Plants for health – food or medicine? June 15 2011.Slide 1

Sweet wormwood (Artemisia annua) and parasitesIda Thøfner, DVM, Ph.D. Student Section for Microbiology, KU LIFE

Torben Wilde Schou, Ph.D., Scientific advisorEnvironment and Toxicology, DHI

Supported by the Danish Council for Strategic Research

Plants for health – food or medicine? June 15 2011.Slide 2

Artemisia project – Objectives

Development of feeding additive and/or veterinary medical concepts based on Artemisia annua (A. annua) for prevention and treatment of coccidiosis, blackhead and necrotic enteritis.

Development of the most cost efficient cultivation methods of A. annua with increased content of bioactive compounds. Identification of A. annua genotypes with optimal levels of bioactive compounds.

Plants for health – food or medicine? June 15 2011.Slide 3

Artemisia project – Overview

WP1

WP2

WP3WP5

WP6

A. annua prototype product

Effect on coccidiosis

and blackhead in

poultry

Effect on C. Perfringensand necrotic enteritis in

poultry

The biological concept of growing A.

annua

Characteri-sation of bioactive

compounds

Economic analysis and development of concept

Eating quality of meat and

eggs

WP4

Plants for health – food or medicine? June 15 2011.Slide 4

Artemisia project partners

Aarhus University• Department of Horticulture• Department of Animal Health and Bioscience

University of Southern Denmark• Department of Chemical Engineering, Biotechnology &

Environmental Technology

DHI• Department of Human Health and Safety

University of Copenhagen• Department of Food and Resource Economics

Danish Meat Research Institute (Danish Technological Institute)

Nutridoc (Regulatory Affairs within the feedstuff sector)Biosynergy A/S (Plant grower)

Plants for health – food or medicine? June 15 2011.Slide 5

Artemisia project- Why?

Coccidiosis and necrotic enteritis are among the most loss causing poultry diseases• Coccidiosis: Estimated annual losses > € 3,8 billion worldwide

(FAOSTAT, 2011; Williams, 1999).

Blackhead is a severe disease in free-range flocks with no possibilities for treatment• Re-emerged since the ban of effective treatments (EU/EEA and

FDA/USA). • Several in vitro and in vivo/field studies in the search for an

efficient therapy

Ionophore coccidiostats with antibiotic effects are presently used as feed additives to control coccidiosis and necrotic enteritis. Likely that these substances will be banned within the EU as soon as alternatives are available

The Danish agriculture is under intense pressure – development of new high value crops are necessary to increase the Danish competitiveness in the global market

Plants for health – food or medicine? June 15 2011.Slide 6

WP1 Parasite infection models in poultryDHI and KU-LIFE

Coccidiosis:

In vitro studies

• Wild type strains will be isolated and maintained in vitro. Artemisinin and other compounds/extractions will be tested

In vivo studies

• Artemisinin and other relevant compounds/extractions will be tested in disease models in chickens.

Clinical signs Damage of the intestinal mucosawith hemorrhages

Plants for health – food or medicine? June 15 2011.Slide 7

WP1 Parasite infection models in poultryDHI and KU-LIFE

Blackhead:H. meleagridis more complicated to culture in vitro.

Collaboration with leading expert in Europe, Dr. M. Hess (University of Veterinary Medicine, Vienna)

In vitro tests and tests on Blackhead infected chickens and turkeys is performed in Vienna

Clinical signs Enlarged hemorrhagic ceca and characteristic liver lesions

Plants for health – food or medicine? June 15 2011.Slide 8

Introduction – Why Sweet Wormwood (Artemisia annua)?

Native to temperate Asia, but naturalized throughout the world

Approx. 2 m tall with a single stem. Fern-like leaves, bright yellow flowers, and a camphor-like scent

Sweet Wormwood has been used as an herbal remedy in traditional Chinese medicine for treatment of fevers, including malaria.

In 1971, scientists demonstrated that plant extracts had antimalarial activity. In 1972 the active ingredient, artemisinin, was isolated and its chemical structure described.

Artemisinin is recommended as first choice treatment of endemic malaria by WHO (combination therapies)

A. annua compounds is demonstrated to have antiprotozoal, antitumor and antibacterial properties

Photo: K. Grevsen

Plants for health – food or medicine? June 15 2011.Slide 9

Artemisinin

Partial synthezisation possible

Artemisinin targets the asexual stages of Plasmodium falciparum

Mode of action is not clearly elucidated• Iron dependant reduction of peroxide

brigde producing free radicals• Destabilising/destruction of the

cellular membrane• SERCA-inhibition (del Cacho, 2010)

(PfATP6)

• Alteration in electron transport with reactive oxygen species (ROS) formation → depolarisation in parastic mitochondria membrane

