Abstract
Shuganjieyu (SGJY) capsule is a classical formula widely used in Chinese clinical application. In this paper, an ultra-performance liquid chromatography coupled with electrospray ionization and ion trap mass spectrometry has been established to separate and identify the chemical constituents of SGJY and the multiple constituents of SGJY in rats. The chromatographic separation was performed on a C18 RRHD column (150 × 2.1 mm, 1.8 μm), while 0.1% formic acid–water and 0.1% formic acid–acetonitrile was used as mobile phase. Mass spectral data were acquired in both positive and negative modes. On the basis of the characteristic retention time (Rt) and mass spectral data with those of reference standards and relevant references, 73 constituents from the SGJY and 15 ingredients including 10 original constituents and 5 metabolites from the rat plasma after oral administration of SGJY were identified or tentatively characterized. This study provided helpful chemical information for further pharmacology and active mechanism research on SGJY.
1. Introduction
Shuganjieyu (SGJY) capsule, which contains two medicinal materials, including the dried herbs of Hypericum perforatum L. and the dried roots and rhizomes or stems of Acanthopanax senticosus (Rupr. et Maxim) Harms, is the first approved Chinese herbal medicine for mild to moderate monopolar depression in Chinese. Currently, the research of the chemical components in SGJY has been mainly based on identification of chemical constituents respectively and systematically from individual herb extracts. The constituents of SGJY are numerous and diverse. However, until now, like most traditional Chinese medicine (TCM), there have been few reports on the absorption and efficacy after oral administration of SGJY, which is valuable for further studies on the pharmaceutical effect and mechanism of the SGJY formula. As tandem mass spectrometry (MS/MS) has been proven to be efficient tool for the rapid on-line analysis for the known compounds and elucidation of unknown compounds in complex matrices, in this study, a high-speed and sensitive technique ultrahigh-performance liquid chromatography (UHPLC)–electrospray ionization (ESI)–MS/MS system was adopted to characterize the constituents of SGJY capsule and the metabolic profile in rat plasma after oral administration of SGJY. Moreover, the result of this study was expected to provide helpful chemical information for further pharmacology and active mechanism research on SGJY formula.
2. Experimental
2.1. Materials and Reagents
Shuganjieyu capsule (batch number 150314), the extract of H. perforatum L. (batch number S150402), and the extract of A. senticosus Harms (batch number S150102) were offered by Chengdu Kanghong Pharmaceutical Co. Ltd. (Chengdu, China). The reference standards of rutin, hyperoside, quercetin, isofraxidin, epicatechin, chlorogenic acid, and eleutheroside E were purchased from National Institutes for Food and Drug Control (Beijing, P.R. China), hypericin was purchased from Chengdu Munster biotechnology company (Chengdu, China), and hyperforin was purchased from ChromaDex Corporate (California, USA). (6S,7E,9R)-Roseoside was isolated in our laboratory (purity, >98%), and its chemical structure was identified by spectral analysis. HPLC-grade acetonitrile and methanol were purchased from Honeywell Burdick & Jackson (Ulsan, Korea). Ultrapure water for the preparation of samples and mobile phase was prepared with Milli-Q Biocel water system (Millipore, Massachusetts, USA). Other reagents were of analytical grade.
2.2. Instrumentation and Analytical Conditions
The Agilent 1290 Infinity UHPLC system (Agilent Technologies Inc., California, USA) was equipped with quaternary pump, vacuum degasser, a cooling autosampler, and a diode-array detector. An Agilent Eclipse Plus C18 RRHD column (150 × 2.1 mm, 1.8 μm) was utilized for separation with the column temperature at 30 °C. A binary gradient elution was adopted with mobile phase consisting of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile: 0–3 min, B 5%; 3–15 min, B 5–10%; 15–25 min, B 10–20%; 25–40 min, B 20–40%; 40–45 min, B 40–100%; 45–50 min, B 100%; 50–51 min, B 100–5%; and 51–60 min, B 5%. The flow rate was set at 0.20 mL/min. The autosampler was conditioned at 4 °C, and the injection volume was 10 μL.
ThermoQuest Finnigan LTQ system equipped with an electrospray ionization source (ThermoQuest LC/MS Division, San Jose, CA, USA) was used for mass spectrometric measurements. The ESI–MSn spectra were acquired in both positive and negative ion modes. The mass spectrometry detector (MSD) parameters were as follows: in (+) ESI, spray voltage, 3500 V (sheath gas, 15 arb; auxiliary gas, 5 arb; purge gas, 0 arb); capillary temperature, 275 °C; capillary voltage, 10 V; lens voltage, 80 V; in (−) ESI, spray voltage, 5000 V (sheath gas, 15 arb; auxiliary gas, 5 arb; purge gas, 0 arb); capillary temperature, 275 °C; capillary voltage, −10 V; lens voltage, −100V. Tandem mass spectrometry (MS/MS) was collected with data-dependent mode, and the three highest intensity peaks in full-scan spectra were acquired for MS/MS analysis. Helium was used as collision gas (collision energy, 35 eV). The full-scan range was from 100 to 1000 m/z.
