Cytotoxicity and Antioxidant Activity of Identified Secondary

Metabolites in Myristica simiarum Leaves

INTRODUCTION

Heart diseases, diabetes, malignant neoplasm and cancer are one of the leading

causes of death in the Philippines (Department of Health, 2019). Patients with low socio-

economic status tend to neglect such diseases due to expensive disease management and

treatment in the Philippines thus, relying on herbal remedies instead of paying expensive

hospital fees. More Filipinos tend to rely on natural products as prevention to various kinds

of diseases. Overproduction of oxidants in the human body is responsible for the

pathogenesis of some diseases. The scavenging of these oxidants is thought to be an

effective measure to depress the level of oxidative stress of organisms. Because of

associated side effects and toxicity of synthetic chemotherapeutic drugs, it would be

important to discover new biologic agents that are less toxic for normal cells that possess

potential antiproliferative effects on cancer cells. Screening active compounds from plants,

leads to the discovery of new medicinal drugs that are promising for the treatment of

various diseases including cancer (Graham et al., 2000).

Plants are an important source of bioactive compounds with antioxidant capacity

and during the last years, the interest for the use of natural products has significantly

increased. The protective effects of phytochemicals, also known as plant secondary

metabolites can be attributed to direct scavenging activities against reactive oxygen

species, as well as to the induction of intracellular antioxidant effect (Zhang et al., 2015).

Antioxidant phytochemicals play a key role in oxidative stress control and in the prevention

of related disorders, such as premature aging, degenerative diseases, diabetes, and cancer
 Cytotoxicity and Antioxidant 2

(Faraone, Rai, & Chiummiento et al., 2018). Recent studies also show that phytochemicals

in plants exhibit cytotoxicity against human carcinomas (Aliomrani, Jafarian, &

Zolfaghari, 2017)

In the province of Capiz, Tanghas, identified as Myristica simiarum, an evergreen

plant usually found in forests. Often neglected by locals because of its unknown use but

several sources claim that it does have medicinal applications. It is believed that its plant

parts are used to cure skin diseases. Literature review and studies are yet to be examined

and almost none is known on its phytochemical properties and potential therapeutic use,

thus the researchers aim to identify its phytochemical components, to evaluate the cytotoxic

and antioxidant activities.

MATERIALS AND METHODS

Materials

The materials used in this study were 500g Myristica simiarum leaves, DPPH

powder, Brine Shrimp eggs, Ascorbic Acid Standard, Hydrochloric acid, Mayer’s reagent,

5% FeCl, 2N NaOH, 10% FeCl, Distilled Water, HNO3, Ninhydrin Reagent, Sulphuric

Acid, Barfoed Reagent, and Molisch’s Reagent.

Tools

The tools used in the study were UV-Visible Spectrophotometer, Rotary

Evaporator, Test tubes, 100mL volumetric flask, 50mL volumetric flask, micropipette,

Rectangular Glass Container, 100mL Graduated cylinder, Analytical Balance, Pasteur

Pipette, Light Source, Microtip pipette, and Magnifying glass.

 Cytotoxicity and Antioxidant 3

General Procedure

Gathering of Plant Samples

The leaves of Myristica simiarum were randomly

collected from Poblacion Takas, Cuartero, Capiz. The

plant samples collected was brought to the Office of the

Provincial Agriculturist and was identified and

authenticated by Dr. Audie B. Belargo, Senior

Agriculturist. Figure 1. M. simiarum Leaves

Photo taken by the Researcher

Plant Sample Preparation

The plant samples were washed thoroughly to remove extraneous matter. The

plants were dried using the method described by Romero-Buenavides et al. (2018). Briefly,

the collected leaves were subjected to a dehydration process in a drying tray with

airflow at a temperature of 32°C for seven days.

Extraction of Plant Sample

Maceration method of extraction described by

Guevara (2005) was used. Two-hundred fifty grams of

dried plant samples were cut and placed in an Erlenmeyer

flask and treated with sufficient 95% ethanol to completely

submerge the material. The flasks were stoppered and the

plant samples were kept soaked for 48 hours. The filtrate

of the plant sample were decanted while the plant residue Figure 2. Rotary Evaporation
Photo taken by the Researcher

was discarded. The filtrate was concentrated under a

vacuum using rotary evaporator at a temperature below 50 o C. The extracts were stored in
 Cytotoxicity and Antioxidant 4

wide mouth container and kept refrigerated until the actual biological assay. The extracted

sample produced a solid paste, it was isolated and placed in a sterile container

Phytochemical Screening

The extracted plant sample was homogenized in

95% ethanol and used for various phytochemical tests. All

determinations were done in two (2) replications.

