Plant
Plant
Plant
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
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
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
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
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
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.
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.
TABLE OF CONTENTS
CONTENT PAGE
TITLE i
TRANSMITTAL ii
ACKNOWLEDGMENT iv
TABLE OF CONTENTS vi
LIST OF TABLES ix
LIST OF FIGURES x
ABSTRACT xvi
CHAPTER I – INTRODUCTION 1
Summary 38
Conclusions 39
Recommendations 40
viii
LITERATURE CITED 41
APPENDICES 46
ix
LIST OF TABLES
LIST OF FIGURES
4 Structure of cyanidin-3,5-O-diglucoside 11
5 Structure of a pentacyclictriterpene 13
7 Tibig leaf 25
8 Cogon leaf 25
LIST OF APPENDICES
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.)”
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
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
content of tibig crude extract (24.51 ± 2.67 mg QE/g extract) was comparable to that
Results from the anthelmintic assay revealed that cogon and tibig crude extracts
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
CHAPTER I
INTRODUCTION
livestock production in the Philippines. The sudden weight loss and even death caused
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
unsegmented tube-like bodies with anterior mouths and longitudinal digestive tracts.
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).
other parasites have become the sought after solutions of everyone that will help
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
2
has been known after several usage of the said product that makes the management of
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
ii. test the presence of secondary metabolites in the crude extracts using
iii. determine the total phenolics and total flavonoid contents in the
extracts;
iv. evaluate the anthelmintic activity of tibig and cogon leaf extracts
concentrations.
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
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
CHAPTER II
REVIEW OF LITERATURE
Tibig (F. nota Blco.) as shown in Figure 1, is a locally known small tree
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).
substances in Ficus species. From the study of Tsai et al. (2012), the F. odorata
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.)
3S)-2, 3-butanediol, and β-sitosterol (Ragasa et al., 2014). Ficus species are generally
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
Cogon (I. cylindrica L.) as shown in Figure 2, is one of the most dominant and
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
6
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
dysentery, diuretic and tonic (Datta & Banerjee, 1978). This plant is also used for soil
stabilization, thatching, and paper industry. The major chemical constituents include
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
7
Considerable research has shown that some plants not only affect the nutrition
of animals, but also have antiparasitic effects (Waghorn & McNabb, 2003). Plants
toxic and can consequently not be overdosed and is possible to be integrated into
Alkaloids
one or more heterocyclic nitrogen atoms, derived from amino acids and
with more than 12,000 substances isolated. A huge variety of structural formulas,
The alkaloids have been divided into three major groups depending on the
precursor and the final structure (Waterman, 1993). Alkaloids are produced by
wide range of biologically active chemicals which have been extensively explored for
nitrate generation which is useful for protein synthesis, suppresses transfer of sucrose
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,
shown that a group of quinolizidine alkaloids were effective feeding deterrents against
1991). Furthermore, these alkaloids were also toxic against fungi and bacteria.
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
Tannin polyphenols are broadly divided into two categories: hydrolysable and
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
Hydrolysis
Gallotannins
Hydrolysis
Ellagitannins
10
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
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
deposition (e.g. iignin), have been shown to be important in resistance to the root-knot
Saponins
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
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/).
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
Literature studies reported that saponins can destabilize the cell membrane and
cuticle collagen of the parasites. Most saponins are hemolytic and display many
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
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
flavonoids.html).
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
between condensed tannins and two common flavonoids, quercetin and luteolin, in
13
Thus, the presence of these bioactive components in plant samples increases the
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
metabolites that are widely distributed in the plant kingdom and found in leaves, stem
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
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
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,
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
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
CHAPTER III
All chemicals and reagents were of analytical grade and were purchased at
The leaves of tibig, from the mid-part of the tree, and cogon leaves of the
The leaves of tibig and cogon were washed with tap water to remove
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.
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
Equation 1:
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
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.
Mayer’s and Dragendorff’s tests (Appendix II.A) were used for the
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.
acid and then filtered. The filtrates were treated with Dragendorff’s reagent (solution
presence of alkaloids.
The gelatin test (Appendix II.B) was used to test the presence of tannins and
added to the extract solutions of tibig and cogon. After which, the formation of a
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
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.
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
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
(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
Equation 3:
where :
Rf = Retardation factor
The total phenolic content of tibig and cogon leaf extracts was determined by
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
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).
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
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
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
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
exposure at 27 ℃, the number of motile and non-motile larvae was counted under a
as the positive and negative control. Three replications were done and the results were
Equation 4:
23
where:
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
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
% 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
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.
test differences among significant sample means. On the other hand, total phenolic
and total flavonoid content determination results were analyzed using Mann Whitney,
CHAPTER IV
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.
