Speaker Eugenio Perez National Agricultural School

SPEAKER EUGENIO PEREZ NATIONAL AGRICULTURAL SCHOOL
Roxas Boulevard, San Carlos City, Pangasinan 2420 Philippines

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Chapter 1
INTRODUCTION
Rationale
Since ancient times, man used traditional medicines utilizing what are found in
their surroundings for curing different diseases. But as years pass, man significantly
shifted dependence on traditional and natural medicines to commercially manufactured
chemical-containing medicines at high consumed rates (Macaraeg, 2015).
Moreover, these commercial medicines were expensive, hindering access to
effectice sure, let alone the risk of adverse effects.
A wound is an injury, usually involving division of tissue or rupture of the
integument or mucous membrane, due to external violence or some mechanical agency
rather than disease (http://www.dictionary.com/browse/wound).
Healing is an interaction of a complex cascade of cellular events that generates
resurfacing, reconstitution and restoration of tensile strength of injured skin. Wound
healing is a natural restorative response to tissue injury.
In day-to-day living, it becomes almost inevitable that people sustain wound by
accident and then shrug it off if it seems not serious or life-threatening, thinking that the
body’s natural ability to repair itself and protect itself from infection will promote healing
in no time. Instead, the wound won’t heal and it can lead to more serious, complicated,
and severe one due to infection. Infection caused by bacteria which may later become
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worse and may cause Sepsis and worst, death. Antibiotics are expensive, there are people
who receives minimum wage that can’t afford it.
So, the researcher decided to study Balikutkut (Heliotropium indicum) as anti-
bacterial agent especially for external wound healing purposes. The researcher tried to
create and develop a natural or indigenous source of treatment that can be used as natural
medicine to minimize the cost of treating external wounds (Macaraeg, 2015). This
proposed treatment has, according to available literature, chemical property in the form of
pyrrolizidine alkaloids that may cure wounds.
This paper emphasizes the study of Balikutkut scientifically known as
Heliotropium indicum (Family Boraginaceae), a plant that is popularly known as stinker,
which grows especially in Brazilian northern and northeast region. In the Philippines, this
can be found in backyards or even in the fields as weed. It is a sub shrub up that grows to
1 meter high, widely distributed in Brazilian states. Its leaves are displayed alternate,
opposite in the same individual, penduculate inflorescences white to purple corolla,
mitriforme fruit and ellipsoid pale and smooth seed (Rheuters, 2015).
The researcher chose this study to minimize the increasing number of people who
are infected of various diseases caused by a wound and to give a full understanding about
the Balikutkut (Heliotropium indicum).

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Conceptual Framework
The researcher presumed that the Balikutkut may be among the plants that have
anti-bacterial. Hence, health benefits from the isolated leaf extract served as the basis of
this idea.
As shown in Figure 1, the samples of Balikutkut leaves were brought to the
University of the Philippines-Diliman campus for the botanical identification and
authentication to determine the specific species of the plant sample.
After isolating the leaf extract from the Balikutkut leaves, it underwent processing
methods: evaporation – preparation for the test of alkaloids and tannins. The presence of
Alkaloid and Tannins supported the possibility of Balikutkut leaves wound healing
activity.
In the process, the different concentrations of the extract, namely 75% and 95%
were prepared and subjected to further phytochemical screening and wound healing
activity tests. To identify the wound healing activity of the Balikutkut leaf extract, the
two concentrations of the extract were applied in cream form to the laboratory rats.
In the output, the phytochemical constituents as well as wound healing activity of
the leaf extract were determined.

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Conceptual Paradigm
INPUT PROCESS OUTPUT
 Sample plant  Identification of

identification active constituents:
 Collection of leaves
 Preparation of o Alkaloids
Balikutkut leaves for o Tannins
Phytochemical
Screening  Identification of
 Phytochemical wound-healing effect
Screening
Balikutkut of Balikutkut leaf
o Test for
(Heliotropium Alkaloids extract
indicum) leaves o Test for  Identifying the
Tannins Balikutkut leaf
 Preparation of extract concentration
Balikutkut leaf extract with comparable
in cream form with wound-healing effect
these concentrations to positive control
o 75%
o 95%
 Negative control
 Positive control
 Application of
treatments to Rattus
albus rats
 Data Analysis
o One-way
ANOVA
o Tukey’s HSD
Figure1. Research paradigm of the study , showing the entire framework of procedures
and methods within which the study takes place.

