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CHAPTER 1
INTRODUCTION
A. Background of the Study
B. Statement of the problem
The general objective of this study is to find out whether Modulo Art Game help in
determine the intelligence of the students in the different level. Specifically, this study answered
the following questions:
1. Does the Modulo Art game will help the teacher and the students to have a better
communication regarding the different learning areas?
2. Are all the learning areas covered by the game in the different level to enhanced the skill
of the students?
C. Significance of the study
This scientific investigation was envisioned to highlight the potential of Modulo art game
to the students with _____.If proven to be feasible, the findings of this study will benefit the
following:
Students
Teachers
1
Production of medicines. The possible contributions of the findings of this research are
directed towards the improvement of the production of the medicines since it will be a basis for
the making of a treatment regarding cancer cells.
Researchers. The students or researchers may likewise benefit from the study in as much
as the data and information could be used as anchorage on similar or expanded researches.
Lastly, the results of the study could be a material or significant inputs for future related
studies.
D. Research Hypothesis
Null: Modulo Art Game will not enhance the student’s aptitude and learning
ability in solving and analysing word problems.
Alternative:
E. Scope and limitation
This study focused only to determine whether the Modulo Art game
The study was conducted last October, 2012 at Vinzons Pilot High School. Actual
laboratory experimentation was performed by the researchers with the aid of a licensed chemis
The researchers focused on the different topics .
The researchers used 1260 brine shrimp eggs, wherein 10 eggs were assigned per
replicate, for brine shrimp lethality bioassay.
2
F. Definition of terms
Modulo Art-
Modular arithmetic -also known as clock arithmetic. For any given modulus n, the finite integers0, 1, 2 … n-1 are used, coupled with the four basic operations.
CHAPTER II
REVIEW OF RELATED LITERATURE AND STUDIES
Related literature
.
3
Related Studies
http://www.eimacs.com/blog/2011/11/introduction-to-modular-addition/
Theoretical Framework
This study was conducted with the theory that Malatalong (Solanum verbascifolium) and
Suob kabayo (Hyptis suaveolen) exhibit cytotoxic property. Malatalong and Suob kabayo are
endemic plants which contain different chemicals that are being tested through phytochemical
screening.
To test if the subjects were cytotoxic active, the proponents used the Brine shrimp
Lethality Bioassay. This process is used based on the related studies that were reviewed which
also tried to test the cytotoxicity of their subject. Alkaloids, Glycosides, Tannins, Flavonoids,
and Saponins are the chemicals being tested whether present in the experimental subjects. In
order to know the effectiveness, the number of killed brine shrimps in each treatment was
compared to that of the control (Methotrexate) at varying level of concentrations.
4
Evidence of Effectiveness
Factors affecting Effectiveness
Time Pressure Questions Puzzle Pieces
Figure 3: Theoretical Framework of the study
CHAPTER III
METHODOLOGY
5
A. Experimental Design
The study used CRD (Completely Randomized Design) in (2 x 3) factorial experimental
design.
There were two factors in the study:
Factor 1 – Leaf Extract
Malatalong
Suob Kabayo
Factor 2 – Solvent Used
Ethanol
Methanol
Distilled Water
There were seven treatments, each of which was prepared in six different concentrations
(5µg, 10µg, 20µg, 50µg, 100µg, 500µg of crude extract dissolved in 1 mL of Dimethyl
Sulfoxide).
Treatment 1 = Suob Kabayo leaves extract with Methanol as solvent
Treatment 2 = Malatalong leaves extract with Methanol as solvent
Treatment 3 = Suob Kabayo leaves extract with Ethanol as solvent
Treatment 4 = Malatalong leaves extract with Ethanol as solvent
Treatment 5 = Suob Kabayo leaves extract with Distilled Water as solvent
Treatment 6 = Malatalong leaves extracted with Distilled Water as solvent
Treatment 7 = Positive Control (Methotrexate)
6
B. Procedure
I. Preparation of the plant materials
Two kilograms each of Suob Kabayo (Hyptis Suaveolens (L) Poit) and
Malatalong (Solanum Verbascifolium Linn.) leaves were gathered from the nursery of
Vinzons Pilot High School. The hand-picked leaves were then weighed to make sure that
they have a mass of at least two kilograms each. The leaves were washed with tap water
to remove the dirt and other elements which are not needed in the experiment. After they
have been washed, they were kept in a room away from sunlight for air drying to reduce
the weight by 12.5%. The process lasted for 24 hours. After the allotted time for drying,
the leaves were ground.
II. Methanolic, Ethanolic, and Distilled Water extraction of the leaves
The air-dried ground leaves were weighed and measured to be 2 kg. The weighed
leaves were transferred in the bottles each containing .5 kg of ground leaves. In this
process, 3 bottles contained half kilo of the ground Suob Kabayo leaves each and the
other 3 bottles contained half kilo of the ground Malatalong leaves each with mass of .5
kg. After filling the bottles with leaves, 1L of Methanol was added to the bottles
containing .5 kg of Malatalong leaves and Suob Kabayo Leaves, the bottles were
labelled accordingly. This process was done over the remaining solvents namely
Ethanol and Distilled Water. The soaked leaves were kept in a room for 48 hours.
7
III. Obtaining the crude extracts
The soaked leaves were filtered with filter papers and transferred to separate
containers. The suction machine hastened the process. After the liquid chemicals were
separated from the solid leaves, each flask containing the extracts was then labelled. Each
of the acquired chemicals was subjected to the rotary evaporator wherein they were set on
different boiling points but the same rpm. The boiling points were based from the kind of
chemical or liquid. In this process of subjecting the obtained liquids to the rotary
evaporator, the crude extract was being separated from the chemical where the leaves
were soaked. After the crude extracts were isolated from the chemicals, they were
removed from the flasks and transferred to the vials which were secured by a cork.
Afterwards, the vials were labelled. To avoid the contamination of the crude extracts they
were placed inside a refrigerator where they were preserved for 24 hours.
IV. Culturing of the Brine Shrimp (Artemia salina)
In the process, 3.8 grams of sea salt was dissolved in 150 mL water, it was filtered
afterwards. The Brine Shrimp eggs were placed in the solution. The eggs were
incubated in a small tank for 48 hours at 28˚C.
V. Phytochemical screening
The cytotoxic property of a specimen may be well proven through the existence
of phytochemicals. The proponents were the ones who conducted the test for the presence
of phytochemicals that might be responsible for the cytotoxicity of the specimens. The
procedures are as follows:
8
A. Test for Alkaloids
After preparing the plant extracts, 8 mL of each of seven plant extracts were
acidified with 1% HCl solution. The extract was then divided into 4 portions in test
tubes labelled A-D. 1 mL test solutions were then applied to each sample - for test
tube A, Dradendorff’s reagent; test tube; Mayer’s reagent; test tube C, Vasler’s
reagent; and test tube D, Wagner’s reagent.
