MS and UPLC-MS profiling of triterpenoid saponins from leaves and roots of four red beet (Beta vulgaris L.) cultivars

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MS/MS and UPLC-MS profiling of triterpenoid saponins from leaves and roots of four red beet (Beta vulgaris L.) cultivars Agnieszka Mroczeka, , Ireneusz Kapustac, Anna Stochmalb, Wirginia Janiszowskaa ⁎

a

Department of Plant Biochemistry, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-960, Warsaw, Poland Department of Biochemistry, Institute of Soil Science and Plant Cultivation, State Research Institute, Czartoryskich 8, 24-100, Puławy, Poland c Faculty of Biology and Agriculture, University of Rzeszów, Ćwiklińskiej 2, 35-601 Rzeszów, Poland b

ARTICLE INFO

ABSTRACT

Keywords: Beta vulgaris Amaranthaceae Pentacycylic triterpenoids Oleanolic acid Saponins

Triterpene saponins from the leaves of red beet (Beta vulgaris L.) were qualitatively profiled using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Results indicated that red beet leaves contain 11 saponins, including oleanolic acid aglycone and a varying number of sugars plus acetyl or methyl groups. We identified and studied two additional saponins other than the nine reported previously from red beets. Relative quantification was undertaken of saponins in leaves, compared to those in the roots of four cultivars (Egyptian, Forono, Red Sphere, and Round Dark Red). Saponins were quantitated using ultra-performance reverse-phase liquid chromatography (UPLC) coupled with ESI-MS/MS. Leaves of Egyptian and Round Dark Red contained higher total amount of saponins than the roots, whereas relatively higher levels of saponins were observed in roots in cultivars Red Sphere and Forono. The differential accumulation of specific triterpene saponins is indicative of spatially differentiated biosynthesis and/or biological function.

1. Introduction Triterpenoid saponins are a widespread class of natural compounds. They are glycosylated secondary metabolites of plant found in many food and pharmaceutical products. These saponins comprise a triterpenoid aglycone and sugar side chains of variable length that are attached to the sapogenin at C-3 and/or C-28 position. Triterpenoid saponins are generally associated with plant defense against pathogens and antifeedant activity (Sparg et al., 2004). Moreover, numerous reports have described the anti-inflammatory, immunomodulatory, cytotoxic, antitumor, antimutagenic, antihepatotoxic, antidiabetic, hemolytic, antiviral, antibacterial, trypanocidal, and antiparasitic activities of saponins (Sparg et al., 2004; Mroczek, 2015; Podolak et al., 2010, Doligalska et al., 2011). Furthermore, saponins have found wide applications in beverages and confectionery manufacturing, as well as in cosmetics and pharmaceutical products (Sparg et al., 2004). Despite the significant biological and commercial importance of these compounds, their distribution or biosynthesis in plants is incompletely understood. Recently, saponins were found in the roots of four red beet (Beta vulgaris L.) cultivars – Red Sphere, Rocket, Wodan (Mroczek et al., 2012), and Nochowski (Mikołajczyk-Bator et al., 2016). The red beet is a biennial cultivated throughout the world for its roots, untilized as food and a source of natural dye. Moreover, the subterranean parts of ⁎

the red beet are used in salads and as a base in soups. Further, the hepatoprotective action of red beet leaf extracts was reported against ethanol-mediated hepatotoxicity (Jain and Singhai, 2012). Thus far, nothing is known of the saponin composition of red beet leaves that could, at least partially, mediate the biological activities of red beet leaves extracts. In addition, biochemical composition can be important in the context of plant–pathogen interactions and crop productivity. The primary goal of the present study was to conduct a rapid, tentative tandem MS/MS analysis of the saponin structure in red beet leaves. The secondary goal was to quantify total and individual saponins in the leaves and roots of red beet to gain a better understanding of the spatial accumulation of different saponins. 2. Results and discussion 2.1. MS/MS analysis of saponins from red beet leaves MS/MS analyses were conducted on samples obtained from the leaves of all investigated cultivars. Fig. 1 presents the representative MS/MS spectrum of the saponin mixture from the red beet cultivar Egyptian. In addition to the cultivar Red Sphere, which was a part of our previous study on red beet roots (Mroczek et al., 2012), cultivars Egyptian, Forono, and Round Dark Red were analyzed for the first time.

