Toxicological Evaluation of Guyabano (Annona muricata) Seeds

Through Brine Shrimp Lethality Assay (BSLA)

Mary Danengel L. Gimoro, Myle Audrey P. Orbon, Catherine Joy R. Pailden
Antipolo City National Science and Technology High School, Antipolo City, Philippines
ABSTRACT
This study aimed to evaluate the cytotoxicity of Guyabano (Annona muricata) seeds through Brine Shrimp
Lethality Assay (BSLA). Guyabano seeds were used for obtaining crude extract. The seeds were subjected to
ethanolic extraction procedure to obtain the crude extract. The study required Brine Shrimp Lethality Assay to
evaluate the cytotoxicity of Guyabano crude extract. After the experimentation, the crude extract showed strong
cytotoxic effect on the Brine Shrimps (Artemia salina) which suggest bioactive components in the material that can
be used for therapeutic significance.
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INTRODUCTION through the Guyabano seeds and the (+) and (-)
Seeds of Guyabano were commonly thrown away and control?
ignored for they are not food sources. Even if the Guyabano
2. Is there a significant difference between the
fruits were rotten, the seeds were left unaffected, and can last
mortality rates of Brine Shrimp in the different
for years. Some superstitions about Guyabano seeds stated that
concentrations of crude extract after 24 hours?
ingesting Guyabano seeds might kill the person who ingested it
(Kwentong Bicol). 3. Is there an interaction effect between the
Guyabano (Annona muricata) is a popular tropical fruit concentration of the crude extracts from Guyabano
that was believed to have cytotoxic claims and was used as a seeds and the mortality rate of Brine Shrimps?
medication for common illnesses in the ancient times. By this,
many researches were conducted regarding Guyabano but MATERIALS AND METHODS
there was never an actual test using biological organisms. The study requires the evaluation of the toxicological
According to Dr. Ragasa of Derpharmachemica (2015), chemical properties of crude extract from Guyabano seeds through
constituents of the leaves and fruits of Guyabano has certain Brine shrimp lethality bioassay. There are two major
cytotoxic effects on five (5) different cancer cell lines, such as processes in this study, the extraction of crude plant extract
breast, colon, liver, lung, and prostate cancer. from the Guyabano seeds through Ethanolic Extraction
A general bioassay that appears to be capable of Procedure performed at the Department of Science and
detecting a broad spectrum of bioactivity present in plant Technology (DOST) Pharmaceutical Services of Industrial
materials is the Brine Shrimp (Artemia sp.) Lethality Assay Technology Development Institute through the help of Mr.
(BSLA). It is commonly used to detect anti-tumor and pesticidal Romy Estrella, chemist. However, the second procedure was
compounds. It is a process that is affordable and easier to done at University of the Philippines Institute of Biology (UP-
perform which makes it an ideal bioassay. IB) through the help of Mr. Jordan Halili, professor in Institute
In this present study, the seeds of Guyabano was of Biology.
tested in vivo for its cytotoxic effect against Brine Shrimp. In
vivo lethality test has been successfully used as a preliminary Ethanolic
study of cytotoxic and antitumor agents. Thus, the findings of Extraction
this present work would give baseline information on the most done in
promising plant material that can be used as a basis for the DOST
development of new medications and tools of great therapeutic Fig 1. Gathering Fig 2. Preparation of
importance. of Seeds Ethanolic Extraction
Particularly, the study pursued to give answer to the
following questions:
1. What is the mortality rate of Brine Shrimp in the
different concentrations of the crude extract gathered
Fig 5. Hatched Fig 4. Preparation of Fig. 3 Crude
Artemia salina ASW and 48 hours Extract
period hatching time for 1
the Artemia salina
 Preparation of the Control Groups
Fig 7. Preparation Fig 8: Different
for the different Brine Shrimp Toxicity Screening
concentrations of Crude
concentrations The Brine Shrimp Lethality Assay (BSLA) was conducted
Extract and ASW
at the Institute of Biology of University of the Philippines (UP-
IB).
The cytotoxicity of the Guyabano seeds (Annona
muricata L.) was determined through the help of Brine
Shrimps (Artemia salina). The setup used in the BSLA was as
follows. Two (2) well plates were prepared for each of the 5
Fig 10. Setup for BSLA Fig 9. Preparation for concentrations (100µg/mL, 50 µg/ mL, 25 µg/mL, 12.5 µg/mL,
the BSLA 6.25 µg /mL) of the crude extract from Guyabano seeds
(Annona muricata L.). In each well, 13 Brine Shrimps were
Figure 1. Research Methodology
added using a Pasteur pipette with the artificial saline water
(ASW) to make a total of 2.2mL. All setups were incubated for
Plant Material 24 hours at a room with 60 Watts light.
The 865g of Guyabano seeds were dried for 5
days from September 7 to 12, 2018 using a dry-heat oven at the Counting of Brine Shrimps
Pharmaceutical Services of Industrial Technology Development
24 hours later, the test tubes were then inspected
Institute. After drying, the Guyabano seeds went from 870g to
using a magnifying glass. The number of Brine Shrimps which
460g. The 460g of dried Guyabano seeds were then powdered
survived and died were counted and the percent of lethality
to provide a greater surface area. The ground 460g Guyabano
was calculated for each of the concentration.
seeds were then loaded into the thimble, and placed inside the
Soxhlet extractor.
Data Analysis
The percent of lethality was calculated by counting all
Preparation of Artificial Saline Water the dead Brine Shrimps against the total number of Brine
The artificial saline water was used in hatching the Shrimps place in each tube.
Brine Shrimps. 1 L of water was placed in a container and 38g of
salt is added to increase the salinity at a rate suitable for the 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐷𝑒𝑎𝑑 𝑆ℎ𝑟𝑖𝑚𝑝
% 𝑀𝑜𝑟𝑡𝑎𝑙𝑖𝑡𝑦 = × 100
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hatching of the Brine Shrimps (Artemia salina).
RESULTS AND DISCUSSIONS