B. Test for Flavonoids
A small piece of Mg ribbon was added to 2 mL of the plant extract. Then, 1
mL of concentrated HCl solution was administered drop by drop. At the duration,
formation of colors was observed within 1-2 flavones. Flavonols were indicated by
the formation of colors ranging from red to crimson.
C. Test for Glycosides
To 2 mL of each of the plant extract, we put in few drops of lead acetate
solution to the filtrate until the solution was weakly alkaline or neutral. The presence
of glycosides showed turbidity or precipitation.
D. Test for Saponins
9
After placing 5 mL of the plant extract in a test tube, it was sealed and
shaken for 30 seconds. It was then let to stand in a vertical position and was
observed over a period of 30 minutes. The sample was presumed to contain saponins
if “honey-comb” froths greater than 3 cm above the surface of the liquid persisted.
All samples were subjected to this process.
E. Test for Tannins
To 2 mL of the plant extract, a few drops of ferric chloride reagent were
added. A blue-black precipitate indicates a positive result.
VI. Preparation of the concentrations of treatments
Each of the crude extracts was weighed and measured according to the specific
concentration there is to be prepared. 5µg, 10µg, 20µg, 50µg, 100µg, and 500µg of the
crude extracts were prepared and dissolved in 1mL of Dimethyl Sulfoxide (DMSO).
This process was done repeatedly to the remaining solvents. The Methotrexate as a
control was also dissolved in Dimethyl Sulfoxide in the same level of concentrations.
VII. Brine Shrimp Lethality Bioassay
The 126 test tubes were placed in seven separate test tube racks. Each test tube
rack contained 18 test tubes that comprise the six different concentrations that were
repeated to 3 replicates. Each of the 126 test tubes contained 6 mL of simulated sea
water with 10 individual brine shrimps in it. The 1 mL volumes of the treatments of
corresponding concentrations were applied in each test tube and replicates. It was left
10
for 24 hours and observed after the 24 hours span. The numbers of dead brine shrimps
were compared to the other test tubes containing the brine shrimps. The comparison will
be based on the concentration level, the chemical used, its replicates as well as the plant
utilized. Also, the controlled treatment was compared with the other treatments.
VIII. Computing for LC50
To calculate the lethal concentration of the treatments, the researchers computed
for the LC50. Ten test subjects or bioassay species were exposed to different treatments,
and were observed for the number of mortality after 24 hours of exposure. The 24-hour
LC50 value was calculated. The purpose of the control (i.e., 0 mg/L) is to determine
whether any bioassay organism died due to factors other than exposure to the test
chemical. Data showed that, there was 10% control mortality (1 out of 10) so the
researchers have to “correct” observed mortalities in other treatments using Abbott’s
formula:
Corrected Mortality (%) = M obs−M control (100)
100−M control
If the corrected percent mortality data versus the log 10 of concentration was
graphed, there would not be a straight line. The purpose of the probit transformation is to
straighten the line in order to estimate LC50 more easily. The researchers regressed Log10
concentration on probit value then using the regression formula it was estimated that the
Log10 concentration is associated with a probit value of 5 (the probit of 50%). Finally, to
get the LC50 value, we first find for the value of X in the regression equation and after
finding the value of X we solved for the antilog X.
11
IX. Statistical Analysis
The concentration-mortality data were analyzed statistically by using probit
analysis for the determination of LC50 values and linear regression for the plant extracts.
(Persoone, G.et al. 1980, Mayer, B.N. et al. 1982, Mclaughlin, J.L., 1990).
Furthermore, data on brine shrimp mortality on the different level of
concentrations were analyzed using TWO-WAY Analysis of Variance.
12
CHAPTER IV
RESULTS
After conducting actual experimentation, the following are the results gathered by
the researchers.
Table 1: Tabulated Result of the phytochemical screening on the crude extracts of Suob
Kabayo (Hyptis suaveolens L Poit.) and Malatalong (Solanum verbascifolium linn.).
Specimen Extractant Alkaloids Glycosides Tannins Flavonoids Saponins
Suob Kabayo
(Hyptis
suaveolens
poir.)
Methanol + + + - +
Ethanol + + + + +
Distilled
Water
+ + - - +
Malatalong
(Solanum
verbascifolium
linn.)
Methanol + + + - +
Ethanol + + + + +
Distilled
Water
+ + - + +
13
The phytochemical screening was conducted by the proponents. They were provided with a procedure on how to perform the test by the assisting personnel to be used as their guide throughout the determination of phytochemicals present in the crude extracts.
B. Result of Brine Shrimp Lethality Bio-assay
Table 2: The mortality of Brine Shrimp at varying levels of concentrations of Methanolic
leaves extract of Suob Kabayo.
Concentration ReplicatesMeanR1 R2 R3
5 µg/mL 0 0 0 0
10 µg/mL 4 5 6 5
20 µg/mL 5 7 9 7
50 µg/mL 7 8 9 8
100 µg/mL 10 10 10 10
500 µg/mL 10 10 10 10
Note: Highest mortality rate is at 500 microgram/mL. No mortality was recorded at 5 µg/mL
14
Table 3: The mortality of Brine Shrimp at varying levels of concentrations of Ethanolic leaf
extract of Suob Kabayo.
Concentration ReplicatesMeanR1 R2 R3
5 µg/mL 0 0 0 0
10 µg/mL 5 6 7 6
20 µg/mL 5 7 9 7
50 µg/mL 7 8 9 8
100 µg/mL 10 10 10 10
500 µg/mL 10 10 10 10
Note: Highest mortality rate is at 500 microgram/mL. No mortality was recorded at 5 µg/mL
15
Table 4: The mortality of Brine Shrimp at varying levels of concentrations of Distilled
Water Leaf extract of Suob Kabayo.
Concentration ReplicatesMeanR1 R2 R3
5 µg/mL 0 0 0 0
10 µg/mL 2 4 6 4
20 µg/mL 4 7 7 6
50 µg/mL 6 7 8 7
100 µg/mL 8 9 10 9
500 µg/mL 10 10 10 10
Note: Highest mortality rate is at 500 microgram/mL. No mortality was recorded at 5 µg/mL
16
Table 5: The mortality of Brine Shrimp at varying levels of concentrations of Malatalong
Methanolic Leaf Extract.
