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American Journal of  
Life Science and Innovation (AJLSI)

Phytochemical Analyses and Cytotoxicity Activity Using Artemia salinaArtemia salina (Brine Shrimp) 
Lethality Assay of  the Caladium bicolorCaladium bicolor and Alocasia sanderianaAlocasia sanderiana Ethanolic Leaf  Extract

Barnuevo, A. E.1, Abkilan, M. J. M.1, Agan, F. A. A. S.1, Almoete, M. A. B.1, Bastian, J. A. L.1, Brigola, S. D. D.1, Celiz, M. A. F. P.1, 
Contreras, E. B. D.1, Cuizon, B. L. A.1, Dalaga, E. E. A.1, Perez, J. G.1, Jamili, J. J.1, Penaverde, F. V.1, Leong-on, M. S.1*

Volume 4 Issue 2, Year 2025
ISSN: 2833-1397 (Online)

DOI: https://doi.org/10.54536/ajlsi.v4i2.5052
https://journals.e-palli.com/home/index.php/ajlsi

Article Information ABSTRACT

Received: April 12, 2025

Accepted: May 16, 2025

Published: October 14, 2025

Even though a lot of  research has previously been done on both C. bicolor and A. anderiana, 
more has to be done to assess the phytochemicals and cytotoxic potential (LC50 value) 
of  their leaves. This work sought to identify the phytochemicals and cytotoxic activity of  
crude ethanolic leaf  extracts of  C. bicolor and A. anderiana using the A. salina (brine shrimp) 
Lethality Assay. During the phytochemical analysis, the leaves were boiled in distilled water 
and mixed with various reagents. The colors were examined to determine whether the 
various phytochemicals were present or not. A. salina cysts in saltwater were permitted to 
hatch in order to perform the cytotoxicity. The ten (10) nauplii were moved to various crude 
ethanolic leaf  extract concentrations. The sixth (6th) hour death of  nauplii was used to 
calculate the percent mortality rate. According to the results, the leaves of  C. bicolor tested 
positive for flavonoids, terpenoids, tannins, and saponins. On the other hand, C. bicolor leaves 
tested negative for alkaloids and steroids. The presence of  cardiac glycosides, terpenoids, 
and tannins in A. anderiana leaves was found to be positive. The leaves of  A. anderiana, 
however, tested negative for alkaloids, steroids, flavonoids, and saponins. On the sixth (6th) 
hour, the LC50 value of  A. anderiana crude ethanolic leaf  extract was 4,712.4 μg/ml, whereas 
that of  C. bicolor was 874,028.4 μg/ml. In the sixth hour, neither extract was cytotoxic. The 
phytochemicals and cytotoxic levels of  the crude ethanolic leaf  extracts of  C. bicolor and A. 
anderiana were determined in this study. The findings can be applied to future research on the 
anti-inflammatory, antibacterial, antioxidant, and anti-diabetic bioactivities of  C. bicolor and 
A. anderiana, which may yield non-cytotoxic medications.

Keywords

Alocasia Sanderiana, Brine 
Shrimp Lethality Assay, Caladium 
Bicolor, Cytotoxicity, Phytochemical 
Analyses

1 College of  Pharmacy and Medical Technology, University of  San Agustin, Iloilo City, Philippines
* Corresponding author’s e-mail: mleongon@usa.edu.ph 

INTRODUCTION
Over the years,  medicinal plants has provided a 
notable edge over a variety of  ailments. Phytochemicals 
from medicinal plants were thought to be safe, 
bioactive, and biodegradable. Among many other 
pharmacologically significant activities, laboratory-
based in vitro investigations have connected these 
substances to antioxidant, anti-inflammatory, and anti-
diabetic qualities (Mapfumari et al., 2022). Even at low 
concentrations, phytochemicals found in medicinal 
plants have demonstrated varying degrees of  efficacy 
against bacteria, making them a viable reservoir of  
therapeutic characteristics (Maharaj et al., 2022). In 
order to identify different classes of  phytoconstituents 
present in different parts of  the plant for the purpose 
of  drug discovery, phytochemical screening is a scientific 
process that involves analysis, examination, extraction, 
and experimentation. The active components can then be 
taken for further study and research (Sharma et al., 2022).
Apart from phytochemical analysis, which was employed 
as a preliminary test to screen for the therapeutic 
properties of  plants, cytotoxicity assays were employed 
to determine the capacity of  particular chemicals or 
mediator cells to consume living cells. The cytotoxicity 
test employed in this study is the brine shrimp lethality 
test (BSLT). The toxicity test is based on the number 
of  brine shrimp that die after being exposed to varying 