Corresponding author. E-mail address: [email protected] (A. Mroczek).

https://doi.org/10.1016/j.phytol.2019.02.015 Received 14 August 2018; Received in revised form 5 February 2019; Accepted 13 February 2019 1874-3900/ © 2019 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

Please cite this article as: Agnieszka Mroczek, et al., Phytochemistry Letters, https://doi.org/10.1016/j.phytol.2019.02.015

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Fig. 1. Direct injection ESI/MS of the mixture of red beet leaves cultivar Egyptian saponins purified by solid-phase extraction. Table 1 Saponins observed by negative ion ESI/MS/MS in B. vulgaris cultivars Red Sphere, Forono, Egyptian, and Round Dark Red leaves. Saponin

ESI/MS

UPLC Rt (min)

ESI/MS/MS

Saponin structure

1

1117.6 [M-H]−

2.25

Hex + Hex + Hex + HexUA + oleanolic acid

2

−

1087.6 [M-H]

2.34

3

953.5 [M-H]−

2.57

955.4 [M-H-Hex]− 793.3[M-H-Hex]− 631.6 [M-H-Hex-Hex]− 455.3 [M-H-Hex-Hex-Hex-HexUA]− 1) 955.4 [M-H-Pen]− 793.3 [M-H-Pen]− 631.3 [M-H-Pen-Hex]− 455.3 [M-H- PenHex -HexUA]− 2) 925.4 [M-H-Hex]− 763.4 [M-H-Hex]− 631.3 [M-H-Hex-Pen]- 455.3 [M-H- Hex -Pen -HexUA]− 793.3 [M-H-160]− 631.3 [M-H-160-Hex]− 455.3 [M-H-160-Hex-HexUA]−

4

−

925.4 [M-H]

2.67

5 6 7 8

955.5 835.3 793.4 925.4

[M-H]− [M-H]− [M-H]− [M-H]−

2.71 2.71 2.77 2.81

9

969.5 [M-H]−

3.24

10

763.4 [M-H]−

3.70

1) 793.3 [M-H-Pen] 631.3 [M-H-Pen-Hex] 455.3 [M-H-Pen-Hex-HexUA] 2) 763.4 [M-H-Hex]− 631.3 [M-H-Hex-Pen]− 455.3 [M-H-Hex-Pen-HexUA]− 793.3 [M-H-Hex]− 631.6 [M-H-Hex-Hex]- 455.3 [M-H-Hex-Hex-HexUA]− 793.3 [M-H-Ac]− 631.3 [M-H-Ac-Hex]− 455.3 [M-H-Ac-Hex-HexUA]− 631.3 [M-H-Hex]− 455.3 [M-H-Hex-HexUA]− 11) 793.3 [M-H-Pen]− 631.3 [M-H-Pen-Hex]− 455.3 [M-H-Pen- Hex-HexUA]− 2) 763.4 [M-H-Hex]− 631.3 [M-H-Hex-Pen]− 455.3 [M-H-Hex-Pen-HexUA]− 807.4 [M-H-Hex]− 645.3 [M-H-Hex]− 631.3 [M-H-Me-Hex]− 455.3 [M-H-Hex-Me-HexA]− 631.3 [M-H-Pen]− 455.3 [M-H-Pen-HexUA]−

11

631.3[M-H]−

4.71

455.3 [M-H-HexUA]−

−

−

−

Pen + Hex + HexUA + oleanolic acid

Molecular fragment 160 + Hex + HexUA + oleanolic acid Pen + Hex + HexUA + oleanolic acid Hex + Hex + HexUA + oleanolic acid Ac + Hex + HexUA + oleanolic acid Hex + HexUA + oleanolic acid Pen + Hex + HexUA + oleanolic acid Hex + Hex + Me + HexA oleanolic acid Pen + HexUA + oleanolic acid HexUA + oleanolic acid

Hex- hexose; Pen- pentose; HexUA- hexuronic acid; Ac-acetyl moiety; Me-methyl moiety.

