Phytochemical Screening, in Vitro Cytotoxicity and Inhb

PHYTOCHEMICAL SCREENING, IN VITRO CYTOTOXICITY, AND
INHIBITION OF GLUCOSE UPTAKE BY YEAST CELLS

OF THE PHILIPPINE Euphorbia heterophylla
LEAF EXTRACT
AN UNDERGRADUATE THESIS
Presented to
the Faculty of the Department of Chemistry
College of Natural Sciences and Mathematics
Mindanao State University
Marawi City
In Partial Fulfillment
of the Requirements for the Degree of
BACHELOR OF SCIENCE IN CHEMISTRY
GIAN FRANCO C. ABUSEJO
DECEMBER 2022
ii
Chemistry Department
College of Natural Sciences and Mathematics
Mindanao State University
Marawi City
APPROVAL SHEET
The faculty of Chemistry Department, College of Natural Sciences and
Mathematics, Mindanao State University, Marawi City, accepts the
Undergraduate Thesis entitled:
Phytochemical Screening, In Vitro Cytotoxicity, and Inhibition of

Glucose Uptake by Yeast Cells of the Philippine
Euphorbia heterophylla Leaf Extracts
Submitted by Gian Franco C. Abusejo in partial fulfillment of the requirement

for the Degree Bachelor of Science in Chemistry.
ZAKARIYA T. MURIPAGA, MSc. MERELL P. BILLACURA, Ph.D.

Panel Member Panel Member
ROEVE ANN MAE C. MAZO Ph.D. YUSOPH C. MANALUNDONG II, Ph.D.

Thesis Adviser Panel Member
YUSOPH C. MANALUNDONG II, Ph.D.

Department Chairperson
JOHNNY JIM S. OUANO, Ph.D.

Acting Dean
iii
ABUSEJO, GIAN FRANCO C. Phytochemical Screening, In Vitro

Cytotoxicity and Inhibition of Glucose Uptake by Yeast Cells of the
Philippine Euphorbia heterophylla Leaf Extracts. Undergraduate Thesis,
Chemistry Department, College of Natural Sciences and Mathematics, Mindanao
State University, Marawi City.
Adviser: ROEVE ANN MAE C. MAZO, Ph.D.
ABSTRACT
The leaves of Euphorbia heterophylla, collected from the farm of
Lingayao, Las Nieves, Agusan del Norte, Mindanao, Philippines, were air-dried
and evaluated for the presence of secondary metabolites and were subjected to the
brine shrimp lethality test (BSLT), antibacterial, and in vitro evaluation of glucose
uptake by yeast cells assay obtained from crude methanolic and decoction
extracts. Phytochemical screening revealed the presence of alkaloids, flavonoids,
saponins, steroids, tannins, and terpenoids from both methanolic and decoction
extracts. Methanolic and decoction extracts' IR spectra revealed the presence of
functional groups found in the detected secondary metabolites. It was found that
the crude methanolic and decoction extracts exhibited cytotoxicity against
Artemia salina nauplii with LC50 values of 3.63 and 260 ppm, respectively, after
24 hours of treatment suggesting that both extracts are highly toxic. The
antibacterial activity of both extracts did not show any activity against
Escherichia coli using the filter paper disc diffusion method. At 0.250 mM
glucose concentrations, metformin (25 ppm), decoction (25 ppm), and methanolic
iv
(25 ppm) extracts exhibited 44.98%, 44.77%, and 41.80% increase in glucose
transport to yeast cells, respectively.
ACKNOWLEDGEMENT
The completion of this paper would be impossible without the presence of
those who provided assistance and support during the making of this paper. As a
result, I'd like to express my gratitude for the following.
First of all, I'd like to express my heartfelt gratitude to God Almighty for
providing me with the strength and courage to pursue my dreams and face all of
the obstacles and fears that have arisen this semester.
To my ever-supportive thesis adviser, for her unending help and efforts in
completing this work. Even though I hadn't finished my experiments, Ma'am
Roeve Ann Mae Mazo was always there to encourage me and let me hear the
words that I needed the most: "Congratulations, mo graduate na gyud ka."
To all my panel members, Sir Yusoph Manalundong, Sir Zakariya
Muripaga, and Sir Merell Billacura, for sincerely checking my paper and
providing clear and precise honest criticisms that greatly aided in the
improvement of my paper.
To Sir Dexter Canalita, without his DMSO this study would not have been
possible, and Sir Oliver Aromin, for the free pasture pipettes that greatly aided me
during the in vitro evaluation of glucose assay, to Ma'am Nourshamsia Barosa, Sir
v
Cyrollah Disoma, Ma'am Sittie Jamelah R. Gumal, and Sir Mohammad Al-thanie
Paudac for their assistance in my antibacterial assay.
To all of the Chemistry Department's faculty and staff for sharing their
knowledge and wonderful experiences with me.
To my girlfriend, who also encouraged and supported me in finishing my
thesis.
To my friends, the ultimate brothers and sisters, for the good and bad
times we shared, as well as the times we spent together in elementary and high
school
To my parents for properly raising me, which is why I grew up with
respect for others and fear of God.
To the Dapat and Buenafe families for treating me as a real family
member and sending me to school.
Lastly, to Uncle Jun, Ante Rosa, Nanay Inday, Tatay Nonoy, Ate Ann
Ann, Kuya Marjun, Ate Membi and to Jonna Rose for meeting all of my academic
needs. Daghan kaayong salamat sa inyung tanan kung wala mow ala ko kabalo ug
aha ko karon. Salamat sa pag dawat sa ako bisag kaisa wala ko ninyo gipa feel
nga dili kapamilya bisag magpabadlong ko usahay.

vi
I am gratefully dedicating this
work to:
My Families
Mama Egay, Papa Siloy, and my Siblings
Uncle Dominador Buenafe Jr., Ante Rosa O. Buenafe,
Nanay Emilda Dapat, Tatay Marcelino Dapat

TABLE OF CONTENTS
Page
TITLE PAGE…………………………………………………..……… i
CERTIFICATE OF PANEL APPROVAL…………………..…….... ii
ABSTRACT…………………………………………………..……….. iii
ACKNOWLEDGEMENT……………………………………………. iv
DEDICATION ……………………………………………………....... vi
TABLE OF CONTENTS……………………………………..………. vii
LIST OF TABLES……………………………………………………. x
LIST OF FIGURES………………………………………………….... xi
CHAPTER
1 INTRODUCTION…………………………………………. 1
1.1 Background of the 1

Study……………………………………..
1.2 Statement of the Problem and Objectives of the Study. 3
………
1.3 Significance of the Study……………………………….…… 5
1.4 Scope and Limitation………………………………………... 5
2 REVIEW OF RELATED LITERATURE………….…….. 7
2.1 History of Medicinal Plants…………………………………. 7

2.2 Phytochemicals……………………………………………… 7
2.2.1 Phytochemical Screening………………………….. 9
2.2.2 Alkaloids………………………….……………….. 10
2.2.3 Flavonoids…………………………………………. 11
2.2.4 Saponins……………………………………..…….. 12
2.2.5 Tannins……………………………………………. 14
2.2.6 Terpenoids………………………………………… 15
2.2.7 Cardiac Glycosides………………………………... 16
2.2.8 Steroids……………………………………………. 19
2.2.9 Phlobatannins……………………………………… 20
2.3 Euphorbia heterophylla……………………………………... 20
2.4 Bioassay……………………………………………………... 21
2.4.1 Brine Shrimp Lethality Test (BSLT) 21
……………….
2.4.2 Filter Paper Disc Diffusion 23
Method………………..
viii
2.4.2.1 Test Microorganism…………………… 24

2.4.2.2 Escherichia coli……………………...... 24
2.4.2.3 Antibacterial Standard………………… 25
2.4.2.4 Ampicillin………………………........... 26
2.4.3 In Vitro Evaluation of Glucose Uptake by Yeast
Cells………............................................................ 27
2.4.3.1 Metformin………………………........... 28
3 METHODOLOGY………………………...……………… 30
3.1 Experimental Design……………………….......................... 30

3.2 Plant Collection and Preparation………………………...… 31
3.2.1 Extraction………………………...……………… 32
3.3 Phytochemical Screening Process………………………...... 32
3.3.1 Flavonoid Screening………………………........... 28
3.3.2 Alkaloids 33
Screening………………………............
3.3.3 Saponin Screening……………………….............. 33
3.3.4 Tannins Screening……………………......……… 34
3.3.5 Terpenoids Screening……..................................... 34
3.3.6 Steroids Screening………………………...……... 35
3.3.7 Phlobatannins Screening……………………….... 35
3.3.8 Cardiac Glycosides…………………..................... 36
3.4 Brine Shrimp Lethality Test (BSLT) ……………………… 36
3.5 Antibacterial Assay………………………...………………. 38
3.5.1 Preparation of Culture 39
Media……………………..
3.5.2 Preparation of Control 39
Treatment………………...
3.5.3 Preparation of Filter Paper 39
Disc…………………..
3.5.4 Preparation of Agar Plates……………………….. 40
3.5.5 Disc Diffusion Method………............................... 40
3.6 In Vitro Evaluation of Glucose Uptake by Yeast
Cells………………………………………………………... 40
4 RESULTS AND DISCUSSIONS……………..................... 42
4.1 Results of Phytochemical 42

Screening………………………...
ix
4.2 IR Spectroscopic Measurement of the Crude Extracts ... 43

……
4.3 Bioassay………………………...………………………...... 44
4.3.1 Result of Brine Shrimp Lethality Test (BSLT) 44
…...
4.3.2 Antibacterial Assay……………….……………... 48
4.3.2.1 Result of Antibacterial Disc Diffusion
Assay………………………………….. 49
4.3.2.2 Result of In Vitro Evaluation of
Glucose 51
Uptake by Yeast
Cells………………….
5 SUMMARY, CONCLUSIONS, AND

RECOMMENDATIONS…………………………………. 54
5.1 Summary………………………...…….………………….... 54
5.2 Conclusions………………………...………………………. 56
5.3 Recommendation………………………...…………………. 57
REFERENCES…………….…………...………………………........... 58
APPENDIX………………………...………………………...………… 71
CURRICULUM VITAE………………………... 95
……………………..
x
LIST OF TABLES
Table Page
1 Phytochemical Screening Results from the Crude Methanolic and

Decoction Extracts of Euphorbia heterophylla 42
Leaves…………….
2 IR Results of the Crude Methanolic and Decoction 43

Extracts……….
3 Lethal Concentration LC50 After 6 Hours in the BSLT of the

Extracts at Different Concentrations 45
………………………………
4 Lethal Concentration LC50 After 24 Hours in the BSLT of the

Extracts at Different Concentrations 46
………………………………
5 Average Inhibition in Diameter of the Tested Concentrations

(mm) in the Antibacterial Assay Against E. 50
coli………………………….
6 Nauplii Deaths After 6 Hours in the BSLT of the Extracts at

