bioactive components from berries: human health...
TRANSCRIPT
Gordon J. McDougall Crop Productivity and Utilization
The James Hutton Institute
[email protected] Abertay MSc students 281012
Bioactive components from berries: Human Health Effects
Berry research at JHI
We breed market-leading varieties
Blackcurrants – the “Ben” series
Raspberries – the “Glen” series
Strawberry – “Symphony, Rhapsody….”
Research into Health Benefits of Berries
Feedback and direct breeding of new varieties
Scheme of talk
Background
Biological activities of berry components
MODEL SYSTEMS
Cardiovascular Health
Cancer
Control of nutrient availability
Diabetes & Obesity
Analytical studies
• Correlate bioactivity with polyphenol composition
“Insufficient intake of fruit and vegetables
increases the chances of developing cancers,
cardiovascular disease and strokes” - World
Health Organisation (2003)
The 3 main causes of premature death in Scotland
Led to the “5 a day” programme - Government-led Mass Intervention to alter our diet and improve health
Minerals (Zinc)? Vitamins (C and E)?
Fibre? Displacement? Lower Fat?
Antioxidants? Phytochemicals?
How do FAV affect health?
Living with oxygen & free radicals
We “burn” our food with Oxygen to
release energy
By-products include free radicals
which are VERY reactive. They can
damage the body and cause
disease.
Our bodies work hard to remove
these radicals and prevent damage
Dietary antioxidants are proposed
to “top-up” protection
A simplified version? Polyphenols as antioxidants
O
OR2
OH
HO
R1
OH
O
Flavonols
O
OH
HO
OH
OH
HR1R2
O+
R3
R4
R4
R1
R4
R2
flavanols/PACs
Anthocyanins
O
OHHO
HO
HO
Vitamin C
OO
OO
O
HO
OH
OH
O
HO
OH
OH
O
OH
OH
HO
OHO
HO
HO
O
OH
OH
O
OO
OO
OH
O
HO
OH
OH
O
HO
OH
OH
O
OH
OH
HO
OHO
HO
HO
O
O
Berries contain a diverse and species specific mixture of
antioxidants – the two main types are Polyphenols and Vitamin C
Tannins
Cardiovascular disease (CVD)
Protection of vasodilatory responses of rat aorta against inflicted oxidative damage
Protective effect of polyphenols
not predicted by in vitro
antioxidant activity
Anthocyanins particularly effective
Prof. Ian Megson, Univ. Highland and Islands
O+
R3
R4
R4
R1
R4
R2
Intervention trial – assess effects of six week ingestion of • blackcurrant berries with low vitamin C content • blackcurrant berries with high vitamin C content • blueberries (No vitamin C) • coloured flavoured water (control)
Effects on cardiovascular function
Positive effects on blood vessel flexibility (intima media thickness) and in vivo markers for endothelial cell function and oxidative stress
Cardiovascular function and intake of soft fruit: Effects of qualitative and
quantitative variation in berry antioxidant status
Effect on Alzheimers?
