Done by: Frank Song Jr. Kyra Brower Shi Guan Ming

Comparing the larvicidal activity of phytoecdysteroids present in Spinicia Oleracea

subspecies

Done by: Frank Song Jr. Kyra Brower

Shi Guan Ming Zhao Xing Liang

Background• Molting is a process that arthropods undergo in order to

develop and grow which occurs by the shedding and re-growth of a new exoskeleton (The Insect Process of Molting, 2010)

• Ecdysteroids are insect molting hormones produced by arthropods that regulate growth, reproduction, and development (Grebenok, Galbraith, Benveniste, Feyereisen, 1996).

• Hence, plants, as a protective mechanism, produce the exact replica of ecdysteroids, called phytoecdysteroids, which disrupt the development of insects.

Background

• Mealworms (Tenebrio Molitor) are insects from the order Coleoptera, and are viewed as pests in countries such as Mexico. (University of California, 2009).

• Spinacia oleracea (spinach) is a plant from the Chenopodiaceae family that produces ecdysteroids structurally similar to those produced by arthropods; they are known as phytoecdysteroids (Alder, Grebenok, 1999)

Objectives

• To test phytoecdysteroid extraction methods to find the most effective way of obtaining the purest amounts from Spinach plants.

• To investigate the larvicidal activity of phytoecdysteroids that can be found in different sub-subspecies of Spinacia Oleracea by using Tenebrio Molitor larvae as indicators of the effectiveness of the phytoecdysteroid extractions from the sub-species.

Rationale

• A novel larvicide may kill larva more effectively, since some larva have acquired resistance against conventional methods (Rangel et al, 2009). Therefore we want to optimize phytoecdysteroid extraction and obtainment to finding another effective larvicide.

• Larvicide that contains organic molecules are also biodegradable, hence it is environmentally-friendly.

Hypothesis

• If two different extraction methods are tested for greatest phytoecdysteroid yield and purification, then the most efficient can be found and adopted into this research.

• If the larvae of Tenebrio Molitor are exposed to phytoecdysteroids from different S. Oleracea sub-species, then the plant with the most effective larvacidal capability can be found

Variables

Constant Independent Dependent

AOS

• Species of larvae• Environmental

conditions• Extraction Method

(after determination)

• Extraction Method (before

determination) • S. Oleracea sub-

species.• Concentration of

extracts

• Mortality Rate of T. molitor

• Observable deformities of T.

Molitor

HCI

Buy grocery spinach and perform Fractionated precipitation

Solvent- Solvent Distribution

ChromatographyPurification of ecdysteroids

Bio- Assay

HCI’s methodology

MaterialsResources:• Spinach plants (Spinacia oleracea seeds)• Water• Soil• Soil mixture: Potting soil, vermiculite, peat, perlite• Methanol• Butanol

Hexane• Dichloromethane• Acetone• Ethanol of 96% purity• Alumina • Octyl Silane

Extraction Method #2

Fractionated precipitation:1. Dried plant of 6g is extracted with Methanol at a mass-

volume ratio of 1:10

2. After extraction, the methanolic solution is split into 3 parts.

3. The first part of the solution is mixed with half the volume of acetone while the second part is mixed with same volume of acetone and the last part is mixed with twice the volume of acetone.

Extraction Method #2 (cont’d)

4. The resulting solution is then filtered and the residue is removed.

5. The washing solution is then added to the filtrate.

6. The solutions are then evaporated.

7. The crude extracts are redissolved in methanol at the same mass-volume ratio of 1:10.

8. Step 2-8 is repeated 2 more times.

Extraction Method #2 cont.

Solvent-solvent distribution:1. After precipitation, the crude extracts are dissolved in 50%

aqueous methanol.2. Hexane is added to the solution to extract the non-polar

compounds in the precipitate.3. The aqueous methanol phase (bottom) is separated and then

evaporated to dryness.4. The resulting residue is dissolved in pure methanol.5. The methanolic solution is mixed with aluminum oxide and

the suspension was evaporated to dryness with a rotary evaporator.


Chromatography:1. The alumina is eluted with a hybrid of

Dichloromethane- 96% Ethanol solution of ratio 9:1 and 8:2.

2. 70-90g (subject to experimental changes) of Alumina is mixed with the eluent.

3. The mixture of alumina and eluent was stirred and poured into the column until is 75% full.

4. The bands in the mobile phases are collected in different beakers for further tests.

Extraction Method #2 cont.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.

• The compounds will be used against the bioassay.

AOS’s methodology

Buy organic spinach and seeds

determine best extraction method

Plant Spinach and freeze-dry when matureHPLC

Bio- Assay

Materials

Equipment:• Cotton Wool• Vacuum Pump• Fluorescent and incandescent bulbs• Pots• C18 Reverse-phase chromatography machinery• Temperature monitor. • Column • Wiley mill (800 micrometer mesh)

Extraction Method #1

1. Dissect spinach into roots and shoots and freeze them.

2. After freezing, lyophilize them to dryness.

3. Grind each sample into fine powder using a Wiley mill (800 micrometer mesh)

4. Take a weighed portion (50-60mg) and place in 10ml of methanol for 48 hours.


5. Take a 7-ml aliquot and mix with 3ml water and partition against 10ml hexane.

6. After the clear-phase separation, remove 8ml of the aqueous methanol layer and evaporate to dryness.

7. After drying, place residue in 5ml water and partition against 5ml butanol.

8. Evaporate 4ml-aliquot of the butanol phase and re-suspend in 400 microliters of methanol before analysis.


9. Use reverse-phase high liquid performance liquid chromatography (RP-HPLC) with a C18 Alltech Spherisorb ODS-2, 4.6-mmx150-mm, 5 micrometer particle column.

10. Use an isocratic 2-propanol-water (12:88 v/v) mobile phase to separate the 20E from the polypodine B.

11. Measure the amount of 20-hydroxyecdysone (20E) by comparing with independent weighing of 20E.

Methodology (Bio-Assay)

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.

HCI TimelineProposal 23 - 28 Oct

Discussion with AOS 29 Oct – 7 Nov

Finalisation of Proposal 8 Nov

Preliminary Experimentation – to determine the best method of extraction

9 Nov – 7 Dec & 16 Jan - Prelims

Preliminary Round of Projects Day Competition March

Experimentation on Mealworms Between Prelims and Semi finals

Semi-Finals July

Final Experimentation on mealworms Between semifinals – 2 weeks before final judging

Compilation of Results with AOS Before Finals, after final experimentation

Research paper After finalizing results

Finals Judging Aug

AOS TimelineBackground research 23 - 28 Oct

Discussion with HCI 29 Oct – 7 Nov

Finalisation of Proposal November

Testing Extraction methods to determine thebest extraction method

Growing and harvesting of spinach November

Extracting Phytoecdysteroids and determining the best extraction method

November - January

Experimentation on Mealworms February to April

Travel to Singapore for finals presentation and finalization

August

Spread of TestingNumber of Mosquito Larvae used

Solvent Control Petri Dish 1 Petri Dish 2 Petri Dish 3

Concentration 1 10 10 10 10



Negative Control 10

Work cited • 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). Within-species 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, (204- 221).

• 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

Thank You

Documents

Done by: Frank Song Jr. Kyra Brower Shi Guan Ming