Molting
• Process that arthropods undergo
• The shedding and re-growth of a new exoskeleton (The
Insect Process of Molting, 2010)
• Insect molting hormones
• regulate growth, reproduction, and development
Ecdysteroids
(Grebenok, Galbraith, Benveniste, Feyereisen, 1996)
• Exact replica of ecdystroids produced by plants
• Protective mechanism
Phytoecdysteroids • Disrupt development of insects
- Spinacia oleracea
(Spinach)
- Chenopodiaceae family
- Produces ecdysteroids
structurally similar to those
produced by arthropods
- Tenebrio molitor (yellow
mealworm)
- Order Coleoptera
- Viewed as pests in
countries such as Mexico
(University of California,
2009).
Find the most
effective way in
extracting
phytoecdysteroids
from Spinacia
oleracea
Investigate the
larvicidal activity
of
phytoecdysteroids
by using Tenebrio
molitor as an
indicator
Develop a novel
insecticide that is
both effective
and
environmentally
friendly
Biodegradable,
environmentally
friendly
Phytoecdysteroids are
beneficial to human
health (Mária Báthori et
al, 2005).
Effective
Therefore
So
Some larvae have
acquired resistance
against conventional
methods (Rangel et al,
2009).
Larvicide
could be used to
exterminate
mealworm larvae in
food items
Controlled
Independent
• Mortality Rate of
T. molitor
• Species of
mealworm larvae
• Culture conditions
• Extraction Method
Dependent
•Concentration of
extracts
• Observable
deformities of T.
molitor
Materials
•
•
•
•
•
•
•
•
Methanol
Hexane
Dichloromethane
Acetone
Ethanol of 96% purity
Alumina
Octyl silane
Cotton wool
Apparatus
• Vacuum pump
• Column
Materials
Apparatus
• Boxes
• Mealworm feed
• Cotton wool
• Petri dishes
• Spraying apparatus
• Micro syringe
Bioassay
HPLC
Solventsolvent
distribution
Purchase,
cutting and
drying of
Spinacia
oleracea
Fractionated
precipitation
Reverse phase
chromatography
Column
chromatography
Concentration
of phytoecdysteroids
1
2
3
Negative
Control
Method for
administration
Number of Meal worms
Solvent
Control
Petri Dish 1
Petri Dish 2
Petri Dish 3
Injection
10
10
10
10
Spraying
10
10
10
10
Ingestion
10
10
10
10
Injection
10
10
10
10
Spraying
10
10
10
10
Ingestion
10
10
10
10
Injection
10
10
10
10
Spraying
10
10
10
10
Ingestion
10
10
10
10
N/A
10
•
Adler, J. H., Grebenok, R. J. (1999). Occurrence, biosynthesis, and putative role of ecdysteroids in plants.
Critical
Reviews in Biochemistry and Molecular Biology, 34(4), 253-264.
•
Bakrim, A., Maria, A., Sayah, F., Lafont, R., Takvorian, N. (2008). Ecdysteroids in spinach (Spinacia
oleracea L.):
Biosynthesis, transport and regulation of levels. Plant Physiology and Biochemistry,
844-854.
•
Grebenok, R.J., Galbraith, D.W., Benveniste, I., & Feyereisen, R. (1996). Ecdysone 20-monooxygenase, a
cytochrome
p450 enzyme from spinach, Spinacia oleracea. Phytochemistry, 42(4), 927-933.The
Insect Process of Molting. (2010). Retrieved from http://www.insectidentification.org
•
Malausa, T., Salles M., Marquet V., Guillemaud T., Alla, S., Marion-Poll, F., Lapchin L. (2006). Withinspecies
variability of the response to 20-hydroxyecdysone in peach-potato aphid (Myzus
persicae sulzer), Phytochemistry, 52, 480-486.
•
Savolainen, V., Wuest, J., Lafont, R., Connat, J. L. (1995). Effects of ingested phytoecdysteroids in the
female soft
tick Ornithodoros moubata. Phytochemistry. 51, 596-600.