concentrations because brine shrimp are cytotoxic to a 
variety of  chemicals and natural compounds.
In traditional medicine, C. bicolor and A. anderiana are 
frequently used to cure a variety of  conditions, such as 
jaundice, boils, snake bites, and hyperglycemia (Arbain et 
al., 2022). However, according to research by Ezea (2022), 
C. bicolor, also referred to as “Ede Umuagbara,” is a wild 
cocoyam found in southeast Nigeria.
Numerous species of  Alocasia have been found to 
have medicinal uses, including acute toxicity tests and 
anti-inflammatory, anti-diabetic, antihyperglycemic, 
antioxidant, antibacterial, antiparasitic, and anti-cancer 
effects (Arbain et al., 2022).
Despite the fact that both C. bicolor and A. anderiana have 
been the subject of  numerous investigations, more research 
is required to assess the phytochemicals’ cytotoxicity. 
Nevertheless, there was a gap in determining the extracts’ 
lethal dose. Only the widely recognized phytochemicals 
have been the subject of  the few research that have been 
conducted on both plants for the phytochemical analysis. 
The presence of  other phytochemical kinds in the plants 
used in this investigation has not yet been investigated.
The study sought to identify the phytochemicals and 
cytotoxicity activities using the brine shrimp lethality 
assay  of  C. bicolor and A. anderiana. The study’s specific 
goal was to identify the phytochemicals such as tannins, 
saponins, terpenoids, flavonoids,  steroids, and alkaloids 



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found in the leaf  extracts of  C. bicolor and A. anderiana. 
The study also determined the degree of  cytotoxicity 
of  C. bicolor and A. sanderiana, as well as the median 
lethal concentration (LC50) of  their crude ethanolic leaf  
extracts on the sixth hours.
The results of  the phytochemical analyses and cytotoxicity 
activity are useful to potential pharmaceutical researchers 
that will use the plant-derived constituents of  C. bicolor 
and A. sanderiana for future bioactivity screening.

MATERIALS AND METHODS
Research Design
The phytochemical analyses of  the two plants’ crude 
ethanolic leaf  extracts were described using a descriptive 
study design. However, an experimental design was 
employed to use the various amounts of  crude ethanolic 
leaf  extracts to evaluate the degrees of  cytotoxicity.

Research Setting
At a private institution in Iloilo City, the phytochemical 
and cytotoxicity investigations were carried out utilizing 
the phytochemical screening (color test) and brine shrimp 
lethality assay.

Ethical Considerations
The accredited Ethics Review Committee of  a private 
university had reviewed the research.

Data Collection Procedures
Plant Identification and Collection: The leaves of  C. bicolor 
(Figure 1.) and A. sanderiana (Figure 2) were gathered 
from Buhang Jaro in Iloilo City and Balabag in Pavia, 

Figure 1: Photograph of  C. bicolor Plant

Figure 2: Photograph of  A. sanderiana Plant

respectively. For accurate identification, the Department 
of  Agriculture identified the plant samples. The mature 
leaves that were gathered were pest-free and devoid of  
any dry or browning areas (Leong-on, 2022b).

Phytochemical Analyses
Using conventional protocols, tannins, saponins, 
terpenoids, cardiac glycosides, flavonoids, steroids, and 
alkaloids were screened. Leong-on (2020) mentioned the 
works of  Tariq et al. (2012) and Tiwari et al. (2011), and 
all of  the processes were taken from those works (Leong-
on, 2022c).

