Characterization and Identification of Prenylated Flavonoids from Artocarpus heterophyllus Lam. Roots by Quadrupole Time-Of-Flight and Linear Trap Quadrupole Orbitrap Mass Spectrometry.

In this study, a combination of quadrupole time-of-flight mass spectrometry (Q-TOF-MS) and linear trap quadrupole orbitrap mass spectrometry (LTQ-Orbitrap-MS) was performed to investigate the fragmentation behaviors of prenylated flavonoids (PFs) from Artocarpus plants. Fifteen PFs were selected as the model molecules and divided into five types (groups A-E) according to their structural characteristics in terms of the position and existing form of prenyl substitution in the flavone skeleton. The LTQ-Orbitrap-MSn spectra of the [M - H]- ions for these compounds provided a wealth of structural information on the five different types of compounds. The main fragmentation pathways of group A were the ortho effect and retro Diels-Alder (RDA), and common losses of C4H10, CO, and CO2. The compounds in group B easily lose C6H12, forming a stable structure of a 1,4-dienyl group, unlike those in group A. The fragmentation pathway for group C is characterized by obvious 1,4A-, 1,4B- cracking of the C ring. The diagnostic fragmentation for group D is obvious RDA cracking of the C ring and the successive loss of CH3 and H2O in the LTQ-Orbitrap-MSn spectra. Fragmentation with successive loss of CO or CO2, ·CH3, and CH4 in the LTQ-Orbitrap-MSn spectra formed the characteristics of group E. The summarized fragmentation rules were successfully exploited to identify PFs from Artocarpus heterophyllus, a well-known Artocarpus plant, which led to the identification of a total of 47 PFs in this plant.

1. Introduction

Prenylated flavonoids (PFs) are a class of structurally distinct chromone derivatives characterized by several prenyl units linked to a flavone nucleus by C–C and/or C–O bonds. PFs possess high structural diversity due to various prenyl substitution patterns on the flavone skeleton, which correspond to their wide range of biological activities such as cytotoxicity [1], tyrosinase inhibition [2], and cathepsin K inhibition [3,4], as well as antiplasmodial [5], antitrypanosomal [5], and anti-inflammatory [6] activity, and inhibition of neutrophil respiratory burst [7]. Due to their complex chemical diversity and wide range of biological activities, the search for structurally interesting and biologically active PFs from natural products has attracted the intense attention of natural product chemists and pharmacologists in the past few decades. Interestingly, the occurrence of PFs appears to have a significantly chemotaxonomical tendency and is limited to within only a few plant families, including Moraceae. The genus Artocarpus of Moraceae has been reported as a rich source of PFs, and more than 300 have been isolated from Artocarpus plants to date [8,9].

Artocarpus heterophyllus Lam., a well-known member of Artocarpus, is an evergreen arbor widely distributed in tropical and subtropical regions of Asia, such as Thailand, Vietnam, the Philippines, and other countries [10]. In China, A. heterophyllus is popularly cultivated for its popular edible fruits, while its root has been used in folk medicine for subduing swelling and detoxicating [11]. A variety of bioactive PFs were previously identified from A. heterophyllus using the conventional process of isolation and purification on a preparative scale followed by structure elucidation via spectroscopic methods [4,12,13]. However, the purification of PFs from the crude extract by this conventional method is usually time-consuming, tedious, and labor-intensive due to their thermal sensitivity and low abundance in A. heterophyllus.

The combination of quadrupole time-of-flight mass spectrometry (Q-TOF-MS) and linear trap quadrupole orbitrap mass spectrometry (LTQ-Orbitrap-MS) is a powerful analytical strategy to rapidly screen and identify trace compounds. The combined method possesses high resolution, high mass accuracy, high sensitivity, and abundance fragment information. It is frequently used for the structural characterization of natural products in crude extracts, especially when the reference compounds are unavailable [14,15,16]. Despite there being vast numbers of known PFs, their fragmentation behaviors and corresponding mechanisms remain relatively unexplored.

In the present work, 15 PFs previously isolated and identified [17,18,19,20] from the extract of Artocarpus plants were selected as model molecules and divided into five types according to the substitution pattern of prenylated groups in the flavone nucleus. The fragmentation behaviors of all the five types of PFs were systematically investigated by Q-TOF-MS and LTQ-Orbitrap-MSn. Based on these summarized fragmentation rules, a total of 47 trace PFs were identified. In this work we developed an effective mass spectrometric method for the rapid identification of PFs in mixtures; this could also provide a valuable aid for the characterization of PFs in other PF-containing medicinal herbs.

2. Results and Discussion

The chemical structures of the 15 reference PFs are shown in Figure 1. According to the positions and existence of prenylated substitution in the flavone skeleton, these compounds fall into five categories: group A: free 6- or 8-prenylated substituted flavones (PFs 1–3); group B: free 8-geranyl substituted flavones (PFs 4 and 5); group C: free 3-prenylated substituted flavones (PFs 6–9); group D: 3-partial heterocyclization-prenoid substituted flavones (PFs 10 and 11); and group E: dihydrobenzoxanthone derivatives (PFs 12–15).

Figure 1

Structures of the five groups of prenylated flavonoids (PFs) selected as model molecules from Artocarpus plants.

All the investigated reference compounds were initially analyzed by electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) mass spectrometry in both positive and negative ion modes. Each group of PFs exhibited a strong molecular ion peak ([M − H]− in negative ion mode) with a very small percentage of fragments in the Q-TOF-MS spectrum, and yielded a wealth of product ions in the subsequent TOF-MS/MS and LTQ-Orbitrap-MSn spectra. Accordingly, the [M − H]− ions were selected as the precursor ions for collision-induced dissociation (CID) fragmentation to produce MS/MS spectra, and the relatively prominent product ions were then chosen for further MSn analysis. As summarized in Table 1, these MSn spectra provided abundant fragment ions for the structural characterization and identification of PFs. Using this rule, a total of 47 PFs in the extract of the A. heterophyllus were rapidly identified by ultra-high performance liquid chromatography (UPLC)-Q-TOF-MS/MS.

Table 1

Precursor ions and main product ions observed in the electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MSn) and linear trap quadrupole orbitrap mass spectrometry (LTQ-Orbitrap-MSn) spectra of the protonated PFs 1–15.

