Screening for Triterpenoid Saponins in Plants Using Hyphenated Analytical Platforms

molecules

Article Screening for Triterpenoid Saponins in Plants Using Hyphenated Analytical Platforms

Bekzod Khakimov 1,2,*, Li Hong Tseng 3, Markus Godejohann 3, Søren Bak 2 and Søren Balling Engelsen 1

1 Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 16, DK-1958 Frederiksberg C, Denmark; [email protected] 2 Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Rolighedsvej 16, DK-1958 Frederiksberg C, Denmark; [email protected] 3 Bruker BioSpin GmbH, 76287 Rheinstetten, Germany; [email protected] (L.H.T.); [email protected] (M.G.) * Correspondence: [email protected]; Tel.: +45-353-281-84

Academic Editors: Vassilios Roussis and Efstathia Ioannou Received: 14 October 2016; Accepted: 19 November 2016; Published: 24 November 2016

Abstract: Recently the number of studies investigating triterpenoid saponins has drastically increased due to their diverse and potentially attractive biological activities. Currently the literature contains chemical structures of few hundreds of triterpenoid saponins of plant and animal origin. Triterpenoid saponins consist of a triterpene aglycone with one or more sugar moieties attached to it. However, due to similar physico-chemical properties, isolation and identification of a large diversity of triterpenoid saponins remain challenging. This study demonstrates a methodology to screen saponins using hyphenated analytical platforms, GC-MS, LC-MS/MS, and LC-SPE-NMR/MS, in the example of two different phenotypes of the model plant Barbarea vulgaris (winter cress), glabrous (G) and pubescent (P) type that are known to differ by their insect resistance. The proposed methodology allows for detailed comparison of saponin profiles from intact plant extracts as well as saponin aglycone profiles from hydrolysed samples. Continuously measured 1D proton NMR data during LC separation along with mass spectrometry data revealed significant differences, including contents of saponins, types of aglycones and numbers of sugar moieties attached to the aglycone. A total of 49 peaks were tentatively identified as saponins from both plants; they are derived from eight types of aglycones and with 2–5 sugar moieties. Identification of two previously known insect-deterrent saponins, hederagenin cellobioside and oleanolic acid cellobioside, demonstrated the applicability of the methodology for relatively rapid screening of bioactive compounds.

Keywords: time slice LC-SPE-NMR/MS; GC-MS; LC-MS/MS; triterpenoid saponins; Barbarea vulgaris

1. Introduction Triterpenoid saponins consist of a triterpene aglycone, with 30 carbon atoms, with one or more sugar moieties (including hexoses, methylpentoses, and pentoses) attached to the aglycone [1]. Triterpenoid saponins are secondary metabolites synthesized in plant and mammalian cells. Several studies have reported on the role of triperpenoid saponins as natural defence compounds in plants [2–5], and some members of triterpenoid saponins have also been found to possess beneﬁcial pharmacological properties [6–8]. The literature contains structures of several hundred saponins isolated from various sources including plants and animals. Saponins exhibit diverse biological activities, and a number of studies investigating biosynthetic pathways and roles of this class of metabolites in the growth, development and physiology of host organisms has increased [9–11]. Recently triterpenoid saponins also became central target compounds for developing natural pesticides and insecticides for crop management in agriculture. Saponins are amphiphilic in nature, due to the

Molecules 2016, 21, 1614; doi:10.3390/molecules21121614 www.mdpi.com/journal/molecules Molecules 2016, 21, 1614 2 of 19 hydrophilic sugar moieties and the lipophilic triterpenoid aglycone. For this reason, the extraction and qualitative and quantitative analysis of saponins might be challenging from complex sample matrices such as plants and must be optimized prior to qualitative and quantitative analysis. This often includes a selection of an appropriate solvent composition depending on the physico-chemical properties of the investigated saponins. Despite the fact that LC-MS is the method of choice for the analysis of saponins, often the chromatographic resolution power is not sufficient to separate structurally similar saponins. The LC-MS analysis of saponins may become even more challenging when samples contain a wide range of hyper-glycosylated saponins (e.g., saponins with three and more sugar moieties) with a similar molecular weight (MW) and/or glycosylation patterns. Nevertheless, LC-MS provides the richest information about the chemical structures of saponins obtained directly from complex mixtures. Structural characterization of saponins can be further improved when tandem mass spectrometry, for example LC-MS/MS, is applied [12]. This will provide unique fragmentation patterns of saponins including the number and type of sugar moieties attached to the aglycone. While GC-MS analysis of saponins is impossible due to high MW and boiling points of the molecules, the triterpenoid aglycones are commonly analysed using the GC-MS. The lipophilic nature and thermal stability of triterpenoid aglycones makes them suitable for GC-MS analysis providing high detection sensitivity and a characteristic electron impact-mass spectrum (EI-MS). In order to screen for triterpenoid aglycone molecules, samples are often hydrolysed to cleave off sugar moieties attached to the aglycone. This procedure is usually performed either by chemical, using acidic or alkaline, or enzymatic hydrolysis. Such protocols are known to be complex, time consuming and require careful optimization in order to improve method reproducibility. Following hydrolysis, the free and liberated aglycone containing samples are derivatized using trimethylsilylation before injection into a GC-MS [13]. Nuclear magnetic resonance (NMR) spectroscopy is another complementary analytical platform to GC-MS and LC-MS for the analysis of saponins. Despite the fact that the sensitivity of NMR is inferior to GC-MS and LC-MS, it can, with practically no sample preparation, quantitatively detect all metabolites, irrespective of their volatility, polarity, size, and chemical structure, provided that they possess chemical elements with non-zero spin quantum number, such as 1H, 13C, 31P, and 15N. Since the proton is the most suitable nucleus (most sensitive, producing sharp and informative NMR signals and allowing rapid data acquisition) for NMR and it is a part of the majority of metabolites, NMR based metabolomics is most often performed using proton nuclei. The main advantages of NMR over mass spectrometry based methods are that it is non-destructive, fast, reproducible and requires much less labour for sample preparation. However, due to the relatively low concentration of saponins when compared to other metabolites present in complex plant tissue matrices, the detection of saponins using NMR can be challenging without prior sample cleaning and saponin enrichment. This study demonstrates the application of hyphenated analytical platforms, including GC-MS, LC-MS/MS, NMR and time slice LC-SPE-NMR/MS, for the qualitative and relative quantitative analysis of triterpenoid saponins in plant leaf extracts of B. vulgaris. Two different genotypes of B. vulgaris plants, glabrous (G-type) and pubescent (P-type), that differ by their saponin content and insect resistance toward Phyllotreta nemorum, large striped flea beetle, were screened using a global metabolite extract and a saponin enriched extract. The study shows that the complete structure elucidation of saponins directly from complex samples mixtures is challenging even when multiple hyphenated platforms are used. However, a significant amount of structural information, MW of aglycones and number and types of sugar moieties can be gained by combining the information obtained from the different platforms.

2. Results and Discussion

2.1. Untargeted Metabolomics for Analysis of Triterpenoid Saponins in B. vulgaris In this study, it was attempted to develop a single metabolomics protocol covering as broad range of metabolites as possible, including saponins, from a leaf tissue of B. vulgaris. A single metabolite Molecules 2016, 21, 1614 3 of 19 extraction method based on 85% methanol was developed and employed for three different analytical platforms,Molecules including 2016, 21, 1614GC-MS, LC-MS and NMR (Figure1)[ 14]. A previous LC-MS metabolomics3 of 19 study performed on the same methanol extracts of 160 F2 plants derived from a crossing of parental G- andperformed P-type onB. the vulgaris same methanolplants resulted extracts inof 160 the F2 identiﬁcation plants derived of from saponins a crossing that of wereparental related G- and to the plants’P-type resistance B. vulgaris against plants the resultedP. nemorum in thelarvae identification [2]. The of follow saponins up studythat were further related scrutinized to the plants’ the same LC-MSresistance data using against a differentthe P. nemorum approach larvae based [2]. The on follow PARAllel up study FACtor further Analysis scrutinized 2 (PARAFAC2) the same LC-MS [15 ,16], whichdata allowed using a the different deconvolution approach based of even on PARAllel very elusive FACtor LC-MS Analysis peaks 2 (PARAFAC2) directly from [15,16], the which raw data. allowed the deconvolution of even very elusive LC-MS peaks directly from the raw data. This approach This approach enabled the tentative identiﬁcation of saponins responsible for discrimination of F2 enabled the tentative identification of saponins responsible for discrimination of F2 plants based on plantstheir based insect on resistance their insect and further resistance insights and into further interactions insights between into the interactions plants’ metabolome between and theinsect plants’ metabolomeresistance and [17]. insect Among resistance these metabolites, [17]. Among hederagenin these metabolites, cellobioside, hederagenin oleanolic cellobioside,acid cellobioside, oleanolic acid cellobioside,4-epihederagenin 4-epihederagenin cellobioside, gypsogenin cellobioside, cellobio gypsogeninside, and a cellobioside, few other unknown and a few saponins other unknownwere saponinsfound were to be found the most to bedeterrent the most towards deterrent the larvae towards of P. thenemorum larvae. This of P. analysis nemorum was. Thisbased analysis on PLS was basedregression on PLS regression relating the relating metabolomics the metabolomics and insect resistance and insect data. resistance data.

