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PHYTOCHEMICAL SCREENING AND EVALUATION OF


ANTHELMINTIC ACTIVITY OF “TIBIG” (Ficus nota Blco.) AND “COGON”
(Imperata cylindrica L.) LEAF EXTRACTS AGAINST STRONGYLE
(Haemonchus contortus Cobb.)

A Thesis Manuscript
Presented to the Faculty of the
DEPARTMENT OF PURE AND APPLIED CHEMISTRY
College of Arts and Sciences
Visayas State University
Visca, Baybay City, Leyte

In Partial Fulfillment
of the Requirements for the Degree of
BACHELOR OF SCIENCE IN CHEMISTRY

MARY JANE MANINGO GONATO


JUNE 2018
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iii
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ACKNOWLEDGMENT

This piece of work will not be possible without You, Lord. Thank you so

much for the guidance, wisdom and courage that you’ve given me throughout the

way. Thank you for giving me such wonderful people that had really supported and

strengthened me during tough times.

I would like to express my deepest gratitude to my family. To my Mama

Mayang and Papa Jaime, thank you so much for all the heartwarming support,

morally, physically and financially. To my ever loving and caring siblings Esay, Jr

and Kuya Jimboy for reminding and uplifting my confidence always. You guys are

my precious gems and nothing compares to the love and appreciation you gave me all

the time.

To Tita Reina, Tita Ann, Tito Frank, Tita Malou, Tita Melda and Tito

Jason, thank you for imparting your thoughts and advices to me and supporting me in

my chosen course. To Lola Ping, thank you for helping me without any hesitations

and supporting me financially.

To Ms. Mary Annilyn L. Villar, for sharing your knowledge, time, efforts

and help as our thesis adviser. I am very grateful ma’am for your support, kindness

and patience especially during overnights. Thank you also ma’am for patiently

checking my manuscript and believing in my capabilities as your advisee.

Thank you also to the staff of DoPAC especially to Sir Glenn, Ma’am

Elizabeth and Sir Ronald for their time in checking my thesis outline and

manuscript. A special thanks to Dr. Jesusito L. Lim and the staff of DPM for

allowing us to conduct our thesis in your department.


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I would like to extend my appreciation to Ma’am Jane and Ate Teng of the

stockroom for allowing us to borrow materials and chemicals during the conduct of

our thesis. I am also thankful to Ma’am Rosie for the assistance inside the instrument

room.

I am also very thankful to my thesis buddies, Reshia and Khrystin. Thanks

for the cooperation, help and support. Thank you to my Team Pangasugan friends

Daisy, Russel, Aldrin, Resh, Jeric and Emie for their undying support, good vibes,

laughter and for the genuine friendship and good memories. To my classmates, thank

you for the strong bond, cooperation and inspiration towards success.

May God bless us all!!


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TABLE OF CONTENTS

CONTENT PAGE

TITLE i

TRANSMITTAL ii

GENERAL EVALUATION OF THE STUDENT THESIS iii

ACKNOWLEDGMENT iv

TABLE OF CONTENTS vi

LIST OF TABLES ix

LIST OF FIGURES x

LIST OF APPENDICES xii

LIST OF APPENDIX TABLES xiii

LIST OF APPENDIX FIGURES xv

ABSTRACT xvi

CHAPTER I – INTRODUCTION 1

Nature and Importance of the Study 1


Objectives of the Study 2
Scope and Limitation of the Study 3
Time and Place of the Study 3

CHAPTER II – REVIEW OF LITERATURE 4

Tibig (F. nota Blco.) 4


Cogon (I. cylindrica L.) 5
Secondary Metabolites in Plants 7
Alkaloids 7
Tannins and Polyphenolic Compounds 9
Saponins 11
Flavonoids 12
Triterpenes and Triterpenoids 13
Strongyle (H. contortus Cobb.) 14
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CHAPTER III – MATERIALS AND METHOD 16

Chemicals and Reagents 16


Collection of Plant Samples 16
Preparation of Plant Samples for Extraction 16
Moisture Determination of Plant Samples 17
Preparation of Plant Extracts 17
Phytochemical Screening of Ethanolic Extracts 18
Screening for Alkaloids 18
Screening for Tannins and Polyphenols 18
Screening for Saponins 19
Screening for Flavonoids 19
Screening for Terpenoids 19
Characterization of Extracts using Thin Layer Chromatography 19
Total Phenolic Determination of Extracts 20
Total Flavonoid Content Determination of Extracts 21
Preparation of H. contortus Cobb. Larvae 22
Preliminary Anthelmintic Evaluation of Extracts 23
Confirmation of Nematode Mortality 23
Evaluation of the Anthelmintic Activity of the Most Active
Extract at Different Concentrations 23
Experimental Design and Statistical Analysis 24

CHAPTER IV – RESULTS AND DISCUSSION 25

Characteristics of the Tibig and Cogon Leaves 25


Extraction of Secondary Metabolites 26
Phytochemical Screening of Tibig and Cogon Crude Extracts 27
Characterization of Extracts using Thin Layer Chromatography 29
Quantification of Total Phenolics in Tibig and Cogon Crude
Extracts 32
Quantification of Total Flavonoids in Tibig and Cogon Crude
Extracts 32
In vitro Anthelmintic Assay of Extracts against H. contortus
Cobb. 33
Evaluation of the Anthelmintic Activity of Tibig Extract at
Different Concentrations 36

CHAPTER V – SUMMARY, CONCLUSIONS AND


RECOMMENDATIONS 38

Summary 38
Conclusions 39
Recommendations 40
viii

LITERATURE CITED 41

APPENDICES 46
ix

LIST OF TABLES

Table No. Title Page

1 Secondary metabolites present in Tibig and Cogon crude


extracts 27

2 Thin-layer chromatographic separations of ethanol extracts


using hexane : ethyl acetate (20:80 % v/v) solvent
system 30
3 Total phenolic content (mg GAE/g extract) of Tibig and Cogon
crude extracts 32

4 Total flavonoid content (mg QE/g extract) of Tibig and


Cogon crude extracts 33

5 Average anthelmintic activity of Tibig and Cogon extracts


against H. contortus Cobb. after 24 hours of treatment 34

6 Percent mortality of Tibig extract and positive control


(Ivermectin) at different concentrations 37
x

LIST OF FIGURES

Figure No. Title Page

1 Image of Tibig (F. nota Blco.) (A) tree (B) fruits


(C) cross-section of the fruit (D) leaf 5

2 Image of cogon grass 6

3 Structure of quinolizidine alkaloid 8

4 Structure of cyanidin-3,5-O-diglucoside 11

5 Structure of a pentacyclictriterpene 13

6 Life cycle of H. contortus Cobb. 15

7 Tibig leaf 25

8 Cogon leaf 25

9 The crude extracts of (A) Cogon (B) Tibig leaf 26

10 Test for tannins in Tibig (TLE) and Cogon (CLE) extracts 28

11 Test for saponins in Tibig (TLE) and Cogon (CLE) extracts 28

12 Test for flavonoids in Tibig (TLE) and Cogon (CLE)


extracts 28

13 Test for terpenoids in tibig (TLE) 28

14 Thin layer chromatogram of Tibig extract viewed when


(A) charred (B) under UV light (365 nm) (C) schematic
diagram 31

15 Thin layer chromatogram of Cogon extract viewed when


(A) charred (B) under UV light (365 nm) (C) schematic
diagram 31

16 Dead L3 larvae of H. contortus Cobb. as viewed under a


scanning microscope 35
xi

Figure No. Title Page

17 Comparison between percent mortality of Tibig extract


and Ivermectin at varying concentrations 37
xii

LIST OF APPENDICES

Appendix No. Title Page

I Preparation of Reagents and Solutions 47

II Phytochemical Screening of Tibig and Cogon


Extracts 48

III Total Phenolic Content Determination 50

IV Total Flavonoid Content Determination 52

V Standard Calibration Curves 54

VI Raw data of the total phenolic content and


statistical analysis 55

VII Raw data of the total flavonoid content and


statistical analysis 57

VIII Raw data of the preliminary anthelmintic assay and


statistical analysis 59

IX Raw data of the evaluation of the most active extract at


different concentrations and statistical analysis 61
xiii

LIST OF APPENDIX TABLES

Appendix Table No. Title Page

1 Raw data of total phenolic content


(mg GAE/g extract) of Tibig and Cogon
leaf extracts 55

2 Mean rank of total phenolic content


(mg GAE/g extract) of Tibig and Cogon
leaf extracts 55

3 Test statistics using Mann Whitney


non-parametric test 56

4 Raw data of total flavonoid content


(mg QE/g extract) of Tibig and Cogon
leaf extracts 57

5 Mean rank of total flavonoid content


(mg QE/g extract) of Tibig and Cogon
leaf extracts 57

6 Test statistics using Mann Whitney


non-parametric test 58

7 Raw data of the percent mortality of Tibig


and Cogon leaf extracts against H. contortus
Cobb. 59

8 ANOVA results of the percent mortality of


Tibig and Cogon leaf extracts against
H. contortus Cobb. 59

9 Multiple comparison of the percent mortality


of Tibig and Cogon leaf extracts against
H. contortus Cobb using Tukey’s HSD 60

10 Raw data of the percent mortality of the


most active extract at varying
concentrations against H. contortus Cobb. 61

11 ANOVA results of the percent mortality of the


most active extract at varying
concentrations against H. contortus Cobb 61
xiv

Appendix Table No. Title Page

12 Multiple comparison of the percent mortality


of the most active extract at varying
concentrations against H. contortus Cobb
using Tukey’s HSD 62

13 Raw data of the percent mortality of the positive


control (Ivermectin) at varying concentrations
against H. contortus Cobb. 63

14 ANOVA results of the percent mortality of the positive


control at varying concentrations against
H. contortus Cobb. 63

15 Multiple comparison of the percent mortality


of the positive control at varying concentrations
against H. contortus Cobb. using Tukey’s HSD 64
xv

LIST OF APPENDIX FIGURES

Appendix Figure No. Title Page

1 Standard calibration curve of Gallic acid 54

2 Standard calibration curve of Quercetin 54


xvi

ABSTRACT

GONATO, MARY JANE M., June 2018. Visayas State University, Visca,
Baybay City, Leyte. “PHYTOCHEMICAL SCREENING AND EVALUATION
OF ANTHELMINTIC ACTIVITY OF “TIBIG” (Ficus nota Blco.) AND
“COGON” (Imperata cylindrica L.) LEAF EXTRACTS AGAINST
STRONGYLE (Haemonchus contortus Cobb.)”

