Berry components inhibit digestive enzymes: A source of health benefits? Ashley Boath, Dominic Grussu, Derek Stewart & Gordon J. McDougall The James Hutton Institute [email protected] 1st International Conference on Food Digestion, Cesena, 19th March 2012

Berry research at the James Hutton We breed market-leading varieties • Blackcurrants – the “Ben” series • Raspberries – the “Glen” series • Strawberry and Blueberries • Research into Health Benefits of Berries • Feedback to direct breeding of new varieties

Outline of talk Introduction Berry polyphenols and digestive enzymes • MODEL IN VITRO SYSTEMS • Polyphenol-enriched extracts • Inhibition of enzymes relevant to

• Diabetes & Obesity • Correlate bioactivities with polyphenol composition using LC-MS techniques

 “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 How do FAV alter our diet and improve health

affect health? Minerals (Zinc)? Vitamins (C and E)? Fibre? Displacement? Lower Fat? Phytochemicals? Antioxidants?

Berries contain a diverse and species specific mixture of antioxidants – the two main types are Polyphenols and Vitamin C R1

HO HO

OH

Flavanols/PACs R4

OH OH

O+

R4

O

R2

HO

O H R R2 1

R3

HO

R4

Anthocyanins

Vitamin C

OH

HO

OH

HO

HO

OH

O

O

O

HO R1

O HO

OH HO

HO O

O

O

O

HO OR2 OH O

O O

HO

O

O

O

HO OH

O

Tannins OH OH

O

O

OH O

OH

Flavonols HO

O

O

O O

OH

OH

HO

OH OH

OH OH

HO

OH OH

How can polyphenols affect human health? Antioxidant theory? Low serum bioavailability! Majority of polyphenols remain in gut Are these components inactive? Possible roles Modulating colonic microbiota? In-gut antioxidants? Benefit gut epithelia function / colon cancer Modulate digestive processes

Control of nutrient availability • Polyphenols can inhibit digestive processes and slow or modulate nutrient release from food • Inhibition of lipid digestion – control of hyperlipidemia, CVD, diabetes and obesity • Inhibition of starch digestion – blood glucose control and type 2 diabetes

Lipid digestion and lipase 120

X

% Lipase Activity

100 80

Inhibitory at 50 g/ml

X

Orlistat

60

Fat uptake

40 20 0 Control

BC

ROW

BB

LB

AB

CB

SB

RB

Lipase inhibition 110 100

OH HO

90 OH

80

OH

HO

HO

OH

O

O

HO

HO

O

60

O

O

HO O

HO

50 40

O

OH

O

O

O

O O

OH O O

O

OH

O

O O

HO

30

O

O

OH

O

HO

70 % Control

Inhibition by cloudberry extracts is saturable

OH HO OH

O

OH HO

OH

OH HO

OH OH

Caused by ellagitannins (ETs) in cloudberry, arctic bramble and raspberry and procyanidins and ETs in strawberry

OH OH

20

Mainly procyanidins in lingonberry

10 0 0

10

20

30

Phenols (mg)

40

50

Ties in with animal studies on obesity

McDougall, Kulkarni & Stewart (2009) Food Chemistry 115, 93–199

Inhibition of starch digestion H

HO H

H

HO H

H

O

HO

OH

H

OH

H

H

O

H

OH

H

H

O

HO

OH

H

H

H

O

HO

OH

H

O

H

O H H

OH

H

O HO

HO

H

O

OH

H H

H

OH

H

O

H

H O

H H

H OH

H

O

H

HO

H

OH

H H

OH

O H

O HO

OH

H

H

H

OH H

H

O

HO H

H

O HO

H

H

OH

O H

H

O HO

H

H

OH H

OH OH

H

H

HO

H

Amylase chops into fragments -glucosidase nibbles off glucose

O

HO H

OH OH

HO

H

H

H

OH

H

H

O

O

HO

H HO

OH

O

H

HO H

H

O

H

HO

O

O

H

H

OH

H

HO

H

HO

H

HO

H

HO

H

HO

H

O

H

HO

OH

HO

H

O

OH

H

HO

HO

H

H

O

O

O

HO

HO

H H

H

H

H

O

H

HO

HO

OH

H

HO

H

O

H

H

HO

HO

HO

H

O

H H

H

OH

H

HO

H

H

H

O

H HO

H

H

O

H H

O

H

H

HO

HO

H

O

H

OH

H

H

H

O

HO

H HO

HO

H

O

O

H H

HO

H

O HO

HO

H

HO

H

H

HO HO

HO

H

O

OH OH

-amylase inhibition 120 Red cabbage Strawberry

100

Blackcurrant Blueberry Raspberry

% Inhibition

80

Green tea

60

Strawberry and raspberry most effective

40 20 0 0

100

200

300

-20

Phenols (mg) McDougall et al (2005) JAFC 53, 2760-2766

400

500

OH

Previous work suggested that the inhibitory components in raspberry were ellagitannins