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Statement of the Problem
The main objective of the study was to determine the wound-healing effect
property of Balikutkut (Heliotropium indicum) leaf extract.
Specifically, it sought to answer the following:
1. What are the phytochemical constituents present in Balikutkut (Heliotropium
indicum) leaf extract?
2. What is the wound healing effect of Balikutkut leaf extract in ointment form in
terms of time?
3. Is there significant difference in the wound healing activity of Balikutkut leaf
extract?
4. What leaf extract concentration exhibits greatest effect of wound healing activity
base on zone of inhibition?
Null Hypothesis
The following null hypothesis was tested at the 0.05 level of significance:
There is no significant difference in the wound healing activity of Balikutkut leaf
extract.
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Scope and Limitation of the Study
The study investigated the wound healing effect of Balikutkut (Heliotropium
indicum) ethanolic leaf extract on Albino rats (Rattus albus). The study used 12 Albino
rats which were bought in Virgen Milagrosa University Foundation – College of
Pharmacy, located in Martin P. Posadas Avenue, San Carlos City, Pangasinan.
One hundred grams of fresh leaves of Balikutkut was collected from Barangay
Mamarlao and Bacnar, San Carlos City, Pangasinan.
The study did not consider parameters like heterogeneity of the rats (e.g., sex,
color, blood typing). The researchers limited this study on determining any
phytochemical properties of the ethanolic leaf extracts of Balikutkut as well as its the
wound healing effect without isolating any bacterial specimen or testing its antibiotic
properties.
Significance of the Study
Since people face day to day wear and tear including accidental cuts and bruises
on the skin as a result of physical activity, accidents in our daily lives, people tend ignore
these not until major infection has set in. A few others would likely apply topical
treatments in an attempt to prevent infection and promote faster healing. Thus, a
scientific search for an effective and low-cost remedy for such cases is a must. This study
provides new insights over the assumption that a safe and effective medication for
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wounds is important. Thus, the findings on this study would be beneficial for the
following:
Department of Health. The agency may gain important insights about new health
alternatives available in nature as regards natural remedies that can serve as a baseline of
further studies to discover more benefits from Balikutkut plant and aid in the
dissemination of information to the public.
Health Education. Individual alertness and knowledge with regard to minor or
severe wounds which may lead to infections and the health benefits of Balikutkut as a
wound healing
Health Care Practitioners. This research study will contribute to the current pool
of knowledge on the anti-bacterial activity of Balikutkut. Further, this study will help
health care providers to gain knowledge and practice needed in determining appropriate
and best response in curing external wounds.
Science Research Students. In line to the profession through discovery of new
drugs, we, the researchers motivate them to conduct more studies through the use of
herbal plants for a cheaper and effective source of medicine like Balikutkut leaf and to
discover more therapeutic actions of the said herbal plant.
Pharmaceutical Companies. This study can enhance the conduct of research on
the anti-bacterial effect of Balikutkut leaves. Further, this will encourage them to develop
a drug from Balikutkut that may be beneficial to the community.

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Future Researchers. This research could also enhance their knowledge in
connection with their courses as well as in the continuity of this study. Further, this study
may initiate the practical application of theories, concepts and principles of research
process that they have learned in the classroom.

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Chapter 2
METHODOLOGY
This part of the study presents the research methods and data gathering
procedures that was used by the researcher for acquiring the needed data. It includes
discussion regarding the sources of information, data collection and research instruments
that is used for the study. This chapter also deals with formulation of the research design
that would serve as the groundwork of the study.
Research Design
The study used a descriptive experimental research design as it deals with the
observation of an experimental research wound healing effect of the Balikutkut
(Heliotropium indicum) ointment applied on albino rats (Rattus Albus). Descriptive
research uses systematic observation to describe a specific behavior. It creates very
detailed records and observed many subject. In experimental research, one variable is
intentionally manipulated by the researchers. The participants are put into groups through
a random assignment.
The objectives of the study and the review of pertinent literature were the primary
bases of the research methodology. The researcher applied a systematic and scientific
approach in dealing with data and information. The data and information gathered from
the research were used for the attainment of the objectives and solutions to the problems
engaged in the study.

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Duration and Locale of the Study
The study was conducted at the laboratories of the College of Pharmacy building
of Virgen Milagrosa University Foundation (VMUF), San Carlos City, Pangasinan
because of the availability of good facilities and services which helped the researchers
attain objective of determining the wound healing effect of Balikutkut leaves. Direct
information on achieving the objective of the study was provided to the researchers from
the procedures they themselves conducted. The places where other requisite processes of
the study were as follows:
The dried Balikutkut (Heliotropium indicum) sample plant species were submitted
for authentication at the University of the Philippines, Diliman, Quezon City through the
help of Dean Sheila De Vera Miranda, of Virgen Milagrosa University Foundation-
College of Pharmacy.
The researchers conducted the phytochemical analysis and the preparation of the
Balikutkut (Heliotropium indicum) leaf extract and ointment at the College of Pharmacy
with the help of technical assistance laboratory technicians of the said College.
Actual experimentation involving incising wounds on laboratory rats to test the
wound healing effect of Balikutkut leaves extract in ointment form was conducted at the
Pathology-Microbiology laboratory of the College of Medicine, Virgin Milagrosa
University Foundation, San Carlos City, Pangasinan.

