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 .
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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
roberto.consonni@ismac.cnr.it
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
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
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