Test for Alkaloids

2 mL of concentrated Hydrochloric acid (HCl) was

Figure 3. Phytochemical
Screening
added to 2 mL plant extract. Then a few drops of Mayer’s
Photo taken by the Researcher

reagent was added using a microtip pipette. The presence

of green color or white precipitation indicates the presence of alkaloids.

Test for Tannins

1 mL of ferric chloride (5% FeCl) was added to 1 mL of plant extract. The

formation of dark blue or greenish black color indicates the presence of tannins.

Test for Saponins

2 mL distilled water was added to 2 mL plant extract. Homogenized using a vortex

and shaken manually for 15 minutes. Formation of 1 cm layer of foam indicates presence

of saponins.

Test for Flavonoids

1 mL of 2N sodium hydroxide (NaOH) was added to 2 mL plant extract. Formation

of dark yellow color indicates the presence of flavonoids.

 Cytotoxicity and Antioxidant 5

Test for Phenols

2mL distilled water followed by few drops of 10% ferric chloride was added to 1

mL plant extract. Formation of blue/green color indicates the presence of phenols.

Test for Proteins and Amino Acid:

Xanthoproteic Test

2 mL of plant extract and 0.5 mL of concentrated nitric acid were added by the side

of the test tube. Presence of a dark yellow colour indicates the presence of proteins and

amino acids.

Ninhydrin Test

A few drops of Ninhydrin reagent was added to the plant extract. The formation of

a violet or purplish coloured ring indicates the presence of amino acids.

Test for sugars:

Molisch’s Test

Molisch’s reagent was prepared. 2 drops of the reagent was added to the plant

extract. To this solution, 1 mL of concentrated sulphuric acid was added to the side of the

inclined test tube. Reddish violet ring at the junction of the two layers indicates the presence

of sugars.

Barfoed’s Test

Barfoed reagent was prepared by dissolving 13.3 g of crystalline neutral copper

actetate in 200mL of 1% acetic solution. The plant extract was dissolved in distilled water

and heated with the reagent. A red color precipitate indicates the presence of

monosaccharides.
 Cytotoxicity and Antioxidant 6

Determination of the Different Treatments

There will be four (4) concentrations as treatments for the Brine Shrimp Lethality

Assay (BSLA) namely: Treatment A – 1 µg/ mL of plant extract; Treatment B – 10 µg/

mL of plant extract; Treatment C – 100 µg/ mL of plant extract; and Treatment D – 1000

µg/ mL of plant extract. There will be four (4) treatments for DPPH radical scavenging

assay namely: Treatment A – 5 µg/ mL of plant extract; Treatment B – 10 µg/ mL of plant

extract; Treatment C – 25 µg/ mL of plant extract; and Treatment D – 50 µg/ mL of plant

extract

Brine Shrimp Lethality Assay

Brine Shrimp Lethality Assay (BSLA) was used to

measure preliminary cytoxicity of the plant extract as

described by Olowa and Oneza (2013).

Brine shrimp eggs were obtained from the Aqua

Laboratory of the Office of the Provincial Agriculturist.

Artificial seawater was prepared by dissolving 38 g of sea

Figure 4. Induction of Different
Concentrations for BSLA
salt in 1 liter of distilled water for hatching the shrimp Photo taken by the Researcher

eggs. The seawater was placed in a small plastic container with a partition for dark and

light areas. Shrimp eggs were added into the dark side of the chamber while a light source

above the other side will attract the hatched shrimp. Two days were allowed for the shrimp

to hatch and mature as nauplii (larva). After two days, when the shrimp larvae were ready,

4 mL of the artificial seawater was added to each test tube and 10 brine shrimps were

introduced into each tube. Thus, there were a total of 30 shrimps per concentration. Then

the volume was adjusted with artificial seawater up to 5 mL per test tube. The test tubes
 Cytotoxicity and Antioxidant 7

were left uncovered under the lamp. The number of surviving shrimps were counted using

a magnifying glass and recorded after 24 hours. A brine shrimp was considered dead if it

is immotile and settles at the bottom of the test tube. The percentage mortality was

computed using the formula:

Number of Nauplii Dead

% Lethality = x100
Total Number of Nauplii

This is to ensure that the death (mortality) of the nauplii is attributed to the bioactive

compounds present in the plant extracts.

