Delimitation

This study will be delimited on the following:

Problem.This study is entitled “Brine Shrimp Lethality Assay of the Plant Extract

of Anonang (Cordia dichotoma) leaves and stem bark.”

 This study aims to determine the toxicity level of the plant extract of leaves and

stem bark of the Anonang (Cordia dichotoma) found in Purok Sandiwa, Barangay

Tiniguiban, Puerto Princesa City by the use of Brine Shrimp Lethality Assay.

The study relies on the statements that were given by the selected medicine

men. The researchers will carefully observe the brine shrimp under the lethality assay

so that the results will be clear to the residents along with the medicine men.

Locale. The study will conduct in the Vivant Delta-P STEM Laboratory of

Palawan National School. The specimens will be collect at Purok Sandiwa, Barangay

tiniguiban, Puerto Princesa City. The map of Purok Sandiwa, Barangay tiniguiban and

the is presented on the next page.

Time Frame.The study started from June 2017 and will end on October 2017.
 Figure 2

Map of Purok Sandiwa, Barangay Tiniguiban

Figure 2 shows the research locale of Purok Sandiwa, Barangay Tiniguiban,

Puerto Princesa City

 Figure 3

Map of Vivant Delta-P STEM Laboratory in Palawan National School

Figure 2 shows the research locale of Vivant Delta-P STEM Laboratory

in Palawan National School

Significance of the Study

The investigations of the Brine shrimp lethality assay of the plant extract of

Anonang (Cordia dichotoma) leaves and stem bark deemed significant to the following:

The Academic Community. The result of the investigation will be helpful for

having prior knowledge and understanding regarding the toxicity effects of Anong

(Cordia dichotoma) leaves and stem bark.

The Local Residents of Puerto Princesa City. This study will give a piece of

information for deeper understanding of the effects of Anonag (Cordia dichotoma) and it

will promote awareness about the different uses of Anonang (Cordiadaichotoma) leaves

and bark to the people of Puerto Princesa City especially for those people who have

Anonang (Cordia dichotoma) somewhere near where they resides.

The Traditional Medicine Practitioners. This study will give information to the

medicine men in Purok Sandiwa, Barangay Tiniguiban that uses Anonang (Cordia

dichotoma) for their practices.

The Health and Medicine Students. This study will served as a basis for their

field of health and medicine about the different uses and effects of Anonang (Cordia

dichotoma).

The Department of Health. This study served as a basis about the effects of

Anonang (Cordia dichotoma) and help to promote the awareness regarding this tree.

Health Workers. This will serve as a basis about the different uses of Anonang

(Cordia dichotoma) and to promote the health benefits of Anonang.

 Palawan Conservation for Sustainable Development. This study can be used

as another reference about the toxicity of Anonang (Cordia dichotoma) and to maintain

and secured the number of Anonang that can be found in the said research locale.

Palawan National School. This study will served as a reading material and

reference for the students regards to the background, effects, uses and toxicity of

Anonang (Cordia dichotoma) and can be used in joining research contest.

Researchers. This study will serve as the final output and part of grades of the

researchers in their subject Research in Daily Life 2.

Future Researchers. This study can/will be used as reference for investigating

the effects of Anonang (Cordia dichotoma) and other studies that is related to this study.

Definition of Terms

In order to facilitate common understanding of information conveyed in this

research study, these terms are hereby defined operationally and theoretically.

Toxic. Containing or being poisonous material especially when capable of

causing death or serious debilitation

Medicine men. This refers to an unlicensed person who practices the art of

healing using traditional practices, herbal remedies and even the power of suggestion. It

is commonly called as “folk healer”.

Albularyo. They are known as the "general practitioner," the more

knowledgeable in terms of using medicinal herbs.

 Manghihilot. They use massaging techniques to treat sprains, fractures, and

other similar conditions that affect the skeletal system and the musculatory system,

including ligaments.

Babaylan. Usually a women, is someone who specializes in the fields of culture,

tradition, medicine, and people.

Traditional Medicine. Any system of healthcare that has ancient roots or is

formed and influenced by traditions of a certain ethnic group.