Compounds	MSn	Precursor Ions	Product Ions m/z (Relative Abundance, %)
PF 1	MS/MS	367 [M − H]−	175 (3.4); 233 (3.9); 265 (4.2); 297 (18.9); 309 (77.7); 337 (1.6); 352 (1.7); 367 (100.0)
	MS3	233	133 (20.4); 165 (61.7); 189 (23.7); 217 (100.0)
		309	175 (61.7); 265 (100.0); 291 (26.7)
	MS4	265	173 (100.0); 193 (36.4); 215 (62.1); 221 (75.1); 237 (70.1)
PF 2	MS/MS	353 [M − H]−	133 (6.8); 297 (15.5); 309 (15.5); 353 (100.0)
	MS3	297	225 (20.1); 227 (21.4); 253 (97.5); 255 (83.4); 269 (100.0)
		309	265 (18.8); 267 (19.9); 309 (100.0)
	MS4	269	225 (53.4); 227 (35.2); 241 (75.7); 251 (100.0); 269 (39.3)
		267	211 (10.4); 223 (17.9); 239 (44.7); 252 (20.5); 267 (100.0)
PF 3	MS/MS	353 [M − H]−	133 (2.0); 219 (9.8);309 (3.1); 353 (100.0)
	MS3	219	133 (80); 151 (39.4); 175 (100.0)
	MS4	175	65 (13.4); 133 (100.0)
PF 4	MS/MS	421 [M − H]−	133 (12.7); 161 (8.9); 255(9.4); 269 (8.7); 297 (100.0); 323(12.4); 337 (12.0); 421 (21.9)
	MS3	297	163 (9.8); 225(22.8); 227 (22.1); 253(100.0); 255 (84.4); 269 (95.9)
	MS4	253	211 (10.7); 225 (100.0); 253 (22.9)
PF 5	MS/MS	435 [M − H]−	133 (6.7); 269 (8.); 297(84.5); 309 (30.1); 311 (13.5); 323(21.1); 351 (100.0); 420 (35.4); 435 (32.8)
	MS3	297	163 (9.0); 225(20.9); 227 (21.0); 253(88.6); 255 (75.6); 269 (100.0)
		351	307 (9.2); 309 (19.1); 333 (24.0); 351 (100.0)
	MS4	253	211 (8.38); 225 (100.0); 253 (20.2)
PF 6	MS/MS	353 [M − H]−	109 (16.7); 125 (30.6); 151 (24.8); 231 (100.0); 243 (13.2); 283 (11.6); 284 (62.0); 353 (50.5)
	MS3	231	145 (23.1); 147 (23.2); 149 (24.0); 151 (76.7); 163 (23.0); 187 (100.0); 189 (28.2)
		284	256 (100.0)
	MS4	256	122 (100.0); 212 (56.7); 227 (43.6); 228 (38.3)
PF 7	MS/MS	421 [M − H]−	193 (8.91); 219 (1.9); 227 (1.8); 283 (2.4); 297 (7.7); 299 (20.9); 309 (38.8); 311 (3.1); 421 (100.0)
	MS3	193	125 (43.1); 149 (100.0); 151 (36.2)
		309	175 (92.2); 265 (100.0)
	MS4	149	107 (100.0)
		265	173 (59.8); 175 (44.4); 215 (74.5); 221 (76.7); 223 (100.0); 237 (56.2)
PF 8	MS/MS	421 [M − H]−	193 (7.1); 293 (7.7); 299 (27.7); 309 (47.1); 421 (100.0)
	MS3	299	244 (90.0)
		309	175 (10.2); 244 (100.0); 256 (19.7); 299 (78.7)
	MS4	299	244 (100.0); 299 (58.0)
		244	123 (100.0); 149 (35.3); 150 (69.0); 203 (53.3); 215 (35.8); 216 (77.4); 229 (99.11)
PF 9	MS/MS	489 [M − H]−	261 (6.1); 351 (6.0); 365 (10.3); 367 (100.0); 377 (75.9); 489 (90.4)
	MS3	367	257 (10.6); 269 (24.5); 312 (100.0); 367 (71.2)
		377	269 (10.1); 312 (31.5); 367 (100.0)
	MS4	312	257 (51.4); 269 (100.0)
PF 10	MS/MS	417 [M − H]−	199 (2.3); 217 (3.0); 333 (7.3); 347 (2.4); 361 (11.7); 373 (6.29); 387 (2.8); 401 (3.3)
	MS3	361	319 (7.5); 334 (13.5); 343 (100.0); 346 (59.4)
	MS4	343	199 (22.1); 287 (36.2); 299 (10.0); 315 (24.2); 343 (100.0)
PF 11	MS/MS	501 [M − H]−	285 (3.8); 429 (3.7); 457 (43.22); 473 (3.3); 501 (100.0)
	MS3	285	285 (5.2); 243 (22.0); 241 (100.0); 217 (8.5)
		457	402 (20.1); 439 (100.0); 442 (10); 457 (18.7)
	MS4	241	133 (67.5); 157 (32.2); 173 (77.2); 193 (26.6); 197 (100.0); 213 (32.5)
		439	357 (58.70); 383 (21.73); 395 (53.51); 411 (100.0); 424 (20.26)
	MS5	411	329 (35.9); 341 (87.8); 354 (73.8); 356 (87.3); 367 (54.4); 383 (76.9); 393 (58.3); 396 (100.0); 411 (22.5)
PF 12	MS/MS	501 [M − H]−	415 (8); 417 (5.0); 429 (7.3); 445 (14.8); 457 (4.2); 471 (13.0); 483 (4.4); 501 (100.0)
	MS3	445	355 (25.3); 373 (38.8); 399 (39.0); 403 (26.7); 417 (100.0); 427 (94.2); 445 (74.6)
		485	429 (100.0); 442 (60.3); 457 (29.9); 469 (46.5); 471 (85.7)
	MS4	427	355 (53.9); 383 (10.5); 399 (100.0); 427 (34.5)
		429	401 (100.0); 411 (31.5); 414 (20.5)
	MS5	399	343 (52.5); 355 (100.0); 356 (41.6)
		401	357 (88.5); 373 (100.0); 383 (26.6); 386 (70.6); 401 (19.3)
PF 13	MS/MS	433 [M − H]−	373 (11.3); 389 (37.0); 390 (63.2); 405 (25.3); 417 (11.2); 433 (100.0)
	MS3	389	361 (15.2); 372 (100.0); 373 (34.3); 375 (17.5); 389 (27.9)
		405	335 (17.4); 350 (32.9); 361 (27.9); 360 (30.4); 372 (21.9); 389 (15.1); 405 (100.0)
	MS4	372	326 (34.9); 327 (35.9); 343 (71.7); 371 (100.0)
		350	309 (21.6); 335 (100.0); 349 (22.2)
	MS5	343	299 (13.9); 343 (100.0)
		335	276 (16.0); 291 (68.1); 308 (25.6); 317 (34.2); 335 (100.0)
	MS6	299	263 (14.0); 276 (100.0); 291 (16.7)
		291	263 (14.0); 276 (100.0); 291 (16.7)
PF 14	MS/MS	435 [M − H]−	349 (34.2); 363 (5.0); 377 (20.7); 379 (12.4); 391 (12.4); 393 (6.3); 419 (6.3); 435 (100.0)
	MS3	349	277 (21.4); 305 (15.0); 321 (32.9); 349 (100.0)
		391	337 (29.5); 349 (100.0); 363 (23.9); 377 (17.8)
	MS4	321	264 (100.0); 276 (40.7); 279 (62.5); 293 (90.0)
		337	309 (100.0)
	MS5	309	237 (21.1); 263 (32.6); 365 (23.3); 279 (27.0); 281 (70.3); 291 (100.0); 309 (31.4)
PF 15	MS/MS	501 [M − H]−	417 (5.0); 429 (5.5); 443 (5.8); 445 (12.2); 457 (7.3); 471 (7.7); 485 (14.8); 501 (100.0)
	MS3	445	355 (27.4); 373 (41.3); 399 (38.9); 417 (99.6); 427 (100.0); 445 (80.8)
		485	429 (100.0); 442 (60.8); 457 (34.2); 469 (47.9); 471 (88.0)
	MS4	427	355 (26.4); 371 (56.0); 385 (27.2); 399 (100.0)
		429	357 (26.3); 374 (19.6); 401 (100.0); 411 (29.9)
	MS5	399	355 (100.0); 384 (18.3)
		401	357 (91.0); 373 (100.0); 383 (32.7); 385 (48.8); 401 (21.8)

