Berry components inhibit digestive enzymes: A source of health benefits? Ashley Boath, Dominic Grussu, Derek Stewart & Gordon J. McDougall The James utton Institute gordon.mcdougall@hutton.ac.uk 1st International Conference on Food Digestion, Cesena, 19 th March 2012
Berry research at the James utton We breed market-leading varieties Blackcurrants the Ben series Raspberries the Glen series Strawberry and Blueberries Research into ealth Benefits of Berries Feedback to direct breeding of new varieties
utline of talk Introduction Berry polyphenols and digestive enzymes MDEL IN VITR SYSTEMS Polyphenol-enriched extracts Inhibition of enzymes relevant to Diabetes & besity 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 ealth rganisation (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 ow do FAV 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 R 4 + R 1 R 4 R 2 Flavanols/PACs Vitamin C R 4 R 3 Anthocyanins R1 R 2 R 1 R 2 Flavonols Tannins
ow 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 120 100 X % Lipase Activity 80 60 40 rlistat Inhibitory at 50 g/ml Fat uptake X 20 0 Control BC RW BB LB AB CB SB RB
Lipase inhibition % Control 110 100 90 80 70 60 50 40 30 20 10 0 0 10 20 30 40 50 Phenols (mg) Inhibition by cloudberry extracts is saturable Caused by 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 McDougall, Kulkarni & Stewart (2009) Food Chemistry 115, 93 199
Inhibition of starch digestion Amylase chops into fragments -glucosidase nibbles off glucose
-amylase inhibition 120 Red cabbage 100 Strawberry Blackcurrant Blueberry % Inhibition 80 60 40 20 Raspberry Green tea Strawberry and raspberry most effective 0-20 0 100 200 300 400 500 Phenols (mg) McDougall et al (2005) JAFC 53, 2760-2766
Previous work suggested that the inhibitory components in raspberry were ellagitannins Tannins bind 3.0 to amylase and prevent 2.5 Absorbance at 280 nm 2.0 1.5 1.0 0.5 0.0 starch digestion? 0 10 20 30 40 50 60 Time (min) McDougall et al (2005) JAFC 53, 2760-2766 Inhibitors
-amylase inhibition % Amylase Activity 60 50 40 30 20 10 0-10 All assays at 100 g/ml Berry polyphenols inhibit to different extents Rowan Raspberry Strawberry Lingonberry Cloudberry Grussu et al (2010) JAFC 53, 2760-2766 Black currant Blackberry Arctic Bramble Pomegranate Blueberry Red wine
Yellow vs. Red Raspberries Re-examine inhibition by raspberry by comparing extracts of red raspberry (Glen Ample) with yellow raspberry (selection 97134B1) R 4 + R 3 R 1 R 4 R 2 These have similar polyphenol profiles but yellow raspberries effectively lack anthocyanins R 4
-amylase inhibition % Amylase activity 110 100 90 80 70 60 50 40 30 20 10 0-10 Yellow Red 0 10 20 30 40 50 Phenol content (mg GAE) Yellow and red raspberry extracts are equally effective This supports ellagitannins as active ingredients and suggests that anthocyanins are less important owever, ellagitannins are proportionally increased in yellow raspberry extracts
Rowan fractionation & amylase inhibition Sephadex L-20 step elution with decreasing polarity 1. Chlorogenic acids 2. Mainly chlorogenic acid (CGA) 3. Anthocyanins + CGA 4. Quercetin hexoses 5. Undefined Flavonols 6. Unknowns 7. Quercetin coumaroyl hexoses 8. Procyanidins By LC-MS analysis
