Antinociceptic Activity of Talisay Extract

Republic of the Philippines
Department of Education
Region 02(Cagayan Valley)
ROXAS NATIONAL HIGH SCHOOL
San Antonio, Roxas, Isabela 3320
ANTINOCICEPTIC ACTIVITY OF ETHYL/CRUDE OIL INDIAN ALMOND
(TERMINALIA CATAPPA ) EXTRACT TO ALBINO RATS
_______________
A Research
Presented to the Teachers of

Roxas National High School
Senior High School Department
Roxas, Isabela
_______________
In Partial Fulfilment
Of the Subject Requirement for

Practical Research 1
By:
PIS-ING, AISHERA JAPHRODITE
CONSOLACION, JOHN ISRAEL
PALAGANAS, JAYSON
PALAGANAS, JOHN MARK
VICITACION, FRECHELLE
VELASQUEZ, ANGELO
CAMPOS, BEBERLY
GARAZA JOSHUA
ORDANZA, ELLA
2019-2020
Chapter 1
INTRODUCTION
A. OVERVIEW OR BACKGROUND OF THE STUDY
Terminalia catappa Linn. is known for its nutritional fruit and possesses
medicinal benefits as well. T. catappa has been recognized for its medicinally
essential phytoconstituents, such as phenol, flavonoid, and carotenoid. Numerous
pharmacological investigations have confirmed this plant's ability to exhibit
antimicrobial, anti-inflammatory, antidiabetic, antioxidant, hepatoprotective, and
anticancer activities, all of which support its traditional uses. (A. Anand, N. Divya, P.
Kotti, 2015)
Terminalia catappa fruit is rich in phytochemicals and nutrients which
contribute to the therapeutic potential. Fruit as a whole is very tasty but the curiosity
in analyzing the flesh and seed separately made an attempt to do this work.
(Krishnaveni, 2015)
Terminalia Catappa fruit is rich in phytochemicals and nutrients which
contribute to the therapeutic potential. Fruit as a whole is very tasty but the curiosity
in analyzing the flesh and seed separately made an attempt to do this work. The
present study was carried out for the presence of phytochemicals, phytonuutrients,
behavior with different chemicals, fluorescence as well as percent yield.(Krishaveni,
2015)
The juice of its fresh leaves is used in the preparation of medicinal lotion for
leprosy and scabies, and it is taken internally for stomachache and headache.
Scientific studies on the medicinal benefits of T. catappa began untimely and the
reports uphold a tradition of repeating the data for each decade. It is necessary to
develop a system to use the plant continuously for the most effective health care
purposes. (A. Anand, N. Divya, P. Kotti, 2015)
T. Catappa’s anti-inflammatory and analgesic properties may lead to a more
complex antinociceptive activity to expand the medicinal and pharmaceutical uses of
this plant for the most effective health care purpose.
Recently, special importance has been given to the role of inflammation in the
pathogenesis of various ailments. Medicinal plants are used worldwide as a remedy
for various inflammatory disorders. The various polyphenolic compounds,
triterpenoids, and other chemical compounds found in the plants may be responsible
for the anti-inflammatory activities. (A. Anand, N. Divya, P. Kotti, 2015)
The undesired effects of the analgesic compounds available in the market
provided an opportunity for new and better analgesic compounds. (Katzung, 2007)
Bioactive compounds derived from plants have been utilized since from the earlier
time for the various purposes including the treatment of pain. Moreover, numerous
plant species were found to possess analgesic and anti-inflammatory activities. (Gill,
2010, Jothimanivannan, 2010, Mills and Bone, 2002)
Terminalla catappa leaves posseses analgesic activity which may mediated
through both central and peripheral mechanism. Earlier researchers have reported the
nociceptive potential of aqueous juice prepared by maceration from the tender leaves
of terminalia catappa to support its folklore use. (S. Arjariya, N. Nema, S. Tiwari, R.
Dubey, 2013) Hence, this study aims to be able to give opportunity on the production
of a naturally occurring anesthesia on which creates a new medical breakthrough.
Pain is an unpleasant sensory or emotional experience associated with actual
or potential tissue damage, or described in terms of such damage. The Nociception
involves the activation of specific primary sensory neuron subpopulations that
transmit the nociceptive information to the spinal cord from where it is relayed to
supra spinal levels. Tissue damage occurs by activation of nociceptors through the
release of several mediators, including excitatory amino acids, peptides, protons,
lipids and cytokines, which bind to receptors and activate signaling pathways. Thus,
pain can be a subject to multiple levels of biochemical and pharmacological controls,
involving a diversity of cell types and soluble mediators. As a result, compounds that
present antinociceptive effect are of potential therapeutic interest for the treatment of
human and animal pain. (S. Arjariya, N. Nema, S. Tiwari, R. Dubey, 2013)
The management of pain using currently available analgesics could not
completely thrive in relieving pain due to the fact that pain modulation is an intricate
process involving many mediators and receptors at the peripheral and central levels.
The sensitivity of nociceptive neuron is adjusted by a large variety of mediators in the
extracellular space. These mediators, either neurotransmitters or neuromodulators,
activate a large number of receptor classes, which in turn activate a plethora of
signaling cascades that are responsible for controlling the perception of pain. Thus,
attempts are being made worldwide to identify the components involved in this
complex process and to develop new agents that act on these components. (I. Suntar,
2018)
Thus, the antinociceptive potential of T. catappa provide related studies that
suggest the possibility of developing an anesthetic substance which is sustainable and
natural. A natural anesthesia has several advantages such as: it is more affordable and
conventional, easier to obtain than synthetic prescription medicines, provides natural
antinociception, and has fewer side effects.
These evidences urged the researchers to study the antinociceptic activity of T.
catappa extract using various nociceptive assays.
B. CONCEPTUAL FRAMEWORK
INDEPENDENT VARIABLE DEPENDENT VARIABLE
Acute Toxicity Test Tested the Acute Toxicity of T.
Catappa
Antinociceptive Analysis Analyzed antinociceptive properties
Saponin Test Tested presence of saponin
Flavonoid test Tested flavonoid content
Tannin and phenolic compound test Tested presence of tannin and
phenolic compound
Alkaloid testing Tested presence of alkaloid
Sterol and Steroid Testing Tested presence of sterols and
steroids
Phytonutrient analysis Estimated Phytonutrients