Alternative Medicine. Any type of therapy that is not considered or outside the

range of traditional medicine.

Brine Shrimp Lethality Bioassay. This term refers to consider a useful tool for

preliminary assessment of toxicity. The method is attractive because it is very simple,

inexpensive and sensitive brine shrimp.

Cytotoxicity. This terms refers are commonly used to monitoring the response

and health of cells in culture after treatment with varius stimuli.

Lethality. The probability that a person threatening suicide will succeed, based

on the method described, the specificity of the plan, and the availability of the means.

Artificial Seawater. A combination of salt and water.

Assay. This term is an investigative procedure in laboratory medicine,

pharmacology environmental, and molecular biology for qualitatively assessing or

quantitatively measuring the presence or amount or the functional activity of a target

entity. Which can be a drug or biochemical substance or a cell in an organism or

organic sample.
 Medicinal Plant. A plant that has similar properties as conventional

pharmaceutical drugs. Humans have used them throughout history to either cure or

lessen symptoms from an illness.

Therapeutic. This refers to the treatment of disease or disorders by remedial

agents or methods.

Concentration. The relative amount of a given substance contained within a

solution or in a particular volume of space.

 Chapter 2

REVIEW OF RELATED LITERATURES AND STUDIES

This chapter presents the relevant literature and studies local or foreign taken

from some books, journals, published thesis and dissertation, and from the internet.

Related Literatures

A. Local Literatures

For about a hundred of thousands species of plants we have on earth, 1,064,035

have scientific plant name as they ranked, 350,699 are accepted as it species name,

642 plant families and 17,020 of plant genera it has. 8 Cordia dichotoma or also known

Anonang in Philippines is a large pantropical genus that have for about 250 – 300

species and can grow to a height of 5 to 10 meters and does have many function and

properties about its part. Mostly found in an open forest, beaches, coastal hills, inland

bounded with mangroves, and sometimes on roadside. Anonang (Cordia dichotoma)

does not grow well on dry land so it is usually found in a place that has enough water or

an area that is almost always dry.

The bark of Anonang (Cordia dichotoma) contains a large amount of tannic acid

and uses as astringent and tonic. The bark of Anonang (Cordia dichotoma) was also

used for tumors, headaches, stomachaches, ulcers, as a gargle, for colicky pains, and

for strengthening teeth. It fruits yields saponin, amino acids, flavonoids, sugar, gum,
8
The Plant List, retrieved from http://www.theplantlist.org/ in July 31, 2017
proteins, palmitic, stearic, linoleic acids, oleic acids behenic acids and was used as a

glue, laxatives, for coughs, ailments particularly to the chest, uterus, and urethra and

also used for healing of wounds. In the Philippines, the leaves of Anonang (Cordia

dichotoma) were used for cooking. Wrapped to fish when cooking. And also used for

wound healing, ulcer, teeth, headaches and stomachaches. 9

In the Philippines where traditional medicine and its method of healing is widely

spread which our ancestors or Filipino ancient healer believes in a manner that states

the imbalances that is caused by different environmental factors that affect our health

and the ability to regain and recover about the concept that states the imbalances that

our ancestors in their time are dealing with. Later on, our Filipino ancestors organized

traditional practitioners to help people find its way to recover up diseases and illnesses

using different herbal plants they have in their place.

On the mean time researchers started to arise and investigate about the liability

of different types of plant and trees that can be used in the field of medicine or medicinal

purposes. Among them is Anonang or also known for its scientific name Cordia

dichotoma which is considered as one of the native tree in the Philippines for its

characteristics and places where it can be found.

