Saint Louis University, Baguio City

School of Natural Sciences

Bachelor of Science in Biology

Cytotoxic Activity of Nephrolepis cordifolia using Brine shrimp (Artemia salina) Lethality
Assay

A Research
Submitted to the Faculty of the Department of Biology
School of Natural Sciences, Saint Louis University
In Partial Fulfillment of the Requirements for the Degree,
Bachelor of Science in Biology

ABEYA, Richard Cai T.

DESIERTO, Jeah Deanne N.
GONZALES, Imee Clarisse F.
IGNACIO, Jannel Olaien N.
MESINA, Andrew James L.
NAVARRO, Ayna Suzane C.
TAPIZ, Andreanina C.

Ms. Tomasa P. Colallad

DECEMBER 17, 2018

 ENDORSEMENT

In partial fulfillment of the requirements for the degree

Bachelor of Science in Biology
This research entitled,

“Cytotoxic activity of Nephrolepis cordifolia using the Brine shrimp (Artemia salina) assay”

Prepared and submitted by:

S1: Abeya, Richard Cai T . S5: Mesina, Andrew James L. .
(Signature over printed name) (Signature over printed name)

S2: Desierto, Jeah Deanne N. . S6: Navarro, Ayna Suzane C. .

(Signature over printed name) (Signature over printed name)

S3: Gonzales, Imee Clarisse F. . S7: Tapiz, Andreanina C. .

(Signature over printed name) (Signature over printed name)

S4: Ignacio, Jannel Olaien N. . S8: ________________________________

(Signature over printed name) (Signature over printed name)

Is hereby endorsed for implementation.

This endorsement implies that this research proposal has been thoroughly read and
reviews and have seen to it that it is scientifically and ethically bound.

Ms. Tomasa P. Colallad .

Faculty Research Promoter
(Signature over printed name)

Date: _______________________
 Table of Contents

I. Abstract………………………………………………………………… 1
II. Introduction…………………………………………………………….. 2
III. Methodology…………………………………………………………… 5
IV. Results and Discussion………………………………………………… 8
V. Conclusion and Recommendations…………………………………… 17
VI. References…………………………………………………………...... 18
VII. Appendix A…………………………………………………………… 20
VIII. Appendix B……………………………………………………………. 21
IX. Appendix C …………………………………………………………… 22
X. Appendix D …………………………………………………………… 24
XI. Appendix E …………………………………………………………… 26

2
 Cytotoxic activity of Nephrolepis cordifolia using Brine shrimp (Artemia salina) assay
Colallad, T.P.(1), Abeya, Richard T.(2), Desierto, Jeah Deanne Shanaiah N.(2), Gonzales, Imee
Clarisse F.(2), Ignacio, Jannel Olaien N.(2), Mesina, Andrew James L.(2), Navarro, Ayna Suzane C.
(2)
, Tapiz, Andreanina C.(2)
Faculty Research Promoter
(1)

Student Researchers, Biology Department, School of Natural Sciences, Saint Louis University
(2)

ABSTRACT

Around half of the drugs currently in clinical use as anticancer drugs are of natural
product origin. Thus, screening of traditional medicinal plants is of paramount importance in
discovering and identifying new medicinal plants and properties as well as isolating new
cytotoxic compounds for life-threatening diseases. Brine shrimp (Artemia salina) lethality (BSL)
assay is a rapid (24 hours), simple, easily mastered and inexpensive method commonly used to
check the cytotoxic effect of bioactive chemicals based on their ability to kill laboratory cultured
larvae (nauplii) (Sarah, Q.S, et al, 2017). A plant that contains several active compounds that
could be tested using BSL assay is Nephrolepis cordifolia (L.) Presl. The study involved the use
the Brine Shrimp Lethality Assay to test the cytotoxicity of methanolic and chloroformic extracts
of Nephrolepis cordifolia of different concentrations; 1000 µg/mL, 500 µg/mL, 250 µg/mL, 125
µg/mL, 62.5 µg/mL which were diluted in Dimethyl Sulfoxide. For each concentration, three
replicates were prepared. This study aimed to determine the cytotoxic activity of N. cordifolia
extracts using the Brine shrimp (Artemia salina) assay. In comparison to capecitabine, with a
lethality period at 1 hr. 27 mins, all concentrations from both the N. cordifolia Methanolic and
Chloroformic extracts were determined as highly cytotoxic against the A. salina nauplii. The
absence of a linear pattern among the results indicated that the lethality of the A. salina was
independent of the concentrations which violates the assumptions needed by most statistical tools
thus limiting the analysis of the results to descriptive method. Dead A. salina nauplii were
subjected to microscopic examination after the experimental exposure to the treatment groups
and were observed to have swelled after exposure to the N. cordifolia Methanolic and
Chloroformic extracts while dead A. salina nauplii exposed to the control groups compared were
observed to have normal morphology. In conclusion, Nephrolepis cordifolia methanolic extract
causes faster lethality compared to its chloroformic extract, implying a potential application as an
anti-metabolite.