2.1. Fragment Naming Rules

In this study, the C–C bond cleavages of different groups occurred at different positions of the C ring. In general, the fragment ions formed by the C–C bond cleavage on the C ring of flavonoids are named i,jA and i,jB [21], where A and B represent benzene ring A and benzene ring B, respectively, on the parent nucleus of the flavonoid. “i” and “j” represent the fracture position of the C–C bond cleavage on the C ring, as shown in Figure 2.

Figure 2

Nomenclature of the different C ring cleavages [21] occurring in the present study, illustrated by a typical flavonoid skeleton.

2.2. Fragmentation of PFs –

PFs 1–3 in group A represent a type of general prenylated flavones with coplanar structure and bearing a free prenyl moiety at the C-6- or -8 position. PFs 1–3 mainly showed the neutral loss of C4H8 or C5H10 via the ortho effect at the C-8 position to yield a prominent ion at m/z 297. This fragmentation pathway was found for the first time in PFs. In addition, the product ions 1,3A− and/or 1,3B− could be deduced, and their fragmentation pathway was retro-Diels–Alder (RDA) cleavage from the 1,3-position of the C-ring. Common losses of other neutral molecules, such as C4H10, CO, and CO2, were also observed.

PF 1 was selected as an example to illustrate the fragmentation characteristics of group A. Figure 3, Figures S1–S5 exhibits the typical ESI-MS spectra and fragmentation pathways of PF 1 in negative ion mode by Q-TOF-MS/MS and LTQ-Orbitrap MSn (n = 2–4) (Supplementary Materials). The Q-TOF-MS spectrum (Figure 3A) of PF 1 showed a precursor [M − H]− ion at m/z 367.1205 (error 4.90 ppm). In the Q-TOF-MS/MS spectrum (Figure 3B), the prominent ion at m/z 297.0412 ([M − H − C5H10]−, error 2.36 ppm) was from the loss of C5H10 via the ortho effect at the C-8 position, and the fragmentation at m/z 309.0417 ([M − H − C4H10]−, error 3.88 ppm) was from the loss of C4H10 at the same position. Simultaneously, a minor ion at m/z 233.0852 was also detected, owing to RDA fragmentation at the 1,3-position of the C-ring. As shown in Figure 3C, further loss of CO2 from the ion at m/z 233.0852 generated the product ion at m/z 189.0938 in the LTQ-Orbitrap-MS3 spectrum. As seen from the MS3–4 spectra (Figure 3D,E), a fragment ion at m/z 309.0417 was observed with the initial loss of the CO2 group at the C ring; subsequently, loss of the CO group at the B ring position yielded prominent ions at m/z 265.0599 [M − H − C4H10 − CO2]− and m/z 237.0466 [M − H − C4H10 − CO2 − CO]−. In addition, the fragment ion at m/z 175.0082 from m/z 309.0417 via the classical RDA cleavage pathway was also observed. Figure 3F summarizes the above fragmentation pathway of PF 1.

Figure 3

(−) ESI-Q-TOF-MS/MS and LTQ-Orbitrap-MSn (n = 3–4) spectra of PF 1 in group A and the fragmentation pathways proposed for PF 1. Q-TOF-MS (A), Q-TOF-MS2 (B), LTQ-Orbitrap-MS3 of the ions at m/z 233.0852 from m/z 367.1205 (C), LTQ-Orbitrap-MS3 of the ions at m/z 309.0417 from m/z 367.1205 (D), LTQ-Orbitrap-MS4 of the ions at m/z 265.0599 from m/z 309.0417 (E), and the proposed fragmentation pathways for PF 1 (F). RDA: retro Diels–Alder.

The fragmentation pathways of PFs 1–3 are almost identical, which is in full support of the structural characterization of this type of PF. The characteristic fragment ions described above can be regarded as the important information for the qualification of group A. There are still some differences in the cleavage pathways of the prenyl moieties at C-6 (PF 3) and C-8 (PFs 1 and 2). The fragment ions of PF 3 are relatively few, and the ortho effect was not observed. PF 3 exhibited RDA cleavage at the C-6 position, resulting in a prominent ion at m/z 219.0673 ([M − H − 1,3B−], error 4.57 ppm) and a fragment ion at m/z 133.0230 ([M − H − 1,3A]− error 3.76 ppm). However, only one fragment ion appeared via RDA cleavage for PFs 1 and 2. These features could help us to differentiate the compounds bearing a free prenoid moiety at the C-6 position from those bearing the same at the C-8 position.