Concentration ReplicatesMeanR1 R2 R3
5 µg/mL 0 1 2 1
10 µg/mL 3 5 7 5
20 µg/mL 6 7 8 7
50 µg/mL 8 9 10 9
100 µg/mL 10 10 10 10
500 µg/mL 10 10 10 10
Note: Highest mortality rate is at 500 microgram/mL. Presence of mortality was recorded at 5
µg/mL
17
Table 6: The mortality of Brine Shrimp at varying levels of concentrations of Malatalong
Ethanolic Leaf extract.
Concentration ReplicatesMeanR1 R2 R3
5 µg/mL 0 0 0 0
10 µg/mL 5 6 7 6
20 µg/mL 6 7 8 7
50 µg/mL 7 8 9 8
100 µg/mL 10 10 10 10
500 µg/mL 10 10 10 10
Note: Highest mortality rate is at 500 microgram/mL. Presence of mortality was recorded at 5
µg/mL
18
Table 7: The mortality of Brine Shrimp at varying levels of concentrations of Distilled
Water Extract of Malatalong Leaves.
Concentration ReplicatesMeanR1 R2 R3
5 µg/mL 0 1 2 1
10 µg/mL 3 5 7 5
20 µg/mL 6 7 8 7
50 µg/mL 7 8 9 8
100 µg/mL 9 9 9 9
500 µg/mL 10 10 10 10
Note: Highest mortality rate is at 500 microgram/mL. Presence of mortality was recorded at 5
µg/mL
19
Table 8: The mortality of Brine Shrimp at varying levels of concentrations of the control,
Methotrexate.
Concentration ReplicatesMeanR1 R2 R3
5 µg/mL 0 0 0 0
10 µg/mL 8 8 8 8
20 µg/mL 8 9 10 9
50 µg/mL 10 10 10 10
100 µg/mL 10 10 10 10
500 µg/mL 10 10 10 10
Note: No mortality at 5 µg/mL, but an increased mortality rate can be observed from 10 µg/mL
up to 500 µg/mL.
20
C. Comparison of the percent mortality of Brine Shrimps using Suob Kabayo and
Malatalong leaf extract using the different solvents
Figure 4: Percent mortality of Suob Kabayo leaf extract using the different solvents.
5 10 20 50 100 5000
20
40
60
80
100
120
Suob Kabayo
MethanolEthanolDistilled Water
Concentration (µg/mL)
Mor
talit
y (%
)
21
Figure 5: Percent mortality of Malatalong leaf extract using the different solvents.
5 10 20 50 100 5000
20
40
60
80
100
120
Malatalong
MethanolEthanolDistilled Water
Concentration (µg/mL)
Mor
talit
y (%
)
22
D. Comparison of the percent mortality of Brine Shrimps using Suob Kabayo and
Malatalong leaf extract using the different solvents (Solvent vs. Leaf extract)
Figure 6: Percent mortality Brine Shrimp in Methanolic extracts.
5 10 20 50 100 5000
20
40
60
80
100
120
Methanol
MalatalongSuob Kabayo
Concentration (µg/mL)
Mor
talit
y (%
)
23
Figure 7: Percent mortality Brine Shrimp in Ethanolic extracts.
5 10 20 50 100 5000
20
40
60
80
100
120Ethanol
MalatalongSuob Kabayo
Concentration (µg/mL)
Mor
talit
y (%
)
24
Figure 8: Percent mortality Brine Shrimp in Aqueous extracts.
5 10 20 50 100 5000
20
40
60
80
100
120
Distilled Water
MalatalongSuob Kabayo
Concentration (µg/mL)
Mor
talit
y (%
)
25
E. Observation of average mortality and LC50 value of Suob Kabayo and Malatalong in
different solvents
Table 9: Methanolic Leaf Extract of Suob Kabayo Observation of average mortality
Concentration(μg/ml)
Replicates
No. Of Shrimp
Test
Average Mortality after
24 hours% Average Mortality
LC50(μg/ml)
5
R1 - a 10
00 00
10
R2 - a 10
R3 - a 10
10
R1 - b 10
05 50R2 - b 10
R3 - b 10
20
R1 - c 10
07 70R2 - c 10
R3 - c 10
50
R1 - d 10
08 80R2 - d 10
R3 - d 10
100
R1 - e 10
10 100R2 - e 10
R3 - e 10
500
R1 - f 10
10 100R2 - f 10
R3 - f 10
Note: LC50 of 10 indicates that the extract is toxic.
26
Figure 9: LC50 of Suob Kabayo Methanolic Leaf Extract
0.700000000000001
1 1.3 1.7 2 2.70
20
40
60
80
100
120
LC50 of Suob Kabayo Methanolic Leaf Extract
Log of Concentration (µg/mL)
Mor
talit
y (%
)
Table 10: Ethanolic Leaf Extract of Suob Kabayo Observation of average mortality
27
Concentration(μg/ml)
Replicates
No. Of Shrimp
Test
Average Mortality after
24 hours% Average Mortality
LC50(μg/ml)
5
R1 - a 10
00 00
4.08
R2 - a 10
R3 - a 10
10
R1 - b 10
07 70R2 - b 10
R3 - b 10
20
R1 - c 10
08 80R2 - c 10
R3 - c 10
50
R1 - d 10
09 90R2 - d 10
R3 - d 10
100
R1 - e 10
10 100R2 - e 10
R3 - e 10
500
R1 - f 10
10 100R2 - f 10
R3 - f 10
Note: LC50 of lower than 10 μg/ml, signifies promising toxicity potential
28
Figure 10: LC50 of Suob Kabayo Ethanolic Leaf Extract
0.700000000000001
1 1.3 1.7 2 2.70
20
40
60
80
100
120
LC50 of Suob Kabayo Ethanolic Leaf Extract
Log of concentration (µg/mL)
Mor
talit
y (%
)
Table 11: Distilled Water Leaf Extract of Suob Kabayo Observation of average mortality
29
Concentration(μg/ml)
Replicates
No. Of Shrimp
Test
Average Mortality after
24 hours% Average Mortality
LC50(μg/ml)
5
R1 - a 10
00 00
19.95
R2 - a 10
R3 - a 10
10
R1 - b 10
04 40R2 - b 10
R3 - b 10
20
R1 - c 10
06 60R2 - c 10
R3 - c 10
50
R1 - d 10
07 70R2 - d 10
R3 - d 10
100
R1 - e 10
09 90R2 - e 10
R3 - e 10
500
R1 - f 10
10 100R2 - f 10
R3 - f 10
Note: LC50 of 19.95 means that the extract is moderately toxic
30
Figure 11: LC50 of Suob Kabayo Aqueous Extract
0.700000000000001