It was stated that all tested cultivars exhibited the same saponin patterns, with 11 saponins. In all analyzed leaf samples, we detected nine saponins from the 12 previously described in red beet roots (Mroczek et al., 2012). The fragmentation patterns of saponins showing molecular ions at m/z 631 (11), 763 (10), 793 (7), 935 (3), 955 (5), 925 (3 and 8), 1087 (2), and 1117 (1) present in leaves were coherent with the fragmentation patterns of saponins from the roots of red beet obtained in previous studies (Mroczek et al., 2012, Mikołajczyk-Bator et al., 2016). Moreover, our results revealed the presence of two compounds that exhibited 969 (9) and 835 (6) [M−H]− ions in leaves, which were detected for the first time in red beet plant. On the basis of MS/MS analysis, as well as the interpretation of red beet root saponin structures from the roots of red beet, the structure of red beet saponins can be predicted as shown in Table 1. Table 1 presents the detailed fragmentation patterns of 11 compounds bearing oleanolic acid as the aglycone. The analyzed compounds underwent fragmentation with the loss of sugars (uronic acid, pentose, and hexose) and short aliphatic fragments; however, different monosaccharide epimers cannot be distinguished by MS/MS. All saponins present in red beet roots can be 3-O-glucuronides of oleanolic acid with different constituents from the hexose, pentose, and methyl or acetyl groups. This pattern of fragmentation, except for two saponins, is similar to what has been described previously in red beet root; moreover, they have a structural pattern similar to that of saponins isolated from sugar beet (Ridout and Price, 1994; Massiot et al., 1994;

Yoshikawa et al., 1996; Murakami et al., 1999). According to these findings, saponins from sugar beet are derivatives of oleanolic acid that contain not only sugars but also acetal- and dioxolane-type substituents, which were presumed to be biosynthesized through the oxidative degradation of a terminal monosaccharide moiety. Nevertheless, it cannot be excluded that red beet leaves contain saponins with one of these unique moieties that can generate a pattern of fragmentation similar to that of typical saccharide derivatives. In our study, none of the saponins analyzed by MS/MS contained these types of constituents; however, it cannot be excluded that saponin 3, with a molecular mass of 954 and for which MS/MS identified an undetermined 160-Da fragment, contains one of these unique moieties. Initially, we elaborated on the structure of the two saponins detected in red beet for the first time. In the MS/MS spectrum of a saponin 6 (m/z 835), after the initial loss of 42 Da, losses of 162 and 176 Da were observed, corresponding to the fragmentation of acetylated oleanolic diglycoside comprising Hex and HexUA. This compound could be presumed to be a saponin 7 derivative with n acetyl fragment linked to the carboxyl group at the C-28 of aglycone or at the carboxyl group of HexUA. Compound 6 has not been identified in the roots of red beet, and seems characteristic for saponins from the leaves; however, acetylated saponins have been found earlier in other plant species (Hamburger et al., 1992). The MS2 spectra of a second novel compound (9) detected in plant leaves yielded ions at m/z 807, 645, 631, and 455, which suggested the 2