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Instrumentation and Data Collection
In this research, albino rats were used as study subjects to test if Balikutkut
(Heliotropium indicum) leaves can indeed heal wounds. In this laboratory experimental
research, 12 albino rats were randomly assigned for the control and experimental groups.
These was three replication (rats) per group: positive control group with Betadine® as
control treatment, non-intervention as negative control; and Balikutkut leaf extracts at
75% and 95% concentration as experimental treatments. The 12 albino rats came from
the same litter that minimized it not eliminated, potential variations and bias.
Material and Procedures
The research focused on the wound healing effect of Balikutkut (Heliotropium
indicum) ethanolic leaf extract. Fresh leaves were collected from Barangay Mamarlao
and Bacnar, San Carlos City, Pangasinan. Twelve laboratory albino rats were used as
laboratory animal subjects of this study.
Phytochemical Screening
One hundred grams of fresh leaves was used in phytochemical screening as well
as in the preparation of ointment for wound healing. In performing the phytochemical
analysis, screening of alkaloids was the first method. These test involved several
processes.
Several millilitres of 1% HCl was mixed with cooled 70 ml of Balikutkut leaf
extract.
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Screening for Alkaloids
Procedures:
The evaporated seventy (70) ml of balikutkut leaf extract must be cooled and
poured the seven (7) ml of one (1) % hydro chloric acid. The evaporated leaf extract with
one (1) % hydro chloric acid was stir; it was put on the electric hot plate where it was
filtered. Another seven (7) ml of 1 % HCL was put on small plate where it was performed
in stirred type. The second batch of stirred leaf extract was filtered and was divided into
four. On the four test tubes containing leaf extract three drops of the reagents was added,
these reagents are Mayer, Valler, Wagner and Bouchardat test. The four test tubes was
then put in the centrifuge machine for few minutes.
Screening for Unsaturated Sterols and Triterpenes
The 30 ml of the ethanolic leaf extract was evaporated to dryness on a water bath.
The residue was cooled and 15 ml of light petroleum ether was added. The procedure was
mixed and filtered. The additional volume of petroleum ether was repeated as needed
until the last volume of petroleum was colorless. He ethereal filtrates were combined and
the defatted residues were set aside for screening for flavonoids and leucoanthocyanins.
The chloroformic solution were dried over anhydrous sodium sulphate and were filtered
and divided equally into 3 dry test tubes.

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Lieberman-Buschard test
Seven ml of the filtrate was poured in a suitably dry test tube, then added 0.3 ml
of acitic anhydride and were mixed gently. One drop of concentrated sulphuric acid was
added. The color changes were immediately observed every five minutes then after over
sixty minutes. The test was concurrently with 5 ml portion on standard solution prepared
from plants known to contain unsaturated sterols or triterpenes.
Salkowski test
The 5 ml of the filtrate was transferred to a dry test tube. The ring test with
concentrated sulphuric acid was performed and was shook after 1-2 minutes and then
color changes were observed. A cherry red color is indicative of the presence of
unsaturated sterols. The same test was also conducted with the standard solution.
Screening for Flavonoids
Defatted residue from a steam bath in 30 ml was dissolved in 50% ethanol. Two
ml of the filtrate were then filtered and placed on three test tubes. On test tube number 1,
0.5 ml of concentrated hydrochloric acid was added and put in a steam bath for about five
minutes and the color changes was observed. The test solutions were allowed to stand at
room temperature for one hour before recording the result. On test tube number 2, 0.5 ml
of concentrated HCl and 3-4 magnesium ruminas was added. A careful observation of the
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color changes was done. Definite color is formed within ten minutes. It was cooled with
an equal volume of water. A 1.0 ml of octyl alcohol was added.
Screening for Steroid (Cardio Active) Glycosides
Presence of unsaturated steroil (Liebermann-Burchard test)
The result of the preceding test was used as basis for this test.
Presence of unsaturated lactones
On 5 ml of the ethanolic leaf extract in an evaporating dish, 5 ml of Keede reagent
was added and mixed well with a glass stirring rod. To the mixture, 2 ml of 1N sodium
hydroxide was added. It was mixed and observed for color development.
Presence of 2- dioxysugars (Ketter Killiani test)
A 10 ml of the ethanolic extract in an evaporating dish was dry on the steam bath.
A 3 ml of the ferric chloride reagent was added and stirred and mixed well and was
transferred in a small test tube. The test tube was held at 45° angle layer, 1 ml of
concentrated sulphuric acid allowing it to get down the inside wall of the test tube, at this
point, the shaking of the test tube was avoided.
A volume of the ethanolic extract was taken. For control, 2 ml of 10% Balkutkut
leaf extract was used in a separate test tube. A 10 ml of distilled water was added to each
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test tube. A stopper was put and the test tubes were shaked vigorously for 30 seconds. It
was allowed to stand and was observed for 30 minutes.
Screening for Tannins and Phenolic Compound
A 100 ml ethanolic extract was evaporated to dryness and on steam bath.
Evaporating dish from the steam bath was removed and 25 ml of hot distilled water was
added to residue. It was mixed with a stirring rod and allowed to cool at a room
temperature spontaneously. The cooled extract was centrifuged for several minutes and
the upper half from each tube used has decanted. 3-4 drops of 10% sodium chloride
solution was added to the decantated supernatant. Precipitation at this point is indicative
of salting-out reaction probably due to non-tannin component. Any precipitate was
filtered and 3 ml of filtrated was added to each of three test tubes. On tube number 1, 3
drops of 1% gelatin solution was added. On tube number 2, same amount of gelatin salt
reagent was added. And on tube number 3, several drops of ferric chloride TS was added.
Screening for Anthraquinone Heterosides
Borntrager Test
A 5 ml of ethanolic leaf extract was transferred to an evaporating dish and dry
over a steam bath. The residue in the dish with 5-10 ml of petroleum ether was defatted
and 50 ml of water was added to the defaulted residue. It was mixed and filtered into a
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small separator funnel. A 10 ml of benzene was added and shaked to mix well. The two
phases were allowed to separate. The aqueous layer (Bottom layer) was drain out and the
benzene phase (Upper layer) was transferred to a test tube. 5 ml of ammonia was mixed
and the benzene layer for the changing of color was observed.
Modified Borntrager Test
A 0.3 g rams of the Balikutkut leaf extract was place in a test tube. It was moisten
with water and a few drops of chloroform was added to enhance enzyme activity for a
firm stopper on the tube, cork was used from which was suspended a piece of picrate
paper. The tube was warm at 35-40° Celsius at room temperature for 3 hours.
Screening for Cynogenic Glycosides
Gulgnard Test
Two and a half grams of the crushed plant sample was placed in a test tube. Then
moistened with water and added with a few drops of chloroform to enhance enzyme
activity for a firm stopper on the tube. A cork was used in which was suspended a piece
of picrate paper. The researcher ensured that the paper strip not touch the inner side of the
test tube. The tube was then warmed between 35-40 degrees Celsius (kept at room
temperature for three hours.