Adviser: Ms. MARY ANNILYN L. VILLAR

This study was conducted to extract the secondary metabolites from tibig and

cogon leaf samples using ethanol as solvent; phytochemically screen the bioactive

components and partially separate the components using thin layer chromatography

(TLC); quantify the total phenolic and flavonoid contents of the extracts; and evaluate

the anthelmintic activity of both extracts against H. contortus Cobb. and determine the

mortality of the most active extract at varying concentrations.

Results showed that both extracts have found to contain bioactive components

such as tannins, saponins, flavonoids and terpenoids. TLC revealed that tibig and

cogon crude extracts resolved four and five spots, respectively. Tibig crude extract

quantitatively showed higher total phenolic content (662. 18 ± 5.00 mg GAE/g

extract) compared to cogon (76. 54 ± 2.00 mg GAE/g extract). Total flavonoid

content of tibig crude extract (24.51 ± 2.67 mg QE/g extract) was comparable to that

of cogon crude extract (29.50 ± 4.33 mg QE/g).

Results from the anthelmintic assay revealed that cogon and tibig crude extracts

exhibited an anthelmintic activity against H. contortus Cobb. at 50 ppm. Moreover,

tibig crude extract showed a significantly higher percent mortality (65 ± 21.21)

compared to cogon crude extract (40 ± 0.00). While, the effect of tibig extract at
xvii

different concentrations was not dose dependent since the results obtained were not

significantly different from each other. Thus, ethanolic extracts of both tibig and

cogon leaf can be a potential anthelmintic agent against H. contortus Cobb.

Keywords: Anthelmintic activity, Ficus nota Blco., Imperata cylindrica L.,

strongyle, H. contortus Cobb.


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CHAPTER I

INTRODUCTION

Nature and Importance of the Study

Parasitism among animals is said to be the major problem concerning

livestock production in the Philippines. The sudden weight loss and even death caused

by gastrointestinal parasites are frequently encountered in grazing livestock

production (Allonby & Urquhart, 1975) and particularly in small ruminants in

developing countries (Coles, 2005; Waller, 1997).

One of the parasitic species that is dominant in ruminant animals and said to

be the causative agent of the decrease in the rate of livestock production is the

Haemonchus contortus Cobb. It is a parasitic roundworm that has long thin

unsegmented tube-like bodies with anterior mouths and longitudinal digestive tracts.

H. contortus Cobb., which belongs to the family of nematodes or nemathelminths, is

one of the three assemblages of parasitic helminthes. This type of nematode is

commonly known as barber’s pole worm that infects cattle, sheep, goat and other wild

ruminants. It lives in the abomasum of the hosts and its eggs are shed with feces of

ruminant animals. The infection may lead to the decrease in growth, anemia and, in

serious scenarios, even cause death to the ruminant host (Hood, 2004).

Preventive measures in order to control H. contortus Cobb. infections and

other parasites have become the sought after solutions of everyone that will help

reduce the risk of the animals to become infected. Nevertheless, production of

anthelmintic drugs has become the most effective way to get rid of such parasite by

controlling the parasite at an early stage. Yet, this is not widely adapted due to the
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cost involve in acquiring such products. Moreover, resistance to anthelmintic drugs

has been known after several usage of the said product that makes the management of

intestinal parasites more difficult (Hood, 2004).

This study promotes the feasibility of using extracts from plants that contain

several bioactive components that will help in controlling such parasites in ruminant

animals. Tibig (Ficus nota Blco.) and cogon (Imperata cylindrica L.) are the possible

plants that can be of good use in preventing the growth of H. contortus Cobb. at its

larvae stage (L3). No study has been conducted on Ficus nota Blco. while cogon, only

its roots had been fully optimized for anthelmintic study. Moreover, cogon leaves

were not fully studied for its anthelmintic activity against parasite specifically H.

contortus Cobb. Furthermore, this study aimed to optimize these plants for the control

of parasites in ruminants at its larvae stage since matured ones are more difficult to

penetrate and control. The results of this study can be a good step in the promotion of

utilizing plant extracts as an effective natural anthelmintic product and as an

alternative to commercially known anthelmintic products.

Objectives of the Study

This study aimed to:

i. extract the secondary metabolites of tibig and cogon leaves;

ii. test the presence of secondary metabolites in the crude extracts using

phytochemical screening and thin layer chromatography;

iii. determine the total phenolics and total flavonoid contents in the

extracts;

iv. evaluate the anthelmintic activity of tibig and cogon leaf extracts

against H. contortus Cobb.; and


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v. evaluate the anthelmintic activity of the most active extract at different

concentrations.

Scope and Limitation of the Study

This study focused on the phytochemical screening of the crude extracts from

the leaves of tibig and cogon and further subjected to Thin Layer Chromatography.

The total phenolics and total flavonoid contents in the extracts were determined

quantitatively. Anthelmintic activity of tibig and cogon leaf extracts was also

evaluated against H. contortus Cobb. at different concentrations.

Time and Place of the Study

The study was conducted from January, 2018 to May, 2018 at the Department

of Pure and Applied Chemistry and Department of Pest Management, Visayas State

University, Baybay City, Leyte.


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CHAPTER II

REVIEW OF LITERATURE

Tibig (F. nota Blco.)

Tibig (F. nota Blco.) as shown in Figure 1, is a locally known small tree

endemic to the Philippines. It is considered as a medicinal plant that belongs to the

family Moraceae. It is an erect, spreading, dioecious tree of about 8 meters in height,

with fruits around 2-3.5 cm in diameter, fleshy and becoming yellowish-white at the

base. The leaves are simple, alternate, obovate, pubescent, and are approximately 24-

28 x 12-14 cm in size (Tolones, 2003). The fruit can be eaten raw when ripe, while

the young leaves are cooked as vegetable (Polinag, 2003) A decoction of the roots

and bark is used for urinary tract infection, hypertension and diabetes (Ragasa et al.,

1999).

There are several studies conducted that showed different chemical

constituents of Ficus species found in the Philippines. Chemical investigation of the

dichloromethane extracts of the leaves led to the discovery of some chemical

substances in Ficus species. From the study of Tsai et al. (2012), the F. odorata

leaves afforded β-sitosteryl-3β-glucopyranoside-6’-O- palmitate, squalene, lutein, α-

amyrin acetate, lupeol acetate, and β- carotene. Among these components, β-

sitosteryl-3β-glucopyranoside-6’-O- palmitate exhibited cytotoxicity against AGS cell

line with 60.28 % growth inhibition.


5

B C

A D

Figure 1. Image of tibig (F. nota Blco.) (A) tree (B) fruits (C) cross-section
of the fruit and( D) leaf
(http://www.farmersnotebookph.com/wpcontent/uploads/2014/10/
farmers notebook_warrentan_04.jpg.)

Furthermore, a study on the chemical composition of unripe fruits has shown

the presence of 4-(2-hydroxyethyl)-2-metoxyphenol, (2R, 3R)-2, 3-butanediol, (2S,

3S)-2, 3-butanediol, and β-sitosterol (Ragasa et al., 2014). Ficus species are generally

composed of flavonoid, glycosides, alkaloids, phenolic acids, steroids, saponins,

coumarins, tannins, and triterpenoids (Sirisha et al., 2010). NMR spectroscopy

revealed that the dichloromethane extract from the leaves of F. pseudopalma and F.

ulmifolia contains squalene, polyprenol, β-amyrin fatty acid ester, α-amyrin fatty acid

ester, lutein, phytol, sitosterol, and stigmasterol (Ragasa et al., 2009).

Cogon (I. cylindrica L.)

Cogon (I. cylindrica L.) as shown in Figure 2, is one of the most dominant and

noxious weeds in agricultural and non-agricultural fields. It is also known as japgrass,

bladygrass, speargrass, alang- alang and lalang-lalang (Dozier et al., 1998). Cogon

grass belongs to the family of Poaceae, formerly known as Graminae comprising 600
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genera and 9000-10,000 more species of grasses which includes lawn and forage

grasses, staple food grains, cereal crops and bamboo which is widely used in

construction (Singh et al., 2002). This grassy weed spreads by seed and vegetatively.

It is an aggressive and perennial grass that is distributed worldwide in the tropical and

subtropical region (MacDonald, 2004). It is an erect, tufted grass, 30-80 cm high with

prominent underground stem. Stems are solis, rather slender; nodes glabrous or

bearded and the leaves are flat.

Figure 2. Image of cogon grass (http://www.stuartxchange.org/Kogon.html)

I. cylindrica L. is effective in conditions like arthritis, diarrhea, cancer,

dysentery, diuretic and tonic (Datta & Banerjee, 1978). This plant is also used for soil

stabilization, thatching, and paper industry. The major chemical constituents include

carbohydrates, glycosides, flavonoids and terpenoids (Asolkar, 2005). According to

Parvathy et al. (2012), the methanolic root extracts of cogon was found to exhibit

anthelmintic activity against Indian earthworms Pheretima posthuma. The root extract

of Imperata cylindrica not only demonstrated paralysis, but also caused death of
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worms especially at higher concentration of 80 mg/50 ml, in shorter time as compared

to reference drug albendazole.

