analytical methods for analyse secondary metabolites · biological activity of secondary plant...
TRANSCRIPT
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Technische Universität München
Analytical methods for analyse secondary
metabolites
Susanne Rühmann
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bioactive compounds in plants(polyphenols: flavonoids)
O
OH
HO
OH
OH
OH
OH
OHO
OH
HO
OH
O
OH
HO
OH
OH
OH
4
8
4
8
OHO
OH O
O
OH
OH
OH
O
O
O
OH
O
OH
OH
HO
OH
OH
O
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Technische Universität München
Polyphenols (flavonoids)
Catechins
Enzymes
Enzymes
Enzymes
Enzymes
Enzymes
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Technische Universität München
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Technische Universität München
Functions in plants
• Colour component (Insects)
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Technische Universität München
Why polyphenols?
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Technische Universität München
Insects like bees and butterflies are
able to notice light till into UV-spectra
area
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Functions of flavonoids at the interface plant and
environment (TREUTTER, 2007).
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Enemies
Stress factors
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Protection against sunlight
Leaf profile
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Protection against sunlight
UV-light affect
DNA in nucleus of
plant cells
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Secondary plantmetabolites
for example
Polyphenols / Flavonoids
Protection against sunlight
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Technische Universität München
Protection against sunlight
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Technische Universität München
Protection against sunlight
Polyphenols = suncreme of plants
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Plant-Pathogen/ElicitorInteraction
Receptor
Nucleus
Pathogen
Plant cell
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Nucleus
Pathogen
Accumulation of phenolics Plant cell
Plant-Pathogen/ElicitorInteraction
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Technische Universität München
Functions in plants
• Colour component (Insects)
• UV-protection (anthocyanidins, flavonols)
• Against pathogens
• Against stress factors (elicitors)
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Technische Universität München
Elicitor
=> inductor
• UV-light
• Enzymes of microorganisms
• Dry-stress
• Saline stress
• High or low temperature
• Acids / alkalins
• Heavy metal
• Ozon (free radicals)
• Toxins (pesticides)
• Phytohormones
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Technische Universität München
Functions in plants
• Colour component (Insects)
• UV-protection (anthocyanidins, flavonols)
• Against pathogens
• Against stress factors (elicitors)
• Hormonal influence
• DNA influence
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Technische Universität München
Why analysis (identification and quantification) of
secondary metabolites?
• Functions in plants
• Polyphenols: a marker for food-quality
• Understanding of mechanisms for human health
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Technische Universität München
human health
In fruits and vegetables are:
• Mineral nutrients
• Vitamins
• Dietary fibres (Ballaststoffe)
• Bioactive compounds ????
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Technische Universität München
Bioactive compounds:
Polyphenols for human health
• Well-known
since 1936 known as human health protector “Vitamin P”
Szent-Györgii
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Technische Universität München
Bioactive compounds:
Polyphenols for human health
• New evaluate
”Bioactive plant compounds”Food against
cancer
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Technische Universität München
Bioactive compounds:
Industry react with products
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Technische Universität München
Bioactive compounds:
Dietary supplement
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Technische Universität München
Bioactive compounds:
New juice creations
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Technische Universität München
Bioactive compounds:
MOGUER, Cuna de Platero S.C.A., Camino de Montmayor, s/n., R.S. 21.0001352/H; R.I.A.21/4086/, HUELVA
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Technische Universität München
Possible mechanisms of polyphenols in human health
• Cardio protective activity
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Technische Universität München
Possible mechanism of cardio protective activity of polyphenols
Endothelzelle
Membran
O2-
Superoxid
Peroxynitrit
Aldini G., M. Carini, E. Bombardelli, R. Maffei Facino:
XXI International Conference on Polyphenols, Marrakech 2002
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Technische Universität München
Possible mechanism of cardio protective activity of polyphenols
Endothelzelle
Membran
O2-
Superoxid
Peroxynitrit
Aldini G., M. Carini, E. Bombardelli, R. Maffei Facino:
XXI International Conference on Polyphenols, Marrakech 2002
OH
OH
OHOH
OH
OH
OH
OHOH
OH
OH
OH
OHOH
OH
OH
OH
OHOH
OH
OH
OH
OHOH
OH
OH
OH
OHOH
OH
OH
OH
OHOH
OH
OH
OHOH
OH
O
OHOH
OH
OH
OH
OH
OHOH
OH
OH
OH
OHOH
OH
OH
OH
OHOH
OH
OH
OHOH
OH
OH
OH
OH
OHOH
OH
OH
OHOH
OH
O
OHOH
OH
OH
OH
OH
OHOH
OH
OH
OH
OHOH
OH
Protection against
radicals
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Technische Universität München
Possible mechanism of cardio protective activity of polyphenols
Inhibition of carcinoma-development in mouse caused by Apigenin (flavon)
Birt et al. (1997).
Carcinoma-
development under
UV-light
Protection by
polyphenols
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Inflammation and developmemt of tumor
normal Epithel
StromaFibroblast
Inflammation
FibroblastOH-O2
- H2O2
NOcankered cells
Tumor-tissue
Tumor-cells
Inhibition of immun-regulationCankered, inflammation protective signals
Inh
ibit
ion
of
infl
amm
atio
nce
nte
r
Different reactors of inflammation
Prof. D. Treutter
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Different signal-chains => Inflammation
Catalysts are wounds, oxidative stress, UV-light …
RTK
Signalacceptor
PLC
JAK
STAT3
Transcriptions-factors
CytosolSTAT3
TranslocationInflammation genes
Nucleus
Cankered inflammation protection signalsiNOS, COX-2, IL-6, IL-1β, TNF-α, VEGF …
Transcription
Pan et al. 2009, J. Agr. Food Chem.