Keywords: Brine Shrimp Lethality Assay, Nephrolepis cordifolia, Cytotoxicity, Lethality

3
INTRODUCTION

Around half of the drugs currently in clinical use as anticancer drugs are of natural product

origin. (Avendaño, C., and Menéndez, J. .; (2008). Thus, screening of traditional medicinal

plants is of paramount importance in discovering and identifying new medicinal plants and

properties as well as isolating new cytotoxic compounds for life-threatening diseases.

Brine shrimp (Artemia salina, fairy shrimp or sea monkeys) lethality (BSL) assay is a

rapid (24 hours), simple, easily mastered and inexpensive method commonly used to check the

cytotoxic effect of bioactive chemicals based on their ability to kill laboratory cultured larvae

(nauplii) (Sarah, Q.S, et al, 2017).

A plant that contains several active compounds that was tested using BSL assay is

Nephrolepis cordifolia (L.) Presl. This plant is commonly known as sword fern or narrow fern

and locally known as bayabang, is classified in the Division Tracheophyta, Class

Polypodiopsida, Order Polypodiales, Family Lomariopsidaceae.

Nephrolepis cordifolia is a well-known remedy in the folkloric system of medicine for the

treatment of various types of diseases. Decoction of its rhizomes, pinnae, fresh frond and root

tuber are used to treat cough and colds, fever, headache, indigestion, enteritis-diarrhea,

rheumatism, chest congestion and etc. The medicinal effects of Nephrolepis cordifolia can be

attributed to its antibacterial and antifungal properties. (Rajasekaran, A., & Sivakumar, V., 2009;

Xiao-qing, C., Yu-Cai, S., et al.,2006; Rani, D., et al., 2010; Karunyal, J., and Andrews, B.,

2010; Singh, B.P., and Upadhyay, R., 2014; Gewali M.B., 2008)

In the study of In Vitro Antibacterial and Antifungal Properties of Aqueous and Non-

Aqueous Frond Extracts of Psilotum nudum, Nephrolepis biserrata and Nephrolepis cordifolia,

4
the water extract of N. cordifolia showed antimicrobial properties. (Rani, D., et al, 2010) This

antimicrobial activity of N. cordifolia can be attributed to the secondary metabolites

concentrated in the fern frond. (Stuart, G., 2014)

Nephrolepis cordifolia contains secondary metabolites like alkaloids, steroids, tannins,

flavonoids, terpenoids, cardiac glycosides, and phenolic compounds in the crude extract. (Xavier,

G.S.A., Selvaraj, P., et. al., 2016) These secondary metabolites contribute to the cytotoxic

property of Nephrolepis cordifolia.

The cytotoxic property of the fern could produce several potentials in the field of medicine

in producing anti-cancer drugs. Cytotoxins also have a potential in the field of agriculture as

insecticides. Insecticidal property was also observed in the N. cordifolia crude extract in several

studies done by Xavier, G.S.A, Selvaraj, P., et al. wherein the crude extract and Fe-AgNP (Fe-

AgNP; fern-silver nanoparticles) formulations significantly reduced the developmental period,

pupation rate, pupal weight and adult emergence of Spodoptera litura (Lepidoptera: Noctuidae)

larvae (Xavier, G.S.A., Selvaraj, P., et al, 2016)

The study aimed to assess the cytotoxic activity of methanolic and chloroformic extracts at

different solvent soluble fractions of N. cordifolia extract. The study also described the

morphological changes brought about by the lethal effects of the extracts.