Test for Tannins (Ferric chloride test)
A 0.5g of  dry leaves were boiled for three minutes in a 
test tube with 20 mL of  distilled water. After cooling, 
the mixture was filtered. Three milliliters of  filtrate were 
then mixed with five drops of  0.1% ferric chloride. The 
presence of  tannins was detected by the development of  
a brownish-green to blue-black coloring.

Test for Terpenoids (Salkowski Test)
A 0.5g of  dried plant material was dissolved in 10 mL of  
distilled water. After cooling, the mixture was filtered. A 
layer was created by adding 0.75 mL of  concentrated sulfuric 
acid after 1.25 mL of  extract and 0.5 mL of  chloroform had 
been combined. The presence of  terpenoids was detected 
by the interface turning reddish-brown.

Test for Flavonoids (Aluminum chloride colorimetric 
technique)
A 0.5 gram of  dried plant material was added to 20 
milliliters of  distilled water and brought to a boil. After 
cooling, the mixture was filtered. Three drops of  a 1% 
aluminum solution were added to three milliliters of  
filtrate. Flavonoids were detected by the emergence of  
a yellow tint.

Test for Steroids (Salkowski test)
One milligram of  crude plant extract was dissolved in ten 
milliliters of  chloroform. The test tube was then filled by 
the sides with an equivalent volume of  pure sulfuric acid. 
The test tube’s top layer glowing red and the sulfuric acid 
layer changing yellow with green fluorescence were signs 
that steroids were present.

Test for Alkaloids (Wagner’s test)
A0.5g of  dried plant material was cooked in 10 mL of  
distilled water. After cooling, the mixture was filtered. 
Next, three milliliters of  filtrate were mixed with three 
drops of  Wagner’s reagent. A white precipitate appeared, 
indicating the presence of  alkaloids.

Extraction of  Plant Compounds
Each plant’s fresh leaves were cleaned, dried, and then 
pulverized into a powder. Specimen vials were then 
used to keep the powdered leaves. The extractant 



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employed was ninety-five percent (95%) ethanol at room 
temperature. The extractant was poured until it was one 
inch higher than the plant material’s overall height. To 
prevent the plant material from oxidizing, a black cloth 
was placed over the container holding the mixture. For 
48–72 hours, the extract was homogenized, and it was 
stirred every 8–12 hours. A fresh cloth or filter paper 
was then used to filter the extract. Evaporation using a 
rotary evaporator set at 40–45 degrees Celsius was used 
to dry and remove the extractant. After that, half  of  the 
extract was put on a petri dish and covered with gauze 
to dry it (Leong-on, 2022a). Before being utilized for a 
cytotoxicity assay and a test for steroids, this was left for 
three to four days.

Cytotoxic Analyses Using Brine Shrimp Lethality 
Assay
To ascertain the plants’ potential for cytotoxicity, they 
were analyzed using the brine shrimp lethality assay. 
Compared to more complex and costly in-vivo and in-
vitro experiments, this bioassay was able to detect a wide 
range of  bioactivity that was present in the extract.

Preparation of  Seawater
Thirty-eight grams (38 g) of  rock salt was dissolved in 1 
L of  distilled water.

Hatching of  Brine Shrimps
For this technique, brine shrimp eggs, also known as 
Artemia salina leach, were gathered. The prepared 
seawater was poured into a shallow rectangular dish, filling 
it 34% of  the way. The jar was filled with fifty milligrams 
(50 mg) of  brine shrimp cysts. Black cartolina paper was 
used to darken a section of  the container. For two days, 
the nauplii cysts were let to hatch in the seawater tank and 
grow into nauplii-like adults. The nauplii without eggshells 
were gathered in the lit section of  the tiny tank after the 
freshly born shrimp were attracted to a light source. To 
make the fresh, pure saltwater more visible, these were 
pipetted and filtered. For every sample concentration, 
ten (10) nauplii were employed (Suryawanshi et al., 2020; 
Leong-on, 2022).

Preparation of  Test Samples
Every sample concentration was measured using small 
container cups. After adding 100 mg of  the sample to 5 
mL of  the produced saltwater, various concentrations of  
crude ethanolic leaf  extracts, ranging from 0 to 10 mg, 
were added to the cups. One drop of  seawater was added 
to the container for 0 mg. Each addition contained 1 mg 
of  the plant material, and the solution was utilized for the 
remaining concentrations (Leong-on, 2022).

