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. 2024 Jun 14;14(6):333. doi: 10.3390/metabo14060333

Metabolite Profiling Analysis of the Tongmai Sini Decoction in Rats after Oral Administration through UHPLC-Q-Exactive-MS/MS

Xianhui Zheng 1,2, Yingying Zhan 1, Mengling Peng 1, Wen Xu 1,2, Guanghai Deng 1,2,*
Editors: Hirokazu Kawagishi, Junsong Wang
PMCID: PMC11205536  PMID: 38921468

Abstract

Tongmai Sini decoction (TSD), the classical prescriptions of traditional Chinese medicine, consisting of three commonly used herbal medicines, has been widely applied for the treatment of myocardial infarction and heart failure. However, the absorbed components and their metabolism in vivo of TSD still remain unknown. In this study, a reliable and effective method using ultra-performance liquid chromatography coupled with hybrid quadrupole-Orbitrap mass spectrometry (UHPLC-Q-Exactive-MS/MS) was employed to identify prototype components and metabolites in vivo (rat plasma and urine). Combined with mass defect filtering (MDF), dynamic background subtraction (DBS), and neutral loss filtering (NLF) data-mining tools, a total of thirty-two major compounds were selected and investigated for their metabolism in vivo. As a result, a total of 82 prototype compounds were identified or tentatively characterized in vivo, including 41 alkaloids, 35 phenolic compounds, 6 saponins. Meanwhile, A total of 65 metabolites (40 alkaloids and 25 phenolic compounds) were tentatively identified. The metabolic reactions were mainly hydrogenation, demethylation, hydroxylation, hydration, methylation, deoxylation, and sulfation. These findings will be beneficial for an in-depth understanding of the pharmacological mechanism and pharmacodynamic substance basis of TSD.

Keywords: Tongmai Sini decoction, metabolites profiles, metabolic pathways, UHPLC-Q-Exactive-MS/MS

1. Introduction

The classical prescriptions of traditional Chinese medicine (TCM) have originated from the fixed combination of certain kinds of herbal medicines recorded in the ancient classics, which have been still widely used in East Asia and exhibited precise clinical efficacy, with obvious characteristics and advantages [1]. For a long time, some classic prescriptions have been developed into modern Chinese medicinal preparations through the optimization of preparation technology and drug development research [2,3]. Most classical prescriptions have existed for at least hundreds of years, and with time, classic prescriptions may have changed to some extent, while their core characteristics (e.g., composition of herbs, proportion of herbs, etc.) have not changed significantly [4,5]. The reasons for the inheritance of classical prescriptions to the present day can be attributed to the high safety of the prescriptions and their proven efficacy due to a large number of clinical applications.

Tongmai Sini decoction (TSD) is a classic formula from the Chinese medical masterpiece “The Treatise on Typhoid Fever”, written 1800 years ago. It consists of three herbal concoctions of Radix Aconiti Lateralis Preparata (RALP), Rhizoma Zingiberis (RZ), and Radix Glycyrrhizae Preparata (RGP) and is commonly used in modern times for myocardial infarction and heart failure, atherosclerosis, shock, diarrhea, etc. [6,7]. TSD has the effects of raising blood pressure, strengthening the heart, anti-hypoxia, anti-shock, anti-thrombosis, anti-myocardial ischemia, anti-slowing arrhythmia, and so on [8,9]. The main chemical constituents of TSD include alkaloids (from RALP), phenolic acids and saponins (from RGP), and volatile oils (from RZ). At present, chemical composition [10], pharmacological, pharmacokinetic [11,12,13], and metabolomics [8,9,14] studies have been preliminarily conducted on TSD, especially on its cardiovascular activities. Most of the studies on TDS concentrated on the pharmacokinetics of diterpene alkaloids after oral administration of TDS, and some of the studies focused on the changes in the in vivo metabolome or lipidome profile against myocardial ischemia, heart failure, hypothyroidism. There is a lack of systematic and in-depth in vivo chemical and metabolite studies of TDS.

The components of different botanicals enter the body and produce metabolites, which exert therapeutic effects through multiple pathways [15]. To fully understand the therapeutic components, it is necessary to first analyze the blood-entering components and their metabolites, as well as to study the metabolites distributed in plasma, urine, feces, and tissues, which is conducive to analysis of the potential components and pathways of action in the body [16,17].

