tandem mass spectrometry.

276

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)

Comprehensive Study of the Phenolics and Saponins from Helleborus niger L. Leaves and Stems by Liquid Chromatography/Tandem Mass Spectrometry by Sarina M. Duckstein and Florian C. Stintzing* WALA Heilmittel GmbH, Department of Analytical Development & Research, Section Phytochemical Research, Dorfstrasse 1, DE-73087 Bad Boll/Eckwlden (phone: þ 49-7164930-6665; fax: þ 49-7164930-5699; e-mail: [email protected])

The aerial parts of the medicinal plant Helleborus niger L. comprise a substantial number of constituents with only few of them identified so far. To expand the knowledge of its secondary metabolite profile, extracts from H. niger leaves and stems were investigated by liquid chromatography/tandem mass spectrometry (LC/MSn ). Specific identification strategies using LC/MS are established and discussed in detail. The leaves turned out to contain acylated and non-acylated quercetin and kaempferol oligoglycosides, protoanemonin and its precursor ranunculin, b-ecdysone, and a variety of steroidal saponins, mainly in the furostanol form. The sapogenins were elucidated as of sarsasapogenyl, diosgenyl, and macranthogenyl structures, and confirmed by comparison with the respective reference compounds. The secondary metabolite profiles were almost identical in both plant parts except that the stems lacked kaempferol derivatives and some saponins. The ranunculin derivatives and b-ecdysone were found in both plant parts. Correlations between the location of the compound groups and the plants defense strategies are proposed. Additionally, the role of the detected secondary metabolites as protective substances against exogenic stress and as a defense against herbivores is discussed.

Introduction. – As a member of the Ranunculaceae family, the genus Helleborus comprises ca. 20 species including Helleborus niger, popularly known as Christmas rose. H. niger grows mainly in the European Alps and some adjacent areas. The perennial plant builds its foliage directly from the planar grown rootstock, resulting in basal leaves with dominant stems [1] [2]. Its rhizome Hellebori nigri rhizoma, traditionally used for treatment of constipation, nausea, worm infections, and nephritis, is known for its substantial toxicity, which had led to a considerable change in todays application of this medicinal plant [2]. Already being in use in Europes complementary medicine as supportive therapy for treatment of different tumors, a recent in vitro study demonstrated the whole H. niger plant extract to possess remarkable cytotoxic activity on a leukaemia cell line [3]. Nevertheless, recent investigations on the secondary plant compounds of the genus Helleborus are still scarce or dating back up to 30 – 50 years, and mostly focusing on the rhizome. It is recognized that the roots from H. niger contain b-ecdysone and steroidal saponins [4], of which only macranthosid I had unambiguously been identified so far [5]. Furthermore, the presence of the lactone protoanemonin [6] and contradicting information about a cardiac glycoside, hellebrin, have been reported [5] [7]. Among the published data, only very limited information on the secondary-metabolite composition of the aerial plant organs is available: Acylated flavonoid glycosides, namely, quercetin 3-O-(2-trans-caffeoyl)-a-l-arabinopyranosyl-(1 ! 2)-b-d-glucopyranoside in H. foetidus L. [8] and H. viridis L. [9] as well 2014 Verlag Helvetica Chimica Acta AG, Zrich


277

as quercetin 3-O-2-((E)-caffeoyl)-a-l-arabinopyranosyl-(1 ! 2)-b-d-glucopyranoside7-O-b-d-glucopyranoside in H. viridis [9] and quercetin 3-O-2-((E)-caffeoyl)-a-larabinopyranosyl-(1 ! 2)-b-d-glucopyranoside-7-O-b-d-glucopyranoside in H. niger [10] have hitherto been identified. Additional non-acylated flavonoid glycosides detected in H. viridis and H. niger leaves are quercetin 3-O-a-l-arabinopyranosyl-(1 ! 2)-b-d-galactopyranoside and kaempferol 3-O-a-l-arabinopyranosyl-(1 ! 2)-b-d-galactopyranoside-7-O-b-d-glucopyranoside, respectively [9] [10]. Also, some furostanol saponins with aglycones of the sarsasapogenyl, diosgenyl, and ruscogenyl type were reported from H. viridis [9] [11]. Furthermore, additional secondary metabolites, glycosyl-hydroxyphenylethyl alcohol, 1-O-caffeoyl-glucose, and phenyllactic acid-2-Oglucoside were described [8] [10]. The main objective of the present investigation was to broaden our knowledge on the constituents of the H. niger aerial parts by analyzing extracts from leaves and stems via liquid chromatography/tandem mass spectrometry (LC/MSn ). By comparison with reference compounds and previous literature, the collected data were interpreted to provide a general overview on its active compounds. Furthermore, the role of the secondary metabolites distributed over the stems and leaves is discussed. Since whole plant extracts from Helleborus roots and the aerial parts are already in the focus of clinical studies and tumor therapies [3] [12] [13], it is important to improve the knowledge of its constituents to unveil the whole potential of the plant. Results and Discussion. – Since only scattered information on the constituents of H. niger leaves is available, an acetone/H2O mixture 1 : 1 (v/v) was used for extraction of polar hydroxycinnamic acid glycosides, medium-polar acylated flavonol glycosides, and saponins, followed by the removal of acetone prior to chromatographic and massspectrometric analyses. Further sample treatment, except centrifugation and filtering, was consciously omitted to obtain an unaltered fingerprint. LC/MSn Analyses were performed in positive- and negative-ionization mode to obtain maximum information on the composition of the leaf and stem constituents for evaluation of their distribution and role in the plant. Identification of Flavonol Glycosides. Among the already described phenolic constituents in Helleborus leaves, acylated quercetin and kaempferol 3-O- and 7-Oglycosides have been reported [8 – 10]. Of particular interest is the repeating structural unit common to the above mentioned quercetin and kaempferol glycosides: a 3-Oconnected hexose linked to a pentose (arabinose), followed by caffeic acid. Therefore, with a sufficient degree of probability, it can be assumed that additional flavonol glycosides should show the same molecular architecture. Due to the identical MS neutral loss of hexose and caffeic acid (162 amu), the possibility of acylated or nonacylated glycoside peaks was examined by verifying their specific UV maxima: the first and second maximum of quercetin (ca. 266 and 350 nm, resp.) and kaempferol (ca. 266 and 346 nm, resp.) confirmed the presence of a non-acylated glycoside, whereas a displaced broad second maximum at ca. 330 nm originating from overlapping absorption with a hydroxycinnamic acid indicates an acylated flavonol glycoside [14]. This indication is useful for identification of other likewise UV-shifting hydroxycinnamic acids, e.g., ferulic acid (176 amu) and coumaric acid (146 amu), to