1 1.3 1.7 2 2.70
20
40
60
80
100
120
LC50 of Suob Kabayo Aqeous Leaf Extract
Log of Concentration (µg/mL)
Mor
talit
y (%
)
Table 12: Methanolic Leaf Extract of Malatalong Observation of average mortality
31
Concentration(μg/ml)
Replicates
No. Of Shrimp
Test
Average Mortality after
24 hours% Average Mortality
LC50(μg/ml)
5
R1 - a 10
01 10
11.93
R2 - a 10
R3 - a 10
10
R1 - b 10
05 50R2 - b 10
R3 - b 10
20
R1 - c 10
07 70R2 - c 10
R3 - c 10
50
R1 - d 10
09 90R2 - d 10
R3 - d 10
100
R1 - e 10
10 100R2 - e 10
R3 - e 10
500
R1 - f 10
10 100R2 - f 10
R3 - f 10
32
Figure 12: LC50 of Malatalong Methanolic Extract
0.700000000000001
1 1.3 1.7 2 2.70
20
40
60
80
100
120
LC50 of Malatalong Methanolic Leaf Extract
Log of Concentration (µg/mL)
Mor
talit
y (%
)
Table 13: Ethanolic Leaf Extract of Malatalong Observation of average mortality
33
Concentration(μg/ml)
Replicates
No. Of Shrimp
Test
Average Mortality after
24 hours% Average Mortality
LC50(μg/ml)
5
R1 - a 10
00 00
6.61
R2 - a 10
R3 - a 10
10
R1 - b 10
06 60R2 - b 10
R3 - b 10
20
R1 - c 10
07 70R2 - c 10
R3 - c 10
50
R1 - d 10
08 80R2 - d 10
R3 - d 10
100
R1 - e 10
10 100R2 - e 10
R3 - e 10
500
R1 - f 10
10 100R2 - f 10
R3 - f 10
34
Figure 13: LC50 of Malatalong Ethanolic Extract
0.700000000000001
1 1.3 1.7 2 2.70
20
40
60
80
100
120
LC50 of Malatalong Ethanolic Leaf Extract
Log of Concentraion (µg/mL)
Mor
talit
y (%
)
Table 14: Distilled Water Extract of Malatalong Leaves Observation of average mortality
35
Concentration(μg/ml)
Replicates
No. Of Shrimp
Test
Average Mortality after
24 hours% Average Mortality
LC50(μg/ml)
5
R1 - a 10
01 10
11.48
R2 - a 10
R3 - a 10
10
R1 - b 10
05 50R2 - b 10
R3 - b 10
20
R1 - c 10
07 70R2 - c 10
R3 - c 10
50
R1 - d 10
08 80R2 - d 10
R3 - d 10
100
R1 - e 10
09 90R2 - e 10
R3 - e 10
500
R1 - f 10
10 100R2 - f 10
R3 - f 10
36
Figure 14: LC50 of Malatalong Aqueous Extract
0.700000000000001
1 1.3 1.7 2 2.70
20
40
60
80
100
120
LC50 of Malatalong Aqueous Leaf Extract
Log of Concentration (µg/mL)
Mor
talit
y (%
)
Table 15: Positive Control - Observation of average mortality
37
Concentration(μg/ml)
Replicates
No. Of Shrimp
Test
Average Mortality after
24 hours% Average Mortality
LC50(μg/ml)
5
R1 - a 10
00 00
3.09
R2 - a 10
R3 - a 10
10
R1 - b 10
08 80R2 - b 10
R3 - b 10
20
R1 - c 10
09 90R2 - c 10
R3 - c 10
50
R1 - d 10
10 100R2 - d 10
R3 - d 10
100
R1 - e 10
10 100R2 - e 10
R3 - e 10
500
R1 - f 10
10 100R2 - f 10
R3 - f 10
38
Table 16: Result of TWO WAY ANALYSIS OF VARIANCE as to the presence of
significant difference between the Mean mortality of Brine Shrimps at different
concentrations of Malatalong and Suob Kabayo leaf extracts.
5 μg/ml level of concentration (mortality) Ethanol Methanol Distilled Water
Suob Kabayo0 0 00 0 00 0 0
Malatalong0 0 00 1 10 2 2
Anova: Two-Factor With Replication
SUMMARY Ethanol Methanol Distilled Water TotalSuob Kabayo
Count 3 3 3 9Sum 0 0 0 0Average 0 0 0 0Variance 0 0 0 0
Malatalong Count 3 3 3 9Sum 0 3 3 6Average 0 1 1 0.666667Variance 0 1 1 0.75
Total
Count 6 6 6
Sum 0 3 3
Average 0 0.5 0.5
Variance 0 0.7 0.7
39
ANOVA Source of Variation SS df MS F P-value F crit
Sample 2 1 2 6 0.030622 4.747225Columns 1 2 0.5 1.5 0.262144 3.885294Interaction 1 2 0.5 1.5 0.262144 3.885294Within 4 12 0.333333333 Total 8 17
Note:
a) There is a significant difference in the performance of the leaves extract using three
solvents. This means that Methanol and Distilled Water are better than Ethanol as solvents
at 5 μg/ml level of concentration.
b) There is no significant difference on the mean mortality of brine shrimps using
Malatalong and Suob Kabayo leaf extracts.
c) Interaction effect is not present between the leaf extracts and the solvents.
10 μg/ml level of concentration (mortality)
Ethano
lMethano
l Distilled Water
Suob Kabayo
5 4 2
6 5 4
7 6 6
40
Malatalong
5 3 3
6 5 5
7 7 7
Anova: Two-Factor With Replication
SUMMARY E M DW TotalSuob Kabayo Count 3 3 3 9Sum 18 15 12 45Average 6 5 4 5Variance 1 1 4 2.25
Malatalong Count 3 3 3 9Sum 18 15 15 48
Average 6 5 55.33333
3Variance 1 4 4 2.5
Total Count 6 6 6Sum 36 30 27Average 6 5 4.5Variance 0.8 2 3.5
ANOVA Source of Variation SS df MS F P-value F crit
Sample 0.5 1 0.5 0.2 0.662686 4.747225Columns 7 2 3.5 1.4 0.28413 3.885294
Interaction 1 2 0.5 0.2 0.821405 3.885294Within 30 12 2.5
Total 38.5 17
Note:
41
a) There is no significant difference in the performance of the leaves extract using three
solvents at 10 μg/ml level of concentration.
b) There is no significant difference on the mean mortality of brine shrimps using
Malatalong and Suob Kabayo leaf extracts.
c) Interaction effect is not present between the leaf extracts and the solvents.
