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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07.02.2018

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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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