Artemisinin ExtractionArtemisinin ExtractionUsing DesignedUsing DesignedPolymer ResinsPolymer Resins

Dr Yi GeDr Yi GeCranfield Health

Cranfield University, UK

Artemisinin Forum 200824-26/Nov/2008

Cranfield University& Cranfield Health

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Postgraduate-only University

Emphasis on Business, Science and Technology

On two sites in southern England

Cranfield main campus adjacent to Cranfield village

The Ministry of Defense college at Shrivenham

Cranfield Health is a school of the University offering

“Total solutions for health and wellbeing”

Our Cranfield Team

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Development of world class material/polymer science forsolving practical problems in separation and sensing

Turnover of £1 million by 2009

World Leaders in Molecular Imprinting, National Leadersin Synthetic receptors and Smart Materials

Area of Expertise

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Synthetic receptors

Molecularly imprinted and conjugated polymers

Computational design

Nanotechnology

Molecular recognition

Drug development

Membrane technology

Sensor technology

Design of ImprintedPolymers

For the design of imprintedpolymers, Cranfield pioneered anew computational approachwhich allows fast and effectiveoptimisation of polymercomposition.

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State-of-the-artFacilities

Equipped with modern instruments forthe polymer design, preparation andcharacterisation: NMR, HPLC-MS-MSsystem, IR-spectrophotometer, HPLCs,UV spectrophotometer, porosimeter,particle size analyser, electronscanning microscope, atomic forcemicroscope and confocal microscope.

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Structure ofPresentation

Project Outline

Methodology & Outcomes

Conclusions

Future Work

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Project Outline

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Computational design of polymers with specificity forartemisinin

Optimisation of the artemisinin quantification methods

Polymers synthesis and testing

Evaluation of artemisinin purification from the plantextracts

Computational Designof Polymers

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Design of functional monomer database

Design of molecular model of artemisinin

Screening using a LEAPFROGTM algorithm

Library of FunctionalMonomers

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F21 2-(TRIFLUOROMETHYL)ACRYLIC ACID

OH

OCF3

F20 N,N-DIETHYLAMINO ETHYL METHACRYLATE

(DEAEM)

O

CH3 O

N

O

HO

N

N

F19 UROCANIC ACIDF18 STYRENE

OH

OCH3

F17 METHACRYLIC ACID

OH

OHO

O

F14 ITACONIC ACID

NH

O

NH

O

F15 m-DIVINYLBENZENE F16 N,N-METHYLENE BIS

ACRYLAMIDE

O

O

N

N

OO

CH3

O

O

CH3

F12 ETHYLENE GLYCOL

DIMETHACRYLATE

(EGDMA)

F13 UROCANIC ACID

ETHYL ESTER

O

NH

CH3

CH3

SO3HH

NNN

F1 1-VINYLIMIDAZOLE F2 2-VINYLPYRIDINE F3 ACRYLAMIDO-2-METHYL-1-

PROPANESULFONIC ACID

(AMPSA)

N

O

CH3 O

OH

F4 2-HYDROXYETHYL

METHACRYLATE

F5 4-VINYLPYRIDINE

OH

OH

NH2

OH

H

OH

F6 ACROLEIN F7 ACRYLAMIDEF8 ACRYLIC ACID

C N

H

F9 ACRYLONITRILE

NH2

F10 ALLYAMINEF11 p-DIVINYLBENZENE

F22 ETHYLENE GLYCOL METHACRYLATE PHOSPHATE

(EGMP)

Molecular Structure ofArtemisinin

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O

O

O O

O

H

H

H

Artemisinin - MBAAMolecular Complex

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MBAA:N,N'-methylenebisacrylamide

Artemisinin - AMPSAMolecular Complex

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AMPSA:2-acrylamido-2-methylpropane sulfonic acid

Leapfrog Table

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Rank Monomer Binding Energy(kcal mol-1)

1 DEAEM -30.78

2 MBAA -27.90

3 Acrylamide -25.73

4 AMPSA -20.53

5 TFMAA -17.50

Polymer Synthesis

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Polymerisation:

5 g of one functional monomer(DEAEM, MBAA, AMPSA, TFMAA)

20 g of cross-linker (ethyleneglycol dimethacrylate, EGDMA) 25 g of porogen (dimethylformamide, DMF), 500 mg of initiator (1,1-azobis(cyclohexanecarbonitrile))