Different 86
Concentrations…………………………………………...
7 Nauplii Deaths After 24 Hours in the BSLT of the Extracts at

Different Concentrations 86
…………………………………………..
xi
8 Finney’s Table for Probit 87

Analysis…………………………………
9 Zones of Inhibition at Varying Concentrations Against E. 91

coli…….
LIST OF FIGURES
Figures
Page
1 Structure of the alkaloids (a) Morphine and (b) Nicotine. 11

…………..
2 Structure of the two most well-known flavonoids Quercetin and

Kaempferol………………………………………………………… 12
3 Structure of the saponins (a) Spirostanol and (b) Furostanol. 14

………
4 Structure of (a) hydrolysable tannins and (b) condensed

tannins............................................................................................... 15
5 Structure of the terpenoids (a) Carvacrol and (b) 16

Thymol…………..
6 Structure of the cardiac glycosides (a) Digoxin and (b)

Bufalin .............................................................................................. 18
.............
xii
7 Structure of steroid……………………………. 19
……………............
8 Adult Artemia 23
salina………………………………………………..
9 Escherichia coli ……………….…………………………………... 25
10 Chemical structure of Ampicillin…………………………………. 27
11 Research schematic diagram…….………………………………… 30
12 Satellite view of Lingayao, Las Nieves, Agusand del 31

Norte………..
13 The Plot of LC50 values of the two extracts after 6 47

hours……………
14 The Plot of LC50 values of the two extracts after 24 hours………… 47
15 Graphical representation of the zones of inhibition of the different

extracts at varying concentrations vs. zone of inhibition of
standard 51
drugs………………………………………………………………..
16 Effects of the methanolic and decoction extracts vs. Metformin on

the uptake of glucose by yeast cells at 25 ppm
concentration………………………………………………………. 52
17 Effects of the methanolic and decoction extracts vs. Metformin on

the uptake of glucose by yeast cells at 100 ppm
concentration………………………………………………….…… 53
18 Euphorbia heterophylla plant……………………………………… 74
19 Collected leaves of E. heterophylla ……………………………….. 74
20 Air drying of the leaves of Euphorbia heterophylla 75

……………….
21 Homogenization of the leaves of Euphorbia 75

xiii
heterophylla………….
22 Maceration of the powdered leaves of Euphorbia heterophylla

with methanol for 72 hours…………………………………. 76
…………...
23 Filtration, evaporation of the solvent, and weighing of the extracts. 76
24 Decoction and filtration of the powdered leaves of Euphorbia

heterophylla…………...………….................................................... 77
25 Results of the phytochemical screening of the decoction and

methanol extracts of the leaves of E. 78
heterophylla…………………
26 Hatching the brine shrimp………………………………. 79

………….
27 Treatment of the brine shrimp…………….……………………….. 79
28 Preparation of the assay 80

…………………………………………….
29 Results of the antibacterial 81

assay…………………………………...
30 Metformin drug……………………………………………………. 82
31 Washing of yeast through repeated centrifugation……. 82

……………
32 Preparation of the in vitro evaluation of glucose uptake by yeast

cells………………………………………………………………… 83
33 Some of the UV-Vis 84

results………………………………………...
34 IR spectrum of decoction extract……... 85

……………………………
35 IR spectrum of methanolic 85
xiv
extract…………………………….........
36 Plot of Probits against log of concentrations to produce calibration

curve after 6 hours of treatment using decoction …………... 88
………

curve after 6 hours of treatment using methanol extract…. 88
…………
38 Plot of Probits against log of concentrations to produce

calibration curve after 24 hours of treatment using decoction 89
extract………….

curve after 24 hours of treatment using methanol extract. 89
………......
40 Absorbance of varying decoction extract concentrations against

varying concentrations of 92
glucose…………………………………..
41 Absorbance of varying methanol extract concentrations against

varying concentrations of 92
glucose…………………………………..
42 Absorbance of varying Metformin concentrations against varying

concentrations of 93
glucose…………………………………………...
43 Effect of decoction extracts on the uptake of glucose by yeast cells

at varying concentrations 93
…………………………………………..
44 Effect of Metformin on the uptake of glucose by yeast cells at

varying 94
concentrations…………………….......................................
45 Effect of methanol extracts on the uptake of glucose by yeast cells

at varying concentrations ………………………………….. 94
………
xv
Chapter 1
Introduction
1.1 Background of the study
Herbal medicines and plant-derived medicines have long been utilized in
traditional civilizations around the world, that is, since humans seek a tool in their
surroundings to recover from a condition, plants have been their only option
(Ugbogu & Akukwe, 2009). This wisdom has been passed on in verbal and in
written form from generation to generation (Sofowora, 2008). They are
increasingly being employed today as natural replacements for synthetic
medicines. The great majority of people on the planet still rely on traditional
medicine for day-to-day health care (Van & Wink, 2018).
The word medicinal plant encompasses a wide range of plants with
medical properties. These plants have a lot of chemicals that can be exploited in
medicine development (Rasool Hassan, 2012). Various seeds, roots, leaves, fruit,
skin, flowers, or even the entire plant may be used as therapeutic plant
components. Most portions of medicinal plants contain active chemicals called
phytochemicals that have overt or covert therapeutic benefits and are employed as
medicinal medicines (Srinivasahan et al., 2014). Plant metabolites that protect
them from biotic and abiotic challenges have been transformed into medicines
that can treat various ailments (Njoku & Obi, 2009; Kocabas, 2017). They also
have significant value in the perfume, agrochemical, and cosmetic industries
(Rasool Hassan, 2012).

2
Phytochemicals are naturally occurring chemical substances in plants that
have beneficial or bad health effects (De Silva et al., 2017). The phytochemical
elements of plants determine their therapeutic qualities (Ezeonu et al., 2016).
Alkaloids, terpenoids, phenylpropanoids phenolics, tannins, saponins, steroids,
glycosides, terpenes, and others are among the secondary metabolites being
studied for therapeutic development (Sanchita & Sharma, 2018; Sheel & Kumar,
2014). All of these secondary metabolites have been linked to the treatment of
various ailments. As an example, alkaloids have antispasmodic, antimalarial,
analgesic, and diuretic properties. Terpenoids have antiviral, anticancer,
antibacterial, antimalarial, anthelmintic, and anti-inflammatory effects, according
to studies. They are also useful for keeping agricultural products since they
prevent cholesterol formation and have insecticidal qualities. Saponins have anti-
inflammatory, antiviral, and plant-defense properties, as well as cholesterol-
lowering properties. Astringent characteristics are seen in phlobatanins.
Antifungal and antibacterial activity have been documented for glycosides.
Antioxidant, anti-allergic, antibacterial and other properties of phenols and
flavonoids are well documented (Padalia & Chanda, 2015; Moteriya et al., 2015).
This research work took off based on the fact that a lot of medicinal plants
in the Philippines are still to be explored. Mexican fire plant or Euphorbia
heterophylla is native to Central and South America, but it can currently be found
in tropical and subtropical regions as well. E. heterophylla is a grassy, erect plant

3
that grows to a height of a 0.5 to 1.5 m tall herb or shrubby plant that is upright,
branching, smooth, and partially woody. Different species have arisen because of
variations in the form of the leaf. The creamy latex is pungent, and the toxicity is
likely a resin rather than an alkaloid or glycoside, which can be lethal (James &
Friday, 2010). Friedelin, ellagic acid, myricyl alcohol, β-sitosterol, benzoic acid,
diterpenes, quercetin, and various amino acids are among the chemical
components found in E. heterophylla (Adnan & Hölscher, 2010). Despite its
toxicity, this plant has several medical qualities, including the treatment of
respiratory tract infections, gonorrhea, malaria, eczema, asthma, and warts (Fred-
Jaiyesimi & Abo, 2010).
Euphorbia heterophylla is found present in Lingayao, Las Nieves, Agusan
del Norte. It can be found almost everywhere on the farm area of the said place.
Furthermore, Euphorbia heterophylla is one of the medicinal plants that are being
used by some of the people of this place. The leaf of this plant is boiled with
water and serves as a medicinal drink that according to the people who use them,
lowers the body fat. Therefore, it is imperative to explore the medicinal use of the
said plant.
1.2 Statement of the Problem and Objectives of the Study
This study was designed to screen the secondary metabolites obtained
from the decoction and methanol extracts of the leaves of Euphorbia heterophylla
4
collected from the farm of Lingayao, Las Nieves, Agusan del Norte, Mindanao,
Philippines. The crude extracts were partially tested for toxicity, and antibacterial
properties, and subjected to in vitro evaluation of glucose uptake by yeast cells
assay.
The specific objectives of the study were as follows:
1. To perform decoction and methanol extraction of the leaves of Euphorbia
heterophylla;
2. To qualitatively assess the various phytochemical qualities (flavonoids,
alkaloids, saponins, tannins, terpenoids, steroids, phlobatannins, and
cardiac glycosides) of the crude methanolic and decoction extracts using
appropriate phytochemical tests;
3. To evaluate the cytotoxicity of the crude methanolic and decoction
extracts using the brine shrimp lethality test (BSLT) at 10-, 100-, and 1000
ppm concentrations and also to determine the LC50;
4. To evaluate the antibacterial properties of the crude methanolic, decoction,
and freshly picked leaf extracts against Escherichia coli using the filter
paper disc diffusion method at 10,000-, 20,000-, and 30,000 ppm; and to
5. To determine the antidiabetic potential of the crude methanolic and
decoction extracts of the leaves of Euphorbia heterophylla using the in
vitro evaluation of glucose uptake by yeast cells assay.