FigureFigure 1. An 1. An overview overview of of the the studystudy design. design. A global A global metabolite metabolite extract extract based on based 85% onmethanol 85% methanol was was employedemployed to screen for for triterpenoid triterpenoid saponins saponins us usinging three three different different analytical analytical platforms, platforms, GC-MS, GC-MS, LC-MS/MSLC-MS/MS and and NMR. NMR. Saponin Saponin enriched enriched extractsextracts base basedd on on SPE SPE based based purificati puriﬁcationon of the of 80% the 80%methanol methanol extractextract were were screened screened using using LC-SPE-NMR/MS, LC-SPE-NMR/MS, LC-MS/MS, and and GC-MS. GC-MS.

Despite the fact that untargeted LC-MS analysis provides rich metabolomics data, the sensitivity Despiteand selectivity the fact of thata given untargeted method vary LC-MS for analysisdifferent chemical provides classes. rich metabolomics Quantification data, of metabolites the sensitivity and selectivitymay often be of a achallenge given method due to ion vary suppression for different and/or chemical complex classes. sample matrix Quantification effects. The of physico- metabolites maychemical often be properties a challenge of triterpenoid due to saponins, ion suppression including their and/or chemical complex structure, sample molecular matrix size and effects. The physico-chemicalpolarity largely influence properties the ionization of triterpenoid efficiency saponins, of molecules including during their the electrospray chemical structure, ionization molecular(ESI) size andin LC-MS, polarity which largely in turn influence determines the the ionization sensitivity efficiencyof the analysis. of molecules Thus, the duringuse of an the untargeted electrospray ionizationLC-MS (ESI) metabolomics in LC-MS, protocol which may in turn not determinesbe an optimal the approach sensitivity for ofquantification the analysis. of Thus,triterpenoid the use of saponins from complex plant extracts without prior method optimization. The LC-MS method applied an untargeted LC-MS metabolomics protocol may not be an optimal approach for quantification of in this study was developed to emphasise detection of triterpenoid saponins from the 85% methanol triterpenoid saponins from complex plant extracts without prior method optimization. The LC-MS extract of B. vulgaris plant leaves. Tandem mass spectrometry was used for the characterization of methodthe appliedtype and innumber this study of sugar was moieties developed as well to emphasiseas the MW detectionof the triterpenoid of triterpenoid aglycone saponins [14]. This from the 85%approach methanol allowed extract tentative of characterizationB. vulgaris plant of a leaves. total of 73 Tandem saponin masspeaks from spectrometry G- and P-type was B. usedvulgaris for. the characterizationMost of these ofsaponins the type had and an aglycone number MW of sugarof 448, moieties456, 458, 472, as wellor 474 as Da the and MW included of the two triterpenoid to five aglyconesugar [moieties,14]. This primarily approach hexose allowed (162 Da), tentative methylpentose characterization (146 Da), and of pentose a total (132 of Da) 73 saponin[14]. peaks from G- andIn order P-type to B.screen vulgaris for .triterpenoid Most of these aglycones saponins using had GC-MS, an aglycone B. vulgaris MW plant of 448,extracts 456, were 458, 472, or 474hydrolysed Da and included using hydrochloric two to five sugaracid and moieties, the liberated primarily aglycones hexose subsequently (162 Da), methylpentose trimethylsilylated (146 Da), and pentoseusing a new (132 derivatization Da) [14]. method developed based on trimethylsilyl cyanide (TMSCN) [13]. Prior to the GC-MS analysis of B. vulgaris triterpenoid aglycones, the derivatization method was tested on In order to screen for triterpenoid aglycones using GC-MS, B. vulgaris plant extracts were the dilution series of several known triterpenoid aglycone standards including hederagenin, oleanolic hydrolysed using hydrochloric acid and the liberated aglycones subsequently trimethylsilylated acid and betulinic acid, which showed a correlation coefficient (r2) of 0.95 or higher [14]. The unique usingEI-MS a new patterns derivatization and retention method indices developed (RIs) of based triterpenoid on trimethylsilyl aglycones obtained cyanide from (TMSCN) the GC-MS [13]. Prior to theanalysis GC-MS allow analysis for unambiguous of B. vulgaris identification.triterpenoid A aglycones, design of an the experiment derivatization (DoE) approach method waswas testedthen on the dilutionused to seriesoptimize of severalthe protocol known including triterpenoid extracti aglyconeon (solvent standards composition, including time hederagenin,and temperature), oleanolic acid andhydrolyzation betulinic acid,(time, whichtemperature) showed and a correlation trimethylsilylation coefficient (time, (r2 )temperature) of 0.95 or higher parameters. [14]. The A 2 unique7-3 EI-MSfractional patterns factorial and retention experiment indices was (RIs)designed of triterpenoid and all seven aglycones parameters obtained of the pr fromotocol the were GC-MS varied analysis at

Molecules 2016, 21, 1614 4 of 19 allow for unambiguous identiﬁcation. A design of an experiment (DoE) approach was then used to optimize the protocol including extraction (solvent composition, time and temperature), hydrolyzation (time, temperature) and trimethylsilylation (time, temperature) parameters. A 27-3 fractional factorial experimentMolecules was 2016, designed 21, 1614 and all seven parameters of the protocol were varied at three levels4 of resulting 19 in a totalthree of levels 19 experiments resulting in a coveringtotal of 19 asexperiments broad experimental covering as broad domain experime as possiblental domain [14]. as The possible S/N ratio and detection[14]. The S/N reproducibility ratio and detection of the reproducibility aglycones, of hederagenin the aglycones, and hederagenin oleanolic and acid oleanolic were acid evaluated were for all experimentsevaluated for performed all experiments within performed the DoE within and the DoE parameters and the parameters of the optimal of the protocoloptimal protocol were found as follows:were found extraction as follows: (85% extraction methanol, (85% 5methanol, min and 5 min 100 and◦C), 100 acidic °C), acidic hydrolyzation hydrolyzation (1 h(1 andh and 99 ◦C), and trimethylsilylation99 °C), and trimethylsilylation parameters parameters (40 min and (40 40min◦ C).and More 40 °C). detailed More detailed results results on aglycones on aglycones proﬁles of B. vulgarisprofilesare of discussed B. vulgaris inare Section discussed 2.3 in. Section 2.3.