The study sought to answer the following the questions: (1) What concentration of the N.

cordifolia methanolic extract exerts a cytotoxic activity: (a) 1000 µg/mL (b) 500 µg/mL (c) 250

µg/mL (d) 125 µg/mL (e) 62.5 µg/mL (2) What concentration of the N. cordifolia chloroformic

extract exerts a cytotoxic activity? (3) Is there a significant difference in the lethality rate of

Brine shrimp (Artemia salina) nauplii as affected by N. cordifolia methanolic and chloroformic

5
extract? (4) What are the effects of N. cordifolia on the Brine shrimp nauplii in terms of

morphological changes?

The N. cordifolia fronds were collected along Baguio-Bauang Road, Sablan, Benguet at

16ᵒ30’12” N, 120◦29’10”E and 580m elevation. Furthermore the study was performed at The

Natural Sciences Research Unit and the Fr. Gerard Braeckman Museum of Natural History

Museum. The study started from January to November 2018.

The significance of the study includes the exploration and recognition of new medicinal

plants and sources of cytotoxic compounds for life-threatening diseases such as cancer.

Moreover, the results of this study will help with the revelation of the potential of Nephrolepis

cordifolia as a cytotoxicity drug and maybe used by future researches on cancer related

researches.

6
METHODOLOGY

Research Design

The study employed the 5x2 factorial experimental design to determine the cytotoxic

activity of Nephrolepis cordifolia extract using Brine shrimp (Artemia salina) nauplii.

Nephrolepis cordifolia collection

Vegetative Nephrolepis cordifolia samples were collected along Baguio-Bauang Road,

Sablan, Benguet at 16◦30’12” N, 120◦29’10”E and 580m elevation.

Test organism

The Brine shrimp was used as the test organism in this study to determine the cytotoxic

activity of Nephrolepis cordifolia. Brine shrimp is a model organism for eukaryotic cells used in

research and toxicology. In toxicological tests, A. salina assay can be used to screen a large

number of extracts for drug discovery in medicinal plants. This is because in this case, aseptic

techniques are not required, and thus A. salina assays could replace the more ethically

challenging assays that require animal serum. Their freeze-dried cysts (A. salina eggs) can last

for several years and can be hatched into larvae without specialized equipment.

7
Figure 1. Flowchart of the study

8
Materials and methods

Preparation of crude extracts

The mature vegetative lamina of Nephrolepis cordifolia were thoroughly washed with

flowing tap water. The washed lamina were air-dried at room temperature and were cut into

pieces. The cut plant parts were oven-dried and pulverized. 365g of plant material were evenly

divided into two according to mass then macerated with methanol and chloroform for seven

days. The methanolic and chloroformic extract were concentrated using water bath set at 40˚C

were diluted with dimethyl sulfoxide to come up with different concentrations: 1000 µg/mL, 500

µg/mL, 250 µg/mL, 125 µg/mL and 62.5 µg/mL.

Brine Shrimp Lethality Bioassay

Artemia salina cysts hatching

Simulation of sea water was done by dissolving 112 grams of rock salt in four liters of

distilled water and was placed in a 43cm x 21cm x 25cm aquarium containing an aerator on one

side and an incandescent lamp on the other side to create an artificial environment for the brine

shrimp cysts. The aerator supplied enough oxygen for the A. salina cysts. While the incandescent

bulb served as the heat source. This artificial environment was necessary for the survival of the

cysts. A teaspoon of prepared A. salina cysts was obtained from University of the Philippines

Diliman were placed inside the aquarium.

Aliquot preparation of solutions

After 24 to 48 hours of hatching period, nauplii (larvae of A. salina) were isolated

individually with the use of a long-nose Pasteur pipette. Isolated nauplii were placed in small

9
plastic containers along with three mL artificial seawater. Each container contained 20 Artemia

salina nauplii. 10 mL of Nephrolepis cordifolia extract were added to each container containing

twenty Artemia salina nauplii. Time of complete annihilation were recorded.

RESULTS AND DISCUSSION

This study was conducted to determine the cytotoxic activity of the methanolic and

chloroformic extract of Nephrolepis cordifolia at different concentrations using the Brine shrimp

(Artemia salina) assay. The cytotoxicity bioassay against Artemia salina is a simple and

inexpensive tool test cytotoxicity to bio-direct fractionation of natural products and as a predictor

of antitumor and pesticidal properties. It also indicates antiviral, aniplasmodial, antifilarial and

antimalarial activities.