20 μg/ml level of concentration (mortality) Ethanol Methanol Distilled Water
Suob Kabayo
5 5 4
7 7 7
9 9 7
Malatalong
6 6 6
7 7 7
8 8 8
Anova: Two-Factor With Replication
SUMMARY E M DW TotalSuob Kabayo Count 3 3 3 9Sum 21 21 18 60
Average 7 7 66.66666
7Variance 4 4 3 3
Malatalong Count 3 3 3 9Sum 21 21 21 63
42
Average 7 7 7 7Variance 1 1 1 0.75
Total Count 6 6 6Sum 42 42 39Average 7 7 6.5Variance 2 2 1.9
ANOVASource of Variation SS df MS F P-value F crit
Sample 0.5 1 0.5 0.214286 0.651711 4.747225Columns 1 2 0.5 0.214286 0.81014 3.885294
Interaction 1 2 0.5 0.214286 0.81014 3.885294Within 28 12 2.333333
Total 30.5 17
Note:
a) There is no significant difference in the performance of the leaves extract using three
solvents at 20 μg/ml level of concentration.
b) There is no significant difference on the mean mortality of brine shrimps using
Malatalong and Suob Kabayo leaf extracts.
c) Interaction effect is not present between the leaf extracts and the solvents.
43
50 μg/ml level of concentration (mortality) Ethanol Methanol Distilled Water
Suob Kabayo
7 7 6
8 8 7
9 9 8
Malatalong
7 8 7
8 9 8
9 10 9
Anova: Two-Factor With Replication
SUMMARY E M DW TotalSuob Kabayo Count 3 3 3 9Sum 24 24 21 69
Average 8 8 77.66666
7Variance 1 1 1 1
Malatalong Count 3 3 3 9Sum 24 27 24 75
Average 8 9 88.33333
3Variance 1 1 1 1
Total Count 6 6 6Sum 48 51 45Average 8 8.5 7.5Variance 0.8 1.1 1.1
44
ANOVA Source of Variation SS df MS F P-value F crit
Sample 2 1 2 2 0.182717 4.747225Columns 3 2 1.5 1.5 0.262144 3.885294
Interaction 1 2 0.5 0.5 0.618625 3.885294Within 12 12 1
Total 18 17
Note:
a) There is no significant difference in the performance of the leaves extract using three
solvents at 50 μg/ml level of concentration.
b) There is no significant difference on the mean mortality of brine shrimps using
Malatalong and Suob Kabayo leaf extracts.
c) Interaction effect is not present between the leaf extracts and the solvents.
100 μg/ml level of concentration (mortality) Ethanol Methanol Distilled Water
Suob Kabayo
10 10 8
10 10 9
10 10 10
45
Malatalong
10 10 9
10 10 9
10 9 9
Anova: Two-Factor With Replication
SUMMARY E M DW TotalSuob Kabayo Count 3 3 3 9Sum 30 30 27 87
Average 10 10 99.66666
7Variance 0 0 1 0.5
Malatalong Count 3 3 3 9Sum 30 29 27 86
Average 10 9.666667 99.55555
6
Variance 0 0.333333 00.27777
8
Total Count 6 6 6Sum 60 59 54Average 10 9.833333 9Variance 0 0.166667 0.4
ANOVA Source of Variation SS df MS F P-value F crit
Sample 0.055556 1 0.055556 0.25 0.626117 4.747225Columns 3.444444 2 1.722222 7.75 0.006904 3.885294
Interaction 0.111111 2 0.055556 0.25 0.782758 3.885294Within 2.666667 12 0.222222
Total 6.277778 17
46
Note:
a) There is no significant difference in the performance of the leaves extract using three
solvents at 100 μg/ml level of concentration.
b) There is a significant difference on the mean mortality of brine shrimps using
Malatalong and Suob Kabayo leaf extracts.
c) Interaction effect is not present between the leaf extracts and the solvents.
500 μg/ml level of concentration (mortality) Ethanol Methanol Distilled Water
Suob Kabayo
10 10 9
10 10 10
10 10 10
Malatalong
10 10 10
10 10 10
10 10 10
Anova: Two-Factor With Replication
SUMMARY E M DW TotalSuob Kabayo Count 3 3 3 9Sum 30 30 29 89
Average 10 109.66666
79.88888
9
Variance 0 00.33333
30.11111
1
47
Malatalong Count 3 3 3 9Sum 30 30 30 90Average 10 10 10 10Variance 0 0 0 0
Total Count 6 6 6Sum 60 60 59
Average 10 109.83333
3
Variance 0 00.16666
7
ANOVA Source of Variation SS df MS F P-value F crit
Sample 0.055556 1 0.055556 1 0.337049 4.747225Columns 0.111111 2 0.055556 1 0.396569 3.885294
Interaction 0.111111 2 0.055556 1 0.396569 3.885294Within 0.666667 12 0.055556
Total 0.944444 17
Note:
a) There is no significant difference in the performance of the leaves extract using three
solvents at 500 μg/ml level of concentration.
b) There is no significant difference on the mean mortality of brine shrimps using
Malatalong and Suob Kabayo leaf extracts.
c) Interaction effect is not present between the leaf extracts and the solvents.
48
CHAPTER V
DISCUSSION
A. Phytochemical screening of crude extracts of Malatalong and Suob Kabayo
The crude extracts of Suob Kabayo and Malatalong were evaluated for qualitative
determination of major phytoconstituents such as alkaloids, flavonoids, tannins, saponins and
glycosides;
Phytochemical screening of the crude extracts of Suob Kabayo Leaves revealed the
presence of the following compounds:
Ethanolic Extract – Alkaloids, Glycosides, Tannins, Flavonoids and Saponins
Methanolic Extract - Alkaloids, Glycosides, Tannins, and Saponins
Distilled Water Extract - Alkaloids, Glycosides, and Saponins
On the other hand, phytochemical screening of the crude extracts of Malatalong Leaves
revealed the presence of the following compounds:
Ethanolic Extract – Alkaloids, Glycosides, Tannins, Flavonoids and Saponins
Methanolic Extract - Alkaloids, Glycosides, Tannins, and Saponins
49
Distilled Water Extract - Alkaloids, Glycosides, Flavonoids and Saponins
Presence of tested secondary metabolites in the crude extracts is in line with earlier
reports as mentioned in the analytical framework of this study. The phytoconstituents detected in
the plant materials could be responsible for their cytotoxic activity though their exact mode of
action is not within the scope of this study.
B. Brine Shrimp Lethality Bio-assay
The brine shrimp lethality assay represents a rapid, inexpensive and simple bioassay for
testing plant extracts bioactivity which in most cases correlates reasonably well with cytotoxic
and anti-tumor properties.