9
Garg J.M, “Philippines Medicinal Plants: Anonang (Cordia dichotoma)” retrieved from www.stuartxchange.org/Anonang.html
in July 31, 2017
 B. Foreign Literatures

For about more than half of the world`s population relies on the traditional

medicine and major role of the traditional medicine including the use of plant extract and

their active constituents. Researchers defined Cordia dichotoma as a small moderate

size plant of family Boragenaceae, commonly called bhokar, lasura, gonda, Indian

cherry, shlesmataka, anonang and etc. Cordia dichotoma uses leaves, fruit, bark, and

seed and have been reported for possessing anti-diabetic, anti-ulcer, anti-inflammatory,

immune-modulator, and analgesic activity. These are just some traits about the uses of

Cordia dichotoma for means of medicine. Screening of fruit, leaves and seed shows the

presence of pyrrolizidine alkaloids, coumarins, flavonoids, saponins, terpenes and

sterols.

According to Samy RP, et. al (2008), plant derived medicines are considered to

be the first line defense in maintaining health and combating diseases and even today

plant source is principal source of new drug therapeutic property. Cordia dichotoma

belong to the concept of positive health means metabolically well-balanced human

beings or also known as Ayurveda which means “science of life”. For about 700 species

of plant that is used in Ayurveda. 10 Cordia dichotoma is one of the traditional medicines

that is very important for the deciduous of plant. Its fruit has been reported to be rich in

polysaccharide. Ripe fruit produces a jelly-like sticky mass that can be used as a glue.

In India, Cordia dichtoma is commonly used for treating common cold, catarrh, cough,

respiratory distress and fevers.

10
Samy RP, et. al, “A compilation of bioactive compunds from Ayurveda”, Bioinformation retrieved from
https/www.ncbi.nlm.nih.gov/PMC3805104/#bl. 2008;3(3):100-110
 In ancient time, leaves and stem bark are used in the treatment of dyspepsia,

fever, diarrhea, leprosy, gonorrhea and burning sensation. Leaf of plant traditionally

shows the therapeutic uses and actions such as anthelmintic, astringent, diuretic,

demulcent, purgative, expectorant, tonic, ulcer and cough. 11

As discussed by Khond (2009), the whole plant of Cordia dichotoma is edible and

is used as food. Immature fruits are pickled and are also as vegetable. Mixture of flower

and curd applied two times in a day used to protect body against heavy sun heat waves.

The seed contain high quantity of fatty oils and proteins which has potential as cattle

feed. The polysaccharide gum obtained from plant used for various pharmaceutical

purposes. Chromium present in the fruit has therapeutic value in diabetes. Fruit also

contain some anti-nutritional factors such as phytic acid, phytate phosphorus and oxalic

acid. Branches of Cordia dichotoma has natural cellulose fabrics that is identified later

on.

Since then, the plant Cordia dichotoma was widely used and accepted in the field

of medicine. Researchers simply tested the liability and effectiveness of Cordia

dichotoma in order to help more the society specifically to the people who is still don’t

have the knowledge about the effects and benefits of this plant, Cordia dichotoma and

also to promote the beauty of Cordia dichotoma as a thing for treating illnesses and

diseases and also for the security of the plants.

11
Khond M, et. al, “Screening of some medicinal plants extracts for invitro antimicrobial activity” Middle-east J Sci Res.
retrieved from https/www.ncbi.nlm.nih.gov/PMC3805104/#bl. 2009;4(4):271-278
Related Studies

A. Local Studies

According to work of Reizl P. Jose et.al, many plants have been used as

traditional medicines with brines shrimp lethality assay. Philippines is believed to have

medicinal properties was conducted in determine whether this medicinal plants has

potential pharmacological activities. The leaf plant toxicity against brine shrimp is

chronically toxic. This findings support the traditional medicinal alternative use of this

plants. Based on this study its possible relationship between brine shrimp lethality and

plant, was could serve for further ethnobotanical and phytochemical research.

Moreover, this study isolation through Gravity Column Chromatography (GCC) and high

Performance Liquid Chromatography (HPLC) with Nuclear Magnetic Resonance (NMR)

for the specific organic compounds determination. The study showed 100% mortality of

brine shrimp in all concentration at six hours and 24 hours with in exposure in all extract

preparation among the different types of extract the 50-50 ethanol extract had the most
12
toxic concentration with less than 10 pmm toxic and LC50 value.