Prof. D. Treutter
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Different signal-chains => Inflammation
Catalysts are wounds, oxidative stress, UV-light …
Signalacceptor
Transcriptions-factors
Cytosol
TranslocationInflammation genes
Nucleus
Cankered inflammation protection signalsiNOS, COX-2, IL-6, IL-1β, TNF-α, VEGF …
Transcription
PLC
JAK
STAT3
STAT3, NFκB, NFAT,
DAG
PKC
NIK
IKK
NFκBIκBα /
Prof. D. Treutter
Pan et al. 2009, J. Agr. Food Chem.
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Different signal-chains => Inflammation
Catalysts are wounds, oxidative stress, UV-light …
Signalacceptor
Transcriptions-factors
Cytosol
TranslocationInflammation genes
Nucleus
Cankered inflammation protection signalsiNOS, COX-2, IL-6, IL-1β, TNF-α, VEGF …
Transcription
RTK
MAPK
PLC
JAK
STAT3
STAT3, NFκB, NFAT, AP-1, C/EBP,
DAG
PKC
NIK
IKK
NFκBIκBα /
TLR
MAP3K
MAP2K
Prof. D. Treutter
Pan et al. 2009, J. Agr. Food Chem.
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Different signal-chains => Inflammation
Catalysts are wounds, oxidative stress, UV-light …
Signalacceptor
Transcriptions-factors
Cytosol
TranslocationInflammation genes
Nucleus
Cankered inflammation protection signalsiNOS, COX-2, IL-6, IL-1β, TNF-α, VEGF …
Transcription
RTK
MAPK (p38, ERK1/2, JNK ½)
PLC
JAK
STAT3
STAT3, NFκB, NFAT, AP-1, C/EBP, CBP/p300, ATF …
DAG
PKC
NIK
IKK
NFκBIκBα /
TLR
MAP3K
MAP2K
G protein
Raf
Prof. D. Treutter
Pan et al. 2009, J. Agr. Food Chem.
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Different signal-chains => Inflammation
Catalysts are wounds, oxidative stress, UV-light …
Signalacceptor
Transcriptions-factors
Cytosol
TranslocationInflammation genes
Nucleus
Cankered inflammation protection signalsiNOS, COX-2, IL-6, IL-1β, TNF-α, VEGF …
Transcription
RTKCytokinreceptor
MAPK (p38, ERK1/2, JNK ½)
PLC
JAK
STAT3
STAT3, NFκB, NFAT, AP-1, C/EBP, CBP/p300, ATF …
DAG
PKC
NIK
IKK
NFκBIκBα /
TLR
MAP3K
MAP2K
G protein
Raf
Ras
PI3K
Akt
Prof. D. Treutter
Pan et al. 2009, J. Agr. Food Chem.
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Different signal-chains => Inflammation
Catalysts are wounds, oxidative stress, UV-light …
Nucleus
Cankered inflammation protection signalsiNOS, COX-2, IL-6, IL-1β, TNF-α, VEGF …
Transcription
RTKDAG
TLR
G protein
Cytokinreceptor
Ras
NIK
IKK
NFκB
PI3K
Akt
MAP3K
MAP2K
MAPK (p38, JNK ½)
JAK
STAT3
OO
O
O O
CH3
CH3
CH3
O
OCH3
CH3
OOH
OH
OH O
OH
OH
OH
OOH
OH
OH O
X
Prof. D. Treutter
Pan et al. 2009, J. Agr. Food Chem.
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Technische Universität München
Biological activity of secondary plant metabolites
• Interaction with enzymes
– Reversible/irreversible binding on proteins
– Chelatisation of metal-ions (Fe-III, Al-III, Cu-II)
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Technische Universität München
Biological activity of secondary plant metabolites
• Interaction with enzymes
– Reversible/irreversible binding on proteins
– Chelatisation of metal-ions (Fe-III, Al-III, Cu-II)
• Antioxidative activity and radical interceptor potential
– Protection of DNA, nucleotids, enzymes, membrans, lipids, vitamins
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Technische Universität München
Biological activity of secondary plant metabolites
• Interaction with enzymes
– Reversible/irreversible binding on proteins
– Chelatisation of metal-ions (Fe-III, Al-III, Cu-II)
• Antioxidative activity and radical interceptor potential
– Protection of DNA, nucleotids, enzymes, membrans, lipids, vitamins
• Modulation of gene activity
– Interaction of transkriptions-factors
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Technische Universität München
Biological activity of secondary plant metabolites
• Antimicrobial activity
– Inhibition of microbial enzymes (lyases)
– Protection of substrates (Glucans, Proteins, Nucleic acids)
– Cell-toxicity
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Technische Universität München
Biological activity of secondary plant metabolites
Human health activity of natural phenolic
compounds
Scientific papers
An
tho
cya
nid
ins
Ch
loro
gen
ic
acid
Ell
ag
icacid
Iso
fla
vo
no
ids
OP
C a
nd
Ca
tec
hin
s
Ph
lori
dzin
Re
sve
ratr
ol
Ru
tin
/ F
lavo
no
ls
Chlorogenic acid Rutin (flavonol)
Anthocyanidin
OPC (oligomeric
procyanidin)
Resveratrol
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Technische Universität München
Biological activity of secondary plant metabolites
Human health activity of natural phenolic
compounds
Scientific papers
An
tho
cya
nid
ins
Ch
loro
gen
ic
acid
Ell
ag
icacid
Iso
fla
vo
no
ids
OP
C a
nd
Ca
tec
hin
s
Ph
lori
dzin
Re
sve
ratr
ol
Ru
tin
/ F
lavo
no
ls
anti-oxidative capacity x x x x x x x x
Potential of radical protection x x x x x x x
Anti-cancer-causing effect x x x x x
Protection of blood vessels x x x x x
Protection of blood plates x x x x x
Regulation of blood sugar x x x x x
Protection of neuronal cells x x x x x
Anti-mutagen potential x x x
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Technische Universität München
Biological activity of secondary plant metabolites
Human health activity of natural phenolic
compounds
Scientific papers
An
tho
cya
nid
ins
Ch
loro
gen
ic
acid
Ell
ag
icacid
Iso
fla
vo
no
ids
OP
C a
nd
Ca
tec
hin
s
Ph
lori
dzin
Re
sve
ratr
ol
Ru
tin
/ F
lavo
no
ls
Anti-viral effect x x x x
Anti-microbial effect x x x x
Protection of cell membranes x x x
Affect to fatty acid metabolism x x x
Affect NO-level in plasma x x
Anti-allergenic effect x x
Immunmodulation x x
Anti-tumor activity x x
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Technische Universität München
Biological activity of secondary plant metabolites
Human health activity of natural phenolic
compounds
Scientific papers
An
tho
cya
nid
ins
Ch
loro
gen
ic
acid
Ell
ag
icacid
Iso
fla
vo
no
ids
OP
C a
nd
Ca
tec
hin
s
Ph
lori
dzin
Re
sve
ratr
ol
Ru
tin
/ F
lavo
no
ls
arteriosclerosis x x x
Estrogenic effect x x
Influence of cholesterol level x x
Protection of vitamin C x x
Regulation of blood preasure x
Protection of DNA x
Dissolving of anxiety state (psychic) x
Against climacteric trouble x
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Technische Universität München
