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