2.3. Animals, Drug Administration, and Blood Sampling
Ten male Sprague-Dawley (SD) rats (160–220 g) were obtained from Chengdu Dashuo Laboratory Animal Co., Ltd. (Sichuan, China). The animals were acclimatized to the facilities for 5 days, and then fasted, with free access to water for 12 h prior to the experiment. All procedures were in accordance with the Guidelines on the Care and Use of Animals for Scientific Purposes 2004.
2.4. Sample Preparation
2.4.1. Preparation of SGJY Extract Samples
SGJY capsule was ground into fine powder. A total of 100 mg was accurately weighed, and 10 mL distilled water was added. Each extract of medicinal material contained in SGJY was 50 mg accurately weighed and dissolved in 10 mL distilled water. All the samples were ultrasonically extracted for 10 min and then filtered through a syringe filter (0.45 μm). Filtrate (10 μL) was subjected to UPLC–ESI–MS/MS analysis.
2.4.2. Preparation of Plasma Samples
Capsule contents of SGJY were dispersed with distilled water as stock solution (0.5 g/mL). The above suspension was orally administered to five rats (1.0 mL/100 g body weight). An equal volume of distilled water was orally administered to the other five rats as control; 60 min after drug administration, the animals were anesthetized by the injection of 7% chloral hydrate. The blood was collected from the abdominal aorta and then centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant (0.5 mL) was added into polypropylene test tube, and 1.5 mL methanol was added. The mixture was vortexed for 60 s and then centrifuged at 10,000 rpm for 10 min. Supernatant was collected and dried under nitrogen gas at 25 °C. The residues were redissolved in 200 μL of methanol and centrifuged at 10,000 rpm for 10 min, and an aliquot of supernatant was subjected to UPLC–ESI–MS/MS analysis.
3. Results and Discussions
3.1. UPLC–MS/MS Analysis and Identification the Constituents of SGJY
Figures 1 and 2 show the ion chromatogram of SGJY in both positive and negative ion modes. A total of 73 peaks were identified or tentatively characterized including 14 organic acids, 37 flavonoids, 8 prenylated phloroglucinols, 2 naphthodianthrones, 5 lignans, 3 phenylpropanoids, and 4 other compounds, on the basis of the ultraviolet (UV) spectra, MS spectra, and MS/MS spectra with fragmentation patterns of reference standards or literature data. All the detailed data are shown in Table 1.
Base peak intensity chromatogram of SGJY (A), dosed plasma (B), and control plasma (C) in positive mode
Citation: Acta Chromatographica Acta Chromatographica 30, 2; 10.1556/1326.2017.00094
Base peak intensity chromatogram of SGJY (A), dosed plasma (B), and control plasma (C) in negative mode
Citation: Acta Chromatographica Acta Chromatographica 30, 2; 10.1556/1326.2017.00094
Characterization of compounds in SGJY by UPLC–ESI–MS/MS (MS in m/z, Rt in min)
| Peak | Name | Origin | R t | MW | MS (+) MS/MS | MS (−) MS/MS |
|---|---|---|---|---|---|---|
| 1 | Neochlorogenic acid | A, H | 11.67 | 354 | 355 [M + H]+ 163 | 353 [M − H]− 191 [M − H − Caffe]− 179 [M − H − Quinic]− |