Polyphenols: a marker for food-quality
• Controlling right description of a product
• Evaluating inner quality of food
• Capturing influence of handling procession in food
industry (heating, cooking, washing, …) on oxidation
processes of polyphenols in basis products
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Technische Universität München
Controlling right description of a product
• Creation of polyphenol-profiles (pattern)
profile cranberry juice
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Technische Universität München
Controlling right description of a product
• Creation of polyphenol-profiles (pattern)
profile cranberry juice
profile apple juice
profile mixed juice
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Technische Universität München
Processions in food industry
• Any thermic procedures =>
degradation of some polyphenols
(creation of cereals)
• Other thermic procedures =>
little degradation of polyphenols
(backing of bread)
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Technische Universität München
Secondary metabolites (polyphenols) in plants
![Page 52: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/52.jpg)
Technische Universität München
Influence factors of polyphenol concentration in plants
• Primary metabolism
• Cultivar
• Crop cultivation methods
• Nutrients
• Time of harvest
• Storage
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Technische Universität München
Influence factors of polyphenol concentration in plants
Cultivar
• Different plant organs show
– Different compound concentration (mostly)
– Different phenol profiles (sometimes)
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Technische Universität München
Chl
orog
ensä
ure
Phl
orid
zin
Hyp
erin
Isoq
uerc
itri
nR
utin
Avi
cula
rin
Que
rcet
in
Hyd
roxy
zim
tsäu
re-D
eriv
at
Hyd
roxy
zim
tsäu
re-D
eriv
at
Hyd
roxy
zim
tsäu
re-D
eriv
at
Hyd
roxy
zim
tsäu
re-D
eriv
at
Fla
vano
l
Fla
vano
l
Fla
vano
l
Fla
vano
l
Käm
pfer
ol-D
eriv
ate
Phl
oret
in-2‘-
xylo
gluc
osid
Apple phenolics „Golden Delicious“
Apple leaf
Apple fruit skin
![Page 55: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/55.jpg)
Technische Universität München
Crop cultivation methods
Change of polyphenol content caused by
• Cutting of the trees => changing in hormone content
• Condition of soil
• Climate conditions
• Light condition (UV-B light)
• Plant protection management (pesticides)
• Level of pathogen attack
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Technische Universität München
Fertilization
High N-fertilization indice mostly
• Increasing of primary metabolism (plant growth)
• Decreasing of secondary metabolism (plant resistance)
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Technische Universität München
Time of harvest / changes during storage
• Red pigments (anthocyanidins) increase during ripening
• Catechins decrease during ripening (strawberry)
![Page 58: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/58.jpg)
Technische Universität München
Analytical methods for secondary metabolites
• Gas chromatography (volatile compounds)
• Liquid Chromatography (low- or not-volatile compounds)
– Thin layer chromatography
– HPLC (High-performance-liquid-chromatography; High-pressure-liquid-chromatography)
![Page 59: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/59.jpg)
Technische Universität München
Liquid Chromatography
Definition of physical method:
Separation of compounds caused by:
• An inactive (stationary), hard phase
And
• A moving (mobile), liquid phase
Preconditions:
• Mixture of compounds is dissolved in a solvent
• Compounds are low-volatile or not volatile
![Page 60: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/60.jpg)
Technische Universität München
Liquid Chromatography
Separation processes:
Types of separation mechanisms differ in interaction between stationary
phase, mobile phase and compound
• Absorption
• Allocation
• Ion-change
• Exclusion
• Affinity
How to quantify and identify plant metabolites
![Page 61: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/61.jpg)
Technische Universität München
Liquid Chromatography
• Thin layer chromatography
• HPLC (High-performance-liquid-chromatography;
High-pressure-liquid-chromatography)
![Page 62: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/62.jpg)
Technische Universität München
HPLC (high-performance-liquid-chromatography)
Separation processes of HPLC:
Types of separation mechanisms differ in interaction between stationary
phase, mobile phase and compound
• Absorption
• Allocation
![Page 63: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/63.jpg)
Technische Universität München
HPLC (high-performance-liquid-chromatography)
Separation processes of HPLC:
Types of separation mechanisms
• Absorption
– Sample molecule is bound reversible on stationary phase by dipol-
dipol-interactions
– Exposer time depends on different intensity of interaction
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Technische Universität München
HPLC (high-performance-liquid-chromatography)
Separation processes of HPLC:
Types of separation mechanisms
• Allocation: Normal-phase-chromatography:
– Stationary phase is more polar than mobile phase
• Allocation: Reverse-phase-chromatography:
– Mobile phase is more polar than stationary phase
![Page 65: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/65.jpg)
Technische Universität München
HPLC (high-performance-liquid-chromatography)
Field of application:
• Is used for identifying, quantifying and purification the individual
components of a mixture
• Is used in:
– Analytical chemistry
– Biochemistry
– Medicine and pharmaceutical industry
» Vitamin D in blood serum
» Drugs in urine
» Purification of substances from a complex biological sample
» Manufacturing pharmaceutical (control)
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Technische Universität München
HPLC (high-performance-liquid-chromatography)
Column:
• Separation column is filled with solid particles
– Silica gel
– Polymers
– sorbents
• Sample mixtures is separated into single compounds as it interacts with