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Preparation of Balikutkut Leaf Extract in Cream Form
Compounding Cream Base Procedure
The formula for making the cream base required the following ingredients and
amounts:
Ingredients Original Amount
Spermaceti 125 g
White wax 120 g
Mineral oil 560 g
Sodium base 5g
Purified water 190 ml
The cream base was prepared using the above formula and the following
procedure:
The spermaceti and white wax were cut into small pieces. Then, the spermaceti
and white wax were melted on the steam bath with the mineral oil and heating is
continued until the temperature of the mixture is 70⁰C. The experimental extract is then
mixed thoroughly with the cream base and applied sparingly on the induced superficial
wounds in the rats assigned in the treatment groups.
Once the cream form is ready, the experimentation procedures followed.

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Experiment Procedures
The albino rats were randomly assigned into the control experiment groups: three
rats for each of the negative control group, positive control group, experimental group
with BLEE at 75% and at 95% concentration. All in all, 12 albino rats were used in the
study, with three replicates for each group.
All rats were incised with wounds approximately 2 inches after being shaved of
its hair on the dorsolateral part of the left lumbar region, making sure that the wound did
not reach the hypodermal layer of the skin.
The test animals in the control group were left untreated of their wound during the
entire observation period. The test animals in the positive control group were treated with
Povidone Iodine, and those assigned in the experimental groups were treated with the two
experimental concentrations of BLEE in cream form.
Observations on the wounds for any healing were done on a daily basis every
morning. The rate of wound healing were graded as follows:
+++ - Completely healed
++ - moderately healed
+ - slightly healed
- - no reaction/healing effect yet

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Data Analysis
This study employed a descriptive documentary type statistics. The researchers
used descriptive statistics to provide summaries about the samples, particularly mean
healing effect. The descriptive ratings using plus and minus symbols were converted to
numeric values 0 (for no healing effect) up to 3 (complete healing) to enable statistical
analysis. To statistically compare the wound healing effects of the experimental leaf
extracts with those of the controls, one-way ANOVA was used. If statistically significant
differences were found to exist, Tukey’s HSD was conducted to determine what specific
treatment or treatments caused the statistical significance.
The formula for one way ANOVA is:
MSSb
F = -------------
MSSw
Where: F = ANOVA statistic
MSSb = mean sum of squares between
MSSw = mean sum of squares within
A statistical software was used throughout the study in the analyses of all
gathered data.
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Chapter 3
RESULTS AND DISCUSSION
This chapter presents the various results from the overall preliminary,
phytochemical screening and the confirmation of the wound healing activity of the
Balikutkut (Heliotropium indicum) leaves in cream form in Albino rats (Rattus albus).
What are the phytochemical constituents present in Heliotropium indicum
(Balikutkut) leaf extract?
I. Phytochemical Screening
Table I presents the result of the phytochemical screening for determining the
presence of chemical constituents.
The findings revealed that there are chemical constituents present in the ethanolic
extracts, namely alkaloids, tannins and phenolic compounds, deoxy sugars and cardiac
glycosides, unsaturated sterols and flavonols. Meanwhile, the extract does not contain
saponins, anthraquinone heterosides and cyanogenic glycosides, since there were no
precipitates obtained upon the addition of any of the said active constituents. The
confirmatory tests revealed that the leaf extract is composed of different chemical
constituents that indicate many potential health-related benefits. This implies that aside
from the known therapeutic activity of Balikutkut, this plant has the potential of being a
wound healing agent as manifested by the presence of different chemical constituents.