2.3. Fragmentation of PFs and

A detailed comparison revealed that the flavones in groups B and A exhibited much structural similarity. The structural difference lies in the side chains at the C-8 position: geranyl for group B, and prenyl for group A. Compared with the fragmentation behavior of group A, the ortho effect and RDA cleavage in group B exhibited similar characteristics. The difference is that the geranyl group of group B easily loses C6H12, forming a stable structure of a 1,4 dienyl group.

PF 4 was selected as an example to illustrate the fragmentation pathways of this class of compounds. Figure 4, Figures S20–S24 shows the typical ESI-MS spectra and fragmentation pathways of PF 4 in negative ion mode by Q-TOF-MS/MS and LTQ-Orbitrap-MSn (n = 2–4). Q-TOF-MS (Figure 4A) of PF 4 gave a precursor [M − H]− ion at m/z 421.1644 (error 3.09 ppm). In the Q-TOF-MS/MS spectrum, Figure 4B shows that the prominent ion at m/z 297.0395 ([M − H − C9H16]−, error 3.37 ppm) could be attributed to the loss of C9H16 from the precursor at m/z 421.1644 via the ortho effect. The precursor at m/z 421.1644 is able to experience RDA cleavage to form m/z 133.0301 ([M − H − 1,3A]−, error 4.51 ppm), similarly to group A. In comparison with group A, owing to the presence of the geranyl (instead of prenyl) side chain at the C-8 position, group B shows obvious cleavage of the geranyl group and thus loses a C6H12 group. This tendency is more obvious when there is a methoxy group in the mother nucleus, such as in PF 5. Figure 4C,D shows the successive loss of CO2 and CO groups at the C ring, yielding a prominent ion at m/z 225.0474 [M − H − C9H16 − CO2 − CO]− from m/z 297.0395. In addition, the primary daughter ion [M − H − C9H16 − CO]− was observed owing to the loss of CO from the product ion at m/z 297.0395. The fragmentation patterns of PF 5 are almost identical to those of PF 4, in full support of the structural characterization of this type of flavone. Figure 4E summarizes the fragmentation pathways to help identify these compounds, taking PF 4 as an example.

Figure 4

(−) ESI-Q-TOF-MS/MS and LTQ-Orbitrap-MSn (n = 3–4) spectra of PF 4 in group B and the fragmentation pathways proposed for PF 4. Q-TOF-MS (A), Q-TOF-MS2 (B), LTQ-Orbitrap-MS3 of the ions at m/z 297.0395 from m/z 421.1644 (C), LTQ-Orbitrap-MS4 of the ions at m/z 253.0483 from m/z 297.0395 (D), and the proposed fragmentation pathways for PF 4 (E).

2.4. Fragmentation of PFs –

Group C contains four free 3-prenylated substituted flavones, with three of them bearing another one or more prenyl moieties at the C-6, -8, and -3′ positions, but not PF 6. For these PFs in group C, non-coplanarity is the common structural feature and results from the free 3-prenylated group in the flavone nucleus forcing the 2-phenyl group out of plane with the chromone ring. This unique stereo configuration of the free 3-prenylated substituted flavones in group C leads to significantly different fragmentation behaviors from the other four groups. The most prominent difference between group C and the other groups is in the fragmentation of the C ring. The C ring of this kind of compound shows obvious 1,4A−, 1,4B− cracking, while the common flavonoids show RDA cracking (1,3A−, 1,3B−).

As shown in Figure 5, Figures S30–S35 PF 6 was taken as an example to illustrate the typical cleavage pathways. Figure 5A shows the molecular ion ([M − H]−, m/z 353.1028, error 0.79 ppm) corresponding to the molecular formula C26H28O6. The product ions [M − H − B1,4]− at m/z 125.0242 (error 1.60 ppm) and [M − H − A1,4]− at m/z 231.1021 (error 2.60 ppm) were from the cracking of the C ring (Figure 5B). The other three compounds all have a similar fragmentation pathway. This kind of cracking was observed only in group C, and not in the other four groups.

Figure 5

(−)ESI-Q-TOF-MS/MS of PF 6 in group C and the fragmentation pathways proposed for PF 6. Q-TOF-MS (A), Q-TOF-MS2 (B), and the proposed fragmentation pathways for PF 6 (C). RDA: retro Diels–Alder.

PF 6 exhibited a prominent product ion ([M − H − C5H9]−, m/z 284.0326, error 0 ppm) which was from the loss of C5H9 from the precursor ion (Figure 5B). In addition, the strong product ion peak at m/z 151.0038 ([M − H − 1,3B]−, error 0.62 ppm) was easily obtained via RDA cleavage from the precursor [M − H]−. PF 7 has the same cleavage pathway with a very weak peak ([M − H − 1,3B]−), but PFs 8 and 9 do not have this fragmentation pathway. This characteristic fragmentation pathway can be used to explore the position and number of prenylated groups of these compounds.

Remarkably, these observations indicated that PFs 7–9 are apt to lose the C4H8 group at the free 3-prenylated group, forming a stable furan ring closed with the hydroxyl at the C-2′ position, and to successively eliminate a C4H8 group at the C-8 position, forming a double bond between C-7 and C-8 via the ortho effect. Taking PF 8 as an example, this cracking pathway eventually produced a prominent ion at m/z 309.0432 ([M − H − C8H16]−, error 3.88 ppm). The characteristic fragmentation pathway of group C is of great significance for the identification of these compounds containing more than two prenyl groups.

2.5. Fragmentation of PFs and

The two compounds in group D are also 3-prenylated flavones similar to those in group C, but they possess a coplanar structure in which the prenyl substituent at the 3-position is partially heterocyclized to form a pyran ring by C–O linkage between C-11 of the prenylated moiety and the hydroxyl group at the C-2′ position of the B ring. The existence of the C-11/C-2′ pyran ring in group D resulted in a wealth of product ions in the MSn (n = 2–5) spectra, which is notably different from group C. The most significant difference between group D and C is the cracking behavior of the C ring: RDA cleavage mainly occurred in group D, and 1,4A−/1,4B− cracking occurred in group C. The difference could be used to differentiate the two groups.

