Biodiversity in Rubus polyphenol content and composition

MethodsSpecific raspberry crosses were constructed from a mix of advanced breeding lines and well known industry leading varieties by Graminor. Father - N 91-29-24 and N 91-42-3; Mother – RU974 07002, Glen Ample and Tulameen. The fruit (including the mother parents and other comparators) were harvest at maturity, frozen and shipped to the JHI for analysis. Optimised polyphenol extracts were prepared by extracting the thawed fruit with 50% acetonitrile/1% formic acid on a w/w basis. The extracts were concentrated, filtered and analysed by Orbitrap LC-FT-MS. Data were processed using on board Xcalibur™ software and quantified using a cocktail of different polyphenol standards.

Results

Sean Conner1, Inger Martinussen2, Dag Røen3, Derek Stewart*1

1Enhancing Crop Productivity and Utilisation, The James Hutton Institute, Invergowrie, DD2 5DA, Dundee, Scotland, UK.2Bioforsk Nord, Tromsø, Norway 3Graminor AS - Njøs, Leikanger, Norway

*Derek.Stewart@hutton.ac.uk

AcknowledgementsWe acknowledge funding from the following:• EU Interreg IVB (ClimaFruit - www.climafruit.com)• Graminor AS (www.graminor.no) • Bioforsk (www.bioforsk.no) • The Scottish Government (www.scotland.gov.uk).

IntroductionThere is an accruing body of evidence to support the health benefits of soft fruit consumption. Although some of these are derived from the constituent vitamins, fibre and mineral intakes the more novel and delicate influence appear to rest with the polyphenol classes and individual components. Model and in vivo studies by our group (1-4) have shown these components to impact beneficial on cancer, cardiovascular disease and obesity. Consequently polyphenols have become traits to be targeted for in breeding programmes and subsequently the search is on for germplasm with diverse polyphenol content and composition. As part of an ongoing study into Rubus diversity we have taken an LC-MS metabolomic approach and mined an extensive germplasm collection hosted between the collaborators

References1. Grussu et al., (2011). Berry polyphenols inhibit alpha-amylase in

vitro: identifying active components in rowanberry and raspberry (Review). J. Agric. Food Chem. (In Press).

2. Brown et al (2010). Anti-proliferative effects of berry components in models of colon cancer (Review). Current Pharmaceutical Biotechnology (In Press).

3. Gill et al (2010), Profiling of phenols in human faecal water after raspberry supplementation. J. Agric. Food Chem. 58, 10389-10395.

4. Whitson et al., (2010). Bioactive berry components: Potential modulators of health benefits. Func. Plant Sci. Biotech. 4, 34-38.

Conclusions

• Even in this limited set of crosses polyphenol diversity is significant.

• C-Soph, C-R and C-G-R are the dominant anthocyanins but their relative proportions vary considerably.

• N 91-29-24 (M) x RU974 07002 (F) yielded the best mean anthocyanin contents (0.454 mg/ml). However some of the progeny (*) were completely deficient in C-G-R.

• Sanguiin H6 and Lambertianin C were the dominant ellagitannins and the relative proportions of these varied significantly both within and across the crosses.

• All crosses with N91-42-3 as the father exhibited greater comparative (to N 91-29-24) mean ellagitannin levels.

• An as yet uncharacterized and minor ellagitannin was quantified (ellagic acid equivalents).

• All individual polyphenols were characterized and quantified for each line using a metabolomic approach . This yielded a significant reduction resource and man-time

requirements and delivered highly detailed data that can very quickly be translated through to breeding programmes.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Pel-GC-GPel-G-RC-RPel-RC-SophC-G-R

Mean=0.454 Mean=0.337 Mean=0.260

*

****

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8 Pel-G

C-G

Pel-G-R

C-R

Pel-R

C-Soph

C-G-R

Mean=0.303 Mean=0.330 Mean=0.273

RU974 07002

RU974 07002

RU974 07002

RU974 07002

RU974 07002

RU974 07002

Glen Ample

Glen Ample

Glen Ample

Glen Ample

Glen Ample

Glen Ample

Glen Ample

Glen Ample

Glen Ample

Tulameen

Tulameen

Tulameen

Tulameen

Tulameen

Tulameen

Tulameen

Tulameen

Tulameen

Tulameen

Mother Tulameen

Mother Glen Ample

0.00

0.05

0.10

0.15

0.20

0.25sanguiin H6

lambertianin C

sanguiin H10

uk_783

Mean=0.085 Mean=0.078 Mean=0.088

RU974 07002

RU974 07002

RU974 07002

RU974 07002

RU974 07002

RU974 07002

RU974 07002

Glen Ample

Glen Ample

Glen Ample

Glen Ample

Glen Ample

Glen Ample

Glen Ample

Glen Ample

Trulameen

Trulameen

Trulameen

Trulameen

Trulameen

Trulameen

Trulameen

TrulameenVeten

Mother RU974 07002

Mother Glen Ample

0.00

0.05

0.10

0.15

0.20

0.25

sanguiin H6

lambertianin C

sanguiin H10

uk_783

Mean=0.106 Mean=0.091 Mean=0.094

O+

ROH

HO

HOH

HO+

ROH

HO

HOH

OH

GlucoseG

RutinoseR

SophoroseSoph

Glucose-RutinoseG-R

Cyanidin (C) C-G C-R C-Soph C-G-R

Pelargonidin (Pel) Pel-G Pel-R - Pel-G-R

Cyanidin Pelargonidin

Anthocyanin levels (mg/ml) in raspberry crosses

Mother (and comparators)Mother (and comparators)

Father – N 91-29-24 Father – N 91-42-3

Father – N 91-29-24 Father – N 91-42-3Ellagitannin levels (mg/ml) in raspberry crosses

O

OO

O O

O

HO

HO OH

O

HO OH

OH

O

OHHO

HO

OHO

HOOH

O

OH

OH

O

O

OO

O O

O

HO

HO OH

O

HO OH

OH

O

OHHO

HO

OHO

HOOH

O

O

OHOH

OHO

OO

OO

O

HOOH

OH

O

HOOH

OH

O

OHOH

HO

OHO

HO

HOO

O

OO

OO

O

HOOH

OH

O

HOOH

OH

O

OHOH

HO

OHO

HO

HOO

OH

OHO

OOO

OO

O

HOOH

OH

O

HOOH

OH

O

OHOH

HO

OHO

HO

HOO

O OH

OH

OHO

OOHO

OH

O

HOOH

OH

O

HO

HO

HOO

OH

OHO

OO

OO

O

O

HOOH

OH

O

HOOH

OH

O

OHOH

HO

OHO

HO

HOO

O OH

OH

OHO

Lambertianin C Sanguiin H6 Sanguiin H10

Unknown Ellagitannin

Unknown EllagitanninUnknown Ellagitannin