NMR based plant metabolomics: saffron as a source of ... · NMR based plant metabolomics: saffron as a source of bioactive compounds Roberto Consonni . National Research Council .

NMR based plant metabolomics: saffron as a source of bioactive

compounds

Roberto Consonni

National Research Council

Institute for Macromolecular Studies, ISMAC

Lab. NMR,

v. Corti, 12,

20133, Milano

[email protected]

W.W.W. Nuclear Magnetic Resonance

What is NMR

Why NMR

When NMR

High resolution NMR

NMR Tomography

Low resolution NMR

0,20- 0,47 T

Minispec/TD NMR

7,04- 28,18 T

0,2 – 4,0 T

Mouse NMR / Unilateral NMR

93.25 46.67 3/2 39 K

75.76 97.99 3/2 35 Cl

100.0 404.86 1/2 31 P

100.0 264.55 3/2 23 Na

100.0 941.06 1/2 19 F

0.36 101.38 1/2 15 N

99.64 72.27 1 14 N

1.07 251.48 1/2 13 C

0.012 153.53 1 2 D

99.98 1000.13 1/2 1 H

Natural abundance (%)

Resonance frequency

at 23,49 T, in MHz

Spin

(m) Nuclei

Nuclei with I>1/2 show a non symmetric charge distribution leading to a quadruple electric moment

Nuclear Magnetic Resonance NMR properties of the most commonly used nuclei

8,8’ C=O

H10/10’

H1 G+gC

Crocine

HSQC/HMBC

Multidimensional & Multinuclear experiments

1H

1H 1H

13C

TOCSY

HSQC-TOCSY

1H

13C

1H

Nuclear Magnetic Resonance W

intelligent bucketing

Bucketing

fixed bucketing

Nuclear Magnetic Resonance

W.W.W. Nuclear Magnetic Resonance

Why NMR

Qualitative and quantitative determination

Simultaneous detection of different classes of chem. cmpds

Non invasive technique (sample is not destroyed)

No sample derivatization

High stability and reproducibility

Small sample quantity (few L or mg)

WHY Nuclear Magnetic Resonance?

Direct interlaboratory comparison of metadata

Chemometrics

W

W.W.W. Nuclear Magnetic Resonance

When NMR

When Nuclear Magnetic Resonance

1. Structural characterization 2. Micro-structural characterization

Sudan IV

Nuclear Magnetic Resonance

3. Compositional characterization of mixtures: Metabolomics

non-targeted identification and quantification of all metabolites within an organism or system under a given set of conditions

Food Plants Biofluids/tissues

W

Metabolomics: applications

Geographical characterization

Fraud identification

Food

Species identification

Ageing (maturation, storage etc)

Plants Humans

System Biology System Biology

Metabolic changes

Discovery of new bioactive compounds

Integrated personal omics

Phenotyping

The non-targeted identification and quantification of all metabolites within an organism or a system under a given set of conditions.

Quality

European cooperation in science and technology

www.cost.eu/fa

Omics Technologies for Crop Improvement, Traceability,

Determination of Authenticity, Adulteration and

Origin in Saffron (SAFFRONOMICS)

Food and Agricolture COST Action no FA1101

2011-2015

COST is supported by the EU Framework Programme Horizon 2020

Crocus sativus L.

Stigmas

Stamens

Tepals Family: Iridacee

Gender: Crocus

Specie: C.sativus

400 Tons in 2016 1 Kg=250.000 flowers 2000-1000$/Kg 1120M$

Saffron: stigmas

Obtained from dried stigma of Crocus Sativa L.

Mainly produced (90%) in Asia (Iran) and in other countries within the

Mediterranean area.

In Italy the larger production area were located in the south part of Italy and in

Sardinia, with a limited production in Tuscany and Umbria.