High-resolution mass spectrometry (HRMS), in combination with chromatography technology, has provided useful structural information about chemical components, offering strong support for the characterization of in vivo and in vitro metabolic components of botanicals [18,19,20]. In recent years, in order to improve the sensitivity and selectivity of obtaining MS/MS or MSn data for trace components in vivo, some acquisition and identification strategies have been developed, combined, and applied, for instance, the extracted ion chromatogram (EIC), mass defect filter (MDF), dynamic background subtraction (DBS) and neutral loss filter (NLF) [21,22,23].

In this paper, the established UHPLC-Q-Exactive-MS/MS methods have great advantages for the qualitative analysis of bioactive samples in rats after oral doses of TSD, and a variety of post-data processing techniques, including EIC, MDF, DBS, and NLF was applied for quickly screen and systematically identify the metabolites. These metabolic studies can provide the chemical foundation and an in-depth understanding of metabolic transformation for further research on effective substances and the action mechanism of TSD.

2. Materials and Methods

2.1. Chemicals and Plant Materials

Aconitine, mesaconitine, hypaconitine, liquiritin, liquiritigenin, ononin, and formononetin, with purities greater than 98%, were purchased from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). HPLC-grade acetonitrile, methanol, and formic acid were purchased from Sigma Aldrich (St. Louis, MO, USA). Ultrapure Water (18.2 MΏ) was produced by a Milli-Q water system (Millipore, Bedford, MA, USA). The herbal pieces of RALP, RZ, and RGP were purchased from Kangmei Pharmaceutical Co. Ltd. Of Guangdong, China, and identified by Prof. Zhi-hai Huang, The Second Clinical College, Guangzhou University of Chinese Medicine, Guangzhou, China.

2.2. Plant Extract Preparation

According to the documentary records of TSD, the TSD pieces that included RALP (30 g), RZ (20 g), and RGP (30 g) were soaked with 8 times the amount of water for 30 min and decocted to boil (100 °C) for 2 h. The filtrate was collected, and the residue was decocted in 8 times the amount of water for 1.5 h again. The hot filtrate was combined and concentrated to 80 mL (1 g herbal pieces/1 mL aqueous solution). The obtained TSD extract was stored at −20 °C before use.

2.3. Animal and Drug Administration

Male Sprague–Dawley rats (220–260 g) were obtained from Guangdong Provincial Medical Laboratory Animal Center (Guangdong, China). All animal experiments were performed at the SPF animal laboratory [experimental animals license number SYXK (Guangdong, China) 2008–0094]. The Institutional Animal Ethics Committee of Guangdong Provincial Hospital of Chinese Medicine approved all experimental protocols (No. 2023131).

Six SD rats were randomly divided into two groups (Urine and plasma groups) and adapted to the metabolic cage for a week before the experiment. Blank urine and plasma samples were collected under abrosia state ahead of gastric gavage. The rats were fasted for 14 h with water ad libitum before oral administration of TSD extract and underwent 4 h of water deprivation after that. TSD extract was orally administered to rats of urine and plasma groups twice at an interval of 1 h, and the dosage was 2 mL per 100 g bodyweight per time.

2.4. Sample Collection and Pretreatment

Urine samples from 0 to 24 h after the second dosing were collected and stored at −80 °C prior to analysis. Plasma samples were obtained at 1, 2, 4, 8, and 12 h after the second administration in heparinized 1.5 mL polythene tubes under diethyl ether anesthesia, respectively. All plasma samples were centrifuged at 4000 rpm for 10 min, and the plasma supernatants were then merged in equal volume and frozen at −80 °C prior to analysis.

The collected urine and plasma samples (200 μL) were added with 4× the volume of acetonitrile-methanol (3:1) to precipitate protein, respectively. All separate supernatants were dried under N2 flow, and the residues were resuspended in 200 μL acetonitrile and centrifuged at 15,000× g for 8 min. Finally, a 5 μL sample was injected into the UHPLC-Q-Exactive-Orbitrap MS system for further analysis.