| 2 | 3-(4-O-Glucosylferuloyl) quinic acid | A | 13.35 | 530 | 529 [M − H]− 367 [M − H − Glu]− 191 [M − H − Glu − Ferul]− | |
| 3 | 1,3-Di-O-caffeoylquinic acid | A, H | 14.91 | 516 | 515 [M − H]− 353 [M − H − Caffe]− 191 [M − H − 2Caffe]− | |
| 4 | 3-O-p-Coumaroylquinic acid | H | 15.82 | 338 | 339 [M + H]+ 147 | 337 [M − H]− 163 [M − H − Quinic]− 191 [M − H − Couma]− |
| 5 a | Chlorogenic acid | A, H | 17.39 | 354 | 355 [M + H]+ 163 372 [M + NH4]+ 355, 163 | 353 [M − H]− 191 [M − H − Caffe]− |
| 6 | 5-Feruloylquinic acid | H | 18.51 | 368 | 367 [M − H]− 193 [M − H − Quinic]− | |
| 7 | 1-O-Caffeoylquinic acid | A, H | 19.26 | 354 | 355 [M + H]+ 163 | 353 [M − H]− 179 [M − H − Quinic]− 191 [M − H − Caffe]− |
| 8 | Eleutheroside B1 | A | 19.97 | 384 | 402 [M + NH4]+ 223 [M + H − Glu]+ 223 [M + H − Glu]+ 208, 163, 135, 107 | 429 [M + HCOOH − H]− 221 [M − H − Glu]− |
| 9 a | Roseoside | H | 21.14 | 386 | 387 [M + H]+ 207 [M + H − Glu − H2O]+ 225 [M + H − Glu]+ 189, 369 | 431 [M + HCOOH − H]− 385 [M − H]− 223 [M − H − Glu]− 205 [M − H − Glu − H2O]− |
| 10 | Savinin | A | 21.41 | 352 | 351 [M − H]− 249, 267, 333 | |
| 11 | 5-O-p-Coumaroylquinic acid | H | 21.98 | 338 | 339 [M + H]+ 147 | 337 [M − H]− 191 [M − H − Couma]− 163 [M − H − Quinic]− |
| 12 | 5-Methoxylariciresinol-4-O-glucoside | A | 22.44 | 552 | 551 [M − H]− 389 [M − H − Glu]− 341, 193 | |
| 13 a | Epicatechin | H | 22.66 | 290 | 291 [M + H]+ 123, 139, 165, 273 | 335 [M + HCOOH − H]− 289 [M − H]− 289 [M − H]− 245, 205, 271, 179 |
| 14 | Quercetin 3,7-diglucoside | H | 23.44 | 626 | 625 [M − H]− 463 [M − H − Glu]− 301 [M − H − 2Glu]− | |
| 15 | 3,4-Di-O-caffeoylquinic acid | A, H | 23.57 | 516 | 515 [M − H]− 353 [M − H − Caffe]− 335 [M − H − Caffe − H2O]− 179 [M − H − Caffe − Quinic]− | |
| 16 | 4-Feruloylquinic acid | H | 23.78 | 368 | 369 [M + H]+ 177 | 367 [M − H]− 191 [M − H − Ferul]− |
| 17 | Quercetin 3,4′-diglucoside | H | 24.08 | 626 | 625 [M − H]− 463 [M − H − Glu]− 301 [M − H − 2Glu]− | |
| 18 | Quercetin 4′,7-diglucoside | H | 24.57 | 626 | 625 [M − H]− 463 [M − H − Glu]− 301 [M − H − 2Glu]− | |
| 19 | 4-O-p-Coumaroylquinic acid | H | 24.94 | 338 | 337 [M − H]− 191 [M − H − Couma]− 163 [M − H − Quinic]− | |
| 20 a | Eleutheroside E | A | 25.23 | 742 | 760 [M + NH4]+ 597 | 787 [M + HCOOH − H]− 579 [M − H − Glu]− 417 [M − H − 2Glu]− 741 [M − H]− |
| 21 | Secoisolariciresinol-9′-O-glucoside | A | 25.77 | 524 | 523 [M − H]− 361 [M − H − Glu]− | |
| 22 | 3-O-p-Coumaroyl-4-O-caffeoylquinic acid | H | 25.89 | 500 | 501 [M + H]+ 193 [M + H − Caffe]+ 355 [M + H − Couma]+ | 545 [M + HCOOH − H]− 499 [M − H]− |
| 23 | Ochroside | H | 26.12 | 596 | 595 [M − H]− 463 [M − H − Ara]− 433 [M − H − Glu]− 301 [M − H − Ara − Glu]− | |
| 24 | Astragaline | H | 26.49 | 448 | 447 [M − H]− 285 [M − H − Glu]− | |
| 25 | Quercetin 3-O-glucoside-7-O-arabionoside | H | 26.58 | 596 | 595 [M − H]− 463 [M − H − Ara]− 433 [M − H − Glu]− 301 [M − H − Ara − Glu]− | |
