molecules

Article Phytochemical Differentiation of ( sativus L.) by High Resolution Mass Spectrometry Metabolomic Studies

Evangelos Gikas 1 , Nikolaos Stavros Koulakiotis 2 and Anthony Tsarbopoulos 2,3,*

1 Department of Chemistry, National and Kapodistrian University of Athens, 15771 Athens, Greece; [email protected] 2 GAIA Research Center, Bioanalytical Department, The Goulandris Natural History Museum, 14562 Kifissia, Greece; [email protected] 3 Department of Pharmacology, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece * Correspondence: [email protected]; Tel.: +30-210-7462-702

Abstract: The metabolite profiling of saffron (Crocus sativus L.) from several countries was measured by using ultra-performance liquid chromatography combined with high resolution mass spectrometry (UPLC-HR MS). Multivariate statistical analysis was employed to distinguish among the several samples of C. sativus L. from Greece, Italy, Morocco, , India, and Kashmir. The results of this study showed that the phytochemical content in the samples of C. sativus L. were obviously diverse in the different countries of origin. The metabolomics approach was deemed to be the most suitable in order to evaluate the enormous array of putative metabolites among the saffron samples studied, and was able to provide a comparative phytochemical screening of these samples. Several markers have been identified that aided the differentiation of a group from its counterparts. This  can be important for the selection of the appropriate saffron sample, in view of its health-promoting  effect which occurs through the modulation of various biological and physiological processes. Citation: Gikas, E.; Koulakiotis, N.S.; Tsarbopoulos, A. Phytochemical Keywords: Crocus sativus L. (); saffron; reversed-phase UPLC; metabolomics; HR MS Differentiation of Saffron (Crocus sativus L.) by High Resolution Mass Spectrometry Metabolomic Studies. Molecules 2021, 26, 2180. https:// 1. Introduction doi.org/10.3390/molecules26082180 Crocus sativus L. is a of flowering of the Crocus which grows in the Mediterranean, east Asia and the Irano-Turanian region. Crocus sativus L. is a member Academic Editor: Nikolaos Pitsikas of the Iridaceae family, and is cultivated worldwide due to the use of its dried styles (the uppermost colored part of which is referred to as ), not only as a spice (saffron), Received: 6 March 2021 but also in health management since ancient times [1,2]. Saffron is a perennial spicy herb Accepted: 5 April 2021 Published: 10 April 2021 which is difficult to be cultivated since it demands a special climate and soil conditions. The earliest apparent reference to Crocus sativus L. cultivation goes back to around 2300

Publisher’s Note: MDPI stays neutral BC, and a saffron harvest is shown in a Minoan fresco painting in the Knossos palace of with regard to jurisdictional claims in Minoan Crete dated from 1600–1500 BC [3]. It is also seen in a fresco in Akrotiri on the published maps and institutional affil- Greek island of Thera dated back in 1627 BC [4], which depicts the flowers being picked iations. by young girls and monkeys. The most plausible ancestor of Crocus sativus L. was [5–7], as derived from morphological [5], cytological [8] and molecular analyses [9]. Saffron is the spice derived from the flower of Crocus sativus which is comprised of the three red stigmas included in the flower that are consequently collected and dried Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. under special conditions to produce the final saffron as a spice. Crocus contains more This article is an open access article than 150 volatile aromatic substances that afford its distinctive aroma, and a large number distributed under the terms and of non-volatiles such as carotenoids including , lycopene, as well as various conditions of the Creative Commons α- and β-carotenes, , monoterpenes, aldehydes, flavonoids, anthocyanins, vita- Attribution (CC BY) license (https:// mins (especially riboflavin and thiamine) and amino acids [10]. The four main bioactive creativecommons.org/licenses/by/ constitutes of saffron stigma are crocetin, crocins, picrocrocin and [11]. In previ- 4.0/). ous studies, the volatile compounds of saffron samples have been characterized by gas

Molecules 2021, 26, 2180. https://doi.org/10.3390/molecules26082180 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 2180 2 of 16

chromatography–mass spectrometry (GC–MS) methods [12,13], and have been evaluated as markers of geographic differentiation [14]. In a recent study, the metabolite profiling of three different parts of Crocus sativus L., i.e., tepals, stigmas and stamens, was measured by ultra-performance liquid chromatography (UPLC) coupled to hybrid quadrupole time-of- flight mass spectrometry (QqTOF MS), which provided the diverse chemical characteristics of the parts of the flower [15]. In the last 20 years, there has been an increasing amount of scientific data on saffron extract or its constituent’s biological activity and health-promoting properties, including use as an anticonvulsant [16], anti-inflammatory [17], anti-tumor [18], anti-oxidant [19], antiatherogenic [20] and has shown antidepressant [21] activity, as well as enhancement of learning and memory capacity [22,23]. Therefore, saffron and its ingre- dients can be useful in the treatment of a variety of diseases such as neurodegenerative disorders, blood pressure abnormalities, acute and/or chronic inflammatory disease and coronary artery disease. The main bioactive constituents in saffron are the crocins existing mainly in the stigmas, and they are mono- and bis-esters of crocetin with glucose, gen- tiobiose and/or gentiotriose [24]. The esterification of crocetin with varying number of hydrophilic gentiobiose(s), or any other sugar precursors, renders the carotenoid derivative water soluble, a property that gives crocus its pigmentation properties. The complexity of crocins may result from the incorporation of various sugars connected to crocetin such as glucose, gentiobiose, gentiotriose and neapolitanose, the number carbohydrate units (1–5), the number of glycosylation sites (1,), their linkage to the acid moiety of the carotenoid, or even by the varying number of repeating units in crocetin [25]. Therefore, there is a huge range of crocins, with most of them being found only in trace amounts. To date, the majority of studies performed support the therapeutic potential of crocins in treating aging and age-related neurodegenerative disorders. The major crocin component, trans-crocin-4 (TC4; bis-gentiobiosyl-E-crocetin) possesses numerous pharmacological activities including antihypertensive [26], anxiolytic [27] and neuroprotective [28] activity. In particular, TC4 has shown the highest inhibitory potential towards reducing or even preventing amyloid oligomerization [29], which is considered as one of the main causes of Alzheimer disease (AD) progression [30]. Conversely, the characteristic flavor of saffron is due to picrocrocin, the of the terpene aldehyde safranal, which comprises more than 4% of the dry weight of saffron. Safranal is produced by the oxidative cleavage of the carotenoid zeaxanthine, and accounts for more than 70% of the volatile fraction of the spice. Saffron is likely the most expensive spice, owing to its very limited geographical spread and the difficulty of its collection. The higher content of the analyzed saffron samples in crocin, picrocrocin and safranal indicates the higher value of saffron. We know that plant secondary metabolites are a group of naturally occurring com- pounds biosynthesized by differing biochemical pathways, and their plant content and regulation is strongly amenable to environmental influences as well as to potential herbal predators [31–33]. Moreover, the level and type of secondary metabolites is strongly influ- enced by the geoclimatic characteristics of the cultivation area as well as the preparation procedures and traditions followed in that area. Therefore, there is a necessity to identify the content of bioactive compounds in collected saffron samples and explore whether there is any correlation between different geographical regions and the contents of the bioactive compounds. The aim of the current study is to explore the chemical space of Crocus sativus L. from different geographical regions in order to spot chemotaxonomic differences in the indigenous species. This could potentially aid towards our understanding of the plant’s biochemistry but also the precise evaluation of its cultivation in diverse environments. We employ a metabolomics methodology based on UPLC high resolution mass spectrometry (HR–MS) in order to provide detailed information on the metabolite profiling of several samples of C. sativus L. from Greece and six other countries/areas: Italy, Morocco, Iran, India, Afghanistan and Kashmir. The metabolomics approach was deemed to be the most suitable choice in order to evaluate the enormous array of putative metabolites among the Molecules 2021, 26, x FOR PEER REVIEW 3 of 16

samples of C. sativus L. from Greece and six other countries/areas: Italy, Morocco, Iran, India, Afghanistan and Kashmir. The metabolomics approach was deemed to be the most suitable choice in order to evaluate the enormous array of putative metabolites among the Molecules 2021, 26, 2180 saffron samples studied, and thus provide a comparative phytochemical screening of3 ofthe 16 saffron samples studied.

2.saffron Results samples and Discussion studied, and thus provide a comparative phytochemical screening of the 2.1.saffron UPLC-MS samples Analysis studied. A representative base peak LC–MS chromatogram of the stigma extract is shown in 2. Results and Discussion Figure 1. Several peaks have been annotated in the early eluting part of the chromatogram with2.1. UPLC-MSthe names Analysisof the putative metabolites shown in Table 1. A representative base peak LC–MS chromatogram of the stigma extract is shown in Figure1. Several peaks have been annotated in the early eluting part of the chromatogram with the names of the putative metabolites shown in Table1.

FigureFigure 1. 1. CharacteristicCharacteristic ultra-performance ultra-performance liquidliquid chromatographychromatographycoupled coupled to to high high resolution resolution mass mass spectrometry spectrometry (UPLC-HR (UPLC- HRMS) MS) base base peak peak chromatogram chromatogram of of the the stigma stigma extract extract from from the theC. C. sativus sativusL. L.sample sample fromfrom India.India. This represents represents the the early early elutingeluting part part of of the the chromatogram chromatogram shown shown in in the the inset. inset. Several Several peaks peaks have have been been annotated annotated in in the the early early eluting eluting part part of of the the chromatogramchromatogram with with the the names names of of the the putative putative metabolites metabolites shown shown in in Table Table 1.1. * * X-Pyr: 3,4,5-Trimethoxyphenol-1-O-[ 3,4,5-Trimethoxyphenol-1-O-[ββ-D--D- → apiofuranosyl-(1apiofuranosyl-(1→6)]]-6)]]-ββ-D-glucopyranoside.-D-glucopyranoside.

The mapping of the chemical potential of saffron according to its geographical spread is of significant importance in the effort to understand evolutional pressure exerted on the species, as well as to provide a chemotaxonomic tool towards the distribution of variants around the world (Figure2). The content of the active secondary metabolites has an apparent effect on the quality as well as the medical properties of saffron. Furthermore, the extreme cost of the Crocus sativus L. stigmas, considered as the most valuable spice, mandates their accurate fingerprinting in order to control its quality. In order to capture the chemical space involved, as well as to compare the species in a holistic manner, an HRMS metabolomics approach was taken. Specimens from seven representative regions capturing saffron’s biodiversity around the world, namely Iran, Greece, Italy, Afghanistan, Kashmir, Morocco, and India were analyzed. The pairwise comparison of all possible combinations of the saffron specimen was employed, as this approach effectively highlights the differences between species regardless of their magnitude. In the case of a “total” comparison, the model would be dominated by the largest variance, and therefore would be biased towards the species with the largest pairwise differences. Figure2 depicts the regions of collected specimens of saffron samples. The associated clusters are illustrated and interconnected in different colors, such as those between Greece and Morocco with saffron samples in orange, whereas Molecules 2021, 26, x FOR PEER REVIEW 4 of 16

Molecules 2021, 26, x FOR PEER REVIEW 4 of 16 Table 1. Cumulative table of the A. Multivariate statistics B. Identifying discriminating features and C. Putative metabolite Moleculesannotation 2021, 26, withx FOR the PEER calculated REVIEW protonated molecular ion (MH+) or the adduct ion mass signals shown in parenthesis,4 of 16