5
1.3 Significance of the Study
This study was conducted to discover and harness the therapeutic potential
of compounds from Euphorbia heterophylla of Lingayao, Las Nieves, Agusan del
Norte, Mindanao, Philippines. The results of this study would provide a baseline
for the future study of this plant. Futhermore, the results of this study would
provide useful information about the medicinal value of this plant and to the claim
that it can lower cholesterol levels.
1.4 Scope and Limitation
The study was conducted to obtain plant extracts using methanol from air-
dried leaves of Euphorbia heterophylla. The plant materials were collected from
one of the farms of Lingayao, Las Nieves, Agusan del Norte. Crude methanolic
and decoction extracts were subjected to phytochemical screening using the
method described by Njoku & Obi (2009), and Iqbal & Lim (2015). The
cytotoxicity of the extracts was determined using Miller's Brine Shrimp Lethality
Test (1972) at 10-, 100-, and 1000 ppm concentrations against Artemia salina.
The extract’s antibacterial property was determined using the filter paper disc-
diffusion method described by Kirbey-Bauer (2004) and was performed at the
microbiology room of the Biology Department at Mindanao State University
using different concentrations of the crude methanolic and decoction extracts at

6
10,000-, 20,000-, and 30,000 ppm against the available microorganism
Escherichia coli. The rate of increase of glucose uptake by yeast cells was
determined using the methods described by Jijith & Jayakumari (2017) and
Pitchaipillai and Ponniah (2016) with minor modifications. All experiments were
conducted in the laboratory of the Biology Department and Chemistry
Departments of the campus of Mindanao State University in Marawi City, Lanao
del Sur.
Chapter 2
Review of Related Literature
2.1 History of medicinal plants
The foundation for treating human ailments is natural compounds derived
from plants, animals, and minerals (Firenzuoli & Gori, 2007). Humans learned to
detect and categorize plant resources suitable for use in supplying the demands of
life over time and with the emergence of communities. The usage of herbs and
herbal extracts for their therapeutic properties can be traced back to the oldest
stories, traditions, and literature that were used to define those plants that can
relieve pain and treat ailments (Mamedov, 2012). In countries like China, Greece,
Egypt, and India, medicinal plants have become one of the oldest disciplines.
Plants were widely employed as a medicine and disinfectant in ancient Persia and
as well as an aromatic agent (Hamilton, 2004). The use of herbal medicines for
disease treatment stretches back to the dawn of human history, that is, since
humans seek a tool in their surroundings to recover from a condition, plants have
been their only option (Ugbogu & Akukwe, 2009).
2.2 Phytochemicals
Phytochemicals are bioactive nutritional plant compounds found in fruits,
vegetables, cereals, and other plant foods that may have health advantages in
8
addition to basic nutrition, such as lowering the risk of major chronic diseases.
Shah and Hossain (2014) stated that non-nutritive plant bioactive chemical
elements known as phytochemicals have a distinct physiological function on the
human body. Phytochemicals are produced in plant cells via unique metabolic
pathways which are divided into two categories, the primary and secondary
metabolites. The secondary metabolites which include phenolic compounds,
alkaloids, flavonoids, terpenoids, tannins, saponins, cardiac glycosides, essential
oils, and essential oils are important in plant defense against herbivory and
adaptation to environmental stress, while primary metabolites such as chlorophyll,
sugars, proteins, lipids, amino acids, and others are required for plant growth and
development (Saxena et al., 2014; Edeoga et al., 2005; Hill, 1952). In addition,
Srinivasahan and Durairaj (2014) stated that medicinal plants contain a variety of
phytochemicals or secondary metabolites that can promote health separately,
additively, or synergistically. For healing diseases, different plant extracts have
diverse modes of action and can be used as food preservatives as well (Bernhoft,
2010).
All of the secondary metabolites have been linked to the treatment of
various ailments. As an example, alkaloids have antispasmodic, antimalarial,
analgesic, and diuretic properties. Terpenoids have antiviral, anthelmintic,
antibacterial, anticancer, antimalarial, and anti-inflammatory effects, according to
studies. They are also useful for keeping agricultural products since they prevent
9
cholesterol formation and have insecticidal qualities. Saponins have anti-
inflammatory, antiviral, and plant-defense properties, as well as cholesterol-
lowering properties. Astringent characteristics are seen in phlobatanins.
Antifungal and antibacterial activities have been documented for glycosides.
Antioxidant, anti-allergic, antibacterial and other properties of phenols and
flavonoids are well documented (Padalia & Chanda, 2015; Moteriya et al., 2015).
2.2.1 Phytochemical Screening
Chemical screening also referred to as phytochemical screening, is a
method of determining the presence of chemicals in a sample. Aqueous and
organic extracts are made from plant samples that include secondary metabolites,
such as bark, roots, stems, or leaves, in this procedure. The presence of secondary
metabolites such as alkaloids, terpenes, and flavonoids is then determined in the
plant extract (Srivastava, 2020).
Farnsworth (1966) reported that several researchers believe that initial
screening of investigational plants must be made based on the presence of certain
chemicals in the plant, relatives of which are usually associated with biological
activity, rather than on evidence that extracts elicit a specific and interesting
biological activity. As a result, some researchers will initially focus on alkaloid-
containing plants for research based on the assumptions that alkaloids usually
have some pharmacologic activity, generally on the central nervous system, but
10
not always, that the vast number of natural products being used in medicine today
are alkaloidal in nature, that the tests for the presence of these constituents of
plants are simple, quick, and reasonably reliable, and that the alkaloids are more
easily controlled due to their chemical composition, making extraction and
separation less difficult.
Furthermore, Farnsworth (1966) stated that a technique for screening
phytochemicals should be basic, quick, tailored to a minimum technology, and
moderately selected for the class of chemicals being researched.
2.2.2 Alkaloids
Manske and Holmes (2014) stated that the study of opium alkaloids began
in the seventeenth century when researchers attempted to identify the principal
responsible for opium's activity. Many extracts were obtained during the
experiments and used in therapies under the name Magisterium opii. Derosne, a
pharmacist practicing in Paris, was the first to isolate a component of opium in
crystalline form in 1803. He diluted his opium syrup with water and used
potassium carbonate to precipitate the opium salt. Sèguin extracted morphine and
meconic acid from opium in 1804 and recognized the power of morphine to
neutralize acids in his following study. Morphine was the first of a vast range of
naturally occurring compounds that were later dubbed alkaloids.

11
Alkaloids are more or less toxic substances that have a basic character,
contain heterocyclic nitrogen, and are synthesized in plants from amino acids or
their immediate derivatives. They have a limited distribution in the plant kingdom
in most cases (Farnsworth, 1966). Examples of alkaloids are morphine and
nicotine (Figure 1).
(a) (b)
Figure 1. Structure of alkaloids (a) morphine and (b) nicotine.
2.2.3 Flavonoids
Szalay (2015) reported that flavonoids are phytonutrients that can be
found in practically all fruits and vegetables. They are accountable for the vibrant
hues in fruits and vegetables, together with carotenoids. With over 6,000 different
varieties, flavonoids are the most diverse category of phytonutrients. Quercetin
and kaempferol (Figure 2) are two of the most well-known flavonoids.
Flavonoids, like other phytonutrients, are potent antioxidants with anti-

12
inflammatory and immune-boosting properties. Flavonoid-rich diets have been
linked to a lower risk of cancer, neurological illness, and cardiovascular disease.
However, it's unclear whether the flavonoids are responsible. Flavonoids are
phytonutrients that belong to the polyphenol family. According to the Global
Healing Center (2014), polyphenols have been utilized in Chinese and Ayurvedic
medicine for centuries and are linked to skin protection, brain function, blood
sugar, and blood pressure management.
(a) (b)
Figure 2. Structure of the two most well-known flavonoids (a) Quercetin

and (b) Kaempferol.
2.2.4 Saponins
Xu and Yu (2021) stated that saponins are a wide group of amphiphilic
glycosides of steroids and triterpene present in plants and some marine creatures.
Saponins demonstrate a wide range of biological and pharmacological activities
by expressing a huge diversity of configurations on both sugar chains and

13
aglycones, and they are important active principles in folk medicine, particularly
traditional Chinese medicine. Due to the microheterogeneity of saponins in
nature, isolating saponins from natural sources is usually difficult. Furthermore,
Xu and Yu (2021) reported that saponins are intriguing prospects for novel
medication design and development due to their extensive history of folk
medicinal usage.
Moreover, Ezzat et al. (2021) stated that saponins have antioxidant
properties and protect the skin from UV damage by blocking extracellular matrix
disintegration and acting as an anti-irritant due to their anti-inflammatory
properties. Saponins may also be utilized to treat acne because of their
antibacterial properties. Saponins also strengthen skin capillaries, reducing the
appearance of couperose and cellulite.
In addition, Picincu (2018) reported that saponins help to maintain
cardiovascular health by lowering cholesterol and body fat levels. According to
researchers, these compounds are thought to decrease cholesterol absorption by
binding to bile salts. Plant saponins have been found to lower plasma cholesterol
concentrations by inhibiting the absorption of cholesterol from the small intestines
in experimental animals (Liwa et al., 2017). Saponins from ginseng and ginger
have been demonstrated in clinical trials to lower total and low-density
lipoprotein (LDL) bad cholesterol without affecting high-density lipoprotein
(HDL) good cholesterol levels.

14
The most common steroid saponins in plants are spirostan-type saponins.
They have a hexacyclic aglycone, such as diosgenin or tigogenin, in their
structure. The 3-OH group is typically decorated with an oligosaccharide chain
(Yang, 2014). Examples of saponins are shown in Figure 3.
(a)
(b)
Figure 3. Structure of the saponins (a) Spirostanol and (b)

Furostanol.
2.2.5 Tannins
Tannins are chemical compounds made up of phenolic acids. They are
called phenolic compounds, and they're found in a wide range of plant species
15
from all climates and places of the world. They are big molecules that bind to
proteins, cellulose, carbohydrates, and minerals easily. These chemicals are
insoluble and resistant to degradation. The word tannin is derived from the
German word “Tanna”, which means oak. It refers to the procedure of turning
animal skins into the leather using wood tannins generated from oak trees.
Different formulations of plant tannins known as "tanning liqueurs" were used to
achieve different hues, textures, and durability of leathers. Tannins are also
accountable for several of the lovely hues found in flowers and the final splendor
of fall leaves (U. S. Forest Service, n. d.).
(a) (b)
Figure 4. Structure of (a) hydrolysable tannins and (b) condensed

tannins.
2.2.6 Terpenoids
Terpenoids, also known as isoprenoids, are a vast (60 percent of all known
natural products) and diversified collection of lipids made up of five-carbon
isoprene units organized in thousands of different ways. A terpenoid is an

16
oxygen-containing compound, whereas a terpene is a hydrocarbon. Both names
are frequently used interchangeably to refer to both groups (CHEMISTRY
LibreText™, 2020).
Perveen (2021) stated that terpenoids are a type of terpene that has had
their functional groups and oxidized methyl groups changed or deleted at different
places. Depending on the number of carbon units, terpenoids are classified as
monoterpenes, diterpenes, sesterpenes, sesquiterpenes, and triterpenes. The
majority of terpenoids, despite their structural differences, are biologically active
and are utilized to treat a variety of ailments around the world. Many terpenoids,
including taxol and its derivatives, suppressed many human cancer cells and are
utilized as anti-cancer medicines. Examples of terpenoids are Carvacrol and
Thymol (Figure 5).
(a) (b)
Figure 5. Structure of terpenoids (a) Carvacrol and (b) Thymol.
2.2.7 Cardiac glycosides

17
A large family of naturally occurring substances known as cardiac
glycosides (CG) bind to and inhibit the enzyme Na+/K+-ATPase. Members of this
family have been used clinically for many years to treat atrial fibrillation and
heart failure, and the mechanism underlying their beneficial inotropic action is
well understood. Exciting new research has proposed additional Na +/K+-ATPase
signaling mechanisms, implicating cardiac glycosides in the control of numerous
critical cellular processes, and emphasizing potential novel therapeutic
applications for these substances in a variety of disorders. The fact that cancer
cells are more susceptible to these substances suggests that they may be used as
cancer therapeutics, and the first generation of glycoside-based anticancer
medicines is currently undergoing clinical testing. The most well-known CGs
include bufalin (Figure 6b), oleandrin, ouabain, and digoxin (Figure 6a). They
share a steroid ring, a lactone ring with five or six carbons, and a sugar moiety in
their chemical makeup (Prassas & Diamandis, 2008).