Specifically, the study sought to answer the following the questions: (1) What

concentration of the N. cordifolia methanolic extract exerts a cytotoxic activity: (a) 1000 µg/mL

(b) 500 µg/mL (c) 250 µg/mL (d) 125 µg/mL (e) 62.5 µg/mL (2) What concentration of the N.

cordifolia chloroformic extract exerts a cytotoxic activity? (3) Is there a significant difference in

the lethality rate of Brine shrimp (Artemia salina) nauplii as affected by N. cordifolia methanolic

and chloroformic extract? (4) What are the effects of N. cordifolia on the Brine shrimp nauplii in

terms of morphological changes?

The study also described the morphological changes brought about by the lethal effects of

the extracts.

10
Brine Shrimp Lethality Assay

Table 1: Lethality to N. cordifolia Methanolic Extracts

Concentration R1 R2 R3 Ave.

1000 g/ mL 34 min. 29 min. 32 min. 31 min. 6 sec.

500 g/ mL 45 min. 1 hr. 1hr. 55 min.

250 g/ mL 12 min. 20 min. 25 min. 19 min.

125 g/ mL 55 min. 46 min. 52 min. 51 min.

62.5 g/ mL 31 min. 53 min. 54 min. 46 min.

The lethality effect of different concentrations of N. cordifolia methanolic extracts was

determined on the length of time of the manifestation of signs of death after exposure. From

Table 1, the extract with a concentration of 250 g/ mL had the fastest lethality period among the

concentrations and the extract with a concentration of 500 g/ mL had the slowest lethality

periods. All concentrations are highly toxic as they only required a small fraction of the 24-hour

suggested length of exposure time for Brine Shrimp Lethality Assay. The results display no

linear pattern indicating the absence of a relationship between the concentrations and the length

of time the signs of death manifested. This probably indicates that the concentrations used in the

experiment were highly toxic for the test organisms and were way above the threshold level of

which appreciable relationship between the concentration and the lethality may be observed.

Table 2: Lethality to Chloroformic Extracts

11
 Concentration R1 R2 R3 Ave.

1000 g/ mL 56 min. 54 min. 1 hr. 14 min. 1 hr. 1 min.

500 g/ mL 24 min. 1 hr. 4 min. 49 min. 45 min 6 sec.

250 g/ mL 22 min. 24 min. 22 min. 22 min. 6 sec.

125 g/ mL 1 hr. 8 min. 1 hr. 21 min. 1 hr. 43 min. 1 hr. 24 min.

62.5 g/ mL 25 min. 38 min. 1 hr. 17 min. 46 min. 6 sec.

Regarding the length of time of the manifestation of signs of death after exposure to

Chloroformic Extracts (Table 2), the extract with a concentration of 250 g/ mL had the fastest

lethality period among the concentrations and the extract with a concentration of 125 g/ mL had

the slowest lethality periods. All concentrations are highly toxic as they only required a small

fraction of the 24-hour suggested length of exposure time for Brine Shrimp Lethality Assay. The

results display no linear pattern indicating the absence of a relationship between the

concentrations and the length of time the signs of death manifested. This indicates that the

concentrations used in the experiment were highly toxic and were way above the threshold level

of which the concentration and the lethality may present relationships.

12
 90

80

70

60
Time (minutes)

50

40

30

20

10

0
62.5 125 250 500 1000
Concentration (µg/mL)

Methanolic Extract Chloroformic Extract

Figure 1. Comparison between the lethality rates of Brine shrimp exposed to methanolic and

chloroformic extracts

Comparing the lethality of the fern extracted with methanol and chloroform, Figure 1

displays the average lethality per concentration. Studies on the bioactive components present in

N. cordifolia confirmed the presence of alkaloids, steroids, tannins, flavonoids, terpenoids, and

phenolic compounds (Xavier, G.S.A., Selvaraj, P. and Nida John. 2016). Ronnie (2010) also

confirmed the presence of flavonoids, triterpenoids, alkaloids, tannins, reducing sugars and

steroids. The presence of the mentioned metabolites with their known effects of interfering with

basic cellular and nuclear functions, inhibiting immune responses as well as inducing cell death

through apoptosis and necrosis may be responsible for the cytotoxic activity of the crude

extracts. The graph shows that the N. cordifolia methanolic extract had displayed faster lethality

periods as compared to the N. cordifolia chloroformic extract. Dixon Dhawan and Jeena Gupta