the column particles (absorbance)
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Technische Universität München
HPLC (high-performance-liquid-chromatography)
Column:
• Analytic HPLC columns => analysis of extract
• inside diameter (ID) 2 - 4.6 mm
• length till 250 mm
• max. flow 2 ml/min
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Technische Universität München
HPLC (high-performance-liquid-chromatography)
Column: Analytic HPLC columns (institute of fruit science)
• C-18-column
• Chemical modified silica gel C-18-chains are bonded at surface
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Technische Universität München
HPLC (high-performance-liquid-chromatography)
Column:
• Semipräparative column => cleaning of extract
• ID 10 - 50 mm
• length 250 mm
• max. flow 100 ml/min
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Technische Universität München
HPLC (high-performance-liquid-chromatography)
Column:
• Präparative column => getting clean single compounds
• ID more than 75 mm
• Flow more than 100 ml/min
![Page 71: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/71.jpg)
Technische Universität München
HPLC (high-performance-liquid-chromatography)
Column:
• Pre-columns => before head column: protection against
waste in extract (same material)
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Method:
Sample mixture is carried to a
separation columncolumn
sample-loop
Injection outlet
hand injection by syringe
Auto-sampler
HPLC (high-performance-liquid-chromatography)
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Method:
mechanical pumps transport
liquid solvent (eluent) through
a separation column
column
outlet
HPLC (high-performance-liquid-chromatography)
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Method:
Single compounds remove the solid
particles at different moments and cross
the column with different speeds
column
eluent
HPLC (high-performance-liquid-chromatography)
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column
eluent
HPLC (high-performance-liquid-chromatography)
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column
eluent
HPLC (high-performance-liquid-chromatography)
![Page 77: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/77.jpg)
Method:
Column leaving compound is transported
to detector
detector
HPLC (high-performance-liquid-chromatography)
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column
eluent
detector
HPLC (high-performance-liquid-chromatography)
![Page 79: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/79.jpg)
Technische Universität München
lamp vessel photo cell Data collector
(computer)
cover
![Page 80: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/80.jpg)
Technische Universität München
lamp vessel photo cell Data collector
(computer)
cover
Light (e.g. 280
nm)
![Page 81: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/81.jpg)
Technische Universität München
100
%100
%
100
%
lamp vessel photo cell Data collector
(computer)
cover
Light (e.g. 280
nm)
![Page 82: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/82.jpg)
Technische Universität München
compound
lamp vessel photo cell Data collector
(computer)
cover
![Page 83: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/83.jpg)
Technische Universität München
Compound absorbs light
100
%20 % 80 %
80 %
lamp vessel photo cell Data collector
(computer)
cover
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Technische Universität München
100
%50 % 50 %
50 %
lamp vessel photo cell Data collector
(computer)
cover
Higher concentration =>
more absorbtion
![Page 85: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/85.jpg)
Technische Universität München
Absorption
concentr
ation
lamp Vessel photo cell Data collector
(computer)
cover
![Page 86: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/86.jpg)
column
eluent
detector
chromatogram
absorption
Retention
time
HPLC (high-performance-liquid-chromatography)
![Page 87: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/87.jpg)
column
eluent
detector
chromatogram
absorption
Retention
time
only eluent for 10 minutes
0
10
HPLC (high-performance-liquid-chromatography)
![Page 88: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/88.jpg)
Retention
time
First molecules of a separated
compound leaves the column
0
10
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
![Page 89: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/89.jpg)
Retention
time
Information:
little absorption => software
0
10
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
![Page 90: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/90.jpg)
Retention
time
0
10
More molecules of the separated
compound leaves the column
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
![Page 91: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/91.jpg)
Retention
time
0
10
Information:
More absorption => software
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
![Page 92: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/92.jpg)
Retention
time
0
10
Rest of molecules leave the column
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
![Page 93: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/93.jpg)
Retention
time
0
10
Information:
Again lesser absorption => software
13
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
![Page 94: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/94.jpg)
Retention
time
0
10
All molecules of compound
have leave the column
13
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
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Retention
time
0
10
Information:
No absorption => software
13
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
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Retention
time
0
10
Same with the next compound
13 20
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
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Retention
time
0
10 13 20
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
Same with the next compound
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Retention
time
0
10 13 20 22
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
Same with the next compound