2.5. Instrumentation and Conditions

LC analyses were conducted on a Thermo UltiMate 3000 UHPLC system (Thermo Fisher Scientific, San Jose, CA, USA) equipped with a quaternary pump, a cooling autosampler, and a thermostatically controlled column oven. An ACQUITY UPLC HSS T3 Column (2.1 × 100 mm, 1.8 μm) was used. The mobile solvents were composed of acetonitrile (A) and water with 0.02% formic acid (B), and the gradient elution profile was employed as follows: 5% A, 0 min; 16% A, 12 min; 55% A, 23 min; 90% A, 35 min; 95% A, 40 min; returning to initial conditions in 4 min at a flow rate of 200 μL/min at room temperature. The injection volume was 5 μL. The temperatures of the sample tray and the column oven were set at 4 and 35 °C, respectively.

A Q-Exactive hybrid quadrupole-orbitrap mass spectrometer was connected to an LC system via an electrospray ionization source as an interface. Data acquisition and processing were calculated using Compound Discoverer 3.2 software. The optimized parameters for MS analysis were as follows: the mass spectrometer parameters were positive (PI) and negative (NI) ion mode; the resolution of the Orbitrap mass analyzer was set as 30,000; ion spray voltage was −3.8 kV; the capillary temperature was 325 °C; the sheath gas flow rate was 40 psi; the auxiliary gas flow rate was 8 psi; and the mass range was m/z 150–1500. The properties of data-dependent MS2 scanning (DDS) parameters and events were as follows: resolution, 17,500; HCD, 35 eV; repeat count, 2; exclusion list, 50; repeat duration, 5 s; and exclusion duration, 30 s. The mass error for molecular ions of all compounds identified was within ±5 ppm.

3. Results and Discussion

3.1. Systematic Analytical Strategy for Online Metabolite Analysis

Based on our previous research on the cleavage patterns of components in RALP and RGP and a review of the literature [24,25,26,27,28], the metabolite profiling of TSD was systematically investigated by UHPLC-Q-Exactive-MS/MS methods. The workflow of the analytic procedure was carried out and shown in Figure 1. Figures S1 and S2 (Supplementary Materials) displayed the detailed workflow for the identification of prototype components and metabolites, respectively.

Figure 1.

Figure 1

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Workflow of the analytic strategy for the metabolite identification of TSD.

The strategy consisted of the following steps: (1) First, the chemical database (Table S1, Supplementary Materials) was constructed, including mass weights, elemental compositions, and structure information of chemical compositions originating from RALP, RGP, and RZ based on our previous research and the related literature [24,25,26,27,28,29]. (2) Then, an online full-scan and MS/MS data acquisition was processed in both negative and positive modes based on the DBS and DDS techniques for potential metabolite detection. (3) Next, the data files were imported into the Compound Discoverer 3.2 software, and the data-mining tools of EIC, NLF, and MDF were applied to screen the possible metabolites of TSD. Table S2 (Supplementary Materials) showed the detailed parameters of data processing. The main compounds with mass spectral peak areas greater than 108 in the decoction (shown in Table 1) were used as parent compound templates for MDF data screening (±50 mDa) (4) Next, based on the chemical database, acquired accurate mass data, retention time, and characteristic fragment ions, the identification of prototype components was elucidated (shown in Table 2). In addition, the Clog p values calculated by ChemDraw 14.0 were used to distinguish isomers at different retention times. (5) Finally, the mass information of potential metabolites, as well as their possible biotransformation pathways and composition change given by Compound Discoverer 3.2, were compared by the data of prototype components and the related literature to verify the metabolites and their metabolic pathways (shown in Table 3).

Table 1.

Main prototype components as parent compound templates for MDF data screening. (mass spectral peak areas greater than 108 in the decoction).

Alkaloids (from RALP) Phenolic and Saponin Compounds (from RGP and RZ)
Karakolidine Liquiritigenin Formononetin
Fuziline Isoliquiritigenin Ononin
Neoline Liquiritin Glycyrrhizic Acid
Songorine Licochalcone B Glycyrrhetinic Acid
14-Benzoylhypaconine Licochalcone C Uralsaponin C
Talatizamine Licochalcone D Licoricesaponin G2
Karakoline Licoflavone C Glycycoum-Arim
14-Benzoylmesaconine Licoflavone A Glycyrol
Mesaconitine Licoricidin Glycyrin
Hypaconitine Licoleafol 6-Gingerol
Aconitine Gancaonin M
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Table 2.

Prototype compounds identified or tentatively characterized in the urine and plasma samples after oral administration of TSD.