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presence of two Hex, one methyl, and one HexUA moieties connected to oleanolic acid. Saponin 9 exhibited a pseudomolecular ion [M−H]− at m/z 969.5 (i.e., 14 Da greater than that of compound 5), and its MS/MS spectrum showed similar fragmentation patterns; thus, this compound can be established as a methyl derivative of oleanolic acid triglycoside that was reported previously in red beet roots (Mroczek et al., 2012). Furthermore, the structures of saponin previously identified in red beet roots were confirmed. The fragmentation of the m/z 631 of compound 11 resulted in m/z 455 corresponding to the [M – H – 176]− ion, which is consistent with the loss of HexUA. The retention time (Rt) of 11 was identical to that of 3-O-β-D-glucuronopyranosyl in oleanolic acid isolated from Calendula officinalis. Moreover, this structure corresponds to the 3-O-β-D-glucuronopyranosyl of oleanolic acid isolated earlier from fresh sugarbeet roots and detected in the roots of red beet, cultivar Red Sphere (Mroczek et al., 2012). In the MS2 spectrum of saponin 10 (m/z 763), after an initial loss of 152 Da, a loss of 176 Da was observed, which corresponded to the fragmentation of oleanolic diglycoside comprising Pen and HexUA. In the second diglycoside (7) detected in plants after the initial loss of hexose residue, the sugar sequence of 7 was determined as Hex-HexA (m/z 631-455). The MS2 spectra of saponin 5 differed from 10 by the 162-Da fragment and yielded ions at m/z 793, 631, and 455, which suggested the presence of two Hex and one HexUA moieties connected to oleanolic acid. Compounds 4 and 8 generated pseudomolecular ions [M−H]− at m/z 925, and are a pair of isomers with the same number of sugar moieties, although linked in a different position. The fragmentation of m/z 925 resulted in the simultaneous loss of pentose and hexose. This indicates that both sugar moieties are linked to the HexUA moiety, or that one of them is linked to the C-28 of aglycone. According to the difference in their retention times on UPLC-MS analysis, we expect that, probably, one of them has a branched sugar chain (4) and the other a blocked carboxylic group in the aglycone (8). Furthermore, two tetraglycosides previously found in roots were observed in all leaf samples. The fragmentation of saponin 1 (m/z 1117) resulted in the loss of three 162 fragments, generating a compound with additional hexose in comparison to saponin 5. The mass difference between saponin 2 and its product ions 925 [M−H]− and 955 [M−H]− was 162 and 132, which corresponds to the simultaneous loss of hexose and pentose. Further fragmentation is consistent with the fragmentation of saponins 4, 8, and 5. This preliminary structural characterization of saponins from red beet is tentative, and further structural analyses, including nuclear magnetic resonance, of saponins are required to elucidate the final structure.

Fig. 2. Selected ion chromatograms for extracts from leaves of the B. vulgaris cultivar Red Sphere illustrate the quantitative and qualitative profiling of representative saponins.

was necessary to prepare a standard calibration curve. To this purpose, oleanolic acid was used as a standard reference in a series of concentrations ranging between 80 and 560 ng/mL. The utilization of the calibration curve, based on the aglycone, for glycoside quantitation could be less accurate than that based on separated standards of individual saponins; however, it provides quick and sufficient quantitation without the time-consuming and challenging separation of compounds. Therefore, the use of the calibration curve seems beneficial in terms of quality control and monitoring of the phytochemical content in plant materials. For the evaluation of the instrumental precision and extraction/ purification repeatability, three samples from the same plant powder were independently extracted and purified within the solid phase extraction (SPE) procedure. For each sample, two independent LC–MS runs were conducted. It was shown that, for repetition of the same sample (n = 2), the relative standard deviation was 4–5.3% (with some exceptions where it was higher). However, the variation of the data caused by the instrument was satisfactory. Moreover, the accuracy and precision were calculated, and the % relative standard deviation (RSD) was determined to be ≤2.01% (intraday) and ≤5.1% (interday). Relative standard deviations between results obtained for three independent extractions ranged from 2.3% to 14%. Considering that it is

2.2. UPLC-MS quantitative analysis In a previous study, we used a high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLCESI/MS/MS) for the identification and quantitation of saponins present in the roots of the B. vulgaris cultivars Red Sphere, Rocket, and Wodan (Mroczek et al., 2012). In the present study, UPLC-ESI/MS/MS was utilized for the separation and quantitation of saponin compounds from leaves, in comparison to those from the roots of red beet. UPLC-MS, the method used more frequently in recent times for monitoring various bioactive compounds in plant material (Chenlei et al., 2017; Foubert et al., 2010; Kowalczyk et al., 2011, Kapusta et al., 2005, Medina-Meza et al., 2016; Ożarowski et al., 2017; Verza et al., 2012), provided rapid quantification of the analyzed compounds. An optimized UPLC-MS procedure using acetonitrile/0.1% formic acid in a water gradient allowed satisfactory separation of red beet saponins with good peak resolution (Fig. 2). The quantitative analysis was conducted for 11 saponins on the basis of the single-ion respond (SIR) detection mode. For the determination of individual saponins, it 3