In the study of Consolation Y., et. al, many parts of tree are well known to have

different medicinal uses: like antidiabetic, anthelmintic, antilarvicidal, hepatoprotective,

immuno-modulator, antidysentery, antidyspepsia, anti-inflammatory, anti-ulcer, diuretic,

and laxative. The report of study on the result isolation of compounds from the

dichloromethane extracts of the leaves and stem. Compounds have been reported to

possess anticancer and cytotoxic properties. The antioxidant property , cholesterol

lowering effect and wound healing effect have also been reported. Thus medicinal

12
Jose, Reizl, P. et al. “Brine Shrimp Lethality Assay of the Ethanolic Extracts of AntidesmaghaesembillaGaertn” retrieved from
http://bepls.com/aug_2015/15.pdf in August 02, 2017
properties of Cordia dichotoma maybe partly attributed which were reported exhibit

diverse biological activities.13

As discuss in study of Mae A. Responte et.al, Brine Shrimp of Lethality Assay

(BSLA) is an important method in the evaluation of plant bioactivity for the subsequent

isolation of bioactive compounds from the plant extracts, which may lead to the

development of new drugs.14

Stated by Cindy D. Arquion et. al, their study was conducted to test for in vevo

brine shrimp lethality of Ficus nota. The plant extract obtained through extraction of the

stem samples with water and absolute ethanol, the three concentrations (100 ppm, 500

ppm, and 1000 ppm) of the extracts were tested and mortality of Artemia salina was

noted after 24 hours exposure. The result showed that both decoction and ethanpo;lic

extracts were active against the brine shrimp with LC50 values of 991.00 ppm and

852.22 ppm. While indicated were both extract may have substance that are cytotoxic

and that active components of the plants are better extracted with absolute ethanol. The

Artemia salina eggs were obtained from Mindanao State University Naawan Campus

were the eggs dehydrated in distilled water for 30 minutes. The tank filled with filtered

sterile water was then divided into two compartments. The hatching chamber was

covered with black cardboard before adding the dehydrated eggs of A. Salina and the

other half of the tank was illuminated to attract with separate the hatched shrimp from

13
Consolacion, Y.et al.“Chemical Constituent of Cordia Dichotoma G. Forst” retrieved from
https://www.researchgate.net/publication/281589867_Chemical _Constituents_of_Cordia_dichotoma_G_Forst in August 02,
2017
14
Responte, Mae, A. et al. “Brine Shrimp Lethality Assay of whole plant extaract of Eleusineindica” retrieved from
http://www.aab.bioflux.com.ro/docs/2015.90-95.pdf in August 02, 2017
the unhatched shrimp eggs. The light is important to simulate the temperature of the

natural habitat of the shrimp were then used for bioassay 24 hours. 15

Foreign Studies

Thirupathi, et. al (2008) stated in their study that even if Cordia dichotoma does

have many uses for treating various diseases and disorder conditions. Plant Cordia

dichotoma have been reported for various pharmacological activities. Several chemicals

have been identified from seeds of Cordia dichotoma. It contains aamyrins, botulin,

octacosanol, lupeol-3rhamnoside, B-sitosterol, B-sitosterol-3glucoside, hentricontanol,

hentricontane, taxifolin-3-5-dirhamnosie, hesperitin-7rhamnoside and fatty acids such

as palmitic acid, stearic acid, arachidicaci, behenic acid, oleic acid and linoleic acid. 5

flavonoid glycosides was found such as robininmrutin, rutoside, datiscoside and

hesperidin. Flavonoid aglyconne and 2 phenolic derivatives such as chlorogenic acid

and caffeic acid were isolated from seed. This signifies the anti-inflammatory activity of

seed beause of a-amyrins and taxifolin-3-5-dirhamnoside. The bark of Cordia dichotoma

is medicinal and contains several chemicals including allantoin, B-sitosterol and 3’, 5-

dihydroxy-4”-methocy flavanone-7-O-alpha-L-rhamnopyranoside. Fruit and leaves

showed presence of pyrrolizidine alkaloids, coumarins, flavonoids, saponins, terpenes,