PF 10 was selected as an example to illustrate the fragmentation pathways of group D. Figure 6, Figures S51–S56 exhibits the typical ESI-MS spectra and fragmentation pathways of PF 10 in negative ion mode by Q-TOF-MS/MS and LTQ-Orbitrap-MSn (n = 2–4). The mass spectrum of PF 10 in Figure 6A showed a precursor ion [M − H]− at m/z 417.1358 ([M − H]−, error 3.41 ppm). PF 10 is mainly cracked via RDA, producing the fragment ions at m/z 217.0516 ([M − H − 1,3B]−) in the Q-TOF-MS/MS spectrum (Figure 6B). In addition, the fragment ion at m/z 361.0737 ([M − H − 4·CH3]−, error 3.60 ppm) came from the loss of ·CH3 from the precursor ion [M-H]-. In the LTQ-Orbitrap-MS3–4 spectra of PF 10 (Figure 6C,D), the product ion at m/z 343.0523 was from the loss of one H2O, and the fragment ion at m/z 199.0301 was obtained from the subsequent cracking of the C ring. Figure 6E summarizes the fragmentation pathways of PF 10 in the Q-TOF-MS/MS and LTQ-Orbitrap-MSn (n = 2–4) results.

Figure 6

(−)ESI-Q-TOF-MS/MS and LTQ-Orbitrap-MSn (n = 3–4) spectra of PF 10 in group B and the fragmentation pathways proposed for PF 10. Q-TOF-MS (A), Q-TOF-MS2 (B), LTQ-Orbitrap-MS3 of the ions at m/z 361.0731 from m/z 417.1358 (C), LTQ-Orbitrap-MS4 of the ions at m/z 343.0523 from m/z 361.073 (D), and the proposed fragmentation pathways for PF 10 (E). RDA: retro Diels–Alder.

It is noteworthy that the compounds containing a free prenylated group at the C-8 position show some different fragmentation pathways. For the compound PF 11, the main fragment pathway is the loss of CO2 from the C ring, generating the product ion at m/z 457.2002 ([M − H − CO2]−, error 3.94 ppm). However, for PF 10, the main pathway is the loss of H2O. This difference can help us to determine the number and location of prenylated groups in compounds of this group.

2.6. Fragmentation of PFs –

The four flavones in group E, with a dihydrobenzoxanthone skeleton, represent the characteristic constituents of the Artocarpus species, and this type of flavone has never been found in any other species. It was observed that dihydrobenzoxanthone-type flavone derivatives have a unique structure in which C–C linkage takes place between the C-6′ position of the B ring and the C-12 position of the prenyl moiety located at the C-3 position. Because of this unique structure, the cleavage behavior of this type of flavonoid is different from that of other groups.

PF 12 was selected as an example to illustrate the fragmentation characteristics of this type of compound. Figure 7, Figures S65–S74 shows the typical ESI-MS spectra and cleavage pathways of PF 12 in negative ion mode for Q-TOF-MS/MS and LTQ-Orbitrap-MSn (n = 2–4). The precursor ion [M − H]− of PF 12 at m/z 501.1943 (error 4.00 ppm) is shown in Figure 7A. The fragment ions at m/z 445.1296 ([M − H − 4·CH3]−, error 0.47 ppm) and m/z 485.1626 ([M − H − CH4]−, error 4.12 ppm) correspond to the loss of 4·CH3 or CH4 from the molecular ion (Figure 7B). In the LTQ-Orbitrap-MS3–4 spectra (Figure 7C,D), we observed the initial loss of the H2O group at the C-4’ position from the fragment ion (m/z 445.1296), and subsequently the loss of the CO group at the C-2’ position, yielding prominent ions at m/z 399.1040 [M − H − 4·CH3 − H2O − CO]−. In addition, Figure 7E,F illustrates the successive losses of CO and another four ·CH3 in the fragmentation [M − H − CH4]−, producing the product ions [M − H − CH4 − CO]− (m/z 457.1961), [M − H − CH4 − 4·CH3]− (m/z 429.1201), and [M − H − CH4 − 4CH3 − CO]− (m/z 401.1094). The three other compounds in this group also showed the same fragmentation patterns as PF 12. Thus, Figure 7G summarizes the possible fragmentation pathway of PF 12. This fragmentation pathway makes it easier to determine the structural characteristics of these compounds.

Figure 7

(−)ESI-Q-TOF-MS/MS and LTQ-Orbitrap-MSn (n = 3–4) spectra of PF 12 in group B and the fragmentation pathways proposed for PF 12. Q-TOF-MS (A), Q-TOF-MS2 (B), LTQ-Orbitrap-MS3 of the ions at m/z 445.1296 from m/z 501.1939 (C), LTQ-Orbitrap-MS4 of the ions at m/z 427.0927 from at m/z 445.1296 (D), LTQ-Orbitrap-MS3 of the ions at m/z 485.1631 from m/z 501.1939 (E), LTQ-Orbitrap-MS4 of the ions at m/z 429.1201 from m/z 485.1631 (F), and the proposed fragmentation pathways for PF 12 (G).

2.7. Rapid Identification of PFs in the Extract of A. heterophyllus Roots

The PFs in the extract of A. heterophyllus roots were analyzed by UPLC-Q-TOF-MS/MS, and the total ion chromatogram (TIC) of the sample in negative ion mode was obtained, as shown in Figure 8A. Based on the existing literature and our previous studies, a total of 47 PFs were identified in the extract of A. heterophyllus roots. The formulae of the 47 compounds were accurately assigned within a relative theoretical mass error of 5 ppm, and the relative theoretical mass errors of the characteristic fragment ions were all within 10 ppm. The identification process of the detected compounds was as follows [22]. Firstly, an accurate molecular mass was obtained via the high-resolution Q-TOF-MS technique according to the deprotonated ion [M − H]−. Secondly, the formula was obtained by Peakview software according to the accurate molecular mass, constituent elements, and isotope abundance. Thirdly, the differentiation and characterization of the analytes were completed by considering the product ions, fragmentation pathways, and literature data. The related information is summarized in Table 2, including the compound name, molecular formula, precursor ion, characteristic fragment ions, and the error. The identified compounds were validated by extracting the relative characteristic fragmentations, such as precursor ions, nuclear parent ions, and daughter ions (Figure 8). The following examples are given to illustrate the identification process of the PFs.