PDO:

1. Saffron from Aquila - Abruzzo (Reg. CE n. 205 of 4.2.2005)

2. Saffron from San Gimignano - Toscana (Reg. CE n. 205 of 4.2.2005)

3. Saffron from Sardegna (Reg. CE n. 98 of 2.2.2009)

COLOR: crocin (glycosyl diesters of gentiobiose/glucose/neapolitanose)

crocetin (carotenoids)

BITTERNESS TASTE: picro-crocin (glycosyl-safranal)

AROMA: safranal (monoterpenic bis-unsaturated aldehyde )

Quality assessment: state of the art

ISO 3632-2 (2010) and

ISO 3632-1 (2011)

procedures:

Definition of three

commercial categories ( I, II

and III) according to

quantification of Crocines,

Picrocrocin e Safranal by

UV-vis measuring the

assorbance at 440, 257 e 330

nm respectively in water

solutions

Thin layer

Chromatography to highlight

the presence of synthetic

dyes (tartrazine E 102)

Picrocrocin

7

3

5

8

9

P5’

P5 P3’ P3

P7,8

P9

gH1 - P FA

FA

FA FA

FA P4

GH1 –C/ GH1

DMSO

FA

C19,19’ C20,20’

G + g

Saffron: 1H NMR aliphatic region

DMSO extracts

C10,10’ C15,15’

C12,12’

C11,11’

C14,14’

FA

K2’,6’ K3’,5’

K8 K6

GH1,gH1 - C

Crocins

Kaempferol

GH1,gH1

P10

Saffron: 1H NMR aromatic region

HSQC/HMBC

8,8’ C=O

H10/10’

H1 G+g

Crocins

HSQC/HMBC H1 g

H4

4 1

Pycrocrocin

C4 4

State of the art

Source of anti-oxidant molecules

Pharmacological applications

Stigmas Stamens Tepals

About 300 articles (2017-2013) WASTE

Effects of safranal on CNS Stigmas

AMD age related macular degeneration

New pharmacological applications

Stigmas Stamens Tepals

New prospectives: NMR metabolomics

Nutritional properties

Stigmas Stamens Tepals

New functional foods

New prospectives

New prospectives: NMR metabolomics

Quality assessment

Stigmas Stamens Tepals

Differentiation between Italian PDO saffron and commercial products. Identification of bio-adulterants Identification of synthetic dyes such as Sudan I-IV Definition of the storage period during which saffron can be yet

considered as fresh

Saffron Quality

Stigmas Stamens Tepals

Identification of bio-adulterant

New prospectives: NMR metabolomics

Pharmacological applications

Stigmas Stamens Tepals

Phytodrugs

New prospectives: NMR metabolomics

Nutritional properties

New functional foods

Hepatoprotective Anti-inflammatory Anti-hyperglycemic Vascular protective

Italian PDO Saffron

Saffron of Sardinia (Reg. CE n°98 02/02/09 e GUCE L 33 03/02/09)

Saffron of L’Aquila (Reg. CE n° 205 04/02/05 e GUCE L 33 05/03/05)

Saffron of S. Gimignano (Reg. CE n° 205 04/02/05 e GUCE L 33 05/03/05)

R2X=98.4% R2Y=98.8% Q2=84.6%

PDO vs Commercials OPLS-DA

4 mg 600 uL DMSO

Cagliani, L.R., Culeddu, N., Chessa, M., Consonni, R. “NMR investigations for a quality assessment of Italian PDO saffron (Crocus sativus L.)”, Food Control, 2015, 50, 342-348.

OPLS-DA Line plot

crocins

picrocrocin

picrocrocin

Fatty acids

saccharides

b Glucose moiety of picrocrocin

b glucose/

b Gentiobiose bound to crocetin

Cagliani, L.R., Culeddu, N., Chessa, M., Consonni, R. “NMR investigations for a quality assessment of Italian PDO saffron (Crocus sativus L.)”, Food Control, 2015, 50, 342-348.

PDO vs Commercials

Curcuma longa L. powdered rhizomes (turmeric)

Gardenia Jasminoides Ellis fruits extracs

Crocus sativus stamens

Carthamus tinctorius L. petals (safflower)

Saffron and BIO-Adulterants

Pure safffron

Turmeric

Stamens

Safflower

Gardenia

Adulteration 20% (w/w)

Saffron and BIO-Adulterants

OPLS-DA

Pc1p=14.2% Pc1o=29.5% R2X=82.4% R2Y=94.5% Q2=92.3%

50 samples

Petrakis, E.A., Cagliani L.R., Polissiou, M.G., Consonni, R. “Evaluation of saffron (Crocus sativus L.) adulteration with plant adulterants by 1H NMR metabolite fingerprinting”, Food Chemistry, 2015, 173, 890-896.