(2017) studied the efficiency of multiple extracting solvents and identified that methanol works

13
best with the extraction of phenolic compounds and flavonoids producing higher concentrations

of bioactive components as compared to the concentrations of similar bioactive components

within chloroformic extracts which were described as at minimal levels. Methanol, a polar

compound is a solvent capable of extracting polar compound but certain group of nonpolar

compounds are fairly soluble in methanol if not readily soluble, therefore, methanol is commonly

used for extraction of bioactive compounds (Agra, 2013). The absence of a linear pattern

indicates the absence of a relationship between the lethality and the extract concentrations. This

is due to the concentration of all chloroformic and methanolic extracts being way above the

threshold level of which the concentration and lethality may present relationships.

Table 3: Length of time of the manifestation of signs of death after exposure to control

groups

R1 R2 R3 Ave.

DMSO 19 hrs. 11 min. 20 hrs. 16 min 20 hrs. 30 min. 21hrs. 1 min

ASW -- -- -- --

Capecitabine 1 hr. 38 min 1 hr. 35 min 1 hr. 8 min 1 hr. 27 mins

(positive control)

As expected of the cytotoxicity of capecitabine, it displayed such rapid lethality to brine

shrimp assay at concentrations of 500 g/mL. Artificial sea water displayed no lethality to the

brine shrimp and DMSO demonstrated slight lethality to brine shrimp, according to a study done

by Sahgal Geethaa, et.,al (2013) DMSO is the safest dissolving agent as it presents the least

14
cytotoxic effects but will start presenting slight cytotoxicity against brine shrimp at

concentrations above 5% which may explain why the brine shrimp died after being exposed to

higher concentration of DMSO.

Table 4: Statistical Analysis of the Treatment Groups and Control Groups

Groups P-value

Concentrations of Methanolic Extract 0.0013

Concentrations of Chloroformic Extract 0.0195

Treatment Groups & Control Groups 2.11 × 10-11

Using Single Factor ANOVA, a p-value of 0.0013 was obtained for the different

concentrations of methanolic extract and a p-value of 0.0195 for the different concentrations of

chloroformic extract, this signifies that there is a significant difference in the length of time the

test specimens manifested signs of mortality as affected by the different concentrations of the

treatment and control groups, implying that the lethality of the different concentrations of the N.

cordifolia methanolic and chloroformic extracts are effected by different toxic phytochemical

agents extracted by the two different extracting solvents.

ANOVA was also used to identify is there is a significant different between the N.

cordifolia extracts and the treatment groups. For the statistical analysis, the concentration with

the least lethality (500 g/ mL concentration for the methanolic extract and the 125 g/ mL

concentration for the chloroformic extract) were selected to be compared with Capecitabine

(Positive Control), and DMSO (vehicular control). A p-value of 2.11 × 10-11 was obtained, which

signifies that there is a significant difference between and among the groups. Post-F-test analysis

15
was not performed due to absence of linear relationship between the independent variable

concentrations of extracts and the dependent variable length of time of onset of lethality.

1400
1200
1000
800
600
400
Time (minutes)

200
0
ic ic l) l)
ol m tro tro
ha
n or n n
et rof Co rC
o
M lo ve la
Ch siti cu
Po ehi
( ( V
ne
a bi SO
ci t DM
pe
Ca

Groups

Figure 2. Average lethality for the 500 g/ mL concentration for the methanolic extract, the

125 g/ mL concentration for the chloroformic extract, Capecitabine (Positive Control),

and DMSO (vehicular control).

Figure 2 shows average lethality for the 500 g/ mL concentration for the methanolic

extract, the 125 g/ mL concentration for the chlorformic extract, Capecitabine (Positive

Control), and DMSO (vehicular control). The 500 g/ mL concentration of the N. cordifolia

methanolic extract shows the least average time for which the signs of death of the brine shrimp

nauplii manifested. This implies that the methanolic extract causes faster lethality than the

chloroformic extract, the positive control and the DMSO. Based from the graph, the

chloroformic extract and capecitabine (positive control) has almost the same lethality. The figure

also shows that DMSO causes the least lethality.

