Elicitation of secondary metabolites in
cotyledonary callus of Ziziphus spina-christi
and measurement of antioxidant activity in
different lines using FRAP method
Wattan Basheer
Supervised by: Dr. Rami Arafh
Introduction
• Ziziphus spina-christi ,
(Rhamnaceae) is a tropical
evergreen tree from Sudanese
origin. known as Christ's Thorn
and its Arabic name is "Sidder“
• In Palestine, it is found in the Jordan Valley and the
Dead Sea area.
•
It is known in traditional medicine for treatment of
several illnesses like dermatitis, diuretic, eye
inflammation, stomachache, rheumatoid arthritis and
has anti-diabetic activity
• The plant is rich in pharmaceutical compounds
including alkaloids, glycosides, flavonoids, and
volatile oils.
Introduction
• Due to the increasing demand on Ziziphus spina-christi in
traditional medicine, there is a need for sufficient plant
material to satisfy the market needs
• Alternative methods are used for the propagation and
culture of Z. spina-christi including seed and stem cutting
propagation.
• In vitro propagation method
offer a powerful alternative for
production of large amount of
plants
• Plant tissue culture also offers an
effective alternative for
utilization of explants that are
not possible to be produced with
conventional methods like root
culture and cells from
cotyledons.
Introduction
Advantages of in vitro plant culture
• In the micropropagation method, only a small amount of
tissue is required to regenerate millions of clonal plants in
a year
• Some plant parts such as cotyledons and hypocotyls
which cannot be found in nature are utilized
• In vitro stock can be quickly proliferated as it is season
independent
• Enables long term storage of valuable germplasm
Introduction
Advantages of in vitro plant culture
• The production of huge amount of plants away from
conventional methods (by seeds or rooted cuttings)
• Pest free plants are quickly multiplied for various
agricultural uses
• Production of secondary metabolites which is difficult
to be synthetically produced or by intermediate agents
like bacteria
Aim of the study
 Examine chemical factors and test their
effect on secondary metabolites
production in cotolydonary callus
 Explore antioxidant properties of callus
extract
Objectives of the study
 Examine the effect of media type and chemical
elicitation on secondary metabolites production
in cotolydonary callus
 Explore antioxidant activity with FRAP
method
Methodology :
1
2
3
• Establishment of callus from
cotyledon
• Extraction of different callus cell line
and compare the UV absorbance of
different extracts
• Measure antioxidant potential of
callus extract using FRAP assay
Methodology
:
Establishment of callus from cotyledonary
tissue
1- The dried fruits of Ziziphus spina-christi (L.) Desf.
were collected in October 2012 from Jericho city then
seeds were isolated manually
2- Seeds were germinated in vitro on PGR-free water-agar
media
Methodology :
3- Cotyledons were excised and used for callus induction
on MS, B5, Mcc or QL media with 1.3 mg/l BA + 0.3 mg/l
NAA
MS
QL
B5
MCC
Methodology :
Extraction of different callus cell line and
compare the UV absorbance of different
extracts
5- Aqua extract of callus obtained from each media
Methodology :
6- Ultraviolet–visible spectroscopy used for secondary
metabolites detection
7- FRAP assay used to measure the antioxidant power of
three media calli “MS, QL, B5”.
Results :
1-The preliminary results indicate significant
differences in the crude secondary metabolites
extracted when using MS,QL and B5 media
B5
MS
QL
Results :
2- The antioxidant power of the three
extract B5, QL, MS also differ with an
absorbance value (0.189), (0.327)
and (0.380) respectively at 593nm
Using FRAP assay .
Discussion
• The change in the amount of secondary
metabolites produced from different media
lines indicate that the micro and macro
nutrients influenced the growth and affected
the type of biochemical's produced.
• The difference in the amounts of these
compounds was clear in changing the
oxidation power of different media
depending on the secondary metabolites
they produce.
Discussion
• Calli grown on B5 has the highest antioxidant
power this correlate with a peak observed at
333nm only in the extract from B5 media
B5
QL
MS
Future work :
• Characterization of the compound
absorbed at 359 nm using NMR .
• Use HPLC to separate the produced
compound .
• Different other chemical and electrical
stress will be used to enhance other
secondary metabolites .