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Table 2 Concentration of individual saponins and total saponins in four B. vulgaris cultivars in μg/g DW. Values are expressed as the means of three independent samples. cv. Red Sphere

cv. Forono

cv. Egyptian

cv. Round Dark Red

No.

roots

leaves

roots

leaves

roots

leaves

roots

leaves

μg/g DW 1 2 3 4 5 6 7 8 9 10 11 Total

172.3 ± 8.3 736.8 ± 39.1 142.4 ± 14.5 349 ± 38.4 9543.1 ± 116.4 nd 403 ± 14.9 157.8 ± 7.6 nd 291.4 ± 10.5 219.2 ± 21.3 9835

632.5 ± 58.8 1987.9 ± 117.3 9.4 ± 0.32 1126.8 ± 89.1 2537.7 ± 83.7 932.6 ± 78.9 820.4 ± 37.7 1123.4 ± 84.3 17.7 ± 0.7 350.3 ± 10.2 129.5 ± 7.5 11555.9

49.4 ± 1.5 750.1 ± 53.3 6.5 ± 0.4 189.3 ± 9.8 3671.6 ± 172.6 nd 557.7 ± 68.1 87.5 ± 8.6 28.9 ± 1.4 469.2 ± 17.8 124.3 ± 6.8 4109.7

60.6 ± 2.2 1515.1 ± 22.7 34 ± 2.5 563.5 ± 62 662.2 ± 54 378 ± 24.2 343.4 ± 24.2 170.3 ± 5.8 nd 172.8 ± 9.5 46.9 ± 2.2 5905.6

58 ± 3.9 389.6 ± 14.8 257 ± 15.9 753.4 ± 24.9 5045.5 ± 771.9 nd 595 ± 26.8 1052.8 ± 102.1 77.4 ± 3.2 336.4 ± 26.2 234.9 ± 10.3 16363.4

780 ± 52.2 3933 ± 562.3 19.7 ± 1.3 1774.6 ± 191.7 4536.9 ± 240.5 1436.9 ± 76.1 2081.5 ± 72.9 988.8 ± 75.1 nd 437.5 ± 19.7 63.6 ± 2.7 8722.5

63.1 ± 4.4 3098 ± 111.5 70.7 ± 3.8 785 ± 76.2 6526 ± 228.4 nd 547 ± 18.1 88.8 ± 7.4 58.8 ± 1.6 401 ± 18.4 482 ± 18.3 11781

37.6 ± 1.4 200.6 ± 9.2 10.3 ± 0.9 2954.4 ± 278.7 2278.8 ± 143.6 866.2 ± 26.9 777.6 ± 124.6 545.1 ± 40.3 nd 623 ± 32.4 67,9 ± 7.5 9138.7

nd – not detected, DW- dry weight.

