abstract

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

http://www.eimacs.com/blog/2011/11/introduction-to-modular-addition/

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.


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


15

Table 4: The mortality of Brine Shrimp at varying levels of concentrations of Distilled

Water Leaf extract of Suob Kabayo.


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


16

Table 5: The mortality of Brine Shrimp at varying levels of concentrations of Malatalong

Methanolic Leaf Extract.


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


Ethanolic Leaf extract.


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


µg/mL

18


Water Extract of Malatalong Leaves.


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


µg/mL

19

Table 8: The mortality of Brine Shrimp at varying levels of concentrations of the control,

Methotrexate.


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


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


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



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



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


Replicates

No. Of Shrimp

Test



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


Replicates

No. Of Shrimp

Test



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


Mor

talit

y (%

)

Table 12: Methanolic Leaf Extract of Malatalong Observation of average mortality

31


Replicates

No. Of Shrimp

Test



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


Mor

talit

y (%

)

Table 13: Ethanolic Leaf Extract of Malatalong Observation of average mortality

33


Replicates

No. Of Shrimp

Test



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


Replicates

No. Of Shrimp

Test



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


Mor

talit

y (%

)

Table 15: Positive Control - Observation of average mortality

37


Replicates

No. Of Shrimp

Test



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


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


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.





Suob Kabayo

5 5 4

7 7 7

9 9 7

Malatalong

6 6 6

7 7 7

8 8 8


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


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:






43


Suob Kabayo

7 7 6

8 8 7

9 9 8

Malatalong

7 8 7

8 9 8

9 10 9



Average 8 8 77.66666

7Variance 1 1 1 1


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


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:







Suob Kabayo

10 10 8

10 10 9

10 10 10

45

Malatalong

10 10 9

10 10 9

10 9 9



Average 10 10 99.66666

7Variance 0 0 1 0.5


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


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:



b) There is a significant difference on the mean mortality of brine shrimps using




Suob Kabayo

10 10 9

10 10 10

10 10 10

Malatalong

10 10 10

10 10 10

10 10 10



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


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:






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

http://www.sciencedirect.com/science/article/pii/0305197878900261

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://en.wikipedia.org/wiki/Cytotoxicity

http://www.noabbiodiscoveries.com/assays/invitro/cytotoxicity_studies.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.diahome.org/productfiles/8357/diaj_12496.pdf

http://www.moleculardevices.com/Applications/Cellular-Function/Cytotoxicity.html

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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.


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.


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


Water Leaf extract of Suob Kabayo.


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


Methanolic Leaf Extract.


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


Ethanolic Leaf Extract.


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


Water Extract of Malatalong Leaves.


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.


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


Replicates

No. Of Shrimp

Test



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


Replicates

No. Of Shrimp

Test



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


Replicates

No. Of Shrimp

Test



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


Replicates

No. Of Shrimp

Test



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


Replicates

No. Of Shrimp

Test



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


Replicates

No. Of Shrimp

Test



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


Replicates

No. Of Shrimp

Test



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

abstract

Documents

modulo art

solanum verbascifolium

replication

distilled

ethanolic

highest mortality

methanolic

suob kabayo