Secondary Metabolites in Plants

Considerable research has shown that some plants not only affect the nutrition

of animals, but also have antiparasitic effects (Waghorn & McNabb, 2003). Plants

secondary metabolites are known to have anti-parasitic properties. Generally non-

toxic and can consequently not be overdosed and is possible to be integrated into

normal diet of ruminants (Thamsborg, 2001a).

Alkaloids

Alkaloids are basic compounds synthesized by living organisms containing

one or more heterocyclic nitrogen atoms, derived from amino acids and

pharmacologically active. It constitutes a very large group of secondary metabolites,

with more than 12,000 substances isolated. A huge variety of structural formulas,

coming from different biosynthetic pathways and presenting very diverse

pharmacological activities are characteristics of the group (Brielmann et al., 2006).

The alkaloids have been divided into three major groups depending on the

precursor and the final structure (Waterman, 1993). Alkaloids are produced by

secondary metabolism of primary metabolites, usually amino acids. Plants produce a

wide range of biologically active chemicals which have been extensively explored for

nematode-antagonistic properties. It possess anti-oxidating effects, thus reduces

nitrate generation which is useful for protein synthesis, suppresses transfer of sucrose

from stomach to small intestine, diminishing the support of glucose to the

helminthes, intercalates into cell wall and DNA of parasites.


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Quinolizidine as shown in Figure 3 is a nitrogen-containing heterocyclic

compound. The quinolizidine alkaloids can be toxic and in some cases very toxic to

other organisms. The bio-toxicity of alkaloids has for some time been considered to

be connected with their bitter taste. It can cause depression, labored breathing,

trembling, convulsions and respiratory paralysis in sheep

(http://www.sciencedirect.com/topics/chemistry/quinolizidine-alkaloid). It has been

shown that a group of quinolizidine alkaloids were effective feeding deterrents against

a number of herbivores including insects, mollusks and mammals (Petterson et al.,

1991). Furthermore, these alkaloids were also toxic against fungi and bacteria.

Figure 3. Structure of quinolizidine alkaloid


(Maloney & Danheiser, 2005)
9

Tannins and Polyphenolic Compounds

Tannins are bitter astringent polyphenol chemicals of plant origin that are

capable of binding and shrinking proteins. Their astringent quality is what causes the

puckery and dry feeling in the mouth after eating foods rich in this type of

polyphenols or drinking teas and red wine.

Tannin polyphenols are broadly divided into two categories: hydrolysable and

condensed (proanthocyanidins). Hydrolysable tannins are molecules with a polyol

(generally D-glucose) as a central core. The hydroxyl groups of these carbohydrates

are partially or totally esterified with phenolic groups like gallic acid or ellagic acid.

These are usually present in low amounts in plants. Hydrolysable tannins have

properties such as hydrolyzed by mild acids or mild bases to yield carbohydrate and

phenolic acids. Hydrolysable tannin is potentially toxic to ruminants (Reed, 1995).

Hydrolysis

Gallotannins

Hydrolysis
Ellagitannins
10

Proanthocyanidins (condensed tannins) on the other hand, are more widely

distributed than hydrolysable tannins. They are relatively stable in the digestive tract

of the animal, and rarely have toxic effects (Reed, 1995). At high concentrations, the

condensed tannins act as anti-feedants because they bind in tight chemical complexes

with proteins, as well as microbial enzymes, thereby reducing fermentation and

degradation of fibrous tissue in the rumen (Reed, 1995; Singh & Bhat, 2003).

Additionally, they may also bind to digestive enzymes, thus reducing their activity,

and also have an astringent taste (D'Mello, 1992; Reed, 1995; Min & Hart, 2003). The

most common anthocyanidins produced are cyanidin (Fig. 4) and delphinidin (from

prodelphinidin).

According to Jones et al. (1994), condensed tannins have been found to bind

cell walls of ruminants, preventing growth and protease activity. Tannins in plants

also inhibit insect growth and disrupt digestive events in ruminant animal (Cowan,

1999). In addition, tannin-rich herbs have direct anti-parasitic activity against internal

nematodes in ruminants and can indirectly increase host resistance. The

hypersensitive response (HR) and elevated phenolic levels, leading to barrier

deposition (e.g. iignin), have been shown to be important in resistance to the root-knot

nematode Meloidogyne incognita (Paulson & Webster, 1972).


11

Figure 4. Structure of cyanidin-3,5-O-diglucoside


(https://en.wikipedia.org/wiki/Cyanidin-3, 5)

Saponins

Saponins are glucosides with foaming characteristics. Saponins consist of a

polycyclic aglycones attached to one or more sugar side chains. The aglycone part,

which is also called sapogenin, is either steroid (C27) or a triterpene (C30). The

foaming ability of saponins is caused by the combination of a hydrophobic (fat-

soluble) sapogenin and a hydrophilic (water-soluble) sugar part. Saponins have a

bitter taste and some saponins are toxic and thus, also known as sapotoxin.

This phytochemical can be found in most vegetables, beans and herbs. The

best known sources of saponins are peas, soybeans, and some herbs with names

indicating foaming properties such as soapwort, sap root, soapbark and soapberry.

(https://www.globalhealingcenter.com/natural-health/what-are-saponins/).

Saponins can have an irritating effect on mucous membranes of the respiratory

and digestive tract, potentially causing sneezing, bloating, gastroenteritis, nausea,

diarrhea, and vomiting. Saponins have also been noted for their hemolytic

properties as they can effectively dissolve the cell walls of red blood cells and disrupt
12

them when taken intravenous or intramuscularly. When taken orally however they are

comparatively harmless or they are not absorbed at all.

Literature studies reported that saponins can destabilize the cell membrane and

cuticle collagen of the parasites. Most saponins are hemolytic and display many

biological activities, such as anti-inflammatory and hypochlosterolimic effects. The

hypocholesterimic mechanism of saponins included their ability to form insoluble

complex with sterols (Malinow et al., 1997). Triterpene saponins from Quillaja

saponaria are used to control insect and nematode development (D’Addabbo et al.,

2005).

Flavonoids

Flavonoids are a diverse group of phytonutrients (plant chemicals) found in

almost all fruits and vegetables. Along with carotenoids, they are responsible for the

vivid colors in fruits and vegetables. Flavonoids are the largest group of

phytonutrients, with more than 6,000 types (https://www.livescience.com/52524-

flavonoids.html).

Most flavonoids occur in edible plants and foods as β-glycosides, bound to

one or more sugar molecules). They can be subdivided into different subgroups

depending on the carbon of the C ring on which B ring is attached, and the degree of

unsaturation and oxidation of the C ring. Flavonoids are part of the polyphenol class

of phytonutrients that are known to possess anthelmintic activity when in high

amounts (Saddique et al., 2013). Flavonoids enhanced especially the anthelmintic

activities of procyanidin tannins. According to Hoste et al. (2015), synergistic effect

between condensed tannins and two common flavonoids, quercetin and luteolin, in
13

terms of inhibiting the in vitro exsheathment of H. contortus L3 larvae was reported.

Thus, the presence of these bioactive components in plant samples increases the

anthelmintic activity due to its synergistic effect when combined.

Triterpenes and Triterpenoids

The triterpenes are one of the most numerous and diverse groups of plant

natural products. As the result of their cyclization and oxidation, various structures are

formed. Transformations occur in two ways, one producing tetra- and

pentacyclictriterpenes (Fig. 5) and the other one leading through cycloartenole to

cucurbitacines or to cholesterol and farther to phytosterols, cardiac glycosides and

steroid saponins. Triterpenes, especially pentacyclic ones, represent secondary

metabolites that are widely distributed in the plant kingdom and found in leaves, stem

bark, fruits and roots (Jäger et al., 2009).

Figure 5. Structure of a pentacyclictriterpene


(http://www.google.com/patents/US20010018459)
14

Numerous in vitro and in vivo studies have revealed their multidirectional

properties: anti-inflammatory, anti-atherosclerotic (Sudhahar et al., 2007) or antiviral

(Baltina et al., 2003). Terpenes and related plant secondary metabolites

(sesquiterpenoids and sterols) have been shown to be important factors in resistance to

several insect pests and pathogens (Harborne, 1991). The insecticidal activity of the

terpenes are either due to their action as antifeedants (or deterrents), toxins, or as

modifiers of insect development, example of terpenes are sterols such as the

phytoecdysones (Harborne, 1991). The study of the anthelmintic terpenoid

components from the Fiji sponge Axinyssa fenestratus and two Thailand sponges,

Acanthella cavernosa and Topsentia sp., has yielded several new nitrogen-containing

sesqui- and diterpenes of known carbon skeletons that are reported to have

anthelmintic activities (Alvi et al., 1991).

Strongyle (H. contortus Cobb.)

H. contortus Cobb. is also known as barber pole worm or red worm which is a

pathogenic nematode that uses ruminant animals as a host and causes haemonchosis,

an infection characterized by anemia and digestive disturbances. It is an invertebrate

nematode that belongs to the family of Trichostrongylidae and lives in the abomasums

of sheep and goats. Adult worms colonize the abomasal mucosa of the sheep and feed

on their blood. The eggs they produce are secreted in the feces, hatch, and are

ingested by the sheep through the consumptiom of grasses (Marchem et al., 1998;

Burke, 2005). Haemonchosis, in its worst case, can lead to protein deficiency, anemia,

and bottle jaw- the swelling of the lower jaw as a result of anemia, and death

(Williams, 2010).
15

The life cycle (Fig. 6) of H. contortus Cobb. takes 21 days to complete. It

begins when larvae in the infective (L3) stage are ingested by a sheep on pasture. The

larvae then travel to the animal’s abomasum, or fourth stomach. It takes three weeks

for the development of the larvae to its adult stage; the worms then attach to the

sheep’s abomasal mucosa and feed on their blood. The eggs formed during this stage

are secreted in the animal’s feces, hatch if the conditions are right, and develop

through the immature developing (L1 and L2) stages.