18
(a)
(b)
Figure 6. Structure of the cardiac glycosides (a) digoxin

and (b) bufalin.
2.2.8 Steroids
Steroids (Figure 7) have cyclopenta[a]phenanthrene scaffolds that are
fully or partially reduced and occasionally have methyl groups at C-10 and C-13.
But distinct steroid skeletons result from differences in the side chain's backbone,
length, and stereochemistry of some of its chiral centers at C-17 (Sandjo & Kuete,
2013). Dinan et al. (2001) stated that according to their biological significance,
the diverse range of steroid compounds that plants make can be grouped into three
19
groups. The first group is the substances that play physiological roles in the plant,
such as hormones or pheromones. Thus, antheridiol and oogoniol are pheromones
in an aquatic filamentous fungus, but brassinosteroids are phytohormones that
promote growth. The second group is the animal hormone-related allelochemical
compounds. While androgens, oestrogens, progestagens, corticosteroids, and
cholecalciferols are connected to vertebrate hormones, ecdysteroids are analogs of
insect molting hormones. The third group is the plant-specific allelochemical
compounds that frequently function protectively against phytophagous animals or
parasitic fungi that act as repellent, antifeedant, and toxic. These include steroidal
alkaloids, sapogenins, withanolides, cardenolides, and bufadienolides, among
others.
Figure 7. Structure of steroid.
2.2.9 Phlobatannins
In the middle of the 1980s, it was shown that a novel class of C-ring
isomerized oligomeric avanoids, known as phlobatannins, occurs naturally and

20
can be synthesized. The discovery of phlobatannins also aids in providing a
logical justification for the significantly decreased solubility of aged
proanthocyanidins in aqueous solvents (ScienceDirect, n. d.).
2.3 Euphorbia heterophylla
According to Falodun et al. (2006), the secondary metabolites present in
Euphorbia heterophylla include tannins, flavonoids, carbohydrates, and saponins.
These metabolites have been examined and found to be effective in improving the
functionality of plant corrosion inhibitors. According to Ajuru et al. (2017), the
family Euphorbiaceae includes the pintado (Mexican fireplant), Euphorbia
heterophylla. Mexican fire plant is a 0.5 to 1.5 m tall herb or shrubby plant that is
upright, branching, smooth, and partially woody. Constipation, bronchitis,
asthma, cough, catarrh, insect bites, and colon cleanser are among the conditions
it is used to treat. To encourage milk flow in nursing women, a decoction of
leaves is applied to the breasts.
Bioactive alkaloids, phenolic chemicals, steroids, tannins, and terpenoids
are present in E. heterophylla L. leaf extract. Due to the presence of phenolic and
alkaloid compounds, ethyl acetate extract, which is semi-polar in nature, has been
found to have a significant level of antioxidant activity (IC 50 = 129,79 ppm).
Based on analysis using UV, IR, 1H-NMR, 13C-NMR, and MS spectroscopy, ethyl
acetate extracts from E. heterophylla L leaves demonstrated that the pure

21
bioactive chemicals included in the extracts were diterpenoid groups. According
to the results of an in vitro test for antidiabetic activity, the ethyl acetate extract's
IC50 value was 138.63 g/mL and the Diterpenoid DS-01 compound's IC 50 value
was 191.82 g/mL (Hilma et al., 2019).
2.4 Bioassay
Natural product extracts have been used as a valuable source of molecular
diversity in many drug discovery programs, and several important drugs have
been isolated from them. Any natural product isolation program in which the end
product is to be a drug or a lead compound must include some form of bioassay
screening or pharmacological evaluation to guide the isolation process toward the
pure bioactive component (Choudhary et al., 2001)
Bioassay comes from the word Bio means living, and assay means to
analyze or to understand. Bioassay is a biological screening procedure that uses
living organisms to estimate the potency or nature of a substance. This bioassay is
performed both in vivo (within the living organism) and in vitro (outside the
living organism in the laboratories using Petri dishes or test tubes) (PharMSkool,
2020).
2.4.1 Brine Shrimp Lethality Test

22
Any substance, even those found in nature, can be toxic based on the
body's tolerance for that substance's amount. Therefore, additional research is
required to comprehend the safety standard of a specific commodity by detecting
its acute poisonous to identify the LC50 rate, various toxic signs, the toxic
spectrum, and the death effect (Akroum et al., 2009).
Brine Shrimp Lethality Test (BSLT) is one of the techniques used to
evaluate a plant's toxic properties. This technique is simple, rapid, and affordable,
allowing it to be employed as a Bioassay Guided Fraction from renewable
resources. The first development of this technique was to assess the toxicity of
seawater, observe anesthetic chemicals, and identify pesticide residues. The other
technique used Artemia salina larvae to create the technique for identifying active
substances. Through probit analysis, the value of LC50 is counted to establish the
toxicity threshold. Comparing the extract's LC50 value, which must be less than or
equal to 1,000 ppm, yields information on its potency (Marzuki et al., 2019).
This is a quick and thorough test for the bioactive component, whether it is
natural or synthesized. Since no aseptic methods are necessary, the test is also
affordable and straightforward. It is simple to use a large number of organisms for
statistical validation, requires no specialized equipment, and only needs a small
sample size (2–20 mg) (Sarah et al., 2017).
The A salina larvae (Figure 8) (nauplii; singular nauplius), which are
around 22 mm in length, are both large enough to see without a magnifying glass
23
and small enough to hatch in huge numbers without requiring a lot of room in a
lab.
Figure 8. Adult Artemia salina (https://www.warrenphotographic.co.uke,

accessed on December 2022).
2.4.2 Filter Paper Disk Diffusion Method
One of the numerous tests used in antimicrobial testing is disc diffusion.
To determine whether bacteria are impacted by antibiotics, a wafer impregnated
with antibiotics is used in the test. In this test, antibiotic-containing wafers are put
on an agar plate with bacteria on them, and the plate is allowed to incubate. There
will be a region of the wafer where the bacteria have not grown sufficiently to be
visible if an antibiotic prevents the bacteria from developing or kills the bacteria.
The zone of inhibition is what we refer to as this. The extent to which the
24
antibiotic successfully halts bacterial growth determines the size of this zone.
Because a lesser concentration of the antibiotic is sufficient to stop development,
a stronger antibiotic will produce a large zone (Kirby, 1959).
2.4.2.1 Test Microorganism
The antibacterial test uses Escherichia coli as the test organism. At
Mindanao State University's College of Natural Science and Mathematics Biology
Department's Microbiology Laboratory Room, the microbe was acquired and
grown. Nutrient Agar was employed as the bacterial cultures' medium to
culture E. coli.
2.4.2.2 Escherichia coli
One of the most adaptable bacterial species is Escherichia coli (Figure 9).
It alternates between living as a commensal in the gut of vertebrates, which is its
natural home (Tenaillon et al., 2010), as well as water, sediment, and its
secondary habitat (Savageau, 1983). Warm-blooded creatures' lower intestines are
frequently home to the bacteria Escherichia coli (E. coli). However, some E. coli
strains can result in life-threatening food poisoning and can occasionally cause the
recall of products due to tainted food (Dippold et al., 2005). The benign strains
are a typical component of the gut flora and provide their hosts with vitamin K
(Bently, 1982), having a symbiotic relationship, and inhibiting the invasion of the
intestine by harmful microorganisms (Hudault, 2001).

25
Figure 9. Escherichia coli (https://healthnews2me.com/, accessed on December

2022).
2.4.2.3 Antibacterial Standards
Antibacterial medications are either made from scratch or obtained from
bacteria or mold. Although the term "antibiotic" is technically exclusively used to
describe antimicrobials originating from bacteria or molds, it is sometimes used
interchangeably with "antibacterial medication." Antibacterial medications kill
bacteria. Drugs that inhibit or stop the development of bacteria in vitro. These
definitions do not necessarily apply. Medications that are bactericidal may only
stop some susceptible bacterial species from growing while bacteriostatic drugs
may eliminate some sensitive bacterial species. The minimum in vitro dose at
which an antibiotic can stop the growth or can kill is the minimum inhibitory
26
concentration (MIC) and can be determined by more accurate quantitative
approaches minimum bactericidal concentration (MBC). When the host's defenses
are damaged locally as in meningitis or endocarditis or systemically, an antibiotic
with bactericidal activity may help to kill bacteria more effectively in cases of
patients who are neutropenic or immunocompromised. Rather than whether a
medicine has bactericidal or bacteriostatic action, the selection of a drug for a
given situation should be based on how the dosage form changes over time
concerning the MIC (MSD MANUAL Professional version, n. d.).
2.4.2.4 Ampicillin
Ampicillin (Figure 10), a penicillin antibiotic, is used to cure or prevent a
wide range of illnesses, including infections of the stomach and intestines,
pneumonia, gonorrhea, bladder infections, and meningitis. Serious adverse
reactions to ampicillin are possible, it includes severe abdominal pain, bloody or
watery diarrhea (even if it happens months after your previous dose), mouth pain,
ulcers, or blisters, irritation, redness, or skin rash, symptoms such as a fever,
chills, sore throat, swollen glands, joint discomfort, or general malaise, chilly
hands and feet and pale skin, or feeling dizzy or having difficulty breathing.
Ampicillin's typical negative effects include the following: diarrhea, vomiting,
and stomach pain, rash, a tongue that is large, black, or "hairy", or itching or
leaking from the vagina (Drugs.com, n. d.).