| 26 | Quercetin 3-O-glucoside-7-O-rhamnoside | H | 26.70 | 610 | 611 [M + H]+ 303 [M + H − Glu − Rha]+ 449 [M + H − Glu]+ 465 [M + H − Rha]+ | 609 [M − H]− 447 [M − H − Glu]− 463 [M − H − Rha]− 301 [M − H − Glu − Rha]− |
| 27 | Multinoside A | H | 27.07 | 610 | 609 [M − H]− 447 [M − H − Glu]− 301 [M − H − Rutino]− | |
| 28 | Quercetin 3-O-rhamnoside-7-O-glucoside | H | 27.25 | 596 | 595 [M − H]− 433 [M − H − Glu]− 463 [M − H − Ara]− 301 [M − H − Ara − Glu]− | |
| 29 a | Rutin | H | 27.44 | 610 | 611 [M + H]+ 303 [M + H − Rutino]+ 465 [M + H − Glu]+ | 609 [M − H]− 301 [M − H − Rutino]− |
| 30 a | Isofraxidin | A | 27.69 | 222 | 223 [M + H]+ 208, 163, 107, 135 | |
| 31 a | Hyperoside | H | 28.01 | 464 | 465 [M + H]+ 303 [M + H − Glu]+ | 463 [M − H]− 301 [M − H − Glu]− |
| 32 | Quercetin 3-O-glucuronide | H | 28.36 | 478 | 477 [M − H]− 301 [M − H − GluA]− | |
| 33 | Isoquercitrin | H | 28.36 | 464 | 465 [M + H]+ 303 [M + H − Glu]+ | 463 [M − H]− 301 [M − H − Glu]− |
| 34 | Sinapaldehyde 4-O-glucoside | A | 29.08 | 370 | 371 [M + H]+ 209 [M + H − Glu]+ 133, 191, 353 | |
| 35 | Quercetin 3 (2-glucosylrhamnoside) | H | 29.22 | 610 | 609 [M − H]− 301 [M − H − Glu − Rha]− 447 [M − H − Glu]− | |
| 36 | Nicotiflorin | H | 29.41 | 594 | 593 [M − H]− 285 [M − H − Rutino]− | |
| 37 | 3,5-Di-O-caffeoylquinic acid | H | 29.64 | 516 | 515 [M − H]− 353 [M − H − Caffe]− 335 [M − H − Caffe − H2O]− | |
| 38 | Quercetin 3-O-arabinoside | H | 29.64 | 434 | 435 [M + H]+ 303 [M + H − Ara]+ | 433 [M − H]− 301 [M − H − Ara]− |
| 39 | Guaijaverin | A | 30.22 | 434 | 435 [M + H]+ 303 [M + H − Ara]+ | 433 [M − H]− 301 [M − H − Ara]− |
| 40 | Quercitrin | H | 30.49 | 448 | 449 [M + H]+ 303 [M + H − Rha]+ | 447 [M − H]− 301 [M − H − Rha]− |
| 41 | Quercetin 3-O-(6-acetylglucoside) | H | 30.72 | 506 | 505 [M − H]− 301 [M − H − Acetylglu]− | |
| 42 | Apigenin 4′-O-glucuronide | H | 31.03 | 446 | 447 [M + H]+ 271 [M + H − GluA]+ | |
| 43 | 4,5-Di-O-caffeoylquinic acid | H | 31.34 | 516 | 515 [M − H]− 353 [M − H − Caffe]− | |
| 44 | Quercetin 3-O-(6-acetylgalactoside) | H | 31.69 | 506 | 507 [M + H]+ 303 [M + H − Acetylgal]+ | 505 [M − H]− 301 [M − H − Acetylgal]− 463 [M − H − Acetyl]− |
| 45 | Quercetin 3-O-(2-acetylglucoside) | H | 31.90 | 506 | 507 [M + H]+ 303 [M + H − Acetylglu]+ | |
| 46 | Quercimetrin | H | 32.04 | 464 | 465 [M + H]+ 303 [M + H − Glu]+ | 463 [M − H]− 301 [M − H − Glu]− |
| 47 | Juglanin | H | 32.37 | 418 | 419 [M + H]+ 287 [M + H − Ara]+ | 417 [M − H]− 285 [M − H − Ara]− |
| 48 | Quercetin 3-O-(2-acetylgalactoside) | H | 32.54 | 506 | 507 [M + H]+ 303 [M + H − Acetylgal]+ 205, 187 | 505 [M − H]− 301 [M − H − Acetylgal]− 463 [M − H − Acetyl]− |
| 49 | Cynaroside | H | 32.96 | 448 | 447 [M − H]− 285 [M − H − Glu]− | |
| 50 | Scutellarin A | H | 33.20 | 446 | 447 [M + H]+ 271 | 445 [M − H]− 269 [M − H − GluA]− 269 251, 241 |
| 51 | Cedrurin | H | 33.90 | 346 | 347 [M + H]+ 332, 314 | 345 [M − H]− 330 [M − H − Me]− |
| 52 | Kaempferol 3-O-(6-acetylglucoside) | H | 34.07 | 490 | 489 [M − H]− 285 [M − H − Acetylglu]− 429, 447 | |
| 53 | Licochalcone A | H | 34.29 | 338 | 337 [M − H]− 322 [M − H − Me]− 257 | |