Thermo-polymerisation at 80 ºC

Quantification ofArtemisinin UsingHPLC-MS

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HPLC-MS protocol

500 ng/ml Ch ME

Time2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

%

0

100

Arte134 SIR of 2 Channels ES+TIC

5.14e69.91

Gradient: 1 mM ammonia acetate buffer pH 5.0/methanolFlow rate: 0.2 ml/minColumn: Luna 50x3 mm (Phenomenex, UK)500 ng/ml Artemisinin

Artemisinin

Quantification ofArtemisinin UsingHPLC-MS

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17-Aug-2007no fragm 50:50 me:amm acetate 1mM

m/z200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400

%

0

100

ART11 1 (1.017) Scan ES+1.29e8305.323

209.260

219.213

214.173263.181247.244

229.291 237.228261.291

257.260

265.260

269.291 281.260 301.354

306.268

345.134

321.260307.276 329.197 365.291355.276

371.276397.354387.402

Artemisinin + Na+

Solid Phase Extraction(SPE)

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1ml SPE cartridges are packed with 100 mg of pulverizedand sieved polymers (30-125 mm ø)

Conditioning: 1 ml of hexane

Loading: 1-15 ml of hexane spiked with artemisinin

Washing: 2 ml of hexane

Elution: 3 x 1ml of the corresponding eluent

Polymerresins

Primary Screening

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MBAA 85% TFMAA 83% DEAEM 79% AMPSA 76%

Artemisinin binding from a model solution in hexane

☻ MBAA-based polymer was selected for future investigation

Capacity of MBAA–Based Polymer

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For the artemisinin in hexane:

Model solution: 1 mg/ml of artemisinin in hexane

Capacity: 120 mg/g of the polymer resin

Total adsorption: 80%

*Effective bed volume: 150

* Ratio between volume of extract from which ≥ 50% could beadsorbed and volume of polymer

Capacity of MBAA–Based Polymer

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For the artemisinin in plant extract:

Plant extract in hexane (0.7 mg/ml)

Capacity: 70 mg/g of the polymer resin

Total adsorption: 94%

*Effective bed volume: 130

* Ratio between volume of extract from which ≥ 50% could beadsorbed and volume of polymer

Elution Optimisation

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keeping balance between elution of artemisinin andimpurities using the plant extract

2.590Hexane/THF (5/1)

455.6Hexane/IPE (1/1)

288Methanol

383.4Acetonitrile

Degree ofpurification*

Recovery, %Eluent

*Degree of purification: dry weight of the loaded sample divided by the dry weight of the

eluted sample.

RegenerationProtocol

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The protocol:

Regeneration solvents: ethyl acetate or THF (2-3 ml)

It was shown that polymer resins are stable and could be re-used at least for 10 regeneration cycles

The regeneration should be conducted immediately afterelution before resin is dried

UV-Vis Spectra ofDifferent Stepsin SPE

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0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

190 240 290 340 390

Wavelength, nm

O.D

.,a.u

.

Non-purified hexane extract (Art 6.5 mg)

FiltrateWash

Elution (Art 6.46 mg)Regeneration

Not applicable for the evaluation of sample purity when working with the plant extract

Conclusions

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Monomer MBAA was selected for the polymer preparation. The HPLC-MS method for quantification of artemisinin was

developed. The capacities of the MBAA-based polymer resins were

obtained (120 mg/g for artemisinin in model hexanesolution and 70 mg/g for artemisinin in plant extract,respectively).

Methanol or hexane/THF (5/1) was selected as theoptimised elution.

Regeneration of the polymer resins was achieved usingethyl acetate or THF.

The resulting polymer resins are stable and could be re-used for many times (>10).

Future Work

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Further optimisation of purification process for commercialsamples using industrial volumes and larger size ofcolumns is needed.

It would be interesting to combine our polymeric materialswith other techniques such as ionic liquids and HFCdeveloped by other experts (Bhupinder Khambay,Rothamsted & Neil Sullivan, SensaPharm).

There would be a need to optimise polymer purificationstage with following crystallisation.

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Special thanks to Prof. Sergey PiletskySpecial thanks to Prof. Sergey Piletsky& Dr. Elena Piletska !& Dr. Elena Piletska !

Sergey Piletsky Mike Whitcombe

Elena Piletska

Kal Karim

Iva Chianella

Yildiz Uludag

Vasiliki FragkouAnna Biela

Dimitris Kyprianou Ewa MoczkoAntonio Guerreiro

Paula PizarroDe Sousa Brito

Tony TurnerAlessandra Bossi

Sreenath Subrahmanyam

Yi Ge

Steve Fowler

Georgios Stavroulakis

Andrew Spooner

Israel Sanchez

Dhana Lakshmi

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