27
Figure 10. Chemical structure of ampicillin.
2.4.3 In Vitro Evaluation of Glucose Uptake by Yeast Cells
All known yeasts can use one or more sugars as their primary source of
carbon and energy. This sugar is converted to ethanol by the yeast. Inhibiting such
glucose uptake by yeast cells is an important tool for assessing the anti-diabetic
properties of synthetic or traditional plant-based compounds (Jijith & Jayakumari,
2017).
Yeast cells' absorption of glucose may differ from that of other eukaryotic
organisms or the human body. Facilitated diffusion may be used to transport
glucose across the yeast membrane in place of the phosphotransferase enzyme
system or some other unidentified method. The concentration of glucose within
the cells or the following metabolism of glucose are two examples of factors that
may have an impact on the yeast cells' ability to absorb glucose. Several in vitro
assays can be used to evaluate a drug's signs of prospective antidiabetic activity,
giving information about that drug's potential in vivo. Other indicator tests besides
28
antioxidant assays include the capacity of glucose uptake across cell membranes,
such as that of yeast cells (Rehman et al., 2018).
A measure of glucose intake by the yeast cells is the amount of glucose
that is still present in the medium after a particular amount of time. Only when
intracellular glucose is effectively decreased or used does glucose transfer take
place (Pitchaipillai & Ponniah, 2016).
2.4.3.1 Metformin
The most often prescribed medication for type 2 diabetes globally is
Metformin, which can be used alone or in combination with other glucose-
lowering treatments such as insulin. Since the 1950s, biguanides, a class of drugs
with herbal origins, have been used extensively to treat diabetes, including
metformin. Due to their ability to produce lactic acidosis, two further biguanides
were taken off the market. Due to worries about lactic acidosis, metformin was
similarly pulled off the market in the US. However, it was later shown to be safe
and effective at lowering blood sugar levels and was reintroduced in 1995 (Flory
& Lipska, 2019).
The commonly used medication Metformin has definite advantages in
terms of glucose metabolism and problems from diabetes. Although Metformin
has been proven physiologically to decrease the generation of hepatic glucose, not
all of its effects can be accounted for by this route, and there is mounting evidence
29
that the gut plays a significant role. Metformin has been demonstrated to act by
inhibiting mitochondrial respiration but also possibly by inhibiting mitochondrial
glycerophosphate dehydrogenase and a mechanism involving the lysosome, as
well as AMP-activated protein kinase (AMPK)-dependent and AMPK
independent mechanisms (Rena et al., 2017).

Chapter 3
Methodology
3.1 Experimental Design
The systematic flow of the investigation is shown in the schematic illustration
below.
Plant Leaves Sample

(Euphorbia heterophylla)
4,910 grams of samples

(Air dried and powdered)
490 grams powdered leaves

495 grams of powdered leaves
Boiled for 10 minutes, Soaked in Methanol
filtered for 72 hours,
filtered
Plant materials Plant materials

Filtrate Filtrate
discarded discarded
freeze in Concentrated in rotavap
refrigerator at 200 rpm and 40℃
the solvent was evaporated at
room temperature
Decoction Extract
Methanolic Extract
Phytochemical screening Bioassays
Antibacterial In Vitro Evaluation of Glucose

(Disc diffusion method) BSLT Uptake by Yeast Cells
Figure 11. Research schematic diagram.

31
3.2 Plant material collection and preparation
Fresh leaves of Euphorbia heterophylla were collected at the farm of
Lingayao, Las Nieves, Agusan del Norte located in Mindanao, Caraga, Region
XIII with coordinates of 8.7460, 125.5852 (8° 45' North, 125° 35' East) and with
an estimated elevation 12.4 meters (40.7 feet) above sea level. The leaves of
Euphorbia heterophylla were washed properly with tap water to remove any
impurities and were air-dried for three days. The dried leaves were homogenized
to a fine powder and stored in an airtight bottle.
Figure 12. Satellite view of Lingayao, Las Nieves, Agusand del Norte.
3.2.1 Extraction
Euphorbia heterophylla dried and powdered leaves (985 g) were divided
for decoction (490 g) and methanol extraction (495 g) preparations.

32
Four hundred ninety grams of powdered leaves were boiled for 10
minutes, cooled, and then filtered to prepare the decoction extract. The residue
was discarded and the filtrate was collected and chilled.
Methanol was used to soak 495 grams of powdered leaves for 72 hours
while being stirred occasionally. It was filtered after soaking, and the filtrate was
evaporated in a rotary evaporator at 200 rpm and 40 ℃ and chilled.
Phytochemical screening was then performed on both extracts.
3.3 Phytochemical Screening Process
The methods used in this study for phytochemical screening were the
methods described by Njoku et al. (2009) and Iqbal et al. (2015).
3.3.1 Flavonoids Screening
3.3.1.a E. heterophylla Decoction Extract
A 10% lead acetate solution was mixed with one mL of a decoction extract.
The presence of a yellow precipitate indicated the presence of flavonoids.
3.3.1.b E. heterophylla Methanolic Extract
A 10% lead acetate solution was mixed with 100 mg of methanolic extract.
The presence of a yellow precipitate indicated the presence of flavonoids.

33
3.3.2 Alkaloids Screening
In a steam bath, 1 % HCl and 3 mL of decoction extract were mixed. After
that, the solution was treated with Mayer's and Wagner's reagents. Alkaloids were
assumed to be present based on the color change of the solution. With Mayer’s
reagent, the solution turned into a slightly creamish color. With Wagner’s reagent,
the solution turned slightly darker.
A weight of 15 mg of extracts was mixed with HCl for 5 minutes in a
water bath, then filtered and divided into two for Mayer’s and Wagner’s tests.
For Mayer's test, Mayer’s reagent was introduced to a part of the filtrate.
A creamish color change was taken as an indicator of the presence of alkaloids.
For Wagner’s test, the filtrate was treated with a few drops of wager's
reagent. The color change to orange-red indicates the presence of alkaloids.
3.3.3 Saponins Screening
In a test tube, 5 mL of the decoction extract and 5 mL of distilled water
were warmed and briskly shaken. The development of stable foam was a sign that
saponins were present.

34
In a test tube, 0.5 g of extracts were mixed with distilled water. The
development of foaming that lasts after warming in a water bath for five minutes
indicates the existence of saponins.
3.3.4 Tannins Screening
3.3.4.a E. heterophylla Decoction extract
A few drops of the ferric chloride solution were added after stirring 2 mL
of the decoction extract with 2 mL of distilled water. The development of the
black color was a sign that tannins were present.
Filtration was performed after stirring 0.5 g of the extract with distilled
water and addition of a few drops of FeCl3 solution. Black pigmentation was
interpreted as a sign that tannins were present.
3.3.5 Terpenoids Screening
In 2 mL of chloroform, 2 mL of decoction extract was dissolved. After
that, 2 mL of concentrated sulfuric acid was added. The presence of terpenoids
was indicated by a reddish-yellow coloring at the junction.

35
A volume of 2 mL of concentrated sulfuric acid was added after mixing
100 mg of the extract with 2 mL of chloroform.
3.3.6 Steroids Screening
A volume of 2 mL of decoction extract was mixed with 2 mL chloroform
and then 2 mL of concentrated sulfuric acid was added. The appearance of
reddish-brown color at the lower layer shows the existence of steroids.
A weight of 0.5 g of the extract and 2 mL of H 2SO4 were combined with 2
mL of acetic anhydride. In certain samples, the color shifted from violet to blue or
green, indicating the presence of steroids.
3.3.7 Phlobatannins
The mixture was heated after 2 mL of decoction extract and 2 mL of 1 %
HCl were added. Phlobatannins were assumed to be present based on the red
precipitate's deposition.
36
A weight of 0.5 g of extract was boiled with distilled water and 2 % HCl.
Phlobatannins were assumed to be present based on the red precipitate's
deposition.
3.3.8 Cardiac glycosides
In the test tube, 1 mL of sulfuric acid was added to the mixture of 2 mL of
extract and 2 mL of glacial acetic acid with FeCl3. Cardiac glycoside is positively
indicated by the development of a brown ring at the interface, and a violet ring
may emerge beneath the brown ring.
A volume of 5 mL of distilled water and 0.5 g were mixed. Then 2 mL of
glacial acetic acid bearing a few drops of ferric chloride was added. Alongside the
test tube, 1 mL of sulfuric acid was added. Cardiac glycoside is positively
indicated by the development of a brown ring at the interface and a violet ring
may emerge beneath the brown ring.
3.4 Brine Shrimp Lethality Test (BSLT)
Employing Miller's method, the cytotoxicity of the extracts was assessed
using the Brine Shrimp Lethality Test (BSLT) (1972). A neighborhood pet store
37
in Iligan City provided the test organism's eggs, an easy-to-grow biological
creature called Artemia salina nauplii. Using artificial seawater generated by
dissolving 38 g of NaCl in 1 L of distilled water, the eggs were made to hatch.
After that, the fluid was filtered to remove any remaining solid particles. The
mixture was then poured into a glass chamber that had two unequal divisions with
small holes. Brine shrimp eggs were then sprinkled into the larger compartment,
which had a black covering to keep it dark, while a light bulb illuminated the
smaller compartment. The eggs hatch after 48 hours, and the nauplii or young
brine were gradually transferred to the illuminated compartment because they are
drawn to light through the divisions' holes.
Using dimethyl sulfoxide as a solvent, a 4000-ppm stock solution was
prepared from the methanolic extract. Solutions in 1000-, 100-, and 10-ppm
concentrations were prepared from this stock solution by diluting 2.5-, 1.0-, and
0.5-mL of the stock solution to 5 mL with saline solution in separate test tubes. A
4000-ppm stock solution was made from the decoction. From this stock solution,
1000- and 100-ppm solutions were prepared by taking 40- and 4.0 µL and diluting
them to 5 mL in separate test tubes with brine solution. A 10-ppm solution was
made from a 100-ppm solution by taking 50 µL and diluting it to 5 mL.
To each test tube with different concentrations of two extracts (methanolic
and decoction), 10 nauplii and a drop of yeast were added. Two separate test tubes
containing only saline solution and 10 nauplii with a drop of yeast were prepared
38
that served as negative controls for each extract. All these test tubes were kept
with light on it. The number of dead nauplii was counted after 6 and 24 hours, and
the experiment was repeated three times.
Using the following formula, the morbidity percent of brine shrimp
Artemia salina was calculated:
Percent (%) mortality = No. of dead brine shrimp x 100

10
The following equation was used to correct the data if there is more than
10 % mortality in the control:
Corrected = % mortality - % mortality in control x 100

100 - % mortality in control
Using Probit analysis, the compound concentration required to kill 50 % of
the subjects (LC50) was determined from 6- and 24-hour death nauplii counting.
The Probits were calculated by looking up the percentage in Finney's (1952)
Table. The log of doses was calculated and plotted against the probits in a graph.
The LC50 was then calculated using the linear regression method.
3.5 Antibacterial Assay
Guevara’s paper disc diffusion method (2004) was used for antibacterial
treatment. Escherichia coli was used as a test organism.

39
3.5.1 Preparation of the Culture Media
All procedures were carried out in an aseptic manner. The nutrient broth
(NB) was used for bacterial subculture. NB solution was made by dissolving 0.48
grams of NB in distilled water in a clean 50 mL Erlenmeyer flask. The ratio
should be 23 g of NB dissolved to 1000 mL of distilled water. Fifteen milliliters
of the prepared broth were transferred and cotton was plugged into the pre-labeled
test tubes. The tubes were then sterilized in an autoclave for 30 minutes at a
pressure of 12 at 15 psi. The sterilized broth tubes were allowed to cool before
being aseptically inoculated with one loopful of bacteria. The broth cultures were
then incubated for 24 hours at 28 °C.
3.5.2 Preparation of Control Treatment
As the stock solution, Ampicillin at a concentration of 500 mg/mL was
prepared. Distilled water was used to dilute 9 mL of the stock solution to 15 mL,
yielding 30 mg/mL. As a negative control, the distilled water was used.
3.5.3 Preparation of Filter Paper Disc
Whatman filter paper no. 1 was cut with a paper puncher in this method.
These paper discs with a diameter of 6 mm were wrapped in aluminum foil and
autoclaved for 30 minutes at 121 °C and 15 psi.