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
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
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
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.
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
The crude extracts of tibig and cogon were subjected to thin layer
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.
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.
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
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
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
1
1
A B C
A B C
The total phenolic content of tibig and cogon ethanolic crude leaf extracts was
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
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
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
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
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.
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
activity of tibig crude extracts which was significantly higher (65 % mortality)
compared to cogon crude extracts (40 % mortality). Moreover, the percent mortality
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
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
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
concentration and the time of exposure. In addition, flavonoids are part of the
Dead H.
contortus
Cobb.
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
inoculating loop and disturbing the media or the petri plate by slightly shaking.
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.
ppm), the mortality rate of the extract also increased with 56.48 ± 16.28 at 30 ppm,
Figure 18 shows the comparison between the positive control (Ivermectin) and
tibig extract. Results show that the positive control (Ivermectin) exhibited a
effect was dose-dependent. On the other hand, the effect of tibig extract was not dose-
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
% 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
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
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
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
Based on the results for the anthelmintic assay, tibig crude extracts showed
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
both tibig and cogon extracts can be an anthelmintic agent against H. contortus Cobb.
Conclusions
1. Tibig and cogon ethanolic extracts showed the presence of secondary metabolites
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.
5. The effect of tibig extract at varying concentrations against H. contortus Cobb. was
not dose-dependent.
40
Recommendations
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.
4. Isolation of the secondary metabolite responsible for the anthelmintic activity of the
5. The effect of tibig and cogon extracts to another nematode species should be
evaluated.
41
LITERATURE CITED
ASOLKAR, L. 2005. Glossary of Indian Medicinal Plants with active principle, Part 1
National Institute of Science and Communication and Information Resources.
New Delhi: CSIR.
BURKE, J. 2005. Management of barber pole worm in sheep and goats in the
southern U.S Booneville, AR. Dal. Bum. Sma. Far. Res. Up. 120:177-178.
DATTA, S. and A. BANERJEE. 1978. Useful plants of West tropical Bengal rice
fields. Eco. Bot. 32(4):297-310.
42
LORKE, D. 1983. A new approach to practical acute toxicity testing. Arch. Toxicol.
54(4):275-287.
RAGASA, C. Y., P. W. TSAI and C. C. SHEN. 2009. Terpenoids and sterols from the
endemic and endangered Philippine trees, Ficus pseudo palma and Ficus
ulmifolia. Phil. J. of Sci. 138(2):205-209.
http://www.farmersnotebookph.com/wpcontent/uploads/2014/10/farmersnotebook_w
arrentan_04.jpg. Date accessed: August 25, 2017.
https://www.globalhealingcenter.com/natural-health/what-are-saponins/.Date
accessed: October 16, 2017.
http://naturalchemistry.utu.fi/research/tannin-and-polyphenol-
chemistry/tannindefinition-and structures/. Date accessed: October 17, 2017.
APPENDICES
47
APPENDIX I
b. 1% Ferric Chloride
c. 1% Tannic acid
solution.
volumetric flask.
volumetric flask.
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
Mayer’s Test
3. The filtrates of the ethanolic extracts of tibig and cogon were added with
samples.
Dragendorff’s Test
3. The filtrates of the ethanolic extracts of tibig and cogon were added with
extracts.
Gelatin Test
Froth Test
1. 1 ml of the extracts was added with 2-3 drops of lead acetate solution.
in the extracts.
E. Terpenoids
Salkowski’s Test
2. The filtrates were added with few drops of concentrated sulfuric acid and
were shaken.
terpenoids.
50
APPENDIX III
volumetric flask.
2. 80 ppm – Measure 8.00 ml from 100 ppm and dilute to mark with 40 %
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.
10 ml volumetric flask.
10 ml volumetric flask.
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.
volumetric flask.
APPENDIX IV
volumetric flask.
2. Weigh 2.5 g of AlCl3 and dissolved with distilled water and dilute to mark in a
25 ml volumetric flask.
1. 100 ppm – Measure 5 ml from stock quercetin solution (200 ppm) and dilute
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.
10 ml volumetric flask.
10 ml volumetric flask.
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.
volumetric flask.
APPENDIX V
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)
0.1400
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 VI
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
Total 6
Test Statisticsa
Mann-Whitney U .000
Wilcoxon W 6.000
Z -1.964
APPENDIX VII
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
Total 6
Test Statisticsa
Mann-Whitney U 2.000
Wilcoxon W 8.000
Z -1.091
APPENDIX VIII
Appendix Table 7. Raw data of the percent mortality of Tibig and Cogon leaf
extracts against H. contortus Cobb
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.
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
APPENDIX IX
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
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
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.
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
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
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.
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