Professional Documents
Culture Documents
Final Final Research 2
Final Final Research 2
Final Final Research 2
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
“Cytotoxic activity of Nephrolepis cordifolia using the Brine shrimp (Artemia salina) assay”
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.
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
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
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
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
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
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-
pupation rate, pupal weight and adult emergence of Spodoptera litura (Lepidoptera: Noctuidae)
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
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
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.
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
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
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
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
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
The study also described the morphological changes brought about by the lethal effects of
the extracts.
10
Brine Shrimp Lethality Assay
Concentration R1 R2 R3 Ave.
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.
11
Concentration R1 R2 R3 Ave.
125 g/ mL 1 hr. 8 min. 1 hr. 21 min. 1 hr. 43 min. 1 hr. 24 min.
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
12
90
80
70
60
Time (minutes)
50
40
30
20
10
0
62.5 125 250 500 1000
Concentration (µg/mL)
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
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.
ASW -- -- -- --
(positive control)
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
Groups P-value
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
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),
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
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
Figure 3. Microscopic observation of Brine shrimps in (A) Artificial sea water, (B) DMSO, (C)
Capecitabine
17
D E
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
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/
Antimicrobial Activity and Brine Shrimp Lethality Bioassay of the Leaves Extract of Dillenia
Avendaño Carmen, and Menéndez J. Carlos. (2008) Medicinal Chemistry of Anticancer Drugs.
Comparison of Different Solvents for Phytochemical Extraction Potential from Datura metel
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,
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)
http://docsdrive.com/pdfs/academicjournals/ajpp/2012/41-46.pdf
Somayeh Rajabi, Ali Ramazani, Mehrdad Hamidi, and Tahereh Naji. (2015) Artemia salina as a
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
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
A. Proposal preparation
1. Project brainstorming
4. Writing of introduction
5. Writing of methodology
B. Conduction of research
1. Fern collection
2. Fern extraction
24
4. Brine shrimp hatching
2. Dry-run presentation
D. Presentation
1. Presentation of research
APPENDIX D
25
Budget Item Particulars Estimated Cost
1. Materials/ Supplies
Boxes/Bags(10.00*5pcs) 50.00
a. Diesel 1500.00
3. Meals
a. Lunch 2000.00
4. Others
26
a. Miscellaneous fees 1500.00
Protocol to use the brine shrimp assay to test the potency of medicinal plant extracts
Extraction step
27
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)
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
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
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
Combine water, sea salt and brine shrimp larvae (BSL) in tank. BSL should be floating in the tank for 24
1. Combine 470 ml of water with 1 tbsp of Instant Sea Salt (for marine aquariums)
28
5. Turn on the bubbler (cheap aerators can be found at Pet Stores).
Protocol for testing plant extracts using BSL (brine shrimp larvae) in 24 well trays
(Methanol) to add to 15 ml conical tube and to determine volume of sea water necessary to bring
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
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
6. Label the lid of the 24 well tray. Write with lab marker on lid, so it is clear which wells are getting
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).
29
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.
30