HO OH

HO

HO

OH

O

O

HO O

O

HO

HO

HO

OH

HO

OH HO O HO O

HO

O

O

O

O O

OH

HO OH

OH HO OH

O O O

HO

OH

OH HO OH

OH

OH

OH HO

OH OH

OH

O OH

O O O

HO

O O O O OH HO OH

OH

OH

OH

OH

O

O

HO

O

O

OH

O O

HO O

O

HO

OH

O

HO

O

O

O

OH

HO OH

O

OH HO

OH OH

3.0 Tannins bind to amylase and prevent 2.5 starch digestion? 2.0

Inhibitors

1.5 1.0 0.5 0.0 20

30

40

Time (min)

McDougall et al (2005) JAFC 53, 2760-2766

50

60

O

O

O

O O

O

OH O OH O O

OH

OH

OH OH

10

O

O

O

HO

0

O

HO

O

HO HO

OH

O

O

HO

HO

HO

O

HO

OH

OH

HO

O O

HO

Absorbance at 280 nm

OH O O

O

HO

OH

O

HO HO

O O

OH O

HO

O

O O

O HO

O

O

O

OH O

O

O

OH

HO

O

O

HO

HO

OH

OH

OH

OH HO

OH OH

-amylase inhibition

% Amylase Activity

60 50

All assays at 100 g/mL

40

Berry polyphenols inhibit to different extents

30 20 10

Grussu et al (2010) JAFC 53, 2760-2766

Red wine

Blueberry

Pomegranate

Arctic Bramble

Blackberry

Black currant

Cloudberry

Lingonberry

Strawberry

Raspberry

-10

Rowan

0

Yellow vs. Red Raspberries Re-examine inhibition by raspberry by comparing extracts of red raspberry (Glen Ample) with yellow raspberry (selection 97134B1)

R1 R4 O+

R4

R2 R3

R4

These have similar polyphenol profiles but yellow raspberries effectively lack anthocyanins

-amylase inhibition 110 100

Yellow

% Amylase activity

90

Red

Yellow and red raspberry extracts are equally effective This supports ellagitannins as active ingredients and suggests that anthocyanins are less important

80 70 60 50 40

However, ellagitannins are proportionally increased in yellow raspberry extracts

30 20 10 0 -10 0

10

20

30

Phenol content (mg GAE)

40

50

8. Procyanidins

7. Quercetin coumaroyl hexoses

6. Unknowns

5. Undefined Flavonols

4. Quercetin hexoses

3. Anthocyanins + CGA

2. Mainly chlorogenic acid (CGA)

1. Chlorogenic acids

Rowan fractionation & amylase inhibition Sephadex LH-20 – step elution with decreasing polarity

By LC-MS analysis

Inhibition by procyanidin-rich fraction 110

OH OH

100 HO

O OH

90

OH

HO OH HO

O

80

OH OH

% Amylase activity

HO

IC50 values for whole Rowan extract < 1 g/ml GAE and Rowan PAC fraction 8 = 1 g/ml GAE

70 60 50

OH HO

O

HO OH

Does this confirm that PACs wholly explain amylase inhibition?

40 30 20 10 0 -10

1

2

3

4

5 Fraction

6

7

8

Rowan

Co-incubation with acarbose Co-incubations at ratios of IC50 – rowanberry PACs first 50 OH OH

% Control amylase activity

45 40 35

HO

Rep 1 Rep 2 Rep 3

O OH OH

HO OH HO

O OH OH

HO OH HO

O

30

HO OH

25 20 15 10 5 0 100/100

50/50

75/25

25/75

75/25

25/75

IC50 ratios

Each at IC50

At half IC50

Addition of protein reduces inhibition

% Control Amylase activity

120 100 80 60 40 20 0 Control

Rowan 1 Rowan + Control + BSA 1 BSA 1

Rowan 2 Rowan + Control + BSA 2 BSA 2

Order of addition assays 110

Normal = amylase + inhibitors then starch to start assay

100

% Control activity

90 80

Revised assay = Amylase added to start reaction – reduced inhibition

70 60 50

Supports amylase-binding mechanism

40 30 20 10 0 CON

Rowan1 R

Rowan1

Rowan2 R

Rowan2

Amylase inhibition • Berry polyphenols inhibit amylase activity in vitro at low levels • The degree of inhibition depends on the polyphenol composition • Tannins seem important but inhibition influenced by other components • Proteins interfere – astringency/enzyme binding mechanism? • Polyphenols can potentiate inhibition caused by acarbose & could substitute for acarbose and maintain inhibition