278


avoid confusion with other possible glycosidic extensions such as glucuronic acid (176 amu) and deoxyhexose (146 amu). The fingerprint of a representative H. niger leaf extract including 15 flavonol glycosides (Peaks 10 – 24) is depicted in Fig. 1. Additional chromatographic, spectroscopic, and spectrometric data are compiled in Table 1. For structural assignments, the MS negative-ion mode was applied exclusively. By using an MS ion-trap instrument, it was possible to cleave the supplemental structural units attached to the flavonol in such a way to detect the aglycone mass peak (MS4 ) and its fragments (MS5 ). By comparison with the respective reference compounds, the aglycones quercetin and kaempferol were assigned. Their side-chain attachments were verified as follows: first, the UV maxima were checked for presence of a hydroxycinnamic acid moiety as discussed above. Nearly half of the detected flavonol glycosides (Peaks 12, 14, 16, 19, 20, 22, and 23; Table 1) revealed hydroxycinnamic acid attachments, including mainly kaempferol derivatives (Peaks 12, 16, 19, 22, and 23; Table 1). Further structure elucidation by tandem MS was identical for each acylated flavonol and is demonstrated by one example, kaempferol 3-O-hex-pent-caf-7-O-hex-hex (Peak 12), the first eluting acylated representative (Fig. 2). After ionization of the molecule yielding a pseudomolecular-ion peak at m/z 1065, the first fragmentation step in the ion trap led to the cleavage of the most labile side chain, located at C(7) of the flavonol skeleton [15]. The resulting MS2 fragments with peaks at m/z 903 as well as at m/z 741 (base peak) originating from two short consecutive neutral losses of 162 amu most probably represent two hexoses. Theoretically, one or both of these mass losses could also be caffeic acid(s), but the report about a 7-O-monoglucosylated quercetin glycoside from H. viridis and H. niger leaves [9] [10] and the fact that acylated flavonol glycosides from other plant sources were not acylated at C(7), but glycosylated instead [14] [15], was evidence enough to conclude the presence of a dihexoside side chain attached to the 7O-position of the kaempferol aglycone. Further fragmentation of the ion with the peak at m/z 741 cleaved another 162 amu yielding the prevailing mass peak m/z 579 in MS3, followed by subsequent abstraction of 132 and 162 amu preliminarily resulting in the aglycone peak at m/z 284/285 in MS4. The connection point of the three units could also be assigned by considering the structural data of the already published acylated quercetin and kaempferol structures from Helleborus [8] [10] which all have a 3-Oconnected side chain containing a hexose-pentose-caffeic acid moiety. Additional evidence for the 3-O-connection point is the presence of the peaks at m/z 284/285 representing the heterolytically (Y0 ) and the homolytically ([Y0 H] . ) cleaved aglycone product ions [16]. Other flavonol glycosides, mostly quercetin derivatives, were not acylated (Peaks 10, 11, 13, 15, 17, 18, 21, and 24; Fig. 1, Table 1). This could be discerned in an unaltered classical position of the UV maximum of quercetin and kaempferol. Further structural elucidation was conducted as described above affiliating the connection points of the side-chain glycosides. Additionally, the already described kaempferol 3-Ohex-pent-7-O-hex from H. niger leaves [10] was detected (Peak 13) and confirmed. The acylated quercetin 3-O-hex-pent-caf-7-O-hex (Peak 14) reported for H. viridis [9] had also been found in H. niger leaves. An overview of the flavonol glycoside structures in H. niger, including the repeating 3-O-hexose-pentose unit, is presented in Fig. 3.

Fig. 1. LC/MS Fingerprint of a representative H. niger leaf extract, depicted as follows: TIC, recorded at positive-ion mode (a, main image) including chromatogram section 0 – 10 min (inset), TIC, negative-ion mode (b), DAD chromatogram, recorded at 360 nm (c, main image) and 300 as well as 200 nm (inset). For peak assignment and chromatographic as well as spectrometric data, see Tables 1 and 2.

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014) 279

22.5

26.2

27.6

28.5

28.7

30.9

2

3

4

5

6

7

Ferulic acid

Caffeic acid

Caffeic acid

Ferulic acid

Coumaric acid

Caffeic acid

hex-hex

hex

hex-hex II

hex-hex I

hex-hex II

hex-hex I

) 533

g

277

487

306

517

298; 322 341

298; 322 503

298; 322 563

302

d

[ M H]

[ M H]

341 179 179 161 355 193

325 163 517 355

[ M H] [Mþ HCOOH H] [ M H]

341 (100); 179 (26)

[ M H]

(100); (7) (100); (16) (100); (16)

(100); (15) (28); (100)

487 (37); 325 (100)

115 (100); 97 (21)

MS [m/z]

2

(2); (45); (100); (66); (10) (8); (100); (30); (11) (100); (14) (34); (100); (21) (100); (16) (100) 193 (100)

265 187 163 145 119 203 179 161 135 163 119 217 193 175 179 135 135

97 (100); 69 (4)

MS [m/z]

3

Fragments ( BPI [%])

[Mþ HCOOH H]

[ M þ H] þ

Pseudo- Species ) molecular ion [m/z]

MS Data a )

298; 322 503

Hydroxycinnamic acids and further polar compounds f R 2.6 5-Hydroxy) hex methyl-2(5H )furanone e ) P 7.7 Protoanemonin 260 1 20.2 Coumaric acid hex-hex I 308

Peak tR [min] Peak assignment lmax no. Aglycone Side chain c ) [nm]

(60); (62); (100) (100)

178 (80); 149 (100); 134 (82)

178 149 134 135

119 (100)

135 (100)

119 (100)

69 (100)

MS [m/z]

4

MS [m/z]

5

þ

þ

þ

–

þ þ

þ

þ

þ

þ þ

þ

þ

þ

þ

þ þ

þ

Leaves Stems

Detection b )

Table 1. Overview of the Flavonol and Hydroxycinnamic Acid Glycosides as well as Further Polar Compounds Detected in H. niger Extracts from Leaves and Stems, Including Their Characteristic Chromatographic and Mass Spectrometric Data

280 CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)

35.5

9

Phenyllactic acid

Ferulic acid

36.4

37.5

37.9

11

12

13

Kaempferol

Kaempferol

Quercetin

Flavonol glycosides 10 35.9 Quercetin

33.2

8

3-O-hexpent-7-Ohex

3-O-hexpent-caf-7O-hex-hex

3-O-hexpent-7-Ohex II

3-O-hexpent-7-Ohex I

hex

hex

266; 318sh; 348

238; 298sh; 328

256; 266sh; 354

256; 266sh; 354

292; 316

Peak tR [min] Peak assignment lmax no. c Aglycone Side chain ) [nm]

Table 1 (cont.)