Using probit analysis, the lethality concentration (LC50) was assessed at 95%

confidence intervals. LC50 of less than 100 µg/mL was considered as potent. LC50 value

of less than 1000 µg/mL is toxic while LC50 value of greater than 1000 µg/mL is non-

toxic.

DPPH Radical Scavenging Activity

The antioxidant activity was measured using DPPH

free radical scavenging assay as described by Saha et al.

(2008) with slight modifications.

Reagent Preparation for DPPH Assay

The DPPH stock solution and plant extracts was

prepared in varying concentrations. Dilution method was

Figure 5. DPPH Stock Solution
used in formulating different concentrations from the stock Photo taken by the Researcher

solution by the formula:

C1 V1 =C2 V2

Where, C1 = Initial Concentration of the Stock solution; V1 = Initial Volume of

solution needed; C2= Final Concentration (Treatment); V2 = Final Volume of the solution.
 Cytotoxicity and Antioxidant 8

DPPH (0.1 mM 1, 1- diphenyl-2- picrylhydrazyl) - Ethanol Solution

The 0.00394 grams of DPPH was dissolved in 10 mL 95% ethanol. The 1 mL from

stock solution was measured and added to ethanol to come up with 10 mL solution.

Treatment A: 5 ug/mL Extract

In order to make 100 mL of solution, 10 mL from the stock solution of 50ug/mL

was measured and added to 90 mL of distilled water to obtain 100 mL.

Treatment B: 10 ug/mL Extract

In order to make 100 mL of solution, 20 mL from the stock solution of 50 ug/mL

was measured and added to 80 mL of distilled water to obtain 100 mL.

Treatment C: 25 ug/mL Extract

In order to make 100 mL of solution, 50 mL from the stock solution of 50ug/mL

was measured and added to 50 mL of distilled water to obtain 100 mL.

Treatment D: 50 ug/mL Extract

The 0.1 g of extract was diluted on 2 L of distilled water.

DPPH Assay

The 0.1 mmol/L solution of DPPH in ethanol was prepared and 1mL of this solution

will be added to 3mL of extract's solution at different

concentrations (5, 10, 25 and 50 μg/mL). After 30 min,

absorbance was measured at 517nm using

spectrophotometer.

Vitamin C (ascorbic acid) was used as a reference

drug following the same concentration of the plant extracts

Figure 6. Reading of
for comparison of the extent of free radical scavenging Absorbance using
Spectrophotometer
Photo taken by the Researcher
 Cytotoxicity and Antioxidant 9

activity of the plant extracts. The percentage inhibition activity was calculated from:

A0 - A1
% inhibition= × 10
A0

Where A0 is the absorbance of the control (DPPH), and A1 is the absorbance of the

extract/ standard. Median inhibitory concentration (IC50) value was calculated from the

equation of line obtained by plotting a graph of concentration (μg/ml) versus % inhibition.

The median inhibitory concentration measures the potency/effectiveness of a drug in

inhibiting a specific biological or biochemical function, in this assay – the free radical

scavenging activity. The average IC50 (7.65 ug/mL) lies between TA (5ug/mL) and TB

(10ug/mL) of the positive control. This means that the standard - ascorbic acid’s highest

antioxidant potency was found in between the two treatments. In other words, the positive

control is most effective when 7.65 ug/mL dosage was used. This explains why the results

of this study was compared only to 5ug/mL and 10ug/mL ascorbic acid.

Data Gathering

The phytochemical analyses, percent lethality, and percent inhibition was

computed and the corresponding LC50 and lC50 values of the bioassays.

Proper Disposal of Tools Used

Tools used were washed with a commercial disinfectant and some were disposed

properly in assigned bins.

Statistical analysis

The data were analyzed on Statistical Tool for Agricultural Research (STAR)

released by the International Rice Research Institute (IRRI). One-way ANOVA was used

as test statistic at 1% level of significance.

 Cytotoxicity and Antioxidant 10

RESULTS

Table 1 shows the different phytochemical tests to determine if different

phytochemicals present in Myristica simiarum.

Results show that the phytochemicals present are Tannins, Flavonoids, Phenols,

Proteins and Amino Acids. While Alkaloids and Saponins are absent. Phytochemical

analysis show that there is a presence of phytochemicals in Myristica simiarum leaves.