and sterols. Fruit has been identified for arabinoglucan, D-glucose and L-arabinose.
16
Leaves also have quercetin and quercitrin.
15
Arquion, Cindy, D. Et al.”Evaluating the Potential Cytotoxic Activity of Ficus nota” retrieved from
http://s3.amazonaws.com/academia.edu.ducuments/42018937/Evaluating_the_Potential_Cytotoxic_Activity_of_Ficus_nota_U
sing.pdf?AWSAccessKeyld=AKIAIWPOWYYGZ2Y53UL3A&Expires=1501635959&Signature=2RM6o6rvGjFYP0acGr1PNKwkR3SQ
%3D&responsecontentdisposition=inline%3B%20filename%3DEvaluating_the_potential_Cytotoxic_Active.pdf in August 3, 2017
16
Thirupathi K, et. al, “A review of medicinal plants of the genus Cordia: their chemistry and pharmacological uses” retrieved
from https/www.ncbi.nlm.nih.gov/PMC3805104/#bl. 2008;8(1):1-10
 At the work of Meyer et al. (1982), LC50 lower than 1000ppm is considered

cytotoxic (active) on the evaluation of plant extract by BSLA, but while non-toxic

(inactive) if it is greater than 1000ppm. The result of the decoction plant preparation

exhibited inactivity where in very low mortality among the brine shrimp. The best

extraction method for the whole plants sample is through mixture of ethanol and water

as it demonstrated maximum mortalities 100% and high LC50 values against the brine

shrimp.17

According to Gaurav S. et. al (2009), the effect of the aqueous extract of Cordia

dichotoma on alloxan induced and normoglycemicWistar rats has been investigated.

Three doses of the extract about 250mg/kg or ppm; 500mg/kg and 1000mg/kg were

considered for study by oral route. The effects of 500 mg/kg extract of Cordia dichotoma

did not show any significant change in blood glucose levels in normoglycemic rats.

Same effect was also observed at 50 mg/kg dose in alloxan induced diabetic wister rats,

when compared to untreated control. The significant P<0.5) decreaces in blood glucose

levels after 4, 8 and 24 h was observed at 500 and 1000 mg/kg dose of extract. The

dose of 1000 mg/kg of plant extract shows that the blood glucose levels at 8 and 24 h.

in overall, the study shows that the doses of extract has shown both significant P<0.05

hupoglymic and antihyperglycemic effects on wister rats. 18

As discussed by Sharker S Md, et. al (2009), crude ethanolic extract of Cordia

dichotomawas evaluated for the analgesic, antibacterial and cytotoxic activity. The

extract produced significant writhing inhibition in acetic acid induced writhing in mice at

17
Meyer B.N., et.al, “Brine shrimp: A convenient general bioassay for active plant constituents”, Plant Med, 45, p. 31-34
18
Gaurav, et. al, “Effects of aqueous leaves extract of Cordia dichotoma on blood glucose levels of normoglycemic and alloxan
induced diabetic wister rats” retrieved from https/www.ncbi.nlm.nih.gov/PMC3805104/#bl
the oral dose of 500 mg/kg body weight respectively which was compared to the

standard drug diclofenac sodium at the dose of 25 mg/kg of body weight. When tested

for antibacterial effects by disc diffusion method, it showed dominant zone of inhibition

of against both gram and positive bacteria such as Streptococcus pyogenes, vibrio

cholerae, streptococcus epiderimis, hafnia and Escherichia coli which is comparable

with kanamycin 30 mg/ml. moreover, when tested for toxicity using brine shrimp lethality

bioassay, the extract showed potent against the brine shrimp artemiasalina. The result

represent the significant analgesic, antibacterial and cytotoxic activities of the extract. 19

Moreover, the researchers will identify the toxicity of plant Cordia dichotoma by

the means of brine shrimp lethality assay in giving specific amount of solution of the

need materials and by knowing the effects of the 3 different amount of the decoction

which will be later on added on the said process for finding the exact number that is

needed in identifying the toxicity of plant Cordia dichotoma. This will help the

researchers to attain the society`s needed in treating different diseases and illnesses

regarding with the different uses of this plant, Cordia dichotoma and also for the liability

and effectiveness in treating diseases and illnesses.