741

1065

757

757 595 (100); 463 (3); 301 (5)

595 (100)

903 (76); 741 (100)

579 (100)

[ M H]

[ M H]

[ M H]

327 (100)

[2M H]

655

[ M H]

355 (100)

[2M H]

711

447 (9); 429 (27); 285 (11); 284 h ) (100)

463 (11); 445 (17); 301 (62); 300 h ) (100); 179 (3) 463 (20); 445 (25); 301 (50); 300 h ) (100); 179 (4) 579 (100)

165 (11); 147 (100); 119 (6)

193 (100)

MS3 [m/z]

Fragments ( BPI [%]) MS2 [m/z]

d


MS Data a )

447 (20); 429 (70); 327 (10); 285 (100); 284 h ) (89) 255 (100); 229 (29); 51 (27)

271 (100); 255 (66); 243 (83); 229 (69)

271 (100); 255 (74); 179 (40); 151 (40)

178 (83); 149 (100); 134 (62) 119 (40); 103 (100)

MS4 [m/z]

255 (100); 151 (8)

255 (77); 201 (100)

MS5 [m/z]

þ

þ

þ

þ

þ

þ

–

–

þ

þ

þ

–

Leaves Stems

Detection b )


38.1

38.5

38.8

39.1

40.3

41.0

14

15

16

17

18

19

Kaempferol

Kaempferol

Quercetin

Kaempferol

Quercetin

Quercetin

3-O-hexpent-caf-7O-hex

hex-hex I

hex-hex

3-O-hexpent-cou-7O-hex-hex

3-O-hexpent-7-Ohex-hex

3-O-hexpent-caf-7O-hex

246; 268; 322

266; 344

256; 266sh; 352

324

266; 348

272; 296sh; 328

Peak tR [min] Peak assignment lmax no. Aglycone Side chain c ) [nm]

Table 1 (cont.)

d

903

609

625

1049

919

919 757 (100); 595 (46)

757 (100); 595 (49)

887 (100); 725 (49)

463 (100); 301 (42)

489 (14); 447 (100)

741 (100)

[M H]

[M H]

[M H]

[M H]

[M H]

MS2 [m/z]

327 (15); 285 (67); 284 h ) (100); 255 (24) 579 (100); 429 (3); 285 (7)

301 (100); 300 h ) (32)

725 (100); 579 (13)

595 (100); 301 (2)

595 (100); 301 (2)

MS3 [m/z]


[M H]


MS Data a )

(100); (2); (39); (32) (100); (12)

447 (16); 429 (46); 285 (100); 284 h ) (69); 255 (20)

271 243 179 151 255 227

463 (15); 445 (18); 301 (60); 300 h ) (100); 271 (19) 463 (20); 445 (25); 301 (49); 300 h ) (100); 271 (17) 579 (100); 561 (25); 429 (5); 458 (8)

MS4 [m/z]

(100); (63); (25); (28)

(100); (58); (43); (25)

255 (100); 151 (23)

227 (100); 211 (41)

447 (5); 429 (27); 285 (100); 284 h ) (54); 255 (11)

271 255 179 151

271 255 179 151

MS5 [m/z]

þ

þ

þ

þ

þ

þ

–

–

þ

–

–

þ

Leaves Stems

Detection b )


42.4

44.0

45.5

49.8

21

22

23

24

Quercetin

Kaempferol

Kaempferol

Kaempferol

Quercetin

3-O-hexpent

3-O-hexpent-cou-7O-hex

3-O-hexpent-fer-7O-hex

hex-hex II

3-O-hexpent-fer-7O-hex 609

933

256; 266sh; 354

595

266; 316 887

266; 332 917

266; 344

256; 266; 336

d

771 (100); 757 (34); 595 (47) 489 (17); 447 (100); 285 (19) 755 (100)

725 (100)

463 (14); 445 (20); 301 (50); 300 h ) (100)

[M H]

[M H]

[M H]

[M H]

MS2 [m/z]

271 255 179 151

(100); (63); (32); (25)

579 (100); 561 (23); 285 (8)

327 (21); 285 (54); 284 h ) (100); 255 (28) 593 (100); 579 (94); 561 (44)

609 (20); 595 (100); 577 (19)

MS3 [m/z]


[M H]


MS Data a )

447 (11); 429 (31); 285 (100); 284 h ) (59) 447 (13); 429 (38); 285 (100); 284 h ) (67); 255 (17)

463 (22); 445 (22); 301 (49); 300 h ) (100) 255 (100); 227 (12)

MS4 [m/z]

255 (100); 151 (18)

255 (100); 151 (33)

227 (59); 211 (39)

271 (88); 255 (100); 151 (24)

MS5 [m/z]

þ

þ

þ

þ

þ

þ

–

–

–

–

Leaves Stems

Detection b )

a ) The m/z values of dominant precursor ions are given in bold. b ) þ , Present; , not present. c ) hex, hexose; pent, pentose; caf, caffeic acid; cou, coumaric acid; fer, ferulic acid. d ) The pseudo-molecular ion also occurs as adduct or cluster ion. e ) Does not show absorption characteristics. f ) 5-Hydroxymethylfuran2(5H )-one-hex corresponds to ranunculin. g ) Does not ionize in the ESI ion source. h ) Homolytically cleaved aglycone fragment [ Y0 H] . .

41.4

20

Peak tR [min] Peak assignment lmax no. c Aglycone Side chain ) [nm]

Table 1 (cont.)


Fig. 2. Tandem mass spectrum of kaempferol 3-O-hex-pent-caf-7-O-hex-hex (Peak 12, for further information, see Table 1) detected in H. niger leaves. Fragmentation characteristics are depicted in negative-ion mode (left), including interpretation of the neutral losses and designation of the corresponding ion peaks. A description of each fragmentation step is outlined in the right segment.