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Table 1. Phytochemical Screening Results of Balikutkut Ethanolic Leaf Extract (BLEE)
Qualitative Test Positive Result Actual result Remark

A. Screening for
Alkaloids
1. Mayer’s Reagent Production of ppt. Production of ppt. Positive
2. Wagner’s Reagent Production of ppt. Production of ppt. Positive
3. Bouchardat’s Reagent Production of ppt. Production of ppt. Positive
4. Valser’s Reagent Production of ppt. Production of ppt. Positive
B. Screening of
Unsaturated Sterols,
and Triterpenes
1. Lieberman’s Burchard Color change/ blue or Green color solution Positive
Test green sol.
2. Salkowski Test Color change/ cherry Light green color Negative
red solution solution
C. Screening for
Flavonoids
1. Bate-Smith Matcalf Red violet color Light green color Negative
Test
2. Cyanidin Test Color change/green Green solution Positive
red
D. Screening for Steroid
(Cardio active
glycosides)
1. Kedde Reaction Purple clor No development of Negative
purple color
2. Keller- Killiani Test Purple ring Development of Positive
purple ring color
E. Screening for Saponins
1. Froth Test Formation of froth and No formation of Negative
Foam froth and foam
F. Screening for Tannin
and Phenolic
Compound
1. Gelatin Test Production of ppt. Production of ppt. Positive
2. Gelatin Black Test Production of ppt. Production of ppt. Positive
3. Ferric Chloride Test Greenish Greenish black color Positive
Blue/Greenish Black solution
color
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G. Screening for
Anthraquinone
Heterosides
1. Borntrager Test Color change/cherry Colorless solution Negative
red or pink sol.
2. Modified Borntrager Color change/cherry Colorless solution Negative
Test red or pink sol.
H. Sceening for
Cyanogenic
Glycosides
1. Gulgnard Test Appearance of various No appearance of Negative
shades of Red with 15 various shades of
minutes Red within 15
minutes
As the table shows, the plant extract possesses alkaloids, tannins, and unsaturated
sterols, and triterpenes. These observations are attributed to the presence of metabolites
like alkaloids and tannins whose anti-bacterial activity has been previously attributed
(Osungumma, Adedeji, 2011). Additionally, balikutkut leaf extract does not contain
saponins, heterosides and cyanogenic glycosides.
Table 2 presents the wound healing effects of the different treatments employed in
this investigation.
Table 2. Effect of Balikutkut (Heliotropium indicum) leaf Extracts, positive and

Negative Controls on the Wounds Induced on Skin of Albino Rats (Rattus Albus)
Treatment Wound Healing Effect (Day Wound Healing Effect on
1 to Day 3) Day 4 to Day 7)
Experimental Group (75%
Balikutkut Leaf Extract +++ +++
concentration
Experimental Group (95%
Balikutkut leaf Extract +++ +++
Concentration
Positive Control (Betadine) +++ +++
Negative Control - +
Legend: +++ - completely healed + - slightly healed
++ - moderately healed - - np reaction/effect yet
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The table shows that the mean wound healing in albino rats (Rattus albus) treated
with Balikutkut leaf extracts at 75% concentrations was 3+, indicating complete healing
as early as the first three days, and such effect persisted consistently from the fourth until
the last day of observation. Likewise, the same observation was recorded on all seven
days of observation on superficial wounds in albino rats treated with 95% Balikutkut leaf
extract concentration.
As expected, Povidone Iodine proved effective in healing epithelial wounds in
albino rats, with the healing effect described as completely healed. Thus, the wound
healing effects of the two leaf extract concentrations of Balikutkut seemingly simulated
the effect of using Povidone iodine being the gold standard in treating superficial
wounds.
On the other hand, the wounds induced on the skins of albino rats randomly
assigned in the negative control (no treatment) group demonstrated spontaneous wound
healing on the fourth day with only a slight degree of healing on the seventh and last day
of observation. This spontaneous wound healing on the negative control rats is
attributable to the physiologic ability of the animal skin in regenerating new skin cells
replacing dead cells and tissues.
The positive control group, 75% and 95% concentrations of Balikutkut leaves
were compared to determine any difference in the effectiveness of the treatments
conducted with those of the negative and positive controls. One-way ANOVA was used
to determine any significant difference.