FigureFigure 2. 1D 2. proton 1D proton NMR NMR analysis analysis of of global globalmetabolome metabolome of of leaves leaves of ofG- G-and and P-type P-type B. vulgarisB. vulgaris using using 85% methanol85% methanol extracts. extracts (A. )(A Mean) Mean of of 1D 1D proton proton NMR spectra spectra ofof 10 10G-type, G-type, 10 P-type, 10 P-type, and 3 and mixture 3 mixture (50% G- and 50% P-type plants) samples; (B) Interval based extended canonical variate analysis (iECVA) (50% G- and 50% P-type plants) samples; (B) Interval based extended canonical variate analysis (iECVA) of the raw NMR data containing 23 spectra. The iECVA analysis depicted spectral regions that were of the raw NMR data containing 23 spectra. The iECVA analysis depicted spectral regions that were most discriminative between G- and P-type plants. iECVA was performed on 100 equal size intervals most discriminativeacross the entire betweenNMR spectra. G- and P-type plants. iECVA was performed on 100 equal size intervals across the entire NMR spectra. 1D proton NMR spectra recorded on 85% methanol extracts of G- and P-type B. vulgaris plants 1Das well proton as on NMR their spectracombined recorded samples (50% on 85% G- and methanol 50% P-type) extracts revealed of G- spectral and P-type regionsB. that vulgaris clearlyplants as welldifferentiate as on their the combined two genotypes samples (Figure (50% 2A). G- The and NMR 50% spectra P-type) measured revealed on spectral complex regions plant extracts that clearly represent signals from the most abundant metabolites while signals of low level metabolites remain differentiate the two genotypes (Figure2A). The NMR spectra measured on complex plant extracts undetected or largely hidden by dominating metabolites. In addition, due to the complexity of the representsample signals matrix, from the theNMR most signals abundant derived metabolites from different while molecules signals overlap of low and level may metabolites hamper data remain undetectedinterpretation. or largely Thus, hidden it is almost by dominating impossible to metabolites. identify well resolved In addition, NMR due signals to theof saponins complexity from of the samplesuch matrix, a complex the sample NMR signalsmixture. derivedHowever, fromsignificant different differences molecules in the NMR overlap spectra and of mayG- and hamper P-type data interpretation.plants were Thus, pronounced it is almost in the impossible spectral regions to identify that correspond well resolved to chemical NMR shifts signals of characteristic of saponins from such asignals complex of saponins. sample mixture.These include However, the aliphatic significant region differences (0.7–1.2 ppm in) thewith NMR singlets spectra derived of G-from and the P-type plants were pronounced in the spectral regions that correspond to chemical shifts of characteristic Molecules 2016, 21, 1614 5 of 19 signals of saponins. These include the aliphatic region (0.7–1.2 ppm) with singlets derived from the methyl groups of triterpenoid aglycones and the anomeric region (4.3–4.5 ppm) with doublets correspondingMolecules 2016, to21, anomeric1614 protons of sugar moieties (Figure S1). Despite being crowded and5 of difficult19 to interpret, the spectral region corresponding to 3.6–3.9 ppm, which represent the protons of sugar moietiesmethyl in groups saponins, of triterpenoid also show aaglycones clear difference and the betweenanomeric theregion two (4.3–4.5 genotypes. ppm) Employmentwith doublets of the intervalcorresponding based extended to anomeric canonical protons variate of sugar analysis moieties (iECVA) (Figure S1). [18 Despite] on the being raw crowded NMR dataset and difficult enabled the identificationto interpret, of the spectral spectral regions region thatcorresponding can discriminate to 3.6–3.9 between ppm, which the two represent plant genotypes.the protons of The sugar resulting discriminativemoieties in saponins, spectral regionsalso show mostly a clear represent difference saponinbetween peaksthe two (Figure genotypes.2B). InEmployment addition toof aliphaticthe interval based extended canonical variate analysis (iECVA) [18] on the raw NMR dataset enabled the and anomeric regions, the spectral region from 5.2–5.3 ppm was found to be the most powerful region identification of spectral regions that can discriminate between the two plant genotypes. The resulting β α for discrimination.discriminative spectral In NMR regions spectra mostly of saponinsrepresent derivedsaponin peaks from (Figure-amyrin, 2B). In-amyrin addition and to aliphatic other similar triterpenoidand anomeric aglycones, regions, the the spectral spectral region region of from 5.2–5.3 5.2–5.3 ppm ppm corresponds was found to to the be tripletthe most from powerful the methine groupregion at C12 for positiondiscrimination. (Figure In S1). NMR This spectra suggests of saponins that one derived of the mainfrom β differences-amyrin, α-amyrin between and G- other and P-type plantsimilar saponins triterpenoid might beaglycones, a special the type spectral of aglycone region of and 5.2–5.3 the ppm data corresponds presented in to thisthe triplet study from indicates the that saponinsmethine of group the G-type at C12 plantposition contain (Figure primarily S1). This suβ-amyrin,ggests thatα one-amyrin, of the main and/or differences other similar between aglycones. G- Thisand finding P-type is plant in agreement saponins withmight previousbe a special studies type of that aglycone showed and isolation the data andpresented identification in this study of several saponinsindicates from that the saponins G-type ofB. the vulgaris G-type, whereplant contain all saponins primarily were β-amyrin, based α on-amyrin,β-amyrin and/or such other as hederageninsimilar aglycones. This finding is in agreement with previous studies that showed isolation and identification cellobioside and oleanolic acid cellobioside [3,4,19]. of several saponins from the G-type B. vulgaris, where all saponins were based on β-amyrin such as 2.2.hederagenin Time Slice LC-SPE-NMR/MS cellobioside and oleanolic Experiment acid oncellobioside Saponin Enriched [3,4,19]. Extracts from G- and P-Type B. vulgaris 2.2.In thisTime experimentSlice LC-SPE-NMR/MS 1D proton Experi NMRment spectra on Saponin were measuredEnriched Extracts from from extracts G- and of P-Type post column B. vulgaris enriched SPE fractionsIn this thatexperiment have been 1D proton collected NMR at spectra 1 min were intervals measured during from the extracts entire of LCpost separation column enriched along with massSPE spectrometric fractions that data have obtained been collected from at a parallel1 min inte detectionrvals during using the a entire flow splitter.LC separation A 2D along visualization with of the timemass slicespectrometric LC-SPE-NMR/MS data obtained data from is illustrateda parallel detec in Figuretion using3 in a the flow example splitter. of A the2D saponinvisualization enriched extractof the of thetime G-type slice LC-SPE-NMR/MSB. vulgaris. data is illustrated in Figure 3 in the example of the saponin enriched extract of the G-type B. vulgaris.

Figure 3. A 2D visualization of the time slice LC-SPE-NMR/MS data illustrated in the example of the Figure 3. A 2D visualization of the time slice LC-SPE-NMR/MS data illustrated in the example of the saponin enriched extract of the G-type B. vulgaris. saponin enriched extract of the G-type B. vulgaris. The data acquired on saponin enriched extracts of G- and P-type plants were evaluated for the contentThe data of saponins. acquired Visual on saponin inspection enriched of the extractsmass spectral of G- data and and P-type corresponding plants were NMR evaluated data for the contentmeasured of on saponins. the same Visualchromatogr inspectionaphic interval of the resulted mass spectral in 27 and data 22 andpeaks corresponding from G- and P-type NMR data measuredplants, respectively, on the same that chromatographic were tentatively identified interval as resulted being saponins in 27 and (Tables 22 peaks1 and 2). from The G-time and slice P-type plants,LC-SPE-NMR/MS respectively, thatdata were of the tentatively G-type plant identiﬁed was more as complex being saponins and relative (Tables intensities1 and2 ).of Thesaponins time slice LC-SPE-NMR/MSwere more abundant data when of the compared G-type plantto the wasP-type more plant complex (Figure 4). and Tentatively relative intensitiesidentified saponin of saponins peaks were characterized by their mass spectral fragmentation patterns that illustrated loss of were more abundant when compared to the P-type plant (Figure4). Tentatively identiﬁed saponin adduct ions corresponding to sugar moieties including hexose (162 Da), methylpentose (146 Da), peaksand were pentose characterized (132 Da). by their mass spectral fragmentation patterns that illustrated loss of adduct ions corresponding to sugar moieties including hexose (162 Da), methylpentose (146 Da), and pentose (132 Da).

Molecules 2016, 21, 1614 6 of 19

Table 1. List of tentatively annotated triterpenoid saponins from the time slice LC-SPE-NMR/MS experiment performed on saponin enriched extract of the glabrous (G) type B. vulgaris.

No. RT (min) Aglycone (Da) A Sugar Moieties B Mass Spectra Fragmentation C Anomeric Protons (ppm) D × 2 Methylpentose − − − − − − − 1 27.3 448 × [M1225 H 162 146 162 146 162] = 447 3 Hexose 4.13 (d, J = 7.8) 2 × Methylpentose 4.17 (d, J = 7.8) − 2 27.7 478 1 × Pentose [M1225 − H − 162 − 132 – 162 − 146 − 146] = 477 4.29 (d, J = 7.8) 2 × Hexose 4.21 (d, J = 7.8) 1 × Methylpentose 4.41 (d, J = 7.8) 3 49.5 504 [M − H − 146 − 162 − 162]− = 503 2 × Hexose 973 4.42 (d, J = 7.8) 5.25 * (t, J = 3.4) 4.21 (d, J = 7.8) 1 × Methylpentose 4.41 (d, J = 7.8) 4 50.5 504 [M − H − 146 − 162 − 162]− = 503 2 × Hexose 973 4.42 (d, J = 7.8) 5.25 * (t, J = 3.4) 4.21 (d, J = 7.8) 1 × Methylpentose 5 51.2 504 [M − H − 162 − 146 − 162]− = 503 4.39 (d, J = 7.8) 2 × Hexose 973 5.25 * (t, J = 3.4) 4.41 (d, J = 8.0) − 6 52.5 649 ** 3 × Hexose [M1135 − H − 162 − 162 − 162] = 649 4.43 (d, J = 8.0) 5.55 (d, J = 8.2) 4.41 (d, J = 8.0) 7 56.2 649 ** 2 × Hexose [M − H − 162 − 162]− = 649 973 5.52 (d, J = 8.2) 4.41 (d, J = 8.0) 8 59.5 649 ** 2 × Hexose [M − H − 162 − 162]− = 649 973 5.52 (d, J = 8.2) 4.34 (d, J = 7.8) 4.41 (d, J = 8.0) 9 60.6 472 4 × Hexose [M − H − 162 − 162 − 162 − 162]− = 471 1119 4.43 (d, J = 7.8) 5.55 (d, J = 8.0) 4.34 (d, J = 7.8) 4.41 (d, J = 8.0) 10 61.2 633 3 × Hexose [M − H − 162 − 162 − 162]− = 633 1119 4.43 (d, J = 7.8) 5.55 (d, J = 8.0) Molecules 2016, 21, 1614 7 of 19

Table 1. Cont.

