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 7,04-28,18 T NMR Tomography 0,2 4,0 T Low resolution NMR Minispec/TD NMR 0,20-0,47 T Mouse NMR / Unilateral NMR
Nuclear Magnetic Resonance NMR properties of the most commonly used nuclei Nuclei Spin (m) Resonance frequency at 23,49 T, in MHz Natural abundance (%) 1 H 1/2 1000.13 99.98 2 D 1 153.53 0.012 13 C 1/2 251.48 1.07 14 N 1 72.27 99.64 15 N 1/2 101.38 0.36 19 F 1/2 941.06 100.0 23 Na 3/2 264.55 100.0 31 P 1/2 404.86 100.0 35 Cl 3/2 97.99 75.76 39 K 3/2 46.67 93.25 Nuclei with I>1/2 show a non symmetric charge distribution leading to a quadruple electric moment
W Nuclear Magnetic Resonance Multidimensional & Multinuclear experiments 13 C 1 H 1 H HSQC-TOCSY H 1 G+gC H 10/10 1 H 13 C Crocine 8,8 C=O 1 H TOCSY 1 H HSQC/HMBC
Nuclear Magnetic Resonance Bucketing fixed bucketing intelligent bucketing
W.W.W. Nuclear Magnetic Resonance Why NMR
W WHY Nuclear Magnetic Resonance? Non invasive technique (sample is not destroyed) Small sample quantity (few L or mg) No sample derivatization High stability and reproducibility Simultaneous detection of different classes of chem. cmpds Qualitative and quantitative determination Direct interlaboratory comparison of metadata Chemometrics
W.W.W. Nuclear Magnetic Resonance When NMR
When Nuclear Magnetic Resonance 1. Structural characterization 2. Micro-structural characterization Sudan IV
W 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
Metabolomics: applications The non-targeted identification and quantification of all metabolites within an organism or a system under a given set of conditions. Food Quality Geographical characterization Fraud identification Plants System Biology Metabolic changes Humans System Biology Integrated personal omics Species identification Discovery of new bioactive compounds Phenotyping Ageing (maturation, storage etc)
European cooperation in science and technology www.cost.eu/fa COST is supported by the EU Framework Programme Horizon 2020 Food and Agricolture COST Action n o FA1101 Omics Technologies for Crop Improvement, Traceability, Determination of Authenticity, Adulteration and Origin in Saffron (SAFFRONOMICS) 2011-2015
Tepals Crocus sativus L. Family: Iridacee Gender: Crocus Specie: C.sativus Stamens Stigmas 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)
Saffron: 1H NMR aliphatic region P9 7 8 5 P7,8 DMSO C19,19 C20,20 3 Picrocrocin 9 G + g gh1 - P GH1 C/ GH1 FA P4 FA P3 FA P3 FA P5 FA P5 FA DMSO extracts
Saffron: 1H NMR aromatic region P10 GH1,gH1 - C Crocins C12,12 C10,10 C15,15 C11,11 C14,14 Kaempferol FA GH1,gH1 K2,6 K3,5 K8 K6
H 10/10 HSQC/HMBC H 1 G+g Crocins 8,8 C=O
HSQC/HMBC H 1 g H 4 4 C 4 Pycrocrocin 1 4
State of the art Stigmas Stamens Tepals About 300 articles (2017-2013) Pharmacological applications Source of anti-oxidant molecules WASTE
Stigmas Effects of safranal on CNS
New prospectives: NMR metabolomics Stigmas Stamens Tepals New pharmacological applications AMD age related macular degeneration
New prospectives Stigmas Stamens Tepals Nutritional properties New functional foods
New prospectives: NMR metabolomics Stigmas Stamens Tepals Quality assessment 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
New prospectives: NMR metabolomics Stigmas Stamens Tepals Saffron Quality Identification of bio-adulterant
New prospectives: NMR metabolomics Stigmas Stamens Tepals Pharmacological applications Phytodrugs Nutritional properties New functional foods Hepatoprotective Anti-inflammatory Anti-hyperglycemic Vascular protective
Italian PDO Saffron Saffron of S. Gimignano (Reg. CE n 205 04/02/05 e GUCE L 33 05/03/05) Saffron of L Aquila (Reg. CE n 205 04/02/05 e GUCE L 33 05/03/05) Saffron of Sardinia (Reg. CE n 98 02/02/09 e GUCE L 33 03/02/09)
PDO vs Commercials OPLS-DA 4 mg 600 ul DMSO R 2 X=98.4% R 2 Y=98.8% Q 2 =84.6% 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 OPLS-DA Line plot picrocrocin picrocrocin b glucose/ b Gentiobiose bound to crocetin crocins b Glucose moiety of picrocrocin saccharides Fatty acids 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.