Synthesis

19
Sharker S Md, et. al , “Analgesic, antibacterial and cytotoxic activity of Cordia dichotoma”, Pharmacology online, retrieved
from https/www.ncbi.nlm.nih.gov/PMC3805104/#bl. 2009;2:195-202.
 Cordia dichotoma also known as Anonang in the Philippines has much type of

therapeutic properties which became as basis for medical purposes and application that

is use in many places around the world. It has been use for such a long period of time

as a traditional medicine such as cicatrizant, astringent, anti-inflammatory,

anthelminthic, antimalarial, diuretic, febrifuge, appetite suppressant, cough suppressant,

treating urinary infections, lung diseases and leprosy. Cordia dichotoma was also use

for gastro-intestine disorder thoroughly internal and external appearances.

This review represents the brief information about the botanical description,

chemical constituents and the specific function of this plant as well as in the field of

medicine. Anonang (Cordia dichotoma) has many pharmaceutical applications that may

be used as excipient in many indigenous people mostly in the Philippines and around

the world.

Since then, traditional medicine here in the Philippines have made it way for such

a long period of time. It is believe that traditional medicine does work well in treating

different illnesses and diseases than what the hospital can gave for treating their

illnesses and diseases. Therefore, the researchers have conducted a study about the

liability of Anonang (Cordia dichotoma) for the reason that it`s said to be ingested,

applied by means of poultice and other therapeutic practices.

The researchers will use brine shrimp lethality assay and plant extraction of

Anonang (Cordia dichotoma) to test its toxicity and prove how safety it is for treating

disease and illnesses in different therapeutic application specifically the ingestion of its

plant extraction.
 Chapter 3

RESEARCH DESIGN AND METHODOLOGY

This chapter presents the research design, instrumentation, experimental

procedure and research gathering procedure which was used in this study.

Research Design

The researchers employed naturalistic research, ethnographic, use-inspired,

quantitative research and pre-experimental research.

Use-Inspired Basic Research

As discussed by the Swiss National Science Foundation, the use-inspired basic

research method recognizes that research which combines the aspect of gaining

scientific knowledge with an orientation towards application is becoming increasingly

important both nationally and internationally; it ensures the adequate evaluation of use-

inspired applications by taking into account their broader impact as an evaluation

criterion and by engaging external reviewers from the practical realm

The use-inspired basic research in this study involves the use of previous

research study as the guide of the researchers regarding the medicinal plants and

traditional therapies used by the medicine men in treating disease and illnesses.
Quantitative Research

According to Church and Rogers, quantitative research is a study involving the

use and analyses of numerical data using statistical techniques. They pose questions of

who, what, when, where, how much, how many, and how. 20

The quantitative research in this study involves the numerical data regarding the

concentration of the ethanolic extract of the Anonang (Cordia dichotoma) leaves and

stem bark, the results of the number of surviving brine shrimp after 24 hours and the

percent molarity of the number of surviving brine shrimp after 24 hours.

Pre-Experimental Research

As stated by Prof. Son-Ung Kim, pre-experimental design is a research design

which does not fit the standards of an authentic experiment. It is usually undertaken for

exploratory purposes. Either a single group or multiple groups are observed subsequent

to some agent that’s presumed to cause change. 21

The pre-experimental research part in this study is the lethality assay of the

Anonang (Cordia dichotoma) leaves and stem bark by using the brine shrimp in which

the researchers will determine the toxicity level of the ethanolic extract of leaves and

stem bark of the Anonang (Cordia dichotoma).