In case of brine shrimp lethality bioassay, the lethality of the crude extracts prepared
through the use of different solvents was evaluated against A. salina for 24 hours of exposure to
the samples and the positive control, Methotrexate.
The LC50 were found to be 6.61, 11.93 and 11.48 μg/ml for Ethanolic, Methanolic and
Distilled Water Extract of Malatalong leaves respectively, on the other hand LC50 were found to
be 6.17, 10.0, and 19.95 μg/ml for Ethanolic, Methanolic and Distilled Water Extract of Suob
Kabayo leaves respectively. This clearly indicates the presence of potent bioactive principles in
these extractives, which might be very useful as antiproliferative, antitumor, pesticidal and other
bioactive agents.
The brine shrimp lethality bio-assay results in this study were interpreted as follows: LC50
<1.0 μg/ml – highly toxic; LC50 1.0‐10.0 μg/ml – toxic; LC50 10.0‐30.0 μg/ml – moderately
50
toxic; LC50 >30<100 μg/ml – mildly toxic, LC50 > 100μg/ml as non‐toxic. (Meyer, B.N.,
Ferrigini, R.N., Putnam, J.E., Jacobsen, L.B., Nichols, D.E., McLaughlin, J.L. (1982) Brine
shrimp: A convenient general bioassay for active plant constituents. Planta Medica 45, 31‐35.)
Based on this we can deduce that T1, T3, and T4, are all toxic while T2, T5 and T6 are all
moderately toxic. With the positive control also considered as toxic.
The degree of lethality was found to be directly proportional to the concentration of the
extract. Maximum mortalities took place at a concentration of 500 μg /ml whereas least
mortalities were at 5 μg/ml concentration. In other words, mortality increased gradually with the
increase in concentration of the test samples.
The brine shrimp lethality assay also indicates various degrees of toxicity. Aqueous
(water) extract showed a level of toxicity from 11.14 to 19.95 μg/ml. Ethanolic extract had a
range from 6.17 to 6.61 μg/ml while Methanolic extract registered a range of 10.0 to 11.93
μg/ml. Thus ethanolic extract of both Suob Kabayo and Malatalong Leaves showed a higher
level of toxicity. The result of these study revealed that the toxic activity of the plant extract
decreases with increase in polarity of solvent. This can be seen in terms of the polarity of
compound extracted by each solvent in addition to their intrinsic bioactivity and by their ability
to dissolve or diffuse in different solvent media used in the assay. The presence of these
compounds is of paramount importance in the pharmaceutical industry.
51
The LC50 values of the plant extracts were obtained by a plot of percentage of the shrimp
nauplii killed against the concentrations of the extracts and the best-fit line was obtained from
the data by means of regression analysis.
This significant lethality of several plant extracts to brine shrimp is an indicative of the
presence of potent cytotoxic components which warrants further investigation. Although the
brine shrimp lethality assay is rather inadequate regarding the elucidation of the mechanism of
action, it is very useful to assess the bioactivity of the plant extracts.
Our results indicate the plant extract to be toxic. There is need for further studies on this
plant to ascertain the active compound(s) and its true toxicity so as to maximize the use of these
endemic plants in development of future pharmaceutical products. However, the result of the
phytochemical screening is essential in interpreting the result of the brine shrimp lethality bio-
assay in this particular study.
The presence of alkaloid in both Malatalong and Suob Kabayo leaf extracts is a
manifestation that these plants are naturally toxic. It has been proven already in literatures that
the alkaloid content of leaves of plants tends to be a positive function of alkaloid toxicity.
(Donald A. Levin: The toxicity of plant alkaloids: an Ecogeographic perspective). In fact,
the toxicity of alkaloids from tropical plants is much greater than those from temperate plants.
Moreover, the presence of flavonoids and saponins in the ethanolic extracts of
Malatalong and Suob Kabayo strengthens the claim for its toxicity. Flavonoids are widely
distributed in edible plants and beverages and have been previously used in traditional
medicines, so they are believed to be nontoxic. However, this family of compounds possess a
52
diverse range of activities in mammalian cells. Related studies showed that flavonoids are found
to be toxic to cancer or immortalized cells but are less toxic to normal cells.
On the other hand, saponins in plants may serve as anti-feedants, and to protect the plant
against microbes and fungi. Some plant saponins are often bitter to taste and so can reduce plant
palatability or even imbue them with life-threatening animal toxicity. Data make clear that some
saponins are toxic to cold-blooded organisms and insects at particular concentrations. (Francis,
George; Zohar Kerem, Harinder P. S. Makkar and Klaus Becker: 2002. The biological
action of saponins in animal systems: A Review)
53
CHAPTER VI
SUMMARY, CONCLUSION AND RECOMMENDATION
A. Summary of the Study
Nearly all plants are associated with some medicinal value. The use of plants
especially in traditional medicine is currently well acknowledged and accepted in our
country.
Extraction of bioactive compounds from medicinal plants permits the demonstration
of their physiological activity. It facilitates pharmacological studies and leads to the synthesis
of pure and potent compounds with decreased toxicity. Furthermore, the active components
of herbal remedies have the advantage of being combined with many other substances that
may be contraindicated with conventional foods or drugs.
The main objectives of this study were to determine the phytochemical constituents
and cytotoxic activities of leaf extract of Suob Kabayo and Malatalong plants.
Brine shrimp lethality bioassay was carried out to investigate the cytotoxic properties
of the leaf extracts using three different solvents.
54
Six different concentrations of sample extracts were made, using Dimethyl sulfoxide
in triplicates. Ten brine shrimp larvae (10 nauplii,) were introduced into each test tube.
B. Summary of Findings
The summary of the research findings are hereby enumerated:
1. The different experimental treatments showed varying levels of cytotoxic
activities ranging from moderately toxic to toxic.
2. Ethanolic Leaf extract of Suob Kabayo plant exhibited the highest LC50 among
the treatments (4.08 microgram/mL) with 100%, 80%, 70%, and 60% of
percentage mortality rate in various levels of concentration prepared.
3. Treatments 1, 3 and 4 showed the same level of cytotoxicity with that of the
positive control.
4. The degree of lethality was found to be directly proportional to the concentration
of the extract. Maximum mortality rate of 100 % was observed with 500μg/ml
while the minimum or least mortality rate was recorded at 5μg/ml.
5. There is no significant difference between and among the LC50 of the different
treatments.
55
C. Conclusion
Malatalong and Suob Kabayo leaf extract possess cytotoxic compounds as they
exhibit cytotoxic properties when tested using Brine Shrimp Lethality Bio-assay. The
researchers believe that these plants are cytotoxic active and are potential sources of
pharmaceutical agents for producing medicinal products in the future.