No. RT (min) Aglycone (Da) A Sugar Moieties B Mass Spectra Fragmentation C Anomeric Protons (ppm) D 4.27 (d, J = 7.8) 4.34 (d, J = 7.8) − 11 63.4 472 3 × Hexose [M957 − H − 162 − 162 − 162] = 471 4.41 (d, J = 7.8) 5.24 * (t, J = 3.4) 5.53 (d, J = 8.0) 4.27 (d, J = 7.8) − 12 64.0 458 3 × Hexose [M943 − H − 162 − 162 − 162] = 457 4.34 (d, J = 7.8) 4.41 (d, J = 7.8)

− 4.17 (d, J = 7.8) 13 66.2 619 ** 2 × Hexose [M943 − H − 162 − 162] = 457 4.27 (d, J = 7.8) 4.34 (d, J = 7.8) 4.35 (d, J = 7.8) − 14 66.6 472 3 × Hexose [M957 − H − 162 − 162 − 162] = 471 4.41 (d, J = 7.8) 5.20 * (t, J = 3.4)

− 4.18 (d, J = 7.8) 15 67.2 458 3 × Hexose [M943 –H − 162 − 162 − 162] = 457 4.26 (d, J = 7.8) 4.35 (d, J = 7.8) 4.41 (d, J = 7.8) − 16 67.6 472 4 × Hexose [M1119 − H − 162 − 162 − 162 − 162] = 471 5.25 * (t, J = 3.4) 5.38 (d, J = 8.3)

− 17 68.2 458 3 × Hexose [M943 − H − 162 − 162 − 162] = 457 4.18 (d, J = 7.8) 4.22 (d, J = 7.8) 4.27 (d, J = 6.0) × − − − − 18 68.3 472 3 Hexose [M957 H 162 – 162 162] = 471 4.30 (d, J = 6.0) 4.35 (d, J = 6.9) 4.41 (d, J = 7.8) 2 × Methylpentose 19 68.5 472 [M − H − 146 − 146 − 162]− = 471 5.20 * (t, J = 3.4) 1 × Hexose 925 5.25 * (t, J = 3.4) Molecules 2016, 21, 1614 8 of 19

Table 1. Cont.

No. RT (min) Aglycone (Da) A Sugar Moieties B Mass Spectra Fragmentation C Anomeric Protons (ppm) D × 1 Methylpentose − − − − − 4.16 (d, J = 6.6) 20 69.1 472 × [M941 H 146 162 162] = 471 2 Hexose 4.35 (d, J = 7.8) 4.41 (d, J = 7.8) 21 69.5 458 3 × Hexose [M − H − 162 − 162 − 162]− = 457 943 5.20 * (t, J = 3.4) − 5.25 * (t, J = 3.4) 22 69.7 456 3 × Hexose [M941 − H − 162 − 162 − 162] = 455 1 × Methylpentose 23 70.2 472 [M − H − 146 − 162 − 162]− = 471 2 × Hexose 941 4.35 (d, J = 7.8) 4.41 (d, J = 7.8) 24 70.4 456 3 × Hexose [M − H − 162 − 162 − 162]− = 455 941 5.25 * (t, J = 3.4) 2 × Methylpentose 5.30 * (t, J = 3.4) 25 70.4 472 [M − H − 146 − 162 − 146]− = 471 1 × Hexose 925 4.41 (d, J = 7.8) 472 26 72.8 2 × Hexose [M –H − 162 − 162]− = 471 4.42 (d, J = 7.8) Hederagenin cellobioside 795 5.24 * (t, J = 3.4) 4.34 (d, J = 7.8) 456 27 75.8 2 × Hexose [M − H − 162 − 162]− = 455 4.41 (d, J = 7.8) Oleanolic ac. cellobioside 779 5.23 * (t, J = 3.4) A A molecular weight of a saponin aglycone observed from the mass spectral fragmentation pattern of a corresponding peak. B Tentatively identiﬁed number and type of sugar moieties. C Mass spectral fragmentation patterns illustrated a loss of sugar moieties including hexose (162 Da), methyl-pentose (146 Da), and pentose (132 Da) that are possibly attached to the corresponding triterpenoid aglycone. Adduct ions of sugar moieties correspond to the molecular weights of sugars minus a water molecule. D Chemical shift (ppm) and spin-spin coupling constants (Hz) of anomeric protons of possible sugar moieties. Note: LC-MS was performed in (-) negative mode and 1D proton NMR spectrum (number of scans = 128) with water suppression was recorded for each interval of 1 min during the entire run. * Triplet derived from the methine group at the C12 position of an aglycone (e.g., hederagenin and oleanolic acid). ** Loss of the last sugar moiety was not observed. Molecules 2016, 21, 1614 9 of 19

Table 2. List of tentatively annotated triterpenoid saponins from the time slice LC-SPE-NMR/MS experiment performed on saponin enriched extract of the pubescent (P) type B. vulgaris.

No. RT (min) Aglycone (Da) A Sugar Moieties B Fragmentation Pattern in Mass Spectra C Anomeric Protons (ppm) D − 1 45.2 448 5 × Hexose [M1257 − H − 162 − 162 − 162 − 162 − 162] = 447 4.13 (d, J = 7.8) 3 × Methylpentose 5.20 (d, J = 7.8) 2 45.7 464 [M − H − 162 − 146 − 162 − 146 − 146]− = 463 2 × Hexose 1225 5.28 (d, J = 7.8) 1 × Pentose 4.13 (d, J = 7.8) − 3 46.4 478 1 × Hexose [M917 − H − 132 − 162 − 146] = 477 4.63 (d, J = 7.8) 1 × Methylpentose 5.17 (d, J = 7.8) 2 × Methylpentose 4 54.1 448 [M − H − 146 − 146 − 162]− = 447 1 × Hexose 901 4.35 (d, J = 8.0) 4.39 (d, J = 8.0) 4.58 (d, J = 8.0) 4.60 (d, J = 8.0) 5 54.8 464 3 × Methylpentose [M − H − 146 − 146 − 146]− = 463 901 5.12 (d, J = 7.8) 5.23 (d, J = 7.8)

4.04 (d, J = 8.0) 2 × Methylpentose 6 56.8 448 [M − H − 146 − 146 − 162]− = 447 4.42 (d, J = 8.0) 1 × Hexose 901 4.75 (d, J = 7.8) 4.29 (d, J = 7.8) 2 × Methylpentose 7 57.7 448 [M − H − 146 − 146 − 162]− = 447 4.43 (d, J = 7.8) 1 × Hexose 901 4.66 (d, J = 7.8) 4.19 (d, J = 7.8) 1 × Methylpentose 8 58.4 609* [M − H − 146 − 162]− = 609 4.31 (d, J = 7.8) 1 × Hexose 917 5.14 (d, J = 7.8) 4.19 (d, J = 7.8) 1 × Methylpentose 9 59.6 448 [M − H − 162 − 162 − 146]− = 447 4.39 (d, J = 7.8) 2 × Hexose 917 4.53 (d, J = 7.8)

4.37 (d, J = 7.8) × 2 Methylpentose − − − − − 4.42 (d, J = 8.0) 10 61.2 448 × [M901 H 146 146 162] = 447 1 Hexose 4.60 (d, J = 8.0) 4.62 (d, J = 8.0) 2 × Methylpentose 5.09 (d, J = 7.8) 11 61.8 448 [M − H − 146 − 146 − 162]− = 447 1 × Hexose 901 5.20 (d, J = 7.8) J − 4.19 (d, = 7.8) 12 62.7 472 2 × Hexose [M795 − H − 162 − 162] = 471 5.14 (d, J = 7.8) Molecules 2016, 21, 1614 10 of 19

Table 2. Cont.

No. RT (min) Aglycone (Da) A Sugar Moieties B Fragmentation Pattern in Mass Spectra C Anomeric Protons (ppm) D 4.42 (d, J = 8.0) 2 × Methylpentose 13 63.1 448 [M − H − 146 − 146 − 162]− = 447 4.60 (d, J = 8.0) 1 × Hexose 901 4.62 (d, J = 8.0)

1 × Methylpentose 4.15 (d, J = 8.0) 14 64.1 623* [M − H − 146 − 162]− = 623 1 × Hexose 931 4.19 (d, J = 7.8) 4.62 (d, J = 8.0) 4.68 (d, J = 8.0) 5.13 (d, J = 7.8) 2 × Methylpentose 15 64.6 448 [M − H − 146 − 146 − 162]− = 447 5.28 (d, J = 7.8) 1 × Hexose 901 5.57 (d, J = 8.0)

4.30 (d, J = 7.8) 4.40 (d, J = 7.8) 16 77.4 474 4 × Hexose [M − H − 162 − 162 − 162 − 162 ]− = 473 1121 4.41 (d, J = 7.8) 4.71 (d, J = 8.0) 4.27 (d, J = 7.8) − 17 78.4 474 3 × Hexose [M959 − H − 162 − 162 − 162 ] = 473 4.41 (d, J = 7.8) 4.41 (d, J = 7.8)