20
Church, Cheyanne and Rogers, Mark, “Quantitative Research Module”, retrieved from
http://dmeforpeace.org/sites/default/files/1.3%20Quantitative%20Research.pdf in July 31, 2017.
21
Son-Ung Kim, “Sociology Index: Pre-experimental Design”, retrieved from
http://www.sociologyindex.com/pre_experimental_design.htm
Sampling Techniques

1. Plant Sample

2. Brine Shrimp Eggs

3. Different Concentrations
 Variables

The researchers anchored the hypothesis on the following variables used:

Constant Variable Independent Variable Dependent Variable

 Amount of Brine shrimp

 Different concentrations  Lethality Assay of Brine
of the ethanolic extract shrimp
 Time of Anonang (Cordia
dichotoma) leaves and
 Temperature stem bark
Instrumentations

Figure 1. Plant Extraction (Ethanolic Extraction)

Figure 1.1 Figure 1.2

Hot Plate 250ml Erlenmeyer Flask

Figure 1.1 Hot plate will be Figure 1.2 250ml Erlenmeyer

needed for the boiling of plant Flask will be used for measuring

extract. the amount of water in 0-250ml.

Figure 1.2 Figure 1.3

Mortar and Pestle Stirring Rod

Figure 1.2 Mortar and Pestle will be Figure 1.3 Stirring Rod will be
used to grinding the plant. used in mix the solution in a

certain container.
Figure 1.4 Figure 1.5
 Ethanol 600ml Beaker

Figure 1.4 Ethanol will be used for Figure 1.5 600ml Beaker will be

the extraction of plant. used as a container for the needed

liquid materials

Figure 1.6 Figure 1.7

Filter Paper Triple Beam Balance

Figure 1.6 Filter paper will be used Figure 1.7 Triple beam balance

in filtering the liquid materials. will be used for measuring the

precise mass of the materials.

Figure 2. Brine Shrimp Lethality Assay

Figure 2.1 Figure 2.2

 150mmx15mm Petri Dish Test Tube Holder

Figure 2.1 150mmx15mm Petri Dish Figure 2.2 Test tube holder
will be used as a container where the will be used for holding the
brine shrimp will be placed. test tube.

Figure 2.3 Figure 2.4

10ml Test Tube Microscope

Figure 2.3 10ml Test Tube will Figure 2.4 Microscope will be

be used to transport a small used to see the small object

amount of water that is needed specifically the brine shrimp.

and where the assay is placed.

Figure 2.5 Figure 2.6

Pipette 100ml Graduated Cylinder

 Figure 2.5 Pipette will be used Figure 2.6 100 ml Graduated

to transport the measured Cylinder will be used to

amount of brine shrimp to measure the amount of water

another container. that is needed.

Experimental Procedures

Collection of Plant Material

Fresh leaves and stem bark of Anonang (Cordia dichotoma) will be collect

randomly from Purok Sandiwa, Barangay Tiniguiban, Puerto Princesa City. This plant

was selected because of its traditional and medicinal usage as a cure in poison, sprain,

and body and/or muscle pain by the process of tincture and is applied through poultice

and ingestion with a dosage depending on the extent of the damage, as gathered from

the interview with the medicine men in Purok Sandiwa, Barangay Tiniguiban, Puerto

Princesa City.

Preparation of the Plant Extract

The leaves and stem bark will be wash with water and air dried in shade. Then,

the air-dried leaves and stem bark will be pound using mortar and pestle and will be

keep in closed plastic containers. Twenty grams of the fine powder of each plant
samples will be weigh and added into an Erlenmeyer flask containing 250 mL of 95%

ethanol. The solution was covered and shaken every 30 min for about six (6) hours and

allowed to stand for about 48 hours in room temperature. Then, it was shaken and

filtered using filter paper. After filtration, the solvent will be remove by evaporation. An

alternative dilution procedure developed by McLaughlin et al. will be adopt in the

preparation of the different dilutions of the plant extracts for Brine Shrimp Lethality

Assay (BSLA) where 20 mg of each extract was dissolved in 2 mL of the solvent. 22 The

final concentrations were 1000, 100, and 10 ppm (μg/mL). There were three (3)

replicates in eachconcentration.

Brine Shrimp Lethality Assay (BSLA)

Brine shrimp eggs were obtained from a friend of one of the researchers, as a gift

sample for the research work. 350 ml of seawater was prepared for hatching the shrimp

eggs. The seawater was put in a small plastic container (hatching chamber. Shrimp

eggs were added into the dark side of the chamber while the lamp above the other side

(light) will attract the hatched shrimp. Two days were allowed for the shrimp to hatch

and mature as nauplii (larva).