C. RESEARCH OBJECTIVES/ QUESTIONS
In testing the antinociceptic activity of ethyll/crude oil extract, the
questions to be answered are the following:
 To analyze the phytochemical properties of Ethanol T. Catappa Extract
Includes; saponin, flavonoid, tannin and phenolic compound, alkaloid,
sterol and steroid tests.
 To test the acute toxicity of Ethanol T. Catappa Extract
 To analyze antinociceptive activity of Ethanol T. Catappa Extract
 To estimate phytonutrients in Ethanol T. Catappa Extract
D. HYPOTHEIS
Null: The properties of Terminalia Catappa have no capabilities on
antinociceptic activity.
E. SIGNIFICANCE OF THE STUDY
This study aims to attain the most effective healthcare purpose of Ethanol T.
Catappa Extract in antinociceptic activity. Generally, provide an affordable,
more accessible, conventional, sustainable and natural anesthetic compound
with less side effects; to help the future researchers, to contribute to the
medical industry with a new discovery, to provide functional knowledge to the
BS Chemical Engineering, Medical Technology, BS Pharmaceutical, and the
present researchers.
The researchers because this study would help them build themselves
up, learn more about the deeper concepts of natural science and to also learn to
appreciate the importance of naturally occurring medicine. This study will
help them grow more on their fields of interest, believe in themselves, develop
a stronger sense of responsibility, manage time wisely, and have more
knowledge on the importance of discovering and innovating.
The Medical Technologists who will benefit with the production of
sustainable antinociceptic for they are in charge in chemical handling inside
hospitals. This study will benefit them by giving a wider range of medicine to
be used on needing patients.
The BS Pharmaceutical may use this study to develop more complex
anesthesia with the use of a naturally occurring substance. This will lead to an
easier, more convenient, and cheaper alternative to synthetic actinociceptics.
The demanding patients who suffer intolerable and non-stop pain. A
dose with stronger antinociceptic property that costs less, natural, and has less
side effects will let them take the dose they need to fully thrive the pain.
Sometimes, currently available analgesics could not give the range of pain
modulation as how antinociceptics do.
The future researchers may use this study as an open reference if
they want to conducts further studies regarding the other potential uses of T.
Catappa. This can become their open basis to save time, money, and effort in
gathering data themselves. This will guide them to the result that would want
to achieve.
F. SCOPES AND LIMITATIONS
This study is being conducted in the Roxas National High School-
Senior High School, San Antonio, Roxas, Isabela from November 2019 to
February 2020`
The researchers all came from Roxas, Isabela and are all students of
Roxas National Highschool-SHS from the Science Technology Engineering
and Mathematics strand aged 16-17 years of age.
The researchers will follow Ethanol Extraction using mature Indian
Almond fruits (1000 Grams, dried) macerated in 2000-3000 mL Ethanol
(70%) and left on ultralow temperature for 24 hours.
THE METHODS FOR THE STUDY:
1. Finding and Harvesting of T. Catappa Fruits
This includes the harvesting of T. Catappa fruits and putting them in a
clean transparent plastic bag. T. Catappa is not just endemic to one area so it
can be found everywhere. The T. Catappa fruits are collected from T.
Catappa trees in Roxas, Isabela. Only mature fruits are collected since they
are the ones capable for the extraction. About 1 kg of mature fruits is needed
to be harvested.
2. Cleaning and Husking of Fruits
The collected fruits are cleaned and husked using a hammer. After
husking, the husks are put on a tray and dried under direct sunlight for 3 days,
6 hours per day. The husks were obtained and set aside for extraction.
3. Soaking Ethanol on Husks
2000-3000 mL Ethanol (70%) is mixed with 1000 grams of T. Catappa
fruit husks (0.5 mg/mL). The material should be fully submerged. After
soaking the husks, the mixture will be left on ultralow temperature freezer (-2
℃ ) for 24 hours.
4. Filtration of Solid Materials from the mixture
Removing all the solid materials form the mixture with simple filtration
process is done with a vacuum pump, Buchner funnel, filter paper and a
vacuum flask. After the material has been filtered, the solution of ethanol and
plant extract remains.
5. Separation of the T. Catappa extract from the ethanol
To separate the two, use a rotary evaporator. When connected to a
vacuum pump, the rotary evaporator uses the vacuum to lower the boiling
point of ethanol, resulting in a quicker evaporation. The resulting extract or oil
will be completely free of ethanol. This process also allows ethanol to be
recovered for additional extraction. A recirculating chiller is also needed as