Inhibition by procyanidin-rich fraction 110 100 90 80 % Amylase activity 70 60 50 40 30 IC 50 values for whole Rowan extract < 1 g/ml GAE and Rowan PAC fraction 8 = 1 g/ml GAE Does this confirm that PACs wholly explain amylase inhibition? 20 10 0-10 1 2 3 4 5 6 7 8 Rowan Fraction
Co-incubation with acarbose Co-incubations at ratios of IC 50 rowanberry PACs first 50 % Control amylase activity 45 40 35 30 25 20 15 10 Rep 1 Rep 2 Rep 3 5 0 100/100 50/50 75/25 25/75 IC50 ratios Each at IC 50 At half IC 50 75/25 25/75
Addition of protein reduces inhibition 120 % Control Amylase activity 100 80 60 40 20 0 Control Rowan 1 Rowan + BSA 1 Control + BSA 1 Rowan 2 Rowan + BSA 2 Control + BSA 2
rder of addition assays % Control activity 110 100 90 80 70 60 50 40 30 20 10 Normal = amylase + inhibitors then starch to start assay Revised assay = Amylase added to start reaction reduced inhibition Supports amylase-binding mechanism 0 CN 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
-glucosidase inhibition by berries % Activity 110 100 90 80 70 60 50 40 30 20 10 0-10 -20 Inhibition by black currant IC 50 = 20 g/ml 0 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 % Activity 110 100 90 80 70 60 50 40 30 20 10 0 0 20 40 60 80 100 Phenol content (µg/ml) IC 50 = 30 g/ml
Rowanberry proanthocyanidins 120 100 % Activity 80 60 40 20 0 IC 50 >100 g/ml 0 20 40 60 80 100 Phenol content (mg/ml)
Polyphenol composition 100 FSD 6.93E5 80 60 40 20 100 0 80 3 2 5-6 9 1 10-11 12-13 4 3 R 1 R 4 60 R + 4 R 2 40 R 3 20 R 4 100 0 R6 R7 80 R2 R4/5 R9 60 R8 R1 R3 40 4 7-8 Anthocyanins R10 R11 R12 BC 4.83E5 BC 1.80E6 Rowan LC-MS analysis shows that the black currant & rowanberry extracts differ greatly in their polyphenol composition BC is rich in anthocyanins Rowan in chlorogenic C acid derivatives 20 C 0 0 5 10 15 20 25 30 Time (min)
Co-incubation with acarbose 100 % Activity 50 0 AC 40 AC 30 AC 20 AC 10 BC 20 BC 15 BC 10 BC 20/AC 40 BC 20/AC 20 BC 15/AC 10 BC 10/AC 30 Black currant/acarbose (mg/ml)
Co-incubation with acarbose 100 % Activity 50 0 AC 40 AC 30 AC 20 AC 10 Row 30 Row 22.5 Row 15 Row 30/AC 40 Row 30/AC 20 Row 22.5/AC 10 Row 15/AC 30 Rowanberry/Acarbose (mg/ml)
Mixing of berry extracts % Activity 110 100 90 80 70 60 50 40 30 20 10 0 BC 20 BC 15 Lack of additive effect suggests components are operating at same site on enzyme? BC 10 BC 5 Row 30 Row 22.5 Row 15 Row 7.5 Row 30/BC 20 Row 15/BC 10 Row 22.5/BC 5 Row 7.5/BC 15
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?
uman trial modified glycemic response 7.0 Volunteers given sucrose-loaded black currant (BC) juice or sucrose-loaded BC juice supplemented with crowberry juice Plasma glucose (mmol/l) 6.5 6.0 5.5 5.0 * The supplemented juice ( ) caused a reduction in peak height of plasma glucose and extended the area under the curve 4.5 0 15 30 45 60 90 120 150 180 Törrönen et al. submitted Time (min)
uman trial insulin response Plasma insulin (pmol/l) 200 150 100 50 The insulin responses showed a similar pattern to the glucose response Possible role for inhibition of glucosidase/glucose transport? * 0 0 15 30 45 60 90 120 150 180 Time (min)
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 ellström, Pirjo Mattila, Juha-Matti Pihlava & Reijo Karjalainen Nikki Jennings (MRS Ltd) for yellow raspberries; Dr arri Kokko (Kuopio) for Nordic berries; Pat Dobson (JI) for technical help
Thank you for your attention JI at Invergowrie on the north bank of the River Tay Questions? Visit http://www.hutton.ac.uk