Figure 6. Life cycle of H. contortus Cobb.


(https://www.pinterest.com/pin/98797785547868200/)
16

CHAPTER III

MATERIALS AND METHOD

Chemicals and Reagents

All chemicals and reagents were of analytical grade and were purchased at

different chemical companies.

Collection of Plant Samples

The leaves of tibig, from the mid-part of the tree, and cogon leaves of the

same maturity were collected from the vicinity of Matag-ob, Leyte.

Preparation of Plant Samples for Extraction

The leaves of tibig and cogon were washed with tap water to remove

contaminants followed by air-drying. After drying, samples were ground using a

blender (Nutribullet). Powdered samples were sieved using a 40-mesh sieve and were

stored in a dry container. Moisture content of the powdered samples was determined

by getting the weight difference of the oven dried sample (3 g) from its initial weight.

Moisture Content Determination of the Plant Samples

The moisture present in the dried samples was quantified by oven-drying

method (AOAC, 2000). Three grams of well-mixed finely powderized tibig and

cogon samples were placed separately into a previously tared aluminum foil with

three replicates. The samples were put in the oven and the temperature was

maintained at 105 oC for at least 5 hours. It was removed from the oven and cooled in

a desiccator. The weight of dried samples and aluminum foil was obtained. The

samples were subjected again to oven drying for another 30 minutes and were
17

repeated until there is a difference of not more than 0.001 g in weight. The moisture

content was calculated using equation 1.

Equation 1:

Preparation of Plant Extracts

The homogenized leaf samples (100 g) were primarily defatted in n- hexane in

1:6 sample-solvent ratio for tibig and a 1:5 sample-solvent ratio for cogon. The

samples were soaked for about 24 hours at room temperature and then filtered using a

silk screen. Filtrates were stored in a container while the residue was dried in the

fumehood until hexane was totally removed prior to another round of extraction. The

residues (about 100 g) of tibig and cogon were soaked separately in 300 mL 80 % v/v

ethanol for 24 hours with occasional stirring. It was then primarily filtered using a silk

screen and filtrate was further filtered using a Whattman # 1 filter paper and dried in

vacuo using rotary evaporator at 30-40 ℃. Extraction process was repeated two more

times for optimum extraction. The % yield of crude leaf extracts was computed using

equation 2:

Equation 2:

The crude ethanolic extracts of tibig and cogon were transferred in a tightly-

sealed vial with proper labelling and stored in the refrigerator prior to analysis.
18

Phytochemical Screening of Ethanolic Extracts

The 0.0200 g of ethanolic extracts of tibig and cogon were dissolved in 80 %

v/v ethanol and then subjected to phytochemical screening using the method

described by Tiwari et al. (2011) as also shown in Appendix II. The phytochemicals

that were tested include alkaloids, flavonoids, saponin, terpenoids, tannins and

polyphenols.

A. Screening for Alkaloids

Mayer’s and Dragendorff’s tests (Appendix II.A) were used for the

determination of alkaloids in the crude extracts of tibig and cogon.

Mayer’s Test. The extract solutions were treated with 1 M hydrochloric acid

and then filtered. The filtrates were treated with Mayer’s reagent (potassium mercuric

iodide). A positive result for this test showed the formation of a yellow precipitate.

Dragendorff’s Test. The extract solutions were treated with 1 M hydrochloric

acid and then filtered. The filtrates were treated with Dragendorff’s reagent (solution

of potassium bismuth iodide). The formation of a red precipitate indicated the

presence of alkaloids.

B. Screening for Tannins and Polyphenols

The gelatin test (Appendix II.B) was used to test the presence of tannins and

polyphenolic compounds. A 1 % gelatin solution containing sodium chloride was

added to the extract solutions of tibig and cogon. After which, the formation of a

white precipitate indicated the presence of tannins in the extracts.


19

C. Screening for Saponins

The Froth test was used to detect the presence of saponins in the plant extracts

(Appendix II.C). In Froth test, extracts of tibig and cogon were diluted with 20 ml

distilled water. This was shaken for 15 minutes and the formation of 1 cm foam

indicated the presence of saponins.

D. Screening for Flavonoids

The plant extract solutions were screened for flavonoids using Lead Acetate

test (Appendix II.D). Primarily, extracts were treated with few drops of lead acetate

solution. A positive result for this test was the formation of a yellow precipitate.

E. Screening for Terpenoids

Salkowski’s test (Appendix II.E) was used to detect the presence of terpenoids

in tibig and cogon crude extract solutions. In this test, extracts were treated with 2 mL

chloroform and then filtered. The filtrates were added with few drops of concentrated

sulfuric acid. It was allowed to stand for several minutes. The presence of a golden

yellow precipitate in the extracts indicates the presence of triterpenes.

Characterization of Extracts using Thin Layer Chromatography

Crude extracts were further characterized using thin layer chromatography to

qualitatively determine the compounds present in the extracts. A commercially

available aluminum TLC plate coated with silica 60 F254 was used. The sheets were

cut into small plates with 2 cm x 8 cm dimension. A 1 cm mark was measured from

the bottom of the plate and a horizontal line was drawn at this mark. The extracts

reconstituted with 80 % v/v ethanol were spotted in a thin layer chromatography

(TLC) plate using a capillary tube. The spotted plate was placed vertically in a
20

developing tank pre-equilibrated with hexane and ethyl acetate (20: 80 % v/v) solvent.

The TLC plate was removed from the developing tank when the solvent was about 0.5

cm away from the upper edge of the plate. The solvent front was then marked using a

pencil. Then the plate was dried using a blow dryer at low speed. The spots in the

plate was then viewed at daylight and under UV lamp at 365 nm then the resolved

components viewed as spots were marked with a pencil. The distance of the resolved

components and the solvent front was measured. The plate was sprayed with 20 %

sulfuric acid in methanol and charred to resolve other components not seen under UV

light. The Rf values were computed using equation 3.

Equation 3:

where :

Rf = Retardation factor

Total Phenolic Content Determination of Extracts

The total phenolic content of tibig and cogon leaf extracts was determined by

using Folin-Ciocalteu reagent following a slightly modified method of Odabasoglu et

al. (2014). Gallic acid was used as the reference standard for creating the calibration

curve. Varying standard concentrations (2, 4, 6, 8, 10, 20, 40, 60, 80, 100 ppm) were

prepared using 40 % ethanol as solvent. Extract solutions of 100 ppm were also

prepared using the same solvent. A 200 µL of each extract and standard was added

with 3 mL water and mixed with 0.5 mL Folin-Ciocalteu reagent. The resulting

mixture was further neutralized with 2 % Na2CO3. The reaction mixture was

incubated at room temperature in the dark for 60 minutes. The absorbance was
21

measured at 740 nm using double beam UV-Vis spectrophotometer (Shimadzu UV-

1800) with the reagent blank as the reference. The total phenolic contents were

determined from the linear equation of a standard curve prepared with gallic acid. The

content was expressed as mg gallic acid equivalent per gram of the dry extract (mg

GAE/g).

Total Flavonoid Content Determination of Extracts

Aluminum chloride assay was used to determine the total flavonoid content of

the tibig and cogon extracts as reported by Piyanete et al. (2009) with slight

modification. Quercetin was used as the standard for creating the calibration curve.

Varying concentrations (2, 4, 8, 10, 20, 40, 80, 100 ppm) of standard solutions were

prepared using 40 % ethanol as the solvent. Extract solutions of 100 ppm were also

prepared using the same solvent. A 0.5 mL of each sample and standard was mixed

with 0.3 mL of 5 % sodium nitrite and was allowed to stand for 6 minutes. After

which, 0.3 mL of 10 % aluminum chloride solution was added and the mixture was

allowed to stand for another 5 minutes. Then, 2 mL of 1 M sodium hydroxide was

added to the mixture and the absorbance was measured at 510 nm using double beam

UV-Vis spectrophotometer (Shimadzu UV-1800). Reagent blank was also used as the

reference. The total flavonoid content was calculated from the linear equation of the

standard curve and expressed as mg Quercetin equivalents (QE)/g dry extract. All

samples were analyzed in triplicates.


22

Preparation of H. contortus Cobb. Larvae

Fecal samples which contained H. contortus Cobb. eggs were collected from

the Department of Animal Science, Visayas State University, Baybay City, Leyte.

The gathered fecal samples were macerated to release the trapped eggs. Flotation

technique was employed to isolate the eggs. Macerated fecal samples were placed in a

shot glass with moist cotton. It was placed inside the culture glass and then filled with

enough tap water up to the brim of the shot glass. The top of the cultured glass was

covered with aluminum foil with small holes while the side was covered with carbon

paper to avoid direct exposure to sunlight. The larval culture set-up was incubated for

6 days at room temperature.

Preliminary Anthelmintic Evaluation of Extracts

The larvae (L3) of H. contortus Cobb. were subjected to an anthelmintic

evaluation following the protocol of Ferreira et al. (2013) with slight modification.

Ten (10) µL suspensions containing 10 L3 were distributed in petri plates and the

reconstituted solutions of crude extracts of tibig and cogon with 1 % DMSO were

added obtaining the final concentration of extract to 50 ppm. After 24 hours of

exposure at 27 ℃, the number of motile and non-motile larvae was counted under a

stereomicroscope. Ivermectin, a known anthelmintic drug and 1 % DMSO were used

as the positive and negative control. Three replications were done and the results were

expressed as % larval mortality and computed using equation 4.