D. Recommendations
Based on the result and findings of the study, the following recommendations are
given.
1. Extraction, isolation and characterization of the active principles of these endemic
plants should be conducted to determine specific principles present in the plants.
2. Other tests should be conducted on the plants as to their possible anti-bacterial
and anti-fungal properties.
3. Further analysis on the toxicity of the combined effect of Malatalong and Suob
Kabayo leaf extract should be conducted.
56
BIBLIOGRAPHY
Mohammad A.A., Rubina A., Khondoker A. & Mohammad S. A. (2009) “In Vitro Cytotoxic
Properties of Ethanolic Extracts of Various Parts of Swietenia Mahagoni”. European
Journal of Scientific Research. ISSN 1450-216X Vol.32 No.4 pp.541-544 ©
EuroJournals Publishing, Inc.
Angayarkanni J., Ramkumar K.M., Poornima T. & Priyadarshini U. (2007) “Cytotoxic Activity
of Amorphophallus paeopiifolius Tuber Extracts In Vitro”. American – Eurasian J. Agric.
& Environment. Sci., 2(4) 395 – 398 ISSN 1818-6769
Md. Sekendar Ali, Md. Saiful Islam, Md. Rabiul, Mohammed Aktar Sayeed and Md. Rakiful
Islam. “Antibacterial and Cytotoxic Activity of Ethanol Extract of Mikania Cordata
(Burm. F.) B.L. Robinson Leaves”.
“Antimicrobial Activity and Brine Shrimp Lethality Bioassay of the Leaves Extract of Dillenia
indica Linn” DOI: 10.4103/0975-1483.62213 PMID: 21331191
57
Sarder Fahim Hossain, Md. Siddiqul Islam, Salma Parvin ,Tahiatul Shams,Mohammad Fahim
Kadir, S. M. Ashraful Islam, A. G. M. Mostofa , Muhammad Shahdaat Bin Sayeed
“Antimicrobial Screening and Brine Shrimp Lethality Bioassay of Calotropis gigantean”,
Danmalam, U. H., Abdullahi, L. M., Agunu, A. and Musa, K. Y. “Acute toxicity studies and
hypoglycemic activity of The methanol extract of the leaves of hyptis Suaveolens poit.
(lamiaceae)” Nigerian Journal of Pharmaceutical Sciences Vol. 8, No. 2, October, 2009,
ISSN: 0189-823X
Farhana Alam Ripa, Mahmuda Haque, Laizuman Nahar, and Laizuman Nahar. “Antibacterial,
Cytotoxic and Antioxidant Activity of Passiflora Edulis Sims” European Journal of
Scientific Research ISSN 1450-216X Vol.31 No.4 (2009), pp.592-598.
Jerry l. Mclaughlin, Ph.D., et.al. “The use of biological assays to evaluate botanicals” Drug
Information Journal, Vol. 32, pp. 513–524, 1998 0092-8615/98.
Internet sources:
http://www.diahome.org/productfiles/8357/diaj_12496.pdf
http://webcache.googleusercontent.com/search?q=cache:syPUGrFZw3AJ:www.lumiia.com/
download.php?filename%3Ddescargas/
studies_on_extracts_abstract.pdf+solanum+verbascifolium+study&hl=fil
http://www.noabbiodiscoveries.com/assays/invitro/cytotoxicity_studies.pdf
http://en.wikipedia.org/wiki/Cytotoxicity
58
http://www.moleculardevices.com/Applications/Cellular-Function/Cytotoxicity.html
http://www.eurojournals.com/ejsr_32_4_11.pdf
http://www.idosi.org/aejaes/jaes2(4)/12.pdf
http://www.jbclinpharm.com/volume2issue2articles/pdf/jbcp_v2_iss2_pr_47.pdf
APPENDIX A
Raw Data
Table 1: The mortality of Brine Shrimp at varying levels of concentrations of Methanolic
leaves extract of Suob Kabayo.
Concentration ReplicatesMeanR1 R2 R3
5 µg/mL 0 0 0 0
10 µg/mL 4 5 6 5
20 µg/mL 5 7 9 7
50 µg/mL 7 8 9 8
100 µg/mL 10 10 10 10
500 µg/mL 10 10 10 10
59
Table 2: The mortality of Brine Shrimp at varying levels of concentrations of Ethanolic leaf
extract of Suob Kabayo.
Concentration ReplicatesMeanR1 R2 R3
5 µg/mL 0 0 0 0
10 µg/mL 5 6 7 6
20 µg/mL 5 7 9 7
50 µg/mL 7 8 9 8
100 µg/mL 10 10 10 10
500 µg/mL 10 10 10 10
60
Table 3: The mortality of Brine Shrimp at varying levels of concentrations of Distilled
Water Leaf extract of Suob Kabayo.
Concentration ReplicatesMeanR1 R2 R3
5 µg/mL 0 0 0 0
10 µg/mL 2 4 6 4
20 µg/mL 4 7 7 6
50 µg/mL 6 7 8 7
100 µg/mL 8 9 10 9
500 µg/mL 10 10 10 10
61
Table 4: The mortality of Brine Shrimp at varying levels of concentrations of Malatalong
Methanolic Leaf Extract.
Concentration ReplicatesMeanR1 R2 R3
5 µg/mL 0 1 2 1
10 µg/mL 3 5 7 5
20 µg/mL 6 7 8 7
50 µg/mL 8 9 10 9
100 µg/mL 10 10 10 10
500 µg/mL 10 10 10 10
62
Table 5: The mortality of Brine Shrimp at varying levels of concentrations of Malatalong
Ethanolic Leaf Extract.
Concentration ReplicatesMeanR1 R2 R3
5 µg/mL 0 0 0 0
10 µg/mL 5 6 7 6
20 µg/mL 6 7 8 7
50 µg/mL 7 8 9 8
100 µg/mL 10 10 10 10
500 µg/mL 10 10 10 10
63
Table 6: The mortality of Brine Shrimp at varying levels of concentrations of Distilled
Water Extract of Malatalong Leaves.
Concentration ReplicatesMeanR1 R2 R3
5 µg/mL 0 1 2 1
10 µg/mL 3 5 7 5
20 µg/mL 6 7 8 7
50 µg/mL 7 8 9 8
100 µg/mL 9 9 9 9
500 µg/mL 10 10 10 10
64
Table 7: The mortality of Brine Shrimp at varying levels of concentrations of the control,
Methotrexate.