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Table 3 presents the one way Analysis of Variance comparing the wound healing
effect of Balikutkut leaf extracts at 75% and 95% concentrations with those of the
negative and positive controls.
The Balikutkut leaf extract with concentrations of 75% and 95% were compared
to the negative control group and statistical significance was found (F 3,4 = 25.000, p<.05).
There is significant difference in the wound healing effect of Balikutkut leaf extract in the
form of ointment on superficial wounds induced in Albino rats. The wound induced on
the positive control was completely healed by the Balikutkut leaf extracts at 75% and
95% concentration in the same way as how Betadine® completely healed the wounds
treated with it on the third day of the said observation on the wounds induced in the three
(3) rats completely healed.
Table 3. ANOVA Table on Wound Healing Activity of Balikutkut Leaf Ethanolic Extract
(BLEE)
Sum of Squares df Mean Square F Sig.

Between Groups 9.375 3 3.125s 25.000 .005
Within Groups .500 4 .125
According to a study (2017), the wound healing contracting ability of ethanolic
leaf extract of Heliotropium indicum in different concentration on excision wound model
was significantly greater than the control group.
Tukeys’ HSD analysis (Table 3) reveals that based on pairwise comparison, the
induced wound on the albino rats in the negative control group showed significant
difference in terms of wound healing when compared against the Betadine® (positive
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control) group of animal subjects and those in the experimental group of rats treated with
75% and 95% concentrations of Balikutkut leaf extracts.
On the other hand, there is no significant difference between the Balikutkut leaf
extracts at 75% and 95% concentrations when compared against each other in terms of
their wound healing effect, and when compared with that of the positive control group
using Betadine® (p>.05), indicating that the wound healing effect of the experimental
leaf extract in the form of cream were comparable to the wound healing property of
Betadine® (Povidone Iodine).
To determine which treatment group caused the statistical significance, psot hoc
Tukey’s HSD analysis was conducted.
Pairwise comparison of the various treatments presented in Table 4a reveal that
negative control consistently differed statistically with those of the positive control group
(Povidone Iodine) and with the two Balikutkut leaf extract cream application in terms of
their wound healing effects (p<.05).
However, 75% Balikutkut leaf extract in cream form did not significantly differ
with that of the positive control (Povidone Iodine) and with the 95% Balikutkut leaf
extract in cream form (p>.05). Considering the efficacy of Povidone Iodine in wound
healing, this means to say that the two concentrations of Balikutkut leaf extracts were
comparable to Povidone iodine in their efficacy in promoting wound healing. Harmonic
mean values (Table 4b) further amplify this consistent wound healing effects of the
various treatment applications.

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Table 4a. Tukey’s HSD post hoc analysis on the wound healing effect of Balikutkut leaf
extract
(I) Treatment (J) Treatment Mean Std. Sig. 95% Confidence

Difference Error Interval
(I-J) Lower Upper
Bound Bound
Positive
-2.50000* .35355 .007 -3.9393 -1.0607
control
Negative Experimental
-2.50000* .35355 .007 -3.9393 -1.0607
control 75%
Experimental
-2.50000* .35355 .007 -3.9393 -1.0607
95%
Negative
2.50000* .35355 .007 1.0607 3.9393
control
Positive Experimental
.00000 .35355 1.000 -1.4393 1.4393
control 75%
Experimental
.00000 .35355 1.000 -1.4393 1.4393
95%
Negative
2.50000* .35355 .007 1.0607 3.9393
control
Experimental Positive
.00000 .35355 1.000 -1.4393 1.4393
75% control
Experimental
.00000 .35355 1.000 -1.4393 1.4393
95%
Negative
2.50000* .35355 .007 1.0607 3.9393
control
Experimental Positive
.00000 .35355 1.000 -1.4393 1.4393
95% control
Experimental
.00000 .35355 1.000 -1.4393 1.4393
75%
*. The mean difference is significant at the 0.05 level.
Harmonic mean analysis reveals that indeed, the two experimental preparations of
Balikutkut leaf extracts were comparable to the wound healing effect of the positive
control treatment (Betadine).

27
Table 4b. Harmonic mean comparison of wound healing effects of Control and
Experimental Treatments
Treatment N Subset for alpha = 0.05