![Page 99: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/99.jpg)
Retention
time
0
10
First compound: Ret. Time max: 11.5 min (10 – 13 min); Abs. : 30 (mAU)
Second compound: Ret. Time max: 21 min (20 – 22 min); Abs.: 20 (mAU)
13 20 22
20
3011.5
21
HPLC (high-performance-liquid-chromatography)
chromatogram
absorption
![Page 100: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/100.jpg)
Retention
time
0
10
System calculate from peak high and peak duration => area of peak
13
3011.5
HPLC (high-performance-liquid-chromatography)
chromatogram
absorption
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Retention
time
0
10
System calculate from peak high and peak duration => peak area
We calculate later from peak area => real concentration
13
3011.5
HPLC (high-performance-liquid-chromatography)
chromatogram
absorption
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Method:
Most digital analyte detector can measure at
different selectable wavelength (channels)
Channels in fruit science lab:
640 nm (catechins after post-column-derivatization)
280 nm (simple phenolics, catechins, …)
320 nm (hydroxycinnamic acids)
350 nm (flavonols)
540 nm (anthocyanidins)
UV-vis (spectra)
detector
HPLC (high-performance-liquid-chromatography)
![Page 103: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/103.jpg)
Method:
digital analyte detector =>
quantitative analysis of compounds
detector
HPLC (high-performance-liquid-chromatography)
![Page 104: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/104.jpg)
Diodenarray-detector
Method:
Diodenarray-detector =>
qualitative analysis of compounds
and
Identification of compounds
HPLC (high-performance-liquid-chromatography)
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Light for identification
UV: 100 – 380 nm
Visible light: 380 - 780 nm
wavelength
Infra red
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Absorbed colorAbsorbed wavelength[nm]
Reflected wavelength (visible color)
blue 446 yellow
green 500 red
yellow 562 blue
orange 595 Blue-green
red 660-668 green
complementary colors
![Page 107: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/107.jpg)
leaf
Leaf absorb red light for photosynthesis > 600 nm
complementary colors
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Leaf reflects green light (short wavlength) < 550 nm
leaf
complementary colors
![Page 109: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/109.jpg)
Green and blue receptors in human eyes are activated
=> Green leaf
leaf
eye
complementary colors
![Page 110: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/110.jpg)
Absorption green light < 550 nm Reflection red light > 600 nm
complementary colors
![Page 111: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/111.jpg)
Absorption Reflection
complementary colors
![Page 112: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/112.jpg)
Diodenarray-detector
prism
lampvessel photo cell Data collector
(computer)
cover
![Page 113: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/113.jpg)
wavelength
abso
rpti
on
prism
lampvessel photo cell Data collector
(computer)
cover cover
Diodenarray-detector
![Page 114: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/114.jpg)
wavelength
abso
rpti
on
prism
lampvessel photo cell Data collector
(computer)
cover cover
In our lab: 250 – 600 nm
Diodenarray-detector
![Page 115: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/115.jpg)
prism
lamp vessel photo cell Data collector
(computer)
cover cover
wavelength
abso
rpti
on
550 – 600 nm
Diodenarray-detector
![Page 116: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/116.jpg)
prism
lamp vessel photo cell Data collector
(computer)
cover cover
wavelength
abso
rpti
on
480 – 550 nm
Diodenarray-detector
![Page 117: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/117.jpg)
prism
lamp vessel photo cell Data collector
(computer)
cover cover
wavelength
abso
rpti
on
400 – 480 nm
Diodenarray-detector
![Page 118: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/118.jpg)
prism
lamp vessel photo cell Data collector
(computer)
cover cover
wavelength
abso
rpti
on
380 – 400 nm
Diodenarray-detector
![Page 119: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/119.jpg)
prism
lamp vessel photo cell Data collector
(computer)
cover cover
wavelength
abso
rpti
on
250 – 380 nm
Diodenarray-detector
![Page 120: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/120.jpg)
wavelength
abso
rpti
on
Anthocyanidin 278, 515 nm
250 nm 500 nm
UV-Vis-spectrum
![Page 121: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/121.jpg)
abso
rpti
on
250 nm 500 nm
Flavonol 258, 354 nm
R1
R2A C
B
wavelength
UV-Vis-spectrum
![Page 122: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/122.jpg)
abso
rpti
on
250 nm 500 nm
Hydroxycinnamic acids 309 - 329 nm
wavelength
UV-Vis-spectrum
![Page 123: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/123.jpg)
Method relies on:
High-performance liquid chromatography (HPLC)
Diodenarray
detector
(DAD)
column
sample-loop
Injection outlet
autosampler
outlet
Eluent pumpEluent
HPLC
software
regulate
![Page 124: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/124.jpg)
Technische Universität München
High-performance liquid chromatography (HPLC)
HPLC separation is influenced by:
• the liquid solvent’s condition
• Pressure
• temperature
• chemical interactions between the sample mixture and the
liquid solvent
• Hydrophobicity
• Protonation, …
• chemical interactions between the sample mixture and the solid
particles packed inside of the separation column
• Ligand affinity
• Ion exchange, …
![Page 125: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/125.jpg)
Technische Universität München
Many different types of columns
varying in:
• Size
• Type (i.e. chemistry) of solid packed particle types
![Page 126: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/126.jpg)
Technische Universität München
Eluent (solvent) process in HPLC
• Isokratic solvent process
• One solvent (eluent): interaction between stationary phase and
mobile phase is the same all the time of separation
• Good separation of similar compounds
![Page 127: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/127.jpg)