285

Fig. 3. Overview of acylated and non-acylated flavonol glycosides detected in H. niger leaves and stems. Left: Typical structural patterns are depicted as a molecular formula. Right: The characteristics of the varying attachments. Abbreviations: hex, hexose, caf, caffeic acid, cou, coumaric acid, fer, ferulic acid. For peak assignment, and chromatographic as well as spectrometric data, see Table 1.

Identification of Saponins. The second dominant compound class present in H. niger leaves are steroidal saponins. Apart from the furostanol saponin macranthosid I (3-O[glucopyranosyl-(1 ! 6)-glucopyranosyl]-26-O-glucopyranosyl-furost-25-en-3,22,26triol), already identified in H. niger roots [5], only related saponins in H. foetidus and H. viridis leaves with an aglycone structure akin to sarsasapogenin, diosgenin and ruscogenin in their corresponding furostanol form have been reported earlier [8] [9] [11]. Hence, that the saponins in H. niger leaves may be at least partially constituted in a similar way appeared plausible. Of course, sapogenin skeletons possess a specific stereodiversity and, therefore, additional designations. Since these cannot necessarily be detected via MS, the identified sapogenin aglycons were labeled according to the most common structure (e.g., sarsasapogenin and diosgenin). Generally, UV/VIS detection of most of the saponins is difficult due to their lack of chromophores [17]. Slight absorption above 200 nm is achieved due to the presence of C¼C bonds in the steroid skeleton. However, it should be noted that sarsasapogenyl glycosides exhibit hardly any UV absorption above 190 nm due to their lack of C¼C bonds. Therefore, the saponins were exclusively detected by means of positive- and negative-ion tandem mass spectrometry. The positive and negative total ion currents (TIC) obtained from a representative H. niger leaf extract are depicted in Fig. 1, a and b, including the saponin fraction (60 – 78 min). In contrast to phenolics, both ionization modes produced signals with substantial intensities. This offers a valuable basis for structure elucidation as both ionization polarities provide different clues. Steroidal saponins occur as furostanol- and spirostanol-type structures. Whereas the pentacyclic furostanol saponins possess an open side chain attached to C(22) of the steroid skeleton, the spirostanol form contains an additional C(22)-connected ring (ring F) derived from condensation of the side chain lacking a sugar moiety at C(26) [18]. In the ESI ion source, ionization of spirostanol saponins resulted in [M þ H] þ ions in positive, and [M H] or [(M þ HCOOH) H] ions in the negative-ion mode. While the furostanol saponins also form [M H] or their formic acid adduct ions in the negative-ion mode, in the positive-ion mode their ionized form differs significantly

286


from that of the spirostanols. Due to the free and rather labile HOC(22) group, which is absent in the spirostanol type, ion formation in MS1 took place by cleavage of this element, resulting in a prominent dehydrated pseudo-molecular ion, [(M H2O) þ H] þ [19] [20]. Compared to the negative-ion mode, this mass discrepancy of 16 amu was confirmed by the analysis of the reference macranthosid I (Fig. 4), whereas trillin (diosgenin 3-O-glucoside), the spirostanol standard used, followed the classical way for spirostanol ionization as described above. In all cases of the saponins, no significant Na or K adducts were found, most possibly due to the use of HCOOH as eluent additive. Among the saponins present in H. niger leaves, macranthosid I (Peak 34) and nine additional furostanol representatives were detected (Peaks 26, 28, 30, 31, 34, 36, 38, 39, and 42; Fig. 1, Table 2). Structure elucidations of the unknown furostanol saponins were conducted as depicted of the example of the reference macranthosid I (Fig. 4). Starting at the dehydrated pseudo-molecular ion [(M H2O) þ H] þ and [M H] with peaks at m/z 901 and 917, respectively, sugar abstraction in the positive-ion mode occurred in a single fragmentation step (MS2 ), ending up with the dehydrated aglycone (m/z 415), whereas, in the negative-ion mode, the sugars were cleaved successively (MS2 – MS5 ), until the aglycone mass peak was detected at m/z 413. Therefore, information on the kind and sequence of the attached saccharides is best obtained from negative-ion mode. Receiving additional structural information on the dehydrated aglycone (m/z 413) after sugar cleavage and H2O elimination was not possible in this mode due to only two OH groups remaining. However, in the positive-ion mode, ionization and fragmentation took place in a different way, underlined by the finding that the aglycone mass peak yet occurred in MS2 and was fragmented further (Fig. 4). To date, only a few publications dealt with the fragmentation mechanism of the sapogenin steroid skeleton proceeding in the ion trap after electrospray ionization [19 – 21]. As reported, after cleavage of the sugar moieties from the dehydrated pseudomolecular ion, [(M H2O) þ H] þ , the enol structure at C(22) is converted to the corresponding C¼O structure via tautomerization, followed by fission of the furostanol side chain (144 amu neutral loss) according to McLafferty rearrangement [19]. Further fragmentation proceeds under elimination of H2O resulting in the corresponding diagnostic fragment-ion peaks at m/z 273 and 255 in the case of sarsasapogenyl structures. Diosgenyl sapogenins yield peaks at m/z 271 and 253 due to their C(5)¼C(6) bond. The important sarsasapogenin diagnostic ion peaks at m/z 273 and 255 were also detected for macranthosid I in MS2 (Fig. 4, a), indicating the same molecular steroid ring structure. Nevertheless, a possible side-chain cleavage via the enol structure has not been addressed earlier [19] [20]. Apart from previous reports, macranthosid I produced a prominent mass peak at m/z 397 resulting in m/z 379 (D 18 amu) and the diagnostic peak at m/z 255 (D 124 amu; Fig. 4, a) after further fragmentation. These findings led to an additional postulated fragmentation path based on the detected ions (Fig. 4, a). Starting from the dehydrated C(22) aglycone enol structure [(M H2O 3 hexoses) þ H] þ in MS2, the electron-pair transfer from the Oatom at C(26) to C(22) as well as from C(17) forming a C(16)¼C(17) bond provokes the cleavage of H2O, resulting in a prominent peak at m/z 397. Within this constellation, the positive charge is located at the O-atom at C(26) and stabilized via mesomeric forms, e.g., tautomerization into the keto form. This step induces the formation of a spirostanol-like structure, including further fragmentation related to a spirostanol