•
Schmelz, E. A., Grebenok, R. J., Ohnmeiss, T. E., Bowers, W. S. (2002). Interactions between Spinacia
oleracea and
Bradysia impatiens: a role for phytoecdysteroids. Archives of Insect Biochemistry and
Physiology, 51, (204221).
•
University of Arizona. (1997). Darkling Beetle/Mealworm Information. Retrieved from September 26,
2010 http://insected.arizona.edu/mealinfo.htm
•
University of California (2009). Mealworms and Darkling Beetles (Tenebrio beetle). Retrieved
September 26, 2010
from
http://lhsfoss.org/fossweb/teachers/materials/plantanimal/tenebriobeetles.html
• Fractionated precipitation
• Dried plant of 6g is extracted with Methanol at a mass-volume ratio of
1:10 ( 60ml methanol needed)
• After extraction, the methanolic solution is split into 3 parts. (20 – 21 ml
each)
• The first part of the solution (20ml) is mixed with half the volume of
acetone (11ml) while the second part (20ml) is mixed with same volume
of acetone (22ml) and the last part is mixed with twice the volume of
acetone (40ml).
• The resulting solution is then filtered and the residue is removed.
• The residue is washed with the same ratio of methanol and acetone as
step 1 and 3 respectively
• The washing solution is then added to the filtrate.
• The solutions are then evaporated.
• The crude extracts are redissolved in methanol at the same mass-volume
ratio of 1:10.
• Step 2-8 is repeated 2 more times.
• Solvent-solvent distribution
• After precipitation, the crude extracts are dissolved
in 50% aqueous methanol (specific numbers needed
– add until everything dissolves)
• Hexane (how much?) is added to the solution to
extract the non-polar compounds in the precipitate.
• The aqueous methanol phase (bottom) is separated
(using separating funnel) and then evaporated to
dryness.
• The resulting residue is dissolved in pure methanol.
• The methanolic solution is mixed with aluminium
oxide and the suspension was evaporated to dryness
with a rotary evaporator.
• Chromatography
• The alumina is eluted with a hybrid of Dichloromethane- 96%
Ethanol solution of ratio 9:1 and 8:2. (need to do 2 times)
• A cotton wool of mass of 0.2 g was placed at the bottom of
the column to prevent alumina from flowing out.
• 70-90g (subject to experimental changes) of Alumina is mixed
with the eluent.
• The mixture of alumina and eluent was stirred and poured
into the column until is 75% full.
• The bands in the mobile phases are collected in different
beakers for further tests.
)
• Purification of Ecdysteroids
• For further purification, the ecdysteriods are separated by reversed-phase
chromatography
• In reversed-phase chromatography, octyl silane is used as the stationary
phase (being non-polar) instead of silica/alumina.
• A cotton wool of mass of 0.2 g was placed at the bottom of the column to
prevent alumina from flowing out.
• 70- 90g (subject to experimental changes) Octyl Silane is mixed with the
eluent.
• To control the flow of the mobile phase, a vacuum will be used at the
outlet.
• Different concentrations of methanol are used as eluents in this
chromatography.
• Stepwise gradient elution is used with an increase of 5% of methanol
content in each step.
• Different bands of ecdysteroids will be formed on the stationary phase.
• The specific hormone, 20-hydroxyecdysone is isolated with methanol of
35-40% purity.
• Preparation of phytoecdysteroids for
bioassay
• Evaporate the solution to dryness
• Dissolve the phytoecdysteroids in water to the
desired concentration
1. 10 last-instar mealworm larvae are placed in a box per
setup.
2. A determined amount of extracts are sprayed onto the
mealworms.
3. The mealworm larvae are left to develop for 30 days.
4. The deformities and mortality rate of the mealworm
larvae is recorded after a day, 15 days and 30 days.
5. Step 1 to 4 is repeated with the extracts being injected
or fed to the mealworm larvae.