White , Science (2008)

http://www.biocis.u-psud.fr/spip.php?article175&lang=fr

Photo: K. Grevsen

Plants for health – food or medicine? June 15 2011.Slide 10

The pharmacodynamic properties of the antimalarial drugs in vivo assessed in terms of parasite clearance per asexual cycle in

humans

White (2008), Science 320, 330-334

Artemisinin derivatives

Most antimalarials

Plants for health – food or medicine? June 15 2011.Slide 11

Artemisinin and poultry

Literature is very limited• Few studies regarding Artemisia spp. compounds and poultry

• Field/feeding trials and coccidia (7 studies)• Toxicology in poultry (2 studies)

• No in vitro studies• No PK/PD or bioavailablity information in poultry

• Short T½ in humans (few hours)

Other herbal substances or natural compounds have been investigated at different levels in a variety of poultry infections (e.g. Colibacillosis, coccidiosis, histomonosis)

Plants for health – food or medicine? June 15 2011.Slide 12

Liebhart & Hess, Avian Pat. (2009)

Photo: JPC

Blackhead/HistomonosisHistomonas meleagridis

Clinical appearance• ↑ mortality rates• ↑ overall morbitity

• Concurrent infections (e.g. E.coli)

Pathology• Severe necrotic lesions in the cecum

and liver • Parasite is widely distributed in the host

• Turkeys: proventriculus, duodenum, jejunum, caeca, pancreas, bursa of Fabricius, liver, kidney, spleen, heart, lung, thymus and the brain

• Chickens: caeca, bursa of Fabricius, kidney, heart and the brain

Plants for health – food or medicine? June 15 2011.Slide 13

Histomonas meleagridis

Protozoan parasite (flagellate)• Pleomorphic appearance

• Amoeboid stages• Flagellate stages• Dividing stages• Cyst-like stages

• Asexual multiplication• Binary fission

• Hydrogenosomes• Intercellular invasion?

Xenic culture• Bacteria spp.• Symbiosis?• Pathogenic?

Hess et al., Parasitology, 2006

Adapted from Mielewczik et al.Parasit. Res. (2008)

Plants for health – food or medicine? June 15 2011.Slide 14

New applications for plants with phytomedical properties- evalution of immunoactive phytonutrients

Herbal compounds

In vitro screening

Macrophage stimulation

Lymphocyte stimulation

Cytokine Tumor cytotoxicity

Direct killing of pathogens

Selected synergistic herbal compounds

In vivo (feeding) trial

Nutrigenomics Infection/ disease

challenge

Performance analysis in livestock

Plants for health – food or medicine? June 15 2011.Slide 15

In vitro testing of Artemisia annua compounds on monoeukariotic Histomonas meleagridis

Antiprotozoal effect and antibacterial effect

Compounds to test• Artemisinin• Dry plant• Hexane extracted essential oil fraction (Hex-oil)• Dichloromethane extracted essential oil fraction (DCM-oil)• Methanol extracted essential oil fraction (MeOH-oil)

Clonal cultures of H. meleagridis• 6 different clones• Pathogenic

Plants for health – food or medicine? June 15 2011.Slide 16

Preliminary results

Hess et al., Parasitology, 2006

Plants for health – food or medicine? June 15 2011.Slide 17

Preliminary results – Overview

Histomonas meleagridis Clone 6b,48 hours

No.

of

cells

/ml (x

10

4)

Cont

rol

Dimetrid

azole 0,

4 mg/

ml

Artemisi

nin 5 mg/

ml

Artemisi

nin 10

mg/

ml

Artemisi

nin 20

mg/

ml

Dry plant

5 m

g/ml

Dry plant

10 mg/

ml

Dry plant

20 mg/

ml

Dry plant

40 mg/

ml

Ess.oil (

hex)

0,5

mg/

ml

Ess.oil (

hex)

1,0

mg/

ml

Ess.oil (

hex)

1,5

mg/

ml

Ess.oil (

dcm) 0

,5 m

g/ml

Ess.oil (

dcm)1

,0 m

g/ml

Ess.oil (

dcm) 1

,5 m

g/ml

Ess.oil (

meth)

0,5

mg/

ml

Ess.oil (

meth)

1,0

mg/

ml

Ess.oil (

meth)

1,5

mg/

ml

0

20

40

60

80

100

Plants for health – food or medicine? June 15 2011.Slide 18

New applications for plants with phytomedical properties- evalution of immunoactive phytonutrients

Herbal compounds

In vitro screening

Macrophage stimulation

Lymphocyte stimulation

Cytokine Tumor cytotoxicity

Direct killing of pathogens

Selected synergistic herbal compounds

In vivo (feeding) trial

Nutrigenomics Infection/ disease

challenge

Performance analysis in livestock

Plants for health – food or medicine? June 15 2011.Slide 19

What’s next?

In vivo experiments• Evaluate the effect on experimentally infected turkeys and

chickens• Signal detection of unidentified toxicity

Group Treatment

I DCM-oil 0,2% in drinking water

II Artemisinin 0,01% (100 ppm) in feed

III Artemisinin 0,26% (2600 ppm) in feed

IV Positive control (Infected-non treated)

None

V Negative control(Non infected- non-treated)

None

Plants for health – food or medicine? June 15 2011.Slide 20

Thank you for listening