After two days, when the shrimp larvae are ready, 4 mL of the seawater will be

add to each test tube and 10 brine shrimps will be introduce into each tube. Thus, there

will be a total of 30 shrimps per dilution. Then the volume is adjust with artificial

seawater up to 5 mL per test tube. The test tubes will be left uncovered under the lamp.

The number of surviving shrimps will be count and record after 24 hours.

22
Mclaughlin J.L., et.al, “The use of biological assays to evaluate botanicals”, Drug Information Journal, 32, 513–524 (1998)
Statistical Treatment

After 24 hours, the test tube will be transfer into a petri dish and will be inspect

under a microscope and the number of surviving larvae will be count. The percentage of

mortality will be calculate at each concentration. The concentration mortality data will be

analyze statistically. The effectiveness or the concentration-mortality relationship of the

plant product is usually expressed as a median lethal concentration (LC50) value. The

LC50 value represents the concentration of the chemical that produces death in half of

the subjects after a certain exposure period. The percentage of mortality at each test

dose and the control was determined by using the formula:

% of mortality = No.of dead nauplii x 100

Total number

The toxicity of herbal extracts expressed as LC50 values is commonly valorized

either by comparison to Meyer’s or to Clarkson’s toxicity index. According to Meyer’s

toxicity index, extracts with LC50 < 1000 μg/ml are considered as toxic, while extracts

with LC50 > 1000 μg/ml are considered as non-toxic. 23 Based on Clarkson’s toxicity

criterion for the toxicity assessment of plant extracts classifies extracts in the following

order, extracts with LC50 above 1000 μg/ml are non-toxic, LC50 of 500 - 1000 μg/ml

are low toxic, extracts with LC50 of 100 - 500 μg/ml are medium toxic, while extracts

with LC50 of 0 - 100 μg/ml are highly toxic. 24

23
Meyer, B.N., “Brine Shrimp: A convenient general bioassay for active plant constituents”. Planta Medica. 45, 31-34.
24
Clarkson, C., “In vitro antiplasmodial activity of medicinal plants native to or naturalized in South Africa”, J
Ethnopharm. 92, 177-191.
Data Gathering Procedures

The researchers made a letter of permission and gave it to the barangay to allow

them to manage an ocular survey in Purok Sandiwa, Barangay Tiniguiban, Puerto

Princesa City. This study included interview of the medicine men in Purok Sandiwa,

Barangay Tiniguiban, Puerto Princesa City. The interview used the general knowledge

of the selected medicine men.

After the approval, the researcher visited the research locale for interviews to

medicine men along with the observation and documentation of the medicinal use of

Anonang (Cordia dichotoma) and traditional therapies done by each medicine man.

After the interview, the researchers had observed that traditional medicine and

therapeutic practices are a popular culture among the residents of Purok Sandiwa,

Barangay Tiniguiban. There are herbalists that uses Anonang (Cordia dichotoma) in

curing poison, sprain, and body and/or muscle pain. They prepared through the process

of tincture and is applied through poultice and ingestion with a dosage depending on the

extent of the damage.

With this way of application, the researchers are encouraged wherein, the

researchers will find out the preliminary assessment of toxicity of the plant ethanolic

extract. The researchers will investigate the toxicity level of the leaves and stem bark of

the Anonang (Cordia dichotoma).

To determine the toxicity level of the leaves and stem bark of the Anonang

(Cordia dichotoma), the researchers will collect the plant material in Purok Sandiwa,
Barangay Tiniguiban, Puerto Princesa City. Then, the collected plant material will

undergo plant ethanolic extraction and lethality assay by the use of Brine Shrimp.

After waiting 24 hours of the Brine Shrimp Lethality Assay (BSLA), the

researcher will inspect the number of surviving larvae under a microscope and count.

The percentage of mortality will be calculate at each concentration. The concentration

mortality data will be analyze statistically. The researcher will compute the percentage

of mortality at each concentration.