Figure 8

Total ion chromatogram (A) and extracted ion chromatogram (B) of prenylated flavonoids in the extract of Artocarpus heterophyllus Lam. roots.

Table 2

The 47 prenylated flavonoids identified from the extract of Artocarpus heterophyllus Lam. roots.

Peak No.	Compounds	RT (min)	Molecular Formula	[M − H]− (m/z)	Error (Δppm)	Product Ions (m/z)	Error (Δppm)	Fragment Ions Identified
1	6-(3-Methylbutyl-2-enyl)apigenin [23]	3.49	C20H18O5	337.10903	2.6	219.0681	8.2	[M – H − 1,3B]−
						281.0473	6.4	[M − H − C4H8]−
2	Artonin K [24]	4.14	C21H18O7	381.09924	3.3	337.0738	5.9	[M – H − C2H4O]−
						365.0687	5.5	[M – H − CH4]−
3	Albanin A [25]	4.66	C20H18O6	353.10459	4.3	125.0246	4.8	[M – H − 1,4B]−
						231.0668	2.2	[M – H − 1,4A]−
4	14-Hydroxyartonin E [26]	4.87	C25H24O8	451.14188	4.5	393.0999	4.8	[M – H − C3H6O]−
						433.1289	0.9	[M − H − H2O]−
5	Artoindonesianin P [27]	5.22	C22H20O7	395.11548	4.7	337.0736	5.3	[M – H − C3H6O]−
						351.0515	1.4	[M – H − 3·CH3]−
6	Artonin J [28]	5.47	C25H24O7	435.14663	3.9	379.0839	4.2	[M – H − C4H8]−
						365.0681	3.8	[M – H − C5H10]−
7	Artelastoxanthone [18]	6.67	C25H22O7	433.12925	−0.1	405.1399	−1.2	[M – H − CO]−
						389.1017	3.6	[M – H − CO2]−
8	Artobiloxanthone [29]	6.68	C25H22O7	433.13224	6.8	405.1364	4.9	[M – H − CO]−
						403.0850	6.7	[M – H − 2CH3]−
9	Artonin E [28]	7.23	C25H24O7	435.14663	3.9	379.0841	4.7	[M – H − C4H8]−
						351.0887	3.7	[M – H − C5H4O]−
10	Cyclocommunol [30]	7.31	C20H16O6	351.08818	2.2	199.0777	6.0	[M – H − 1,3A]−
						295.0259	3.7	[M – H − C4H8]−
11	Artoindonesianin Q [4]	7.47	C22H22O7	397.12891	−0.9	137.0247	−2.2	[M – H − 1,4B]−
						313.0345	2.9	[M – H − CH3 − C5H9]−
						311.0195	0.6	[M – H − OCH3 − C4H7]−
						365.1021	−2.2	[M – H − OCH3]−
12	Artoindonesianin R [31]	7.49	C22H22O7	397.13136	5.1	313.0357	1.0	[M – H − C6H12]−
						137.0242	−1.5	[M – H − 1,4B]−
13	Artonol E [32]	7.91	C26H24O7	447.14548	1.2	415.1155	−7.7	[M – H − OCH4]−
						391.0831	2.0	[M – H − C4H8]−
14	Artoindonesianin T [31]	8.31	C22H20O7	395.11548	4.7	339.0518	2.4	[M – H − C4H8]−
						365.0655	−3.3	[M – H − 2CH3]−
15	Cycloartobiloxanthone [27]	10.48	C25H22O7	433.13134	4.7	391.0824	0.3	[M – H − C3H6]−
						415.1177	−2.4	[M – H − H2O]−
16	Artoindonesianin B [33]	10.92	C26H28O8	467.17322	4.4	261.0758	−3.8	[M – H − 1,4A]−
						451.1399	0.2	[M – H − CH4]−
17	Artocarpetin A [34]	13.07	C21H20O6	367.11769	−2.8	233.0829	−4.3	[M – H − 1,3B]−
						297.0410	−1.7	[M – H − C5H10]−
						309.0763	1.6	[M – H − C3H4O]−
18	Heteroflavanone C [35]	13.44	C23H26O7	413.16078	0.5	247.0975	−0.4	[M – H − C9H10O3]−
						235.0982	2.6	[M – H − 1,2B]−
19	Artocarpesin [36]	13.48	C20H18O5	337.10903	2.6	133.0294	−0.8	[M – H − 1,3A]−
						281.0477	7.8	[M – H − C4H8]−
20	6-(3-Methylbut-2-enyl) Apigenin [18]	13.5	C20H18O5	337.10809	−0.2	281.0464	−3.2	[M – H − C4H8]−
						117.0349	−2.6	[M – H − 1,3A]−
21	Styracifolin D [4]	13.88	C30H32O8	519.20258	0.3	461.1583	5.0	[M – H − C3H6O]−
						491.2075	0.0	[M – H − CO]−
						501.1919	0.0	[M – H − H2O]−
22	Cycloartocarpesin [37]	14.28	C20H16O5	335.09274	0.7	133.0301	4.5	[M – H − 1,3A]−
						305.0462	2.3	[M – H − 2CH3]−
23	Artonin U [38]	15.17	C21H20O5	351.12462	2.4	281.0469	5.0	[M – H − C5H10]−
24	Norartocarpin [39]	17.16	C25H26O6	421.16567	0	227.0725	4.8	[M – H − 1,4A]−
						309.0391	−4.5	[M – H − C8H16]−
						365.1009	-6.0	[M – H − C4H8]−
25	Mulberrochromene [40]	17.50	C25H24O6	419.15040	0.9	199.0781	8.0	[M – H − 1,3A]−
						349.0726	2.3	[M – H − C5H10]−
26	Dihydroisocycloartomunin [31]	17.79	C26H26O7	449.16080	0.5	191.0715	0.5	[M – H − 1,4B]−
						365.0694	7.4	[M – H − C6H12]−
27	Morusin [41]	18.48	C25H24O6	419.15040	0.9	191.0728	7.3	[M – H − 1,4B]−
						217.0515	4.1	[M – H − 1,3B]−
						227.0725	4.8	[M – H − 1,4A]−
28	Heterophyllin [17]	20.55	C30H32O7	503.20842	2.1	217.0879	4.1	[M – H − 1,3A]−
						243.0662	−0.4	[M – H − 1,4A]−
						259.1351	4.2	[M – H − 1,4B]−
						285.1112	5.6	[M – H − 1,3B]−
29	Artocarpetin B [42]	20.72	C22H22O6	381.13518	2.2	233.0806	−5.6	[M – H − 1,3B]−
						311.0549	−3.9	[M – H − C5H10]−
30	Artelastofuran [43]	21.07	C30H34O7	505.22363	0.9	277.1455	3.6	[M – H − 1,4B]−
						285.1147	5.3	[M – H − 0,3B]−
31	5’-Hydroxycudraflavone A [17]	21.81	C25H22O7	433.12925	−0.1	389.1372	5.7	[M – H − CO2]−
						415.1192	−1.2	[M – H − H2O]−
						217.0516	−4.6	[M – H − 1,3A]−
						377.0663	1.1	[M – H − C3H4O]−
32	Artonin F [44]	22.04	C30H30O7	501.19231	0.9	429.1343	−0.2	[M – H − C4H8O]−
						473.1945	−5.3	[M – H − CO]−
33	Isocycloheterophyllin [45]	22.23	C30H30O7	501.19290	−2.0	445.1267	−2.2	[M – H − CH4]−
						485.1615	−1.9	[M – H − C3H4O]−
						443.1511	−2.5	[M – H − C3H6O]−
34	Artoindonesianin I [46]	22.70	C30H34O7	505.22363	0.9	285.1137	1.8	[M – H − 0,3B]−
						447.1806	−1.6	[M – H − C3H6O]−
35	Artocarpin [47]	23.48	C26H28O6	435.18167	0.8	207.1039	5.8	[M – H − 1,4B]−
						233.0818	−0.4	[M – H − 1,3B]−
36	Cannflavin C [20]	23.59	C26H28O6	435.18022	−2.5	351.0878	−1.1	[M – H − C6H12]−
						323.0939	−4.3	[M – H − C6H7 − OCH3]−
						313.1452	−2.2	[M – H − C9H14]−
37	Artonin H [44]	25.10	C30H34O7	505.22363	0.9	191.0728	7.3	[M – H − 1,4B]−
						217.0512	2.8	[M – H − 1,3B]−
38	Artonin S [38]	25.36	C26H28O7	451.17621	0.0	393.1357	3.3	[M – H − C3H6O]−
						421.1311	4.3	[M – H − 2CH3]−
39	Artonin G [44]	26.21	C30H32O7	503.20862	2.2	447.1422	−6.0	[M – H − C4H8]−
						475.2103	−4.8	[M – H − CO]−
40	Artonin A [48]	28.15	C30H30O7	501.19231	0.9	457.1286	−1.5	[M – H − C3H8]−
						473.1950	−4.2	[M – H − CO]−
41	Artonin B [48]	28.39	C30H30O7	501.19230	0.8	485.1602	0.8	[M – H − CH4]−
						445.1296	−0.7	[M – H − C3H4O]−
42	Cudraflavone A [49]	28.41	C25H22O6	417.13475	0.9	217.0515	4.1	[M – H − 1,3B]−
						373.1072	−2.4	[M – H − C2H4O]−
43	Artoindonesianin G [46]	29.45	C30H32O6	487.21308	1.0	259.1351	4.2	[M – H − 1,4B]−
						285.1116	−5.6	[M – H − 1,3B]−
						365.1762	1.1	[M – H − C7H6O2]−
44	Cycloartocarpin [50]	30.97	C26H26O6	433.16608	1.0	199.0777	6.0	[M – H − 1,3A]−
						363.0878	1.1	[M – H − C5H10]−
						375.0878	1.1	[M – H − C4H10]−
45	Artoindonesianin H [46]	31.80	C30H32O6	487.21308	1.0	227.0731	7.5	[M – H − C9H18]−
						361.0731	3.6	[M – H − 1,4A]−
						473.1966	−0.8	[M – H − CO]−
46	Cycloheterophyllin [17]	33.05	C30H30O7	501.19231	0.9	285.1145	4.6	[M – H − 1,3B]−
						445.1281	−2.7	[M – H − C4H8]−
						473.1966	−0.8	[M – H − CO]−
47	Artelastochromene [43]	36.01	C30H30O6	485.19690	−0.1	285.1145	4.6	[M – H − 1,3B]−
						469.1659	0.4	[M – H − CH4]−