Fig. 4. Tandem mass spectra of macranthosid I (Peak 35; for further information, see Table 2) detected in H. niger leaves and stems. Fragmentation characteristics are depicted in positive- and negative-ion modes (a and b, resp.), including interpretation of the neutral losses and a proposed fragmentation mechanism of the aglycone macranthogenin (a) based on the diagnostic fragments with peaks at m/z 379 and 255 (positive-ion mode). Description of each fragmentation step is outlined in the middle.


b-Ecdysone

63.3

63.9

27

28

Sarsasapogenin hex-hex-hexhex II

Unknown agly- hex-hex I cone

Saponins (steroid glycosides) 26 62.5 Sarsasapogenin hex-hex-hexhex I

Ecdysteroid 25 50.9

furo

furo

1082 903 (16); 741 (24); 579 (100);

[ M H2O þ H] þ 1065

837 f )

(100); (41); (6) (65); (100)

919 757 595 833 819

[ M H]

1081

(17); (9); (83); (100); (21)

(68); (7); (100); (19)

1082 903 741 579 417 399

480 427 409 371 303

[ M H2O þ H] þ

[M 2 H2O þ H] þ

MS2 [m/z]

(46); (20); (40); (16); (41); (100)

(100); (18); (7) (55); (100); (26); (26) 435 (39); 273 (44); 255 (100)

757 595 433 789 657 495 477

273 (100); 255 (41)

353 335 247 231 215 123

MS3 [m/z]

MW Fragments ( BPI [%])

1065

445

Peak tR [min] Peak assignment MS Data a ) no. c Aglycone Sugar moiety ) Steroid Pseudo- Species e ) typed ) molecular ion [m/z]

627 537 495 407 289 213 199 173

(31); (14); (100); (12); (33) (8); (30); (92);

227 199 185 173 159 147 117 595 (100); 433 433 (44) 255 (100)

(25); (51); (32); (100); (79); (16); (16) (100)

MS4 [m/z] MS5 [m/z]

þ

þ

þ

þ

þ

þ

þ

þ

Leaves Stems

Detection b )

Table 2. Overview of the Steroidal Constituents (mainly steroidal saponins) Detected in H. niger Extracts from Leaves and Stems, Including Their Characteristic Chromatographic and Mass-Spectrometric Data


64.5

64.7

65.5

29

30

31

Sarsasapogenin hex-hex-hexhex IV

Sarsasapogenin hex-hex-hexhex III

Unknown agly- hex-hex II cone

furo

furo

[ M H2O þ H] þ

[ M H] 1081

[ M H]

1065

1081

1082 903 741 579 435 417 399 919 757 595 1082 903 741 579 435 417 399

(12); (15); (100); (24); (46); (32) (100); (41); (10) (26); (14); (100); (28); (60); (53)

835 (100); 685 (27)

[Mþ HCOOH H] [ M H2O þ H] þ

881

1065

836 833 (100)

(66); (42) (100); (39)

[ M þ H] þ

[ M H]

417 399 919 757

MS [m/z]

2

(100); (23); (8) (16); (100); (37)

757 595 433 435 417 273 255

435 417 399 273 255

(100); (18); (6) (24); (9); (27); (100)

(26); (7); (7); (29); (100)

685 (100); 523 (28)

757 595 433 789 671 509

MS [m/z]

3


837

1081

MS Data a ) Peak tR [min] Peak assignment no. c Aglycone Sugar moiety ) Steroid Pseudo- Species e ) typed ) molecular ion [m/z]

Table 2 (cont.)

213 185 173 159 147

227 199 185 173 159 147 595 433

(9); (15); (69); (33); (100)

(6); (20); (21); (99); (64); (100) (100); (42)

(17); (17); (100); (70); (51) (100)

433 (100)

105 (100)

433 (100)

119 (68); 105 (100); 91 (49)

493 465 207 177 150 523 (100); 361 361 (9)

(76); (100) (100); 433 (100) (48) 627 (82); 509 (100)

159 147 595 433

5

MS [m/z] MS [m/z]

4

þ

þ

þ

þ

þ

Leaves Stems

Detection b )


66.4

66.7

67.6

68.2

32

33

34

35

Macranthogenin g )

Diosgenin

hex-hex-hex I

hex-hex-hex

Unknown agly- hex-hex IV cone

Unknown agly- hex-hex III cone

furo

furo

[Mþ HCOOH H]

863

755 593 575 918 739 577 435 415 397

[ M H] 917

[ M H2O þ H] þ

918 739 577 433 415 397

[ M H2O þ H] þ

901

(100); (46); (9) (100); (32); (39) (100)

(100); (11); (6) (24); (100); (61); (72); (76);

(70); (100); (17); (70); (67)

819 (100); 675 (86); 513 (19)

919 757 595 818 657 495 477 817

MS [m/z]

2

(100); (16); (5) (100); (45)

593 575 431 435 415 397 273 255

433 415 271 253

(100); (82); (16) (33); (10); (15); (34); (100)

(63); (14); (91); (100)

789 (10); 657 (100); 495 (44)

655 (100)

757 595 433 495 477

MS [m/z]

3


901

837 f )

[ M þ H] þ

819


Table 2 (cont.)

213 187 173 159 147

627 495 431 281 191 211 197 185 171 157 145 575 431

(13); (27); (89); (71); (100)

(71); (100); (29); (15); (31) (24); (32); (20); (29); (100); (21) (34); (100)

465 (7); 177 (100)

595 (100); 433 (69)

413 (100); 299 (99)

465 (32); 177 (100)

135 (100)

5

MS [m/z] MS [m/z]

4

þ

þ

þ

þ

þ

þ

þ

þ

Leaves Stems

Detection b )


69.2

70.8

71.5

36

37

38

Macranthogenin f ) hex-hex-hex II

Unknown agly- hex-hex V cone

Sarsasapogenin hex-hex-hex I

furo

furo

[ M H2O þ H] þ

[ M H] 917

[ M H]

817 901

[ M þ H] þ

[ M H]

919

819

[ M H2O þ H] þ

[ M H]

903

917


Table 2 (cont.)

