adsi austrian drug screening institute gmbh vo trennmethoden · - non-invasive spectroscopy methods...
TRANSCRIPT
07.02.2018
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ADSI – Austrian Drug Screening Institute GmbH
VO Trennmethoden
Screening for Bioactive Natural Compounds
in Plants and Algae
Dr. Thomas Jakschitz
7.02.2018
„Screening“ ?!
Warum in Pflanzen und Algen?
Naturstoffe als Vorlage für Wirkstoffe
Benötigte Geräteausstattung
Was macht ADSI
Beispiel – Anti-inflammatorische Komponenten in Algen
Übersicht
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- systematisches Testverfahren - innerhalb eines definierten Prüfbereichs - Komponenten mit bestimmten Eigenschaften herausfiltern
Screening
Herausforderung: „die Nadel im Heuhaufen zu finden“
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Screening
High-Throughput Screening High Content Screening
Sehr hoher Probendurchsatz Hoher Automatisierungsgrad
Einfache Assay´s – meist biochemisch Einfache readouts – Absorption
Lumineszenz Fluoreszenz Findung Aktiver Komponenten
Geringer Probendurchsatz Automatisierung nur bedingt möglich Teilweise komplexe Fragestellungen
Kombination Analytik – Biologie Oftmals Methodenentwicklung Findung aktiver Komponenten
Erklärung Mode Of Action
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Warum in Pflanzen und Algen?
- Überlieferte traditionelle Anwendung
- ca. 400.000 Pflanzenarten weltweit
- 5-10% chemisch / pharmakologisch untersucht
- 200 Pflanzen in österreichischen Phytopharmazeutika
Pflanzenarten weltweit
Chemisch / pharmakologisch untersucht
Phytopharmazeutika in Österreich
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Warum in Pflanzen und Algen?
Pflanzen produzieren „Abwehrstoffe“ gegen
- Umwelteinflüsse - Fraßfeinde - Parasiten - Bakterien - Viren
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Sekundäre Pflanzenstoffe
Primäre Pflanzenstoffe • Hauptbestandteile der Pflanzen • Vom menschlichen Körper als
Nährstoff genutzt Vertreter • Kohlenhydrate, Proteine, Fette, Mineralstoffe, Zucker
Sekundäre Pflanzenstoffe • Kommen meist in geringen
Mengen in der Pflanze vor • Haben häufig pharmakologische
Wirkungen
“sekundär” heisst nicht zweitrangig 7
Bioaktive Komponenten
Beispiele für bioaktive, sekundäre Pflanzenstoffe
Lycopin
Lebensmittelfarbstoff E160d
Diallyltrisulfid
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Naturstoffe als Vorlage
„50 Prozent aller am Markt erhältlichen Arzneimittel sind entweder Naturstoffe, Naturstoffderivate oder haben Naturstoffe als
Vorbild“
Salix alba
Acetylsalicylsäure
Antirheumatikum
Morphin Papaver somniferum
Analgetikum
Artemisia annua Artemisinin
Antiprotozoika
Kokain Erythroxylum coca
Lokalanästhetikum
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Screening at ADSI
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Screening at ADSI
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Extraction - Accelerated Solvent Extraction (Thermo Scientific ASE 350) - Aquasolv® - US-Extraction - MWA-Extraktion - Soxhlet - Mazeration
Concentration - Rotary Evaporator up to 5000ml
Drying - Vacuum compartment dryer
Documentation - Automated moisture analyzer (Mettler Toledo HR-83g)
Enrichment - Phynexus MEA Purification System
Extraktion und Anreicherung
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Analysengeräte - LC/UV (analytical and nano LC, Thermo Ultimate 3000) - hr-qTOF MS (Bruker Maxis Impact) - MALDI-TOF MS (Bruker Autoflex Speed) - Proteineer 2 (Bruker) - Image Prep (Bruker)
Analytik Ausstattung
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Instrumentation - Multiplexing (Luminex Flexmap 3D) - Flow Cytometry (Guava easy cyte) - Microscopy (Confocal laser microscope) - ASAP (Automated Screening Assay Platform)
Biologie Ausstattung
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Biostatistik und Bioinformatik
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Bestätigung der Aktivität
Fractionation - (semi)Preparative Chromatography - Flash Chromatography - Counter Current Chromatography + Further activity testing in biological systems
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Strukturufklärung
Methods - LC-MS/MS - NMR
UV 280nm
MS2 esi-neg
MS2 esi-neg
MS2 esi-neg
1H-NMR
13C-NMR
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Screening at ADSI
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Planung
- Wonach soll gescreent werden? - Welche „Aktivität“ soll getestet werden
- Existieren passende Assaysysteme - Anzahl der Proben (techn. und biolog. Replikate
bedenken!) - Welche Proben sollen getestet werden
- Wildsammlung, Anbau, Probenvorbereitung (Extraktion...)