− 4.27 (d, J = 7.8) 18 79.1 458 4 × Hexose [M1105 − H − 162 − 162 − 162 − 162] = 457 4.34 (d, J = 7.8) 4.38 (d, J = 7.8) 4.39 (d, J = 7.8) − 19 79.4 472 4 × Hexose [M1119 − H − 162 − 162 − 162 − 162 ] = 471 4.41(d, J = 7.8) 4.62 (d, J = 7.8) 5.53 (d, J = 8.2) 1 × Methylpentose 20 79.8 474 [M − H − 146 − 162 − 162 − 162 ]− = 473 5.60 (d, J = 8.2) 3 × Hexose 1105

− 4.27 (d, J = 7.8) 21 80.4 458 4 × Hexose [M943 − H − 162 − 162 − 162 ] = 457 4.31 (d, J = 7.8) 4.34 (d, J = 7.8) 4.38 (d, J = 7.8) 1 × Methylpentose 4.39 (d, J = 7.8) 22 80.8 472 [M − H − 162 − 162 − 146 − 162 ]− = 471 3 × Hexose 1103 4.41 (d, J = 7.8) 4.61 (d, J = 7.8) A A molecular weight of a saponin aglycone observed from the mass spectral fragmentation pattern of a corresponding peak. B Tentatively identiﬁed number and type of sugar moieties. C Mass spectral fragmentation patterns illustrated a loss of sugar moieties including hexose (162 Da), methyl-pentose (146 Da), and pentose (132 Da) that are possibly attached to the corresponding triterpenoid aglycone. Adduct ions of sugar moieties correspond to the molecular weights of sugars minus a water molecule. D Chemical shift (ppm) and spin-spin coupling constants (Hz) of anomeric protons of possible sugar moieties. Note: LC-MS was performed in (-) negative mode and 1D proton NMR spectrum (number of scans = 128) with water suppression was recorded for each interval of 1 min during the entire run. * Loss of the last sugar moiety was not observed. Molecules 2016, 21, 1614 11 of 19 Molecules 2016, 21, 1614 11 of 19

FigureFigure 4. 4.LC-MS LC-MS base base peakpeak chromatogramchromatogram (BPC) (BPC) of of saponi saponinn enriched enriched extracts extracts of ofthe the G- G-and and P-type P-type B.B. vulgaris vulgarisobtained obtained from the the time time slice slice LC-SPE-NMR/M LC-SPE-NMR/MSS experiment. experiment. Peak numbers Peak numbers correspond correspond to the tonumbering the numbering of tentatively of tentatively identified saponins identified listed saponins in Table listed1 and 2 in for Tables G- and1 P-typeand2 plants,for G- respectively. and P-type plants, respectively. The majority of saponins (17 out of 27) detected from the G-type plant contained only hexose moieties, whereas the number of saponins decorated with only hexose moieties in the P-type plant The majority of saponins (17 out of 27) detected from the G-type plant contained only hexose was found to be much less (seven out of 22). Moreover, most of the saponins of the P-type plant (14 out moieties, whereas the number of saponins decorated with only hexose moieties in the P-type plant of 22) contained two types of sugars, methylpentose and hexose. Only one pentose containing saponin waspeak found was todetected be much from less each (seven plant out type. of 22). The Moreover, number of most sugar of themoieties saponins attached of the to P-type the triterpenoid plant (14out ofaglycone 22) contained varied two from types 2 to of5 for sugars, both plants. methylpentose The majority and hexose.of saponins Only contained one pentose three containing sugar moieties. saponin peakA total was of detected 12 P-type from and each16 G-type plant tentatively type. The id numberentified of saponins sugar moieties contained attached three sugar to the moieties. triterpenoid aglyconeMass varied spectral from fragmentation 2 to 5 for both patter plants.ns suggest The majority that all 49 of saponin saponins peaks contained detected three from sugar both plants moieties. Aoriginate total of 12from P-type eight and different 16 G-type triterpenoid tentatively aglycone identifieds with different saponins MW. contained The MW three of all sugar aglycones moieties. and massMass spectral spectral fragmentations fragmentation that patternsshowed loss suggest of su thatgar moieties all 49 saponin matched peaks with detectedan observed from MW both of plantsthe originatecorresponding from eight saponin different peak. triterpenoidA total of 10 saponins aglycones detected with different from the MW.G-type The plant MW corresponded of all aglycones to an and massaglycone spectral with fragmentations a MW of 472 Da, that whereas showed nine loss P-type of sugar saponins moieties were matchedderived from with an an aglycone observed with MW a of theMW corresponding of 448 Da. In total, saponin four peak. aglycones A total with of MW 10 saponins of 448, 458, detected 472, and from 478, the were G-type common plant for corresponded both types toof an plants. aglycone G-type with aglycones a MW of also 472 included Da, whereas MW of nine 456 P-typeand 504 saponins Da, and aglycones were derived with fromMW of an 464 aglycone and with474 aDa MW were of exclusively 448 Da. In detected total, four from aglycones the P-type with plant. MW Tentatively of 448, characterize 458, 472, andd triterpenoid 478, were commonaglycones for bothwith types MW of of 456 plants. and 472 G-type Da correspond aglycones to alsothe oleanolic included acid MW and of hedera 456 andgenin, 504 respectively, Da, and aglycones which were with MWboth of identified 464 and 474from Da the were G-type exclusively B. vulgaris detected in a previous from study the P-type [19]. Other plant. aglycones Tentatively from characterized B. vulgaris triterpenoidplants were aglycones found for withthe first MW time of 456in this and study. 472 DaDespite correspond the fact to that the previous oleanolic studies acid and performed hederagenin, on respectively,B. vulgaris have which suggested were both the presence identified offrom saponins the G-typein P-typeB. plants, vulgaris noin structural a previous information study [19 about]. Other aglycones and their decoration by sugar moieties were available [2]. aglycones from B. vulgaris plants were found for the first time in this study. Despite the fact that The 1D proton NMR spectra that represented a fingerprint of an entire eluent corresponding to previous studies performed on B. vulgaris have suggested the presence of saponins in P-type plants, a 1 min chromatographic interval further assisted in the characterization of tentatively identified no structural information about aglycones and their decoration by sugar moieties were available [2]. saponins. Due to the complexity of the sample matrix and the similar physico-chemical properties of The 1D proton NMR spectra that represented a fingerprint of an entire eluent corresponding saponins, the resolution of the applied LC was not sufficient to separate some of saponins. Accordingly, tothe a 1 NMR min chromatographicspectra for some of interval the chromatographic further assisted intervals in the represent characterization NMR signals of tentatively of more than identified one saponins.saponin. DueIn addition to the complexityto the spectral of thecomplexity, sample matrixthe low and S/N the ratio similar of the physico-chemical NMR signals obtained properties from of saponins,the time theslice resolution LC-SPE-NMR/MS of the applied experiment LC was makes not sufficientstructure elucidation to separate of some saponins of saponins. very challenging. Accordingly, theConsequently, NMR spectra tentative for some identification of the chromatographic of saponins wa intervalss based on represent both their NMR characteristic signals of mass more spectral than one saponin. In addition to the spectral complexity, the low S/N ratio of the NMR signals obtained from