(12); (100); (30); (63); (62); (34); (38) (100); (26); (8) (100); (40) (12); (100) (23); (58); (7); (100); (36) 899 (40); 755 (100)

920 741 579 435 417 399 273 255 757 595 433 818 657 495 771 655 918 739 577 435 415 397

273 (59); 255 (65) 755 (100)

MS [m/z]

2

(28); (8); (32); (100)

(8); (65); (100); (29); (37) 737 (33); 593 (100); 433 (22)

397 273 271 255 253

495 (100); 477 (47) 493 (100)

595 (100); 433 (50)

435 399 273 255

593 (100); 433 (30)

MS [m/z]

3


226 211 197 173 157 147 563 433 431

135 (100)

(13); (41); (38); (33); (100); (37) (23); 415 (95); (100); 346 (64); (48) 289 (100)

465 (10); 177 (100)

415 (100)

(100); (49); (13) (18); 145 (100); (100); 131 (71); (58); 117 (32) (93)

433 (100)

575 433 431 187 173 159 147

5

MS [m/z] MS [m/z]

4

þ

þ

þ

þ

þ

Leaves Stems

Detection b )


72.3

72.8

73.6

74.1

39

40

41

42

Sarsasapogenin hex-hex

Hydroxy-sarsa- hex-hex-hex I sapogenin

Unknown agly- hex-hex VI cone

Sarsasapogenin hex-hex-hex II

furo

spiro

furo

741

917

(6); (68); (17); (100); (58); (53); (40) (100); (17) (100); (39); (9) (100)

755 (100)

758 579 (100); 435 (26)

[ M H2O þ H] þ

918 595 (100); 433 (25); 415 (14)

920 741 579 435 417 399 273 255 757 595 818 657 495 477 817

MS [m/z]

2

(100); (48) (100); (34)

435 417 273 255

577 547 415 397 301 283 593

(25); (2); (33); (100)

(100); (39); (15); (16); (16); (80) (100)

655 (100)

595 433 495 477

273 (100); 255 (45)

MS [m/z]

3


[ M H]

[Mþ HCOOH H] [ M hex þ H] þ

863

757

[ M þ H] þ

[ M H]

919 819

[ M H2O þ H] þ

903


Table 2 (cont.)

563 431 403 187 173 159 147 105

547 415 397 283 241

(100); (86); (34) (21); (100); (50); (94); (11)

(43); (23); (31); (100); (26)

465 (9); 177 (100)

433 (100)

255 (100)

(13); (36); (34); (97); (100); (90); (25) (100)

201 173 149 119

(75); (98); (82); (100)

135 (100)

213 199 185 173 159 147 105 415

5

MS [m/z] MS [m/z]

4

þ

þ

þ

þ

þ

þ

þ

Leaves Stems

Detection b )


76.2

44

Methoxy-sarsa- hex-hex sapogenin

Hydroxy-sarsa- hex-hex-hex II sapogenin

spiro

spiro

[ M þ H] þ

[ M H]

771

769

607 (100); 445 (31)

(95); (63); (100); (39); (14)

755 (100)

[ M H]

917

770 753 609 591 429 283

918 577 (15); 433 (100); 415 (19)

[M2 hex þ H] þ

595

(100); (13); (66); (70); (51); (36)

445 (100)

429 (19); 411 (14); 283 (100)

593 (100)

415 397 385 301 283 259

595 (100); 433 (48)

MS [m/z]

3

415 (100); 385 (87); 345 (67); 289 (68); 273 (52) 397 (49); 255 (60); 385 (96); 241 (38); 301 (91); 227 (24); 283 (100); 201 (100); 259 (46) 173 (97); 149 (76); 95 (56) 563 (100); 431 (56); 401 (67) 255 (22); 227 (61); 201 (66); 187 (66); 173 (100); 147 (61) 415 (100); 383 (25); 293 (36); 263 (26)

433 (100)

5

MS [m/z] MS [m/z]

4

þ

þ

þ

þ

Leaves Stems

Detection b )

a ) The m/z values of dominant precursor ions are given in bold. b ) þ , present, , not present. c ) hex, hexose. d ) furo, furostanol-type saponin, spiro, spirostanol-type saponin. e ) The pseudo-molecular ion also occurs as adduct, partially fragmented or dehydrated ion. f ) MS in positive-ion mode. g ) Aglycone of macranthosid I.

75.4

43

803 757 (100)

MS [m/z]

2


[Mþ HCOOH H]


Table 2 (cont.)


294


sapogenin. Additionally, charge remote elimination of the OH group at C(26), as suggested for a diosgenyl aglycon by Li et al. [19], was considered improbable, because of the naturally occurring C(25)¼C(27) bond of macranthosid I, inhibiting the formation of an elimination-driven C(25)¼C(26) bond. The accrued dehydrated keto steroid with a peak at m/z 397 undergoes H2O elimination (m/z 379) with subsequent scission of the spiroketal structure (D 124 amu; ring F), ending up with the diagnostic fragment peak at m/z 255 in MS3. Further fragmentation in MS4 led to the total breakup of the remaining steroid skeleton yielding various fragments (Fig. 4, a). It should be noted that the prominent neutral loss of 144 amu, as reported for the fragmentation of the furostanol side chain via E-ring cleavage of diosgenyl and sarsasapogenyl saponins [19] [20], was also detected for macranthosid I in MS2 (m/z 415 ! 273 or m/z 397 ! 255) as a neutral loss of 142 amu due to a C¼C bond in the side chain. On this background, in addition to macranthosid I (Peak 35) another macranthogenin triglycoside (Peak 38; Table 2) was detected and identified according to Fig. 4. However, most of the saponins occurring in H. niger leaves were sarsasapogenyl derivatives with two to four sugar moieties (Peaks 26, 28, 30, 31, 36, 39, and 42), the most intensive peak corresponding to sarsasapogenin trihexoside (Peak 36). Additionally, one diosgenyl trihexoside (Peak 34) with diagnostic ion peaks at m/z 415, 271, and 253 (Table 2), as described in [19], was identified. Steroidal tri- and tetraglycosides possessing sarsasapogenyl and diosgenyl aglycones have previously been detected in H. viridis leaves [9] [11], confirming the present findings of similar secondary metabolites within the genus Helleborus. Of these furostanol glycosides, minor peaks of spirostanol saponins (Peaks 41, 43, and 44; Table 2) were also detected, essentially corresponding to sarsasapogenyl glycosides with an additional OH (Peaks 41 and 43) or MeO group (Peak 44). These tentatively assigned structures exhibit slightly different fragmentation characteristics with additional H2O or CH2O cleavage. Hydroxycinnamic Acids and Miscellaneous Compounds. Besides the two main secondary metabolite fractions containing flavonol glycosides and steroidal saponins, some further compounds were detected. One very early eluting peak (R; tR 2.6 min) did not exhibit characteristic UV absorption and produced a pseudo-molecular ion, [M þ H] þ , only in the positive-ion mode (m/z 277), undergoing cleavage of one hexose (m/z 115) together with few additional product ions (m/z 97 and 69; Table 1; and Fig. 1, a, inset). The absence of signals in the negative-ion mode and the unspecific absorption led to the assumption that this structure possesses a low molecular weight, and no or only few double bonds, OH groups, or other UV-bathochromic-shifting features. Ranunculin, as the glycosidic inactive precursor of the lactone protoanemonin and occuring in many Ranunculaceae plants [22], was tentatively assigned to Peak R. By comparison with an authentic reference compound (tR and UV characteristics), the corresponding aglycone protoanemonin (Peak P) was also detected in the leaf extract (Table 1; Fig. 1, a, inset) which is in accordance with earlier reports on H. niger leaves [23]. It should be mentioned that protoanemonin is neither readily ionized in the negative- nor the positive-ion mode, rendering detection via tandem mass spectrometry virtually impossible. Another constituent group, represented by eight compounds (Peaks 1 – 8) was identified as hexose esters of coumaric (Peaks 1 and 3), caffeic (Peaks 2, 5, and 6) and