whereas the fragment ions are denoted in curly brackets. MoleculesMolecules 2021,2021 26, x, 26 FOR, 2180 PEER REVIEW 4 of 164 of 16 Table 1. Cumulative table of the A. Multivariate statistics B. Identifying discriminating features and C. Putative metabolite + Tableannotation 1. Cumulative with the tablecalculated of the A.protonated Multivariate molecular statistics ion B. Identifying(MH ) or the discriminating adduct ion mass features signals and C.shown Putative in parenthesis, metabolite B. Features (ac- * whereas the fragment ions are denoted in curly brackets. + C. Putative Metabolite (Calculated mass) A. Scoresannotation Plot andwith S-plot the calculated protonated molecularcurate ion (MHMH+_re-) or the adduct ion mass signals shown in parenthesis, {fragment} 1 whereasTable 1. the Cumulative fragment table ions ofare the thosedenoted A. Multivariate between in curly India brackets. statistics andtention Afghan B. Identifying time) specimens discriminating are indicated features by green. and These C. Putative interconnections metabolite annotation with the calculatedhave protonated been revealed molecular in the ion multivariate (MH+) or the statistical adduct analysis ion mass (Figure signals3). shown in parenthesis, B. Features (ac- * whereast_filtering_NEW_2_the the latest.M12fragment (OPLS-DA), ions Greece+Morocco are denoted vs BabySaffron+BabySaffr in curly1 o brackets. + C. Putative Metabolite (Calculated mass) A. Scores Plot and S-plot 2 Scaled proportionally to R2X B.curate Features MH (ac-_re- Table 1. Cumulative table of the A. Multivariate statistics B. Identifying discriminating features{fragment} and C. Putative 1 metabolite * Colored according to classes in M12 C. Putative Metabolite (Calculated mass) A. Scores Plot and S-plot curatetention MH time)+_re- annotation with theMorocco calculated protonated molecular ion (MH ) or the adduct ion mass signals shown1 in parenthesis, 40000 {fragment} B.tention Features time) (ac- 30000whereas the fragment ions are denoted in curly brackets. * 1 t_filtering_NEW_2_the latest.M12 (OPLS-DA),Morocco MoroccoGreece+Morocco vs BabySaffron+BabySaffro C. Putative Metabolite (Calculated mass) 20000 Babysaffronsmall + A. Scores Plot and S-plotMoroccoGreece0708Greece0708 2 curate MH _re- ScaledGreece0708 proportionally to R2X Babysaffronsmall Greece0708MoroccoGreece0708 1 10000 Greece0708 BabysaffronsmallAfganBabysaffronAfgan {fragment} ColoredGreece0708Morocco according to classes in M12Babysaffron Afgan 1 t_filtering_NEW_2_the latest.M12Morocco (OPLS-DA),Greece0708 Greece+Morocco vs BabySaffron+BabySaffro B. Features (Accurate Morocco BabysaffronBabysaffronsmallBabysaffron 1 0 MoroccofromUNI BabysaffronAfgan 2 tention time) A. ScoresMoroccoGreece0708 Plot and S-PlotBabysaffronBabysaffronsmallAfgan + C. Putative Metabolite (Calculated Mass) * {Fragment} Scaled proportionallyMoroccofromUNIMorocco toBabysaffronsmall R2XAfgan Babysaffron GreeceNewGreeceNewMoroccofromUNIMoroccofromUNI BabysaffronsmallBabysaffronBabysaffron MH _Retention Time) -1000040000 GreeceNew Afgan Colored accordingMoroccofromUNIMoroccofromUNIGreeceNewMoroccofromUNI to classes in M12 BabysaffronsmallAfgan GreeceNewGreeceNew AfganBabysaffronsmall -2000030000 307.1003_4.95 MoroccofromUNIGreeceNewGreeceNewMoroccofromUNI EGC: C15H14O7 (307.0818) {263, 139} t_filtering_NEW_2_the latest.M12 (OPLS-DA),GreeceNewMoroccoMorocco Greece+MoroccoMorocco vs BabySaffron+BabySaffr1 o 4000020000 Babysaffronsmall -30000 MoroccoGreece0708Greece0708 2 ScaledGreece0708 proportionally to R2X Babysaffronsmall + Greece0708MoroccoGreece0708 365.1196_3.36 -400003000010000 Greece0708 BabysaffronsmallAfganBabysaffronAfgan 21 26 4 ColoredGreece0708 Moroccoaccording to classes in M12Babysaffron Afgan Methyl crocetin: C H O (MNa = 365.1729) {118]} Morocco Greece0708MoroccoMorocco Babysaffron -30000 -20000 -10000Morocco 0 10000BabysaffronsmallBabysaffronBabysaffronsmallAfgan 20000 200000 Greece0708MoroccofromUNI Babysaffron MoroccoMoroccoGreece0708Greece07081.01628 * t[1] BabysaffronBabysaffronsmallAfgan 369.15_2.95 Greece0708 MoroccofromUNIBabysaffronsmallAfganBabysaffronsmallBabysaffron X[1] = 0.0877 R2Xo[1]Greece0708Morocco Greece0708= 0.126GreeceNewGreeceNew MoroccofromUNI MoroccofromUNI Morocco BabysaffronsmallEllipse:Babysaffron Hotelling'sBabysaffron T2 (95%) Tomentogenin: C21H36O5 (369.2641) {232} 10000-1000040000 GreeceNewGreece0708 BabysaffronsmallAfganBabysaffronAfganAfganBabysaffronsmall Greece0708MoroccoMoroccofromUNIMoroccofromUNIGreeceNewMoroccofromUNI Babysaffron AfganAfgan Morocco GreeceNewGreeceNewGreece0708 AfganBabysaffronsmall Morocco BabysaffronBabysaffronsmallBabysaffron -20000300000 MoroccofromUNI BabysaffronAfgan 383.1298_2.85307.1003_4.95 + MoroccoGreece0708GreeceNewGreeceNewMoroccofromUNI BabysaffronBabysaffronsmallAfgan EGC: C15H14O7 (307.0818) {263, 139} MoroccofromUNIGreeceNewMoroccofromUNIBabysaffronsmallAfgan Babysaffron GreeceNewGreeceNewMoroccofromUNIMoroccofromUNIMoroccoMorocco BabysaffronsmallBabysaffronBabysaffron Astragalin: C21H20O11 (MNa = 472.0982) {287, 145} GreeceNew BabysaffronsmallAfgan -10000-3000020000 MoroccofromUNIMoroccofromUNIGreece0708MoroccofromUNI BabysaffronsmallAfgan MoroccoGreeceNewGreeceNewGreece0708 AfganBabysaffronsmall + Greece0708GreeceNew Babysaffronsmall 472.1734_3.24365.1196_3.36 Greece0708MoroccoGreece0708 307.1003_4.95 -20000-4000010000 GreeceNewMoroccofromUNIGreece0708 BabysaffronsmallAfganBabysaffronAfgan Methyl crocetin:15 C1421H726 O4 (MNa = 365.1729) {118]} Greece+MoroccoGreece0708MoroccoMoroccofromUNI GreeceNewvs India +AfghanBabysaffron Afgan Par EGC: C H O (307.0818) {263, 139} -30000 -20000Morocco -10000GreeceNewGreece0708 0 10000 20000 Morocco BabysaffronBabysaffronsmallBabysaffron -30000 0 MoroccofromUNI BabysaffronAfgan MoroccoGreece07081.01628 * t[1] BabysaffronBabysaffronsmallAfgan 369.15_2.95vs + MoroccofromUNIBabysaffronsmallAfgan Babysaffron 365.1196_3.36 + X[1] = 0.0877 R2Xo[1] = GreeceNew0.126GreeceNewMoroccofromUNI MoroccofromUNI Babysaffronsmall Ellipse:Babysaffron BabysaffronHotelling's T2 (95%) Tomentogenin: C21H36O5 (369.2641) {232} -40000-10000 GreeceNew AfganBabysaffronsmall Methyl crocetin:15 C2221H2 26O4 (MNa4 = 365.1729) {118]} MoroccofromUNIMoroccofromUNIGreeceNewMoroccofromUNI Afgan EDO: C H O (NH : 252.1963) {140} -30000 -20000 -10000GreeceNewGreeceNew 0 10000AfganBabysaffronsmall 20000 252.1061_0.59383.1298_2.85 ect_filtering_NEW_2_the-20000 latest.M12 (OPLS-DA), Greece+Morocco vs BabySaffron+BabySaff 307.1003_4.95369.15_2.95 EGC: C1515H1414O7 (307.0818)+ {263, 139} MoroccofromUNIGreeceNewGreeceNew1.01628MoroccofromUNI * t[1] EGC: 21C 20H 11O (307.0818) {263, 139} X[1] = 0.0877 R2Xo[1] = 0.126GreeceNew Ellipse: Hotelling's T2 (95%) Astragalin:Tomentogenin: C H O C 21(MNaH 36O5 =(369.2641) +472.0982) {{287,232} 145} -30000 367.135_3.42472.1734_3.24365.1196_3.36 Methyl crocetin: C21H26O4 (MNa =+ 365.1729) {118} 0.8 383.1298_2.85365.1196_3.36 + -40000 Methyl crocetin: C21H26O4 (MNa+ = 365.1729) {118]} Greece+Morocco vs India +Afghan Par 369.15_2.95 Astragalin:Tomentogenin: C21H2020O2611 C (MNa214H36 O5 =(369.2641) 472.0982) {232} {287, 145} 0.6 -30000 -20000 -10000 0 10000 20000 369.1508_1.49vs Bornyl ferulate: C H O (MK = 369.1470) {266, 239} 1.01628 * t[1] 472.1734_3.24383.1298_2.85369.15_2.95 + X[1] = 0.0877 R2Xo[1] = 0.126 Ellipse: Hotelling's T2 (95%) Tomentogenin: C21H36O5 (369.2641) {232} 0.4Greece+Morocco vs India +Afghan Par EDO: C15H22O2 (NH 4+: 252.1963) {140} 305.0824_0.71252.1061_0.59472.1734_3.24 Astragalin: C21H20O11 (MNa = 472.0982) {287, 145} ect_filtering_NEW_2_the0.2 latest.M12 (OPLS-DA), Greece+Morocco vs BabySaffron+BabySaff 383.1298_2.85vs + vs. Astragalin: C21H20O11 (MNa + = 472.0982) {287, 145} 0 Greece + Morocco vs. India + Afghan Par EDO: C15H22O2 (NH4 : 252.1963) {140} 167.1057_1.49252.1061_0.59472.1734_3.24367.135_3.42 + ect_filtering_NEW_2_the0.8 latest.M12 (OPLS-DA), Greece+Morocco vs BabySaffron+BabySaff 252.1061_0.59 EDO: C15H22O2 (NH4 : 252.1963)+ {140} -0.2 Greece+Morocco vs India +Afghan Par Bornyl ferulate: C20H26O4 (MK = 369.1470) {266, 239} 0.6 369.1508_1.49367.135_3.42vs -0.4 367.135_3.42 + 0.80.4 369.1508_1.49 BornylEDO: ferulate: C15 CH22HO2O (NH(MK4 +:+ 252.1963)= 369.1470) {266,{140} 239} -0.6 305.0824_0.71 Bornyl ferulate: C2020H2626O44 (MK = 369.1470) {266, 239} ect_filtering_NEW_2_the0.60.2 latest.M12 (OPLS-DA), Greece+Morocco vs BabySaffron+BabySaff 369.1508_1.49252.1061_0.59 -0.8 305.0824_0.71 0.40 -1 167.1057_1.49367.135_3.42 0.8 305.0824_0.71167.1057_1.49 0.2-0.2-0.25 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 + 0.6 p[1] Bornyl ferulate: C20H26O4 (MK = 369.1470) {266, 239} -0.40 R2X[1] = 0.0877 167.1057_1.49369.1508_1.49 0.4 -0.2-0.6 305.0824_0.71 0.2 -0.4-0.8 0 -0.6-1 167.1057_1.49 -0.25 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 -0.8-0.2 CROCOMICS ORBI_correct_filtering_NEW_2_the latest.M17p[1] (OPLS-DA), iran vs Italy Par 6 -0.4 R2X[1] = 0.0877 -1 Scaled proportionally to R2X 7 -0.25 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 -0.6 Colored according to classesp[1] in M17 R2X[1] = 0.0877 -0.8 40000 -1 30000-0.25 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 Iran Iran p[1] 6 CROCOMICS20000 ORBI_correct_filtering_NEW_2_the latest.M17Italy (OPLS-DA), iran vs Italy Par R2X[1] = 0.0877 7 Scaled proportionallyIran to R2XItaly 10000 Italy ColoredIran according to classes in M17Italy 6 CROCOMICS0 ORBI_correct_filtering_NEW_2_theIran latest.M17Italy (OPLS-DA), iran vs Italy Par Italy 7 -10000 ScaledIran proportionally to ItalyR2X 40000 Italy Iran Iran Italy -20000 Colored according to classes in M17 139.1108_7.13 30000 Iran Iran CROCOMICS-30000 ORBI_correct_filtering_NEW_2_theIran latest.M17Italy (OPLS-DA), iran vs Italy Par 6 10 16 2 4000020000 169.1212_4.39 HTCC isomer: C H O (169.1228) -40000 7 Scaled proportionallyIran to R2XItaly Italy 3000010000 + -50000 IranIran Italy 0 Colored accordingIran to classes Italyin M17Italy 369.1508_1.49 20 26 4 20000-50000 -40000 -30000 -20000 -10000Iran 0 10000 20000 30000 40000 Bornyl ferulate: C H O (MK = 369.1470) {266, 239} Italy IranIran 1.00094 * t[1] ItalyItaly 10000-10000 Italy Italy X[1] = 0.30540000 R2Xo[1] = 0.235 Ellipse: Hotelling's T2 (95%) Iran Iran Iran ItalyItaly 203.082_4.75139.1108_7.13 -200000 Iran Italy 30000 Iran Iran Italy 139.1108_7.13 -10000-30000 Iran Iran Italy 10 16 2 20000 Italy Italy 171.1005_3.58 HTCC isomer: C H O (169.1228) Iran Iran Italy 169.1212_4.39 -20000-40000 139.1108_7.13169.1212_4.39 HTCC isomer: C10H16O2 (169.1228) Iran Italy Italy 10000 Iran + + -50000 Iran -30000 Italy Italy 369.1508_1.49369.1508_1.49vs Bornyl ferulate: C2020H26 O4410 (MK16 =2 369.1470) {266, 239} -500000 -40000 -30000 -20000 -10000Iran 0 10000 20000 30000 40000 169.1212_4.39 Bornyl ferulate:HTCC isomer: C H O C (MKH O = (169.1228)369.1470) {266, 239} -40000 Italy -10000 Iran 1.00094 * t[1] Italy 203.082_4.75 X[1] = 0.305 R2Xo[1] = 0.235 Italy Ellipse: Hotelling's T2 (95%) 8 8 4 + -50000 IranIran vsIran Italy par Italy 169.1211_3.66139.1108_7.13203.082_4.75 Vanillic acid: C H O (169.0601) {151, 105} -20000 369.1508_1.49171.1005_3.58 Bornyl ferulate: C20H26O4 (MK = 369.1470) {266, 239} -50000 -40000 -30000 -20000 -10000Iran 0 10000 20000 30000 40000 -30000 1.00094 * t[1] 177.1629_9.84171.1005_3.58 HTCC isomer: C 10H16O2 (169.1228) X[1] = 0.305 R2Xo[1]Iran = 0.235 vs. Italy par Ellipse: Hotelling's T2 (95%) 203.082_4.75169.1212_4.39vs. -40000 CROCOMICS ORBI_correct_filtering_NEW_2_the latest.M17 (OPLS-DA), iran vs Italy Par 169.1211_3.66 Vanillic acid: C8H8O4 (169.0601)+ + {151, 105} -50000 353.1548_3.66171.1005_3.58369.1508_1.49vs BornylPicrocrocin: ferulate: C21H C2620OH426 (MNaO4 (MK = 353.1577) = 369.1470) {185, {266, 151} 239} -50000 -40000 -30000 -20000 -10000 0 10000 20000 30000 40000 177.1629_9.84 1.00094 * t[1] X[1] = 0.305 R2Xo[1] = 0.235 Ellipse: Hotelling's T2 (95%) 167.0115_24.08169.1211_3.66 Vanillic acid: C8H8O4 (169.0601)+ {151, 105} 1 Iran vs Italy par 353.1548_3.66203.082_4.75 Picrocrocin: C H O (MNa = 353.1577) {185, 151} 205.0789_4203.082_5.387.1157_4337.1661_1429.2068_4185.1159_3203.082_4. 139.1108_7 vs 21 26 4 140.0094_0167.1054_3171.1005_3 2 339.1393_4293.0614_0449.1046_5125.985_0.167.0115_0 169.1212_4 155.069_3.329.1573_4279.2309_1441.097_0.266.158_0.345.168_6. 372.1092_5191.5794_6305.0953_8381.1862_5167.1056_2102.0326_0155.1056_3104.1057_0118.085_0.327.1577_6369.1508_1 167.0115_24.08 Thiamine (B1): C12H17N4OS (265.1123) {144, 122} 493.2244_2239.1626_4123.1156_4593.2181_7209.1524_6209.1523_5185.1159_4151.1105_4355.1717_2151.034_0. 265.1216_0.58177.1629_9.84 795.1909_1137.0949_3593.2181_8265.0137_0239.9648_0543.1281_0369.1513_2773.2092_1364.1955_1167.1057_1169.1211_4 171.1005_3.58 8 8 4 0.5 Iran vs Italy223.9873_0153.09_3.2515.208_3. 449.105_1.par110.0074_0 169.1211_3.66 Vanillic acid: C H O (169.0601) 2{151, 105} 369.1503_4510.2517_3455.1126_0371.204_5.265.0142_2274.0906_0153.0314_0367.135_3.111.0192_1247.1319_8155.1056_3 395.2022_5185.1158_1305.095_7.461.1231_0247.1316_7316.1795_1327.1572_5198.5693_2102.033_8. 265.1216_0.58 Thiamine (B1): C H N OS (265.1123)+ {144, 122} CROCOMICS ORBI_correct_filtering_NEW_2_the latest.M17347.1677_3383.0741_0167.1055_1102.0329_9168.0148_2381.0766_0 (OPLS-DA), iran vs Italy Par 12 17 4 393.1858_5611.1568_2241.9631_2723.1912_0123.039_0.229.121_8.515.2074_3469.3131_1414.1577_0399.3058_1151.0339_2239.965_24611.157_4.125.985_24 625.1721_4169.1215_3152.0348_0182.9842_3633.1384_4649.2148_4287.0532_4167.0119_2360.1475_0280.9915_2255.9424_2 353.1548_3.66 Picrocrocin: C21H26O4 (MNa = 353.1577) {185, 151} 285.0793_5169.0443_0111.038_24369.1498_3325.1109_0449.1046_4208.0381_2199.168_16531.2029_2453.1712_1138.5098_2102.5343_2163.1108_8169.9763_2619.4352_1367.135_3.575.4094_1487.3576_1443.332_18171.9919_2 177.1629_9.84vs 532.0842_1123.0391_2532.178_5.644.491_17185.0219_2338.3401_2184.9841_2553.1852_5102.0326_4457.3478_1102.0328_3545.3994_1531.3835_1 0 598.4849_1663.4611_1345.1676_4271.9151_2649.4459_1146.4983_2385.8995_2112.0167_2153.0898_4144.9809_2707.4872_1456.1678_0501.3735_1182.9846_2413.3219_1207.205_0.110.0188_2111.019_24 614.4798_1517.3679_1167.0118_1365.1028_0111.0191_1429.3161_1605.4201_1561.394_17482.4017_1327.1565_5496.4184_1371.1666_21033.3688_139.0166_2214.9161_2773.2087_2213.5742_2211.4774_2 154.9892_2554.4593_1570.454_18167.0117_1526.4282_1473.342_17327.1576_4658.5058_1540.4445_1999.3636_6207.1002_5337.1243_4707.217_0.548.2299_51028.4146_366.1418_2205.5768_3325.1111_1104.0292_2 CROCOMICS ORBI_correct_filtering_NEW_2_the510.4333_1466.4076_1102.0329_3556.4389_1525.1702_4589.4255_1422.3817_1512.4128_1184.0268_2370.1528_2693.4715_1296.9688_2171.0044_2272.0717_3553.1856_5273.9153_2 latest.M17 (OPLS-DA), iran vs Italy Par + 182.9839_2102.0326_4345.1674_5871.3162_4523.2156_3246.0591_0553.1856_5161.0948_5 167.0115_24.08 8 8 4 1 Iran vs327.1577_6600.4649_1391.2827_2527.1701_3527.171_3.683.3206_3 680.3127_3122.9233_0350.1473_3155.0691_1Italy139.9866_2186.9795_2 par 353.1548_3.66169.1211_3.66 Vanillic acid:21 26 C 4H O (169.0601) {151, 105} 167.0115_2 715.3105_2339.1041_2353.2286_9205.6135_9181.0848_3295.2257_9276.1422_0515.2295_3207.1001_5525.6729_4680.8146_3110.5403_2461.1983_4432.1679_0837.3112_6376.2306_3247.0561_0457.167_4.311.1623_6 205.0789_4203.082_5.387.1157_4337.1661_1429.2068_4185.1159_3203.082_4. 