1 2
Negative control 2 .5000
Positive control 2 3.0000
Experimental 75% 2 3.0000
Experimental 95% 2 3.0000
Sig. 1.000 1.000
Means for groups in homogeneous subsets are displayed.
a. Uses Harmonic Mean Sample Size = 2.000.
28
Chapter 4
SUMMARY, CONCLUSIONS AND RECOMMENDATIONS
This chapter presents the summary of the study, the conclusions inferred and the
corresponding recommendations advocated.
Summary
This study focused on the determination of the wound healing activity of the leaf
extract of Balikutkut (Heliotropium indicum, Fam. Boraginaceae). The plant was gathered
from the barangays of Mamarlao and Bacnar, San Carlos City, Pangasinan and was
utilized in this study. It underwent plant authentication in the University of the
Philippines. The leaf extracts of Balikutkut was subjected to extraction, phytochemical
analysis and conversion of leaf extract into ointment at the Virgen Milagrosa University
Foundation-College of Pharmacy Instrumentation Laboratory.
The results of the phytochemical analysis and wound healing activity was
accurately observed, recorded and compared. The wound healing activity on induced
wounds in albino rats (Rattus albus) of the two concentrations (75% and 95%) of the
Balikutkut, negative control and one positive control group (Betadine) was compared
and subjected to statistical treatment in which one-way ANOVA was used for the
comparison of results.
29
Findings
The investigation showed that Balikutkut leaf extracts contain alkaloids, tannins
and phenolic compounds, deoxy sugars and cardiac glycocides, unsaturated sterols and
flavonols which are constituents that have the ability to heal external wounds.
Furthermore, the 75% and 95% concentration of Balikutkut leaf extracts
demonstrated wound healing activity comparable to that of the effect of Betadine
treatment in the test animals, based the actual wound healing observations and on the
ANOVA and Tukey’s statistical analyses conducted (p<.05). Meanwhile, the wound
healing effect of both 75% and 95% extract concentrations of Balikutkut and the positive
control were significantly different with that of the negative control group (p>.05).
Conclusion
Based from the experiment done and to the obtained results, the researchers
concluded that the leaf extract from Balikutkut (Heliotropium indicum) leaves has the
ability to heal external wounds that is comparable to that of Betadine.
Recommendations
Based on the conclusions, the following recommendations are hereby suggested:
1. That Balikutkut (Heliotropium indicum) leaf extract be further subjected to more
laboratory test as to phytochemical analysis to determine the phytochemical
constituents that are present

30
2. To evaluate the wound healing activity of Balikutkut leaf extracts in different set
of test animals aside from the Albino rats (Rattus albus).
3. Determination of what is the ideal concentration of Balikutkut leaf extract to be
used in experiment that is capable of exhibiting optimal wound healing effect.
4. Replication of this study is advocated to validate its findngs.

31
ACKNOWLEDGMENT
The success of this study will not be attained without those people who directed
and guided us, and so we, the researchers, offer this part to recognize them and to say
how much we appreciated what they have done for the research.
To Mrs. Carmelita Magleo and Mrs. Evelyn Mendoza, Senior High
Coordinators of Speaker Eugenio Perez National Agricultural School, with their
paramount motivations, approval to conduct this research and love for every researcher
and for sharing their expertise and ascertained their suggestions for the improvement of
this research;
To Mr. Raymundo B. Ambanloc, School Principal IV of Speaker Eugenio Perez
National Agricultural School, for the logistical and moral support to finish the research
with scientific endeavour;
To Mr. Alison Gutierrez, our research adviser who diligently equipped our
knowledge and passions he needs as an outstanding researcher.
To Mr. Alvin C. Franza and Mr. Elmer D. Bernardino of Virgen Milagrosa
University Foundation who helped us perform the laboratory tests.
To our family especially our parents for the moral and financial support,
sacrifices and understanding, they kept on bestowing to us that enable us to pursue this
endeavour.
32
Above all, to our Almighty God, with His unconditional love and mercy, the
knowledge that He has given to us and His Divine Presence will still continue guiding us
in everything we do. The Glory and honour is raise unto You.

33
BIBLIOGRAPHY
Adelaja AA, A. M. (2008). Evaluation of the Histo-Gastroprotective and Antimicrobial Activities Of

Heliotropium indicum Linn (Boraginaceae). Malaysian J. Med. Sci.
Ashoka M, S. C. Stimulation of Immune Functtion Activity of the Extract of Heliotropium indicum

leaves. J. pharmacol.
Dash GK, M. P. (2011). Studies on Wound healing Activity of Heliotropium indicum. ISRN
Pharmacology.
Dupont S, C. N. (2006). In vitro antimacrobial activity of Australian herb Extracts against Food
Relted Bacteria.
Trease GE, E. W. (1989). A Textbook of Pharmacognosy. London: Baillere Tinal.