EU project
BrainHealthFood
Bioactive compounds
from blackcurrant
processing waste for
brain health
Oxidative stress, Alzheimer’s and the Brain Brain = 2 % adult body mass but uses 20 % oxygen
inhaled
Poorer antioxidant mechanisms
High levels of PUFAs, minerals and neuro- transmitters – good targets for free radicals
Brain cells don’t renew by cell division - accumulate FR-induced damage with age & FR damage implicated in AD
MTT Agrifood JHI TTZ Univ. Kuopio & SME partners
Protective effect of anthocyanins in Alzheimer’s model system
New studies confirm effects in mice models
Dr Saila Vepsäläinen . Univ. Kuopio
Effects on cancer cells
0
20
40
60
80
100
Blu
eberry
Pomeg
ranate
Sea B
uckth
orn
Bla
ckberry
Bla
ckcurr
ant
Row
an
Ras
pberry
Lingonberry
Clo
udberry
Arc
tic B
ram
ble
Straw
berry
% C
on
tro
l
All berry extracts tested at 50 mg/ml
Inhibition not related to in vitro antioxidant capacity
McDougall et al. (2008) JAFC 56; 3016-3023
Anti-cancer effects
Cell Number
0.E+00
1.E+06
2.E+06
3.E+06
4.E+06
5.E+06
6.E+06
7.E+06
8.E+06
9.E+06
1.E+07
1 2 3 4 5 6 7
Days
Cel
l num
ber
0
7.5
15
30
Raspberry inhibits cancer cell growth at low levels (mg/ml)
Most effective components are tannins
0
20
40
60
80
100
0 5 10 15 20 25 30
mg
%C
on
tro
l
Raspberry
Tannins
Anthocyanins
Joint projects on berry polyphenols & colon cancer
Emma Brown and Dr Chris Gill, School of Biomedical Sciences, University of Ulster, Coleraine
Matrigel invasion assay
0
20
40
60
80
100
120
50 ug 25 ug 12 ug 6 ug 3 ug control
Phenols (GAE)
Invasion, % from
control
cytotoxicity, % from
control
Invasion by HT115 colon cancer cells was inhibited by raspberry polyphenols in the mg range
Invasion related to ability to spread from initial site
Colon cancer and polyphenols
Coates et al. J. Carcinogenesis (2007) 6, 1-13
Control of nutrient availability
Polyphenols can inhibit digestive processes and slow or
modulate nutrient release from food
Inhibition of starch digestion – blood glucose control
and type 2 diabetes
Inhibition of lipid digestion – control of hyperlipidemia,
CVD and obesity
Inhibition of starch digestion
O
H
O
H
HO
H
OHH
H
HOO
H
HO
H
HO
H
OHH
H
HO
O
H
O
H
HO
H
OHH
H
HOO
H
O
H
HO
H
OHH
H
HO
O
H
O
H
HO
H
OHH
H
O
H
O
H
HO
H
OHH
H
HO
O
H
O
H
HO
H
OH
OHH
H
HOO
H
O
H
HO
H
OHH
H
HO
O
H
O
H
HO
H
OHH
H
HOO
H
HO
H
HO
H
OHH
H
HO
O
H
O
H
HO
H
O
OHH
H
HOO
H
O
H
HO
H
OHH
H
HO
O
H
O
H
HO
H
OHH
H
HOO
H
HO
H
HO
H
OHH
H
HO
O
H
O
H
HO
H
OH
OHH
H
HOO
H
O
H
HO
H
OHH
H
HO
O
H
O
H
HO
H
OHH
H
HOO
H
HO
H
HO
H
OHH
H
HO
O
H
O
H
HO
H
OH
OHH
H
O
H
HO
H
HO
H
OHH
H
HO
O
H
O
H
HO
H
O
OHH
H
HOO
H
HO
H
HO
H
OHH
H
HO
OH
O
H
HO
H
HO
H
OH
OHHH
OH
Amylase chops into fragments
Glucosidase nibbles off glucose
a-amylase inhibition
-20
0
20
40
60
80
100
120
0 100 200 300 400 500
Phenols (mg)
% I
nh
ibit
ion
Red cabbage
Strawberry
Blackcurrant
Blueberry
Raspberry
Green tea
Strawberry and raspberry most effective
– ellagitannins implicated
McDougall et al (2005) JAFC 53, 2760-2766; Grussu et al., 2011
a-glucosidase inhibition by berries
Pharmaceutical inhibition with acarbose ?
Not obvious which polyphenols involved?