Table 1. Phytochemical Screening of Myristica simiarum Leaves

Test Replicate 1 Replicate 2

Alkaloids - -

Tannins + +

Saponins - -

Flavonoids + +

Phenols + +

Xanthoproteic Test (Proteins and - -

Amino Acid)
- -
Ninhydrin Test (Amino Acid)
Molisch’s Test (Sugars) + +

+ +
Barfoed’s Test (Monosaccharides)
( + ) Indicates Presences; ( - ) Indicates Absence
 Cytotoxicity and Antioxidant 11

Table 2 shows the percent lethality of the different concetrations Myristica simiarum

Leaves Extract against Brine Shrimp. The percentage mortality was affected by the

different concentrations, according to the inferred statistical analysis.

The highly significant effect between the different concentrations of Myristica

simiarum leaves extract. The LSD further revealed that Treatment D (1000 ug/mL) got the

highest percent mortality having 96.67%, followed by Treatment C (100 ug/mL), and

Treatment B (100 ug/mL) with 76.67% and 56.67% respectively. While Treatment A (1

µg/mL) got the lowest percent mortality with 10.00%.

Table 2. Percent Lethality of the Different Concentrations of Myristica simiarum Leaves

Extract against Brine Shrimp
Replicates (%)
Treatments Mean ±S.D
1 2 3
A – 1 µg/mL 0 20 10 10.00d 10.00

B – 10 µg/mL 50 60 60 56.67c 5.77

C – 100 µg/mL 80 70 80 76.67b 5.77

D – 1000 µg/mL 100 100 90 96.67a 5.77

LSD= 3.36 p –value = 0.000 significant

*column mean with the same letter superscripts are not significantly different
 Cytotoxicity and Antioxidant 12

Table 3 shows the percentage inhibitions of the different concetrations Myristica

simiarum leaves extract when DPPH assay is used. The percentage inhibited was affected

by the different concentrations, according to the inferred statistical analysis.

The highly significant effect between the different concentrations of Myristica

simiarum leaves extract. LSD further revealed that Treatment D (50 µg/mL) got the highest

percentage inhibited having 86.79%, followed by Treatment C (25 µg/mL) with 65.81%

and Treatment A (5 µg/mL) got the least percentage inhibited which is 18.00%. However,

there is no significant difference among Treatment B (10 µg/mL) and Positive controls: 5

µg/mL and 10 µg/mL with percent inhibitions 45.93%, 42.09%, and 53.33% respectively.

Table 3. Percent Inhibitions of the Different Concentrations of Myristica simiarum

Leaves Extract using DPPH assay.
Replicates (%)
Treatments Mean ±S.D
1 2 3
A – 5 µg/mL 18.52399 17.12177 18.37638 18.00738d 0.77

B – 10 µg/mL 50.92251 42.21402 44.35424 45.83026c 4.54

C – 25 µg/mL 65.38745 65.90406 66.12546 65.80566b 0.38

D – 50 µg/mL 88.48708 86.27306 85.60886 86.78967a 1.51

(+) 5 µg/mL 50.44280 38.67159 40.14760 42.08733c 6.41

(+) 10 µg/mL 54.09594 53.21033 52.69373 53.33333c 0.71

LSD= 6.10 p –value = 0.000 significant

*column mean with the same letter superscripts are not significantly different
 Cytotoxicity and Antioxidant 13

DISCUSSION

The results of the present study can be attributed to the phytochemical analysis

conducted where Myristica simiarum leaves were found to contain tannins, flavonoids, and

phenols. This also conformed to the study of Umesh (2014), the health benefit results from

the secondary metabolites synthesized in the plant that includes the bioactive constituents

like phenolics, flavonoids and tannins.

The cytotoxic activity of Myristica simiarum leaves were measured using Brine

Shrimp Lethality Assay (BSLA). Results show that there is a significant difference among

the varying concentrations of Myristica simiarum leaves. The highest percent lethality was

observed in treatment D (1000 µg/mL) which is the highest concentration. The median

lethal concentration (LC50) computed was found to be less than 1000 µg/mL. Which

means that the plant extracts are toxic against experimental organisms.

The antioxidant activity was measured using DPPH radical scavenging assay.

Results show that there is a significant difference among the treatments. The highest

percent inhibition of the DPPH free radical was observed in treatment D (50 µg/mL), which

is the highest concentration. Additionally, Treatment B (10 µg/mL) is comparable to the

positive controls which means that the plant extract is most effective at 10 µg/mL. Very

low IC50 value computed suggest effective free radical inhibition.