Molecules 2016, 21, 1614 12 of 19 the time slice LC-SPE-NMR/MS experiment makes structure elucidation of saponins very challenging. Consequently, tentative identification of saponins was based on both their characteristic mass spectral fragmentation patterns and NMR features. One of the most characteristic NMR signals corresponding to saponins are the doublets derived from the anomeric protons of the sugar moieties attached to the triterpenoid aglycone. Despite the spectral complexity, it was possible to identify anomeric proton doublets for all tentatively characterized saponins. Most of these doublets were in the chemical shift range of 4.13–4.61 ppm with a spin-spin coupling constant of 7.8 Hz for both types of plants. Saponins that contained methylpentose moieties depicted doublets between 5.09–5.60 ppm with a spin-spin coupling constant of 7.8–8.2 Hz. The later finding is in agreement with a previously published study that showed similar chemical shifts for the methylpentose moieties of saponins [20]. It is worth to mention that spin-spin coupling constants found for all anomeric doublets suggest exclusively the β-configuration of the sugar moieties. This is in good agreement with the literature [19,21]. As mentioned previously, one of the main differences between the 1D proton NMR spectra of the G- and P-type B. vulgaris plants is a triplet, in the range of 5.2–5.3 ppm, which is absent in the P-type plants. This triplet corresponds to the methine group of the double bond at C12 position in the triterpenoid aglycone. Indeed the NMR data obtained from the time slice experiment showed triplet signals between 5.20–5.25 ppm, with a spin-spin coupling constant of 3.4 Hz, but only for the G-type saponins. This finding indicates that P-type saponins are not derived from β-amyrin, α-amyrin or other similar triterpenoid aglycones that possess a double bond at the C12 position. Our previous study performed on identification of genes involved in biosynthesis of saponins also confirmed that P-type B. vulgaris produces lupeol and lupeol derived aglycones, while G-type B. vulgaris accumulates mainly β-amyrin derived aglycones [10]. Figure S1 illustrates the mass spectral fragmentation patterns and 1D proton NMR spectra of some tentatively identified saponins from the G- and P-type B. vulgaris. The mass spectrum of the P-type saponin, which elutes at 78.4 min, exhibits a MW of 959 Da and a loss of 3 hexose moieties. Subsequently a MW of the aglycone was identified as 474 Da (Figure S1A). The NMR spectrum of the same saponin peak shows 5 singlets in the range of 0.7–1.7 ppm, which suggest the presence of 5 methyl groups on the triterpenoid aglycone. Three anomeric doublets corresponding to three hexose moieties were detected at 4.27 and 4.41 ppm with a spin-spin coupling constant of 7.8 Hz. In contrast to most G-type saponins, this peak did not possess a triplet around 5.2 which is supporting evidence for P-type saponins not being derived from β-amyrin or similar aglycones. The second chromatographic interval, at the retention time (RT) range of 79–80 min, for the P-type plant, suggests elution of at least two different saponins (Supplementary Figure S1B). The mass spectral fragmentation pattern that corresponds to the peak at the RT of 79.3 min has a molecular ion with a mass of 1105 Da and shows a subsequent loss of 4 hexose moieties. The corresponding aglycone ion is detected at m/z 457 which suggest that this saponin is based on an aglycone with MW of 458 Da. The mass spectral fragmentation pattern of the peak at RT of 79.7 min, from the same interval, reveals a loss of one methylpentose and three hexose moieties and a MW of the corresponding aglycone of 474 Da. The NMR data measured for this interval was also complex and showed 8 different doublets in the range of 4.27–5.60 ppm, with a spin-spin coupling constant of 7.8 or 8.2 Hz. Although it is difficult to quantify relative ratios of singlets, due to the complexity of the spectra, the five most intense singlets were observed in the range of 0.7–1.7 ppm. In addition to the spectral complexities observed at the aliphatic and anomeric regions, the complexity of the carbohydrate region, around 3.12–3.95 ppm, suggests co-elution of more than one type of saponin at this chromatographic interval. In contrast, the P-type saponins eluting in the RT range of 80.0–81.0 min have notably big differences in their relative concentrations, with the peak eluting at 80.4 min for the most dominant; accordingly the NMR spectra was less complex (Figure S1C). The tentative saponin peak eluting at 80.4 min possessed three hexose moieties, an aglycone with MW of 458 Da, and its molecular ion was detected at 943 m/z. A number of sugar moieties was confirmed from the 1D proton NMR data, which showed three distinct doublets at 4.27, 4.34, and 4.41 ppm, with a spin-spin coupling constant of 7.8 Hz. In addition to this, five singlets, between 0.7–1.7 ppm, that are consistently detected for P-type saponins were also detected for this tentative saponin. Molecules 2016, 21, 1614 13 of 19

Examples of mass spectral fragmentation patterns and NMR data for three tentatively identified MoleculesG-type saponins2016, 21, 1614 are shown in Figure S1D–F. The G-type saponin, which eluted at the RT of 49.513 min,of 19 show a loss of three sugar moieties, one methylpentose and two hexoses, and its molecular ion and showaglycone a loss masses of three were sugar 974 moieties, and 504 Da,one respectively. methylpentose The and corresponding two hexoses, NMR and its data molecular confirmed ion three and aglyconedoublets, masses at 4.27, were 4.41, and974 4.42and ppm,504 Da, with respectively. spin-spin coupling The corresponding constant of NMR 7.8 Hz, data a triplet confirmed at 5.25 three ppm doublets,(J = 3.4 Hz at) which4.27, 4.41, is characteristic and 4.42 ppm, for with the spin-spin G-type saponins, coupling and constant five singlets of 7.8 Hz, in a the triplet aliphatic at 5.25 region. ppm (TwoJ = 3.4 other Hz) G-typewhich is saponins characteristic eluted for at RTthe ofG-type 63.4 andsaponins, 64.0 min and and five possessed singlets in aglycones the aliphatic with region. MWs Twoof 472 other and G-type 458 Da, saponins respectively, eluted and at bothRT of were 63.4 decoratedand 64.0 min with and three possessed hexose aglycones moieties (Figure with MWs S1E,F). of 472The and two 458 major Da, insecticides respectively, [3, 4and] found both inwere the G-typedecoratedB. vulgariswith three, hederagenin hexose moieties cellobioside (Figure and S1E,F). oleanolic The twoacid major cellobioside, insecticides eluted [3,4] at found 72.8 andin the 75.8 G-type min, B. respectively, vulgaris, hederagenin and their cellobioside mass spectral and fragmentation oleanolic acid cellobioside,patterns and eluted 1D proton at 72.8 NMR and 75.8 spectra min, are respectively, shown in and Figures their5 andmass6. spectral The mass fragmentation spectra and patterns NMR anddata 1D obtained proton fromNMR the spectra time are slice shown LC-NMR in Figures experiment 5 and for6. The these mass two spectra saponins and were NMR compared data obtained with fromthe data the time reported slice inLC-NMR the literature experiment and were for these unequivocally two saponins identified were compared [3,4,19]. Thewith massthe data spectrum reported of inhederagenin the literature cellobioside and were shows unequivocally a molecular ident ionified at m/z [3,4,19].795 that The corresponds mass spectrum to the [Mof −hederageninH]− anion, cellobiosideand a lossof shows two hexoses,a molecular in thision at case m/z glucose795 that moieties.corresponds The tom/z the [Mions − atH]− 633 anion, and and 471 a correspond loss of two hexoses,to [M – Hin− thisglucose] case glucose− and moieties.[M − H − The2 × m/zglucose] ions at− 633anions, and 471 respectively. correspondMoreover, to [M – H − the glucose] 1D proton− and [MNMR − H data − 2 of× hederageninglucose]− anions, cellobioside respectively. confirmed Moreover, the presence the 1D proton of 6 singlets NMR deriveddata of fromhederagenin methyl cellobiosidegroups of the confirmed hederagenin the presence at C24, C25, of 6 C26, singlets C27, derived C29, and from C30 methyl between groups 0.7–1.25 of ppm.the hederagenin The doublets at C24,of two C25, anomeric C26, C27, protons C29, and were C30 detected between at 0.7–1.25 4.41 and ppm. 4.42 The ppm, doublets with aof coupling two anomeric constant protons of 7.8 were Hz, detectedand the protonat 4.41 ofand C12 4.42 double ppm, bondwith a was coupling represented constant by of a triplet7.8 Hz, at and 5.24 the ppm proton (J = 3.4of C12 Hz). double In addition, bond wasthe protonrepresented at the by tertiary a triplet carbon, at 5.24 C18, ppm of hederagenin, (J = 3.4 Hz). andIn addition, two methylene the proton groups at the of glucosetertiary moietiescarbon, C18,were of detected hederagenin, at 2.85 (dd,and twoJ = 4.3 methylene and 14.2 Hz)groups and of 3.86 glucose (dd) ppm,moieties respectively. were detected Similarly, at 2.85 oleanolic (dd, J = acid 4.3 andcellobioside 14.2 Hz) was and identified 3.86 (dd) andppm, its respectively. mass spectral Sim andilarly, NMR oleanolic data matched acid cellobioside with the data was reported identified in andthe literature.its mass spectral and NMR data matched with the data reported in the literature.

Figure 5. 5. NegativeNegative mode mode mass spectral mass spectral fragmentation fragmentation pattern (upper pattern figure) (upper and 1D ﬁgure) proton and NMR 1D spectrum proton (lowerNMR spectrumfigure) of (lowerhederagenin ﬁgure) cellobioside of hederagenin (peak 26 cellobioside in Table 1) (peak measured 26 in in Table an LC-SPE-NMR/MS1) measured in experimentan LC-SPE-NMR/MS performed experiment on a saponin performed enriched onextract a saponin of G-type enriched B. vulgaris extract. of G-type B. vulgaris.

Molecules 2016, 21, 1614 14 of 19 Molecules 2016, 21, 1614 14 of 19

Figure 6. 6. NegativeNegative mode mode mass massspectral spectral fragmentation fragmentation pattern (upper pattern figure) (upper and 1D ﬁgure) proton and NMR 1D spectrum proton NMR(lower spectrumfigure) of (loweroleanolic ﬁgure) acid cellobioside of oleanolic (peak acid 27 cellobioside in Table 1) (peak measured 27 in in Table an LC-SPE-NMR/MS1) measured in anexperiment LC-SPE-NMR/MS performed experiment on a saponin performed enriched onextract a saponin of G-type enriched B. vulgaris extract. of G-type B. vulgaris.