16
Morphological Observations

The observed swelling in the specimens subjected to microscopic examination after the

experimental exposure to the treatment groups is may be due to the inherent activity of DMSO as

an organic solvent to increase the permeability of the test cell membranes allowing influx of

extracellular components into the intracellular environment (X. Q. Wang et al., 2011) This

possible explanation could also be the reason why the actual toxic components of the fern

extracts in either extracting solvent were seemingly potentiated thereby bringing about toxic

effects at the lowest tested concentration. In comparison, Brine shrimp nauplii subjected to

microscopic observations after the experimental exposure to the control groups; positive control

(Capecitabine), negative control (salt water), and vehicular control (DMSO) were all observed to

be of normal morphology and were without any traces of swelling.

Figure 3. Microscopic observation of Brine shrimps in (A) Artificial sea water, (B) DMSO, (C)

Capecitabine

17
 D E

Figure 4. Microscopic observation of Brine shrimp in methanolic concentrations (A) 1000

g/mL, (B) 500 g/mL (C) 250 g/mL (D) 125 g/mL (E) 62.5 g/mL

A B C

D E

18
 Figure 5. Microscopic observation of Brine shrimp in Cloroformic concentrations (A) 1000

g/mL, (B) 500 g/mL (C) 250 g/mL (D) 125 g/mL (E) 62.5 g/mL

CONCLUSION

Nephrolepis cordifolia is locally well known for its medicinal properties. The study has

been conducted with an aim to assess the cytotoxic activity of N. cordifolia methanolic and

chloroformic extracts in terms of its lethality to A. salina assay and determine the optimal

concentrations by which the cytotoxic activities will be presented as well as describe the

morphological changes on the A. salina nauplii brought about by exposure to N. cordifolia

Methanolic and Chloroformic extracts.

The results from this study show that the Methanolic N. cordifolia extracts demonstrated

the fastest lethality indicating its high cytotoxicity against A. salina assay. Further, the absence

of a linear correlation between the results points out that the lethality is independent and is

insignificant to the tested concentrations. In addition, dead A. salina nauplii exposed to the

Methanolic and Chloroformic crude extracts were observed to have swelled and were abnormally

bigger. The potent cytotoxic characteristic of the N. cordifolia methanolic and chloroformic

extracts at is a display of its potential as an anti-metabolite and cancer treatment drug and can be

explored further.

RECOMMENDATION

The researchers recommend the use of more low concentrations to identify medicinally

applicable doses of the extracts and the use of other solvents other than Dimethyl sulfoxide for

the dilution of the extracts. It is further recommended that future researches should consider the

extract dissolved in Dimethyl sulfoxide as a whole solution and the use of distilled water to

19
explore the pure potential of Nephrolepis cordifolia for cytotoxic activities. Finally, interested

future researches should isolate thespecific component of N. cordifolia responsible for its

cytotoxic activity and to explore the cytotoxic activity of N. cordifolia with other assays.

REFERENCES

Apurba sarker apu, shakhawat hossan bhuyan, farjana khatun, mahmuda sultana liza, maima

matin, md. Faruq hossain (2013). Assessment of cytotoxic activity of two medicinal

plants using brine shrimp (artemia salina) as an experimental tool. International journal of

pharmaceutical sciences and research. Retrieved May 10, 2018, from http://ijpsr.com/bft-

article/assessment-of-cytotoxic-activity-of-two-medicinal-plants- using-brine-shrimp-artemia-

salina-as-an-experimental-tool/

AS Apu, MA Muhit, SM Tareq, AH Pathan, ATM Jamaluddin, and  M Ahmed. (2010).

Antimicrobial Activity and Brine Shrimp Lethality Bioassay of the Leaves Extract of Dillenia

indica Linn. US National Library of Medicine National Institute of Health. Retrieved

May 10, 2018, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3035885/

Avendaño Carmen, and Menéndez J. Carlos. (2008) Medicinal Chemistry of Anticancer Drugs.

