ANTIMITOTIC EFFECT OF Acromitoides purpurus CRUDE VENOM EXTRACT ON Allium cepa ROOT MERISTEMATIC CELLS

Cabales DF, Catague RA, Celeste KP, Dizor JM, Fuentes EM, Labra PR, Locsin RC, Mangente A, Monje MT, Otico F, Tadlas JH

GROUP 4

Background of the Study

Marine animals produce bioactive compounds that have several modes of action including antimicrobial, ion channel blockage, cytotoxic, and many more.

Background of the Study

Cnidarians such as jellyfishes, anemones, and corals also produce potential compounds. Acromitoides purpurus is a jellyfish species commonly found in coastal areas of Negros and Guimaras.

A. purpurus on the shore of Bacolod City Port

On site cutting of tentacles before transport

Background of the Study

Allium cepa is a model system for most researches because it can demonstrate the cellular mechanics like that of large eukaryotes such as mitosis, metabolism, and DNA replication

Significance of the Study

A. purpurus, does not have any use, aside from being an indicator of water pollution. The results of this study will provide evidence to establish the potential use of A. purpurus as an antimitotic agent.

Significance of the Study

If the biologic activity of the extract be firmly established as a chemotherapeutic agent , this can be a probable economic venture for the said locality. This could propel interest of other researches to investigate on its biochemical properties and institute studies on animal models as well as on human cancer cells.

General Objective
The study aims to evaluate the antimitotic effect of the crude venom extracted from A. purpurus on A. cepa root meristematic cells.

Specific Objectives
1. To determine the mean mitotic index of A. cepa root meristematic cells treated with 10%, 50%, 100% crude venom extract, and distilled water (negative control). To determine if there is a significant difference between the mean mitotic indices of A. cepa root meristematic cells treated with crude venom extract and the negative control. To determine if there is significant difference among the mean mitotic indices of A. cepa root meristematic cells treated with 10%, 50% and 100% crude venom extract.

2.

3.

Conceptual Framework
Distilled Water 10% Crude Venom Extract 50% Crude Venom Extract 100% Crude Venom Extract A. cepa root meristematic cells Mean Mitotic Index (MMI)

Methods
Study Design Experimental Randomized Controlled Trial. Study Setting The extraction of A. purpurus venom was conducted at the Southeast Asian Fisheries Development Aquaculture Department (SEAFDEC-AQD), Tigbauan, Iloilo. The Allium Test was conducted at West Visayas State University Central Science Laboratory. Study Period This study was conducted from August 2012 to February 2014.

Maneuvers
Collection and Identification of the Specimen Isolation of Jellyfish Nematocyst Extraction of the Venom from the Nematocyst

Preparation of Microscope Slide

Treatment Set-up (Allium Test of Levan)

Induction of Onion Root Tips

Evaluation of Mitotic Figures

Maneuvers
Collection and Identification of the Specimen Isolation of Jellyfish Nematocyst Extraction of the Venom from the Nematocyst

Preparation of Microscope Slide

Treatment Set-up (Allium Test of Levan)

Induction of Onion Root Tips

Evaluation of Mitotic Figures

Collection of specimen

On site cutting of tentacles

Maneuvers
Collection and Identification of the Specimen Isolation of Jellyfish Nematocyst Extraction of the Venom from the Nematocyst

Preparation of Microscope Slide

Treatment Set-up (Allium Test of Levan)

Induction of Onion Root Tips

Evaluation of Mitotic Figures

Extraction

. Filtering and centrifugation

Maneuvers
Collection and Identification of the Specimen Isolation of Jellyfish Nematocyst Extraction of the Venom from the Nematocyst

Preparation of Microscope Slide

Treatment Set-up (Allium Test of Levan)

Induction of Onion Root Tips

Evaluation of Mitotic Figures

Onion growth induction

Treatment

Maneuvers
Collection and Identification of the Specimen Isolation of Jellyfish Nematocyst Extraction of the Venom from the Nematocyst

Preparation of Microscope Slide

Treatment Set-up (Allium Test of Levan)

Induction of Onion Root Tips

Evaluation of Mitotic Figures

Fixation, Clearing and Staining

Maneuvers
Collection and Identification of the Specimen Isolation of Jellyfish Nematocyst Extraction of the Venom from the Nematocyst

Preparation of Microscope Slide

Treatment Set-up (Allium Test of Levan)

Induction of Onion Root Tips

Evaluation of Mitotic Figures

Microscopy

Mitotic Index

Data Analysis
The data was entered in SPSS v. 22 and was subjected to statistical analysis using One-way Analysis of Variance (ANOVA) and Scheffe Post-hoc analysis with a level of significance set at p value of <0.05.

Results and Discussion

Mean Mitotic Index (MMI)

0.3 0.25 0.2 0.15

0.25

P value: <0.001

0.1
0.05 0 Distilled Water

0.08 0.01

0.06

10% crude 50% crude 100% crude venom venom venom Treatment Fig 1. Mean Mitotic Index of Root Tip Meristem of Allium cepa treated with Distilled Water and Acromitoides purpurus Crude Venom Extract

0.3 0.25 Difference 0.2 0.15 0.1 0.05 0

P value <0.01

P value <0.01

0.24

P value <0.01

0.17

0.19

10% crude venom 50% crude venom 100% crude venom Treatment Fig 2. Post Hoc Analysis (Scheffe) of Mean Mitotic Indices Between Distilled Water and Different Treatment Concentrations of A. purpurus Crude Venom Extract

Table 1. Post Hoc Analysis (Scheffe) of Different Concentrations of A. purpurus Crude Venom Extract Treatment I Treatment II p value 10% crude venom 50% crude venom 0.16 50% crude venom 100% crude venom 0.38 100% crude venom 10% crude venom 0.95

Discussion
It could be due to cytotoxic proteins being cell cycle specific on A. cepa meristematic cells. Cell cycle specific agents usually increase cell kill by increasing the duration of exposure rather than increasing the drug dose.

Discussion
The antimitotic effect of A. purpurus crude venom extract could be attributed to the cytotoxic proteins contained by the jellyfish as was characterized and isolated by Agriam and colleagues in their local study of this particular jellyfish species in 2008.

Discussion
Cytotoxic proteins contained in the venom of A. purpurus nematocysts may have a role in the cell cycle inhibition of A. cepa root meristematic cells.

Conclusion
The A. purpurus crude venom extract has an antimitotic effect which is dose-independent.

Recommendations
Researchers recommend investigating the effects of increasing the duration of exposure of A. cepa to A. purpurus crude venom extract.

Recommendations
Pure cytotoxic protein isolates of the crude venom extract should also be tested on its effects on A. cepa mitotic cell division in order to investigate its potential antimitotic activity.

Recommendations
The use of a standard anticancer drug as a positive control is also recommended to compare the efficacy of the crude venom extracts to A. cepa. The use of human cancer cell line as a test specimen is highly recommended to assess the efficacy of the A. purpurus crude venom extract on actual cancer cells.

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