40
3.5.4 Preparation of Agar Plates
Mueller-Hinton Agar (MHA) was used as a base for the agar plates. The
MHA was prepared by weighing 4.158 grams of MHA and dissolving it with 135
mL of distilled water in a 500 mL Erlenmeyer flask. The mixture was then placed
on the hot plate and heated to facilitate dissolution. It was constantly stirred until
the liquid became transparent. The mixture was then sterilized in the autoclave at
121 °C and at 15 psi for 30 minutes. After sterilization, 15 mL of the agar was
evenly poured into 9 Petri dishes. The agar was then allowed to solidify on a level
surface.
3.5.5 Disc Diffusion Method
Throughout the assay, the aseptic technique was strictly followed. The
MHA plates were aseptically inoculated with the test microorganism using a
cotton applicator. Filter paper discs were individually impregnated into their
corresponding extracts and placed on the surface of the agar plates using sterile
forceps. The plates were then incubated at 37 °C for 24 hours. The MHA plates
were examined after 24 hours for a zone of inhibition around the filter paper
discs.
3.6 In Vitro Evaluation of Glucose Uptake by Yeast Cells

41
The assay was performed following the method described by Jijith and
Jayakumari (2017) and Pitchaipillai and Ponniah (2016) with minor
modifications.
Commercial baker's yeast was washed in distilled water by repeated
centrifugation (5 min) until the supernatant fluids were clear, and a 10% (v/v)
suspension was prepared. A volume of 2.41 mL from each various extract
concentration (25, 50, 75, and 100 g/mL) was mixed with 2.14 mL of glucose
solution (0.125-, 0.250-, 0.375-, and 0.500 mM) and incubated at 37 °C for 10
minutes. After that, 0.45 mL of yeast suspension was added, and the mixtures
were vortexed and incubated at 37 °C for 60 minutes. After an hour of incubation,
the solutions were centrifuged for 2 minutes and the supernatant layer was drawn
and analyzed in a UV-Visible Spectrophotometer (SPECORD 200 PLUS) for
absorption at 520 nm. Metformin was used as a positive control for lowering
glucose uptake using a yeast model. The following formula was used to calculate
the percentage increase in glucose uptake by yeast cells (Cirillo, 1962):
% Increase in glucose uptake = Anegative control – Asample or drug x 100

Anegative control
Where: A - absorbance
42
The negative control contains all reagents except the test compound or
drug (glucose + yeast), whereas the positive control contains all reagents plus the
metformin (glucose + yeast + metformin) but no test compound.

CHAPTER 4
RESULTS AND DISCUSSION
4.1 Results of Phytochemical Screening
Phytochemicals such as alkaloids, flavonoids, saponins, tannins,
terpenoids, and steroids were determined in the crude decoction and methanolic
extracts of the air-dried leaves of Euphorbia heterophylla. The procedure
described by Njoku and Obi (2009) for decoction extract and Iqbal et al. (2015)
for methanol extract was used in this study. The result of this screening confirmed
the reports of Falodun et al. (2006) and Mary et al. (2020). Table 1 summarizes
the results of the analysis and the supplemental information can be found in
Appendix C.
Table 1. Phytochemical Screening Results from the Crude Methanolic and

Decoction Extract of Euphorbia heterophylla Leaves
Phytochemicals Extracts
Decoction Methanol
Alkaloids (+) (+)
Flavonoids (+) (+)
Cardiac Glycosides (-) (-)
Phlobatannins (-) (-)
Saponins (+) (+)
Steroids (+) (+)
Tannins (+) (+)
Terpenoids (+) (+)
Legends: (+) = presence, (-) = absence
43
The phytochemical screening of the crude methanolic and decoction
extracts of Euphorbia heterophylla leaves revealed the presence of alkaloids,
flavonoids, saponins, tannins, terpenoids, and steroids. See Appendix C for the
results.
4.2 IR Spectroscopic Measurement of the Crude Extracts
The crude methanolic extract and decoction extract from E. heterophylla
were subjected to partial characterization using IR Spectroscopy (Agilent
Technologies Cary 630 FTIR). The prominent peaks were identified in the IR
Spectrum of samples as presented in Table 2.
Table 2. IR Results of the Crude Methanolic and Decoction Extracts

Extract Prominent IR Peaks Functional Groups
(cm )
-1
Decoction 1010 C-O stretch, C-N stretch

1319 O-H bending
1438 O-H bending
1651 Aromatic C-H stretch
3365 O-H stretch, Aromatic
C-H stretch
Methanol 1636 C=O stretch
3265 O-H stretch
The prominent peak at 1651 cm-1 could be attributed to the presence of a
substituted benzene ring while the peak at 3365 cm -1 could be attributed to the
overlapping signal of O-H stretch and aromatic C-H stretch and the signal at 1636
44
cm-1 could be attributed to the presence of carbonyl group (see Appendix G).
These functional groups are present in the structure of the secondary metabolites
detected in the respective extracts. This result is consistent with the report of
Yuan et al. (2018) that a hydroxyl stretching vibration was detected at
approximately 3500 cm-1, and there was a carbonyl group stretching vibration
peak noted at 1717 cm-1 ~ 1616 cm-1.
4.3 Bioassays
Bioassays help determine the therapeutic potential of the test compound to
guide the isolation process toward the pure bioactive component (Choudhary et
al., 2001). The following section reveals the results in the antibacterial using disc
diffusion assay, cytotoxicity using BSLT, and inhibition of glucose using yeast
cells.
4.3.1 Result of Brine Shrimp Lethality Test (BSLT)
To investigate the cytotoxicity of the plant extracts, a brine shrimp lethality
test was performed. Meyer et al. (1982) determined that a crude plant extract is
toxic (active) if its LC50 value is less than 1000 µg/mL and non-toxic (inactive) if
it is greater than 1000 µg/mL. Table 3 and Table 4 summarize the findings from
the brine shrimp lethality test (BSLT). The number of nauplii deaths was counted
45
after 6 and 24 hours of treatment with crude methanolic and decoction extracts at
different concentrations. Percent mortality was computed using the formula;
% mortality = No. of dead larva x 100

total larvae
Corrected % mortality = % death in test vial - % death in control vial x 100

100 - % death in control vial
The probability unit values were obtained from Finney's (1952) table after 6
and 24 hours. According to the findings, the brine shrimp lethality test of all
extracts was concentration-dependent. The degree of lethality was directly
proportional to the concentration of the extract. A higher percentage of the
mortality rate indicates higher toxicity and bioactivity. It can be observed from
the table that the methanolic extract exhibits higher toxicity than the decoction
extract.
Table 3. Lethal Concentration LC50 After 6 Hours in the BSLT of the Extracts at
Different Concentrations
After 6 hours
Average %
Extract Concentration
corrected Probits LC50
(ppm)
mortality
10 0.00 1.03
Decoction 100 13.33 3.87 16,943 ppm
1000 3.33 3.12
10 16.67 4.01
Methanolic 100 13.33 3.87
46
100 43.33 4.80 9,078 ppm
Table 4. Lethal Concentration LC50 After 24 Hours in the BSLT of the Extracts at
After 24 hours
Average %
Extract Concentration
corrected Probits LC50
(ppm)
mortality
10 8.10 3.59
Decoction 260 ppm
100 37.62 4.69
1000 70.00 5.52
10 58.33 5.20
Methanolic 3.63 ppm
100 100.00 8.9538
100 100.00 8.9538
A linear relationship was found when the log value to the base 10 (log 10)
of concentrations was plotted against Probits for each extract at 6 and 24 hours.
The 50% lethal concentration (LC50) of the tested organism was calculated by
setting the y value to 5, representing 50%, in the equation of the line for each
graph. The LC50 was calculated using the inverse logarithm of the x value.
Finney's (1952) procedure served as the foundation for this procedure. The
individual graphs and LC50 are presented in Appendix H while the combined
graph for LC50 values of the extracts is shown in Figure 13 and Figure 14.
47
18,000
16,000
14,000
12,000
10,000
8,000
6,000
4,000
2,000
0
decoction methanol
Figure 13. The plot of LC50 values of the two extracts after 6 hours.
300
250
200
150
260
100
50
0 3.63
decoction methanol
Figure 14. The plot of LC50 values of the two extracts after 24 hours.
Based on Figures 13 and 14, both extracts (decoction and methanol) are
inactive after 6 hours with a concentration of 16,943 ppm and 9,078 ppm
respectively (greater than 1000 µg/mL). However, after 24 hours both extracts
48
showed cytotoxic responses with LC50 values of 260 ppm and 3.63 ppm (lower
than 1000 µg/mL). Based on these findings, it is safe to assume that both extracts
are toxic or bioactive after 24 hours of treatment and contain active or potent
components.
Preliminary phytochemical screening revealed the presence of saponins
and alkaloids. Thus, the presence of such compounds could explain the observed
cytotoxicity action. The bioactive extracts reported in this study merit additional
pharmacological and phytochemical studies to determine what kind of
antibacterial and antioxidant activity they have (if any) and to extract the natural
active constituents responsible for the activity. Such research is required before a
phytotherapeutic agent can be widely recommended for pharmaceutical use.
4.3.2 Antibacterial Assay
The antibacterial assay of crude methanolic, decoction and fresh leaf
extracts of varying concentrations was tested against Escherichia coli using the
filter paper disc diffusion method. Distilled water was used as a negative control
and Ampicillin was used as a positive control. See Appendix E for the
documentation.
49
4.3.2.1 Result of Antibacterial Disc Diffusion Assay
Table 5 presents the data gathered for the antibacterial assay of the
extracts against E. coli. The concentrations used were 10,000 ppm, 20,000 ppm,
and 30,000 ppm for crude methanolic and decoction extracts and 30,000 ppm for
ampicillin. The sap of freshly picked leaves of E. heterophylla was also tested
against the bacteria. Based on the table, it can be observed that only the reference
drug (Ampicillin) showed activity against the test bacteria. All crude extracts,
including the freshly picked leaves, did not show any inhibition according to the
method used. The growth of E. coli was observed in the second trial in the
positive control Ampicillin in the third set of the Petri dish. The growth of the test
bacteria in the zone of inhibition of the standard drug could mean that it had
developed drug resistance in that specific plate. The fresh, methanol extract
(10,000 ppm), and the negative control (distilled water) somehow exhibited a very
small zone of inhibition. This very small inhibition could have resulted from the
poor sterilization of the forceps that were used in picking the filter paper discs.
Parekh et al. (2007) described that the inhibition produced by the plant extracts
against particular organisms depends upon various extrinsic and intrinsic
parameters. Due to variable diffusability in agar medium, the antibacterial
property may not demonstrate a zone of inhibition to commensurate its efficacy.
A graphical representation of zones of inhibition of all extracts and the standard
reference material (Ampicillin) is shown in Figure 15.