Human studies show promise
Whitson et al. Functional Plant Science and Biotechnology (2010)
0
20
40
60
80
100
120
140
160
180
200
Row
an
Pom
egra
nate
Straw
berry
Clo
udber
ry
Ras
pber
ry
Lingonberr
y
Bla
ckcurr
ant
Bla
ckber
ry
Blu
eberry
Red
Win
e
% C
on
tro
l A
cti
vit
y
Lipid digestion and lipase
X
Fat uptake
X
0
20
40
60
80
100
120
Control BC ROW BB LB AB CB SB RB
% L
ipa
se
Ac
tiv
ity
Inhibitory at 50 mg/ml
Summary
Berry polyphenols have bioactivities that may influence
human health
Their mechanisms of action are not well defined
Efficacy not always related to antioxidant capacity
Their stability and bioavailability in vivo is not fully
understood
Acknowledgements
All staff in CPU, JHI
Universities of Dundee, St Andrews and Abertay
Dundee for B.Sc and M.Sc students
Kuopio
Questions?
Visit http://www.hutton.ac.uk
JHI is located in Invergowrie on the north bank of the River Tay
Lipase inhibition
0
10
20
30
40
50
60
70
80
90
100
110
0 10 20 30 40 50
Phenols (mg)
% C
on
tro
l
Inhibition by cloudberry extracts is saturable
Due to ellagitannins (ETs) in cloudberry, arctic bramble and raspberry and
procyanidins and ETs in strawberry
Mainly procyanidins in lingonberry
Ties in with animal studies on obesity
OO
OO
O
HO
OH
OH
O
HO
OH
OH
O
OH
OH
HO
OHO
HO
HO
O
OH
OHO
OO
OO
O
O
HO
OH
OH
O
HO
OH
OH
O
OH
OH
HO
OHO
HO
HO
O
OOH
OH
OH
O
McDougall et al. (2009) Food Chemistry 115, 93–199
Insulin mimicking
+ - X W UB B1 B2 Y UB B
Phosphorylated
FOXO1A
Two different berry extracts stimulate phosphorylation of FOXO1A
but the active ingredients fractionate differently on SPE.
Various polyphenols stimulate the phosphorylation and activation
of FOXO1A, a transcription factor involved in regulating insulin
responses and controlling glucose mobilization.
Sandra Bacon & Graham Rena, University of Dundee
About JHI
Dundee
JHI
Long-established breeding program for
berries
Developing high-through-put methods for assessing inheritance of polyphenols
Link to genetic maps and markers to speed up selection
Improve on traditional means of assessing polyphenol
levels slow
Develop and validate new methods
Use power of mass spectroscopic and metabolic profiling
methods
Stewart et al (2007) Mol.Nutr.Food Res. 51, 645–651
McDougall et al (2008) J. Chromatog. B 871, 362–369
Targeted analysis
Yield, flavour, aroma taste, texture, disease
resistance, bioactivities, antioxidant
capacity, polyphenol content, ascorbate,
anthocyanins
Untargeted analysis: Hi-through-
put metabolic profiling
LC-MS GC-TOF-MS
RT: 0.00 - 20.00
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Tim e (m in)
0
20000
40000
60000
80000
100000
120000
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160000
180000
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220000
240000
260000
280000
300000
320000
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440000
460000
480000
500000
520000
540000
560000
580000
600000
620000
640000
660000
680000
700000
uAU
8.26
8.01
6.05
5.62
12.11
12.3213.26
10.888.75
9.68
13.967.3114.26 19.6418.76
15.42 17.95
2.34 2.481.94 4.341.50
NL:
7.11E5
Total Scan
PDA
NORTHBE
RRY
256_03082
0144520
Chromatogram
Mass spectrum
NORTHBERRY 256_030820144520 #552-580 RT: 8.43-8.86 AV: 16 NL: 8.03E6
T: - c APCI Full m s [ 80.00-800.00]
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800
m /z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rela
tive
Abun
danc
e
191.2
353.0
398.6
236.8
354.1
192.2161.3
355.1 399.7207.0135.3 466.9
85.2 397.3237.8 706.9400.6335.2 436.8 502.0 544.9301.1 578.3 718.1668.1642.7 783.2
Direct Infusion MS No chromatography
Principal component
analysis of MS data
Hierarchical
cluster analysis:
Measure of
(phytochemical)
biodiversity - link to
genetic map
Correlation
Network: Interrelate
metabolite
changes.