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ADSI Services
Cosmetic claim
confirmation
(Phyto)-Nutrition Food Supplements
- High resolution UPLC-qTOF-MS
- GC-FID and GC-MS
- Capillary Electrophoresis
- MALDI-TOF-MS
- Non-invasive spectroscopy
methods (NIR, ATR, MIR)
Phytocosmetic and
phytopharmaceutical
product
development
- Extraction of natural products
- Screening of active compounds
- Efficacy of cosmetic formulations
- Secretomics & metabolomics
- 2D and 3D in vitro cell culture
- Enzymatic and cell-free assays
- Mode-of-action studies
- In vitro safety assays
Austrian Drug
Screening
Institute GmbH
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Phytopharmazeutische Produktentwicklung
Plant extraction Endotoxin quantification Extract solubility Fingerprint screening of plant extracts 2D and 3D in vitro cell culture Enzymatic and cell-free assays Quantification of specific compounds Identification of bioactive components Bioavailability assays - PAMPA Assay - CaCo2-Assay
ADSI Services
Cosmetic claim
confirmation
(Phyto)-Nutrition Food Supplements
- High resolution UPLC-qTOF-MS
- GC-FID and GC-MS
- Capillary Electrophoresis
- MALDI-TOF-MS
- Non-invasive spectroscopy
methods (NIR, ATR, MIR)
Phytocosmetic and
phytopharmaceutical
product
development
- Extraction of natural products
- Screening of active compounds
- Efficacy of cosmetic formulations
- Secretomics & metabolomics
- 2D and 3D in vitro cell culture
- Enzymatic and cell-free assays
- Mode-of-action studies
- In vitro safety assays
Austrian Drug
Screening
Institute GmbH
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Kosmetik
ADSI Services
Cosmetic claim
confirmation
(Phyto)-Nutrition Food Supplements
- High resolution UPLC-qTOF-MS
- GC-FID and GC-MS
- Capillary Electrophoresis
- MALDI-TOF-MS
- Non-invasive spectroscopy
methods (NIR, ATR, MIR)
Phytocosmetic and
phytopharmaceutical
product
development
- Extraction of natural products
- Screening of active compounds
- Efficacy of cosmetic formulations
- Secretomics & metabolomics
- 2D and 3D in vitro cell culture
- Enzymatic and cell-free assays
- Mode-of-action studies
- In vitro safety assays
Austrian Drug
Screening
Institute GmbH
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Nahruns- und Nahrungsergänzungsmittel
Quality control of cacao and other seeds Olive oil: Quality control parameters Anti-oxidative potential EFSA health claims Food process analysis
Screening @ ADSI
Cosmetic claim
confirmation
(Phyto)-Nutrition Food Supplements
- High resolution UPLC-qTOF-MS
- GC-FID and GC-MS
- Capillary Electrophoresis
- MALDI-TOF-MS
- Non-invasive spectroscopy
methods (NIR, ATR, MIR)
Phytocosmetic and
phytopharmaceutical
product
development
- Extraction of natural products
- Screening of active compounds
- Efficacy of cosmetic formulations
- Secretomics & metabolomics
- 2D and 3D in vitro cell culture
- Enzymatic and cell-free assays
- Mode-of-action studies
- In vitro safety assays
Austrian Drug
Screening
Institute GmbH
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Associated partners and technology platforms
Univ. Vienna Antioxidative Metabolism
Univ. Vienna Inflammation assay
Endo-/Epithelial Cells
PBMC
Med. Univ. Innsbruck Toxicology
renal Epithelial- / Livercells
Med. Univ. Innsbruck Immunological assay
dendritic cells / macrophages
Univ. Innsbruck Electrophoretical
techniques CE, CE-MS, CEC
AlNuMed NMR-Zentrum
Bayreuth
Univ. Innsbruck Spektroscopical
Techniques IR, RAMAN
Bioavailabilty GI-Barrier