50
Table 5. Antibacterial Assay Against E. coli Average Inhibition Diameter of the

Tested Concentrations (mm)
Zone of Inhibition (mm)
Extract Concentration Trial 1 Trial 2 Trial 3 Average

(ppm)
Set 1 of the Petri Dish
Decoction 10,000 0 0 0 0
Methanol 10,000 0 0 0 0
Fresh - 0 0 0 0
Ampicillin 30,000 14 16.5 17.5 16
Distilled - 0 0 0 0
water
Decoction 20,000 0 0 0 0
Methanol 20,000 0 0 0 0
Fresh - 0 0 0 0
Ampicillin 30,000 13.5 16.5 17.5 15.8
Distilled - 0 0 0 0
water
Decoction 30,000 0 0 0 0
Methanol 30,000 0 0 0 0
Fresh - 0 0 0 0
Ampicillin 30,000 16 With 18.5 17.25
growth
Distilled - 0 0 0 0
water
51
18
16
zone of inhibition (mm)
14
12
10
8
6
4
2
0
30000 20000 10000
concentration (ppm)
decoction methanol ampicillin

Figure 15. Graphical representation of the zones of inhibition of the different
extracts at varying concentrations vs. zone of inhibition of Metformin
drug.
4.3.2.2 Results of In vitro evaluation of glucose uptake by yeast cells
The increase in glucose uptake by yeast cells of the decoction and
methanolic extracts was evaluated and compared to positive control Metformin.
The highest concentration of Metformin (100 ppm) exhibited maximum activity at
all glucose concentrations and showed a maximum increase (44.98%) relative to
the negative control in the presence of 0.250 mM glucose concentration. The
decoction extract (25 ppm) exhibited a 44.77% increase in the presence of 0.250
mM glucose concentration while the methanolic extract (25 ppm) showed a
41.80% increase in glucose uptake in the presence of 0.250 mM glucose
concentration.
52
The rate of glucose transport across cell membranes in yeast cells system
at 25 and 100 ppm concentrations of decoction and methanolic extracts and
Metformin is shown in Figures 16 and Figure 17.
Effect of Decoction, Methanol Extracts and Metformin

on Glucose Uptake by Yeast Cells at 25 ppm
enhanced glucose transport %
46.00
44.00
42.00
40.00
38.00
36.00
34.00
25 50 75 100
concentration (ppm)
0.250 mM Glucose + Decoction Extract 0.250 mM Glucose + Methanol Extract

0.250 mM Glucose + Metformin
Figure 16. Effects of ethanolic and decoction extracts vs. metformin on the
uptake of glucose by yeast cells at 25 ppm concentration.
53
Effect of Decoction, Methanol Extracts and Metformin

on Glucose Uptake by Yeast Cells at 100 ppm
50.00
40.00
30.00
20.00
10.00
0.00
100
concentration (ppm)
0.250 mM Glucose + Decoction Extract 0.250 mM Glucose + Methanol Extract

0.250 mM Glucose + Metformin
Figure 17. Effects of methanolic and decoction extracts vs. metformin on the
uptake of glucose by yeast cells at 100 ppm concentration.
The test compounds' enhanced transport of glucose molecules inside the
yeast cell membrane indicates that they can lower glucose levels. This mechanism
is due to the ability of the yeast cells to use one or more sugars as their primary
source of carbon and energy and convert this sugar into ethanol. The in vitro
screening method for the inhibition of glucose uptake uses this glucose transport
mechanism across the yeast cell membrane (Cirillo, 1962; Paul & Majumdar,
2020; Teusink et al., 1998).

CHAPTER 5
SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS
5.1 Summary
The leaves of Euphorbia heterophylla were collected at some areas of the
farm of Lingayao, Las Nieves, Agusan del Norte. The leaves were manually
plucked and washed under running water to remove dirt and other unwanted
materials before air-dried. The air-dried leaves were then powdered using a
mechanical blender and stored in an air-tight bottles. An amount of 495 grams of
the powdered leaves were soaked in 1,800 mL of methanol for 72 hours with
occasional stirring. It was then filtered and the solvent was evaporated using a
rotary evaporator at 40 ℃ and 200 rpm. The residue is the methanolic extract. An
amount of 490 grams of the powdered leaves were boiled with 3,400 mL of
distilled water for 10 minutes and was filtered. The filtrate is the decoction
extract. The decoction and methanolic crude extracts of Euphorbia heterophylla
were subjected to phytochemical screening using the procedure described by
Njoku et al. (2009) and Iqbal et al. (2015). Phytochemical screening revealed the
presence of alkaloids, tannins, saponins, steroids, terpenoids, and flavonoids. The
results of IR spectroscopy of both methanolic and decoction extracts showed the
presence of functional groups that are present in the detected phytochemicals.
Crude methanol and decoction extracts were evaluated for their cytotoxic
activity against Artemia salina at 10-, 100-, and 1000 ppm concentrations. Ten
55
brine shrimp nauplii were used and three replicates were made per treatment
under illumination. Death counts at 6 and 24 hours were converted to percent
mortality and corrected if the control extract exceeds 10 % mortality. The percent
mortality of each treatment per concentration was converted to Probit value to
solve for the median lethal concentration LC50. After 6 hours of observation, all
crude extracts of E. heterophylla was found to be non-toxic (inactive) with LC50
values greater than 1000 µg/mL. However, after 24 hours of observations, the
crude methanolic and decoction extracts were found to be toxic (active) against
tested organisms with LC50 values lower than 1000 µg/mL as described by Meyer
et al. (1982). Thus, the leaves of E. heterophylla are toxic and bioactive.
The antibacterial activity of the crude methanol, decoction, and fresh leaf
extracts at varying concentrations was tested against E. coli using the Kirby-Bauer
assay or filter paper disc diffusion assay described by Guevara (2005). All of the
extracts showed no inhibition zones which indicates the inactivity of all extracts.
The in vitro evaluation of glucose uptake by yeast cells of the crude
methanolic and decoction extracts revealed that the decoction extract (25 ppm)
increased the yeast cells’ glucose uptake by 44.77% in the presence of 0.250 mM
glucose while the methanolic extract (25 ppm) increased the glucose uptake of
yeast cells only by 41.80% in the presence of 0.250 mM glucose.

56
5.2 Conclusions
Phytochemical screening revealed the presence of alkaloids, flavonoids,
saponins, steroids, tannins, and terpenoids. The presence of flavonoids in both
extracts indicates the presence of a naturally occurring phenolic compound with
antioxidant and free radical-neutralizing properties in the human diet (Del-Ro et
al., 1997). The presence of saponins in both extracts indicates that the leaves of E.
heterophylla have anti-inflammatory, antiviral, and plant-defense properties as
well as cholesterol-lowering properties (Padalia & Chadna, 2015; Moteriya et al.,
2015). Terpenoids also showed a positive result in the crude methanolic and
decoction extracts which suggests that the plant is useful in keeping agricultural
products since they prevent cholesterol formation and have insecticidal qualities
(Padalia & Chadna, 2015; Moteriya et al., 2015). Of the two extracts, the crude
methanolic extract is the most bioactive after 24 hours of treatment with an LC 50
value of 3.63 ppm. All extracts did not show any antibacterial activity according
to the method used and the results showed the inactivity of the methanol and
decoction extracts against E. coli. Based on the results of the in vitro evaluation of
glucose uptake by yeast cells, both extracts exhibited enhancement relative to the
negative control of glucose uptake by yeast cells which means that the leaves of
E. heterophylla can potentially lower glucose levels.

57
5.3 Recommendations
These are the following recommendations from the results of the study:
1. To conduct a study using other parts of the Euphorbia heterophylla such
as roots, stems, and fruits;
2. To conduct a study using different solvents to extract the bioactive
components which may have not been extracted with decoction and
methanol alone;
3. To test the antibacterial potential of the crude methanolic and decoction
extracts of E. heterophylla other than E. coli;
4. To conduct a study on anti-inflammatory, anti-viral, and other bioassays of
all extracts; and
5. To evaluate the antidiabetic potential of the extracts of the E. heterophylla
using the appropriate antidiabetic assay.

58
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APPENDIX
72
APPENDIX A
Preparation of Reagents
A. 1 % (v/v) Hydrochloric Acid (HCl)
A concentration of 1 % (v/v) HCl was prepared from 5 % stock HCl by
measuring out 6 mL of 5 % HCl and diluting it to 30 mL with distilled water.
B. 2 % (v/v) Hydrochloric Acid (HCl)
A concentration of 2 % (v/v) HCl was prepared from 5 % stock HCl by
measuring out 4 ml of 5 % HCl and diluting it to 10 mL with distilled water
C. 5 % (w/v) FeCl3 solution
Five grams of ferric chloride was dissolved in 100 mL of distilled water.
D. 10 % (w/v) Lead Acetate
A weight of 10 grams of lead acetate was dissolved in 100 mL of distilled
water.
E. Mayer’s reagent
In 60 mL of distilled water, an amount of 1.58 g of mercury chloride
(HgCl2) was dissolved. In 10 mL of distilled water, an amount of 5 grams of
potassium iodide (KI) was dissolved. The two solutions were then combined,
and distilled water was added to make a 100 mL total volume of the mixture.
73
F. Wagner’s reagent
A weight of 2 g of potassium iodide (KI) was dissolved in 5 mL of
distilled water, then 1.27 g of iodine was added and the mixture was
thoroughly mixed. To make a total volume of 100 mL, distilled water was
added.
74
APPENDIX B
Plant Sample Preparation
Figure 18. Euphorbia heterophylla plant.
Figure 19. Collected leaves of E. heterophylla.

75
Figure 20. Air drying of the leaves of Euphorbia heterophylla.
Figure 21. Homogenization of the leaves of Euphorbia heterophylla.

76
Figure 22. Maceration of the powdered leaves of Euphorbia heterophylla with

methanol for 72 hours.
Figure 23. Filtration, evaporation of the solvent, and weighing of the extracts.
77
Figure 24. Decoction and filtration of the powdered leaves of Euphorbia

heterophylla.
78
APPENDIX C
Phytochemical Screening
Figure 25. Results of phytochemical screening of the decoction and methanol

extracts of the leaves of E. heterophylla.
79
APPENDIX D
BRINE SHRIMP LETHALITY TEST (BSLT)
Figure 26. Hatching the brine shrimp.
Figure 27. Treatment of the brine shrimp.

80
APPENDIX E
ANTIBACTERIAL ASSAY
Figure 28. Preparation of the assay.

81
Figure 29. Results of the antibacterial assay.

82
APPENDIX F
Invitro Evaluation of Glucose Uptake by Yeast cells
Figure 30. Metformin drug.
Figure 31. Washing of yeast through

repeated centrifugation.
83
Figure 32. Preparation of the in vitro evaluation of glucose uptake by yeast cells.
84
Figure 33. Some of the UV-Vis results.

