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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
Technische Universität München
Polyphenols (flavonoids)
Catechins
Enzymes
Enzymes
Enzymes
Enzymes
Enzymes
Technische Universität München
Insects like bees and butterflies are
able to notice light till into UV-spectra
area
Technische Universität München
Functions in plants
• Colour component (Insects)
• UV-protection (anthocyanidins, flavonols)
• Against pathogens
• Against stress factors (elicitors)
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
Technische Universität München
Functions in plants
• Colour component (Insects)
• UV-protection (anthocyanidins, flavonols)
• Against pathogens
• Against stress factors (elicitors)
• Hormonal influence
• DNA influence
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
Technische Universität München
human health
In fruits and vegetables are:
• Mineral nutrients
• Vitamins
• Dietary fibres (Ballaststoffe)
• Bioactive compounds ????
Technische Universität München
Bioactive compounds:
Polyphenols for human health
• Well-known
since 1936 known as human health protector “Vitamin P”
Szent-Györgii
Technische Universität München
Bioactive compounds:
Polyphenols for human health
• New evaluate
”Bioactive plant compounds”Food against
cancer
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
Technische Universität München
Possible mechanisms of polyphenols in human health
• Cardio protective activity
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
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
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
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
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
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.
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.
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.
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.
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.
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)
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
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
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
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
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
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
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
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
Technische Universität München
Controlling right description of a product
• Creation of polyphenol-profiles (pattern)
profile cranberry juice
Technische Universität München
Controlling right description of a product
• Creation of polyphenol-profiles (pattern)
profile cranberry juice
profile apple juice
profile mixed juice
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)
Technische Universität München
Influence factors of polyphenol concentration in plants
• Primary metabolism
• Cultivar
• Crop cultivation methods
• Nutrients
• Time of harvest
• Storage
Technische Universität München
Influence factors of polyphenol concentration in plants
Cultivar
• Different plant organs show
– Different compound concentration (mostly)
– Different phenol profiles (sometimes)
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
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
Technische Universität München
Fertilization
High N-fertilization indice mostly
• Increasing of primary metabolism (plant growth)
• Decreasing of secondary metabolism (plant resistance)
Technische Universität München
Time of harvest / changes during storage
• Red pigments (anthocyanidins) increase during ripening
• Catechins decrease during ripening (strawberry)
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)
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
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
Technische Universität München
Liquid Chromatography
• Thin layer chromatography
• HPLC (High-performance-liquid-chromatography;
High-pressure-liquid-chromatography)
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
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
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
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)
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)
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
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
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
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
Technische Universität München
HPLC (high-performance-liquid-chromatography)
Column:
• Pre-columns => before head column: protection against
waste in extract (same material)
Method:
Sample mixture is carried to a
separation columncolumn
sample-loop
Injection outlet
hand injection by syringe
Auto-sampler
HPLC (high-performance-liquid-chromatography)
Method:
mechanical pumps transport
liquid solvent (eluent) through
a separation column
column
outlet
HPLC (high-performance-liquid-chromatography)
Method:
Single compounds remove the solid
particles at different moments and cross
the column with different speeds
column
eluent
HPLC (high-performance-liquid-chromatography)
Method:
Column leaving compound is transported
to detector
detector
HPLC (high-performance-liquid-chromatography)
Technische Universität München
lamp vessel photo cell Data collector
(computer)
cover
Light (e.g. 280
nm)
Technische Universität München
100
%100
%
100
%
lamp vessel photo cell Data collector
(computer)
cover
Light (e.g. 280
nm)
Technische Universität München
Compound absorbs light
100
%20 % 80 %
80 %
lamp vessel photo cell Data collector
(computer)
cover
Technische Universität München
100
%50 % 50 %
50 %
lamp vessel photo cell Data collector
(computer)
cover
Higher concentration =>
more absorbtion
Technische Universität München
Absorption
concentr
ation
lamp Vessel photo cell Data collector
(computer)
cover
column
eluent
detector
chromatogram
absorption
Retention
time
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
Retention
time
only eluent for 10 minutes
0
10
HPLC (high-performance-liquid-chromatography)
Retention
time
First molecules of a separated
compound leaves the column
0
10
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
Retention
time
Information:
little absorption => software
0
10
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
Retention
time
0
10
More molecules of the separated
compound leaves the column
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
Retention
time