Elsevier. Retrieved May 19, 2018

Comparison of Different Solvents for Phytochemical Extraction Potential from Datura metel

Plant Leaves. (n.d.). Retrieved from https://scialert.net/fulltextmobile/?doi=ijbc.2017.17.22

Kim Vincent (2008) Probit Analysis Retrieved May 19, 2018, from

http://userwww.sfsu.edu/efc/classes/biol710/probit/ProbitAnalysis.pdf

20
New world encyclopedia (2008). Brine Shrimp. New world encyclopedia Retrieved May 10,

2018, from http://web.newworldencyclopedia.org/entry/Brine_shrimp

Pharmacological Sciences (2018). Natural Products in Cancer Chemotherapy. Pharmacological

Sciences. Retrieved May 10, 2018 from https://www.pharmacologicalsciences.us/anticancer-

drugs/natural-products-in-cance r-chemotherapy.html

S.K. Biswas, A. Chowdhurry, S.Z. Raihan, M.A Muhit, M.A Akbar, and R. mowla. (2012)

Phytochemical investigation with assessment of cytotoxicity and antibacterial activities of

Chloroform Extracts of the leaves of Kalanchoe pinnata. American Journal of Plant

Physiology Retrieved May 19, 2018 from

http://docsdrive.com/pdfs/academicjournals/ajpp/2012/41-46.pdf

Somayeh Rajabi, Ali Ramazani, Mehrdad Hamidi, and Tahereh Naji. (2015) Artemia salina as a

model organism in toxicity assessment of nanoparticles. US National Library of Medicine

National Institute of Health. Retrieved May 19, 2018 from

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4344789/

Wu gong cao (2014) bayabang. Philippine medicinal plants. Retrieved May 10, 2018 from

http://www.stuartxchange.org/Bayabang.html

Xavier, G.S.A., Selvaraj, P. and Nida John. (2016) Impact of phytoecdysone fractions of the

ferns Cyclosorous interruptus, Christella dentata and Nephrolepis cordifolia on the biology of

21
Spodoptera litura (Fab.). JBiopest 9(2):125-134. Retrieved May 10, 2018 from

http://www.jbiopest.com/users/LW8/efiles/vol_9_2_125-134.pdf

APPENDIX A

Figure 5. Set-up of the experiment

A
B
22
 Figure 6. Materials used: (A) Drying oven, (B) Water bath

APPENDIX B

A B

Figure 7. A. salina with varying concentrations of Chloroformic extract (A) and Methanolic

extract (B)

23
APPENDIX C

GANNT CHART

Activity Month

January February March April May June July

A. Proposal preparation

1. Project brainstorming      

2. Gathering of related literature        

3. Construction of research title    

4. Writing of introduction    

5. Writing of methodology    

August September October November December

B. Conduction of research

1. Fern collection  

2. Fern extraction    

3. Dilution of fern extract    

24
4. Brine shrimp hatching  

5. experimentation and observation    

C. Final output preparation

1. Encoding and editing of research paper  

2. Dry-run presentation  

D. Presentation

1. Presentation of research  

APPENDIX D

BUDGET PLAN OF THE PROJECT

25
Budget Item Particulars Estimated Cost

1. Materials/ Supplies

a. Bond paper 1 Ream (300.00*1 pc) 300.00

b. Writing materials Ball pens (10.00 * 5pcs) 50.00

Marker (30.00*3pcs) 90.00

c. Other paper supplies Notebook (15.00* 1pc) 15.00

Folders (10.00*10pcs) 100.00

d. Collection materials Straws (100.00 * 1 pc) 100.00

Shovel (250.00*2pcs) 500.00

Boxes/Bags(10.00*5pcs) 50.00

e. Cutting materials Knives (100.00*2pcs) 200.00

g. Printing costs 500.00

2. Fare for transportation

a. Diesel 1500.00

b. Jeep and Taxi Fares 500.00

3. Meals

a. Lunch 2000.00

b. Snacks and drinks 1000.00

4. Professional/ Service Fee

a. Professional Fees 3000.00

b. Laboratory Fees 14,000.00

4. Others
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a. Miscellaneous fees 1500.00

b. First aid kit 500.00

APPENDIX E

BRINE SHRIMP ASSAY PROTOCOL

Protocol to use the brine shrimp assay to test the potency of medicinal plant extracts

Author: Dr. Kevin Curran

We have been using this assay in the USD Biology Dept.

1. Collect plants and create extracts.

2. Grow your brine shrimp.

3. Incubate hatched brine shrimp in plant extract.

4. Count lethality rates after 24 hours incubation.

Protocol for performing methanol extraction of medicinal plants

Extraction step

1. Break up plant matter, dry in sun on clean surface or in desiccator.

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 2. Prepare 70% methanol. Use large graduated cylinder (700ml of 100%methanol/300ml water).