this connects to the rotary evaporator to accelerate solvent recovery. After
completing these simple steps, the extract is now ready for the next phase of
the process.
6. Winterization of Extract
Winterization is a process where fats, plant lipids, or any kind of waxes
are removed from the extract. This can be accomplished by adding ethanol to
the extract. Repeat the filtering step and again use the rotary evaporator to boil
off the ethanol, leaving a pure extract that can be dried in a laboratory oven or
readily infused.
7. Conduct of Phytochemical and Phytonutrient Analyses
The crude extract of T. Catappa is assessed qualitatively to detect the
presence of different phytochemicals such as carbohydrates, saponins,
flavonoids, tannins, alkaloids, glycosides, glucosides, reducing sugars,
proteins, gums, and steroids following standard procedures.
8. Conduct of Toxicity Test
Extracts is administered on Albino rats orally, the rats were allowed food
and water ad libitum and all animals were observed for abnormal behaviors,
allergic symptoms, and mortality for the next 72 hours.
9. Conduct of Antinociceptive Analyses
Several test are conducted on Albino rats through hot plate test, tail
immersion test, acetic acid induced writhing test, glutamate induced
nociception, and formalin induced nociception.

G. DEFINITION OF TERMS
Terminalia Catappa – the main subject of the study where analgesic and anti-
inflammatory properties are observed to analyze the antinociceptive activity of
its ethanol extract.
Antinociceptic- the activity that blocks the detection of pain stimulus and is
being analyzed on T. Catappa extract
Ethanol- the solvent used for the Ethanol Extraction of T. Catappa husk.
Ethanol extraction- the process of extracting the essential oils of T. Catappa
husk by using Ethyl alcohol as the solvent.