295

ferulic acids (Peaks 4, 7, and 8; Fig. 1; and Table 1) in comparison with MS and UV literature data [24] [25]. It is noteworthy that these hydroxycinnamic acids are biochemical building blocks of the acylated flavonoids described above. A quite similar finding was reported for Brassica leaves, also containing both, acylated flavonol glycosides and hydroxycinnamic acid glycosides [14]. Moreover, the report of 1-b-Ocaffeoyl-d-glucose as a biosynthetic intermediate from H. foetidus foliage [8] confirms this hypothesis. A phenyllactic acid glycoside peak (Peak 9) and a small peak of the ecdysteroid becdysone (20-hydroxyecdysone; Peak 25) were detected and assigned according to literature data [10] and a reference compound, respectively. Phenyllactic acid 2-O-b-dglucopyranoside has recently been identified in H. niger leaves [10], whereas becdysone was earlier found in H. niger roots [26] and aerial parts [27]. Additionally, some unknown glycosides (Peaks 27, 29, 32, 33, 37, 40; Fig. 1; and Table 2) were detected, eluting in the neighborhood of the saponins. By means of tandem MS analyses, distinct neutral mass losses of 162 amu indicated a glycosidic character, but further fragmentation of the aglycone provided no useful information on the underlying structure, i.e., in contrast to the steroid skeleton only very few fragment peaks appeared in MS3 – MS5 (Fig. 4). Also, no characteristic UV data were detected. Distribution of Secondary Metabolites in H. niger Leaves and Stems. The leaves of H. niger, directly grown from the rootstock, possess dominant stalks, resulting in plant heights of 15 – 30 cm [1]. These stalks were considered separately and compared with leafs regarding their constituents (Tables 1 and 2). Among the detected compounds, substantial differences were observed for the flavonol glycosides and slight variations within the saponin group. The rest of the compounds including ranunculin (Peak R) and protoanemonin (Peak P) were detected in both plant parts. Among others, flavonoid glycosides are considered to possess UV-protective and radical-scavenging properties [28 – 30]. Due to their specific flavan-3-ol structure, quercetin and kaempferol glycosides exhibit considerable radical-scavenging activities, with quercetin being somewhat more efficient due to its additional B-ring OH group [31]. Flavonol glycosides acylated with hydroxycinnamic acids possess particularly high UV-protective qualities compared to their corresponding non-acylated representatives [28]. Therefore, the overall and especially light-protective role of the flavonoid glycosides in H. niger leaves is obvious. The stems also contain flavonol glycosides, but in a less extensive range. As indicated in Table 1, only five of the identified flavonol constituents from the leaves were detected in the stems (Table 1). Interestingly, exclusively quercetin, but no kaempferol glycosides were observed. Similar results were found by Tronchet [32]. Possibly, since the stems are less exposed to sun light and UV radiation, fewer cytoprotective compounds are required. Correspondingly, the occurrence of hydroxycinnamic acid glycosides as possible precursors for acylated flavonol glycosides in both leaves and stems is plausible (Table 1). Additionally, hydroxycinnamic acids are virtually ubiquitous phenolic compounds in plants. Compared to flavonol glycosides, steroid saponins accomplish a different function in plant organs. They typically occur as inactive bidesmosidic precursors in cell vacuoles. In case of injury of the plant tissue, one sugar chain is cleaved enzymatically, resulting in the active, monodesmosidic type [33]. Steroidal saponins possess

296


distinctive cytotoxicity by interacting especially with cholesterol and other biomembrane constituents, resulting in destabilization and leakage of cells [18] [33] [34]. This strategy protects the plant against herbivores. Additionally, also the ranunculin derivatives occur as inactive precursors (ranunculin; Peak R) together with the active fission product protoanemonin (Peak P) to defend herbivores [22] [33]. Both, the ranunculin derivatives and several saponins were detected in leaves and stems. In both cases, the inactive precursors ranunculin and furostanol saponin were primarily found (Fig. 1, and Tables 1 and 2). The finding that these precursors were detected, although the plant material was cut with subsequent break-up of cell compartments, may be due to using an extraction solvent containing high organic proportion (50% acetone) denaturing the endogenous cleaving enzymes. Nevertheless, small peaks of the respective active compounds protoanemonin (Peak P) as well as some spirostanol saponins (Peaks 41, 43 and 44; Table 2) were found in the leaves and also in the stems in the case of protoanemonin (Peak P) indicating a slight enzymatic activity. Compared to H. niger leaves, one striking finding is the absence of the activated spirostanol saponins in the stems in addition to two furostanol saponins (Peaks 31 and 37) despite using identical extraction methods. One may speculate that the role of the stems as a transporting plant organ or the focus of herbivores preferentially attacking the leaves may result in a less active enzyme equipment compared to the foliage. Conclusions. – The aerial parts of Helleborus niger comprise a substantial number of constituents most of them not reported earlier. Detailed analyses of plant extracts from leaves and stems allowed detailed insights into the nature of H. niger secondary metabolites. Using one chromatographic gradient for the diversity of structures present, together with application of tandem mass spectrometry, and the combination of the positive- and negative-ionization mode, turned out to be a powerful tool for structure identification. This instrumental setting made it possible to identify even chromophore-lacking constituents of a variety of compounds. Within this work, hitherto published reports on mass-spectrometric fragmentation characteristics in combination with data on constituents already identified in the aerial parts of Helleborus were considered for reliable structure assessment without time-consuming isolation and NMR analyses. Using this approach, the present work contributes to a better understanding on the plants compound profile and constitutes a basis not only for further phytochemical investigations, but also for pharmacological testing of plant extracts to elucidate possible mechanisms of action. Furthermore, the saponin and flavonol distribution pattern may provide substantial information on the plant defense strategies and the biochemical relevance of individual compounds. Additionally, detailed knowledge on the presence of these constituents shall facilitate finding characteristic marker substances for the chemotaxonomic classification of the genus Helleborus. The authors are grateful to Prof. O. Spring (Department of Botany, Hohenheim University, Stuttgart, Germany) for identification and confirmation of the Helleborus specimens.