139.1108_7 Picrocrocin: C H O (MNa = 353.1577) {185, 151} -0.5 327.1571_5715.3121_2501.1549_1364.1944_2155.1055_4195.1368_9489.1939_3342.1371_0167.1056_3182.5557_3327.1401_2166.0852_0511.1753_3641.1668_3213.1473_9979.3212_1155.0693_2185.1159_2177.1627_8 140.0094_0167.1054_3171.1005_3 2 331.173_3.294.1526_0367.1354_1367.1344_3345.1524_1339.1399_2200.0823_1258.1085_0287.0721_0261.0353_0329.1727_6314.09_0.7522.2155_3511.1749_4355.1345_1271.0796_4458.1571_3207.1001_5 339.1393_4293.0614_0449.1046_5125.985_0.167.0115_0 169.1212_4 252.1061_0317.1579_2496.1993_1339.1401_4234.0956_0183.1005_1302.1581_3329.1579_3707.2487_3633.2481_9620.2085_3369.1497_1148.0591_0139.1104_2353.0821_0497.1956_5355.1705_4161.0949_5317.1364_5 155.069_3.329.1573_4279.2309_1441.097_0.266.158_0.345.168_6. 268.1024_0280.1373_0278.1215_0328.1379_0307.1006_4711.2811_1167.1054_2153.09_1.4327.1572_4370.1533_2284.0972_0183.1005_3138.0535_0510.2509_3289.09_4.9385.1449_1151.1105_3 372.1092_5191.5794_6305.0953_8381.1862_5167.1056_2102.0326_0155.1056_3104.1057_0118.085_0.327.1577_6369.1508_1 Thiamine (B1): C12H17N4OS (265.1123) {144, 122} 353.1548_3155.1057_2279.1583_1345.1676_5348.227_1.348.1994_3185.1163_2282.1166_0513.1908_4633.308_2.357.1861_5303.0456_0307.1003_5 493.2244_2239.1626_4123.1156_4593.2181_7209.1524_6209.1523_5185.1159_4151.1105_4355.1717_2151.034_0. 265.1216_0.58 365.1196_3527.1706_4329.1577_2310.1113_0123.1156_3321.056_0.167.1056_2483.1804_5383.1298_2515.2063_3321.0477_9415.1504_5 795.1909_1137.0949_3593.2181_8265.0137_0239.9648_0543.1281_0369.1513_2773.2092_1364.1955_1167.1057_1169.1211_4 167.0115_24.08177.1629_9.84 0.5 169.1211_3 177.1629_9151.1105_3307.1003_4307.1137_3541.2597_9305.0824_0347.1682_1355.1712_4177.0381_0163.0588_4184.0719_0475.2144_5169.121_3.132.1006_0 223.9873_0153.09_3.2515.208_3.449.105_1.110.0074_0 1 266.1216_0497.1591_4606.229_4.491.2096_4444.1771_4103.0377_0371.1665_1116.0693_0265.0901_0461.199_5.492.2041_4459.183_5.353.1549_4363.0664_0 369.1503_4510.2517_3455.1126_0371.204_5.265.0142_2274.0906_0153.0314_0367.135_3.111.0192_1247.1319_8205.0789_4203.082_5.337.1661_1155.1056_3203.082_4. -1 481.1645_5348.1266_4112.9985_0 395.2022_5185.1158_1305.095_7.461.1231_0247.1316_7316.1795_1327.1572_5198.5693_2102.033_8.387.1157_4167.1054_3429.2068_4185.1159_3171.1005_3 139.1108_7 2 CROCOMICS ORBI_correct_filtering_NEW_2_the latest.M17347.1677_3383.0741_0167.1055_1102.0329_9168.0148_2140.0094_0339.1393_4449.1046_5 381.0766_0(OPLS-DA), iran169.1212_4 vs Italy Par + 393.1858_5611.1568_2241.9631_2723.1912_0123.039_0.229.121_8.515.2074_3469.3131_1414.1577_0399.3058_1151.0339_2239.965_24611.157_4.125.985_24279.2309_1293.0614_0441.097_0.266.158_0.125.985_0.345.168_6.167.0115_0 625.1721_4169.1215_3152.0348_0182.9842_3633.1384_4649.2148_4287.0532_4167.0119_2360.1475_0280.9915_2372.1092_5255.9424_2155.069_3.305.0953_8167.1056_2329.1573_4102.0326_0155.1056_3104.1057_0118.085_0.327.1577_6 21 1226 174 4 285.0793_5169.0443_0111.038_24369.1498_3325.1109_0449.1046_4208.0381_2199.168_16531.2029_2453.1712_1138.5098_2102.5343_2163.1108_8169.9763_2619.4352_1367.135_3.575.4094_1487.3576_1443.332_18171.9919_2593.2181_7209.1524_6209.1523_5185.1159_4191.5794_6381.1862_5151.1105_4151.034_0.369.1508_1 265.1216_0.58353.1548_3.66 ThiaminePicrocrocin: (B1): C CH HO N(MNaOS (265.1123)= 353.1577) {144, {185, 122} 151} 532.0842_1123.0391_2532.178_5.644.491_17185.0219_2338.3401_2184.9841_2553.1852_5102.0326_4457.3478_1102.0328_3545.3994_1531.3835_1493.2244_2239.1626_4123.1156_4265.0137_0239.9648_0543.1281_0773.2092_1355.1717_2364.1955_1167.1057_1169.1211_4 0.50 598.4849_1663.4611_1345.1676_4271.9151_2649.4459_1146.4983_2385.8995_2112.0167_2153.0898_4144.9809_2707.4872_1456.1678_0501.3735_1182.9846_2413.3219_1207.205_0.110.0188_2111.019_24223.9873_0153.09_3.2795.1909_1137.0949_3593.2181_8110.0074_0369.1513_2 614.4798_1517.3679_1167.0118_1365.1028_0111.0191_1429.3161_1605.4201_1561.394_17482.4017_1327.1565_5496.4184_1371.1666_21033.3688_139.0166_2214.9161_2773.2087_2213.5742_2211.4774_2369.1503_4455.1126_0371.204_5.274.0906_0515.208_3.449.105_1. 155.1056_3 -1.5 154.9892_2554.4593_1570.454_18167.0117_1526.4282_1473.342_17327.1576_4658.5058_1540.4445_1999.3636_6207.1002_5337.1243_4707.217_0.548.2299_51028.4146_366.1418_2205.5768_3325.1111_1104.0292_2395.2022_5185.1158_1305.095_7.510.2517_3461.1231_0265.0142_2153.0314_0198.5693_2367.135_3.102.033_8.111.0192_1247.1319_8 510.4333_1466.4076_1102.0329_3556.4389_1525.1702_4589.4255_1422.3817_1512.4128_1184.0268_2370.1528_2693.4715_1296.9688_2171.0044_2272.0717_3553.1856_5273.9153_2167.1055_1247.1316_7316.1795_1102.0329_9327.1572_5168.0148_2 -0.25 -0.2 -0.15 -0.1 -0.05182.9839_2102.0326_4345.1674_5871.3162_4 0523.2156_3246.0591_0553.1856_5161.0948_5393.1858_5123.039_0.347.1677_3229.121_8.414.1577_0383.0741_0 0.05125.985_24381.0766_0 0.1 0.15 0.2 327.1577_6600.4649_1391.2827_2527.171_3.683.3206_3680.3127_3122.9233_0350.1473_3139.9866_2186.9795_2625.1721_4169.1215_3611.1568_2241.9631_2723.1912_0633.1384_4649.2148_4167.0119_2515.2074_3280.9915_2469.3131_1399.3058_1151.0339_2239.965_24255.9424_2611.157_4. 167.0115_24.08 1 167.0115_2 353.2286_9205.6135_9527.1701_3181.0848_3295.2257_9276.1422_0515.2295_3207.1001_5525.6729_4680.8146_3110.5403_2461.1983_4432.1679_0155.0691_1376.2306_3247.0561_0457.167_4.311.1623_6208.0381_2138.5098_2152.0348_0182.9842_3287.0532_4360.1475_0171.9919_2 205.0789_4 327.1571_5715.3121_2715.3105_2501.1549_1364.1944_2339.1041_2155.1055_4195.1368_9342.1371_0p[1]182.5557_3641.1668_3213.1473_9979.3212_1155.0693_2837.3112_6185.1159_2177.1627_8185.0219_2553.1852_5285.0793_5169.0443_0111.038_24102.0326_4369.1498_3325.1109_0449.1046_4199.168_16531.2029_2545.3994_1453.1712_1102.5343_2163.1108_8169.9763_2531.3835_1619.4352_1367.135_3.575.4094_1487.3576_1443.332_18 203.082_5.387.1157_4337.1661_1429.2068_4185.1159_3203.082_4. 139.1108_7 -0.50 294.1526_0345.1524_1200.0823_1261.0353_0329.1727_6489.1939_3314.09_0.7167.1056_3327.1401_2166.0852_0271.0796_4458.1571_3511.1753_3207.1001_5707.4872_1456.1678_0501.3735_1182.9846_2413.3219_1110.0188_2207.205_0.532.0842_1111.019_24123.0391_2532.178_5.644.491_17338.3401_2184.9841_2457.3478_1102.0328_3140.0094_0339.1393_4167.1054_3171.1005_3 169.1212_4 2 331.173_3.R2X[1]367.1354_1339.1401_4367.1344_3234.0956_0183.1005_1 339.1399_2302.1581_3=329.1579_3258.1085_0707.2487_3620.2085_3287.0721_0 522.2155_3511.1749_4355.1345_1161.0949_5317.1364_50.305598.4849_1214.9161_2663.4611_1345.1676_4271.9151_2649.4459_1146.4983_2385.8995_2112.0167_2153.0898_4144.9809_2 293.0614_0449.1046_5441.097_0.125.985_0.167.0115_0 252.1061_0280.1373_0317.1579_2711.2811_1496.1993_1370.1533_2183.1005_3138.0535_0289.09_4.9633.2481_9369.1497_1385.1449_1148.0591_0139.1104_2353.0821_0497.1956_5355.1705_4167.0117_1526.4282_1473.342_17614.4798_1517.3679_1167.0118_1365.1028_0111.0191_1429.3161_1605.4201_1561.394_17337.1243_4482.4017_1327.1565_5496.4184_11028.4146_371.1666_2366.1418_21033.3688_139.0166_2325.1111_1773.2087_2213.5742_2211.4774_2 155.069_3.329.1573_4279.2309_1266.158_0.345.168_6. 268.1024_0278.1215_0279.1583_1328.1379_0348.227_1.307.1006_4282.1166_0167.1054_2153.09_1.4513.1908_4327.1572_4633.308_2.284.0972_0510.2509_3307.1003_5510.4333_1151.1105_3154.9892_2554.4593_1525.1702_4570.454_18327.1576_4658.5058_1272.0717_3540.4445_1999.3636_6207.1002_5553.1856_5707.217_0.548.2299_5205.5768_3104.0292_2372.1092_5191.5794_6305.0953_8381.1862_5167.1056_2102.0326_0155.1056_3104.1057_0118.085_0.327.1577_6369.1508_1 Thiamine (B1): C12H17N4OS (265.1123) {144, 122} 353.1548_3155.1057_2365.1196_3527.1706_4345.1676_5348.1994_3310.1113_0123.1156_3321.056_0.185.1163_2383.1298_2321.0477_9415.1504_5357.1861_5303.0456_0466.4076_1102.0329_3556.4389_1589.4255_1422.3817_1512.4128_1184.0268_2370.1528_2693.4715_1296.9688_2171.0044_2273.9153_2493.2244_2239.1626_4123.1156_4593.2181_7209.1524_6209.1523_5185.1159_4151.1105_4355.1717_2151.034_0. 265.1216_0.58 305.0824_0177.0381_0329.1577_2475.2144_5167.1056_2182.9839_2483.1804_5515.2063_3169.121_3.132.1006_0102.0326_4345.1674_5871.3162_4523.2156_3246.0591_0553.1856_5161.0948_5795.1909_1137.0949_3593.2181_8265.0137_0239.9648_0543.1281_0369.1513_2773.2092_1364.1955_1167.1057_1169.1211_4 0.5 169.1211_3 266.1216_0177.1629_9151.1105_3606.229_4.491.2096_4307.1003_4307.1137_3541.2597_9371.1665_1461.199_5.347.1682_1355.1712_4492.2041_4459.183_5.353.1549_4363.0664_0163.0588_4184.0719_0327.1577_6600.4649_1391.2827_2527.1701_3527.171_3.683.3206_3680.3127_3122.9233_0350.1473_3155.0691_1139.9866_2186.9795_2223.9873_0153.09_3.2110.0074_0 167.0115_2 497.1591_4444.1771_4103.0377_0116.0693_0265.0901_0481.1645_5348.1266_4112.9985_0715.3105_2339.1041_2353.2286_9205.6135_9181.0848_3295.2257_9276.1422_0515.2295_3207.1001_5525.6729_4680.8146_3110.5403_2461.1983_4432.1679_0837.3112_6376.2306_3247.0561_0457.167_4.311.1623_6369.1503_4510.2517_3455.1126_0371.204_5.265.0142_2274.0906_0515.208_3.153.0314_0449.105_1.247.1319_8155.1056_3 -0.5-1 327.1571_5715.3121_2501.1549_1364.1944_2155.1055_4195.1368_9489.1939_3342.1371_0167.1056_3182.5557_3327.1401_2166.0852_0511.1753_3641.1668_3213.1473_9979.3212_1155.0693_2185.1159_2177.1627_8395.2022_5185.1158_1305.095_7.461.1231_0316.1795_1198.5693_2367.135_3.102.033_8.111.0192_1 331.173_3.294.1526_0367.1354_1367.1344_3345.1524_1339.1399_2200.0823_1258.1085_0287.0721_0261.0353_0329.1727_6314.09_0.7522.2155_3511.1749_4355.1345_1271.0796_4458.1571_3207.1001_5 167.1055_1247.1316_7102.0329_9327.1572_5168.0148_2 252.1061_0317.1579_2496.1993_1339.1401_4234.0956_0183.1005_1302.1581_3329.1579_3707.2487_3633.2481_9620.2085_3369.1497_1148.0591_0139.1104_2353.0821_0497.1956_5355.1705_4161.0949_5317.1364_5393.1858_5123.039_0.347.1677_3229.121_8.414.1577_0383.0741_0399.3058_1125.985_24381.0766_0 268.1024_0280.1373_0278.1215_0328.1379_0307.1006_4711.2811_1167.1054_2153.09_1.4327.1572_4370.1533_2284.0972_0183.1005_3138.0535_0510.2509_3289.09_4.9385.1449_1151.1105_3625.1721_4169.1215_3611.1568_2241.9631_2152.0348_0723.1912_0633.1384_4649.2148_4167.0119_2515.2074_3280.9915_2469.3131_1151.0339_2239.965_24255.9424_2611.157_4. 353.1548_3155.1057_2279.1583_1345.1676_5348.227_1.348.1994_3185.1163_2282.1166_0513.1908_4633.308_2.357.1861_5303.0456_0307.1003_5285.0793_5169.0443_0111.038_24208.0381_2453.1712_1138.5098_2163.1108_8182.9842_3367.135_3.287.0532_4360.1475_0443.332_18171.9919_2 365.1196_3527.1706_4329.1577_2310.1113_0123.1156_3321.056_0.167.1056_2483.1804_5383.1298_2515.2063_3321.0477_9415.1504_5 123.0391_2185.0219_2184.9841_2553.1852_5102.0326_4369.1498_3325.1109_0457.3478_1449.1046_4199.168_16102.0328_3531.2029_2545.3994_1102.5343_2169.9763_2531.3835_1619.4352_1575.4094_1487.3576_1 0 169.1211_3 177.1629_9151.1105_3307.1003_4307.1137_3541.2597_9305.0824_0347.1682_1355.1712_4177.0381_0163.0588_4184.0719_0475.2144_5169.121_3.132.1006_0 663.4611_1649.4459_1146.4983_2112.0167_2707.4872_1456.1678_0501.3735_1182.9846_2413.3219_1207.205_0.110.0188_2532.0842_1111.019_24532.178_5.644.491_17338.3401_2 -1.5 266.1216_0497.1591_4606.229_4.491.2096_4444.1771_4103.0377_0371.1665_1116.0693_0265.0901_0461.199_5.492.2041_4459.183_5.353.1549_4363.0664_0 365.1028_0561.394_17482.4017_1327.1565_5496.4184_1371.1666_21033.3688_598.4849_1214.9161_2773.2087_2345.1676_4213.5742_2211.4774_2271.9151_2385.8995_2153.0898_4144.9809_2 -1 481.1645_5348.1266_4112.9985_0167.0117_1526.4282_1473.342_17327.1576_4658.5058_1614.4798_1517.3679_1167.0118_1111.0191_1429.3161_1540.4445_1605.4201_1207.1002_5337.1243_4707.217_0.548.2299_51028.4146_366.1418_2205.5768_3325.1111_1139.0166_2104.0292_2 510.4333_1466.4076_1154.9892_2554.4593_1525.1702_4422.3817_1512.4128_1184.0268_2570.454_18370.1528_2296.9688_2272.0717_3171.0044_2999.3636_6553.1856_5273.9153_2 -0.25 -0.2 -0.15 -0.1 -0.05182.9839_2102.0326_4345.1674_5102.0329_3871.3162_4556.4389_1523.2156_3589.4255_1 0693.4715_1553.1856_5 0.05 0.1 0.15 0.2 600.4649_1527.171_3.680.3127_3122.9233_0350.1473_3246.0591_0139.9866_2186.9795_2161.0948_5 167.0115_2 327.1577_6353.2286_9391.2827_2205.6135_9527.1701_3181.0848_3295.2257_9276.1422_0683.3206_3207.1001_5525.6729_4110.5403_2461.1983_4155.0691_1 327.1571_5715.3105_2501.1549_1364.1944_2339.1041_2342.1371_0182.5557_3515.2295_3680.8146_3641.1668_3p[1]213.1473_9979.3212_1155.0693_2432.1679_0837.3112_6185.1159_2376.2306_3247.0561_0457.167_4.311.1623_6177.1627_8 -0.5 294.1526_0715.3121_2345.1524_1200.0823_1261.0353_0155.1055_4329.1727_6195.1368_9489.1939_3314.09_0.7167.1056_3327.1401_2166.0852_0271.0796_4458.1571_3511.1753_3207.1001_5 331.173_3.R2X[1]367.1354_1339.1401_4367.1344_3234.0956_0183.1005_1339.1399_2 302.1581_3329.1579_3258.1085_0707.2487_3620.2085_3287.0721_0=522.2155_3511.1749_4355.1345_1161.0949_5 317.1364_50.305 -1.5 252.1061_0280.1373_0317.1579_2711.2811_1496.1993_1370.1533_2183.1005_3138.0535_0289.09_4.9633.2481_9369.1497_1385.1449_1148.0591_0139.1104_2353.0821_0497.1956_5355.1705_4 155.1057_2268.1024_0278.1215_0279.1583_1328.1379_0348.227_1.307.1006_4282.1166_0167.1054_2153.09_1.4513.1908_4327.1572_4633.308_2.284.0972_0510.2509_3307.1003_5151.1105_3 -0.25 -0.2 -0.15353.1548_3 -0.1365.1196_3 -0.05527.1706_4345.1676_5348.1994_3329.1577_2310.1113_0123.1156_3321.056_0.185.1163_2167.1056_2383.1298_2515.2063_3321.0477_9415.1504_5357.1861_5303.0456_0 0 0.05 0.1 0.15 0.2 151.1105_3307.1137_3541.2597_9305.0824_0355.1712_4177.0381_0184.0719_0475.2144_5483.1804_5169.121_3.132.1006_0 169.1211_3 266.1216_0177.1629_9606.229_4.491.2096_4307.1003_4103.0377_0371.1665_1265.0901_0461.199_5.347.1682_1492.2041_4459.183_5.353.1549_4363.0664_0163.0588_4 -1 497.1591_4444.1771_4116.0693_0481.1645_5348.1266_4112.9985_0p[1] R2X[1] = 0.305 -1.5 -0.25 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 p[1] R2X[1] = 0.305