Chapter II
REVIEW OF RELATED LITERATURE AND STUDIES
This chapter presents all the related literature and studies that exists from other
sources that digs in the depth of the study done by the researchers. It also includes the
synthesis of ideas, theoretical and conceptual framework to explain further the
importance and novelty of the study for better comprehension.
A. Related Literature
B. Related Studies
C. Synthesis
Chapter III
RESEARCH METHODOLOGY
A. Research Design
For the purpose of this study, experimental design was used. The
research design will ensure accuracy, stability and precision of the data to be
studied and so to organize the framework of variabilities. The results of an
experimental design can be statistically analyzed and so changes, replication
and validation can be easily implemented. Experimental design is used
because it is best served to answer the questions and purposes of the study.
B. Materials/ Ingredients/ Equipment/ Facilities
Terminalia Catappa Fruits (Matured) Buchner Funnel
Stainles Tray Automated Self-Cleaning Vacuum
Pump
Hammer Vacuum Flask
Transparent Bag Ethyl Alcohol
Beaker Filter Paper
Rotary Evaporator Ultralow Temperature Freezer
Vacuum pump Albino Rats
Recirculating chiller Reagent Bottle

Stirring rod Morphine Sulfate
Diclofenac sodium Gloves
C. Research Procedure
COLLECTION OF FACILITIES AND EQUIPMENT
The solvent (Ethyl Alcohol) is collected at Savemore, Roxas, Isabela while
the hammer, stainless tray, and transparent bags were all collected across different
stores in Roxas, Isablea. Gloves, morphine sulfate and diclofenac sodium are
collected in Mercury Drug Store in Roxas, Isabela. Filtration and separation
equipment were barrowed from Roxas National High School-Senior High School,
Laboratory.
COLLECTION OF PLANT MATERIAL
One (1) kilogram of brown and mature T. Catappa are harvested from T.
Catappa trees in Roxas, Isabela and Gamu, Isabela. The harvested fruits are
stored in a transparent plastic bag to get ready for the next phase.
COLLECTION OF TESTING ANIMALS
Six (6) female albino rats are collected at the pet shop in Roxas, Isabela.
The size, weight, and age of the rats are close to each other.
PREPARATION FOR EXTRACTION

The collected T. Catappa fruits are cleaned and husked with the help of a
hammer. The husks are obtained and placed on a stainless tray. Dried under direct
sunlight for three days, six hours per day.
EXTRACT PRODUCTION
The dried husks are fully-submerged in 1000 ml of ethyl alcohol (70% ).
The mixture is stored in reagent bottles and kept inside an ultralow temperature
freezer (-2℃ ¿ for 24 hours. After 24 hours, the solid particles are removed
through the process of filtration using vacuum pump, Buchner funnel, filter paper
and a vacuum flask. This will let the ethanol and husk extract to remain.
After filtration is separating the ethanol from the solution, leaving only the
essential oil of the husk to remain. Using a rotary evaporator and vacuum flask
because the ethanol and the extract have different boiling points. This process will
remove all of the ethanol from the extract.
EXTRACT WINTERIZATION
This process is just repeating the addition of ethanol up to the separation
method. Winterization allows a purer extract by removing fats, plant lipids and
waxes are removed from the extract. After this process, the extract is ready for
tests and analyses.
D. Test and Analysis

TEST FOR PHYTOCHEMICAL PROPERTIES
The phytochemical testing of the Ethanol T. Catappa Extract is
forwarded to the Isabela State University, Cauayan campus’ laboratory as
requested through a letter. The phytochemical testing methods and parameters
are followed.
TEST FOR TOXICITY
The toxicity of the extract is tested through exposure test. The
researchers administered the extract to the rats orally and observed any
abnormal behaviors. The researchers used three (3) female albino rats for they
are easier to handle and are generally less expensive than males. The rats were
allowed food and water ad libitum and all animals were also observed for
allergic symptoms, and mortality for the next 72 hours.
TEST FOR ATINOCICEPTIC ACTIVITY
Hot plate and tail immersion tests are conducted in Isabela State
University, Cauayan Campus as requested by the researchers through a letter.
The next antinociceptive activity test is the acetic acid-induced writhing test
where, three (3) albino rats were treated with T. Catappa extract and then the
writhing was induced with injection of 0.6% acetic acid 15 min after drug or
30 min after T. Catappa extract administration. Five minutes after acetic acid
administration, the mice were observed and the writhing number was counted
for 30 min as described previously. The incidences of contractions of the

abdomen, elongation of the body, twisting of the trunk, and/or pelvis ending
were considered as complete writhing.
Next, 10 μM of glutamate was injected into the ventral surface of the
right hind paw of the rats 30 min after EEAP treatment or 15 min after
injection of diclofenac sodium. The animals were then observed for 15 min
following glutamate injection and the licking number of its injected paw was
recorded as an indication of nociception. Finally, 60 min after T. Catappa
extract or 15 min after treatment with morphine, 20 μL of 2.5% formalin
solution was injected into the subplantar region of the right hind paw of the
rats. The licking or biting of the injected paw was then recorded from 0–5 min
as neurogenic phase and 15–30 min for inflammatory phase.