| 54 | Vincetoxicoside B | H | 34.49 | 448 | 447 [M − H]− 301 [M − H − Rha]− | |
| 55 | Linarin | H | 34.68 | 592 | 593 [M + H]+ 447 [M + H − Rha]+ 285 [M + H − Glu]+ | 637 [M + HCOOH − H]− 591 [M − H]− 283 [M − H − Rha − Glu]− |
| 56 | Corylifolinin | H | 34.91 | 324 | 323 [M − H]− 243 | |
| 57 | Acacetin 7-O-glucuronide | H | 35.66 | 460 | 461 [M + H]+ 285 [M + H − GluA]+ | 459 [M − H]− 283 [M − H − GluA]− 283 268 [M − H − GluA − Me]− |
| 58 | Tilianin | H | 36.37 | 446 | 447 [M + H]+ 285 [M + H − Glu]+ | 491 [M + HCOOH − H]− 283 [M − H − Glu]− 329 |
| 59 a | Quercetin | H | 36.57 | 302 | 301 [M − H]− 179, 151, 257, 273 | |
| 60 | Glychionide B | H | 36.68 | 460 | 461 [M + H]+ 285 [M + H − GluA]+ | 459 [M − H]− 283 283 [M − H − GluA]− 268 [M − H − GluA − Me]− |
| 61 | Apigenin | H | 41.12 | 270 | 271 [M + H]+ 253, 225, 167, 123 | 269 [M − H]− 251 [M − H − H2O]− |
| 62 | Acacetin | H | 44.20 | 284 | 285 [M + H]+ 270 [M + H − Me]+ | |
| 63 | Wogonin | H | 44.51 | 284 | 285 [M + H]+ 270 [M + H − Me]+ | |
| 64 | Garsubellin E | H | 47.36 | 498 | 497 [M − H]− 428, 357, 399 | |
| 65 | 17R,18-Dihydroxyfurohyperforin | H | 47.44 | 586 | 585 [M − H]− 445, 516, 399, 291 | |
| 66 | Pseudohypericin | H | 47.91 | 520 | 519 [M − H]− 503 | |
| 67 | Furohyperforin | H | 48.17 | 552 | 551 [M − H]− 482, 413, 383, 315 | |
| 68 | Hyperfirin | H | 48.86 | 468 | 467 [M − H]− 398 | |
| 69 | Adhyperfirin | H | 49.66 | 482 | 481 [M − H]− 412 | |
| 70 | Oxedhyperforin | H | 50.56 | 554 | 553 [M − H]− 484, 401, 415, 333 | |
| 71 a | Hyperforin | H | 51.16 | 504 | 503 [M − H]− 459 | |
| 72 a | Hypericin | H | 51.39 | 536 | 535 [M − H]− 466, 383, 397, 315 | |
| 73 | Adhyperforin | H | 52.14 | 550 | 549 [M − H]− 480, 411, 397, 329 |
The compounds have been identified by reference standards; A indicates Acanthopanax senticosus Harms; H, Hypericum perforatum L.; Me, methyl; Glu, glucosyl; Ara, arabionosyl; Gla, galactosyl; Rha, rhamnosyl; GluA, glucuronosyl; Caffe, caffeoyl; Quinic, quinic acid; Couma, coumaroyl.
3.1.1. Identification of Components by Standards
Compounds 5, 9, 13, 20, 29, 30, 31, 59, 71, and 72 were respectively attributed to chlorogenic acid, (6S,7E,9R)-roseoside, epicatechin, eleutheroside E, rutin, isofraxidin, hyperoside, quercetin, hypericin, and hyperforin, by comparison with the retention times and mass spectral data of the reference standards.
3.1.2. Identification of Components through Investigating Literatures
3.1.2.1. Organic acids identification
Organic acids are vital compounds found in both H. perforatum L. and A. senticosus Harms. Chlorogenic acid (5) was identified for certain by comparison with the reference standards. Chlorogenic acid, one of the main organic acids in SGJY, could be used to characterize the fragmentation pathways. It gave diagnostic ions at m/z 372 [M + NH4]+, 355 [M + H]+, and 163 [M + H − 192]+ in positive mode and at m/z 353 [M − H]− and 191 [M − H − Caffe]− in negative mode. Based on these fragmentation patterns, compounds 1–4, 6, 7, 11, 15, 16, 19, 22, 37, and 43 were identified.