Molecules 2021, 26, x FOR PEER REVIEW 5 of 16

MoleculesMolecules 2021, 262021, x, FOR26, 2180 PEER REVIEW 5 of 165 of 16

Molecules 2021, 26, x FOR PEER REVIEW 1 5 of 16 2 30000 Table 1. Cont.

MoroccofromUNI 20000 MoroccofromUNIGreeceNew GreeceNew 1 MoroccofromUNI 1 2 MoroccofromUNI GreeceNewGreeceNew 2 B. Features (Accurate 1000030000 GreeceNewGreeceNew MoroccofromUNI 1 30000 A. Scores PlotGreeceNew and S-Plot Iran + C. Putative Metabolite (Calculated Mass) * {Fragment} MoroccofromUNIMoroccofromUNIGreeceNew MoroccofromUNI Iran MoroccoMoroccofromUNI Iran MH _Retention Time) 20000 MoroccofromUNIGreeceNew MoroccofromUNIGreeceNew 0 MoroccofromUNIMorocco GreeceNew Iran Iran 20000 MoroccofromUNIGreeceNew GreeceNewMorocco MoroccofromUNI Iran MoroccofromUNIMorocco MoroccoGreeceNewGreeceNew Iran Iran 1 MoroccofromUNI GreeceNewGreeceNew Iran 10000 GreeceNewGreeceNewMorocco MoroccofromUNIGreeceNew -1000010000 MoroccoGreeceNewGreeceNew MoroccofromUNI Iran 2 MoroccofromUNIMoroccofromUNIGreeceNew MoroccofromUNIGreece0708GreeceNew Iran Crocusatin A: C9H14O2 (155.1073) {159} 30000 MoroccofromUNIGreeceNew MoroccofromUNI Iran Iran 155.1057_2.77 MoroccofromUNIGreece0708 MoroccoGreece0708 Iran 0 Morocco Greece0708Morocco IranIran Iran -200000 Greece0708Morocco Morocco Iran Iran + Morocco MoroccofromUNIMorocco Iran 20000 MoroccofromUNIGreeceNewMorocco GreeceNewGreece0708Morocco Iran Iran Iran HTCC isomer: C10H 16O2 (MH 169.1228) MoroccofromUNIMorocco IranIran 169.1212_4.39 Morocco Greece0708Morocco Iran Iran -30000-10000 MoroccoMorocco -10000 MoroccofromUNIMoroccoGreece0708 Greece0708GreeceNewGreeceNew Crocusatin A: C9H14O2 (155.1073) {159} GreeceNewGreeceNew Greece0708 155.1057_2.77 9 14 2 10000 Greece0708 MoroccofromUNIGreece0708GreeceNew 155.1057_2.77 Crocusatin A: C H O (155.1073) {159} Greece0708 Greece0708Greece0708 Iran 294.1526_0.79 -40000-20000 MoroccofromUNIMoroccofromUNIGreeceNewGreece0708 Greece0708MoroccofromUNI Iran + -20000 Greece0708 Morocco Iran 10 16 2 + -40000 -30000 -20000MoroccoMorocco -10000Greece0708 0 10000Iran 20000 30000Iran HTCC isomer:15 C 24H 7O (MH 169.1228) 0 Morocco Greece0708Morocco 169.1212_4.39 Y-glucopyranoside:HTCC isomer: C CH10HO16O (317.1600)2 (MH 169.1228) {383, 261, 196} 1.00312Greece0708Morocco * t[1] 317.1579_2.81169.1212_4.39 -30000 Morocco Greece0708 Iran Iran X[1] -30000= 0.117 R2Xo[1]Greece0708 = 0.142 Morocco Ellipse: IranHotelling'sIran T2 (95%) MoroccoGreece0708 2 -10000 Morocco 294.1526_0.79 Greece0708 12 179 414 2 -40000 265.1216_0.58294.1526_0.79155.1057_2.77 ThiamineCrocusatin (B1): C A:H C NH OSO (265.1123)(155.1073) {159}{144, 122} -40000 Greece0708 Greece0708 -40000 -30000 -20000 -10000Greece0708 0 10000 20000 30000 15 24 7 -20000-40000 -30000 -20000Greece0708 -10000 0 10000 20000 30000 Y-glucopyranoside: C15H24O7 (317.1600) {383, 261, 196} 1.00312 * t[1] 317.1579_2.81 Y-glucopyranoside: C H O (317.1600)+ {383, 261, 196} Morocco 317.1579_2.81 10 16 2 X[1] = 0.117 R2Xo[1] = 0.1421.00312 Greece0708 * t[1] Ellipse: Hotelling's T2 (95%) 169.1212_4.39155.1057_2.77vs HTCCCrocusatin isomer: A: C 9HH14OO2 (155.1073)(MH 169.1228) {159} X[1] = 0.117 R2Xo[1] = 0.142 Greece0708 Ellipse: Hotelling's T2 (95%) 2 -30000 2 Greece+MoroccoGreece0708 vs Iran Par Thiamine (B1): C12H17N4OS (265.1123)+ {144, 122} 151.1105_3.67265.1216_0.58294.1526_0.79169.1212_4.39 ThiamineHTCCSafranal: (B1): isomer: C C12H10 CH1710N14HO416OS O(151.1123)2 (265.1123)(MH 169.1228) {123} {144, 122} -40000 294.1526_0.79 -40000 -30000 -20000 -10000 0 10000 20000 30000 vs Y-glucopyranoside:Vanillic acid: CC815HH8O244O (169.0601)7 (317.1600) {151, {383, 105} 261, 196} COMICS ORBI_correct_filtering_NEW_2_theGreece+Morocco 1.00312latest.M21 * t[1] vs (OPLS-DA), Iran Gr eece+MoroccoPar vs Iran 169.1211_3.66317.1579_2.81317.1579_2.81 Y-glucopyranoside: C15H24O7 (317.1600) {383, 261, 196} X[1] = 0.117 Greece+Morocco R2Xo[1] = 0.142 vs Iran Ellipse: ParHotelling's T2 (95%) 151.1105_3.67 Safranal: C10H14O (151.1123)2 {123}2 169.1211_4.86151.1105_3.67265.1216_0.58265.1216_0.58 Hydroxy-ThiamineThiamineβ -cyclocitral(B1): (B1): C C1212HH1717 (HTCC):NN4OS (265.1123)(265.1123) C10H16{144,O 2{144, {169.1228} 122} 122} 0.8 Greece + Morocco vs. Iran Par vs. Vanillic acid: C8H8O4 (169.0601) {151, 105} COMICS ORBI_correct_filtering_NEW_2_the latest.M21 (OPLS-DA), Greece+Morocco vs Iran 169.1211_3.66 VanillicTomentogenin: acid: C HC21OH 36(169.0601)O5 (369.2641) {151, {232} 105} COMICS0.6 ORBI_correct_filtering_NEW_2_the latest.M21 (OPLS-DA), Greece+Morocco vs Iran 169.1211_3.66369.15_2.95vs Greece+Morocco vs Iran Par 151.1105_3.67 Hydroxy-β-cyclocitralSafranal: C10H (HTCC):14O (151.1123) C10H {123}16O2 {169.1228} 0.4 169.1211_4.86 Hydroxy-Safranal:β-cyclocitral C10H (HTCC):14O (151.1123)+ C10H16 {123}O2 {169.1228} 0.8 472.1734_3.24169.1211_4.86151.1105_3.67169.1211_3.66 Astragalin:Vanillic C21 acid:H20O C118H (MNa8O4 (169.0601) = 472.0982) {151, 105} {287, 145} 0.20.8 Tomentogenin: C21H36O5 (369.2641) {232} 0.6 169.1211_4.86369.15_2.95 Hydroxy-Tomentogenin:β-cyclocitral8 C (HTCC):821H436O5 C(369.2641)10H16O2 {169.1228} {232} 0.60 169.1211_3.66369.15_2.95 Vanillic acid: C H O (169.0601) {151, 105} COMICS0.4 ORBI_correct_filtering_NEW_2_the latest.M21 (OPLS-DA), Greece+Morocco vs Iran + 0.4 369.15_2.95 Tomentogenin: C21H36O+5 (369.2641) {232} -0.2 472.1734_3.24 Astragalin: C21H20O11 (MNa = 472.0982)10 16 2 {287, 145} 0.2 472.1734_3.24 Hydroxy-Astragalin:β-cyclocitral C21H20O11 (MNa(HTCC):+ = 472.0982) C H O {287,{169.1228} 145} 0.2 169.1211_4.86 -0.40.8 472.1734_3.24 Astragalin: C21H20O11 (MNa = 472.0982) {287, 145} 0 0 21 36 5 -0.60.6 369.15_2.95 Tomentogenin: C H O (369.2641) {232} -0.2 -0.2 -0.80.4 + -0.4 21 20 11 -0.4 472.1734_3.24 Astragalin: C H O (MNa = 472.0982) {287, 145} 0.2-1 -0.6-0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 -0.6 0 p[1] -0.8 R2X[1] = 0.117 -0.8 -0.2 -1 -1 -0.4-0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 p[1] -0.6 R2X[1] = p[1]0.117 MICS ORBI_correct_filtering_NEW_2_theR2X[1] latest.M24 = 0.117 (OPLS-DA), Greece+Morocco vs Iran 1Pa -0.8 Scaled proportionally to R2X 2

-1 Colored according to classes in M24 MICS ORBI_correct_filtering_NEW_2_the-0.4 -0.3 -0.2 -0.1 latest.M24 0 (OPLS-DA), 0.1 Gree 0.2ce+Morocco 0.3 vs Iran 1Pa p[1]Greece0708 1 MICS ORBI_correct_filtering_NEW_2_the latest.M24 (OPLS-DA), Greece+Morocco vs Iran 2Pa 30000 Scaled proportionallyR2X[1] = 0.117 to R2X 2 Scaled proportionallyGreece0708Greece0708 to R2X Morocco Colored according to classes in M24 20000 Colored according toGreece0708 classes in M24Greece0708 Greece0708 Morocco Greece0708MoroccoGreece0708 10000 Italy Greece0708MoroccoGreece0708 30000 Italy Italy Italy MoroccoMorocco 1 MICS30000 ORBI_correct_filtering_NEW_2_theItaly latest.M24Greece0708Greece0708 (OPLS-DA),Greece0708MoroccoMorocco Greece+Morocco vs Iran Pa 0 Greece0708Morocco Morocco ItalyItaly MoroccofromUNIGreece0708Morocco MoroccofromUNI 2 20000 Scaled proportionallyGreece0708 to R2X Greece0708 20000 Italy MoroccofromUNI 123.1156_3.66 MoroccofromUNIGreeceNewGreece0708GreeceNew Greece0708GreeceNewGreece0708GreeceNew -10000 Italy Morocco GreeceNew Colored accordingGreece0708 toMorocco classes inGreece0708 M24 10000 Italy MoroccofromUNIGreece0708GreeceNewMorocco MoroccoGreece0708 10000 Italy Italy Morocco MoroccoMoroccofromUNIMoroccofromUNIGreece0708 -20000 ItalyItaly Italy MoroccoMorocco 10 14 Italy ItalyItaly GreeceNewMoroccoGreeceNewMoroccoGreece0708Greece0708Morocco 151.1105_3.67 Safranal: C H O (151,1123) {123} 0 Italy MoroccofromUNIMorocco Greece0708GreeceNewMoroccoMoroccofromUNI 300000 ItalyItaly MoroccoMorocco MoroccofromUNI -30000 ItalyItaly MoroccofromUNIMorocco MoroccofromUNI Italy MoroccofromUNIGreece0708Greece0708 MoroccoMoroccofromUNI 123.1156_3.66 -10000 Italy GreeceNewGreeceNew GreeceNewGreeceNewGreeceNewMoroccofromUNI 123.1156_3.66 Italy MoroccofromUNIGreeceNewGreeceNew GreeceNewGreeceNew 169.1211_4.86 Hydroxy-β-cyclocitral (HTCC): C10H16O2 (169.1228) -1000020000 Italy GreeceNewGreece0708 GreeceNewGreece0708 -40000 MoroccofromUNIGreeceNew MoroccofromUNIMoroccofromUNI MoroccofromUNI Greece0708MoroccofromUNI -20000-40000 -30000 -20000 -10000Greece0708GreeceNewMorocco 0 10000MoroccofromUNI 20000 30000 151.1105_3.67 Safranal: C10H14O (151,1123) {123} -2000010000 Italy GreeceNewMorocco MoroccoGreece0708 123.1156_3.66 10 14 Italy 1.00123GreeceNewGreeceNew * t[1] GreeceNew 348.1994_3.66151.1105_3.67 Safranal: C H O (151,1123) {123} MoroccoMorocco GreeceNewMoroccofromUNI X[1] -30000= 0.159 Italy Italy Italy R2Xo[1] = 0.134 Greece0708 MoroccoMoroccofromUNI Ellipse: Hotelling's T2 (95%) -300000 MoroccofromUNIMorocco MoroccofromUNI 151.1105_3.67 Safranal: C10H14O (151,1123)10 {123}16 2 ItalyItaly Morocco 169.1211_4.86 Hydroxy-β-cyclocitral (HTCC): C10H16O2 (169.1228) -40000 Italy MoroccofromUNI 169.1211_4.86123.1156_3.66369.15_2.95 Hydroxy-Tomentogenin:β-cyclocitral C 21(HTCC):H 36O5 (369.2641) C H O {232}(169.1228) -40000 MoroccofromUNIGreeceNewGreeceNew GreeceNewGreeceNew -10000-40000Italy -30000 -20000 -10000 0 10000GreeceNew 20000 30000 169.1211_4.86 Hydroxy-β-cyclocitral (HTCC): C10H16O2 (169.1228) -40000 -30000 -20000 -10000GreeceNew 0 10000 20000 30000 MoroccofromUNI1.00123 * t[1] MoroccofromUNIMoroccofromUNI 348.1994_3.66 -20000 1.00123 * t[1] 348.1994_3.66 10 14 X[1] = 0.159 R2Xo[1] = 0.134GreeceNew GreeceNew Ellipse: Hotelling's T2 (95%) 151.1105_3.67348.1994_3.66vs Safranal: C H O (151,1123) {123} X[1] = 0.159 R2Xo[1] = 0.134 GreeceNew Ellipse: Hotelling's T2 (95%) Greece+Morocco vsMoroccofromUNI Italy Par -30000 369.15_2.95369.15_2.95 Tomentogenin:Tomentogenin: C 21H 36OO5(369.2641) (369.2641) {232} {232} 139.1108_7.13169.1211_4.86369.15_2.95 Hydroxy-Tomentogenin:β-cyclocitral C 2121(HTCC):H36 36O55 (369.2641) C10H16O2 {232}(169.1228) -40000 Greece + Morocco vs. Italy Par vs. -40000 -30000 -20000 -10000 0 10000 20000 30000 vs vs 10 16 2 Greece+Morocco1.00123 * t[1] vs Italy Par 169.1212_4.39348.1994_3.66139.1108_7.13 HTCC isomer: C H O (169.1228) X[1] = 0.159 Greece+Morocco R2Xo[1] = 0.134 vs Italy Ellipse: ParHotelling's T2 (95%) 139.1108_7.13 185.1159_3.69139.1108_7.13169.1212_4.39369.15_2.95 Tomentogenin:HTCC isomer: C21 CH10 H36O16O5 2(369.2641)(169.1228) {232} 185.1159_3.69 169.1212_4.39203.082_4.75vs HTCC isomer: C10 H16O2 (169.1228) Greece+Morocco vs Italy Par 203.082_4.75 171.1005_3.58185.1159_3.69139.1108_7.13171.1005_3.58 203.082_4.75 169.1212_4.39203.082_4.75 HTCC isomer: C10 H16O2 (169.1228) 171.1005_3.58185.1159_3.69 203.082_4.75 171.1005_3.58

Molecules 2021, 26, x FOR PEER REVIEW 6 of 16

MoleculesMolecules 2021, 202126, x, 26FOR, 2180 PEER REVIEW 6 of 166 of 16 Molecules 2021, 26, x FOR PEER REVIEW 6 of 16

Molecules_ correct_filtering_NEW_2_the2021, 26, x FOR latest.M27 PEER (OPLS-DA), REVIEW BabySaffron+B abySaffronSmall+Afgha1 6 of 16 Scaled proportionally to R2X 2 Colored according to classes in M27 Table 1. Cont.