Cytotoxicity Assay
Five milliliters of  prepared saltwater were added to the 
cup with the dry extract and swirled. By pouring a drop of  
seawater to a white spoon and counting the brine shrimp 

nauplii until there were ten, ten were added to each cup 
containing the plant extract. After removing superfluous 
water, two drops containing ten brine shrimp nauplii were 
introduced to the cup containing the ten nauplii together 
with five milliliters of  seawater and extract. Their food, 5 
mL of  saltwater, was mixed with 3 mg of  measured yeast 
in a different container. A single drop of  the suspension 
was added to each cup. The light was used to preserve 
these cups. The nauplii were fed using the yeast solution. 
After six hours, the number of  dead shrimp was counted 
under a magnifying lens, and the percentage of  mortality 
was then calculated.

Recording of  Observations
Every six hours, the quantity of  dead brine shrimp was 
tallied, and the subsequent observations were contrasted 
with the test control. The brine shrimp’s mortality 
percentage was determined by counting the number of  
dead shrimp each hour (Kale et al., 2019).
% mortality = (number of  dead nauplii)/(number of  live 
nauplii)
Using brine shrimp lethality assays of  C. bicolor and A. 
sanderiana crude ethanolic leaf  extracts, this study 
created three (3) trials and five (5) replicates to guarantee 
the validity and reliability of  the procedures to ascertain 
the phytochemical and cytotoxicity analyses. The expert 
validated the results.

Statistical Tool
After calculating the percent mortality (%), the median 
lethal concentration (LC50) was determined six hours 
later using the cytotoxicity bioassay instrument. The 
LC50 value was found using the probit analysis.

Waste Disposal
Toxic materials or residues were disposed of  in receptacles 
designated for hazardous chemical wastes and tightly 
sealed to prevent leaks. Dry infectious garbage was placed 
in a red container, while wet infectious waste was placed 
in a yellow bin (Nandy et al., 2022).

RESULTS AND DISCUSSIONS 
The results indicated that the presence of  tannins with 
a brownish-green solution and saponins with a hazy 
appearance was detected. There were also flavonoids with a 
light yellow solution, and terpenoids with a light red-brown 
solution. Nevertheless, C. bicolor tested negative in the test 
for alkaloids, which had a clear, light brown solution, or 
steroids, which had a dark green solution (Table 1).
Regarding A. sanderiana leaves, it showed positive results 
for the presence of  terpenoids with a light red solution, and 
tannins with a brownish-green solution. With a clear, light 
brown solution devoid of  foam, however, it tested negative 
for saponins. A clear, purple solution was produced, and 
the results were negative for flavonoids as well. Alkaloids 
have a clear, light brown solution and steroids have a dark 
green solution, hence those tests are negative (Table 1).



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Table 2: Cytotoxicity of  Crude Ethanolic of  C. bicolor and A. sanderiana Leaf  Extract Using Brine Shrimp (A. salina) 
Assay
Concentrations (mg/5ml) %  Mortality of  A. salinaA. salina in C. bicolorC. bicolor 

(mean+SD)
%  Mortality of  A. salinaA. salina in A. A. 
sanderianasanderiana (mean+SD)

0 0.0 + 0.00 1.6 + 0.08
1 1.0 + 0.00 2.2 + 0.06
2 1.0 + 0.00 2.2 + 0.12
3 1.0 + 0.00 3.2 + 0.18
4 1.2 + 0.45 3.0 + 0.10
5 1.4 + 0.55 3.6 + 0.03
6 1.2 + 0.45 3.8 + 0.12
7 1.4 + 0.55 3.2 + 0.12
8 1.2 + 0.45 3.6 + 0.08
9 1.4 + 0.55 3.8 + 0.06
10 1.6 + 0.55 4.2 + 0.06
LC 50 874,028.4 µg/ml                             4,712.4 µg/ml