CaCo2
Univ. Innsbruck PTR – MS
Biological Screening
Cell-Models
Analytical Screening
Biostatistics Bioinformatics
Med. Univ. Lublin Fractionation, SFC,
CCC
Gazi Univ. Cell free enzyme inhibition assays
Univ. Athens Special extraction
technologies - upscaling
Gazi Univ. Cell free enzyme inhibition assays
Med. Univ. Innsbruck
Scanning Electron microscopy
Kooperation ADSI - MCI
Identification and Characterization of Anti-inflammatory Compounds from
Soil-algae Extracts
Peter D. Leitner
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Kooperation ADSI - MCI
• Estimated >500 Strains, Slant agar cultures
• Soil- and airalgae
• Origin: alpin area central europe
Production of secondary metabolites
Unique Algae collection ASIB, Institute of Botany, University Innsbruck Prof. Georg Gärtner
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Kooperation ADSI - MCI
Bioactivity guided Fractionation
MCI – Biotechnology & Food Engineering
Cultivation
Harvesting
Testing
Extraction
Analytics
Biomass
Stress
Solvent Cryopreservation
Bioactivity Assays
Toxicity
MoA
HPLC- MS
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Kooperation ADSI - MCI
Bubble Columns • Volume 1 l • Optimal light conditions • Gasing in with filtered
air and CO2
• Autoclavable
Variation of culture conditions: Different metabolites are produced
N2 Starvation
Osmolarity
Glucose stress
pH
1 day 3 weeks
Duration Stress Cultivation
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Kooperation ADSI - MCI
Skin inflammation model: NF-kB reporter cell line
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Kooperation ADSI - MCI
Anti-inflammatory screening
Post treatment = restoring effect
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Kooperation ADSI - MCI
Anti-inflammatory screening
Pre treatment = protecting effect
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Kooperation ADSI - MCI
Anti-inflammatory screening - Summary
• Tested Strains: 29
• Tested Extracts: 53
• Cultivation: Nitrogen-Stress, Salt, Mixotrophic
• Extraction: Polar Apolar
• Screen Settings: Pre, Post
Time course, Dilution,
Secondary Screen
Σ 140 Extracts to be tested
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Kooperation ADSI - MCI
Anti-inflammatory screening - Summary
Confirmed results of primary screening
• Anti-inflammatory: 10
• Pro-inflammatory: 5
Further investigation using secondary screening
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Kooperation ADSI - MCI
3D-Assays for Final products – in vitro epidermis model
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Kooperation ADSI - MCI
Bioactivity guided Fractionation
MCI – Biotechnology & Food Engineering
Cultivation
Harvesting
Testing
Extraction
Analytics
Biomass
Stress
Solvent Cryopreservation
Bioactivity Assays
Toxicity
MoA
HPLC- MS
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ADSI Services
Cosmetic claim
confirmation
(Phyto)-Nutrition Food Supplements
- High resolution UPLC-qTOF-MS
- GC-FID and GC-MS
- Capillary Electrophoresis
- MALDI-TOF-MS
- Non-invasive spectroscopy
methods (NIR, ATR, MIR)
Phytocosmetic and
phytopharmaceutical
product
development
- Extraction of natural products
- Screening of active compounds
- Efficacy of cosmetic formulations
- Secretomics & metabolomics
- 2D and 3D in vitro cell culture
- Enzymatic and cell-free assays
- Mode-of-action studies
- In vitro safety assays
Austrian Drug
Screening
Institute GmbH
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