This document describes a study that performed bioassay-guided fractionation of extracts from Codiaeum variegatum against Entamoeba histolytica. The study involved preparing extracts from different parts of C. variegatum using Soxhlet extraction. Fractionation was then performed on the crude extracts. E. histolytica was cultured to perform an in vitro anti-amoebic assay to test the extracts against the parasite and determine their EC50 values after 72 hours of incubation. The goal was to isolate and identify compounds from C. variegatum with anti-amoebic activity.

1. Bioassay-guided fractionation of
extracts from Codiaeum variegatum
against E. histolytica.
Prepared and
Presented by:
Ujjal Naskar
NIPERA1820NP05

2. Plant Profile
Codiaeum Variegatum
 Family: Euphorbiaceae
 Common Name: Garden croton
 Geographical Source:Indonesia,Malayasia,
Australia, Pacific oceans etc.
 Uses: Anti inflammatory, Antifungal, antiamoebic
and anticancerous activities.

3. Preparation of plant extract
Leaf, stem, root and flowers were collected from
Codiaeum variegatum.
They were dried for one week at room temperature
Dried plant parts were grinded in a blender to fine
particles.
 Crude plant extract was prepared by Soxhlet
extraction method.
Filterate are collected by using Whatman filter paper.

4. Fractionation Procedure and Isolation of
Compounds

5. In Vitro Culture Of E. Histolytica
• E. histolytica was grown in 15 ml screw cap glass tubes
at 37degC on TYI-S-33 axenic medium.
• Axenic medium is supplemented with 15% (v/v)
complement-inactivated bovine serum , 3% Diamond
vitamin Tween 80 and 1% Penicillin-Streptomycin.
• Bacterial growth.

6. In Vitro Anti-amoebic Assay
 Plant extracts were prepared using sterile DMSO and culture medium leading to
concentrations of 100, 50 or 10 mg/ml respectively.
 Each mixture was filtered and aliquots of two-fold serial dilutions were prepared
from these stock solutions.
 A fresh culture of 5000 trophozoites per ml was introduced in each well after
allowing the parasite to adhere at the bottom of the well then extract was added.
 Metronidazole was used as the positive control
 The 50% efficient concentrations (EC50)
were determined by plotting the graph of mortality rate versus the
Concentration.
 EC 50≤15.62µg/ml after 72 hrs of incubation.