Compounds 1 and 7 gave precursor ions at m/z 355 [M + H]+ and 353 [M − H]− and fragment ions at m/z 163 in positive mode and 191 in negative mode, which, with the same diagnostic ions of chlorogenic acid (5), were assigned as neochlorogenic acid and 1-O-caffeoylquinic acid [1]. Compounds 4, 11, and 19 gave diagnostic ions 16 Da less than 5, were assigned as p-coumaroylquinic acid [1]. Compounds 6 and 16 gave diagnostic ions 14 Da more than 5 and were assigned as feruloylquinic acid [1]. Compound 2 gave precursor ions at 529 [M − H]−, and fragment ions at 367 and 191 in negative mode; through investigating references, compound 2 was identified as 3-(4-O-glucosylferuloyl) quinic acid [2]. Compounds 3, 15, 37, and 43 with the same MS spectra gave diagnostic ions 162 Da more than 5 and were characterized as di-O-caffeoylquinic acid [1]. Compound 22 gave diagnostic ions 16 Da less than 3 and were identified as 3-O-p-coumaroyl-4-O-caffeoylquinic acid [3].
3.1.2.2. Flavonoids identification
Flavonoids are abundant in H. perforatum L. Rutin (29), hyperoside (31), and quercetin (59) were identified for certain by comparison with the reference standards. Hyperoside, one of the most abundant and well responded flavonoids in SGJY, could be used to characterize the fragmentation pathways. It gave diagnostic ions at m/z 465 [M + H]+ and 303 [M + H − Glu]+ in positive mode, and at m/z 463 [M − H]− and 301 [M − H − Glu]− in negative mode. Based on these fragmentation patterns, compounds 14, 17, 18, 23–28, 32, 33, 35, 36, 38–42, 44–50, 52, 54, 55, 57, 58, and 60–63 were identified.
Compounds 14, 17, 18, 23, 25–28, 32–33, 35, 38, 40, 41, 44–46, 48, and 54 gave diagnostic ions at 303 in positive mode and/or 301 in negative mode, which suggested that these compounds should be quercetin (59) derivatives. Compounds 38 and 39 gave diagnostic ions 132 Da more than 59 and were identified as quercetin 3-O-arabinoside and guaijaverin [4, 5]. Compounds 40 and 54 showed diagnostic ions 146 Da more than 59 and were characterized as quercetin-O-rhamnoside [6, 7]. Compounds 33 and 46 with the same MS spectra were assigned as isoquercitrin and quercimetrin, respectively, for their diagnostic ions 162 Da more than 59 [6, 8]. Compound 32 gave diagnostic ions 176 Da more than 60 and were identified as quercetin 3-O-glucuronide [9]. With the same approach, compounds 26, 27, 28, and 35 were assigned as quercetin-O-glucoside-rhamnoside [10–13]; compounds 23 and 25 were assigned as quercetin-O-glucoside-arabionoside [14, 15]; compounds 41, 44, 45, and 48 were assigned as quercetin-O-acetylglucoside [16–19]; and compounds 14, 17, and 18 were assigned as quercetin diglucoside [20–22].
Compounds 24, 36, 47, 49, and 52 gave diagnostic ions at 287 in positive mode and/or 285 in negative mode, which suggested that these compounds should be kaempferol or luteolin derivatives. Compound 47 gave diagnostic ions 132 Da more than kaempferol and were identified as kaempferol 3-O-arabinoside [23]. Compounds 24 and 49 showed diagnostic ions 162 Da more than kaempferol or luteolin and were characterized as astragaline and cynaroside [24, 25]. Compounds 52 and 36 showed diagnostic ions 204 Da and 308 Da more than kaempferol, respectively, and were characterized as kaempferol 3-O-(6-acetylglucoside) and kaempferol 3-O-rutinoside [16, 24].
Compounds 42 and 50 with the same MS spectra showed precursor ion at m/z 447 [M + H]+ and 445 [M − H]−, which produced prominent ions as compound 61 at m/z 271 in positive mode and 269 in negative mode, owing to loss of a glucuronyl. By comparing with reference, these compounds were characterized as scutellarin A, apigenin 4′-O-glucuronide, and apigenin [26–28].