Saffron and BIO-Adulterants

O2PLS-DA

Pc1p=31.7% Pc2p=18.4% Pc3p=6.8% R2X=95.2% R2Y=97.6% Q2=96%

40 samples

Petrakis, E.A., Cagliani L.R., Polissiou, M.G., Consonni, R. “Evaluation of saffron (Crocus sativus L.) adulteration with plant adulterants by 1H NMR metabolite fingerprinting”, Food Chemistry, 2015, 173, 890-896.

Saffron and BIO-Adulterants

98 samples of different harvest (1999, 2002, 2003, 2004, 2005, 2006, 2008, 2009, 2010, 2011 e 2012)

51 Greek samples stored for 1 - 14 years

24 Iranian samples stored for 1 - 11 years

21 Spanish samples stored for 0 - 11 years

2 Italian samples stored for 1 year

Saffron and Shelf life

PCA

Pc1=68.8% Pc2=17.1% R2X=92.8% Q2=90.4%

Ordoudi S.A., Cagliani L.R., Lalou S., Naziri E., Tsimidou M.Z., Consonni R. “1H NMR-based metabolomics of saffron reveals markers for its quality deterioration”, Food Research International, 2015, 70, 1-6.

4 mg 600 uL DMSO Group A

Group B

Saffron and Storage

Group A (0-4 years) Group B (5-14 years)

PCA

Ordoudi S.A., Cagliani L.R., Lalou S., Naziri E., Tsimidou M.Z., Consonni R. “1H NMR-based metabolomics of saffron reveals markers for its quality deterioration”, Food Research International, 2015, 70, 1-6.

Pc1=68.8% Pc2=17.1% R2X=92.8% Q2=90.4%

4 mg 600 uL DMSO Group A

Group B

Saffron and Storage

Ordoudi S.A., Cagliani L.R., Lalou S., Naziri E., Tsimidou M.Z., Consonni R. “1H NMR-based metabolomics of saffron reveals markers for its quality deterioration”, Food Research International, 2015, 70, 1-6.

1Pc par =63.2% 1Pc ort=19.8% R2X=88.2% R2Y=94.6% Q2=93.7%

OPLS-DA

Saffron and Storage

Group A (0-4 years) Group B (5-14 years)

Ordoudi S.A., Cagliani L.R., Lalou S., Naziri E., Tsimidou M.Z., Consonni R. “1H NMR-based metabolomics of saffron reveals markers for its quality deterioration”, Food Research International, 2015, 70, 1-6.

OPLS-DA / S-Plot

Fatty Acids

Crocins Picrocrocin

a Gentiobiose + a Glucose free

b Glucose moiety of picrocrocin

b Gentiobiose + b Glucose bound to crocetin

Group B (5-14 years)

Group A (0-4 years)

Saffron and Storage

Sudan I

Sudan IV Sudan II

Sudan III

Saffron and Sudan dyes

Referring to internal solvent =100

Saffron and SUDAN III

Concentration by weight of Sudan III (g/kg) in adulterated saffron (in the range of 0.14-7.1 g/kg) in correlation with integral values

New pharmacological application of saffron

Age-related macular degeneration (AMD) is the most common cause of visual impairment in individuals over the age of 55 years in developed countries. AMD is a degenerative disease of the macula characterized in the early stage by large soft drusen and hyper/hypopigmentation of the retinal pigment epithelium (RPE), and a moderate loss of central vision (age-related maculopathy, following the International Classification, Leibowitz H et al. 1980).

6.4mg/g croc 126mg/g kaemp

Kaempferol, 3-OH-butyrolactone derivatives Alcoholic extraction

Thanks to

Fondazione

Antonio De Marco

Dott.ssa Laura Ruth Cagliani

Dott. ssa Erica Cusano

M. Z. Tsimidou, S. A. Ordoudi, E. Naziri, S. Lalou Aristotle University of Thessaloniki

M. G. Polissiou, E. A. Petrakis, K. Astraka Agricultural University of Athens

S. Bisti, M. Maggi