2.3. GC-MS Analysis of Saponin Enriched Extracts of G- and P-Types B.B. vulgarisvulgaris Prior to screening of a triterpenoid aglycone pool of G- and P-type B. vulgaris,, saponinsaponin enrichedenriched extracts were hydrolyzed using hydrochloric acid,acid, then derivatized using trimethylsilyl cyanide and analysed by GC-MS. Total ionion currentcurrent (TIC)(TIC) chromatogramschromatograms of G- and P-type plants are shown in Figure7 7.. AA totaltotal ofof 2828 peakspeaks werewere detecteddetected fromfrom bothboth plants.plants. AsAs waswas alsoalso thethe casecase forfor thethe LC-MSLC-MS data, the GC-MS data of the hydrolysed G-type plantplant was more complex, with additional and higher abundant peaks when compared to the P-type plant. Despite the fact that the majority of peaks were common for both plants, few peaks were exclusivelyexclusively present in only oneone typetype ofof plant.plant. The G-type plant contained U4, U7, U9, and U13 while U10 and U21 were presentpresent onlyonly inin P-type.P-type. Two major peaks that differ between the GC-MS profilesprofiles of G- and P-type plants were oleanolic acid (peak 11) and hederagenin (peak (peak 16) 16) which which are are only only present present in inthe the G-type. G-type. These These two twopeaks peaks were were the only the ones only onesidentified identified at level at level 1 [22] 1 [ 22using] using authentic authentic standards standards of oftriterpenoid triterpenoid aglycones. aglycones. This This finding finding is is in agreement with the LC-MS and 1D proton NMR data described earlier that show the presence of oleanolic acid cellobioside and hederageninhederagenin cellobiosidecellobioside fromfrom thethe G-typeG-type B.B. vulgarisvulgaris.. EI-MS of all GC-MS peaks detected from the hydrolysedhydrolysed G- and P-type plants (Figure S2) were compared against authentic standards of triterpenes and only few peaks matched with a similarity of 75%–95%. The EI-MS spectrum of an unknown peak U9, which is exclusively found in G-type, exhibits 84% similarity with the mass spectrumspectrum of echinocysticechinocystic acid, which is aa ββ-amyrin-amyrin derived triterpene. Despite the fact fact that that the the observed observed spectral spectral simi similaritylarity was was relatively relatively high, high, intensities intensities of some of some m/zm ions/z ions and andretention retention indices indices differed differed significantly. significantly. The The latter latter suggests suggests that that U9 U9 is is most most probably probably not echinocysticechinocystic acid, but probably a structurallystructurally similar triterpene. Two other G-type peaks, U25 and U26, eluting at late RT, 26.726.7 andand 27.327.3 min,min, werewere tentativelytentatively identifiedidentified as two different TMS derivatives of quillaic acid, a β-amyrin derived triterpene, which possesses two hydroxyl,hydroxyl, one carboxylic acid, and one aldehyde functional groups. Moreover, Moreover, an an unknown unknown peak peak obse observedrved only only in in the the P-type P-type plant, plant, U21, U21, displayed displayed a similar but not identical EI-MS pattern (74%) to betulinic acid, a lupeol derived triterpene. Notable differences in relative intensities of m/z ions, including 175, 189, 292, and 320. In addition, GC-MS

Molecules 2016, 21, 1614 15 of 19

Moleculesa similar 2016 but, 21 not, 1614 identical EI-MS pattern (74%) to betulinic acid, a lupeol derived triterpene. Notable15 of 19 differences in relative intensities of m/z ions, including 175, 189, 292, and 320. In addition, GC-MS peakspeaks which which are are detected detected exclusively exclusively in in G-type G-type pl plant,ant, including including U7, U7, U13, U13, U18, U18, and and U23, U23, matched matched withwith EI-MS EI-MS patterns patterns of of protopanaxatriol, protopanaxatriol, caulophyllogenin, caulophyllogenin, madecassic madecassic acid, acid, and and 6- 6-ββ-maslinic-maslinic acid, acid, respectively,respectively, withwith atat least least 75% 75% similarity. similarity. A commonA common GC-MS GC-MS peak peak for both for plants,both plants, U19, possessU19, possess similar similar(77%) EI-MS(77%) EI-MS pattern pattern as betulin, as betu althoughlin, although it contains it contains relatively relati abundantvely abundant unique unique ions ions at m /atz m473/z 473 and and512. 512. A total A total of 10of 10 out out of of 28 28 GC-MS GC-MS peaks peaks detected detected from from both both plants plants depicteddepicted EI-MS similarity of of at at leastleast 75% 75% when when compared toto anan in-house in-house developed developed library. library. However, However, authentic authentic standards standards and/or and/or 1D 1Dand and 2D 2D NMR NMR data data based based comparison comparison will will be requiredbe required prior prior to ato level a level 1 identiﬁcation. 1 identification.

FigureFigure 7. 7. GC-MSGC-MS total total ion ion current current (TIC) (TIC) chromatograms chromatograms of of saponin saponin enriched enriched extracts extracts of of the the G- G- and and P-typeP-type B.B. vulgaris vulgaris afterafter acidic acidic hydrolysis hydrolysis of of the the extracts extracts which which allowed allowed cleave cleave off off sugar sugar moieties moieties attached attached toto aglycones. aglycones. A A total total of of 28 28 peaks peaks were were detected detected from from both both plants. plants. Electr Electronon impact-mass impact-mass spectrum spectrum (EI-MS)(EI-MS) fragmentation fragmentation patterns patterns of of all all 28 28 peaks peaks are are illustrated in Supplementary Figure S2.2. * *Peak Peak 11 11 andand 16 16 were were identified identiﬁed as as oleanolic oleanolic acid acid and and hedera hederagenin,genin, respectively, respectively, using using authentic authentic standards standards of compounds.of compounds.

3.3. Experimental Experimental Section Section

3.1.3.1. Plant Plant Materials Materials and and Metabolite Metabolite Extraction Extraction FreshFresh leaves leaves of of 4–12 4–12 weeks weeks old old G- G- and and P-type P-type BarbareaBarbarea vulgaris vulgaris plantsplants were were used used for for the the metabolite metabolite extraction.extraction. All All plants plants were were grown grown in in a a climate climate chamber chamber as as described previously [2]. [2]. Untargeted Untargeted metabolitemetabolite analysis analysis using using GC-MS, GC-MS, LC-MS, LC-MS, and and NM NMRR was was performed performed on on a a global global metabolite metabolite extract extract basedbased on on 85% 85% methanol. methanol. Ten Ten mg mg of of fresh fresh leave leave di diskssks were were quenched quenched by by rapid rapid cooling cooling using using liquid liquid nitrogen,nitrogen, transferred transferred into into 2.0 2.0 mL mL Ep Eppendorfpendorf tubes, tubes, and and extracted extracted for for 5 min 5 min with with 0.8 0.8mL mL 85% 85% methanol methanol at ◦ 100at 100°C byC bymixing mixing at 1400 at 1400 rpm rpm in a in thermomixer a thermomixer (Eppendorf, (Eppendorf, Hørsholm, Hørsholm, Denmark). Denmark). After After this, this,the extractsthe extracts were werecooled cooled on ice onand ice centrifuged and centrifuged at 16,000 atg for 16,000 3 ming infor order 3 min to inobtain order a clear to obtain supernatant a clear forsupernatant LC-MS and for NMR LC-MS analysis. and NMR Prior analysis. to GC-MS Prior anal toysis, GC-MS the methanol analysis, extract the methanol was hydrolysed extract was as previously described [23], except the final solvent, diethyl ether, was replaced with ethyl acetate in a current study. It is worth to mention that residual acid hampers trimethylsilyl derivatization, so the ethyl acetate fraction must be washed with water.

Molecules 2016, 21, 1614 16 of 19 hydrolysed as previously described [23], except the final solvent, diethyl ether, was replaced with ethyl acetate in a current study. It is worth to mention that residual acid hampers trimethylsilyl derivatization, so the ethyl acetate fraction must be washed with water. Saponin enriched extracts of G- and P-type B. vulgaris plants were obtained from methanol extraction and enrichment of the saponin fraction was made by using solid phase extraction (SPE) cartridges. The freeze dried leaves of the plants were soaked in 55% ethanol (1:10 wt/vol) and boiled in a water bath for 5 min, followed by 10 min of extraction in an ultrasonic bath at 50 ◦C. The obtained extracts were filtered, dried under reduced pressure and re-dissolved in 30% methanol in a 3:1 vol/vol ratio between initial extract and methanol. Then, 60 mL of the 30% methanol extract was passed through a Strata C18 SPE cartridge (10 g, 60 mL) (Phenomenex ApS, Værløse, Denmark) which was pre-conditioned with 30% methanol and fraction 1 (30% methanol fraction) was obtained. Then, the same cartridge was flushed with 60 mL of 90% methanol which resulted in fraction 2 (90% methanol fraction), and finally 60 mL of 100% methanol (fraction 3) recovered the most non-polar metabolites of the plant extract. Fractions 1–3 were concentrated by drying using ScanVac (Labogene, Lynge, Denmark), and re-dissolved in 50% methanol, in a volume-to-volume ratio of 4:1 between the initial extracts and 50% methanol. Then, all fractions were analysed by LC-MS, which showed that fraction 2 (90% methanol) contained the majority of saponins of the B. vulgaris leaves.