Method relies on:
High-performance liquid chromatography (HPLC)
Diodenarray
detctor (DAD)
column
sample-loop
ventile
Eluent pumpEluent
HPLC
software
regulate
![Page 128: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/128.jpg)
Technische Universität München
Isokratic Elution
No changing of mobile phase composition during separation process
100 min
100
%
Solvent A (80%H2O; 20% MeOH)
![Page 129: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/129.jpg)
Technische Universität München
Eluent (solvent) process in HPLC
• Isokratic solvent process
• One solvent (eluent): interaction between stationary phase and
mobile phase is the same all the time of separation
• Good separation of similar compounds
• Gradient between different eluents
• Composition of different eluents (mostly 2 or 3) is changed
continued about the sepatation period: interaction between
stationary phase and mobile phase change all the time
• Good separation of compounds with different polarity
![Page 130: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/130.jpg)
Method relies on:
High-performance liquid chromatography (HPLC)
Diodenarray
detctor (DAD)
column
sample-loop
Eluent pumpEluent B
HPLC
software
regulate
Eluent A
![Page 131: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/131.jpg)
HPLC-gradient
column
Column particles(unpolare surface)
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Extract (mixture of different compounds)=> On the column top
HPLC-gradient
column
Column particles(unpolare surface)
![Page 133: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/133.jpg)
Compounds are fixed on column particle bypolarity power (like magnetic effect)
HPLC-gradient
column
Column particles(unpolare surface)
![Page 134: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/134.jpg)
column
Column particles(unpolare surface)
HPLC-gradient
Eluent 1 (polar) transfer column (e.g. water)
% Solvent A (H2O)
![Page 135: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/135.jpg)
column
Column particles(unpolare surface)
HPLC-gradient
Compounds with similar polarity like water (green) => solve from particles and transfer the column .
Compounds with more unpolar character => stayon partcles
![Page 136: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/136.jpg)
Eluent 1 (polar) (e.g. water) was combinated withEluent 2 (more unpolar) (e.g. methanol)(1:1; v:v) (50% MeOH)=> Mixture is more unpolar than water
column
Column particles(unpolare surface)
HPLC-gradient
% Solvent A (H2O)
% Solvent B (MeOH)
![Page 137: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/137.jpg)
column
Column particles(unpolare surface)
HPLC-gradient
Compounds with similar polarity 1:1 (water : methanol) (blue) => solve from particles and transfer the column .
Compounds with more unpolar character => stay on partcles
![Page 138: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/138.jpg)
column
Column particles(unpolare surface)
HPLC-gradient
Eluent 1 (polar) (e.g. water) was combinated withEluent 2 (more unpolar) (e.g. methanol)(2:8; v:v) (80% MeOH)=> Mixture is more unpolar than (water:methanol) (1:1)
% Solvent A (H2O)
% Solvent B (MeOH)
![Page 139: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/139.jpg)
column
Column particles(unpolare surface)
HPLC-gradient
Yellow coumpounds leave the column
![Page 140: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/140.jpg)
Technische Universität München
Gradient-method
changing of mobile phase composition during separation process
0 2
0
40
60
80
100 % % Solvent A (H2O)
% Solvent B (MeOH)
![Page 141: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/141.jpg)
Technische Universität München
Begining of HPLC-work:
•Creating of a data base of polyphenol standards
•Different compounds
•Different concentrations
•Analyse at different wavelength
What about linearity
Where is the concentration limit of HPLC
![Page 142: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/142.jpg)
Technische Universität München
Extraction methods
• Fresh-extraction
• Dry-extraction
![Page 143: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/143.jpg)
Technische Universität München
Dry-extraction of polyphenols
Ground freeze-dried
plant material
Swing mill
Mortar
![Page 144: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/144.jpg)
Technische Universität München
Dry-extraction of polyphenols
Ground freeze-dried
plant material
fill powder [x mg]
in 2 ml Eppendorf tubes
![Page 145: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/145.jpg)
Technische Universität München
Dry-extraction of polyphenols
Ground freeze-dried
plant material
fill powder [x mg]
in 2 ml Eppendorf tubes
extract with methanol
containing an internal
standard
+
![Page 146: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/146.jpg)
Technische Universität München
Dry-extraction of polyphenols
extract with methanol
containing an internal
standard
MeOH
Plant powder
complex with
anthocyanidins in
vacuoles
![Page 147: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/147.jpg)
Technische Universität München
Dry-extraction of polyphenols
extract with methanol
containing an internal
standard
MeOH
Fresh methanol
transport
anthocyanisins out of
plant material (high
concentration gradient:
inside much outside
nothing))
![Page 148: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/148.jpg)
Technische Universität München
Dry-extraction of polyphenols
extract with methanol
containing an internal
standard
MeOH
anthocyanisins
accumulate around the
plant complex.
Concentration gradient
is smaller => extraction
of anthocyanidins
inside is during longer
![Page 149: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/149.jpg)
Technische Universität München
Dry-extraction of polyphenols
extract with methanol
containing an internal
standard
MeOH
Better extraction when
concentration gradient
stay at high level
=>
![Page 150: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/150.jpg)
Technische Universität München
Dry-extraction of polyphenols
Ground freeze-dried
plant material
fill powder [x mg]
in 2 ml Eppendorf tubes
extract with methanol
containing an internal
standard
Shake the extract!