Equation 4:
23

where:

A = number of dead larvae in the extract

B = number of dead larvae in the solvent

10 = total number of larvae

Confirmation of Nematode Mortality

One of the techniques used in confirming nematode mortality was through

prodding. The prodding was done during inspection under the stereomicroscope by

slightly touching the nematode using the inoculating loop. Any observed motility

upon viewing indicated an alive larva. Furthermore, the media or the petri plate was

also shaken lightly in order to disturb the system of the nematode. Lastly, after 24

hours of treatment the nematodes were washed and resuspended in distilled water. It

was allowed to stand for 2 hours, then after which the plates were viewed again under

stereomicroscope to check and to confirm the nematode mortality.

Evaluation of the Anthelmintic Activity of the Most Active Extract at Different


Concentrations
The method described by Lorke (1983) with modification was employed to

determine the anthelmintic activity of the most active extract at different

concentrations. Five concentrations of the most active extract were prepared with the

following concentrations: 100, 40, 30, 20 and 10 ppm. Ten H. contortus Cobb. were

distributed into the petri plates with three replications for each concentration. The

same concentrations of Ivermectin solutions as positive control were prepared and 1

% DMSO was used as negative control. The H. contortus Cobb. larvae was observed

for signs of toxicity and possible deaths for 12 to 24 hours under stereomicroscope

and scanning microscope.


24

Experimental Design and Statistical Analysis

This study was conducted using Completely Randomized Design (CRD) for

the preliminary anthelmintic assay which comprised of the following treatments: T1=

tibig extract (50 ppm), T2= cogon extract (50 ppm), T3= 1 % DMSO and T4=

Ivermectin (50 ppm). Evaluation of the most active extract at varying concentrations

(100, 40, 30, 20 and 10 ppm) was done using the same experimental design.

The two-way analysis of variance (ANOVA) was utilized for the

determination of the statistical differences between treatments and Tukey’s HSD to

test differences among significant sample means. On the other hand, total phenolic

and total flavonoid content determination results were analyzed using Mann Whitney,

a non-parametric test to determine significant differences between groups.


25

CHAPTER IV

RESULTS AND DISCUSSION

One of the major problems concerning the livestock production in the

Philippines is the infestation of gastrointestinal nematodes specifically H. contortus

Cobb. in small ruminants such as goats and sheeps. In addition, several anthelmintic

products are being used to control the said parasites in small ruminants. Furthermore,

this study promotes the use of plant leaf extracts of tibig (F. nota Blco.) and cogon (I.

cylindrica L.) as an alternative anthelmintic product in controlling parasites such as H.

contortus Cobb. at its larvae (L3) stage.

Characteristics of the Tibig and Cogon Leaves

Tibig leaves are oblong to elliptic in shape with soft texture because of the

hairy covering beneath the leaves (Fig. 7). Its margins are irregular and distinctly

toothed. On the other hand, cogon has slender stems; nodes are bearded and its leaves

are flat (Fig.8). The dried sample of cogon leaves had 9.9 % moisture content which

was less compared to tibig sample (16.23 %).

Figure 7. Tibig leaf Figure 8. Cogon leaf


26

Extraction of Secondary Metabolites

The ground samples of tibig and cogon was primarily defatted with hexane to

remove the non-polar contents from the samples. Ethanol was used for extracting the

polar components in the leaf samples. In a 100 g of powdered leaf sample of tibig and

cogon soaked in 300 ml solvent, 3.73 % and 3.85 % were obtained for tibig and

cogon, respectively.

Ethanolic crude extracts of both tibig and cogon contained mostly of the polar

components. The color characteristics of the extracts were supposedly comparable to

one another since they were both leaf samples. However, the crude extract of tibig

was green while that of cogon was brownish (Fig. 9) which means that some of the

non-polar components that were not removed by hexane were carried by ethanol in

cogon extract.

A B

Figure 9. The crude extracts of (A) Cogon (B) Tibig leaf


27

Phytochemical Screening of Tibig and Cogon Crude Extracts

The ethanolic extracts of tibig and cogon were subjected to phytochemical

screening using the method described by Tiwari et al. (2011). Table 1 shows the

results of the phytochemical screening of tibig and cogon ethanolic crude extracts.

Qualitative tests were done to determine the bioactive components present in both

extracts. Results showed that both extracts were positive for tannins (Fig. 10) which

could either be the condensed or the hydrolysable tannins as indicated by the presence

of white precipitate in Gelatin test. Saponins were also found due to the formation of

froth in the extracts as shown in Figure 11. Both crude extracts had also showed

positive results for flavonoid (Fig. 12) in the Lead Acetate test due to the formation of

yellow precipitate. Moreover, only the tibig crude extract showed a positive result for

the presence of terpenoids because of the golden yellow precipitate formed as shown

in Figure 13.

Table 1. Secondary metabolites present in Tibig and Cogon crude extracts


Phytochemicals Test Observation Tibig Cogon
Extract Extract
Alkaloids Mayer's Test No precipitate - -

Dragendorff's Test No precipitate - -

Tannins and Gelatin Test White ppt + +


Polyphenols

Saponins Froth Test Has froth + +

Flavonoids Lead Acetate Test Yellow ppt + +

Terpenoids Salkowski's Test Golden-yellow + _


ppt
(+) – positive or detected
(--) – negative or not detected
28

Figure 10. Test for tannins in Tibig Figure 11. Test for saponins
(TLE) and Cogon (CLE) in Tibig (TLE) and
extracts Cogon (CLE) extracts

FLAV FLAV
TLE CLE

Figure 12. Test for saponins in Tibig Figure 13. Test for
(TLE) and Cogon (CLE) terpenoids
extracts in Tibig
(TLE)
29

Characterization of Extracts using Thin Layer Chromatography

The crude extracts of tibig and cogon were subjected to thin layer

chromatography to qualitatively determine the compounds in the extracts. Table 2

shows the results for the thin layer chromatographic separations of ethanol extracts

using hexane: ethyl acetate (20:80 % v/v) solvent system. The tibig ethanolic crude

extract (Fig. 14) showed good resolution of separations in 20:80 ratio of the solvent

system used. There were 4 spots observed on the 2 cm x 8 cm TLC plate under UV

light (365 nm) and was also charred with 20 % sulfuric acid in methanol to detect

whether there were other components that were not viewed under UV light. The

appearance of the observed spots under UV light was a mixed of light blue and red

colored spots. The first spot had a noticeable light blue appearance which could also

mean that this component maybe dominantly present in the tibig crude extract.

Moreover, spots 2 and 4 appeared to be in shades of red which suggests that

chlorophyll is present in the extract as well. Chlorophyll reflects green color and when

under UV light it readily absorbs red and blue light (Johnson, 2007). In the charred

TLC plate, only spot 1 was visible and no other additional spots were detected.

Cogon crude extract (Fig. 15) also showed a separation of bioactive

component using the same ratio of the solvent system. There were 4 spots detected

under UV light (365 nm) while an additional single spot was observed when the plate

was charred with 20 % sulfuric in methanol. The color appearances are mostly in faint

blue and a distinct red color. This would suggest that cogon extract was abundant in

chlorophyll constituents compared to other components which appeared in minimal

amount as indicated by its light coloration. However, an overlapping was observed in

spot 4 due to the solvent system used suggesting that further optimization of the
30

solvent system is very necessary to fully resolve the bioactive components in the plant

extracts. A total of 5 spots were visible in the charred TLC plate. The Rf values were

computed by the ratio of the distance traveled by the extract to the distance traveled

by the solvent.

Table 2. Thin layer chromatographic separations of the ethanol extract using hexane:
ethyl acetate (20:80 % v/v) solvent system

Color Characteristics
Extract Spot No. Rf values UV light Charred
Tibig 1 0.05 light blue faint black

2 0.06 faint red Not seen

3 0.61 faint red Not seen

4 0.97 red Not seen

Cogon 1 0.03 very faint blue faint black

2 0.36 not seen faint white

3 0.43 very faint blue faint white

4 0.63 very faint blue faint white

5 0.94 red faint black

Moreover, the pink-colored spots in both extracts were found to be larger in Rf

values and traveled more distance compared to the blue ones. This means that these

are non-polar in nature compared to the blue spots detected in both TLC plates.

Larger values of the distance traveled by the components means that the components

are less polar because it interacts less strongly with the polar adsorbent on the TLC

plate. Based on the observed colors under UV light, blue and red colored spots
31

detected pertain to the membrane-bounded components present in plant leaves such as

the chlorophylls, xanthophyll and carotene (Johnson, 2007).

1
1
A B C

Figure 14. Thin layer chromatogram of Tibig extract viewed when


(A) charred (B) under UV light (365 nm) (C) schematic diagram.

A B C

Figure 15. Thin layer chromatogram of Cogon extract viewed when


(A) charred (B) under UV light (365 nm) (C) schematic diagram
32

Quantification of Total Phenolics in Tibig and Cogon Crude Extracts

The total phenolic content of tibig and cogon ethanolic crude leaf extracts was

determined using Folin-Ciocalteu method (Odabasoglu et al., 2004). Total phenolic

content expressed as mg GAE/g extract was computed based on the equation obtained

from the standard calibration curve of Gallic acid (Appendix 4). Table 3 shows that

the total phenolic content of the two ethanolic leaf extracts from tibig and cogon

differed significantly with ethanolic extract of tibig containing a higher phenolic

content (662.18 ± 5.00 mg GAE/g extract) compared to that of cogon (76.54 ± 2.00

mg GAE/g extract).

Table 3. Total phenolic content (mg GAE/g extract) of Tibig and Cogon crude
extracts

Total Phenolic Content


Extract (mg GAE/ g extract)**
Tibig 662.18* ± 5.00
Cogon 76.54 ± 2.00
1/
** - High significance between the two groups using Mann Whitney non-parametric test.
* - Higher mean response

According to Wang et al. (2010), alcohols are good extracting solvent for

polyphenols in plant samples since it penetrates the cellular membrane easily and

extract the intracellular components from the plant material. In addition, bioactive

compounds which are aromatic and saturated organic compounds are easily extracted

using ethanol as solvent (Cowan, 1999).