Tannins. Anticarcinogenic potentials of tannins may be related to their

antioxidative property, which is important in protecting cellular oxidative damage (Gulcin

et al., 2010). The mechanism of tannins in biological samples inhibit proliferation of cancer

cells and inducing apoptotic activity against various human carcinomas (Yildrim & Cutlu,

2015). This explains the cytotoxicity induced by Myristica simiarum leaves against brine
 Cytotoxicity and Antioxidant 14

shrimp. Also, tannins exhibit antioxidant activity by donating electrons to DPPH free

radicals with the same mechanism as to other plant secondary metabolites according to

Ujwala et al. (2014). Because of the anticarcinogenic and antioxidant properties of tannins,

it may be the influence of tannins present in Myristica simiarum leaves to exhibit cytoxicity

against experimental organisms and antioxidative effect on the DPPH free radical.

Flavonoids. Flavonoids are polyphenolic compounds that are ubiquitously found in

plants that accounts for their ability to prevent cancer and tumor growth (Ren et al., 2003).

According to Ren (2003), flavonoids can be promising anticancer agents. Briefly, many

mechanisms of action have been identified, including carcinogen inactivation,

antiproliferation, cell cycle arrest, induction of apoptosis and differentiation, inhibition of

angiogenesis, antioxidation and reversal of multidrug resistance or a combination of these

mechanisms. Research studies have shown that flavonoids as single electron donors can

stabilize and scavenge the free radicals (Chung, 2006). This implies that the presence of

flavonoids in Myristica simiarum leaves exhibit cytotoxic activities against brine shrimp

and electron donation in an unstable DPPH free radical.

Phenols. Plant phenolics and extracts are able to affect cell signaling associated

with regulated cell-death mechanism (Lantto, 2017). The use of phenolic-based

phytochemicals as chemopreventive agents coupled with the extensive usage and

prevalence of various phenols as industrial chemicals or environmental pollutants warrants

a thorough and systematic study of the variables that define their chemical reactivities and

subsequent biological activities (Selassie et al., 2005). Phenolic compounds which possess

antioxidant property can quench DPPH by providing hydrogen atoms or by electron

donation, via a free-radical attack on the DPPH molecule and converting them to a
 Cytotoxicity and Antioxidant 15

colorless/bleached product a stable diamagnetic molecule (Matthaus, 2002) which result

in decrease in the absorbance at 517 nm. (Mathew et al., 2015). Additionally, phenolic

compounds found in plant extracts exhibit cytotoxicity against experimental organisms like

brine shrimp and human cancer cell lines as studied by Firdaus et al. (2013). The phenolics

found in Myristica simiarum leaves exhibit cell death on brine shrimp as experimental

models and antioxidant mechanism against DPPH free radical.

The cytotoxicity of plant extracts can be related to the antioxidant activity and

synergism effect of multi-component in extract. Due to the ethical issues in toxicological

tests, substituting animals with alternative models is very important. Brine Shrimp

Lethality Assay is a convenient system for monitoring biological activities of various plant

species (Hamidi et al., 2014). Various studies showed that phytochemicals tannins,

flavonoids, and phenols induce cytotoxicity against brine shrimp and human cancer cell.

Thus, an effective phytochemical-mediated cytotoxic activity of plant extracts. Antioxidant

mechanism delays or prevents free radicals formation and thereby suppresses the life-

threatening diseases. Besides, it can prevent cancer progression by maintaining normal cell

cycle regulation, inhibiting cell proliferation and inducing apoptosis. Numerous plants and

their active compounds are reported to possess the antioxidant activity which controls

many disease conditions. Therefore, natural antioxidant compounds have been investigated

for various types of treatment modalities including cancer (Padmapriya, 2017).

The cytotoxic activity was computed with median lethal concentration (LC50). The

LC50 (94 g/mL) computed is lower than 1000 g/mL. This indicates effective toxicity

against experimental organisms according to Olowa et al. (2013). The biochemical half

maximal inhibitory concentration (IC50) is the most commonly used metric for on-target
 Cytotoxicity and Antioxidant 16

activity in lead optimization. The low IC50 (19.47 g/mL) displayed indicates effective

free radical inhibition (Kalliokoski et al., 2013).

The presence of these secondary metabolites and computed LC50 and IC50 values

accounts for the potent cytotoxicity and antioxidant activity of the Myristica simiarum

leaves. Therefore, Myristica simiarum leaves may exhibit toxicity against actual human

cancer cell lines and can be potentially used as a natural antioxidant supplement.

CONCLUSIONS

Therefore, the researchers conclude that:

1. There is a presence of phytochemicals in Myristica simiarum leaves.

2. There is a significant difference in the percent lethality of the different concentrations

of Myristica simiarum leaves against brine shrimp.

3. There is a significant difference in the percent inhibition of the different concentrations

of Myristica simiarum leaves on the DPPH free radical.