By hand
![Page 151: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/151.jpg)
Technische Universität München
Dry-extraction of polyphenols
Ground freeze-dried
plant material
fill powder [x mg]
in 2 ml Eppendorf tubes
extract with methanol
containing an internal
standard
Cool (4°C) ultrasonic
bath for 30 min
![Page 152: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/152.jpg)
Technische Universität München
Dry-extraction of polyphenols
Ground freeze-dried
plant material
fill powder [x mg]
in 2 ml Eppendorf tubes
extract with methanol
containing an internal
standard
centrifuge for 10 min
at 10000 rd/min
Cool (4°C) ultrasonic
bath for 30 min
![Page 153: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/153.jpg)
Technische Universität München
Dry-extraction of polyphenols
Ground freeze-dried
plant material
fill powder [x mg]
in 2 ml Eppendorf tubes
extract with methanol
containing an internal
standard
centrifuge for 10 min
at 10000 rd/min
Fill supernatant in a new
Eppendorf tube =>
Extract is used for HPLC
analysis (soluble phenolics)
Cool (4°C) ultrasonic
bath for 30 min
Storage extract
at -20°C
centrifuge for 10 min
at 10000 rd/min
before filling extract
in sample tubes
![Page 154: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/154.jpg)
Technische Universität München
Dry-extraction of polyphenols
Volume of plant material:
fill powder [x mg]
in 2 ml Eppendorf tubes
plant organ Fresh
weight
[mg]
Dry
weight
[mg]
apple leaves 1000 100
grape flowers 2000 200
![Page 155: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/155.jpg)
Technische Universität München
Dry-extraction of polyphenols
Best extraction-solvent and best concentration
compounds Optimal solvent
flavonoids Methanol 100 %
anthocyanidins Methanol +
Formic acid
95% + 5% (v/v)
carotenoids Aceton
Tetrahydrofuran
Diethylether
Ethylacetat
Hexan
Petrolether
100 %
xanthophylls Ethanol 100 %
![Page 156: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/156.jpg)
Technische Universität München
Dry-extraction of polyphenols
Best extraction-solvent and best concentration
• For extraction of anthocyanidins the formic acid was used
for stabilization of pH and so for protection anthocyanidins
compounds Optimal solvent
anthocyanidins Methanol +
Formic acid
95% + 5% (v/v)
![Page 157: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/157.jpg)
Technische Universität München
Dry-extraction of polyphenols
Best relation between plant material and solvent
plant organ Fresh
weight
[mg]
Dry
weight
[mg]
Volume
MeOH +
Std. [µl]
apple skin 1000 100 1000
apple leaves 1000 100 500
grape flowers 2000 200 500
![Page 158: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/158.jpg)
Technische Universität München
Dry-extraction of polyphenols
Best internal Standard-compound
=> Place in a chromatogram
Retention time [min]
![Page 159: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/159.jpg)
Technische Universität München
Dry-extraction of polyphenols
Best concentration of
internal Standard-compound
y = 348.5x + 2.1166R² = 0.9996
0
50
100
150
200
250
300
350
400
0 0.2 0.4 0.6 0.8 1 1.2
Are
a(m
AU
*min
)
Concentration (mg/ml)
Optimal linearity between 0,02 und 1 mg/ml
![Page 160: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/160.jpg)
Technische Universität München
Dry-extraction of polyphenols
Best concentration of internal Standard-compound
• Take concentration in the middle of linear area => you can dilute or
concentrate sample with results in linear area of standard
y = 348.5x + 2.1166R² = 0.9996
0
50
100
150
200
250
300
350
400
0 0.2 0.4 0.6 0.8 1 1.2
Are
a(m
AU
*min
)
Concentration (mg/ml)
![Page 161: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/161.jpg)
Technische Universität München
Dry-extraction of polyphenols
Best concentration of internal Standard-compound
• Take concentration in the middle of linear area => you can dilute or
concentrate sample with results in linear area of standard
• Take concentration nearly with the same high of the peaks like the biggest
peaks in the sample
![Page 162: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/162.jpg)
Technische Universität München
Dry-extraction of polyphenols
When peaks are too small
• concentrate extract by evaporation (drying under vacuum => protect phenolic
compounds against oxidation)
• Resuspend dried extract in smaller volume of methanol (500 µl initial extract=>
250 µl concentrate extract)
![Page 163: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/163.jpg)
Technische Universität München
Dry-extraction of polyphenols
When peaks are too big
• Dilute extract by addition of more solvent (methanol without standard
compound)
![Page 164: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/164.jpg)
Technische Universität München
Dry-extraction of polyphenols
Best time of extraction
• Extract sample for different time (10, 20, 30, 40, 50, 60 min)
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Technische Universität München
Dry-extraction of polyphenols
Best time of extraction
• Extract sample for different time (10, 20, 30, 40, 50, 60 min)
• Remove extract after centrifugation