Quantification of Total Flavonoids in Tibig and Cogon Crude Extracts

Aluminum chloride test was employed to determine the total flavonoid content

in both tibig and cogon crude extracts. Total flavonoid content expressed as mg QE/g

extract was computed based on the equation obtained from the standard calibration
33

curve of quercetin (Appendix 4). Table 4 shows the total flavonoid content in tibig

and cogon leaf extracts. Results revealed that cogon extract had not significantly

higher flavonoid content (29.50 ± 4.33 mg QE/g extract) than tibig extract (24.51 ±

2.67 mg QE/g extract). Hence, tibig and cogon extracts had comparable total

flavonoid contents

Table 4. Total flavonoid content (mg QE/g extract) of Tibig and Cogon crude extracts

Total Flavonoid Content


Extract (mg QE/ g extract)ns
Tibig 24.51 ± 2.67
Cogon 29.50*± 4.33
1/
*- Higher mean response
ns - No significant difference between the two groups using Mann Whitney non-parametric
test.

In vitro Anthelmintic Assay of Extracts against H. contortus Cobb.

The larvae (L3) of H. contortus Cobb. were subjected to anthelmintic assay

using the method of Ferreira et al. (2013) with slight modification. Table 5 shows the

average anthelmintic activity of tibig and cogon extracts against H. contortus Cobb.

using 50 ppm concentration of extract after 24 hours of treatment.

Based on the results obtained, ethanolic extracts of tibig and cogon had

exhibited anthemintic activity against third stage larvae (L3) of H. contortus Cobb at

50 ppm concentration after 24 hours of treatment. Furthermore, the anthelmintic

activity of tibig crude extracts which was significantly higher (65 % mortality)

compared to cogon crude extracts (40 % mortality). Moreover, the percent mortality

of tibig extract was significantly comparable to that of the positive control

(Ivermectin) at 0.05 level of significance.


34

Table 5. Average anthelmintic activity of Tibig and Cogon crude extracts against H.
contortus Cobb. after 24 hours of treatment

% Corrected
Treatments Extract % Mortality** Mortality**
24 hours 24 hours
T1 Control - 1 % DMSO 25b ± 7.07
T2 Control - Ivermectin 100a ± 0.00
T3 Tibig 90ab ± 14.14 65ab ± 21.21
T4 Cogon 65b ± 7.07 40b ± 0.00
1/
**- Indicates highly significant result at 0.05 level of significance using Tukey’s HSD.
Means in a column having different letters are significantly different from each other.
Means in a column having the same letter are significantly comparable from each other.

The anthelmintic activity of tibig and cogon crude extracts was possibly due to

the present bioactive components in the leaf samples. Ethanolic crude extract of tibig

had qualitatively shown positive results for tannins and polyphenols, saponins,

flavonoids, and terpenoids while the ethanolic extract of cogon was positive to tannins

and polyphenols, flavonoids and saponins only. In addition, tibig crude extract

showed higher total phenolic content (662.18 ± 5.00 mg GAE/g extract) compared to

cogon crude extract (76.54 ± 2.00 mg GAE/g extract), respectively. This implies that

the bioactivity of extracts could be a synergistic effect of the secondary metabolites

present. Also, exposure to extracts from tannin-containing plants caused degeneration

of muscle cells, accumulation of electron-dense vesicles and marked intracellular

disorganization to organism (Brunet et al., 2011). In addition, tannins have been

shown to interfere with coupled oxidative phosphorylation thus blocking ATP

synthesis in parasites (Martin, 1997). This may also bind to the cuticle of the

helminth’s body surface making it immobile causing the parasite to become paralysed

leading to its death (Thompson & Geary, 1995). Another possible anthelmintic effect
35

of tannins is that they can bind to free proteins in the gastrointestinal tract of host

animal or glycoprotein on the cuticle of the parasite and cause death (Patel et al.,

2010). Moreover, tannins are found to reduce the motility of the L3s, which may

indicate paralysis and interference to neuromuscular coordination of the larvae

(Molan et al., 2000).

The tibig and cogon crude extracts also contained saponins which are reported

to destabilize the cell membrane and cuticle collagen of the parasite (D’Addabbo et

al., 2010). Saponins are typically toxic and could cause rupturing and lysis of red

blood cells. However, this mechanism of saponins depends on the level of

concentration and the time of exposure. In addition, flavonoids are part of the

polyphenol class of phytonutrients that are known to possess anthelmintic activity

when in high amounts (Saddique et al., 2013).

Dead H.
contortus
Cobb.

Figure 16. Dead L3 of H. contortus Cobb. as viewed under a scanning microscope

It was also observed that after 12 hours’ time of treatment, several parasites

treated with tibig extract had been paralyzed when viewed under the microscope

compared to cogon extract. After 24 hours’ treatment, there was no observed motility
36

in the parasite and the parasite became thinner and flat in structure (Fig. 16). The dead

H. contortus Cobb was confirmed by slightly prodding the nematode by an

inoculating loop and disturbing the media or the petri plate by slightly shaking.

Evaluation of the anthelmintic activity of Tibig Extract at Different


Concentrations

The anthelmintic activity of the most active extract which was the tibig extract

was evaluated using different concentrations (100, 40, 30, 20 and 10 ppm) and the

results are presented in Table 6 and Figure 17. The percent mortality of the H.

contortus Cobb. at different concentrations of tibig extract did not differ significantly

suggesting that the effect of the extract to the H. contortus Cobb. is not dose

dependent. It was observed that at 100 ppm, the % mortality reached up to 80.09 ±

18.86. Higher mortality rate was expected at higher concentrations of the tibig extract

since the extract might contain the maximum amount of bioactive components present

in the plant extract. In 40 ppm, the % mortality recorded was about 51.39 ± 14.63. At

this concentration, the number of dead L3 was half of the total number (10) of H.

contortus Cobb. per plate. However, as the concentration decreases (from 30 to 10

ppm), the mortality rate of the extract also increased with 56.48 ± 16.28 at 30 ppm,

79.63 ± 8.01 at 20 ppm and 75.93 ± 12.60 at 10 ppm, respectively.

Figure 18 shows the comparison between the positive control (Ivermectin) and

tibig extract. Results show that the positive control (Ivermectin) exhibited a

statistically decreasing mortality as its concentration decreases suggesting that the

effect was dose-dependent. On the other hand, the effect of tibig extract was not dose-

dependent since at lower concentrations, percent mortality increased and was

significantly higher than that of Ivermectin (positive control). The plausible cause for
37

such trend in the percent mortality was primarily attributed to the H. contortus Cobb.

species being subjected for this assay. Although, the L3 larvae were cultured at the

same number of days prior to the assay but the same maturity of the species did not

guarantee that they had acquired the same physical survival nature. Nevertheless, tibig

extract exhibited anthelmintic activity at varying concentrations and can be a potential

alternative for chemically-based anthelmintic products.

Table 6. Percent mortality of Tibig extract and positive control (Ivermectin) at


different concentrations

% Mortality
ns
Concentration (ppm) Tibig Ivermectin
100 80.09 ± 18.86 100.00a ± 0.00
40 51. 39 ± 14.63 90.00ab ± 0.00
30 56.48 ± 16.28 76.67b ± 5.77
20 79. 63 ± 8.01 60.00c ± 10.00
10 75.93 ± 12.60 26.67d ± 5.67
1/
Means in a column having different letters are significantly different from each other.
ns - Means in a column are not significantly different from each other at 0.05 confidence
level using Tukey’s HSD.

120
100
80
% Mortality

60
Tibig
40
Ivermectin
20
0
100 ppm 40 ppm 30 ppm 20 ppm 10 ppm
Concentration

Figure 17. Comparison between percent mortality of Tibig extract and Ivermectin
at varying concentrations
38

CHAPTER V

SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

Summary

This study was conducted to (1) extract the secondary metabolites of tibig and

cogon leaves, (2) test the presence of secondary metabolites in the crude extracts

using phytochemical screening and thin layer chromatography, (3) quantify the total

phenolics and total flavonoids in the extracts, (4) evaluate the activity of the extracts

against H. contortus Cobb. and (5) evaluate the anthelmintic activity of the most

active extract at different concentrations.

Results of the phytochemical screening revealed the presence of bioactive

components in both tibig and cogon extracts. Tibig and cogon were found positive for

tannins and polyphenols, saponins and flavonoid. Only the tibig crude extract showed

a positive result for terpenoids. Moreover, thin layer chromatography was used to

further characterize and separate the bioactive components in the extracts. Four spots

were resolved in tibig extract while five spots in cogon extract using the same solvent

system of hexane to ethyl acetate (20:80 % v/v).

Total phenolic and flavonoid contents of the extracts were determined

quantitatively through different standard procedures. It was accounted that tibig

extract had a total phenolic content of 662. 18 mg GAE/ g extract while cogon extract

has 76. 55 mg GAE/ g extract. In addition, cogon extract had a total flavonoid content

of 29. 50 mg QE/ g extract while tibig has 24.51 mg QE/ g extract.


39

Based on the results for the anthelmintic assay, tibig crude extracts showed

much higher % mortality against larvae of H. contortus Cobb. compared to cogon

extract. Statistically, the % mortality of tibig extract at 50 ppm for 24 hours of

exposure was comparable to that of the positive control (Ivermectin) at 0.05 level of

significance. In addition, the results for anthelmintic assay in which tibig extract was

tested at varying concentrations, revealed that the effect were not significantly

different from each other which means that the mortality of H. contortus Cobb. was

not dose-dependent to the tibig extract. However, the effect of Ivermectin (positive

control) exhibited a dose-dependent effect to the H. contortus Cobb. Nevertheless,

both tibig and cogon extracts can be an anthelmintic agent against H. contortus Cobb.