Both C. bicolor and A. sanderiana have saponins and 
tannins. These compounds from Wrightia tinctoria, 
Euphorbia hirta, Thespesia populnea, and Cassia alata 
were demonstrated efficacy against Bacillus subtilis and 
Pseudomonas aeruginosa at a dose of  0.5 µg (Rji et al., 
2019).
Both C. bicolor and A. sanderiana contain terpenoids. 
These compounds are known to possess antiviral, 
antifungal, antimicrobial, and antiparasitic qualities. They 
also have anti-inflammatory, antiviral, and antioxidant 
lower the risk of  vascular diseases (Ullah, 2020) and 
cancer chemopreventive effects (Ramteke et al., 2021).
Both C. bicolor and A. sanderiana have been found to 
contain cardiac glycosides. This compound had been 
shown to increase cardiac output.
It was discovered that C. bicolor contains flavonoids. 
Flavonoids are effective in preventing the growth of  
tumor cells and inducing the activation of  several vital 
detoxification enzymes.
The cytotoxicity of  plants varies according to their 
constituent parts and the extractant used.  When the 
LC50 value was 500–1000 μg/ml, the cytotoxic activity 
was deemed mild. When the LC50 result falls between 
100 and 500 μg/ml, it indicates moderate toxicity; when it 
falls between 0 and 100 μg/ml, it indicates strong toxicity. 
When the LC50 value exceeds 1000 μg/ml, it is classified 
as non-cytotoxic (Nguta et al., 2013). Strongly poisonous 
plants may provide new scaffolding for the development 

of  anti-cancer medications. Conversely, those with low 
toxicity might be excellent candidates for the creation of  
food supplements, nutraceuticals, or herbal medications. 
C. bicolor exhibited less cytotoxic activity than A. 
sanderiana. However, both crude ethanolic leaf  extracts 
were non-cytotoxic because their LC50 values were 
greater than 1000 μg/ml. Non-cytotoxic medications 
may be derived from plants such as C. bicolor and A. 
sanderiana. When compared to chemically manufactured 
drugs, natural products may have fewer adverse effects 
or be non-cytotoxic, which could explain their growing 
popularity.
The study’s results were accurate only when the crude 
ethanolic leaf  extracts of  C. bicolor and A. sanderiana 
death rate was calculated at the sixth hour and LC50 
values were determined. The mature leaves were gathered 
from San Isidro, Jaro, and Balabag, Pavia,  Iloilo City, 
Philippines, at 6:00 in the morning. This investigation 
served as the foundation for additional bioactivity testing 
of  crude ethanolic leaf  extracts of  A. sanderiana and C. 
bicolor.

CONCLUSIONS
The phytochemicals found in C. bicolor leaves included 
flavonoids, terpenoids, cardiac glycosides, tannins, and 
saponins; alkaloids and steroids did not test positive. 
Tannins,and  terpenoids are found in A. sanderiana 
leaves. Alkaloids, steroids, flavonoids, and saponins all 

Table 1: Phytochemical analyses of  the C. bicolor and A. sanderiana Leaves
Plant Tannins Saponins Terpenoids Flavonoids Steroids Alkaloids
C. bicolor Positive Positive Positive Positive Negative Negative
A. sanderiana Positive Negative Positive Negative Negative Negative
Negative Control Negative Negative Negative Negative Negative Negative

The C. bicolor’s LC50 value was 874,028.4 μg/ml whereas 
the A. sanderiana was 4,712.4 μg/ml (Table 1), according 

to A. salina nauplii death rates. At the sixth hour, both the 
ethanolic leaf  extracts were not cytotoxic.



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had negative results.  The LC50 value for  C. bicolor was 
874,028.4 μg/ml but the value for A. sanderiana was 
4,712.4 μg/ml,. This outcome makes it clear that on the 
sixth (6th) hour, neither the crude ethanolic leaf  extract 
of  C. bicolor nor A. sanderiana was cytotoxic. The study’s 
findings can be applied to future research on C. bicolor and 
A. sanderiana’s various bioactivities, including their anti-
inflammatory, antioxidant, antibacterial, and anti-diabetic 
properties.

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