3.2. GC-MS Analysis A total of 50 µL final metabolite extracts, either the 85% methanol or saponin extract, were completely dried using the ScanVac, operating at 40 ◦C, into 150 µL glass inserts. Immediately after drying, insets were sealed with air tight magnetic lids into GC–MS vials and derivatized by addition of 40 µL trimethylsilyl cyanide (TMSCN) [13]. All steps involving sample derivatization and injection were automated using a Dual-Rail MultiPurpose Sampler (MPS) (Gerstel, Mülheim an der Ruhr, Germany). After reagent addition, the sample was transferred into the agitator of the MPS and incubated at 40 ◦C for 40 min at 750 rpm. This procedure ensured precise derivatization time and reproducible sample injection. Immediately after derivatization, 1 µL of the derivatized sample was injected into a cooled injection system port (CIS4, Gerstel) in splitless mode. The septum purge flow and purge flow to split vent at 2.5 min after injection were set to 25 and 15 mL/min, respectively. Initial temperature of the CIS4 port was 120 ◦C, and heated at 5 ◦C/s to 320 ◦C (after 30 s of equilibrium time), where it was kept for 10 min. After heating, the CIS4 port was gradually cooled to 250 ◦C at 5 ◦C/s, and this temperature was kept constant during the entire run. The GC-MS consisted of an Agilent 7890A GC and an Agilent 5975C series MSD (Agilent Technologies, Glostrup, Denmark). GC separation was performed on an Agilent HP-5MS column (30 m × 250 µm × 0.25 µm) by using hydrogen carrier gas at a constant flow rate of 1.2 mL/min. The GC oven temperature program was as follows: initial temperature, 40 ◦C; heating rate, 12.0 ◦C·min−1; end temperature, 300 ◦C; hold time, 8.0 min; and post-run time, 5 min at 40 ◦C. Mass spectra were recorded in the m/z range of 50–600 with a scanning frequency of 2.3 scan/s, and the MS detector was switched off during the 8.5 min solvent delay time. The transfer line, ion source, and quadrupole temperatures were set to 280, 230, and 150 ◦C, respectively. The mass spectrometer was tuned according to manufacturer recommendations by using perfluorotributylamine. The MPS and GC-MS was controlled using vendor software Maestro (Gerstel). A blank sample containing only derivatization reagent was run in order to monitor reagent derived and column derived non-sample related peaks. An alkane mixture standard sample (all even C10-C40 alkanes at 50 mg/L in hexane) was used prior to calculate retention indices.

3.3. NMR Analysis An aliquot (300 µL) of 85% metabolite extracts from G- and P-type B. vulgaris plant leaves were completely dried in 1.5 mL glass vials and re-dissolved in 500 µL methanol-d4 (99.8%) which contained 5 µL of 20 mg·mL−1 TSP in deuterated water. A total of 10 G-type, 10 P-type biological replicates, and three 50% G-type and 50% P-type mixture samples were investigated. 1D proton NMR spectra were Molecules 2016, 21, 1614 17 of 19 recorded using an Avance DSX 500 NMR spectrometer (11.7 Tesla) (Bruker Biospin GmbH, Rheinstetten, Germany) operating at 500.13 MHz, and equipped with a BBI probe for 5 mm (o.d.) sample tubes. Data acquisition for all the samples was automated using the IconNMR automation software (version 3.5, Bruker Biospin GmbH, Rheinstetten, Germany) and each sample was automatically tuned, matched and shimmed. A total of 512 scans were recorded at room temperature with a relaxation delay of 5 s, a sweep width of 20 ppm, and 32 k complex data points. The obtained spectra were referenced to the 3-(trimethylsilyl)-propionic acid-d4 (TSP) peak at 0.00 ppm. Raw NMR data of 23 samples were imported into MATLAB (Version 7.13.0.564, R2011b, MathWorks Inc., Natick, MA, USA) after baseline and phase correction using TopSpin (version 2.1 Bruker BioSpin). The multivariate data analysis included interval based extended canonical variate analysis (iECVA) which was performed using in-house MATLAB scripts.

3.4. LC-SPE-NMR/MS Analysis The system consisted of an Agilent 1200 HPLC system equipped with a quaternary pump, auto sampler, diode array detector, a Bruker/Spark Prospekt 2 LC-SPE system (Spark, Emmen, The Netherlands) and a MicrOTOF mass spectrometer (Bruker Daltonics, Bremen, Germany) connected via a Bruker NMR MS Interface (BNMI-HP). MS spectra, in negative mode, were acquired between m/z 50 and 1600. The calibration was done with a 20 mM lithium formate automatically which is introduced at the beginning of each chromatographic run. Separation was done on a Prodigy LC column (ODS3, 5 µm particle size, and 250 mm × 4.6 mm; Phenomenex, Aschaffenburg, Germany). The chromatographic conditions were as following; a flow rate of 0.5 mL/min starting with a solvent composition of 90% A (H2O 0.1% formic acid-d2) and 10% B (acetonitrile 0.1% formic acid-d2) with a linear gradient to 70% A at 60 min, followed by another linear gradient to 10% A at 80 min. Starting at 15 min the eluent was collected on SPE cartridges. For this trapping process a makeup-flow of 1.5 mL·min−1 (mobile phase A) was added to the eluent before it passed through the SPE cartridges in order to increase the retention of analytes on the cartridges. For a time slice LC-SPE-NMR/MS experiment, 1 min period of the chromatograms were collected on a cartridge, before the system automatically changed to the next cartridge. Therefore the content of one cartridge contains the compounds eluted during this period of time from the column. For the trapping 10 mm × 2 mm SPE cartridges filled with GP resin were used. Prior to NMR measurements of SPE cartridge eluents, samples were dried with a stream of nitrogen, the fraction from each cartridge being eluted using a total of 250 µL deuterated methanol. The sample was eluted directly into a CryoFit with a 30 µL flow cell placed in a TCI CryoProbe Prodigy with a Bruker Avance III 500 MHz. NMR spectra were acquired with a relaxation delay of 5 s and a sweep width of 10 ppm, and 16 k complex data points. A 1D-WET multiple solvent suppression on two solvent resonances was used (residual OH methanol signal). 128 transients were acquired. 13C satellites of the methanol were selectively decoupled with a low power continuous wave decoupling using the 2nd channel of the spectrometer. After multiplication of the resulting free induction decay with an exponential function leading to a peak broadening of 1.0 Hz, Fourier transformation was performed followed by phase and baseline correction leading to a single NMR spectrum. All spectra were combined into a pseudo-2D file which shows NMR spectrum against chromatographic retention time.

4. Conclusions This study has shown how integrated and hyphenated analytical platforms can be used to elucidate the diversity of saponins (aglycone and sugar decoration) from plant tissues. The LC-SPE-NMR/MS and GC-MS study revealed a total of eight different triterpenoid aglycones with different MWs. Four aglycones with MW of 448, 458, 472, and 478 were found to be common for both types of plants. Two with MWs of 456 and 504 Da was found to be exclusive to G-type plants and two with MW of 464 and 474 Da were exclusive detected from the P-type plant. The average Molecules 2016, 21, 1614 18 of 19 glycosylation of the aglycones were three sugar residues and were exclusively found in β-anomer configuration. The study suggests that P-type B. vulgaris produces lupeol and lupeol derived aglycones, while G-type B. vulgaris produces mainly β-amyrin derived aglycones, in agreement with previous studies. GC-MS of saponin enriched extracts of G- and P-types B. vulgaris confirmed the identity of the saponin aglycones. The G-type extracts were found to be much more complex, including the two major saponin aglycons, oleanolic acid and hederagenin, which are only present in G-type. These two peaks were the only ones with identities confirmed at level 1 [22], using authentic standards of triterpenoid aglycones. In order to elucidate structures of G- and P-type saponins, observed from the LC-SPE-NMR/MS and GC-MS analysis, we are currently in progress with LC fractionation of saponin enriched extracts, from both plants, prior to 2D NMR and GC-MS analysis of pure saponin fractions.

Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/ 12/1614/s1. Acknowledgments: This work was supported by the Danish Council for Independent Research, Technology and Production Sciences (grant No. 09-065899 and 1335-00151), the Villum Kann Rasmussen Foundation to Pro-Active Plants, the European Community’s Seventh Framework Programme (FP7/2007-2013; grant No. 613692 to TriForC). Authors thank former laboratory technician trainee Shirley Myrna Sursigis Nielsen for her assistance to prepare saponin enriched plant extracts. Author Contributions: B.K., S.B.E., S.B. designed the study. B.K. performed the study and wrote the manuscript. L.H.T. and M.G. performed LC-NMR experiment. Conflicts of Interest: The authors declare no financial or commercial conflict of interest.

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Sample Availability: Not Avaiable.

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