1 _correct_filtering_NEW_2_the20000 latest.M27 (OPLS-DA), BabySaffron+BabySaffronSmall+Afgha Babysaffronsmall 1 _correct_filtering_NEW_2_the Scaledlatest.M27Afgan proportionally (OPLS-DA), to BabySaffron+B R2X abySaffronSmall+Afgha2 15000 2 B. Features (Accurate 1 ScaledAfgan proportionallyBabysaffronsmall to R2X A.Colored Scores according Plot to classes and in S-PlotM27 Iran C. Putative Metabolite (Calculated Mass) * {Fragment} 10000 Babysaffron Afgan + Colored according to classes in M27 MH _Retention Time) Babysaffronsmall Iran 5000 Iran Babysaffron 20000 Afgan BabysaffronAfgan Iran 0 BabysaffronsmallBabysaffronsmall 1 _correct_filtering_NEW_2_the20000 latest.M27Afgan (OPLS-DA), BabySaffron+BIranabySaffronSmall+Afgha + 15000 BabysaffronsmallBabysaffronsmall Iran BabysaffronsmallAfganAfgan AfganBabysaffronsmall 2 252.1061_0.59 EDO: C15H22O2 (NH4 : 252.1963) {140} 15000-5000 BabysaffronsmallBabysaffronScaledBabysaffron proportionallyAfgan to R2X Iran IranIran 10000 BabysaffronsmallAfgan Babysaffronsmall ColoredBabysaffronBabysaffron accordingBabysaffronsmallAfgan to classes in M27 Iran Iran -1000010000 BabysaffronsmallBabysaffron Iran Iran 5000 Babysaffron BabysaffronBabysaffronAfganBabysaffronsmallAfgan IranIran 294.1526_0.79 -150005000 BabysaffronBabysaffron Iran 0 Babysaffron Babysaffronsmall 20000 AfganAfgan BabysaffronAfgan Iran Iran + -200000 Babysaffronsmall BabysaffronsmallAfganBabysaffronsmall Iran 15 22 2 4 -5000 BabysaffronsmallAfganAfgan Afgan Iran 252.1061_0.59 EDO: C H O (NH : 252.1963) {140} 15000 Babysaffron Afgan Iran Iran 280.1373_0.73 + Babysaffronsmall BabysaffronsmallAfgan Iran 15 22 2 4 -25000-5000 BabysaffronsmallBabysaffronsmallBabysaffronAfgan Afgan Iran 252.1061_0.59 EDO: C H O (NH : 252.1963) {140} -10000 Babysaffron Afgan Iran Iran Iran 10000-50000 -40000 -30000 -20000Babysaffron -10000AfganBabysaffronsmallBabysaffron 0 10000 20000 30000 40000 BabysaffronsmallBabysaffron 2 -10000 BabysaffronBabysaffronsmall Iran 294.1526_0.79 -15000 Babysaffron 1.00122BabysaffronsmallBabysaffron * t[1] Iran Iran 265.1216_0.58 12 17 4 5000 Babysaffron Babysaffron Thiamine (B1): C H N OS (265.1123) {144, 122} X[1] = 0.204 R2Xo[1]BabysaffronBabysaffron = 0.0573 Ellipse: Hotelling's T2 (95%) 294.1526_0.79 -15000 Afgan BabysaffronAfganBabysaffron Iran -200000 Babysaffronsmall Afgan Iran 280.1373_0.73 -20000 Iran 606.229_4.15 + -25000 Babysaffronsmall AfganBabysaffronsmall 15 22 2 4 -5000 BabysaffronsmallBabysaffron Afgan Iran Iran 280.1373_0.73252.1061_0.59 EDO: C H O (NH : 252.1963) {140} -25000-50000 -40000 -30000 -20000 -10000 0 10000 20000 30000 40000 2 BabysaffronsmallBabysaffron Iran -10000-50000 -40000 -30000 -20000 -100001.00122BabysaffronsmallBabysaffron 0 * t[1] 10000 20000 30000 40000 265.1216_0.58vs 12 17 4 + 2 Babysaffron Thiamine (B1): C H N OS (265.1123) {144, 122} X[1] = 0.204 R2Xo[1]Babysaffron = 0.0573 Ellipse: Hotelling's T2 (95%) 265.1216_0.58294.1526_0.79252.1061_0.59 EDO: C H O (NH : 252.1963) {140} -15000 India + Afghan1.00122Babysaffron * t[1]vs Iran Par Thiamine (B1):15 C1222H172 N4OS4 (265.1123) {144, 122} X[1] = 0.204 R2Xo[1] = 0.0573 Ellipse: Hotelling's T2 (95%) 10 14 -20000 151.1105_3.67606.229_4.15294.1526_0.79 Safranal: C H O (151.1123) 280.1373_0.73606.229_4.15 -25000 280.1373_0.73 8 8 4 -50000 -40000 -30000 -20000 -10000 0 10000 20000 30000 40000 vs Vanillic acid: C H O (169.0601) {151,2 105} BI_correct_filtering_NEW_2_the latest.M27 (OPLS-DA), BabySaffron+BabySaffronSmall+Afgh 169.1211_3.66 2 India + Afghan1.00122 * t[1]vs Iran Par 265.1216_0.58265.1216_0.58vs ThiamineThiamine (B1): (B1): C C1212HH1717NN4OS (265.1123)(265.1123){144, {144, 122} 122} X[1] = 0.204 India R2Xo[1] + Afghan = 0.0573 vs Iran Ellipse: Par Hotelling's T2 (95%) Hydroxy-β-cyclocitralSafranal: C (HTCC):10H14O (151.1123) C10H16O 2 (169.1228) 169.1211_4.86151.1105_3.67606.229_4.15 10 14 0.8 151.1105_3.67606.229_4.15 Safranal: C H O (151.1123) Vanillic acid: C8H218O436 (169.0601)5 {151, 105} 0.6 India + Afghan vs. Iran Par 169.1211_3.66369.15_2.95vs. Tomentogenin: C H O (369.2641) {232} BI_correct_filtering_NEW_2_the latest.M27 (OPLS-DA), BabySaffron+BabySaffronSmall+Afgh 169.1211_3.66vs Vanillic acid: C8H8O4 (169.0601) {151, 105} BI_correct_filtering_NEW_2_the0.4 latest.M27 (OPLS-DA), BabySaffron+BabySaffronSmall+Afgh 151.1105_3.67 Safranal: C10H14O+ (151.1123)10 16 2 India + Afghan vs Iran Par 169.1211_4.86 Hydroxy-β-cyclocitral21 20 11 (HTCC): C H O (169.1228) 0.8 472.1734_3.24 Astragalin:Safranal: C H O C (MNa10H14O =(151.1123) 472.0982)10 16 2 {287, 145} 0.2 169.1211_4.86151.1105_3.67169.1211_3.66 Hydroxy-Vanillicβ-cyclocitral acid: C8H (HTCC):8O4 (169.0601) C {151,H O 105} (169.1228) 0.8 0.6 Tomentogenin: C21H36O5 (369.2641) {232} 0 169.1211_4.86369.15_2.95 Hydroxy-β-cyclocitral8 (HTCC):8 4 C10H16O2 (169.1228) 0.6 169.1211_3.66 VanillicTomentogenin: acid: C HC21OH 36(169.0601)O5 (369.2641) {151, {232} 105} BI_correct_filtering_NEW_2_the0.4 latest.M27 (OPLS-DA), BabySaffron+BabySaffronSmall+Afgh 369.15_2.95 + -0.2 369.15_2.95 Tomentogenin: C21H36O5 (369.2641) {232} 0.4 472.1734_3.24 Astragalin: C21H20O11 (MNa+ = 472.0982) {287, 145} 0.2 Hydroxy-β-cyclocitral (HTCC):+ C10H16O2 (169.1228) -0.4 472.1734_3.24169.1211_4.86472.1734_3.24 Astragalin:Astragalin: C C21HH20OO11 (MNa(MNa == 472.0982)472.0982) {287, {287, 145} 145} 0.20.8 21 20 11 -0.60 0.6 Tomentogenin: C21H36O5 (369.2641) {232} 0 369.15_2.95 -0.8-0.2 -0.20.4 + -0.4-1 472.1734_3.24 Astragalin: C21H20O11 (MNa = 472.0982) {287, 145} -0.40.2-0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 -0.6 p[1] -0.60 R2X[1] = 0.204 -0.8 -0.8-0.2 -1 -0.4-1 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 p[1] correct_filtering_NEW_2_the-0.4 -0.3 -0.2 latest.M30 -0.1 (OPLS-DA), 0 BabySaffron+B 0.1abySaffronSmall+Afgha 0.2 0.3 1 n -0.6 R2X[1] =p[1] 0.204 2 Scaled proportionallyR2X[1] = 0.204 to R2X -0.8 Colored according to classes in M30 -1 correct_filtering_NEW_2_the-0.4 -0.3 -0.2 latest.M30 -0.1 (OPLS-DA),Babysaffronsmall 0 BabySaffron+B 0.1abySaffronSmall+Afgha 0.2 0.3 1 n Afganp[1] 12 correct_filtering_NEW_2_the20000 latest.M30Scaled proportionallyR2X[1] (OPLS-DA), = 0.204 to BabySaffron+B R2X abySaffronSmall+Afghan Afgan Scaled proportionallyBabysaffron to R2X 2 Colored according Babysaffronsmallto classes in M30 10000 Colored accordingAfgan to classes in M30 ItalyItaly Babysaffronsmall Babysaffronsmall AfganAfgan ItalyItaly Babysaffron BabysaffronBabysaffronsmallAfganAfgan Italy correct_filtering_NEW_2_the200000 latest.M30 (OPLS-DA), BabySaffron+BabySaffronSmall+Afgha1 n AfganAfgan BabysaffronAfgan 2 20000 BabysaffronsmallScaled proportionallyBabysaffronsmallBabysaffronsmallBabysaffron to R2X ItalyItaly Italy 139.1108_7.13 BabysaffronsmallBabysaffronAfganAfgan Babysaffronsmall 10 16 2 Babysaffron HTCC isomer: C H O (169.1228) -1000010000 Afgan BabysaffronBabysaffronsmall Italy ColoredBabysaffronsmall according toBabysaffronsmall classes in M30 ItalyItaly Babysaffronsmall Afgan 10000 BabysaffronAfgan AfganAfgan ItalyItaly ItalyItaly 169.1212_4.39 + BabysaffronsmallBabysaffron BabysaffronsmallBabysaffronAfgan Italy -200000 AfganAfgan ItalyItaly EDO: C15H22O2 (MNH4 = 252.1963) {140} Babysaffron Afgan BabysaffronAfganAfgan Italy 200000 Babysaffronsmall BabysaffronsmallBabysaffronsmallBabysaffron ItalyItaly Italy 139.1108_7.13 BabysaffronsmallBabysaffronAfganAfganAfgan 252.1061_0.59 10 16+ 2 Babysaffronsmall BabysaffronsmallBabysaffronBabysaffronsmallBabysaffron Italy Italy HTCC isomer: C H O (169.1228) -30000-10000 BabysaffronBabysaffronsmall Italy Italy 139.1108_7.13 BabysaffronsmallBabysaffronsmallBabysaffronAfgan Bornyl ferulate: C20H26O4 (MK10 16 =2 369.1470) {266, 239} -60000 -40000 -20000BabysaffronAfganBabysaffronsmall 0 20000 40000 HTCC isomer: C H O (169.1228) -1000010000 BabysaffronAfgan BabysaffronBabysaffronsmall Italy + Babysaffronsmall 1.00034 * t[1] ItalyItaly 369.1508_1.49169.1212_4.39139.1108_7.13 BabysaffronsmallBabysaffron Afgan X[1] =-20000 0.266 R2Xo[1] = 0.0637AfganAfgan Ellipse: Hotelling'sItalyItaly T2 (95%) 169.1212_4.39 EDO: C15H22O2 (MNH4+ = 252.1963) {140} Babysaffron Babysaffron Italy 15 22 2 4 -200000 Afgan 169.1212_4.39 EDO: HTCCC H isomer:O (MNH C10H16 O=2 252.1963)(169.1228) {140} Afgan 252.1061_0.59203.082_4.75 + -30000 Babysaffronsmall BabysaffronsmallBabysaffronsmallBabysaffron ItalyItaly Italy 252.1061_0.59139.1108_7.13 20 26 4 + BabysaffronsmallBabysaffronAfgan 252.1061_0.59 Bornyl ferulate:EDO: C CH HO (MNHO 10(MK16=+ 252.1963)=2 369.1470) {140} {266, 239} -30000-60000 -40000 -20000Babysaffron 0 20000 40000 HTCC15 isomer:22 2 C H4 O (169.1228) -10000 BabysaffronBabysaffronsmall Italy 20 26 4 Babysaffronsmall 1.00034 * t[1] 369.1508_1.49 Bornyl ferulate: C H O (MK+ = 369.1470) {266, 239} -60000 -40000 -20000Afgan 0 20000 40000 vs X[1] = 0.266 R2Xo[1]Babysaffron = 0.0637 Ellipse: Hotelling's T2 (95%) 169.1212_4.39369.1508_1.49 Bornyl ferulate: C20H26O4 (MK+ = 369.1470) {266, 239} India + Afghan1.00034 * t[1]vs Italy Par 369.1508_1.49 Safranal: C10H14 O (151.1123) X[1] -20000= 0.266 R2Xo[1] = 0.0637 Ellipse: Hotelling's T2 (95%) EDO: C15H22O2 (MNH4 = 252.1963) {140} 151.1105_3.67203.082_4.75203.082_4.75 India + Afghan vs. Italy Par 252.1061_0.59203.082_4.75 Vanillic acid: C8H8O4 (169.0601)+ {151, 105} -30000 vs. Bornyl ferulate: C20H26O4 (MK = 369.1470) {266, 239} -60000 -40000 -20000 0 20000 40000 169.1211_3.66vs BI_correct_filtering_NEW_2_the latest.M30 (OPLS-DA), BabySaffron+BabySaffronSmall+Afgh 369.1508_1.49151.1105_3.67 Safranal: C H O+ (151.1123) India + Afghan1.00034 * t[1]vs Italy Par vs Picrocrocin:Safranal: C21H26O 4C (MNa10H1414O =(151.1123) 353.1577) {185, 151} X[1] = 0.266 India R2Xo[1]+ Afghan = 0.0637 vs Italy Ellipse: Par Hotelling's T2 (95%) 151.1105_3.67 Safranal: C10H 14O (151.1123) 353.1548_3.66169.1211_3.66 Vanillic acid: C8H8O4 (169.0601)+ {151, 105} 151.1105_3.67203.082_4.75 Vanillic acid: C8H8O4 (169.0601)+ {151, 105} 353.1548_3.66 MethylPicrocrocin:Vanillic crocetin: acid: C21 HC C26218OH4268(MNaOO44 (169.0601)(MNa= 353.1577)= 365.1729) {151, {185, 105} 151} {118} 1 169.1211_3.66 BI_correct_filtering_NEW_2_the latest.M30 (OPLS-DA), BabySaffron+BabySaffronSmall+Afgh 365.1196_3.36vs + + BI_correct_filtering_NEW_2_the latest.M30 (OPLS-DA), BabySaffron+BabySaffronSmall+Afgh 169.1211_3.66365.1196_3.36 Picrocrocin:Methyl crocetin: C21H C2621OH4 2621(MNaO436(MNa+5= 353.1577)= 365.1729) {185, {118} 151} India + Afghan vs Italy Par Picrocrocin:Tomentogenin:Safranal: C21H26O C4 (MNa10H14O =(151.1123) (369.2641) 353.1577) {232}{185, 151} 353.1548_3.66369.15_2.95369.15_2.95 Tomentogenin: C H O (369.2641)+ {232} 0.5 353.1548_3.66151.1105_3.67 21 36 5 MethylVanillic crocetin: acid: C C218H826OO4 4(169.0601) (MNa+ = 365.1729) {151, 105} {118} 1 365.1196_3.36 Methyl crocetin: C21H26O4 (MNa = 365.1729) {118} 1 169.1211_3.66 BI_correct_filtering_NEW_2_the latest.M30 (OPLS-DA), BabySaffron+BabySaffronSmall+Afgh 365.1196_3.36 Tomentogenin: C21H36O+ 5 (369.2641) {232} 0 369.15_2.95 Picrocrocin:Tomentogenin: C21H26O C4 (MNa21H36O5= (369.2641) 353.1577) {232}{185, 151} 0.5 353.1548_3.66369.15_2.95 0.5 + -0.5 Methyl crocetin: C21H26O 4 (MNa = 365.1729) {118} 1 365.1196_3.36 0 0 Tomentogenin: C21H36O5 (369.2641) {232} -1 369.15_2.95 0.5-0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 -0.5 p[1] -0.5 R2X[1] = 0.266 0 -1 -1 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 -0.4 -0.3 -0.2 -0.1p[1] 0 0.1 0.2 0.3 1 orrect_filtering_NEW_2_the-0.5 latest.M33 (OPLS-DA),R2X[1] =p[1] 0.266 Afghan+BabySaffron+BabySaffronSmall v 9 14 2 R2X[1] = 0.266 2 155.1057_2.77 Crocusatin A: C H O (155.1073) Scaled proportionally to R2X Colored according to classes in M33 -1 307.1003_4.95 EGC: C15H14O7 (307.0818) {263, 139} -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 orrect_filtering_NEW_2_the latest.M33 (OPLS-DA), Afghan+BabySaffron+BabySaffronSmall1 v p[1] 155.1057_2.77 Crocusatin A: C9H1514O242 (155.1073)7 orrect_filtering_NEW_2_the latest.M33 (OPLS-DA),R2X[1] = 0.266 Afghan+BabySaffron+BabySaffronSmall12 v 317.1579_2.81 Y-glucopyranoside: C H O (317.1600) 40000 ScaledBabysaffronsmall proportionallyAfgan to R2X 155.1057_2.77 Crocusatin A: C9H14O2 (155.1073) Kashmir 2 ColoredBabysaffronsmallScaled accordingAfgan proportionallyBabysaffronsmallAfgan to classes to R2X in M33 Kashmir 15 14 7 BabysaffronBabysaffron Kashmir 307.1003_4.95331.173_3.66 Picrocrocin:EGC: C HC16HO26 O(307.0818)7 (331.1757) {263, {185, 139} 151} 20000 Colored according to classes in M33 Kashmir 15 14 7 Babysaffron 307.1003_4.95 EGC: C H O (307.0818) {263, 139} Babysaffronsmall 15 24 + 7 orrect_filtering_NEW_2_the0 Babysaffronsmall latest.M33 (OPLS-DA),Babysaffron Afghan+BabySaffKashmirron+BabySaffronSmall1 v 317.1579_2.81 Y-glucopyranoside: C H O (317.1600) 40000 BabysaffronsmallAfganAfganAfgan 364.1944_2.96 Methyl crocetin - C21H26O9 4 (MNa14 2 = 365.1729) {118} 2 155.1057_2.77 Y-glucopyranoside:Crocusatin A: C HC15OH24 (155.1073)O7 (317.1600) 40000 BabysaffronsmallAfgan KashmirKashmir KashmirKashmir 317.1579_2.81 BabysaffronsmallBabysaffronsmallBabysaffronScaledAfgan proportionallyAfganBabysaffronBabysaffronAfganBabysaffronsmallAfgan to R2X KashmirKashmir 16 26 7 -2000020000 BabysaffronsmallBabysaffronAfganBabysaffronBabysaffronsmallAfgan Kashmir KashmirKashmir 331.173_3.66 Picrocrocin: C H O (331.1757) {185, 151} Colored accordingAfgan BabysaffronsmallBabysaffronsmallBabysaffron to classes in M33 Kashmir vs BabysaffronsmallBabysaffron AfganBabysaffron Kashmir 307.1003_4.95 EGC: C15H1614O726 (307.0818)7 {263, 139} 20000 BabysaffronsmallBabysaffronBabysaffron KashmirKashmir 331.173_3.66 Picrocrocin: C H O (331.1757) {185, 151} Babysaffron + -400000 BabysaffronsmallBabysaffronsmall Babysaffron Kashmir 364.1944_2.96 Afgan Afgan 151.1105_3.67 Methyl crocetinSafranal: - C1021H1426O 4(151.1123) (MNa15 24 + 7= 365.1729) {123} {118} 400000 BabysaffronsmallBabysaffronsmallBabysaffron KashmirKashmirKashmir 364.1944_2.96317.1579_2.81 Y-glucopyranoside: C H O (317.1600) BabysaffronsmallBabysaffronAfganAfganAfganBabysaffronBabysaffronAfgan Methyl crocetin - C21H26O4 (MNa = 365.1729) {118} Kashmir Kashmir -60000-20000 BabysaffronsmallAfgan Kashmir Kashmir BabysaffronsmallBabysaffronAfganAfgan BabysaffronBabysaffronsmallAfganBabysaffronBabysaffronsmall Kashmir vs -40000 -30000 -20000BabysaffronsmallBabysaffron -10000Babysaffronsmall 0 10000 20000 30000 151.1105_4.86 -20000 BabysaffronBabysaffronAfgan KashmirKashmir Picrocrocin: C16H26O7 (331.1757) {185, 151} 20000 Afgan BabysaffronsmallBabysaffronsmallBabysaffron KashmirKashmirKashmir 331.173_3.66vs BabysaffronsmallBabysaffron Afgan1.0001 * t[1] X[1] =-40000 0.127 Babysaffronsmall R2Xo[1] = 0.318Babysaffron Ellipse: Hotelling'sKashmir T2 (95%) 169.1211_3.66151.1105_3.67 VanillicSafranal: acid: C810HH8O14O4 (169.0601) (151.1123)+ {151, {123} 105} -40000 Babysaffronsmall Babysaffron Kashmir 0 Afgan Afgan 151.1105_3.67364.1944_2.96 Methyl crocetinSafranal: - C2110H2614O4 (151.1123) (MNa = 365.1729) {123} {118} -60000 Kashmir Kashmir BabysaffronsmallBabysaffron Babysaffron -40000 -30000 -20000Afgan -10000BabysaffronAfgan 0 10000 20000 30000 151.1105_4.86 10 16 2 -60000-20000 Kashmir 169.1211_4.86 Hydroxy-β-cyclocitral (HTCC): C H O (169.1228) Afgan BabysaffronsmallBabysaffronsmallBabysaffron1.0001 * t[1] Kashmir 151.1105_4.86vs -40000 -30000Babysaffronsmall -20000Babysaffron -10000Afgan 0 10000 20000 30000 X[1] = 0.127 R2Xo[1] = 0.3181.0001 * t[1] Ellipse: Hotelling'sKashmir T2 (95%) 169.1211_3.66 Vanillic acid: C8H8O4 (169.0601) {151, 105} X[1] -40000= 0.127 Afghan+ R2Xo[1] India = 0.318 vs Kashmir Ellipse: Hotelling's Par T2 (95%) 611.157_4.14 Kaempferol-di-glucoside: C27H30O16 (611.1611) {287} 169.1211_3.66151.1105_3.67 VanillicSafranal: acid: C108HH814OO4 (169.0601)(151.1123) {151,{123} 105} 10 16 2 -60000 169.1211_4.86 Hydroxy-β-cyclocitral (HTCC): C H O (169.1228) -40000 -30000 -20000 -10000 0 10000 20000 30000 169.1211_4.86151.1105_4.86 Hydroxy-β-cyclocitral (HTCC): C10H16O2 (169.1228) Afghan+ India1.0001 vs * t[1] Kashmir Par 611.157_4.14 Kaempferol-di-glucoside: C27H30O16 (611.1611) {287} X[1] = 0.127 Afghan+ R2Xo[1] India = 0.318 vs Kashmir Ellipse: Hotelling's Par T2 (95%) 169.1211_3.66611.157_4.14 Kaempferol-di-glucoside:Vanillic acid: C8H8O4 C(169.0601)27H30O16 (611.1611) {151, 105} {287}

169.1211_4.86 Hydroxy-β-cyclocitral (HTCC): C10H16O2 (169.1228) Afghan+ India vs Kashmir Par 611.157_4.14 Kaempferol-di-glucoside: C27H30O16 (611.1611) {287}

Molecules 2021, 26, x FOR PEER REVIEW 6 of 16

_correct_filtering_NEW_2_the latest.M27 (OPLS-DA), BabySaffron+BabySaffronSmall+Afgha1 Scaled proportionally to R2X 2 Colored according to classes in M27