3. Weigh out 10 grams of dried plant material from each sample. Chop or tear up plant material to

provide greater surface area for extraction (about 1/4 inch sized pieces)

4. Move dried plant material into 250 ml. graduated cylinders.

5. Add 200 ml of 70% Methanol.

6. Label tubes (plant name, operator initials, date and time of beginning of extraction.)

7. Allow to extract for 3-4 days. Stir often – either manually or on rotating platform.

Filtration Step

1. Fold large Whatcom filter in a cone. Place coned filter inside plastic funnel. Put piece of tape on

filter to attach to filter.

2. Pour methanol extraction into filter cone. Allow extract to drip through.

3. Squeeze out final methanol trapped in plant material. Do this by removing wet plant material from

drained bottle. Move material into coffee filter. Hold coffee filter over filter cone and squeeze

gently. This is important to capture compounds trapped in material.

4. Store extract in Freezer (-20°F), ideally in a brown, tinted bottle. In general, compounds should

store well (not degrade) for a 6-8 month time frame in -20°F. At refrigerator (4 °F) you can expect

half of that. Of course, every compound will be slightly different but these are general conditions

for a broad collection of compounds.

Protocol for hatching the brine shrimp larvae

Combine water, sea salt and brine shrimp larvae (BSL) in tank. BSL should be floating in the tank for 24

hrs (hatching) before testing with extracts begin.

1. Combine 470 ml of water with 1 tbsp of Instant Sea Salt (for marine aquariums)

2. Mix thoroughly in large plastic cylinder using parafilm.

3. Pour into plastic tank (empty coke bottle).

4. Add in 1⁄2 teaspoon of BSL (1 gram weight)

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 5. Turn on the bubbler (cheap aerators can be found at Pet Stores).

6. Place lid on top of tank.

7. Return the following day to harvest your larvae.

8. Hatching is optimal at 70-80 ° F.

Protocol for testing plant extracts using BSL (brine shrimp larvae) in 24 well trays

1. Label a 15 ml. conical tube for each test condition.

2. Perform a C1 x V1 = C2 x V2 equation to determine the volume of plant extract in 70% ME

(Methanol) to add to 15 ml conical tube and to determine volume of sea water necessary to bring

conical tubes up to final volume.

3. Pipette determined volume of plant extract (70% ME) into 15 ml conical.

4. Before bringing test solution to final volume, add in ~50 BSL. These will be in sea water, which is

good. Number of duplicate wells being tested will determine approximate number of BSL. For

example, if you are filling 3 wells, then you need 30 BSL total. Therefore, 50 is a safe number to

use for 3 wells.

5. Bring final volume of 15 ml conical tube up to the 15 ml. You now have about 50 BSL swimming

in 15 ml of your test condition at the correct concentration. The 24 hour period of exposure has

officially begun at this moment.

6. Label the lid of the 24 well tray. Write with lab marker on lid, so it is clear which wells are getting

which test condition.

7. Move 10 BSL into each well from appropriate 15 ml conical tube. This can be done by pouring the

test condition/BSL into a shallow dish. With plastic pipette shuttle 10 BSL into each well. You

may find it easier to pipette up 10 BSL with the unaided eye (as opposed to using a dissecting

microscope).

8. Allow the BSL to swim in test condition for 24 hours total.

9. After incubation period, return and count % lethality.

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Notes:

The internal volume of each well in a 24 well plates is 3 ml. We fill each well with 2 ml of extract/larvae.

The final concentration of extract and methanol that the BSL is exposed to in 24 hour period can be

determined by a percent dilution of the solvent. For example, we are using 70% methanol (ME) as our

solvent. We know that we prepared the raw plant material as a 10gram/200 ml concentration.

Therefore, at an undiluted test condition of 70% ME, we would be exposing the BSL to 1g/20ml, which is

50 ug/ml.

When we perform a 1/10 dilution of 70% ME, we then expose the BSL to 7% ME and 5ug/ml of plant

material.

When we perform a 1/100 dilution of 70% ME, we then expose the BSL to .7% ME and .5ug/ml of plant

material.

So, first decide what treatments (plant type and X ug/ml conc.) you want to test. Then make appropriate

dilutions.

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