Compounds 1-500
Co
mp
ounds 1
-50
0
(102-103 compounds)
NORTHBERRY 256_030820144520 #552-580 RT: 8.43-8.86 AV: 16 NL: 8.03E6
T: - c APCI Full m s [ 80.00-800.00]
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800
m /z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rela
tive
Abun
danc
e
191.2
353.0
398.6
236.8
354.1
192.2161.3
355.1 399.7207.0135.3 466.9
85.2 397.3237.8 706.9400.6335.2 436.8 502.0 544.9301.1 578.3 718.1668.1642.7 783.2
MS spectra
Latham X Moy
Two environments, 5 seasons
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0 10 20 30 40 50 60
Time (min)
Abso
rban
ce a
t 2
80
nm
O
OO
OO
O
HO
HO OH
O
HO OH
OH
O
OHHO
HO
O
HO
HO
OH
O
OH
OH
O
O
OO
OO
O
HO
HO OH
O
HO OH
OH
O
OHHO
HO
O
HO
HO
OH
O
O
OH
OH
OH
O
OO
OO
O
HO
OH
OH
O
HO
OH
OH
O
OH
OH
HO
OHO
HO
HOO
O
OO
OO
O
HO
OH
OH
O
HO
OH
OH
O
OH
OH
HO
OHO
HO
HO
O
OH
OHO
OO
OO
O
O
HO
OH
OH
O
HO
OH
OH
O
OH
OH
HO
OHO
HO
HO
O
OOH
OH
OH
O
Inhibitors
The inhibitory components in raspberry are Ellagitannins
OOHO
OH
O
HO
OH
OH
O
HO
HO
HO
O
OH
OHO
OO
OO
O
O
HO
OH
OH
O
HO
OH
OH
O
OH
OH
HO
OHO
HO
HO
O
OOH
OH
OH
O
Tannins bind to amylase and prevent starch digestion
? Full story ?
The Rhubarb story
Stability and Bioavailability
0.E+00
1.E+08
2.E+08
3.E+08
4.E+08
5.E+08
6.E+08
7.E+08
0 12 24 36 48 60 72
Time (hours)
MS
dete
cto
r re
sp
on
se
0.E+00
1.E+07
2.E+07
3.E+07
4.E+07
5.E+07
6.E+07
7.E+07
Sanguiin H6
Ellagic acid
Raspberry ellagitannins inhibit cancer cell growth
Ellagitannins bind to proteins in media
Not taken up by cells!
Breakdown to release ellagic acid
What is the active anti-cancer component?
OH
OHO
O
OHO
HO
O
Ellagic acid
OO
OO
O
HO
OH
OH
O
HO
OH
OH
O
OH
OH
HO
OHO
HO
HO
O
OH
OHO
OO
OO
O
O
HO
OH
OH
O
HO
OH
OH
O
OH
OH
HO
OHO
HO
HO
O
OOH
OH
OH
O
In vitro digestion
Stability not related to
aglycone or sugar moeity
For Pelargonidin
Glc >GRut >Rut >Soph
For Cyanidin
GRut >Soph >Rut >Glc
Stability dependent on
components in mixture
*Total Recovery wrt gastric figures - McDougall et al. (2005) JAFC 53 5896-5904
Raspberry Anthocyanins
40
45
50
55
60
65
70
75
80
85
Pg Glc
CyG
Rut
Cy S
oph
Pg GRut
Pg Rut
Pg Soph
Cy R
ut
Cy G
lc
% R
EC
OV
ER
Y*
O+
OH
HO
OH
OH
Cyanidin-3-O-glucoside
O
OH
H
H
HO
H
H
OHH
O
OH
In vitro digestion
Simulation of human digestive
system
1. Gastric digestion – 2 hrs at
37°C at pH 1.7 with pepsin
2. Pancreatic digestion – 2 hrs
at 37°C with digestive
enzymes and bile salts
Analyse recovery of
components
Which components stable and bioavailable?