20000 Babysaffronsmall Afgan 15000 Afgan Babysaffronsmall Iran 10000 Babysaffron Afgan Babysaffronsmall Iran 5000 Iran Babysaffron Afgan BabysaffronAfgan Iran 0 Babysaffronsmall Afgan Iran + Babysaffronsmall AfganBabysaffronsmall Iran 15 22 2 4 -5000 BabysaffronsmallBabysaffron Afgan Iran Iran 252.1061_0.59 EDO: C H O (NH : 252.1963) {140} BabysaffronsmallBabysaffron Iran -10000 BabysaffronsmallBabysaffron Babysaffron Babysaffron 294.1526_0.79 -15000 Babysaffron -20000 280.1373_0.73 -25000 -50000 -40000 -30000 -20000 -10000 0 10000 20000 30000 40000 2 1.00122 * t[1] 265.1216_0.58 Thiamine (B1): C12H17N4OS (265.1123) {144, 122} X[1] = 0.204 R2Xo[1] = 0.0573 Ellipse: Hotelling's T2 (95%) 606.229_4.15 vs India + Afghan vs Iran Par 151.1105_3.67 Safranal: C10H14O (151.1123) Vanillic acid: C8H8O4 (169.0601) {151, 105} BI_correct_filtering_NEW_2_the latest.M27 (OPLS-DA), BabySaffron+BabySaffronSmall+Afgh 169.1211_3.66 169.1211_4.86 Hydroxy-β-cyclocitral (HTCC): C10H16O2 (169.1228) 0.8 21 36 5 0.6 369.15_2.95 Tomentogenin: C H O (369.2641) {232} 0.4 + 472.1734_3.24 Astragalin: C21H20O11 (MNa = 472.0982) {287, 145} 0.2 0 -0.2

-0.4

-0.6

-0.8

-1 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 p[1] R2X[1] = 0.204

correct_filtering_NEW_2_the latest.M30 (OPLS-DA), BabySaffron+BabySaffronSmall+Afgha1 n Scaled proportionally to R2X 2 Colored according to classes in M30

Babysaffronsmall Afgan 20000 Afgan Babysaffron Babysaffronsmall 10000 Afgan ItalyItaly Babysaffronsmall AfganAfgan ItalyItaly Babysaffron Babysaffron Italy 0 Afgan Afgan Babysaffronsmall BabysaffronsmallBabysaffronsmallBabysaffron ItalyItaly Italy 139.1108_7.13 BabysaffronsmallBabysaffronAfgan 10 16 2 Babysaffron HTCC isomer: C H O (169.1228) -10000 BabysaffronBabysaffronsmall Italy Babysaffronsmall Babysaffron Afgan 169.1212_4.39 + -20000 EDO: C15H22O2 (MNH4 = 252.1963) {140} 252.1061_0.59 + -30000 20 26 4 -60000 -40000 -20000 0 20000 40000 Bornyl ferulate: C H O (MK = 369.1470) {266, 239} 1.00034 * t[1] 369.1508_1.49 X[1] = 0.266 R2Xo[1] = 0.0637 Ellipse: Hotelling's T2 (95%) 203.082_4.75

vs India + Afghan vs Italy Par Safranal: C10H14O (151.1123) 151.1105_3.67 Vanillic acid: C8H8O4 (169.0601) {151, 105}

BI_correct_filtering_NEW_2_the latest.M30 (OPLS-DA), BabySaffron+BabySaffronSmall+Afgh 169.1211_3.66 + Picrocrocin: C21H26O4 (MNa = 353.1577) {185, 151} 353.1548_3.66 + Methyl crocetin: C21H26O4 (MNa = 365.1729) {118} 1 365.1196_3.36 Tomentogenin: C21H36O5 (369.2641) {232} 369.15_2.95 Molecules0.5 2021, 26, 2180 7 of 16

0

-0.5 Table 1. Cont. -1 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 p[1] R2X[1] = 0.266 B. Features (Accurate A. Scores Plot and S-Plot C. Putative Metabolite (Calculated Mass) * {Fragment} 1 MH+_Retention Time) orrect_filtering_NEW_2_the latest.M33 (OPLS-DA), Afghan+BabySaffron+BabySaffronSmall1 v 155.1057_2.77 Crocusatin A: C9H14O2 (155.1073) Scaled proportionally to R2X 2 Colored according to classes in M33 307.1003_4.95 EGC: C15H14O7 (307.0818) {263, 139} 317.1579_2.81 Y-glucopyranoside: C15H24O7 (317.1600) 40000 BabysaffronsmallAfgan Kashmir BabysaffronsmallAfgan BabysaffronsmallAfgan Kashmir Kashmir 16 26 7 Molecules 202120000, 26, x FOR PEERBabysaffronBabysaffron REVIEW Kashmir 331.173_3.66 Picrocrocin: C H O (331.1757) {185, 151} 7 of 16 Babysaffronsmall Babysaffron + Babysaffronsmall Babysaffron Kashmir 0 Afgan Afgan 364.1944_2.96 Methyl crocetin - C21H26O4 (MNa = 365.1729) {118} Kashmir Kashmir BabysaffronsmallBabysaffronAfgan BabysaffronBabysaffronAfgan -20000 Kashmir Molecules 2021, 26, x FOR PEERAfgan BabysaffronsmallBabysaffronsmallBabysaffronREVIEW Kashmir vs 7 of 16 BabysaffronsmallBabysaffron Afgan Molecules 2021, 26, x FOR PEER REVIEW Kashmir 155.1057_2.77 Crocusatin A: C9H14O2 (155.1073) 7 of 16 -40000 10 14 151.1105_3.67307.1003_4.95 Safranal:EGC: C15 HC14OH7 (307.0818)O (151.1123) {263, 139} {123} -60000 317.1579_2.81 Y-glucopyranoside: C15H24O7 (317.1600) -40000 -30000 -20000 -10000 0 10000 20000 30000 151.1105_4.86 1.0001 * t[1] 331.173_3.66 Picrocrocin: C16H26O7 (331.1757) {185, 151} X[1] = 0.127 R2Xo[1] = 0.318 Ellipse: Hotelling's T2 (95%) 169.1211_3.66 Vanillic acid: C8H8O4 (169.0601)+ {151, 105} 364.1944_2.96 Methyl crocetin-C21H26O4 (MNa = 365.1729) {118} Afghan + India vs. Kashmir Par 169.1211_4.86vs. Hydroxy-β-cyclocitral (HTCC): C10H16O2 (169.1228) 151.1105_3.67 Safranal: C H O (151.1123) {123} Afghan+ India vs Kashmir Par 611.157_4.14 Kaempferol-di-glucoside:10 14 C27H30O16 (611.1611) {287} 151.1105_4.86 169.1211_3.66 Vanillic acid: C8H8O4 (169.0601) {151, 105} 169.1211_4.86 Hydroxy-β-cyclocitral (HTCC): C10H16O2 (169.1228) 611.157_4.14 Kaempferol-di-glucoside: C27H30O16 (611.1611) {287}

ICS ORBI_correct_filtering_NEW_2_the latest.M36 (OPLS-DA), Greece+Morocco vs Kashmir1 Scaled proportionally to R2X 2 Colored according to classes in M36

ICS ORBI_correct_filtering_NEW_2_the30000 latest.M36 (OPLS-DA), Greece+Morocco vs Kashmir1 GreeceNew ICS ORBI_correct_filtering_NEW_2_theMoroccofromUNIGreeceNew latest.M36MoroccofromUNI (OPLS-DA), Greece+Morocco vs Kashmir12 20000 Scaled proportionally to R2X MoroccofromUNI MoroccofromUNI Kashmir 2 ColoredScaledGreeceNewGreeceNew according proportionallyMoroccofromUNI to classes to R2X in M36 10000 GreeceNew GreeceNewMoroccofromUNI Kashmir Kashmir ColoredGreeceNew accordingMoroccofromUNI to classes in M36 MoroccofromUNIMorocco Morocco Kashmir Kashmir 0 GreeceNewMorocco MoroccofromUNIMorocco Kashmir GreeceNew Kashmir 30000 Greece0708Greece0708Greece0708 GreeceNew -1000030000 MoroccofromUNIGreece0708 MoroccofromUNI Kashmir Kashmir MoroccoMoroccoGreeceNewGreece0708Morocco 155.1057_2.77 Crocusatin A: C9H14O2 (155.1073) 20000 GreeceNew Kashmir MoroccofromUNI MoroccofromUNIMoroccofromUNI -20000 GreeceNewGreeceNewMoroccoGreeceNewMoroccoMoroccofromUNIGreece0708 20000 Kashmir Kashmir 10000 MoroccofromUNIGreeceNew GreeceNewMoroccofromUNIMoroccofromUNI KashmirKashmir GreeceNewGreeceNew Greece0708MoroccofromUNIMoroccofromUNI Greece0708 Kashmir 183.1005_3.33 -3000010000 MoroccofromUNIGreeceNewMorocco GreeceNewMoroccoMoroccofromUNI Kashmir KashmirKashmir 0 GreeceNewGreeceNewMorocco MoroccofromUNIMorocco Kashmir Kashmir MoroccofromUNIGreeceNewMorocco MoroccoGreece0708Greece0708 Kashmir Kashmir -400000 GreeceNewMorocco MoroccofromUNIMoroccoGreece0708 Kashmir Kashmir -10000 GreeceNewGreece0708 Greece0708 Kashmir Kashmir 15 14 7 MoroccoMorocco Greece0708Greece0708Greece0708Morocco 307.1003_4.95155.1057_2.77 EGC:Crocusatin C H O A: (307.0818) C9H14O2 (155.1073) {263, 139} -50000-10000 Greece0708 Kashmir Kashmir MoroccoMoroccoMorocco Greece0708MoroccoGreece0708 9 14 2 -20000-50000 -40000 -30000 -20000 -10000Morocco 0 10000 20000 30000Kashmir 40000 155.1057_2.77 Crocusatin A: C H O (155.1073) -20000 Greece0708Morocco Greece0708MoroccoGreece0708 Kashmir 15 24 7 -30000 1.00078 * t[1] 317.1579_2.81183.1005_3.33155.1057_2.77 Y-glucopyranoside:Crocusatin A: C9H C14OH2 (155.1073)O (317.1600) X[1] = 0.158 R2Xo[1]Greece0708 = 0.135Greece0708 Ellipse: Hotelling's T2 (95%) -30000 183.1005_3.33 -40000 183.1005_3.33 331.173_3.66 Picrocrocin:15 C1614H267 O7 (331.1757) {185, 151} -40000 Greece0708 307.1003_4.95 EGC: C H O (307.0818) {263, 139} -50000 Greece0708 307.1003_4.95307.1003_4.95 EGC:EGC: C15 C15HH1414OO7 7(307.0818)(307.0818) {263, {263, 139} 139} -50000-50000 -40000 -30000 -20000 -10000 0 10000 20000 30000 40000 vs 15 24 7 -50000 -40000 -30000 -20000 -100001.00078 0 * t[1] 10000 20000 30000 40000 317.1579_2.81317.1579_2.81 Y-glucopyranoside:Y-glucopyranoside: C C15HH24O7O(317.1600)(317.1600) X[1] = 0.158Greece+Morocco R2Xo[1] = 0.1351.00078 vs * t[1] Kashmir Ellipse: Hotelling's Par T2 (95%) 317.1579_2.81 Y-glucopyranoside: C15H24O7 (317.1600) X[1] = 0.158 R2Xo[1] = 0.135 Ellipse: Hotelling's T2 (95%) 151.1105_3.67331.173_3.66331.173_3.66 Picrocrocin:Picrocrocin:Safranal: C C 161610HH262614OOO7 (331.1757)(151.1123) {185, {123}{185, 151} 151} Greece + Morocco vs. Kashmir Par 331.173_3.66vs. Picrocrocin: C16H26O7 (331.1757) {185, 151} 151.1105_4.86vs MICS ORBI_correct_filtering_NEW_2_theGreece+Morocco latest.M36 vs (OPLS-DA), Kashmir Greece+Morocco Par vs Kashm 151.1105_3.67vs Safranal: C10H14O (151.1123) {123} Greece+Morocco vs Kashmir Par 169.1211_3.66151.1105_3.67151.1105_4.86 VanillicSafranal: acid: C810HH8O14O4 (169.0601) (151.1123) {151, {123} 105} 0.8 151.1105_3.67 Safranal: C10H14O (151.1123) {123} 169.1211_3.66 Vanillic acid: C8H8O4 (169.0601) {151, 105} 0.6 169.1211_4.86151.1105_4.86 Hydroxy-β-cyclocitral (HTCC): C10H16O2 (169.1228) MICS ORBI_correct_filtering_NEW_2_the latest.M36 (OPLS-DA), Greece+Morocco vs Kashm 169.1211_4.86 Hydroxy-β-cyclocitral (HTCC): C H O (169.1228) MICS0.4 ORBI_correct_filtering_NEW_2_the latest.M36 (OPLS-DA), Greece+Morocco vs Kashm 151.1105_4.86 10 16 2 611.157_4.14 Kaempferol-di-glucoside:8 8 4C27H30O16 (611.1611) {287} 0.2 169.1211_3.66611.157_4.14 Kaempferol-di-glucoside:Vanillic acid: C H O C 27(169.0601)H30O16 (611.1611) {151, 105} {287} 0.8 169.1211_3.66 Vanillic acid: C8H8O4 (169.0601) {151, 105} 0.8 0.60 169.1211_4.86 Hydroxy-β-cyclocitral (HTCC): C10H16O2 (169.1228) 0.6 10 16 2 -0.20.4 169.1211_4.86 Hydroxy-β-cyclocitral (HTCC): C H O (169.1228) 0.4 611.157_4.14 Kaempferol-di-glucoside: C27H30O16 (611.1611) {287} -0.40.2 611.157_4.14 Kaempferol-di-glucoside: C27H30O16 (611.1611) {287} 0.2 -0.60 0 -0.8-0.2 -0.2 -0.4-1 -0.25 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 -0.4 -0.6 p[1] R2X[1] = 0.158 -0.6 -0.8 -0.8 -1 -1-0.25 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 OCOMICS-0.25 ORBI_correct_filtering_NEW_2_the -0.2 -0.15 -0.1 -0.05 latest.M39p[1] 0 0.05 (OPLS-DA), 0.1 Italy 0.15 vs Kashmir 0.2 Par 1 R2X[1] = 0.158 Scaled proportionallyp[1] to R2X 2 R2X[1] = 0.158 Colored according to classes in M39

OCOMICS ORBI_correct_filtering_NEW_2_the latest.M39 (OPLS-DA), Italy vs Kashmir Par 1 OCOMICS40000 ORBI_correct_filtering_NEW_2_the latest.M39 (OPLS-DA), Italy vs Kashmir Par 12 Scaled proportionallyKashmir to R2X Kashmir 2 ColoredScaled according proportionally to classes to R2X in M39 20000 Kashmir Colored accordingItaly to classes in M39Kashmir Italy Italy ItalyItaly Kashmir 0 Vanillic acid: C8H8O4 (169.0601) {151, 105} Italy Kashmir 40000 Italy Italy Kashmir KashmirKashmir 169.1211_3.66 40000 Italy Kashmir 15 14 7 -20000 Kashmir Kashmir EGC: C H O (307.0818) {263, 139} 20000 Kashmir Italy Kashmir 20000 Kashmir 307.1003_4.95 Italy Kashmir KashmirKashmir 16 26 7 -40000 ItalyItaly ItalyItaly Kashmir Picrocrocin: C H8 8O 4 (331.1757) {185, 151} 0 Italy Vanillic acid: C H O (169.0601) {151, 105} ItalyItaly ItalyItaly Kashmir Kashmir 8 8 4 0 Italy Kashmir 331.173_3.66 Vanillic acid: C H O (169.0601)+ {151, 105} Italy Italy Kashmir 169.1211_3.66 -60000 Italy Italy Kashmir -20000 Kashmir 169.1211_3.66 Picrocrocin:EGC: C C1615HH2614OO7 7(MNa (307.0818)= 353.1577) {263, 139} {185, 151} -80000 -60000 -40000Italy -20000 0Kashmir 20000 40000 60000 15 14 7 -20000 1.0001 * t[1] 353.1548_3.66 EGC: C H O (307.0818) {263, 139} Kashmir 307.1003_4.95 X[1] = 0.37 R2Xo[1] = 0.205 Ellipse: Hotelling's T2 (95%) -40000 Kashmir 307.1003_4.95 Picrocrocin:Tomentogenin: C16 HC2621OH736 (331.1757)O5 (369.2641) {185, {232} 151} Kashmir -40000 Kashmir Picrocrocin: C16H26O7 (331.1757) {185, 151} 331.173_3.66369.15_2.95 + -60000 331.173_3.66 Picrocrocin: C16H26O7 (MNa+ = 353.1577) {185, 151} -60000-80000 -60000 -40000 -20000 0 20000 40000 60000 353.1548_3.66vs Picrocrocin: C16H26O7 (MNa = 353.1577) {185, 151} -80000 -60000 -40000 -200001.0001 0 * t[1] 20000 40000 60000 HTCC isomer: C10H16O2 (169.1228) X[1] = 0.37 Italy R2Xo[1] vs = 0.205 Kashmir1.0001 * t[1] ParEllipse: Hotelling's T2 (95%) 353.1548_3.66 Tomentogenin: C21H36O5 (369.2641) {232} X[1] = 0.37 R2Xo[1] = 0.205 Ellipse: Hotelling's T2 (95%) 169.1212_4.39369.15_2.95 Tomentogenin: C21H36O+ 5 (369.2641) {232} 369.15_2.95 EDO: C15H22O2 (MNH 4 = 252.1963) {140}

CROCOMICS ORBI_correct_filtering_NEW_2_the latest.M39 (OPLS-DA), Italy vs Kashmir Par 252.1061_0.59vs + Italy vs Kashmir Par vs Bornyl ferulate:HTCC Cisomer:20H26O 4C (MK10H16O = 2369.1470) (169.1228) {266, 239} HTCC isomer: C10H16O2 (169.1228) Italy vs Kashmir Par 369.1508_1.49169.1212_4.39 + 169.1212_4.39 EDO: C15H22O2 (MNH 4+ = 252.1963) {140} 1 252.1061_0.59203.082_4.75 EDO: C15H22O2 (MNH4 = 252.1963) {140} CROCOMICS ORBI_correct_filtering_NEW_2_the latest.M39 (OPLS-DA), Italy vs Kashmir Par 27 + 30 16 CROCOMICS ORBI_correct_filtering_NEW_2_the latest.M39 (OPLS-DA), Italy vs Kashmir Par 252.1061_0.59 BornylKaempferol-di-glucoside: ferulate: C20H26O4 (MK C H+ = O369.1470)(611.1611) {266, {287} 239} 0.5 369.1508_1.49611.157_4.14 Bornyl ferulate: C20H26O4 (MK = 369.1470) {266, 239} 369.1508_1.49 01 1 203.082_4.75 203.082_4.75 Kaempferol-di-glucoside: C27H30O16 (611.1611) {287} -0.50.5 611.157_4.14 Kaempferol-di-glucoside: C27H30O16 (611.1611) {287} 0.5 611.157_4.14 -10 0