0
10
Information:
More absorption => software
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
Retention
time
0
10
Rest of molecules leave the column
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
Retention
time
0
10
Information:
Again lesser absorption => software
13
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
Retention
time
0
10
All molecules of compound
have leave the column
13
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
Retention
time
0
10
Information:
No absorption => software
13
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
Retention
time
0
10
Same with the next compound
13 20
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
Retention
time
0
10 13 20
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
Same with the next compound
Retention
time
0
10 13 20 22
HPLC (high-performance-liquid-chromatography)
column
eluent
detector
chromatogram
absorption
Same with the next compound
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
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
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
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)
Method:
digital analyte detector =>
quantitative analysis of compounds
detector
HPLC (high-performance-liquid-chromatography)
Diodenarray-detector
Method:
Diodenarray-detector =>
qualitative analysis of compounds
and
Identification of compounds
HPLC (high-performance-liquid-chromatography)
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
wavelength
abso
rpti
on
prism
lampvessel photo cell Data collector
(computer)
cover cover
Diodenarray-detector
wavelength
abso
rpti
on
prism
lampvessel photo cell Data collector
(computer)
cover cover
In our lab: 250 – 600 nm
Diodenarray-detector
prism
lamp vessel photo cell Data collector
(computer)
cover cover
wavelength
abso
rpti
on
550 – 600 nm
Diodenarray-detector
prism
lamp vessel photo cell Data collector
(computer)
cover cover
wavelength
abso
rpti
on
480 – 550 nm
Diodenarray-detector
prism
lamp vessel photo cell Data collector
(computer)
cover cover
wavelength
abso
rpti
on
400 – 480 nm
Diodenarray-detector
prism
lamp vessel photo cell Data collector
(computer)
cover cover
wavelength
abso
rpti
on
380 – 400 nm
Diodenarray-detector
prism
lamp vessel photo cell Data collector
(computer)
cover cover
wavelength
abso
rpti
on
250 – 380 nm
Diodenarray-detector
Method relies on:
High-performance liquid chromatography (HPLC)
Diodenarray
detector
(DAD)
column
sample-loop
Injection outlet
autosampler
outlet
Eluent pumpEluent
HPLC
software
regulate
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, …
Technische Universität München
Many different types of columns
varying in:
• Size
• Type (i.e. chemistry) of solid packed particle types
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
Method relies on:
High-performance liquid chromatography (HPLC)
Diodenarray
detctor (DAD)
column
sample-loop
ventile
Eluent pumpEluent
HPLC
software
regulate
Technische Universität München
Isokratic Elution
No changing of mobile phase composition during separation process
100 min
100
%
Solvent A (80%H2O; 20% MeOH)
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
Method relies on:
High-performance liquid chromatography (HPLC)
Diodenarray
detctor (DAD)
column
sample-loop
Eluent pumpEluent B
HPLC
software
regulate
Eluent A
Extract (mixture of different compounds)=> On the column top
HPLC-gradient
column
Column particles(unpolare surface)
Compounds are fixed on column particle bypolarity power (like magnetic effect)
HPLC-gradient
column
Column particles(unpolare surface)
column
Column particles(unpolare surface)
HPLC-gradient
Eluent 1 (polar) transfer column (e.g. water)
% Solvent A (H2O)
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
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)
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
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)
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)
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
Technische Universität München
Dry-extraction of polyphenols
Ground freeze-dried
plant material
Swing mill
Mortar
Technische Universität München
Dry-extraction of polyphenols
Ground freeze-dried
plant material
fill powder [x mg]
in 2 ml Eppendorf tubes
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
+
Technische Universität München
Dry-extraction of polyphenols
extract with methanol
containing an internal
standard
MeOH
Plant powder
complex with
anthocyanidins in
vacuoles
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))
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
Technische Universität München
Dry-extraction of polyphenols
extract with methanol
containing an internal
standard
MeOH
Better extraction when
concentration gradient
stay at high level
=>
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
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
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
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
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
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 %
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)
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
Technische Universität München
Dry-extraction of polyphenols
Best internal Standard-compound
=> Place in a chromatogram
Retention time [min]
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
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)
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
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)
Technische Universität München
Dry-extraction of polyphenols
When peaks are too big
• Dilute extract by addition of more solvent (methanol without standard
compound)
Technische Universität München
Dry-extraction of polyphenols
Best time of extraction
• Extract sample for different time (10, 20, 30, 40, 50, 60 min)
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
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
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
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
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
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
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
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
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)
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
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
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