difficult to obtain homogeneous plant materials and that the preparation of analytical samples included several steps of extraction and purification, this relative standard variation was satisfactory and comparable to others that reported LC–MS data for red beet (Mroczek et al., 2012) as well as other plants (Kapusta et al., 2005). Herein, we report an efficient and rapid analytical method for the simultaneous detection of saponins in the sample. The method of quantitative analysis we used enabled the evaluation of the relative differences in the content of saponins in roots and leaves of various cultivar varieties of the red beet plant (Table 2). Red beet cultivars have been reported to differ in the total concentration of saponins. In leaves, the highest concentrations of saponins were detected in cultivar Egyptian, followed by cultivars Round Dark Red, Red Sphere, and Forono, and were 28%, 40%, and 75% lower. The cultivar with the highest concentration of saponins in roots turned out to be cultivar Red Sphere. In comparison to Red Sphere, the saponin concentration in roots of Forono, Egyptian, and Round Dark Red beets was respectively 49%, 72%, and 75% lower. In the case of Red Sphere and Forono cultivars, it was demonstrated that the total concentration of the saponins in roots dominated the concentration in the leaves in the ratio 1:0.8 and 1:1.7, respectively. In contrast, the ratios of the total content in roots to total content in leaves of cultivars Egyptian and Round Dark Red, equaling 1:1.8 and 1:1.3, respectively, indicate the dominance of saponin content in leaves. Moreover, differences in the relative content of individual saponins were observed. Quantifications were undertaken for nine saponins in roots and leaves, and an additional two saponins in the leaves. The compound which showed a pseudomolecular ion at m/z 955 [M−H]− (5) was the dominant saponin in most of the analyzed samples, except in two. The content of this compound varied from 79.4% of total saponins in Red Sphere roots to 16.1% in Forono leaves; in roots, it was, depending on the sample, 2.1–3.9 times higher than in the leaves. In most of the samples, the second most dominant compound was saponin 1087 [M−H]− (2) and, furthermore, it was dominant in Forono and Round Dark Red leaves. In general, we could observe 2.9–12 times higher levels of this saponin in leaves than in roots. Saponins 969 [M−H]− (9) and 835 [M−H]− (6) were detected only in leaves. All leaf samples contained moderate amounts of saponin 6 (7.4–9.5% of total saponin) and very low amounts of saponin 9 (0.2–0.7%). The investigation of different red beet samples led to the demonstration of some divergences in the total saponin content in the roots and leaves of different red beet cultivars, in plants of the same species and variety. Specific organs contain the same glycosides, albeit in slightly different proportions. Furthermore, studies on saponins occurring in other species showed significant differences between their content in different varieties of other crops. An example of such a plant

is quinoa, whose seeds of sweet varieties contain 23–28 times less saponins than the bitter varieties (Medina-Meza et al., 2016). In turn, in three cultivars of Medicago truncuatula, the differences in saponin content were approximately 30% (Kapusta et al., 2005). Probably, selection leading to the new varieties of plants can have a targeted effect on the content of saponins in a variety if the features related to their presence are subject to selection. Beetroots are selected for such properties as the intensity of the color of the flesh (related to the content of betalain), clarity of rings, sugar content, and reduced tendency to accumulate nitrates, all of which have no relation to the content of saponins. On the other hand, the characteristics of cultivable varieties important in the food industry, such as root durability during storage or leaf durability sold in bunches, may be related to the content of saponins. The relatively high level of saponins in the roots and leaves can affect the durability of the food material, due to the bacteriostatic and fungicidal properties that often characterize saponins. From the tested varieties, cultivars with a relatively high content of saponins in the roots – Red Sphere, Egyptian, and Round Dark Red – are appropriate for long storage (Kołota and Adamczewska-Sowińska, 2006). The Egyptian and Round Dark Red varieties, cultivated for their leaves, are characterized by the highest content of saponin in the leaves. On the other hand, the Forono variety, which is cultivated for fresh root consumption and food processing, has a very low content of saponins in both organs and cannot be stored for a long period of time. Both organs differ in terms of the content of the total sum of saponins as well as of individual compounds. In most cultivars, saponins with a molecular mass 956 (5) is clearly dominant in the roots, whereas, in the case of leaves, its content is reduced significantly or is lower than the content of its potent tetraglycoside derivative with a molecular mass of 1088 (2). This may be related to the biological function of these compounds in the plant. In addition, specific conjugation patterns may be indicative of function, because the level of biological activity of saponins is often associated with the specific sugar composition. The differential accumulation of saponins, both in the roots and in the leaves, may be indicative of specific protective function against pathogens, competitive plants, and herbivores, and these activities are undoubtedly related to the construction of saponins (Oleszek and Biały, 2006). 3. Experimental 3.1. Plant material Red beet plants (Beta vulgaris L.) of cultivars Egyptian, Forono, Red Sphere, and Round Dark Red were collected from fields in central Poland in October 2011 and divided into roots and leaves. The roots 4

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and leaves of 10 plants of every cultivar were pooled, then dried at 40 °C, and finely powdered using a grinder (Moulinex, France).

mixture. Quantitation was based on external standardization by employing the calibration curve of oleanolic acid in the range of 80–560 ng/mL. The quantitative analyses were based on the peak area calculated from the selected ion chromatograms of the corresponding [M−H]− ion, and saponins were identified through a comparison of their retention times and ion mass. Microsoft Excel 2000 was used for statistical analysis.