-1.5-0.5 -0.5-0.25 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 p[1] -1 R2X[1] = 0.37 -1 -1.5 Vanillic acid: C8H8O4 (169.0601) {151, 105} -1.5-0.25 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 -0.25 -0.2 -0.15 -0.1 -0.05p[1] 0 0.05 0.1 0.15 0.2 R2X[1] =p[1] 0.37 169.1211_3.66 EGC: C15H14O7 (307. 0818) {263, 139} R2X[1] = 0.37

307.1003_4.95 Picrocrocin:Vanillic acid: C 16CH8H268OO74 (331.1757)(169.0601) {185,{151, 151}105} Vanillic acid: C8H8O4 (169.0601) {151, 105} 169.1211_3.66 EGC: C15H14O7 (307. 0818) {263, 139} 169.1211_3.66 EGC: C15H14O7 (307. 0818) {263, 139} 307.1003_4.95 Picrocrocin: C16H26O7 (331.1757) {185, 151} 307.1003_4.95 Picrocrocin: C16H26O7 (331.1757) {185, 151}

Molecules 2021, 26, x FOR PEER REVIEW 7 of 16

Molecules 2021, 26, x FOR PEER REVIEW 7 of 16

ICS ORBI_correct_filtering_NEW_2_the latest.M36 (OPLS-DA), Greece+Morocco vs Kashmir1 Scaled proportionally to R2X 2 Colored according to classes in M36

ICS ORBI_correct_filtering_NEW_2_the latest.M36 (OPLS-DA), Greece+Morocco vs Kashmir1 30000 GreeceNew 2 MoroccofromUNIScaled proportionallyGreeceNewMoroccofromUNI to R2X 20000 Kashmir ColoredMoroccofromUNIGreeceNewGreeceNew according MoroccofromUNItoMoroccofromUNI classes in M36 10000 GreeceNew GreeceNewMoroccofromUNI Kashmir Kashmir GreeceNew MoroccofromUNI MoroccofromUNIMorocco Morocco Kashmir Kashmir 300000 GreeceNewMorocco MoroccofromUNIMorocco Kashmir GreeceNew Kashmir GreeceNewGreece0708Greece0708 MoroccofromUNIGreeceNewMoroccofromUNIGreece0708 -1000020000 Greece0708 Kashmir Kashmir MoroccoMorocco Greece0708Morocco Kashmir 155.1057_2.77 Crocusatin A: C9H14O2 (155.1073) MoroccofromUNIGreeceNewGreeceNew MoroccofromUNIMoroccofromUNI -2000010000 GreeceNewMorocco GreeceNewMoroccoMoroccofromUNIGreece0708 Kashmir KashmirKashmir GreeceNew MoroccofromUNI MoroccofromUNIGreece0708Morocco MoroccoGreece0708 Kashmir Kashmir -300000 GreeceNewMorocco MoroccofromUNIMorocco Kashmir 183.1005_3.33 GreeceNew Kashmir Greece0708Greece0708Greece0708 -10000-40000 Kashmir Kashmir Greece0708MoroccoMorocco Greece0708 9 14 2 Greece0708Morocco 307.1003_4.95155.1057_2.77 EGC:Crocusatin C15H14 OA:7 (307.0818)C H O (155.1073) {263, 139} -20000-50000 Morocco MoroccoGreece0708 Kashmir -50000 -40000 -30000Greece0708 -20000 -10000Greece0708 0 10000 20000 30000 40000 -30000 1.00078 * t[1] 317.1579_2.81183.1005_3.33 Y-glucopyranoside: C15H24O7 (317.1600) X[1] = 0.158 R2Xo[1] = 0.135 Ellipse: Hotelling's T2 (95%) -40000 15 14 7 Greece0708 307.1003_4.95331.173_3.66 Picrocrocin:EGC: C HC16HO26 (307.0818)O7 (331.1757) {263, {185, 139} 151} -50000 -50000 -40000 -30000 -20000 -10000 0 10000 20000 30000 40000 15 24 7 1.00078 * t[1] 317.1579_2.81vs Y-glucopyranoside: C H O (317.1600) X[1] = 0.158Greece+Morocco R2Xo[1] = 0.135 vs Kashmir Ellipse: Hotelling's Par T2 (95%) 151.1105_3.67331.173_3.66 Picrocrocin:Safranal: C C1610HH2614OO7 (331.1757)(151.1123) {185,{123} 151} 151.1105_4.86vs MICS ORBI_correct_filtering_NEW_2_theGreece+Morocco latest.M36 vs (OPLS-DA), Kashmir Greece+Morocco Par vs Kashm 169.1211_3.66151.1105_3.67 VanillicSafranal: acid: C108HH814OO4 (169.0601)(151.1123) {151,{123} 105} 0.8

MICS0.6 ORBI_correct_filtering_NEW_2_the latest.M36 (OPLS-DA), Greece+Morocco vs Kashm 169.1211_4.86151.1105_4.86 Hydroxy-β-cyclocitral (HTCC): C10H16O2 (169.1228) 0.4 169.1211_3.66611.157_4.14 Kaempferol-di-glucoside:Vanillic acid: C8H8O4 C(169.0601)27H30O16 (611.1611) {151, 105} {287} 0.20.8

0.60 169.1211_4.86 Hydroxy-β-cyclocitral (HTCC): C10H16O2 (169.1228) 0.4 -0.2 611.157_4.14 Kaempferol-di-glucoside: C27H30O16 (611.1611) {287} Molecules-0.40.2 2021, 26, 2180 8 of 16 -0.60

-0.8-0.2

-0.4-1 -0.25 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 -0.6 p[1] R2X[1] = 0.158 -0.8 Table 1. Cont. -1 -0.25 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 p[1] 1 OCOMICS ORBI_correct_filtering_NEW_2_theR2X[1] latest.M39= 0.158 (OPLS-DA), Italy vs Kashmir Par B. Features (Accurate 2 1 A. ScoresScaled proportionally Plot and to R2X S-Plot + C. Putative Metabolite (Calculated Mass) * {Fragment} Colored according to classes in M39 MH _Retention Time)

OCOMICS ORBI_correct_filtering_NEW_2_the latest.M39 (OPLS-DA), Italy vs Kashmir Par 1 2 40000 Scaled proportionally to R2X Kashmir Kashmir Colored according to classes in M39 20000 Kashmir Italy Kashmir Italy Italy ItalyItaly Kashmir 8 8 4 400000 Vanillic acid: C H O (169.0601) {151, 105} ItalyItaly Kashmir Italy Kashmir Kashmir 169.1211_3.66 Italy Kashmir 15 14 7 -2000020000 Kashmir EGC: C H O (307.0818) {263, 139} Italy Kashmir Italy Kashmir 307.1003_4.95 Italy ItalyItaly KashmirKashmir -400000 Picrocrocin:Vanillic acid: C C168HH268O47 (169.0601)(331.1757) {151,{185, 105}151} ItalyItaly Kashmir Italy Kashmir 169.1211_3.66 Italy 331.173_3.66 + Kashmir 15 14 7 -20000-60000 Picrocrocin:EGC: C C16HH26OO7 (MNa (307.0818)= 353.1577) {263, 139} {185, 151} -80000 -60000 -40000 -20000 0 20000 40000 60000 Kashmir 353.1548_3.66307.1003_4.95169.1211_3.66 Vanillic acid: C H O (169.0601) {151, 105} 1.0001 * t[1] Kashmir 168 268 47 X[1] -40000= 0.37 R2Xo[1] = 0.205 Ellipse: Hotelling's T2 (95%) Picrocrocin:Tomentogenin: C H C21OH 36(331.1757)O5 (369.2641) {185, {232} 151} 307.1003_4.95 EGC: C15H14O7 (307.0818) {263, 139} 331.173_3.66369.15_2.95 + -60000 Picrocrocin: C16H26O7 (MNa = 353.1577) {185, 151} -80000 -60000 -40000 -20000 0 20000 40000 60000 331.173_3.66 Picrocrocin: C16H26O7 (331.1757) {185, 151} + 1.0001 * t[1] 353.1548_3.66353.1548_3.66vs Picrocrocin: C H O (MNa = 353.1577) {185, 151} X[1] = 0.37 Italy R2Xo[1] vs = 0.205 Kashmir Ellipse:Par Hotelling's T2 (95%) Tomentogenin:HTCC isomer:16 26 C7 21 CH1036HO165O (369.2641)2 (169.1228) {232} 169.1212_4.39369.15_2.95369.15_2.95 Tomentogenin: C21H36O+5 (369.2641) {232} Italy vs. Kashmir Par vs. EDO: C15H22O2 (MNH 4 = 252.1963) {140} 252.1061_0.59vs CROCOMICS ORBI_correct_filtering_NEW_2_theItaly vs Kashmir latest.M39 (OPLS-DA), Par Italy vs Kashmir Par 169.1212_4.39 HTCCHTCC isomer: isomer: CC1010HH1616O+O2 (169.1228)2 (169.1228) Bornyl ferulate: C20H26O4 (MK+ = 369.1470) {266, 239} 369.1508_1.49169.1212_4.39252.1061_0.59 EDO: C15H22O2 (MNH4 += 252.1963) {140} EDO: C15H22O2 (MNH4 + = 252.1963) {140} 1 369.1508_1.49 Bornyl ferulate: C20H26O4 (MK = 369.1470) {266, 239} CROCOMICS ORBI_correct_filtering_NEW_2_the latest.M39 (OPLS-DA), Italy vs Kashmir Par 252.1061_0.59203.082_4.75 + 203.082_4.75 BornylKaempferol-di-glucoside: ferulate: C20H26O4 (MK C27H =30 O369.1470)16 (611.1611) {266, {287} 239} 0.5 369.1508_1.49 611.157_4.14611.157_4.14 Kaempferol-di-glucoside: C27H30O16 (611.1611) {287} Molecules 20211 , 26, x FOR PEER REVIEW 8 of 16 0 203.082_4.75 Kaempferol-di-glucoside: C27H30O16 (611.1611) {287} 0.5 611.157_4.14 -0.5 Molecules 20210 , 26, x FOR PEER REVIEW 8 of 16 -1

-0.5 21 36 5 -1.5 331.173_3.66 Tomentogenin: C H O (369.2641) {232} -0.25 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 OCOMICS ORBI_correct_filtering_NEW_2_the latest.M42 (OPLS-DA), Iran vs Kashmir Par 1 p[1] -1 2 369.15_2.95 Scaled proportionallyR2X[1] = 0.37 to R2X Colored according to classes in M42 -1.5 511.1749_4.94331.173_3.66 Tomentogenin: C21H 36O5 (369.2641) {232} -0.25 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 0.2 Vanillic acid: C8H8O4 (169.0601) {151, 105} OCOMICS40000 ORBI_correct_filtering_NEW_2_the latest.M42p[1] (OPLS-DA), Iran vs Kashmir Par 1 + R2X[1] = 0.37 2 369.15_2.95vs EDO: C15H22O2 (MNH 4 = 252.1963) {140} 30000 Scaled proportionally to R2X 169.1211_3.66 EGC: C15H14O7 (307. 0818) {263, 139} Kashmir 20000 Colored according to classes in M42 Iran Kashmir 252.1061_0.59511.1749_4.94 Kashmir Kashmir Vanillic acid: C168H268O47 (169.0601) {151, 105} 10000 Iran IranIran 307.1003_4.95 Picrocrocin: C H O (331.1757) {185, 151} 40000 Kashmir Kashmir + 0 Iran 294.1526_0.79vs EDO: C15H1522O142 (MNH7 4 = 252.1963) {140} 30000 Iran Iran Kashmir 169.1211_3.66 EGC: C H O (307. 0818) {263, 139} -10000 Kashmir Iran Kashmir 20000 Iran 2 -20000 Iran Kashmir 280.1373_0.73252.1061_0.59 1216 1726 47 Kashmir Kashmir 307.1003_4.95 ThiaminePicrocrocin: (B1): CC HH NOOS (331.1757) (265.1123) {185, {144, 151} 122} 10000 Iran Iran -30000 Iran Kashmir Kashmir KashmirKashmir 0 Iran -40000 265.1216_0.58294.1526_0.79 Kaempferol-di-glucoside: C27H30O16 (611.1611) {287} Iran Iran Kashmir -10000 Kashmir -50000 Iran 2 Iran 611.157_4.14 -20000-60000 -40000 -20000 0 20000 40000 280.1373_0.73 Thiamine (B1): C12H17N4OS (265.1123) {144, 122} 1.00206 * t[1] 169.1211_3.66 Vanillic acid: C8H8O4 (169.0601) {151, 105} -30000 Kashmir X[1] = 0.273 R2Xo[1] = 0.156 Ellipse:Kashmir Hotelling's T2 (95%) -40000 265.1216_0.58307.1003_4.95 Kaempferol-di-glucoside:EGC: C15H14O7 (307. C27 0818)H30O {263,16 (611.1611) 139} {287} -50000 331.173_3.66 Picrocrocin: C16H26O7 (331.1757) {185, 151} -60000 -40000 -20000 0 20000 40000 611.157_4.14 1.00206 * t[1] 369.15_2.95 Tomentogenin: C21H36O5 (369.2641) {232} X[1] = 0.273 Iran R2Xo[1] vs = 0.156 Kashmir ParEllipse: Hotelling's T2 (95%) 511.1749_4.94 Iran vs. Kashmir Par vs. + CROCOMICS ORBI_correct_filtering_NEW_2_theIran vs Kashmir latest.M42 (OPLS-DA),Par Iran vs Kashmir Par 252.1061_0.59 EDO: C15H22O2 (MNH4 = 252.1963) {140} 294.1526_0.79 280.1373_0.73 1 2 CROCOMICS ORBI_correct_filtering_NEW_2_the latest.M42 (OPLS-DA), Iran vs Kashmir Par 265.1216_0.58 Thiamine (B1): C12H17N4OS (265.1123) {144, 122}

0.5 611.157_4.14 Kaempferol-di-glucoside: C27H30O16 (611.1611) {287}

1

0

0.5

-0.5

0

-1 -0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15 -0.5 p[1] R2X[1] = 0.273

-1 *-0.2 The calculated -0.15 -0.1 mass -0.05 values 0 correspond 0.05 0.1 to 0.15 the protonated molecular ion (MH+) ion or the adduct ion (e.g., with Na or K) shown in p[1] + * Theparenthesis. calculated1 massThe fragmentR2X[1] values = 0.273 ions correspond for the selected to the precursor protonated ions are molecular shown in curly ion brackets. (MH )2 ionIn case or the of thiamine, adduct the ion M +(e.g.,ion is with observed Na or K) showninstead in parenthesis. of MH+, as the1 The compound fragment has a ions fixed for positive the selected charge. Abbreviations: precursor ions HTCC: are Hydroxy- shownβ -cyclocitral;in curly brackets. EGC: Epigallocatechin; 2 In case of EDO:thiamine, Epoxybisabola-7(14),10-dien-2-one. the* The M +calculated ion is observed mass values instead correspond of MH+, as to the the compound protonated has molecular a fixed positiveion (MH charge.+) ion or Abbreviations the adduct ion: HTCC:(e.g., with Hydroxy- Na or K)β- cyclocitral;shown in parenthesis. EGC: Epigallocatechin;1 The fragment ED ionsO: Epoxybisabola-7(14),10-dien-2-one. for the selected precursor ions are shown in curly brackets. 2 In case of thiamine, the M+ ion is observed instead of MH+, as the compound has a fixed positive charge. Abbreviations: HTCC: Hydroxy-β- cyclocitral; EGC: Epigallocatechin; EDO: Epoxybisabola-7(14),10-dien-2-one. The mapping of the chemical potential of saffron according to its geographical spread is of significant importance in the effort to understand evolutional pressure exerted on the species,The as mapping well as toof providethe chemical a chemotaxonom potential of saffronic tool accordingtowards the to distributionits geographical of variants spread aroundis of significant the world importance (Figure 2). in Thethe effortcontent to understandof the active evolutional secondary pressuremetabolites exerted has anon ap-the parentspecies, effect as well on asthe to quality provide as a well chemotaxonom as the medicalic tool properties towards of the saffron. distribution Furthermore, of variants the extremearound thecost world of the (Figure Crocus sativus2). The L.content stigmas, of consideredthe active secondary as the most metabolites valuable spice, has an man- ap- datesparent their effect accurate on the qualityfingerprinting as well inas orderthe medical to control properties its quality. of saffron. In order Furthermore, to capture the chemicalextreme cost space of involved,the Crocus as sativus well as L. to stigmas, compare considered the species as in the a holisticmost valuable manner, spice, an HRMS man- metabolomicsdates their accurate approach fingerprinting was taken. in order to control its quality. In order to capture the chemicalSpecimens space involved,from seven as well representative as to compare regions the species capturing in a holistic saffron’s manner, biodiversity an HRMS aroundmetabolomics the world, approach namely was Iran, taken. Greece, Italy, Afghanistan, Kashmir, Morocco, and India wereSpecimens analyzed. Thefrom pairwise seven comparisonrepresentative of al lregions possible capturingcombinations saffron’s of the saffronbiodiversity spec- imenaround was the employed, world, namely as this Iran, approach Greece, effect Italy,ively Afghanistan, highlights Kashmir, the differences Morocco, between and India spe- cieswere regardless analyzed. ofThe their pairwise magnitude. comparison In the ofcase all ofpossible a “total” combinations comparison, of the the model saffron would spec- beimen dominated was employed, by the largestas this variance,approach andeffect thivelyerefore highlights would be the biased differences towards between the species spe- withcies regardless the largest of pairwise their magnitude. differences. In Figurethe case 2 depictsof a “total” the regionscomparison, of collected the model specimens would ofbe saffrondominated samples. by the The largest associated variance, clusters and tharereforee illustrated would and be interconnectedbiased towards inthe different species colors,with the such largest as pairwisethose between differences. Greece Figure and 2Morocco depicts thewith regions saffron of collectedsamples inspecimens orange, whereasof saffron those samples. between The Indiaassociated and Afghan clusters specimens are illustrated are indicated and interconnected by green. These in different inter- connectionscolors, such haveas those been betweenrevealed inGreece the multivariate and Morocco statistical with saffronanalysis samples (Figure 3).in orange, whereas those between India and Afghan specimens are indicated by green. These inter- connections have been revealed in the multivariate statistical analysis (Figure 3).