The precursor ion and fragment ions of peaks 3, 7, 17, 33 and 41 in the TIC profile of A. heterophyllus root extract are basically the same as those for PFs 6, 13, 1, 15 and 12 discussed in this experiment. Therefore, compounds 3, 7, 17, 33 and 41 were identified as PFs 6, 13, 1, 15 and 12. The precursor ions of compounds 11 and 28 were m/z 397.1289 ([M − H]−) and m/z 503.2068 ([M − H]−), respectively, affording the molecular formulae C22H22O7 and C30H32O7 with errors of −0.9 and −1.4 ppm, respectively. In the Q-TOF-MS/MS spectra, these two compounds have 1,4A−, 1,4B− fragments. According to the fragmentation pattern of group C, it can be inferred that both of them have a prenylated substitution in the C-3 position. In addition, the fragment ions of compound 10 included [M − H − 2OCH3]− (m/z 335.0935). It can be inferred that compound 11 may contain two OCH3. By comparison with our data [4], compound 11 was identified as artoindonesianin Q. Compound 28 also produced a low abundance of RDA fragments. Based on the fragmentation pattern of group C, the C-8 position of compound 28 may contain a prenylated group, but there is no ortho effect. It is possible that there is no free phenolic hydroxyl at the C-7 position. Based on the related literature [17] and retention time, compound 28 was identified as heterophyllin.