4. In terms of cytotoxic activity, a directly proportional relationship is observed between

the different treatments and the percent lethality.

5. In terms of antioxidant activity, a directly proportional relationship is observed between

the different treatments and the percent inhibitions.

 Cytotoxicity and Antioxidant 17

RECOMMENDATIONS

The researchers recommend:

1. To quantify the phytochemicals present in Myristica simiarum leaves.

2. To utilize treatment D (1000 µg/mL) of the Myristica simiarum leaves against actual

human cancer cell lines.

3. To utilize treatment B (10 µg/mL) of the Myristica simiarum leaves as an alternative

natural antioxidant supplement.

4. To further confirm the antioxidant activity of Myristica simiarum leaves using different

antioxidant assays.

5. To further investigate other therapeutic potentials of Myristica simiarum leaves.

 Cytotoxicity and Antioxidant 18

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 Cytotoxicity and Antioxidant 20

ACKNOWLEDGMENTS

The researchers would like to express their heartfelt gratitude towards the pillars

who helped and present to the extent of this study:

To Mrs. Ma. Rita F. Villareal, Principal IV of Capiz National High School, for

giving the researchers the opportunity to conduct this study;

To Dr. Teresita A. Barrio, SHS Assistant Principal II – Academics of Capiz

National High School, for her consent and boundless support;

To Ms. Debs F. Aguana, Research Adviser, for her assistance, recommendations

and for the motivation she gave to the researchers;

To Dr. Stephen G. Sabinay, Research Consultant, for his time and effort in helping

the researchers in the completion of the study;

To Mrs. Florie May C. Castro, for her technical input, support, encouragement and

remarks that gave insight to this study;

To Ms. Shella Maria B. Luda, Class Adviser, for her never-ending support and

motivation;

To the researchers’ parents, relatives and friends, for their moral, emotional and

financial support throughout the conduct of this study;

Above everything else, greatest thanks to the Lord God Almighty for the strength

and wisdom He provided to finish this research study successfully;

Thank you very much!

The Researchers
 Cytotoxicity and Antioxidant 21

Appendices
 Cytotoxicity and Antioxidant 22

Appendix
Appendices A
Raw Data and Statistical Analyses
 Cytotoxicity and Antioxidant 23

Raw Data

Table 4. Number of Brine Shrimp Dead after 24 hours of exposure to the different
concentrations M. simiarum Leaves extract
Number of A. salina Dead
Concentration
R1 R2 R3 Mean

1 µg/mL 0 2 1 1.0

10 µg/mL 5 6 6 5.7

100 µg/mL 8 7 8 7.7

1000 µg/mL 10 10 9 9.7

Table 5. Percent Lethality of Brine Shrimp after 24 hours of exposure to the different
concentrations of M. simiarum Leaves Extract
Percent Lethality (%)
Concentration Mean LC50
R1 R2 R3 (µg/mL)

1 µg/mL 0 20 10 10.0

10 µg/mL 50 60 60 56.7
94.39
100 µg/mL 80 70 80 76.7

1000 µg/mL 100 100 90 96.7

 Cytotoxicity and Antioxidant 24

Mean Percent Lethality of M. simiarum Leaves Extract

100.00000

80.00000
y = 0.0545x + 44.856
R² = 0.5046
60.00000

40.00000

20.00000

0.00000
0 100 200 300 400 500 600 700 800 900 1000

Figure 7. Mean Percent Lethality of the Different Concentrations of M. simiarum Leaves

Extract
 Cytotoxicity and Antioxidant 25

Table 6. Absorbance (nm) at 517nm of the Different Test Samples of M. simiarum

Leaves Extract and Positive Control (Ascorbic Acid) with DPPH Radical
M.simiarum Leaves Replicates

Concentration 1 2 3

5 µg/mL 1.104 1.123 1.106

10 µg/mL 0.665 0.783 0.754

25 µg/mL 0.469 0.462 0.459

50 µg/mL 0.156 0.186 0.195

DPPH (Control/Blank): 135

Ascorbic Acid

Concentrations 1 2 3

5 µg/mL 0.807 0.831 0.811

10 µg/mL 0.622 0.634 0.641

25 µg/mL 0.252 0.244 0.255

50 µg/mL 0.101 0.104 0.105

Table 7. Percent Inhibition of the Different Concentrations of M. simiarum Leaves

Extract
Replicates IC50 Value
Concentrations Mean
1 2 3 (µg/mL)