% Activity

-glucosidase inhibition by berries 110 100 90 80 70 60 50 40 30 20 10 0 -10 0 -20

Inhibition by black currant

IC50 = 20 g/ml

10

20

30

40

50

60

70

80

90

100

Phenol content (mg/ml)

Boath et al. submitted; & Whitson et al. Funct. Plant Sci. & Biotech. 4, 34-38 (2010)

Inhibition by rowanberry 110 100 90

% Activity

80 70 60 50

IC50 = 30 g/ml

40 30 20 10 0 0

20

40

60

Phenol content (µg/mL)

80

100

Rowanberry proanthocyanidins 120

% Activity

100 80 60

IC50 >100 g/ml

40 20 0 0

20

40

60

Phenol content (mg/ml)

80

100

Polyphenol composition 4 100

3

FSD 6.93E5

7-8

80

2 60

5-6

BC

9

1

10-11 12-13

40 20 0 100

R1

80

4

3

4.83E5

BC

R4 60

O+

R4

Anthocyanins

R2

40

BC is rich in anthocyanins

R3

20

R4

0 100

R6

1.80E6

R7 R2

80

R4/5

R10

Rowan

R9 60

Rowan in chlorogenic acid derivatives O

R8 R1

LC-MS analysis shows that the black currant & rowanberry extracts differ greatly in their polyphenol composition

R3

R11

R12

HO

C

OH

40 O C

20

O

OH OH

0 0

5

10

15 Time (min)

20

25

30

HO

HO

Co-incubation with acarbose

% Activity

100

50

0 C A

40

C A

30

AC

20

C A

10

C B

20

C B

15

BC

10

C B

2

C A / 0

40

C B

2

20

10

C B

C B

C A / 0

Black currant/acarbose (mg/ml)

C A / 15

1

C A / 0

30

Co-incubation with acarbose

% Activity

100

50

0 C A

40

C A

30

C A

20

C A

10

ow R

30 w o R

. 22

5

15

40

20

10

C C C C A A A ow A / / / / R .5 30 30 15 2 2 ow ow ow w R R R o R

Rowanberry/Acarbose (mg/ml)

30

Mixing of berry extracts 110 100 90 70 60 50 40 30 20 10

20 15 /B Ro C w 10 22 .5 /B Ro C w 5 7. 5/ BC 15

7. 5

15

22 .5

30

5

10

C

Ro w

30 /B

Ro w

Ro w

Ro w

Ro w

Ro w

BC

Lack of additive effect suggests components are operating at same site on enzyme?

BC

15 BC

20

0 BC

% Activity

80

Summary – -glucosidase inhibition • Berry polyphenols inhibit glucosidase activity in vitro at low levels • Inhibition depends on polyphenol composition • Tannins are not important and astringency is probably not the main mechanism • Anthocyanin-rich and chlorogenic acid-rich black currant and rowanberry are similarly effective • The active components potentiate effect of acarbose but different berries do not act additively – sites of action?

Human trial – modified glycemic response Volunteers given sucrose-loaded black currant (BC) juice or sucrose-loaded BC juice supplemented with crowberry juice

Plasma glucose (mmol/L)

7.0

6.5

* 6.0

5.5

The supplemented juice (•) caused a reduction in peak height of plasma glucose and extended the area under the curve

5.0

4.5 0

15

30

45

60

Törrönen et al. submitted

90

120

Time (min)

150

180

Human trial – insulin response The insulin responses showed a similar pattern to the glucose response

Plasma insulin (pmol/L)

200

Possible role for inhibition of glucosidase/glucose transport? *

150

100

50

0 0

15

30

45

60

90

120

Time (min)

150

180

Summary • Berry polyphenols inhibit enzymes involved in starch and lipid digestion in vitro • The inhibition occurs at concentrations easily reached in the GIT • The active components are unknown but differ between enzymes and in potential mechanisms (↑ synergy?) • Berry components can potentiate inhibition by acarbose at low levels • Initial human studies show promise

Acknowledgements

*

* Kuopio

Thanks to Nimish Kulkarni, Dominic Grussu & Ashley Boath *Riitta Törrönen, Jarkko Hellström, Pirjo Mattila, Juha-Matti Pihlava & Reijo Karjalainen Nikki Jennings (MRS Ltd) for yellow raspberries; Dr Harri Kokko (Kuopio) for Nordic berries; Pat Dobson (JHI) for technical help

Thank you for your attention

JHI at Invergowrie on the north bank of the River Tay

Questions? Visit http://www.hutton.ac.uk