Compound 31 produced a 433.1293 [M − H]− precursor ion in negative ion mode. It was inferred that its molecular formula is C25H22O7, and the error was −0.1 ppm. The characteristic fragment ions of compound 31 include [M − H − CO2]− (m/z 389.1372), [M − H − H2O]− (m/z 415.1192), and [M − H − 1,3A]− (m/z 217.0516). It can be inferred that in compound 30, there is a 3-prenyl side chain forming a pyran ring with HO-6’ of the B ring, and C-3’ and C-4’ of the B ring contain two free phenolic hydroxyl groups. This is basically consistent with the fragmentation pathway of group D. This information suggested that this compound should be 5’-hydroxycudraflavone [17]. The precursor ion of compound 39 ([M − H]− ion at m/z 503.2086) afforded the molecular formula C30H32O7 with an error of 2.2 ppm. The fragmentations [M − H − C4H8]− at m/z 447.1422 and [M – H − CO]− at m/z 475.2103 indicated that this compound was artonin G [44]. This is basically consistent with the fragmentation pathway of group E.

3. Materials and Methods

3.1. Plant Material

The roots of A. heterophyllus were collected from Nanning city, Guangxi Zhuang Autonomous Region, China, in April 2017 and authenticated by Associate Researcher Lv Shihong of Guangxi Institute of Botany, Chinese Academy of Sciences. The voucher specimen (TCM20170502) was deposited in the Herbarium of the Department of Pharmacognosy, Research Center of Natural Resources of Chinese Medicinal Materials and Ethnic Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang, China.

3.2. Model Molecules and Reagents

A total of 15 PFs (PFs 1–15) were isolated from Artocarpus plants in our previous studies [17,18,19,20]. Their structures (Figure 1) were unequivocally elucidated by spectroscopic methods, including one-dimensional and two-dimensional nuclear magnetic resonance, high-resolution electrospray ionization mass spectroscopy, and infrared spectroscopy. The purity of these compounds was determined to be higher than 98% by normalizing the peak area using high-performance liquid chromatography (HPLC) equipped with a diode array detector (DAD). Chromatographic-grade acetonitrile (ACN) and methanol were purchased from Tedia Co. Inc. Deionized water was prepared using a Milli-Q water purification system (Millipore, USA).

3.3. Sample Preparation

The air-dried and powdered roots of A. heterophyllus (50.0 g) were extracted with 95% EtOH three times (500 mL for each extraction) at room temperature. The filtrate was evaporated in vacuo to produce a residue (4.2 g) which was loaded on an HP-20 macroporous resin column chromatograph and eluted successively with a gradient of EtOH/H2O (0:100 and 95:5, v/v) to give two fractions. The 95% EtOH elution fraction was concentrated to afford a residue. The dried residue was re-dissolved in 50 mL of methanol in a volumetric flask; then, the samples were filtered through a 0.22 μm membrane prior to analysis.

3.4. Ultra-High Performance Liquid Chromatography (UPLC)-Q-TOF-MS/MS and UPLC-LTQ-Orbitrap-MSn Analysis of PFs

A TripleTOF AB 5600+ high-resolution mass spectrometer (AB SCIEX, Framingham, USA) was used to obtain primary and secondary mass spectrometry data by the UPLC-Q-TOF-MS/MS method. The AB mass spectrometer was equipped with an electrospray ionization (ESI) ion source and an LC-30A ultra-high-performance liquid chromatograph (Shimadzu, Japan). The multistage mass spectrometry data were obtained using a Thermo Fisher LTQ-Orbitrap Electrostatic Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany).

In the UPLC-Q-TOF/MS analysis, the chromatographic separation was carried out on an ACQUIT UPLC® BEH (1.7 μm, 2.1 × 50 mm, Water, Milford, MA, USA). The mobile phase was composed of 30% ultrapure water and 70% methanol with a flow rate of 0.3 mL/min, and the injection volume was 2.0 μL. The mobile phase for the extracts was ultrapure water (A) and methanol (B) using a gradient elution program: 45–60% B at 0–2 min, 60–65% B at 2–10 min, 65–70% B at 10–25 min, 70–80% B at 25–30 min, 80–100% B at 30–40 min, then held for 10 min. Q-TOF-MS analysis was performed in negative ion mode using full scan mode with a mass range of 100–1500 Da. The MS parameters were optimized as follows: ion spray voltage, −4500 V; source temperature, 550 °C; curtain gas, 30 psi; nebulizer gas (GS1), 50 psi; heater gas (GS2), 50 psi; de-clustering potential (DP), −100 V; and pre-scan and trigger second-stage scan time of Q-TOF-MS, 250 and 100 ms, respectively. AB Analyst TF Software (AB SCIEX, Framingham, USA) was used to collect data. The collision energy (CE) and collision energy superposition (CES) of each compound were optimized.

The Thermo Fisher LTQ-Orbitrap Electrostatic Mass Spectrometer was used in ESI negative ion mode and multistage scanning mode. The optimized parameters were as follows: capillary temperature, 320 °C; ion source heater, 300 °C; source voltage, 3.6 kV; sheath gas (N2), 35 arbitrary units; aux gas, 10 arbitrary units; sweep gas flow, 0 arbitrary units; S-Lens RF level (%): 60%; CE, 30 eV; and CES, 10 eV. The experiments were performed in negative mode, scanning from m/z 100 to 1000.

3.5. Data Preprocessing Analysis

The data collected via UHPLC-Q-TOF-MS/MS were processed using Peak View 1.2 software (AB SCIEX, version 1.2.0.3) from AB Sciex Company. The empirical molecular formulae were deduced from Peakview by comparing the theoretical masses of molecular ions and/or adductions with the determined values based on the following error limits: mass accuracy, < 5 ppm; retention time, < 5.0%; and isotope abundance, < 10%. The multistage mass spectrometry data collected via ESI-LTQ-Orbitrap-MSn were processed using Thermo Xcalibur 2.2 (Thermo Fisher Scientific).

4. Conclusions

In this work, 15 PFs from Artocarpus plants served as standards and were divided into five types according to their structural characteristics with respect to the position and existence of prenyl substitution in the flavone skeleton. A data acquisition strategy based on the combination of Q-TOF-MS and LTQ-Orbitrap-MSn was used to obtain both [M − H]− and product ion information. Fragmentation rules for the five groups were summarized, and possible fragmentation pathways were proposed. These fragmentation rules were successfully exploited to qualitatively analyze the PFs from A. heterophyllus, a well-known Artocarpus plant; a total of 47 PFs were identified from the extract of this plant. These results indicate that the developed analytical method could be used as a rapid, effective technique for the qualitative characterization of PFs in Artocarpus plants and in other PF-accumulating medicinal herbs.