5 µg/mL 18.52399 17.12177 18.37638 18.00738

10 µg/mL 50.92251 42.21402 44.35424 45.83026

19.47
25 µg/mL 63.38745 65.90406 66.12546 65.80566

50 µg/mL 88.48708 86.27306 85.60886 86.78967

 Cytotoxicity and Antioxidant 26

Table 8. Percent inhibitions of the different concentrations of Positive Control

(Ascorbic Acid)
Replicates
Concentrations Mean IC50 Value
1 2 3 (µg/mL)

5 µg/mL 40.44280 38.67159 40.14760 39.75400

10 µg/mL 54.09594 53.21033 52.69373 53.33333

7.65
25 µg/mL 81.40221 81.99262 81.18081 81.52522

50 µg/mL 32.54613 92.32472 92.25092 92.37392

M. simiarum - Mean % Inhibition (DPPH Assay)

120

y = 1.3577x + 23.559
R² = 0.8764
100

y = 1.1279x + 41.368
R² = 0.8738
80
Mean % Inhibition

Mean % Inhibition
(Tanghas)
60 Mean % Inhibition
(Control)
Linear (Mean %
Inhibition (Tanghas))
Linear (Mean %
40
Inhibition (Control))

20

0
0 10 20 30 40 50 60
Concentrations (ug/mL)

Figure 8. Mean Percent Inhibition of the Different Concentrations of M. simiarum Leaves

Extract
 Cytotoxicity and Antioxidant 27

Statistical Analyses

Brine Shrimp Lethality Assay

Statistical Tool for Agricultural Research (STAR)

Sat Aug 31 14:18:47 2019

Analysis of Variance
Completely Randomized Design

=================================================
ANALYSIS FOR RESPONSE VARIABLE: X..Lethality
=================================================

Summary Information
-----------------------------------------------------------------------
---
FACTOR NO. OF LEVELS LEVELS
-----------------------------------------------------------------------
---
Concentration 4 1 æg/mL, 10 æg/mL, 100 æg/mL, 1000
æg/mL
-----------------------------------------------------------------------
---
Number of Observations Read and Used: 12

ANOVA TABLE
Response Variable: X..Lethality
----------------------------------------------------------------
Source DF Sum of Square Mean Square F Value Pr(> F)
----------------------------------------------------------------
Concentration 3 12400.0000 4133.3333 82.67 0.0000
Error 8 400.0000 50.0000
Total 11 12800.0000
----------------------------------------------------------------

Summary Statistics
---------------------------
CV(%) X..Lethality Mean
---------------------------
11.79 60
---------------------------

Standard Errors
------------------------
Effects StdErr
------------------------
Concentration 5.77
------------------------

Pairwise Mean Comparison of Concentration

 Cytotoxicity and Antioxidant 28

Least Significant Difference (LSD) Test

Alpha 0.01
Error Degrees of Freedom 8
Error Mean Square 50.0000
Critical Value 3.3554
Test Statistics 19.3723

Summary of the Result:

-------------------------------------
Concentration means N group
-------------------------------------
1 æg/mL 10.00 3 d
10 æg/mL 56.67 3 c
100 æg/mL 76.67 3 b
1000 æg/mL 96.67 3 a
-------------------------------------
Means with the same letter are not significantly different.

M. simiarum DPPH Radical Scavenging

Statistical Tool for Agricultural Research (STAR)

Sat Aug 31 08:53:59 2019

Analysis of Variance
Completely Randomized Design

==================================================
ANALYSIS FOR RESPONSE VARIABLE: X..Inhibition
==================================================

Summary Information
----------------------------------------------------
FACTOR NO. OF LEVELS LEVELS
----------------------------------------------------
Concentration 6 C10, C25, ..., PC5
----------------------------------------------------
Number of Observations Read and Used: 18

ANOVA TABLE
Response Variable: X..Inhibition
----------------------------------------------------------------
Source DF Sum of Square Mean Square F Value Pr(> F)
----------------------------------------------------------------
Concentration 5 8026.7191 1605.3438 147.67 0.0000
Error 12 130.4529 10.8711
Total 17 8157.1720
----------------------------------------------------------------

Summary Statistics
----------------------------
CV(%) X..Inhibition Mean
----------------------------
6.32 52.14
----------------------------
 Cytotoxicity and Antioxidant 29

Standard Errors
------------------------
Effects StdErr
------------------------
Concentration 2.69
------------------------

Pairwise Mean Comparison of Concentration

Tukeys's Honest Significant Difference (HSD) Test

Alpha 0.01
Error Degrees of Freedom 12
Error Mean Square 10.8711
Critical Value 6.1011
Test Statistics 11.6141

Summary of the Result:

-------------------------------------
Concentration means N group
-------------------------------------
C10 45.83 3 c
C25 65.81 3 b
C5 18.01 3 d
C50 86.79 3 a
PC10 53.33 3 c
PC5 43.09 3 c
-------------------------------------
Means with the same letter are not significantly different.