297

Experimental Part Chemicals. Eluent and additive for chromatographic analyses were MeCN (LC/MS grade) and HCOOH (98%, for MS), both purchased from SigmaAldrich (D-Steinheim). For extraction, acetone (Merck, D-Darmstadt) and diatomaceous earth (Kieselgur, calcined, purified; SigmaAldrich, DSteinheim) were used. Purified H2O (0.055 mS/cm) from a Purelab Option-Q system (Elga Berkefeld GmbH, D-Celle) was used throughout. The following chemicals served as reference compounds: protoanemonin (Stauris Forschungsreagenzien, D-Jena), anemonin (Phytolab, D-Vestenbergsgreuth), quercetin (USP reference standard, Rockville, USA), kaempferol (Fluka, CH-Buchs), b-ecdysone (Phytolab, D-Vestenbergsgreuth), diosgenin (SigmaAldrich, D-Steinheim), sarsasapogenin and trillin (diosgenin 3-O-glucoside; Phytolab, D-Vestenbergsgreuth). Macranthosid I (3-O-[glucopyranosyl-(1 ! 6)-glucopyranosyl]-26-O-glucopyranosylfurost-25-ene-3,22,26-triol) was isolated from H. niger roots by ReseaChem (CH-Burgdorf), and assigned via LC/MS and NMR according to literature data [5]. Plant Material. Whole H. niger plants were cultivated by Geywitz Christrosenkulturen (D-Illingen) and purchased in March 2010. The leaves and stems were separated from the rest of the plant, immediately sorted, and stored frozen at 808 until analysis. Voucher specimens of H. niger plants were identified by Prof. O. Spring and deposited with the Department of Botany (Hohenheim University, DStuttgart; vouchers: HOH-011279 and HOH-011280). Extraction. Samples of 4 g each of freshly cut plant material (Helleborus niger leaves and stems) were extracted with 30 ml of acetone/H2O 1 : 1 (v/v) for 6 h at r.t. Thereafter, the resulting extract was filtered through a diatomaceous earth filter bed placed in a sintered glassfilter funnel (porosity 3; Robu Glasfilter-Gerte, D-Hattert). After collection of the filtrate, the acetone was removed under reduced pressure by rotovaporation (408). The acetone-free raw extract was transferred into a volumetric flask and made up to a final volume of 25 ml with purified H2O. Before chromatographic analyses, the extracts were centrifuged (19,000 g) and filtered through a 0.2-mm syringe filter (material nylon; Carl Roth, DKarlsruhe). HPLC-DAD and LC/MS/MS Analyses. Chromatographic analyses were performed on an Agilent 1200 HPLC-system consisting of a degasser G1322A, a binary pump G1312A, an autosampler G1329A, a thermostatic column compartment G1316A, and a diode array detector (DAD) G1315B (Agilent, DWaldbronn) connected to an HCTultra ion-trap MS detector fitted with an ESI ion source (Bruker Daltonik, D-Bremen). The following gradient was executed at 108 on the anal. column Kinetex XB C18 (150 2.1 mm; 2.6 mm particle size; Phenomenex, Torrance, USA) with a flow rate of 0.2 ml/min: starting at 100% A (0.1% HCOOH) for a 5-min step, a linear increase up to 50% B (MeCN with 0.1% HCOOH) at 115 min, with a further ramp to 100% B at 120 min was executed, staying isocratically for 5 min, and returning to the initial conditions (100% A) within 5 min, followed by a 10-min re-equilibration step. The DAD UV traces were recorded at 200, 300, and 360 nm. Detection parameters for MSn analysis were set as follows: a) positive-ion mode: cap. voltage, 4000 V; dry gas flow (N2 ), 9 l/min; nebulizer pressure, 40 psi; cap. temp., 3008; b) negative-ion mode: cap. voltage, 4000 V; dry gas flow (N2 ), 9 l/min; nebulizer pressure, 40 psi; cap. temp. 3658. MS: at the ultra scan mode between m/z 50 and 2000. MSn Experiments were carried out with a compound stability and trap drive level of 100% for negative-ion mode as well as 100%, and, resp., 10% for positive-ion mode in the auto MS/MS operation category. Equipment control and data processing software used were ChemStation B.01.03 (Agilent, DWaldbronn) as well as EsquireControl and DataAnalysis V 6.1 (Bruker Daltonik, D-Bremen). Peaks and compounds were identified according to their specific UV spectra (if available), fragmentation patterns, and retention times (tR ) in comparison with literature data as well as reference compounds.

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Tandem mass spectrometry of lithium-attachment ions from polyglycols.

Fucoidan Analysis by Tandem MALDI-TOF and ESI Mass Spectrometry.

Assay for Glycosaminoglycans by Tandem Mass Spectrometry and its Applications.

Crux: rapid open source protein tandem mass spectrometry analysis.

Evaluation of physiological amino acids profiling by tandem mass spectrometry.

Rapid identification of disaccharides by tandem mass spectrometry.

Tandem mass spectrometry: the competitive edge for pharmacology.

Learning Peptide-Spectrum Alignment Models for Tandem Mass Spectrometry.