3.2. Chemicals and reagents Oleanolic acid standard was purchased from Sigma-Aldrich (Steinheim, Germany). The 3-O-b-D-glucuronopyranosyl of oleanolic acid was obtained earlier from Calendula officinalis flowers in the Department of Plant Biochemistry, University of Warsaw, Poland. All other chemicals were from Linegal Chemicals (Warsaw, Poland).

Acknowledgments We thank Professor Wiesław Oleszek from Department of Biochemistry, Institute of Soil Science and Plant Cultivation for giving us opportunity to perform analysis on the HPLC-MS equipment. The study was supported by the Polish government grant N N312 202635.

3.3. Extraction 1 g samples of finely powdered material were extracted three times with the assistance of a microwave oven (300 W) for 10 min, using 50 ml of 80% methanol each time. Three independent extractions were performed for each cultivar. The obtained extracts were filtered and concentrated to dryness under reduced pressure (rotary evaporator Heidolph, Schwabach, Germany).

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3.4. Purification The extracts were diluted in 4 ml water and loaded into Lichrolut 1000 mg RP-18 cartridges (Merck, Darmstadt, Germany) preconditioned with methanol and water in a vacuum manifold. The cartridges were washed with water and then eluted with 40% and 80% methanol in water and, finally, in 100% methanol. 24 ml volume of each solution was used, and elutes were monitored by thin layer chromatography (TLC) on silica gel plates (Merck, Darmstadt, Germany) that were developed in ethyl acetate/acetic acid/water (7:2:2 v/v/v). Triterpenoids were visualized by spraying the TLC plates with 50% sulfuric acid in water, followed by heating at 130 °C. Saponins were eluted with 80% methanol, evaporated under reduced pressure, and redissolved in 2 ml of methanol for direct injection into MS/MS analyses. For UPLC, 0.5-mL samples were was transferred to another vial, evaporated, and redisolved in 2 ml of 25% acetonitrile in water. 3.5. ESI-MS/MS analysis Analyses were conducted in a Thermo Finnigan LCQ Advantage Max ion-trap mass spectrometer with an electrospray ion source. The following instrumental parameters were used for ESI-MS/MS analysis of saponins: spray voltage 4.2 kV; capillary offset voltage to −60 V; capillary temperature 220 °C; and nitrogen ion injection time of 200 ms. The calibration of the mass range (400–2000 Da) was performed in negative ion mode. SIM (selected ion monitoring) in negative ion mode was used as the detection mode. 3.6. UPLC-MS analysis Analyses were carried out using an Acquity ultra-performance liquid chromatograph (Waters) coupled with an Acquity TQD tandem quadrupole mass spectrometer with an ESI source. The separation was undertaken using a 50 × 2.1 mm i.d., 1.7 μm, Acquity UPLC BEH C18 column. A mobile phase consisting of 0.1% formic acid in acetonitrile (B) and 0.1% formic acid in water (A) was used for the separation. The gradient elution was linear from 25% to 60% B, over 0–6 min; isocratic at 60% B, over 6–6.5 min; linear from 60% to 25% B, over 6.5–6.6 min; isocratic at 25% B, 6.6–7 min. The column was maintained at 50 °C at a constant flow rate of 0.4 mL/min. The sample injection volume was 5 μL. The following instrumental parameters were used for ESI-MS analysis of saponins: capillary voltage, 3500 V; cone voltage, 80 V; desolvatation gas, nitrogen 800 L/h; cone gas, nitrogen 100 L/h; source temperature, 120 °C; desolatation temperature, 350 °C; and dwell time, 0.05 s. The detection mode was SIR in negative ion mode. Two independent chromatographic runs were undertaken for each saponin 5