Ascorbic Acid DPPH Radical Scavenging

Statistical Tool for Agricultural Research (STAR)

Sun Sep 01 09:29:01 2019

Analysis of Variance
Completely Randomized Design

==================================================
ANALYSIS FOR RESPONSE VARIABLE: X..Inhibition
==================================================

Summary Information
---------------------------------------------------
FACTOR NO. OF LEVELS LEVELS
---------------------------------------------------
Concentration 4 C10, C25, C5, C50
---------------------------------------------------
Number of Observations Read and Used: 12

ANOVA TABLE
Response Variable: X..Inhibition
----------------------------------------------------------------
Source DF Sum of Square Mean Square F Value Pr(> F)
 Cytotoxicity and Antioxidant 30

----------------------------------------------------------------
Concentration 3 4836.2005 1612.0668 154.17 0.0000
Error 8 83.6489 10.4561
Total 11 4919.8494
----------------------------------------------------------------

Summary Statistics
----------------------------
CV(%) X..Inhibition Mean
----------------------------
4.78 67.58
----------------------------

Standard Errors
------------------------
Effects StdErr
------------------------
Concentration 2.64
------------------------

Pairwise Mean Comparison of Concentration

Least Significant Difference (LSD) Test

Alpha 0.01
Error Degrees of Freedom 8
Error Mean Square 10.4561
Critical Value 3.3554
Test Statistics 8.8590

Summary of the Result:

-------------------------------------
Concentration means N group
-------------------------------------
C10 53.33 3 c
C25 81.53 3 b
C5 43.09 3 d
C50 92.37 3 a
-------------------------------------
Means with the same letter are not significantly different.

% Inhibition of Final Results (Table 3)

Statistical Tool for Agricultural Research (STAR)

Sun Sep 01 15:03:11 2019

Analysis of Variance
Completely Randomized Design

===============================================
ANALYSIS FOR RESPONSE VARIABLE: Inhibition
===============================================

Summary Information
--------------------------------------------------
 Cytotoxicity and Antioxidant 31

FACTOR NO. OF LEVELS LEVELS

--------------------------------------------------
Concentration 6 A5, B10, ..., P5
--------------------------------------------------
Number of Observations Read and Used: 18

ANOVA TABLE
Response Variable: Inhibition
----------------------------------------------------------------
Source DF Sum of Square Mean Square F Value Pr(> F)
----------------------------------------------------------------
Concentration 5 8026.7191 1605.3438 147.67 0.0000
Error 12 130.4529 10.8711
Total 17 8157.1720
----------------------------------------------------------------

Summary Statistics
-------------------------
CV(%) Inhibition Mean
-------------------------
6.32 52.14
-------------------------

Standard Errors
------------------------
Effects StdErr
------------------------
Concentration 2.69
------------------------

Pairwise Mean Comparison of Concentration

Tukeys's Honest Significant Difference (HSD) Test

Alpha 0.01
Error Degrees of Freedom 12
Error Mean Square 10.8711
Critical Value 6.1011
Test Statistics 11.6141

Summary of the Result:

-------------------------------------
Concentration means N group
-------------------------------------
A5 18.01 3 d
B10 45.83 3 c
C25 65.81 3 b
D50 86.79 3 a
P10 53.33 3 c
P5 43.09 3 c
-------------------------------------
Means with the same letter are not significantly different.
 Cytotoxicity and Antioxidant 32

Appendix B
Appendices
Letters and Communication
Cytotoxicity and Antioxidant 33
Cytotoxicity and Antioxidant 34
Cytotoxicity and Antioxidant 35
Cytotoxicity and Antioxidant 36
Cytotoxicity and Antioxidant 37
Cytotoxicity and Antioxidant 38
 Cytotoxicity and Antioxidant 39

Appendix C
Appendices
Scanned Logbook
Cytotoxicity and Antioxidant 40
Cytotoxicity and Antioxidant 41
Cytotoxicity and Antioxidant 42
 Cytotoxicity and Antioxidant 43

Appendix
AppendicesD
Project Evaluation Form
Cytotoxicity and Antioxidant 44