34
APPENDIX A
CERTIFICATION OF AUTHENTICATION
35
APPENDIX B
CERTIFICATION OF WOUND HEALING ACTIVITY RESULTS
36
37
38
A
PPENDIX C
CERTIFICATION OF PHYTOCHEMICAL SCREENING
39
40
41
APPENDIX D
PREPARATION OF THE EXTRACT
42
PREPARATION OF THE EXTRACT

43
APPENDIX E
PHYTOCHEMICAL ANALYSIS
Screening for Alkaloids
Screening for Tannins

44
APPENDIX F
INDUCING OF WOUND ON TEST ANIMALS

45
CURRICULUM VITAE
NAME: Kenneth B. Rosario
AGE: 17 years old
DATE OF BIRTH: January 30, 2000
PLACE OF BIRTH: Brgy. Bacnar San Carlos City,
Pangasinan
CIVIL STATUS: Single
CITIZENSHIP: Filipino
RELIGION: Roman Catholic
PARENTS:
Mr. Arnold P. Rosario
Mrs. Yolanda B. Rosario
EDUCATIONAL BACKGROUND:
ELEMENTARY: Bacnar Elementary School
SECONDARY: Speaker Eugenio Perez National Agricultural School

46
CURRICULUM VITAE
NAME: Airra Mae R. Frias
AGE: 17 years old
DATE OF BIRTH: February 29, 2000
PLACE OF BIRTH: Mamarlao San Carlos City,
Pangasinan
PARENTS:
Mr. Manuel D. Frias
Mrs. Gloria R. Frias
ELEMENTARY: Central 1 Elementary School

47
CURRICULUM VITAE
NAME: Jay Mark M. Domantay
AGE: 17 years old
DATE OF BIRTH: March 1, 2000
PLACE OF BIRTH:Bolingit, San Carlos City, Pangasinan
PARENTS:
Mr. Jimmy V. Domantay
Mrs. Marian M. Domantay
ELEMENTARY: Ano Elementary School

Speaker Eugenio Perez National Agricultural School

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Speaker Eugenio Perez National Agricultural School

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SPEAKER EUGENIO PEREZ NATIONAL AGRICULTURAL SCHOOL

Roxas Boulevard, San Carlos City, Pangasinan 2420 Philippines

shifted dependence on traditional and natural medicines to commercially manufactured

chemical-containing medicines at high consumed rates (Macaraeg, 2015).

Moreover, these commercial medicines were expensive, hindering access to

effectice sure, let alone the risk of adverse effects.

A wound is an injury, usually involving division of tissue or rupture of the

integument or mucous membrane, due to external violence or some mechanical agency

rather than disease (http://www.dictionary.com/browse/wound).

Healing is an interaction of a complex cascade of cellular events that generates

resurfacing, reconstitution and restoration of tensile strength of injured skin. Wound

healing is a natural restorative response to tissue injury.

In day-to-day living, it becomes almost inevitable that people sustain wound by

who receives minimum wage that can’t afford it.

So, the researcher decided to study Balikutkut (Heliotropium indicum) as anti-

pyrrolizidine alkaloids that may cure wounds.

This paper emphasizes the study of Balikutkut scientifically known as

Heliotropium indicum (Family Boraginaceae), a plant that is popularly known as stinker,

opposite in the same individual, penduculate inflorescences white to purple corolla,

the Balikutkut (Heliotropium indicum).

As shown in Figure 1, the samples of Balikutkut leaves were brought to the

University of the Philippines-Diliman campus for the botanical identification and

authentication to determine the specific species of the plant sample.

In the output, the phytochemical constituents as well as wound healing activity of

the leaf extract were determined.

INPUT PROCESS OUTPUT

 Sample plant  Identification of

and methods within which the study takes place.

Statement of the Problem

property of Balikutkut (Heliotropium indicum) leaf extract.

Specifically, it sought to answer the following:

1. What are the phytochemical constituents present in Balikutkut (Heliotropium

indicum) leaf extract?

3. Is there significant difference in the wound healing activity of Balikutkut leaf

base on zone of inhibition?

There is no significant difference in the wound healing activity of Balikutkut leaf

Scope and Limitation of the Study

The study investigated the wound healing effect of Balikutkut (Heliotropium

rats which were bought in Virgen Milagrosa University Foundation – College of

Pharmacy, located in Martin P. Posadas Avenue, San Carlos City, Pangasinan.

Mamarlao and Bacnar, San Carlos City, Pangasinan.

Significance of the Study

treatments in an attempt to prevent infection and promote faster healing. Thus, a

dissemination of information to the public.

Health Education. Individual alertness and knowledge with regard to minor or

and best response in curing external wounds.

Science Research Students. In line to the profession through discovery of new

discover more therapeutic actions of the said herbal plant.

Pharmaceutical Companies. This study can enhance the conduct of research on

a drug from Balikutkut that may be beneficial to the community.

Future Researchers. This research could also enhance their knowledge in

process that they have learned in the classroom.

that would serve as the groundwork of the study.

observation of an experimental research wound healing effect of the Balikutkut

(Heliotropium indicum) ointment applied on albino rats (Rattus Albus). Descriptive

research uses systematic observation to describe a specific behavior. It creates very

engaged in the study.

Duration and Locale of the Study

of Virgen Milagrosa University Foundation (VMUF), San Carlos City, Pangasinan

the study were as follows:

help of Dean Sheila De Vera Miranda, of Virgen Milagrosa University Foundation-

Actual experimentation involving incising wounds on laboratory rats to test the

Pathology-Microbiology laboratory of the College of Medicine, Virgin Milagrosa