85
APPENDIX G
IR SPECTRA
Figure 34. IR spectrum of decoction extract.
Figure 35. IR Spectrum of methanolic extract.

86
APPENDIX H
RAW DATA
H.1 Raw Data od Brine Shrimp Lethality Test and LC50 Calculations
Table 6. Nauplii Deaths After 6 Hours in the BSLT of the Extracts at

Extracts Concentration No. of dead nauplii after 6 Average No.
(ppm) hours of Dead
T1 T2 T3 Nauplii
10 0 0 0 0
Decoction 100 2 1 1.33
1000 0 1 0 0.33
10 5 0 0 1.67
Methanol 100 2 1 1 1.33
1000 6 7 0 4.33
Control 0 0 0 0
Table 7. Nauplii Deaths After 24 Hours in the BSLT of the Extracts at

Extracts Concentration No. of dead nauplii after Average no.
(ppm) 24 hours of dead
T1 T2 T3 nauplii
10 0 4 1 1.67
Decoction 100 4 6 3 4.33
1000 5 10 6 7
Control 0 3 0 1
10 10 5 10 8.33
Methanol 100 10 10 10 10
1000 10 10 10 10
Control 6 6 4 5.33
87
Steps for LC50 Calculation:
Step 1. Convert % mortality to probits (short for probability units)
Use Finney’s table to convert % mortality to probits.
Table 8. Finney’s Table for Probit Analysis
For example, for a 4 % response, the corresponding probit would be 3.25. For
50% response (LC50), the corresponding probit would be 5.00
Step 2. Take the log of the concentrations
Step 3. Graph the probits versus the log of the concentrations and fit a line of
regression as shown in Figure 36 to Figure 39.

88
5.00
4.80
4.50
f(x) = 0.395 x + 3.43666666666667
4.00 4.01R² = 0.62054222457908 3.87
3.50
3.00
probit
2.50
2.00
1.50
1.00
0.50
0.00
1 2 3
log of conc.
Figure 36. Plot of probits against log of concentrations to produce calibration

curve after 6 hours of treatment using decoction.
5.00
4.50
4.00
3.50 f(x) = 1.0433 x + 0.587866666666667
3.00 R² = 0.503818632180055
probit
2.50
2.00
1.50
1.00
0.50
0.00
1 2 3
log of conc.

curve after 6 hours of treatment using methanol extract.
89
10
9
8
7
6
5.52
f(x) = 0.965 x + 2.67
probit
5
R² = 0.993518617305025 4.69
4
3.59
3
2
1
0
1 2 3
log of conc.

curve after 24 hours of treatment using decoction extract.
10
9 f(x) = 1.8769 x + 3.94873333333333
8.9538 8.9538
R² = 0.75
8
7
6
5.2
probit
5
4
3
2
1
0
1 2 3
log of conc.

curve after 24 hours of treatment using methanol extract.
90
Step 4. Find the LC50 using the equation of the line obtained by letting y = 5.0
depicting the death of half of the test organisms then calculating for the value of
x. The inverse log of x is the LC50.
decoction Methanol
y = 0.965x + 2.67; y = 5.0 y = 1.8769x + 3.9847; y = 5.0
5.0 = 0.965x + 2.67 5.0 = 1.8769x + 3.9847
x = 2.415 x = 0.560
LC50 = 102.415 LC50 = 100.560
LC50 = 260 ppm LC50 = 3.63 ppm
91
H.2 Raw Data in Antibacterial Assay
Table 9. Zones of Inhibition at Varying Concentrations Against E. coli

Zone of Inhibition (mm)
Extract Concentration Trial 1 Trial 2 Trial 3 Average
(ppm)
Decoction 10,000 0 0 0 0
Methanol 10,000 0 0 0 0
Fresh - 0 0 0 0
Ampicillin 30,000 14 16.5 17.5 16
Distilled - 0 0 0 0
water
Decoction 20,000 0 0 0 0
Methanol 20,000 0 0 0 0
Fresh - 0 0 0 0
Ampicillin 30,000 13.5 16.5 17.5 15.8
Distilled - 0 0 0 0
water
Decoction 30,000 0 0 0 0
Methanol 30,000 0 0 0 0
Fresh - 0 0 0 0
Ampicillin 30,000 16 With 18.5 17.25
growth
Distilled - 0 0 0 0
water
92
H.3 Raw Data In Invitro Evaluation of Enhancement of Glucose Uptake by
Yeast Cells
Figure 40. Absorbance of varying decoction extract concentrations against

varying concentrations of glucose
Figure 41. Absorbance of varying methanol extract concentrations against

varying concentrations of glucose.
93
Figure 42. Absorbance of varying metformin concentrations against varying

concentrations of glucose.
Effect of Decoction Extract on Glucose Uptake by

Yeast Cells
45.00
40.00
35.00
30.00
25.00
20.00
15.00
10.00
5.00
0.00
-5.00 25 50 75 100
Decoction extract concentration (ppm)
0.125 mM Glucose 0.250 mM Glucose 0.375 mM Glucose 0.500 mM Glucose

Figure 43. Effect of decoction extracts on the uptake of glucose by yeast cells at
varying concentrations.
94
Effect of Metformin Extract on Glucose Uptake by

Yeast Cells
50.00
40.00
30.00
20.00
10.00
0.00
25 50 75 100
-10.00
Metformin extract concentration (ppm)

Figure 44. Effect of Metformin on the uptake of glucose by yeast cells at varying
concentrations.
Effect of Methanol Extract on Glucose Uptake by

Yeast Cells
50.00
40.00
30.00
20.00
10.00
0.00
25 50 75 100
-10.00
Methanol extract concentration (ppm)

Figure 45. Effect of methanol extracts on the uptake of glucose by yeast cells at
varying concentrations.
95
CURRICULUM VITAE
GIAN FRANCO C. ABUSEJO

Purok 2B Lingayao, Las Nieves
Agusan Del Norte
Mobile Numbers: +639613939906
Email address:
gianfrancoabusejo3@gmail.com
abusejo.gc27@s.msumain.edu.ph
Personal Information
Nickname: Gian
Date of Birth: February 11, 1994
Place of Birth: Mambugan, Antipolo Rizal
Citizenship: Filipino
Sex: Male
Civil Status: Single
Formal Education
Bachelor of Science in Chemistry

Mindanao State University -Main Campus, Marawi City
January 2023
95
Thesis:
Phytochemical Screening, In Vitro Cytotoxicity, and Inhibition of Glucose Uptake
by Yeast Cells of the Philippine Euphorbia heterophylla Leaf Extracts
Lingayao, National High School
Lingayao, Las Nieves, Agusan Del Norte2011-2012
Lingayao, Elementary School
Lingayao, Las Nieves, Agusan Del Norte
2007-2008
Casiklan Elementary School
2001-2006

Phytochemical Screening, in Vitro Cytotoxicity and Inhb

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PHYTOCHEMICAL SCREENING, IN VITRO CYTOTOXICITY, AND

INHIBITION OF GLUCOSE UPTAKE BY YEAST CELLS

GIAN FRANCO C. ABUSEJO

Phytochemical Screening, In Vitro Cytotoxicity, and Inhibition of

Submitted by Gian Franco C. Abusejo in partial fulfillment of the requirement

ZAKARIYA T. MURIPAGA, MSc. MERELL P. BILLACURA, Ph.D.

ROEVE ANN MAE C. MAZO Ph.D. YUSOPH C. MANALUNDONG II, Ph.D.

YUSOPH C. MANALUNDONG II, Ph.D.

JOHNNY JIM S. OUANO, Ph.D.

ABUSEJO, GIAN FRANCO C. Phytochemical Screening, In Vitro

Adviser: ROEVE ANN MAE C. MAZO, Ph.D.

The leaves of Euphorbia heterophylla, collected from the farm of

extracts. Phytochemical screening revealed the presence of alkaloids, flavonoids,

extracts. Methanolic and decoction extracts' IR spectra revealed the presence of

the crude methanolic and decoction extracts exhibited cytotoxicity against

transport to yeast cells, respectively.

result, I'd like to express my gratitude for the following.

the obstacles and fears that have arisen this semester.

To my ever-supportive thesis adviser, for her unending help and efforts in

completing this work. Even though I hadn't finished my experiments, Ma'am

words that I needed the most: "Congratulations, mo graduate na gyud ka."

To all my panel members, Sir Yusoph Manalundong, Sir Zakariya

Paudac for their assistance in my antibacterial assay.

knowledge and wonderful experiences with me.

To my girlfriend, who also encouraged and supported me in finishing my

To my parents for properly raising me, which is why I grew up with

respect for others and fear of God.

To the Dapat and Buenafe families for treating me as a real family

member and sending me to school.

nga dili kapamilya bisag magpabadlong ko usahay.

I am gratefully dedicating this

Uncle Dominador Buenafe Jr., Ante Rosa O. Buenafe,

Nanay Emilda Dapat, Tatay Marcelino Dapat

1.1 Background of the 1

2 REVIEW OF RELATED LITERATURE………….…….. 7

2.1 History of Medicinal Plants…………………………………. 7

2.4.2.1 Test Microorganism…………………… 24

3.1 Experimental Design……………………….......................... 30

4 RESULTS AND DISCUSSIONS……………..................... 42

4.1 Results of Phytochemical 42

4.2 IR Spectroscopic Measurement of the Crude Extracts ... 43

5 SUMMARY, CONCLUSIONS, AND

1 Phytochemical Screening Results from the Crude Methanolic and

2 IR Results of the Crude Methanolic and Decoction 43

3 Lethal Concentration LC50 After 6 Hours in the BSLT of the

4 Lethal Concentration LC50 After 24 Hours in the BSLT of the

5 Average Inhibition in Diameter of the Tested Concentrations

6 Nauplii Deaths After 6 Hours in the BSLT of the Extracts at

7 Nauplii Deaths After 24 Hours in the BSLT of the Extracts at

8 Finney’s Table for Probit 87

9 Zones of Inhibition at Varying Concentrations Against E. 91

1 Structure of the alkaloids (a) Morphine and (b) Nicotine. 11

2 Structure of the two most well-known flavonoids Quercetin and

3 Structure of the saponins (a) Spirostanol and (b) Furostanol. 14

4 Structure of (a) hydrolysable tannins and (b) condensed

5 Structure of the terpenoids (a) Carvacrol and (b) 16

6 Structure of the cardiac glycosides (a) Digoxin and (b)

9 Escherichia coli ……………….…………………………………... 25

10 Chemical structure of Ampicillin…………………………………. 27

11 Research schematic diagram…….………………………………… 30

12 Satellite view of Lingayao, Las Nieves, Agusand del 31

13 The Plot of LC50 values of the two extracts after 6 47

14 The Plot of LC50 values of the two extracts after 24 hours………… 47