Conclusions

Based on the results obtained, the following conclusions can be drawn:

1. Tibig and cogon ethanolic extracts showed the presence of secondary metabolites

such as tannins, saponins, flavonoids and terpenoids.

2. The tibig leaf extract exhibited a higher phenolic content compared to cogon.

3. Cogon extract was found to have higher flavonoid content than tibig extract.

4. Tibig extract showed a high potential anthelmintic activity against H. contortus

Cobb. compared to cogon extract.

5. The effect of tibig extract at varying concentrations against H. contortus Cobb. was

not dose-dependent.
40

Recommendations

The following recommendations are made for future studies:

1. Another type of solvent for the extraction of the plant samples should be optimized.

2. Other parts of the plant such as the fruit of tibig should be studied.

3. An effective confirmatory staining agent should be used to fully examine the

mortality of the nematode species.

4. Isolation of the secondary metabolite responsible for the anthelmintic activity of the

most active extract should be pursued.

5. The effect of tibig and cogon extracts to another nematode species should be

evaluated.
41

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46

APPENDICES
47

APPENDIX I

PREPARATION OF REAGENTS AND SOLUTIONS

Test for Tannins


a. 10% NaCl

Dissolve 10 g NaCl crystals with water. Transfer to a 100 mL volumetric

flask and dilute to mark.

b. 1% Ferric Chloride

Dissolve 1g solid ferric chloride with water. Transfer to a 100 mL volumetric

flask and dilute to mark.

c. 1% Tannic acid

Dissolve 1 g of tannic acid with enough distilled water to make 100 mL

solution.

Total Phenolic Content Determination

1. Weigh 0.0075 g of Gallic acid and dissolved with 40 % ethanol in 25 ml

volumetric flask.

Preparation of Folin-Ciocalteu reagent

1. Dilute 2.5 ml of Folin-ciocalteu reagent in 25 ml volumetric flask.

2. Dilute to mark with 40 % ethanol.

Total Flavonoid Content Determination

1. Weigh 0.0005 g of Quercetin and dissolved with 40 % ethanol in 25 ml

volumetric flask.

Preparation of the Aluminum chloride solution

1. Weigh 2.5 g of AlCl3 and dissolved with distilled water and dilute to mark in a

25 ml volumetric flask.
48

APPENDIX II

PHYTOCHEMICAL SCREENING OF TIBIG AND COGON EXTRACTS


(Modified Tiwari et al., 2011)

A. Screening for Alkaloids

Mayer’s Test

1. 1 ml of the extract was treated with 2 ml of hydrochloric acid.

2. The mixture was filtered using a filter paper.

3. The filtrates of the ethanolic extracts of tibig and cogon were added with

2-3 drops potassium mercuric iodide.

4. Formation of a yellow precipitate shows the presence of alkaloids in the

samples.

Dragendorff’s Test

1. 1 ml of the extract was treated with 2 ml of hydrochloric acid.

2. The mixture was filtered using a filter paper.

3. The filtrates of the ethanolic extracts of tibig and cogon were added with

2-3 drops of potassium bismuth iodide.

4. Formation of a red precipitate indicates the presence of alkaloids in the

extracts.

B. Screening for Tannins

Gelatin Test

1. 1 ml of the extracts of tibig and cogon were added with 1 % gelatin

solution containing sodium chloride.

2. The formation of a white precipitate indicates the presence of tannins.


49

C. Screening for Saponins

Froth Test

1. Dilute 5 mL of the filtrate with 15 mL of water and shake vigorously. A

stable froth up on standing indicates the presence of saponins.

D. Screening for Flavonoids

Lead acetate Test

1. 1 ml of the extracts was added with 2-3 drops of lead acetate solution.

2. The formation of a yellow precipitate indicates the presence of flavonoids

in the extracts.

E. Terpenoids

Salkowski’s Test

1. 1 ml of the extracts was treated with chloroform and then filtered.

2. The filtrates were added with few drops of concentrated sulfuric acid and

were shaken.

3. The formation of a golden yellow precipitate indicates the presence of

terpenoids.
50

APPENDIX III

TOTAL PHENOLIC CONTENT DETERMINATION


(Folin-Ciocalteu Method, Odabasoglu et al., 2004)

Preparation of Standard Gallic Acid


1. Weigh 0.0075 g of Gallic acid and dissolved with 40 % ethanol in 25 ml

volumetric flask.

Preparation of Folin-Ciocalteu reagent

2. Dilute 2.5 ml of Folin-ciocalteu reagent in 25 ml volumetric flask.

3. Dilute to mark with 40 % ethanol.

Preparation of Standards by Serial Dilution


1. 100 ppm – Measure 8.33 ml from stock solution and dilute to mark with 40 %

ethanol in 10 ml volumetric flask.

2. 80 ppm – Measure 8.00 ml from 100 ppm and dilute to mark with 40 %

ethanol in 10 ml volumetric flask.

3. 60 ppm – Measure 7.5 ml from 80 ppm and dilute to mark with 40 % ethanol

in 10 ml volumetric flask.

4. 40 ppm – Measure 6.67 ml from 60 ppm and dilute to mark with 40 % ethanol

in 10 ml volumetric flask.

5. 20 ppm – Measure 5 ml from 40 ppm and dilute to mark with 40 % ethanol in

10 ml volumetric flask.

6. 10 ppm – Measure 5 ml from 20 ppm and dilute to mark with 40 % ethanol in

10 ml volumetric flask.

7. 8 ppm – Measure 8 ml from 10 ppm and dilute to mark with 40 % ethanol in

10 ml volumetric flask.
51

8. 6 ppm – Measure 7.5 ml from 8 ppm and dilute to mark with 40 % ethanol in

10 ml volumetric flask.

9. 4 ppm – Measure 6.67 ml from 6 ppm and dilute to mark with 40 % ethanol in

10 ml volumetric flask.

10. 2 ppm – Measure 5 ml from 4 ppm and dilute to mark with 40 % ethanol in 10

ml volumetric flask.

11. 1 ppm – Measure 5 ml from 2 ppm and dilute to mark with 40 % ethanol in 10

ml volumetric flask.

For the Prepared Standard Solutions:

1. Get 200 µL from each dilution.

2. Add 3 ml distilled water and 0.5 ml Folin-Ciocalteu reagent.

3. Add 2 ml 2 % Na2CO3 and stand for 60 minutes in the dark.

4. Read the absorbance at 650 nm using UV-Vis Spectrophotometer.

Preparation of the Extracts

1. Weigh 0.0002 g of the extracts and dissolved with 40 % ethanol in 10 ml

volumetric flask.

2. Get 200 µL from the prepared extracts with three replicates.

3. Add 3 ml distilled water and 0.5 ml Folin- Ciocalteu reagent.

4. Add 2 ml 2 % Na2CO3 and stand for 60 minutes in the dark.

5. Read the absorbance at 650 nm using UV-Vis Spectrophotometer.


52

APPENDIX IV

TOTAL FLAVONOID CONTENT DETERMINATION


(Aluminum chloride complex assay, Piyanete et al., 2009)

Preparation of Standard Quercetin

1. Weigh 0.0005 g of Quercetin and dissolved with 40 % ethanol in 25 ml

volumetric flask.

Preparation of the Aluminum chloride solution

2. Weigh 2.5 g of AlCl3 and dissolved with distilled water and dilute to mark in a

25 ml volumetric flask.

Preparation of Standards by Serial Dilution

1. 100 ppm – Measure 5 ml from stock quercetin solution (200 ppm) and dilute

to mark with 40 % ethanol in 10 ml volumetric flask.

2. 80 ppm – Measure 8 ml from 100 ppm and dilute to mark with 40 % ethanol

in 10 ml volumetric flask.

3. 60 ppm – Measure 7.5 ml from 80 ppm and dilute to mark with 40 % ethanol

in 10 ml volumetric flask.

4. 40 ppm – Measure 6.67 ml from 60 ppm and dilute to mark with 40 % ethanol

in 10 ml volumetric flask.

5. 20 ppm – Measure 5 ml from 40 ppm and dilute to mark with 40 % ethanol in

10 ml volumetric flask.

6. 10 ppm – Measure 5 ml from 20 ppm and dilute to mark with 40 % ethanol in

10 ml volumetric flask.

7. 8 ppm – Measure 8 ml from 10 ppm and dilute to mark with 40 % ethanol in

10 ml volumetric flask.
53

8. 6 ppm – Measure 7.5 ml from 8 ppm and dilute to mark with 40 % ethanol in

10 ml volumetric flask.

9. 4 ppm – Measure 6.67 ml from 6 ppm and dilute to mark with 40 % ethanol in

10 ml volumetric flask.

10. 2 ppm – Measure 5 ml from 4 ppm and dilute to mark with 40 % ethanol in 10

ml volumetric flask.

11. 1 ppm – Measure 5 ml from 2 ppm and dilute to mark with 40 % ethanol in 10

ml volumetric flask.

For the Prepared Standard Solutions:

1. Get 0.5 ml from each dilution.

2. Add 0.3 ml of 5 % sodium nitrite and allowed to stand for 6 minutes.

3. Add 0.3 ml of 10 % AlCl3 and stand for 5 minutes.

4. Finally, add 2 ml of 1 M NaOH.

5. Read the absorbance at 510 nm using UV-Vis Spectrophotometer.

Preparation of the Extracts

1. Weigh 0.0200 g of the extracts and dissolved with 40 % ethanol in a 10 ml

volumetric flask.

