Bioassay guided fractionation of natural product

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.