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 Table 1. Mortality Rate of Artemia salina After 24 Hours interaction effects between the concentration of
Concen- Total no. of Total no. of living Brine Total no. of dead % Mor-
trations Brine Shrimp Shrimp Brine Shrimp tality
crude
6.25 78 5 73 94% extract and mortality rate of Brine Shrimps.
12.50 78 0 78 100% The data mean that the effects of the different
25.00 78 0 78 100% concentrations don’t depend on the value of
concentrations.
50.00 78 0 78 100%
100.00 78 0 78 100%
The high toxicity exerted by the crude
+ Control extract of Guyabano seeds in Brine Shrimp
78 0 78 100%
Lethality Assay suggest bioactive components in
- Control
78 59 19 24% the material.
Table 1 presents the mortality rate of Artemia salina in Wanvoike et al., (2004) investigated the
the different concentrations of Guyabano crude extract as well difference between the mortality rate of varying
as the positive and negative controls in 24 hours. components of crude extract to Brine shrimp and
present work agrees with the findings that there
Table 2. One-Way Analysis of Variance on Mortality Rate in the Different was no significant difference between the
Concentrations mortality rates of the different concentrations.
Source of P- F
Variation SS df MS F value crit
Between CONCLUSIONS
Groups 3.333333 4 0.833333 4.310345 0.00867 2.75871047
Within Guyabano seeds crude extract exhibited
Groups 4.833333 25 0.193333 cytotoxic activity against Brine Shrimps and
Total 8.166667 29
considered as containing active or potent
The result showed that the concentration 12.5
components. The ethno-pharmacological activities
µg/mL, 25 µg/mL, 50 µg/mL and 100 µg/mL of crude
of this plant species is due to the different bioactive
extract has resulted to greater mortality rates than the rest
compounds present in it, such Annonaceous
in 24 hours. The data imply that crude extract has a strong
Acetogenins that can be use as a cure for cancer.
cytotoxicity at high concentrations.
Although, BSLA is inadequate in determining the
The above data affirm the findings of Singh et al.
mechanism of action of the bioactive substances in
2009. The Brine Shrimp Lethality Bioassay shows a
the plant, it is very useful by providing a preliminary
moderate cytotoxicity at high concentrations.
screen that can be supported by a more specific
Table 2 shows the output of the ANOVA Analysis
bioassay, once the active compound has been
on the mortality rate of Artemia salina in the different
isolated. Thus, some useful drugs of therapeutic
concentrations.
importance may be developed out of the research
It can be deduced from the table that there was no
work.
statistically significant difference in the mean mortality
rate of Artemia salina between the concentrations of
RECOMMENDATIONS
crude extract (p=0.00867). This implies that the different
To be more precise, it is suggested to know
concentrations including the positive control have the
the chemical constituents of the crude extract of
same cytotoxic capacity.
Guyabano via NMR spectroscopy and isolate the
This suggest that all the concentrations can be
active compounds present. The author also suggests
used for therapeutic purposes.
to improve the study by performing another
The data further showed that there were no significant
Cytotoxicity assay using Allium cepa.

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