2. Get 0.5 ml of the prepared extracts with three replicates.

3. Add 0.3 ml of 5 % sodium nitrite and allowed to stand for 6 minutes.

4. Add 0.3 ml of 10 % AlCl3 and stand for 5 minutes.

5. Finally, add 2 ml of 1 M NaOH.

6. Read the absorbance at 510 nm using UV-Vis Spectrophotometer.


54

APPENDIX V

STANDARD CALIBRATION CURVES

0.12
y = 0.3609x + 0.0025
0.1 R² = 0.9948

0.08
Absorbance

0.06

0.04

0.02

0
0 0.05 0.1 0.15 0.2 0.25 0.3
Concentration (ppm)

Appendix Figure 1. Standard calibration curve of Gallic acid.

0.1400

0.1200 y = 0.8822x + 0.024


R² = 0.9871
0.1000
Absorbance

0.0800

0.0600

0.0400

0.0200

0.0000
0 0.02 0.04 0.06 0.08 0.1 0.12
Concentration (ppm)

Appendix Figure 2. Standard calibration curve of Quercetin


55

APPENDIX VI

RAW DATA OF TOTAL PHENOLIC CONTENT AND STATISTICAL


ANALYSIS

Appendix Table 1. Raw data of total phenolic content (mg GAE/g extract)
of Tibig and Cogon leaf extracts

Replications
Crude extract 1 2 3 Average
Tibig 763.24 633.33 589.96 662.18
Cogon 12.59 135.07 81.97 76.55

Appendix Table 2. Mean rank of total phenolic content (mg GAE/g extract) of Tibig
and Cogon leaf extracts

RANKS

TPC Group N Mean Mean Rank Sum of Ranks

Tibig 3 662.18* 5.00 15.00

Cogon 3 76.55 2.00 6.00

Total 6

* - Higher mean response


56

Appendix Table 3. Test statistics using Mann Whitney non-parametric test

Test Statisticsa

Mann-Whitney U .000

Wilcoxon W 6.000

Z -1.964

Asymp. Sig. (2-tailed) .050**

**. There is a significant difference between two groups.


57

APPENDIX VII

RAW DATA OF TOTAL FLAVONOID CONTENT AND STATISTICAL


ANALYSIS

Appendix Table 4. Raw data of total flavonoid content (mg QE/g extract) of Tibig
and Cogon leaf extract

Replications
Crude extract 1 2 3 Ave.
Tibig 26.94 21.45 25.12 24.51
Cogon 30.73 24.16 33.61 29.5

Appendix Table 5. Mean rank of total flavonoid content (mg QE/g extract) of Tibig
and Cogon leaf extract

RANKS

TFC Group N Mean Mean Rank Sum of Ranks


Tibig 3 24.51 2.67 8.00

Cogon 3 29.50* 4.33 13.00

Total 6

*- Higher mean response


58

Appendix Table 6. Test statistics using Mann Whitney non-parametric test

Test Statisticsa

Mann-Whitney U 2.000

Wilcoxon W 8.000

Z -1.091

Asymp. Sig. (2-tailed) .275*

*- No significant difference between the two groups.


59

APPENDIX VIII

RAW DATA OF THE PRELIMINARY ANTHELMINTIC ASSAY AND


STATISTICAL ANALYSIS

Appendix Table 7. Raw data of the percent mortality of Tibig and Cogon leaf
extracts against H. contortus Cobb

Extract N R1 % R2 % Average Corrected


Mortality Mortality % %
Mortality Mortality

Tibig 10 10 100 8 80 90 65
Cogon 10 6 60 7 70 65 40
Ivermectin 10 10 100 10 100 100
1 % DMSO 10 2 20 3 30 25

Appendix Table 8. ANOVA results of the percent mortality of Tibig and Cogon leaf
extracts against H. contortus Cobb

Sum of Mean
Squares df Square F Sig.

Between Groups 6450 3 2150 17.2 0.009469**

Within Groups 500 4 125

Total 6950 7
** - Treatments are highly significant (p-value < 0.05)
60

Appendix Table 9. Multiple comparison of the percent mortality of Tibig and Cogon
leaf extracts against H. contortus Cobb using Tukey’s HSD

95% Confidence
Mean Interval
(I) (J) Std.
Difference Sig.
TREATMENT TREATMENT Error Lower Upper
(I-J)
Bound Bound
1 2 25.000 11.180 .089 -6.04 56.04

3 -35.000* 11.180 .035 -66.04 -3.96

4 40.000* 11.180 .023 8.96 71.04

2 1 -25.000 11.180 .089 -56.04 6.04

3 -60.000* 11.180 .006 -91.04 -28.96

4 15.000 11.180 .251 -16.04 46.04

3 1 35.000* 11.180 .035 3.96 66.04

2 60.000* 11.180 .006 28.96 91.04

4 75.000* 11.180 .003 43.96 106.04

4 1 -40.000* 11.180 .023 -71.04 -8.96

2 -15.000 11.180 .251 -46.04 16.04

3 -75.000* 11.180 .003 -106.04 -43.96

*. The mean difference is significant at the 0.05 level.


61

APPENDIX IX

RAW DATA OF THE EVALUATION OF THE MOST ACTIVE EXTRACT


AT DIFFERENT CONCENTRATIONS AND STATISTICAL ANALYSIS

Appendix Table 10. Raw data of the percent mortality of the most active extract at
varying concentrations against H. contortus Cobb.

Replications Corrected
(% Mortality) Mortality
(ppm) N 1 % 2 % 3 % 1 2 3 Ave

100 10 10 100 7 70 8 80 100 62.5 78 80.09

40 10 5 50 6 60 7 70 37.5 50 67 51.39

30 10 6 60 8 80 5 50 50 75 44 56.48

20 10 8 80 8 80 9 90 75 75 89 79.63

10 10 9 90 7 70 8 80 87.5 62.5 78 75.93

1% DMSO 10 2 20 2 20 1 10 16.67

Appendix Table 11. ANOVA results of the percent mortality of the most active
extract at varying concentrations against H. contortus Cobb.

Sum of df Mean F Sig.


Squares Square
Between Groups 2693.333 4 673.333 1.942 .180
Within Groups 3466.667 10 346.667
Total 6160.000 14

Treatments are not significantly different from each other (p-value > 0.05).
62

Appendix Table 12. Multiple comparison of the percent mortality of the most active
extract at varying concentrations against H. contortus Cobb.
using Tukey’s HSD

95%
Mean Confidence
(I) (J) Std.
Difference Sig. Interval
CONC. CONC. Error
(I-J) Lower Upper
Bound Bound
10 20 -30.000 15.202 .077 -63.87 3.87

30 3.333 15.202 .831 -30.54 37.21

40 -3.333 15.202 .831 -37.21 30.54

100 -23.333 15.202 .156 -57.21 10.54

20 10 30.000 15.202 .077 -3.87 63.87

30 33.333 15.202 .053 -.54 67.21

40 26.667 15.202 .110 -7.21 60.54

100 6.667 15.202 .670 -27.21 40.54

30 10 -3.333 15.202 .831 -37.21 30.54

20 -33.333 15.202 .053 -67.21 .54

40 -6.667 15.202 .670 -40.54 27.21

100 -26.667 15.202 .110 -60.54 7.21

40 10 3.333 15.202 .831 -30.54 37.21

20 -26.667 15.202 .110 -60.54 7.21

30 6.667 15.202 .670 -27.21 40.54

100 -20.000 15.202 .218 -53.87 13.87

100 10 23.333 15.202 .156 -10.54 57.21

20 -6.667 15.202 .670 -40.54 27.21

30 26.667 15.202 .110 -7.21 60.54

40 20.000 15.202 .218 -13.87 53.87


63

Appendix Table 13. Raw data of the percent mortality of the positive control
(Ivermectin) at varying concentrations against H. contortus
Cobb.

Replications
[ppm] N 1 % 2 % 3 % Ave

100 10 10 100 10 100 10 100 100

40 10 9 90 9 90 90 90 90

30 10 8 80 7 70 80 80 76.67

20 10 7 70 5 50 60 60 60

10 10 3 30 2 20 30 30 26.67

Appendix Table 14. ANOVA results of the percent mortality of the positive control
at varying concentrations against H. contortus Cobb.

Sum of df Mean F Sig.


Squares Square

Between Groups 9960.000 4 2490.000 74.700 2.079e-07


***

Within Groups 333.333 10 33.333


Total 10293.333 14
*** - Treatments are very highly significant (p-value <0.05).
64

Appendix Table 15. Multiple comparison of the percent mortality of the positive
control at varying concentrations against H. contortus Cobb.
using Tukey’s HSD

95%
Mean Confidence
(I) (J) Std.
Difference Sig. Interval
CONC CONC Error
(I-J) Lower Upper
Bound Bound
10 20 -33.333* 4.714 .000 -43.84 -22.83

30 -50.000* 4.714 .000 -60.50 -39.50

40 -63.333* 4.714 .000 -73.84 -52.83

100 -73.333* 4.714 .000 -83.84 -62.83

20 10 33.333* 4.714 .000 22.83 43.84

30 -16.667* 4.714 .005 -27.17 -6.16

40 -30.000* 4.714 .000 -40.50 -19.50

100 -40.000* 4.714 .000 -50.50 -29.50

30 10 50.000* 4.714 .000 39.50 60.50

20 16.667* 4.714 .005 6.16 27.17

40 -13.333* 4.714 .018 -23.84 -2.83

100 -23.333* 4.714 .001 -33.84 -12.83

40 10 63.333* 4.714 .000 52.83 73.84

20 30.000* 4.714 .000 19.50 40.50

30 13.333* 4.714 .018 2.83 23.84

100 -10.000 4.714 .060 -20.50 .50

100 10 73.333* 4.714 .000 62.83 83.84

20 40.000* 4.714 .000 29.50 50.50

30 23.333* 4.714 .001 12.83 33.84

40 10.000 4.714 .060 -.50 20.50

*. The mean difference is significant at the 0.05 level.

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