Compounds 62 and 63 with the same MS spectra showed precursor ion at m/z 285 [M + H]+, and gave the fragment ion at 270; through investigating references, these two compounds were identified as acacetin and wogonin [29, 30]. Compounds 58 and 60 with the same MS spectra showed diagnostic ions 162 Da more than 62 and 63 and were characterized as tilianin and glychionide B [31]. Compound 55 gave diagnostic ions 308 Da more than 62 and were identified as linarin [32]. Compound 57 gave diagnostic ions 176 Da more than 62 and were identified as acacetin 7-O-glucuronide [33].
3.1.2.3. Prenylated phloroglucinols identification
Prenylated phloroglucinols are one of the most famous natural herbal ingredients which are abundant in H. perforatum L. Hyperforin (71) was identified for certain by comparison with the reference standards. Hyperforin, one of the most abundant and well responded prenylated phloroglucinols in SGJY, could be used to characterize the fragmentation pathways. It gave diagnostic ions at m/z 535 [M − H]−, 466, 383, 397, and 315 in negative mode. Based on these fragmentation patterns, compounds 64, 65, 67–70, and 73 were identified.
Compounds 64, 65, 67–70, and 73 gave the characteristic loss of 69 or 68 Da, the same with hyperforin (72). By investigating reference data, these compounds were identified as garsubellin E (64) [34], 17R,18-dihydroxyfurohyperforin (65) [35], furohyperforin (67) [36], hyperfirin (68) [9], adhyperfirin (69) [9], oxedhyperforin (70) [37], and adhyperforin (73) [9].
3.1.2.4. Naphthodianthrones identification
Naphthodianthrones are another one of the most famous natural herbal ingredients which are abundant in H. perforatum L. Hypericin (72) was identified for certain by comparison with the reference standards. Compound 66 showed precursor ion at m/z 519 [M − H]− and fragment ions at m/z 503, and was identified as pseudohypericin through investigating references [9].
3.1.2.5. Lignans identification
Lignans are one of the main ingredients which are abundant in A. senticosus Harms. Eleutheroside E (20) was identified for certain by comparison with the reference standards. Eleutheroside E, which responded well in SGJY, could be used to characterize the fragmentation pathways. It gave diagnostic ions at m/z 787 [M + HCOOH − H]−, 741 [M − H]− , 579 [M − H − Glu]−, and 417 [M − H − 2Glu]− in negative mode. Based on these fragmentation patterns, compounds 10, 12, 21, and 51 were identified.
Compound 10 produced a precursor ion at m/z 351 [M − H]− and fragment ions at m/z 249, 267, 333. By investigating reference data, it was identified as savinin [38].
Compound 12 showed precursor ion at m/z 551 [M − H]− and fragment ions at m/z 389 [M − H − Glu]−, 341, and was identified as 5-methoxylariciresinol-4-O-glucoside through investigating references [39].
Compound 21 produced a precursor ion at m/z 523 [M − H]−, and fragment ions at m/z 361 [M − H − Glu]−. By investigating reference data, it was identified as secoisolariciresinol-9′-O-glucoside [40].
Compound 51 produced precursor ions at m/z 347 [M + H]+ and 345 [M − H]−, and fragment ions at m/z 332 [M + H − Me]+ and 330 [M − H − Me]−. By investigating reference data, it was identified as cedrurin [41].
3.1.2.6. Phenylpropanoids identification
Isofraxidin (30), identified for certain by comparison with the reference standard, was the main constituent of A. senticosus Harms. It gave diagnostic ions at m/z 223 [M + H]+, 208, 163, 107, and 135 in positive mode. Based on these fragmentation patterns, compounds 8 and 34 were identified.
Compound 8 showed precursor ion at m/z 402 [M + NH4]+, which produced prominent ions as compound 30 at m/z 223 in positive mode, owing to loss of a glucuronyl. By comparing with referenced, compound 8 was characterized as eleutheroside B1 [2].
Compound 34 showed precursor ion at m/z 371 [M + H]+ and fragment ions at m/z 209 [M + H − Glu]+, and was identified as sinapaldehyde 4-O-glucoside through investigating references [2].
3.1.2.7. Other compounds identification
Compound 53 gave precursor ions at m/z 337 [M − H]− and fragment ions at m/z 322 [M − H − Me]−, 247 in negative mode. By comparing with referenced, compound 53 was characterized as licochalcone A [42]. Compounds 56 showed diagnostic ions 14 Da less than 53 and were characterized as corylifolinin [43].