Molecules 2021, 26, x FOR PEER REVIEW 9 of 16

Molecules 2021, 26, x FOR PEER REVIEW 9 of 16 Molecules 2021, 26, 2180 9 of 16

Figure 2. Regions of collected specimens of saffron samples along with their associated cluster.

2.2. Multivariate Statistical Analysis In order to gain insight into the chemical space covered by the genus, as well as to discover trends and spot any possible outliers, a Principal Component Analysis (PCA) analysis was performed employing Pareto scaling. No significant clustering was apparent, but also no outlier values were detected. The R2 was 0.707 with the Q2 being 0.49 (7-fold cross validation). In order to further explore potentially significant markers among the samples, orthogonal Projections to Latent Structures Discriminant Analysis (oPLS-DA) was applied to enhance separation among the groups in PCA. The oPLS-DA algorithm was used in order to explore for underlying associations existing in the data, as it is con- sidered a more efficient discriminating algorithm. Using Par scaling, clear clustering has been observedFigureFigure 2. 2. showingRegions Regions of offive collected collected clusters specimens specimens of samples, of of saffron saffron as depicted samples samples in along along Figure with with 3. their their associated associated cluster. cluster.

2.2. Multivariate Statistical Analysis In order to gain insight into the chemical space covered by the genus, as well as to discover trends and spot any possible outliers, a Principal Component Analysis (PCA) analysis was performed employing Pareto scaling. No significant clustering was apparent, but also no outlier values were detected. The R2 was 0.707 with the Q2 being 0.49 (7-fold cross validation). In order to further explore potentially significant markers among the samples, orthogonal Projections to Latent Structures Discriminant Analysis (oPLS-DA) was applied to enhance separation among the groups in PCA. The oPLS-DA algorithm was used in order to explore for underlying associations existing in the data, as it is con- sidered a more efficient discriminating algorithm. Using Par scaling, clear clustering has been observed showing five clusters of samples, as depicted in Figure 3.

Figure 3. Orthogonal Projections to Latent Structures Discriminant Analysis (oPLS-DA) (Par scaling) of the seven saffron Figurespecies 3. Orthogonal examined. Projections to Latent Structures Discriminant Analysis (oPLS-DA) (Par scaling) of the seven saffron species examined. 2.2. Multivariate Statistical Analysis In order to gain insight into the chemical space covered by the genus, as well as to discover trends and spot any possible outliers, a Principal Component Analysis (PCA) analysis was performed employing Pareto scaling. No significant clustering was apparent, but also no outlier values were detected. The R2 was 0.707 with the Q2 being 0.49 (7-fold cross validation). In order to further explore potentially significant markers among the samples, orthogonal Projections to Latent Structures Discriminant Analysis (oPLS-DA) was applied to enhance separation among the groups in PCA. The oPLS-DA algorithm was used in order to explore for underlying associations existing in the data, as it is considered a more efficient discriminating algorithm. Using Par scaling, clear clustering has been observed showing five clusters of samples, as depicted in Figure3. Figure 3. Orthogonal ProjectionsThus, to Latent each one Structures of the species Discriminant found Analysis in Italy Iran(oPLS-DA) and Kashmir (Par scaling were) allocated of the to unique seven saffron speciesclusters examined. as their only members, while Greece formed a cluster with Morocco, and Indian

Molecules 2021, 26, 2180 10 of 16

and Afghan saffron species formed another cluster. The model exhibited excellent fitting (R2 = 0.896) and predictive power (Q2 = 0.688). In order to focus on differentiating metabolites between species, all pairwise oPLS- DA models between the five groups were constructed. Ten models were constructed as shown in Table1. All models were validated by permutation testing, whereas ANalysis Of VAriance testing of Cross-Validated predictive residuals (CV-ANOVA) was used to verify the statistical significance of the model (p < 0.05). To verify the validity of the multivariate analysis concerning the generated models, the Hottelings T2 and the DModX were evaluated, and were considered as valid when no value exceeds the d-critical level set to 0.05. The residuals normality was also considered and examined for values deviating from normality. In order to discover the most influential features for the construction of each model, the corresponding S-plot was evaluated in every case, where the most differentiated metabolites for each compared group can be distinguished. Therefore, the Paretto based models were considered, whereas the VIP values were also considered in the process. As a rule of thumb, the five major features from each group of each pairwise comparison were investigated. All features identified in the manuscript are at identification level four [34]. The big three MS methodologies employed (MS, MS/MS and HR MS) gave access to corresponding fragmentation used for the assignment of probable structure through MS/MS. Features were attributed to metabolites based on multiple criteria, i.e., that the accurate mass should not deviate more than 10 ppm from its theoretical value, the isotopic pattern should show a score of >90, while the MS/MS fragments should be present with unit resolution (as they were acquired in the linear ion trap using a parallel scan). The second column in Table1 lists the accurate protonated molecular ions MH + with their respective retention times, whereas the third column lists the putative metabolites and the corresponding fragment ions (in brackets) obtained in the linear ion trap using a parallel scan. The metabolites tabulated in Table1 are the ones that are upregulated in the first pair (e.g., Greece and Morocco) and downregulated in the counterpart pair of the comparison (e.g., India and Afghan). It should be noted that there was no need to employ crocin standards available in our laboratory for the identification of putative metabolites, because none of them was identified to be significantly differentiated between the saffron samples analyzed, thus not assisting the discrimination of their geographical origin.

2.3. Geographical Region Differentiation The results show that saffron samples were differentiated according to the geograph- ical region of their collection. Interestingly, crocus from distant areas e.g., Greece and Morocco, exhibit more pronounced similarities compared to neighboring regions such as Greece and Italy. This could be attributed to the pivotal impact of microclimatic conditions rather than considering the wider geographical area of cultivation. The Moroccan climate is typically Mediterranean, resembling the Greek weather, even in the Atlantic coast of the country. Nevertheless, the proximity of Greece to Italy and the fact that they are both Mediterranean countries should indicate a large degree of similarity for the species. The Italian saffron shows the same degree of differentiation to the Greek, Moroccan and Iranian species. This reflects that the geoclimatic characteristics, along with the different prepa- ration practices followed in the cultivation area determine the chemical composition of the final product [14,31]. The Greek saffron is collected in a very narrow area (a village in Macedonia province called Krokos from the name of the plant) where the conditions are likely to be the same when compared to the conditions in the collection area of Morocco. Indeed, this is also reflected to the Asian derived species. Thus, the Indian and Afghan saf- fron are more similar to the Greek and Moroccan species in terms of chemical profile when compared to either Kashmir region or their Iranian counterparts. It should be noted that the Iranian and Kashmir species are more differentiated in terms of chemical components content, despite their proximity. Another issue that should be noted is the genetic profile of the species. Considering that they are cultivated rather than native, it seems that the phylogenetic associ- Molecules 2021, 26, 2180 11 of 16

ation is more closely related to the human intervention than to their historically driven distribution. Thus, an assumption that needs further verification is that the Iranian or the Kashmir branch were transferred by merchants to the countries that were in financial contact. The Iranian/Middle East axis has a strong impact on the human financial rela- tions, and it seems that the same holds true for the Kashmir/India branch. The Crocus sativus L. species were cultivated and integrated in the areas from the Mediterranean basin to Iran/Middle East and India/Kashmir, and both their similarities and differences clearly reflect the international trade and financial relations between countries that were geographically distant.

2.4. Saffron Components in the Saffron Samples from Various Regions Saffron contains more than 150 volatile and aroma-yielding compounds. It also has a number of nonvolatile active components [10], many of which are carotenoids, including zeaxanthin, lycopene, and various α- and β-carotenes. The wealth (plethora) of chemical components in saffron poses complexity for its analysis. Several chromatographic and mass spectrometric methods have been developed for the quantitation of the main bioactive ingredients of saffron, such as crocins and picrocrocin [14,35–38]. The content of the active secondary metabolites has an effect on the quality and efficacy of saffron [14,26]. In view of the limitations of other techniques, ultra-performance liquid chromatography (UPLC-HR MS) has been considered to be the most suitable method to analyze the constituents of saffron extracts. In our study, UPLC-HR MS provided the metabolomic profile of the saffron samples affording high sensitivity and retention time reproducibility. The UPLC-HR MS and multivariate statistical analysis were combined to analyze saffron stigmas. Our results showed that chemical characteristics of saffron were apparently diverse, which mainly arose from the different geoclimatic characteristics inherent to the territory of cultivation. Moreover, changes in the preparation procedures, i.e., flower collection, separation/drying and conservation of stigmas, may strongly modify the final composition of chemical components present in the stigma.

2.5. Marker Discriminating Power In order to discover generalized markers of discriminating the saffron species, the cross-tab (Table2) has been created. The intention was to identify a single feature or even a couple that could differentiate a group from its counterparts. There were two clusters, those of Indian-Afghan and those in the Greek-Morocco saffron that were considered as belonging to the same group. Thus, no generalized marker was found for discriminating the Indian- Afghan saffron from the other groups, however the 252.1061_0.59 and 472.1734_3.24 ions could differentiate the four of the five groups. The former mass signal corresponds to the + (M+NH4) adduct ion of 3,4-Epoxybisabola-7(14),10-dien-2-one (EDO; C15H22O2) marker, + whereas the latter corresponds to the (M+Na) adduct ion of astragalin (C21H20O11). Conversely, the putative metabolite of tomentogenin [12] with MH+ signal 369.15_2.95 could be used to discriminate three of the five groups in the case of the Iranian stigmas. In the case of the Greek-Morocco saffron, the results were even less general, and no molecular species was found to show such a capacity. The Italian varieties could be clearly separated from the others using the 169.1211_3.66 and the 353.1548_3.66 ions corresponding to the MH+ of vanillic acid and the (M+Na)+ adduct ion of picrocrocin, respectively. Finally, the 611.157_4.14 ion corresponding to the MH+ ion of kaempferol-di-glucoside was found it can be employed to distinguish the Kashmir saffron. These results indicate that a combination of markers should be employed which necessitates the use of hyphenated separation methodologies (e.g., LC–MS) for achieving the screening of saffron extracts, as well as probing the saffron for the presence of adulterants [26]. Molecules 2021, 26, 2180 12 of 16

Table 2. Markers discovered towards the differentiation of the Crocus sativus L. stigmas from various destination sources. In bold are marked the common metabolites across at least two destination sources whereas underlined are the ones that could differentiate between all species.

IND_AFG GR_MOR IR IT KSH 307.1003_4.95 151.1105_3.67 151.1105_3.67 151.1105_3.67 365.1196_3.36 169.1211_3.66 169.1211_3.66 151.1105_4.86 IND_AFG 369.15_2.95 169.1211_4.86 353.1548_3.66 169.1211_3.66 383.1298_2.85 369.15_2.95 365.1196_3.36 169.1211_4.86 472.1734_3.24 472.1734_3.24 369.15_2.95 611.157_4.14 252.1061_0.59 123.1156_3.66 169.1211_3.66 151.1105_3.67 367.135_3.42 151.1105_3.67 177.1629_9.84 151.1105_4.86 GR_MOR 369.1508_1.49 169.1211_3.66 353.1548_3.66 169.1211_3.66 305.0824_0.71 169.1211_4.86 167.0115_24.08 169.1211_4.86 167.1057_1.49 369.15_2.95 265.1216_0.58 611.157_4.14 252.1061_0.59 155.1057_2.77 169.1211_3.66 252.1061_0.59 294.1526_0.79 169.1212_4.39 177.1629_9.84 294.1526_0.79 IR 280.1373_0.73 294.1526_0.79 353.1548_3.66 611.157_4.14 265.1216_0.58 317.1579_2.81 167.0115_24.08 280.1373_0.73 606.229_4.15 265.1216_0.58 265.1216_0.58 265.1216_0.58 139.1108_7.13 123.1156_3.66 139.1108_7.13 169.1212_4.39 169.1212_4.39 151.1105_3.67 169.1212_4.39 252.1061_0.59 IT 252.1061_0.59 169.1211_4.86 369.1508_1.49 369.1508_1.49 369.1508_1.49 348.1994_3.66 203.082_4.75 611.157_4.14 203.082_4.75 369.15_2.95 171.1005_3.58 203.082_4.75 155.1057_2.77 155.1057_2.77 169.1211_3.66 169.1211_3.66 307.1003_4.95 183.1005_3.33 307.1003_4.95 307.1003_4.95 KSH 317.1579_2.81 307.1003_4.95 331.173_3.66 331.173_3.66 331.173_3.66 317.1579_2.81 369.15_2.95 353.1548_3.66 364.1944_2.96 331.173_3.66 511.1749_4.94 369.15_2.95

3. Materials and Methods 3.1. Chemical Reagents and Standards Methanol (MeOH) and acetonitrile (ACN) of HPLC grade were supplied from were supplied from Carlo Erba (Milano, Italy) and Fisher Scientific (Pittsburgh, PA, USA), respectively. Trifluoroacetic acid (TFA) was obtained from Acros Organics (Fair Lawn, NJ, USA) and water was purified using Milli-Q (RG) filter systems from Millipore Corporation (Billerica, MA, USA).

3.2. Sample Collection Crocus Sativus L. dried styles (saffron) were kindly supplied by the Cooperative De Safran, (Krokos Kozanis, West Macedonia, Greece). The saffron samples from Morocco, Italy, Iran, India, Kashmir and Afghanistan were purchased from Sahar Saffron Company (Cleveland, OH, USA). All samples were kept refrigerated at 5 ◦C until analysis. Molecules 2021, 26, 2180 13 of 16

3.3. Sample Preparation 50 mg of saffron stigmas were soaked in methanol water 1:1 (v/v) for 200 days in dark under ambient temperature. The stigmas were extracted with 10mL MeOH:water (1:1, v/v) for 24 h at 25 ◦C in the absence of light with continuous stirring, and then centrifuged, filtered through a 0.2-µm filter and evaporated to dryness employing a Speed Vac system (Labconco Corp., Kansas City, MO, USA). Samples were reconstituted in MeOH:water (1:1, v/v), transferred to 1.5 mL autosampler vials and an appropriate volume was injected to the LC–MS system.

3.4. UPLC—HR MS Metabolomics Analysis A quality control sample (QC) taken from all samples was prepared in order to periodically assess the reproducibility of the measurements. The separation of the an- alytes contained in the saffron samples was achieved with a Fortis UPLC C18 column (2.1 mm × 100 mm, 1.7 µm, Fortis Technologies Ltd., Cheshire, UK). The hyphenated LC- HRMS system comprised of an Accela UHPLC equipped with an autosampler, a vacuum degasser, a binary pump and a temperature-controlled column (Thermo Scientific, Ger- many) coupled to an Orbitrap Discovery XL, which was equipped with an IonMAX ion source (Thermo Scientific, Bremen, Germany). The mobile phase consisted of 0.1% aq. formic acid (v/v) (solvent A) and 0.1% formic acid in LC–MS grade ACN (v/v) (solvent B). The gradient program was for solvent B: 5% at 0 min, 5% at 3 min, 95% at 24 min, 95% at 26 min, 5% at 28 min, 5% at 30 min. The overall analysis time spanned for 30 min, whereas the injection volume was 5 µl keeping a flow rate of 400 µl min−1. The positive ioniza- tion ESI mode was used using a mass range of 100–1000 amu. The “big three” approach, employing parallel scans, was used. The samples were centrifuged using a Mikro 200R centrifuge (Hettich Lab Technology, Tuttlingen, Germany), and for the solvent evaporation was performed on a GeneVac HT-4X EZ-2 series evaporator Lyospeed ENABLED (Genevac Ltd., Ipswich, UK).

3.5. Statistical Analysis The raw data were imported to the Mzmine 2.51 [39] and the Automated Data Anal- ysis Pipeline (ADAP) pipeline was employed, using the wavelets methodology for the chromatogram deconvolution as implemented to ADAP [40]. The feature list was analyzed by SIMCA 14.1 (Umetrics, Umea, Sweden) for the construction of the multivariate models, whereas all univariate analyses were performed by Jamovi. The multiple correction for t-testing, which used the false discovery rate approach, was performed by the qvalue R package [41]. All multivariate models were validated by n-fold as well as by permutation testing, employing 100 random permutations. The CV-ANOVA was used with p < 0.05 to verify the validity of the produced models [42].

3.6. Feature Identification The peak list MS features were annotated using the KEGG, CheBI, MetaCyc, LIPIDMAPS, FOR-IDENT, and HMDB libraries. The Met-Frag online version was employed [43] for the annotation and an additional home-assembled MS library was also used.

4. Conclusions The metabolomic profiling of saffron (Crocus sativus L. stigma) from different geo- graphical regions, employing UPLC-HR MS analysis combined with multivariate statistical analysis, provided evidence that the phytochemical content in the samples of C. sativus L. was diverse in the different countries of origin. This diversity apparently arises from the different geoclimatic characteristics of the area of cultivation in combination with the distinct preparation procedures in the respective countries. Our results indicated that there are characteristic ions that could differentiate a certain group from its counterparts such as the Indian-Afghan and the Greek-Morocco saffron samples that could be considered to belong to the same group. The metabolomics approach was deemed to be the most suitable Molecules 2021, 26, 2180 14 of 16

choice in order to evaluate the enormous array of putative metabolites of saffron, and thus provide a comparative phytochemical screening among the saffron samples studied. In addition, this UPLC-HR MS-based metabolomics approach could be also employed for probing possible adulteration of saffron samples. In view of saffron’s health-promoting effects through the modulation of various biological and physiological processes, the se- lection of the appropriate saffron sample guided by the respective metabolomic profiling could be an important step. That, in turn, may aid the emerging popularity and interest in alternative medicine-based treatments within health practices.

Author Contributions: E.G. and A.T.; Conceptualization; E.G., N.S.K. and A.T.; methodology devel- opment, E.G. and N.S.K. formal analysis, E.G.; statistical analysis, E.G. and A.T.; coordinated and supervised the study, E.G., N.S.K. and A.T.; writing—original draft preparation. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the “Large Scale Cooperative Project” (TreatAD, 09SYN-21- 1003) co-financed by the European Social Fund (ESF) and the General Secretariat for Research and Technology in Greece. Institutional Review Board Statement: Not available. Informed Consent Statement: Not available. Data Availability Statement: The data presented in this study are available in this article. Acknowledgments: We would like to thank Günter Allmaier and Ernst Pittenauer from the Institute of Chemical Technologies and Analytics, Vienna University of Technologies and Analytics, Vienna, Austria for their helpful discussions on the crocus project. Conflicts of Interest: The authors declare no conflict of interest. Sample Availability: The samples are not available from the authors.

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