Concentration ReplicatesMeanR1 R2 R3
5 µg/mL 0 0 0 0
10 µg/mL 8 8 8 8
20 µg/mL 8 9 10 9
50 µg/mL 10 10 10 10
100 µg/mL 10 10 10 10
500 µg/mL 10 10 10 10
65
Observation of average mortality and LC50 value of Suob Kabayo and Malatalong in
different solvents
Table 8: Methanolic Leaf Extract of Suob Kabayo Observation of average mortality
Concentration(μg/ml)
Replicates
No. Of Shrimp
Test
Average Mortality after
24 hours% Average Mortality
LC50(μg/ml)
5
R1 - a 10
00 00
10
R2 - a 10
R3 - a 10
10
R1 - b 10
05 50R2 - b 10
R3 - b 10
20
R1 - c 10
07 70R2 - c 10
R3 - c 10
50
R1 - d 10
08 80R2 - d 10
R3 - d 10
100
R1 - e 10
10 100R2 - e 10
R3 - e 10
500
R1 - f 10
10 100R2 - f 10
R3 - f 10
Table 9: Ethanolic Leaf Extract of Suob Kabayo Observation of average mortality
66
Concentration(μg/ml)
Replicates
No. Of Shrimp
Test
Average Mortality after
24 hours% Average Mortality
LC50(μg/ml)
5
R1 - a 10
00 00
4.08
R2 - a 10
R3 - a 10
10
R1 - b 10
07 70R2 - b 10
R3 - b 10
20
R1 - c 10
08 80R2 - c 10
R3 - c 10
50
R1 - d 10
09 90R2 - d 10
R3 - d 10
100
R1 - e 10
10 100R2 - e 10
R3 - e 10
500
R1 - f 10
10 100R2 - f 10
R3 - f 10
Table 10: Distilled Water Leaf Extract of Suob Kabayo Observation of average mortality
67
Concentration(μg/ml)
Replicates
No. Of Shrimp
Test
Average Mortality after
24 hours% Average Mortality
LC50(μg/ml)
5
R1 - a 10
00 00
19.95
R2 - a 10
R3 - a 10
10
R1 - b 10
04 40R2 - b 10
R3 - b 10
20
R1 - c 10
06 60R2 - c 10
R3 - c 10
50
R1 - d 10
07 70R2 - d 10
R3 - d 10
100
R1 - e 10
09 90R2 - e 10
R3 - e 10
500
R1 - f 10
10 100R2 - f 10
R3 - f 10
Table 11: Methanolic Leaf Extract of Malatalong Observation of average mortality
68
Concentration(μg/ml)
Replicates
No. Of Shrimp
Test
Average Mortality after
24 hours% Average Mortality
LC50(μg/ml)
5
R1 - a 10
01 10
11.93
R2 - a 10
R3 - a 10
10
R1 - b 10
05 50R2 - b 10
R3 - b 10
20
R1 - c 10
07 70R2 - c 10
R3 - c 10
50
R1 - d 10
09 90R2 - d 10
R3 - d 10
100
R1 - e 10
10 100R2 - e 10
R3 - e 10
500
R1 - f 10
10 100R2 - f 10
R3 - f 10
Table 12: Ethanolic Leaf Extract of Malatalong Observation of average mortality
69
Concentration(μg/ml)
Replicates
No. Of Shrimp
Test
Average Mortality after
24 hours% Average Mortality
LC50(μg/ml)
5
R1 - a 10
00 00
6.61
R2 - a 10
R3 - a 10
10
R1 - b 10
06 60R2 - b 10
R3 - b 10
20
R1 - c 10
07 70R2 - c 10
R3 - c 10
50
R1 - d 10
08 80R2 - d 10
R3 - d 10
100
R1 - e 10
10 100R2 - e 10
R3 - e 10
500
R1 - f 10
10 100R2 - f 10
R3 - f 10
Table 13: Distilled Water Extract of Malatalong Leaves Observation of average mortality
70
Concentration(μg/ml)
Replicates
No. Of Shrimp
Test
Average Mortality after
24 hours% Average Mortality
LC50(μg/ml)
5
R1 - a 10
01 10
11.48
R2 - a 10
R3 - a 10
10
R1 - b 10
05 50R2 - b 10
R3 - b 10
20
R1 - c 10
07 70R2 - c 10
R3 - c 10
50
R1 - d 10
08 80R2 - d 10
R3 - d 10
100
R1 - e 10
09 90R2 - e 10
R3 - e 10
500
R1 - f 10
10 100R2 - f 10
R3 - f 10
Table 14: Positive Control - Observation of average mortality
71
Concentration(μg/ml)
Replicates
No. Of Shrimp
Test
Average Mortality after
24 hours% Average Mortality
LC50(μg/ml)
5
R1 - a 10
00 00
3.09
R2 - a 10
R3 - a 10
10
R1 - b 10
08 80R2 - b 10
R3 - b 10
20
R1 - c 10
09 90R2 - c 10
R3 - c 10
50
R1 - d 10
10 100R2 - d 10
R3 - d 10
100
R1 - e 10
10 100R2 - e 10
R3 - e 10
500
R1 - f 10
10 100R2 - f 10
R3 - f 10
APPENDIX B
72
Cost Analysis
Quantity Unit Description Cost
2 L Methanol 2,800.00
2 L Ethanol 1,500.00
4 L Distilled Water 280.00
50 Mg Methotrexate 290.00
1 Pack Brine Shrimp Eggs 300.00
9 Hour(s)Usage of Rotary
Evaporator1000.00
2 Test(s)Phytochemical
Screening600.00
8 Piece(s) Bottle 12.00
1 Bottle Methotrexate 290.00
APPENDIX C
73
Duration and Schedule of Activities
1 month
Number of Weeks
0 1 2 3 4
Number of Days
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Legend:
Gathering of Materials
Actual Experimentation and Data Gathering
Statistical Analysis
Writing of Final Papers
APPENDIX D
74
Documentation
Gathering of the Malatalong and Suob Kabayo leaves
Grinding of Malatalong and Suob Kabayo leaves
75
Preparing of the treatments for the 48 hours soaking
Filtration of the prepared treatments soaked for
48 hours in different solvents
76
Subjecting the treatments in the Rotary Evaporator
Gathered crude extracts from the treatments
77
Phytochemical screening of the extracts
The treatments in different reagents for the phytochemical analysis
78
Preparation of the concentration of the treatments
The treatments
79
The crude extracts dissolved in Dimethy Sulfuxide
Cultured Brine Shrimp
80
Test tubes containing 10 Brine Shrimps each
Applying the treatments to the test tubes of 10 nauplii each
81
APPENDIX E
Receipts
82
83
APPENDIX F
Certification
84
APPENDIX G
Communication Letter
85
APPENDIX H
Phytochemical Result
Specimen Solvent Alkaloids Glycosides Tannins Flavonoids Saponins
86