Faecal metabolism of berry polyphenols
Profiling of faecal water metabolites in
10 free-living students after intake of
raspberry puree (200 g/d for 14 d) by
gas-chromatography mass spectrometry
(GC-MS)
Substantial ingestion of anthocyanins,
ellagitannins etc
Focus on major phenolic metabolites
Some patterns noted
Faecal metabolism of berry polyphenols
Phenylacetic acid increased in 7/10 subjects
4-hydroxy phenylacetic acid increased in 6/10 subjects
3-hydroxy phenylacetic acid increased in 5/10 subjects
3-Phenylpropionic acid increased in 6/10 subjects
3(4-hydroxy) phenylpropionic acid increased in 5/10 subjects
3, 4-dihydroxy benzoic acid increased in 7/10 subjects
4-hydroxy benzoic acid increased in 2/10 subjects
Bu
t n
ot th
e s
am
e s
ub
jects
• Fits evidence from model studies with faecal inocula
but shows large inter-individual variation
• Due to differences in diet or microflora?
O
OH
HO
O
OH
HO
HO
OH
O
Gill et al, in press
Other areas
Effect of tea and coffee polyphenols on
neurodegeneration and obesity models resp.
Analysis of carotenoids in sea buckthorn & carrot
Anti-parasitic effects of berry and vegetable extracts
Natural products as anti-inflammatory agents
Availability of nitric oxide (NO) in ex vitro rat carotid arteries
Raspberry extract effective at 50-fold less than Vitamin C or Quercetin derivatives – known effectors
of CV performance
Nitric oxide protection by Raspberry extracts
0
1
2
3
4
5
6
7
Vehicle Vit.C
(18)
RE
(90)
RE
(18)
RE
(1.8)
RE
(0.36)
RE
(0.036)
Vehicle QG (50)
QG (10)
QS (60)
QS (12)
QS (1.5)
Sample (mg/ml)
Nit
ric
Ox
ide
Bio
ava
ila
bilit
y
Whitson et al. Functional Plant Science and Biotechnology (2010)
Protection of NO bio-availability maintains blood vessel flexibility
Prof. Carlene Hamilton, BHF, University of Glasgow
O
O
O
O
O
OH
OHHO
O
OHHO
HOO
OHHO
HO
O
HO
HO
HO
O
OH
OH
O
O
O
O
O
O
O
OH
OH
OH
O
OHHO
HO
O
HO
HO
HO
O
O
Sanguiin H6
OH
OHHO
O
OHHO
HO
O
O
O
O
O
O
O
OH
OHHO
O
OHHO
HO
O
OHHO
HO
O
HO
HO
HO
O
OH
OH
O
O
O
O
O
O
O
OH
OH
OH
O
OHHO
HO
O
HO
HO
HO
O
O
OH
OHHO
O
OHHO
HO
O
O
O
O
O
O
O
OH
OHHO
O
OHHO
HOO
OHHO
HO
O
HO
HO
HO
O
OH
OH
O
O
Lambertianin C
O
O
O
O
O
O
OHHO
O
OHHO
HO
O
OHHO
HO
O
HO
HO
HO
O
O
OH
O
O
O
O
O
OH
OH
HO
OH
OHHO
O
OHHO
HO
O
O
OH
OH
OH
O
OH
OH
OH
O
Nobotanin A
O
O
HO
OH
O
OHHO
HO
O
HO
HO
HO
O
OH
OH
O
O
O
O
O
O
O
OH
OH
OH
O
OHHO
HO
O
HO
HO
HO
O
O
OH
OHHO
O
OHHO
HO
O
Sanguiin H10
OH
OHO
O
OHO
HO
O
Ellagic acid
Ross et al, (2007) Phytochemistry 68, 218
Are these components bio-available?
Do they get from berries - gut – site of action?
Ellagitannins – most effective anti-cancer
polyphenols in raspberry