Saffron and BIO-Adulterants 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 Adulteration 20% (w/w) Turmeric Stamens Safflower Gardenia
Saffron and BIO-Adulterants OPLS-DA 50 samples Pc1p=14.2% Pc1o=29.5% R 2 X=82.4% R 2 Y=94.5% Q 2 =92.3% Petrakis, E.A., Cagliani L.R., Polissiou, M.G., Consonni, R. Evaluation of saffron (Crocus sativus L.) adulteration with plant adulterants by 1 H NMR metabolite fingerprinting, Food Chemistry, 2015, 173, 890-896.
Saffron and BIO-Adulterants O2PLS-DA 40 samples Pc1p=31.7% Pc2p=18.4% Pc3p=6.8% R 2 X=95.2% R 2 Y=97.6% Q 2 =96% Petrakis, E.A., Cagliani L.R., Polissiou, M.G., Consonni, R. Evaluation of saffron (Crocus sativus L.) adulteration with plant adulterants by 1 H NMR metabolite fingerprinting, Food Chemistry, 2015, 173, 890-896.
Saffron and Shelf life 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 Storage PCA Group A 4 mg 600 ul DMSO Group B Pc1=68.8% Pc2=17.1% R 2 X=92.8% Q 2 =90.4% 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. 1 H NMR-based metabolomics of saffron reveals markers for its quality deterioration, Food Research International, 2015, 70, 1-6.
Saffron and Storage PCA Group A 4 mg 600 ul DMSO Group B Pc1=68.8% Pc2=17.1% R 2 X=92.8% Q 2 =90.4% Ordoudi S.A., Cagliani L.R., Lalou S., Naziri E., Tsimidou M.Z., Consonni R. 1 H NMR-based metabolomics of saffron reveals markers for its quality deterioration, Food Research International, 2015, 70, 1-6.
Saffron and Storage OPLS-DA 1Pc par =63.2% 1Pc ort=19.8% R 2 X=88.2% R 2 Y=94.6% Q 2 =93.7% 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. 1 H NMR-based metabolomics of saffron reveals markers for its quality deterioration, Food Research International, 2015, 70, 1-6.
Saffron and Storage OPLS-DA / S-Plot Group B (5-14 years) Fatty Acids Group A (0-4 years) b Glucose moiety of picrocrocin a Gentiobiose + a Glucose free Crocins Picrocrocin b Gentiobiose + b Glucose bound to crocetin Ordoudi S.A., Cagliani L.R., Lalou S., Naziri E., Tsimidou M.Z., Consonni R. 1 H NMR-based metabolomics of saffron reveals markers for its quality deterioration, Food Research International, 2015, 70, 1-6.
Saffron and Sudan dyes Sudan I Sudan III Sudan II Sudan IV
Saffron and SUDAN III Referring to internal solvent =100 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 Dott.ssa Laura Ruth Cagliani Dott. ssa Erica Cusano M. G. Polissiou, E. A. Petrakis, K. Astraka Agricultural University of Athens M. Z. Tsimidou, S. A. Ordoudi, E. Naziri, S. Lalou Aristotle University of Thessaloniki Fondazione Antonio De Marco S. Bisti, M. Maggi