E. Research Flowchart
Collection of
Collection of Plant Collection of
Materials and
Material Testing Animals
Equipment
Cleaning, Husking,
and dryiing of plant
material
Dried Husks
Extracting
Procedures
Soaking Filtration Separation Winterization
Phytochemical Antinociceptic Toxicity Test

Analyses Activity Analyses
Hot Plate Test

Tail Immersion
Test
Acetic acid-induced nociception
Glutamate Induced nociception
Formalin Induced nociception

Chapter IV
RESULTS AND DISCUSSIONS

Chapter V
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
A. SUMMARY
B. COMCLUSIONS
C. RECOMMENDATIONS

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Republic of the Philippines

ANTINOCICEPTIC ACTIVITY OF ETHYL/CRUDE OIL INDIAN ALMOND

(TERMINALIA CATAPPA ) EXTRACT TO ALBINO RATS

Presented to the Teachers of

Of the Subject Requirement for

PIS-ING, AISHERA JAPHRODITE

CONSOLACION, JOHN ISRAEL

PALAGANAS, JOHN MARK

A. OVERVIEW OR BACKGROUND OF THE STUDY

Terminalia catappa Linn. is known for its nutritional fruit and possesses

medicinal benefits as well. T. catappa has been recognized for its medicinally

essential phytoconstituents, such as phenol, flavonoid, and carotenoid. Numerous

pharmacological investigations have confirmed this plant's ability to exhibit

antimicrobial, anti-inflammatory, antidiabetic, antioxidant, hepatoprotective, and

Terminalia catappa fruit is rich in phytochemicals and nutrients which

Terminalia Catappa fruit is rich in phytochemicals and nutrients which

behavior with different chemicals, fluorescence as well as percent yield.(Krishaveni,

purposes. (A. Anand, N. Divya, P. Kotti, 2015)

T. Catappa’s anti-inflammatory and analgesic properties may lead to a more

complex antinociceptive activity to expand the medicinal and pharmaceutical uses of

this plant for the most effective health care purpose.

pathogenesis of various ailments. Medicinal plants are used worldwide as a remedy

for various inflammatory disorders. The various polyphenolic compounds,

for the anti-inflammatory activities. (A. Anand, N. Divya, P. Kotti, 2015)

The undesired effects of the analgesic compounds available in the market

2010, Jothimanivannan, 2010, Mills and Bone, 2002)

Terminalla catappa leaves posseses analgesic activity which may mediated

of a naturally occurring anesthesia on which creates a new medical breakthrough.

Pain is an unpleasant sensory or emotional experience associated with actual

or potential tissue damage, or described in terms of such damage. The Nociception

involves the activation of specific primary sensory neuron subpopulations that

release of several mediators, including excitatory amino acids, peptides, protons,

pain can be a subject to multiple levels of biochemical and pharmacological controls,

The management of pain using currently available analgesics could not

The sensitivity of nociceptive neuron is adjusted by a large variety of mediators in the

extracellular space. These mediators, either neurotransmitters or neuromodulators,

activate a large number of receptor classes, which in turn activate a plethora of

Thus, the antinociceptive potential of T. catappa provide related studies that

suggest the possibility of developing an anesthetic substance which is sustainable and

conventional, easier to obtain than synthetic prescription medicines, provides natural

antinociception, and has fewer side effects.

These evidences urged the researchers to study the antinociceptic activity of T.

catappa extract using various nociceptive assays.

INDEPENDENT VARIABLE DEPENDENT VARIABLE

Acute Toxicity Test Tested the Acute Toxicity of T.

Antinociceptive Analysis Analyzed antinociceptive properties

Saponin Test Tested presence of saponin

Flavonoid test Tested flavonoid content

Tannin and phenolic compound test Tested presence of tannin and

Alkaloid testing Tested presence of alkaloid

Sterol and Steroid Testing Tested presence of sterols and

Phytonutrient analysis Estimated Phytonutrients

C. RESEARCH OBJECTIVES/ QUESTIONS

In testing the antinociceptic activity of ethyll/crude oil extract, the

questions to be answered are the following:

 To analyze the phytochemical properties of Ethanol T. Catappa Extract

Includes; saponin, flavonoid, tannin and phenolic compound, alkaloid,

sterol and steroid tests.