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Technische Universität München
Dry-extraction of polyphenols
Best time of extraction
• Extract sample for different time (10, 20, 30, 40, 50, 60 min)
• Remove extract after centrifugation
• Extract staying plant material again with the same volume of solvent
![Page 167: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/167.jpg)
Technische Universität München
Dry-extraction of polyphenols
Best time of extraction
• Extract sample for different time (10, 20, 30, 40, 50, 60 min)
• Remove extract after centrifugation
• Extract staying plant material again with the same volume of solvent
HPLC-
analysis
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Technische Universität München
We will begin practical work with:
•Creating of a data base of polyphenol standards
•Different compounds
•Different concentrations
•Analyse at different wavelength
What about linearity
Where is the concentration limit of HPLC
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Technische Universität München
Development of Standard-database
Susanne Rühmann
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Technische Universität München
Different concentrations of standard compounds
(0,001; 0,005; 0,01; 0,025; 0,05; 0,25; 0,5; 1,0 mg/ml)
• Catechin
• Epicatechin
• P-coumaric acid
• Chlorogenic acid
• Quercetin
• Rutin
• Kämpferol
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Technische Universität München
Rutin (Croma; Nucleosil; gradient: isokratic 80% MeOH)
260, 351 nm
280 nm320 nm
350 nm
Absortion
mAU*min
Concent
ration
[mg/ml]
280 nm 320 nm 350 nm
0,001 4.8208 5.9552 5.4592
0.005 4.3318 4.6422 4.8743
0.01 6.0476 6.8603 8.1012
0.025 11.4039 15.1045 22.0569
0.05 20.7677 27.508 43.4618
0.25 50.5845 69.7975 115.2529
0.5 113.268 150.7571 236.7213
1 202.579 287.9368 460.4141
Biggest
area
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Technische Universität München
Rutin (Croma; Nucleosil; gradient: isokratic 80% MeOH)
260, 351 nm
280 nm320 nm
350 nm
Absortion
mAU*min
Concent
ration
[mg/ml]
280 nm 320 nm 350 nm
0,001 4.8208 5.9552 5.4592
0.005 4.3318 4.6422 4.8743
0.01 6.0476 6.8603 8.1012
0.025 11.4039 15.1045 22.0569
0.05 20.7677 27.508 43.4618
0.25 50.5845 69.7975 115.2529
0.5 113.268 150.7571 236.7213
1 202.579 287.9368 460.4141
Overview-
chromatogram
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Technische Universität München
Rutin (Croma; Nucleosil; gradient: isokratic 80% MeOH)
260, 351 nm
280 nm
350 nm
Absortion
mAU*min
Concent
ration
[mg/ml]
280 nm 350 nm
0,001 4.8208 5.4592
0.005 4.3318 4.8743
0.01 6.0476 8.1012
0.025 11.4039 22.0569
0.05 20.7677 43.4618
0.25 50.5845 115.2529
0.5 113.268 236.7213
1 202.579 460.4141
![Page 174: Analytical methods for analyse secondary metabolites · Biological activity of secondary plant metabolites Human health activity of natural phenolic compounds Scientific papers dins](https://reader036.vdocuments.mx/reader036/viewer/2022062414/5f0253d27e708231d403b96f/html5/thumbnails/174.jpg)
Technische Universität München
Rutin (Croma; Nucleosil; gradient: isokratic 80% MeOH)
260, 351 nm
280 nm
350 nm
Absortion
mAU*min
Concent
ration
[mg/ml]
280 nm 350 nm
0,001 4.8208 5.4592
0.005 4.3318 4.8743
0.01 6.0476 8.1012
0.025 11.4039 22.0569
0.05 20.7677 43.4618
0.25 50.5845 115.2529
0.5 113.268 236.7213
1 202.579 460.4141
Detection limit
(upper, lower)
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Technische Universität München
Rutin (Croma; Nucleosil; gradient: isokratic 80% MeOH)
260, 351 nm
280 nm
350 nm
Absortion
mAU*min
Concent
ration
[mg/ml]
280 nm 350 nm
0,001 4.8208 5.4592
0.005 4.3318 4.8743
0.01 6.0476 8.1012
0.025 11.4039 22.0569
0.05 20.7677 43.4618
0.25 50.5845 115.2529
0.5 113.268 236.7213
1 202.579 460.4141
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Technische Universität München
Croma; Nucleosil; gradient: isokratic 80% MeOH
HPLC Column gradient RT [min]wavelenght
[nm]Compound-group Compound
conc. [mg/ml]
Area [mAU*min] RF
linear (from .. To)
[mg/ml]
linear (from .. To) [mAU/min] UV max
croma nucleosil 80% MeOH 280 flavonols Rutin 0.25 50.58 4.76E-05 0.25 - 1 50 - 202 256; 358
croma nucleosil 80% MeOH 350 flavonols Rutin 0.25 115.2529 2.15E-05 0.25 - 1 115 - 460 256; 358
croma nucleosil 80% MeOH 280Hydroxy cinnamic acids Chlorogenic acid 0.1 37.5499 2.63E-05 0.005 - 1 1.9 - 350 328
croma nucleosil 80% MeOH 320Hydroxy cinnamic acids Chlorogenic acid 0.1 76.2917 1.31E-05 0.005 - 1 3.6 - 825 328
croma nucleosil 80% MeOH 280 flavan-3-ols Eopicatechin 0.1 21.1954 4.70E-05 0.1 - 1 21 - 208 279
croma nucleosil 80% MeOH 280Hydroxy cinnamic acids p-coumaric acid 0.1 124.5077 8.37E-06 0.025 - 0.25 28 - 299 309
croma nucleosil 80% MeOH 320Hydroxy cinnamic acids p-coumaric acid 0.1 165.4809 6.35E-06 0.05 - 0.25 75 - 393 309
croma nucleosil 80% MeOH 280 flavonols Quercetin 0.1 30.647 3.29E-05 0.01 - 1 3 - 306 255; 372
croma nucleosil 80% MeOH 350 flavonols Quercetin 0.1 63.059 1.62E-05 0.01 - 1 6 - 639 255; 372
croma nucleosil 80% MeOH 280 flavan-3-ols Catechin 0.1 24.2558 4.51E-05 0.1 - 1 24 - 244 279