3.2. UPLC–MS/MS Analysis and Identification the Constituents of SGJY in Rat Plasma
To clarify the active constituents responsible for the pharmacological action, it is necessary to analyze the chemical constituent profile in vivo. Therefore, the rat plasma after oral administration of SGJY capsule was analyzed by the same UHPLC–ESI–MS/MS method used above. By comparing the retention time and mass chromatography of dosed rat plasma with control plasma and SGJY, 15 compounds were observed in dosed rat plasma which did not appear in control plasma. Among them, 10 compounds (29, 32, 53, 56, 66, 67, 70, 71, 72, and 73) were indicated as original constituents of SGJY, compounds M1–5 were tentatively predicted to be metabolites of SGJY. Ion chromatograms of dosed and controlled rat plasma are shown in Figure 2. The MS spectra and retention behavior of 15 peaks for original constituents and metabolites are summarized in Table 2.
Characterization of compounds in SGJY treated rat plasma by UPLC–ESI–MS/MS
| No. | Name | R t (min) | MW | MS (−) | MS/MS (−) |
|---|---|---|---|---|---|
| 29 | Rutin | 27.47 | 610 | 609 [M − H]− | 301 |
| 32 | Quercetin 3-O-glucuronide | 28.28 | 478 | 477 [M − H]− | 301 |
| 53 | Licochalcone A | 34.44 | 338 | 337 [M − H]− | 322, 257 |
| 56 | Corylifolinin | 34.99 | 324 | 323 [M − H]− | 243 |
| 66 | Pseudohypericin | 48.01 | 520 | 519 [M − H]− | 503 |
| 67 | Furohyperforin | 48.01 | 552 | 551 [M − H]− | 482, 413, 383, 315 |
| 70 | Oxedhyperforin | 50.56 | 554 | 553 [M − H]− | 484, 415, 401, 333 |
| 71 | Hyperforin | 51.26 | 536 | 535 [M − H]− | 466, 397, 383, 315, 313 |
| 72 | Hypericin | 51.16 | 504 | 503 [M − H]− | 459, 327 |
| 73 | Adhyperforin | 52.14 | 550 | 549 [M − H]− | 480, 397, 329, 465, 313 |
| M1 | Quercetin bisglucuronide | 25.59 | 654 | 653 [M − H]− | 477, 301 |
| M2 | Kaempferol 3-O-glucuronide | 30.39 | 462 | 461 [M − H]− | 285, 175 |
| M3 | Isorhamnetin glucuronide | 31.01 | 492 | 491 [M − H]− | 315, 300 |
| M4 | Tamarixetin glucuronide | 31.85 | 492 | 491 [M − H]− | 315, 300 |
| M5 | Epicatechin glucuronide | 31.94 | 466 | 465 [M − H]− | 289 |
3.2.1. Identification of Original Constituents in Rat Plasma
Ten compounds were indicated to be original constituents of SGJY. They were identified as rutin, quercetin 3-O-glucuronide, licochalcone A, corylifolinin, pseudohypericin, furohyperforin, oxedhyperforin, hypericin, hyperforin, and adhyperforin, respectively.
3.2.2. Identification of Metabolites of SGJY in Rat Plasma
To identify the metabolites accurately, probable structures were first assumed in accordance with the rules of drug metabolism in vivo. Flavones were the main constituents of SGJY and showed as mentioned above. The main metabolic pathways of flavones were glucuronidation, sulfation, and methylation. In this study, the constituents of SGJY identified as mentioned above may provide guidance for investigating the metabolites of SGJY in rat plasma. The loss of 176 Da could be assigned as a glucuronate in the structure (Figure 3).
Chemical constituents of SGJY capsule
Citation: Acta Chromatographica Acta Chromatographica 30, 2; 10.1556/1326.2017.00094
The metabolite M1 gave a precursor ion at m/z 653 [M − H]− and product ions at m/z 477 and 301 in negative mode; the loss of 176 Da and 176 Da could be assigned as two glucuronate in the structure. By investigating reference data, it was identified as quercetin di-O-glucuronide [44].
The metabolites M3 and M4 produced same precursor ion at m/z 491 [M − H]−, eluted at 30.39 and 31.01 min, respectively. They exhibited the same product ions at m/z 315 and 300 in negative mode, and were identified as isorhamnetin glucuronide and tamarixetin glucuronide through investigating references [45]. The metabolites M2 and M5 also gave the same MS fragmentation patterns [44] (Table 2).
4. Conclusion
UPLC–ESI–MS/MS was proved to be an effective method for the characterization and identification of major components of SGJY capsule. A total of 73 constituents were successfully separated and identified by this method. In vivo, the absorption and metabolism of SGJY capsule were explored. As a result, a total of 15 compounds were identified from rat plasma after oral administration of SGJY, including 10 of the original constituents and 5 of the metabolites. In addition, this study demonstrated that UHPLC–ESI–MS/MS would be a useful tool to investigate the potential effective constituents in SGJY capsule.
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