ID [M+H]+(m/z) Formula tR
(min)		 Error
(ppm)		 ms/ms Identification ClogP Area
Urine Plasma
Alkaloids
1. 394.25839 C22H35NO5 3.51 −1.58 376.2474, 358.2376, 344.2229, 326.2116, 243.2516 Karakolidine +++
2. 394.25820 C22H35NO5 4.02 −1.42 376.2476, 358.2367, 340.2268, 328.2260, 307.4473, 218.6333 Chuanfumine +++
3. 439.25229[M-H]− C23H37NO7 4.25 0.56 392.2438, 344.2226, 295.8235, 193.8604, 146.9375 9-Hydroxysenbusine A +
4. 424.26871 C23H37NO6 6.69 −1.55 406.2584, 388.2478, 356.2207, 154.1227 Senbusine A −2.70 +++
5. 486.26941 C24H39NO9 7.01 −0.72 454.2438, 436.2322, 404.2069, 378.1887, 372.1793, 319.9836 Mesaconine ++
6. 424.26871 C23H37NO6 7.74 −1.55 406.2581, 388.2472, 356.2210, 154.1231 Senbusine B 0.16 ++
7. 378.26306 C22H35NO4 8.20 −2.18 360.2524, 342.2431, 328.2268, 242.3140 Karakoline ++
8. 408.27371 C23H37NO5 8.26 −1.81 390.2631, 372.2533, 358.2367, 340.2271 Isotalatizidine ++
9. 358.23691 C22H31NO3 9.17 −2.13 340.2265 Songorine +
10. 360.25293 C22H33NO3 9.18 −1.09 - Napelline ++
11. 330.20569 C20H27NO3 9.70 −2.07 236.8785, 170.7432, 152.4712 Hetisine ++
12. 470.27435 C24H39NO8 10.51 −1.05 - Hypaconine ++
13. 454.27933 C24H39NO7 11.33 −1.26 436.2685, 418.2609, 404.2422, 154.1227 Fuziline +++
14. 438.28445 C24H39NO6 11.58 −1.28 420.2736, 402.2617, 388.2472, 356.2214, 278.6899 Neoline +++
15. 420.27390 C24H37NO5 12.31 −1.32 402.2632, 384.2512, 370.2359, 342.2414, 324.2322, 251.1396 14-Acetylkarakoline +
16. 484.28937 C25H41NO8 12.41 −2.33 - Deoxyaconine +
17. 342.16931 C20H23NO4 12.89 −1.96 297.1120, 282.0887, 237.0910, 219.0804, 191.0860 N-Methyl-laurotetanine +++
18. 422.28931 C24H39NO5 13.64 −1.88 390.2629, 372.2517, 358.2379, 340.2238, 98.0970 Talatizamine ++++ ++
19. 420.23825 C23H33NO6 15.48 −0.86 402.2268, 370.1989, 293.7002, 154.1224 Giraldine F ++
20. 452.29996 C25H41NO6 15.54 −1.57 420.2740, 388.2465, 356.2219, 209.1644, 154.1228, 114.0916 Chasmanine ++++
21. 464.30038 C26H41NO6 16.933 −0.60 432.2740, 414.2626, 400.2474, 372.2535, 265.1608, 235.1487, 154.1225 14-Acetyltalatizamine ++++ ++
22. 606.28992 C31H43NO11 17.26 −1.60 574.2627, 556.2545, 524.2269, 506.2188, 492.1945, 261.0641, 173.0955, 105.0341 14-Benzoyl-10-OH-mesaconine ++
23. 544.28955 C30H41NO8 19.47 −0.95 512.2635, 494.2548, 480.2364, 462.2258, 390.2286, 270.0846, 105.0340 Gadenine +
24. 590.29490 C31H43NO10 19.51 −1.78 558.2616, 540.2575, 419.7593, 307.8019, 246.8854, 105.0339 14-Benzoylmesaconine ++
25. 540.29486 C31H41NO7 20.01 −1.33 504.2730, 462.2614, 382.2463, 340.2256, 322.2149, 304.2042 Aconicarchamine B +
26. 604.31060 C32H45NO10 20.53 −1.68 572.2811, 554.2750, 522.2495, 490.2176, 340.3151, 105.0341 14-Benzoylaconine ++
27. 574.30010 C31H43NO9 21.20 −1.65 542.2744, 510.2461, 304.5384, 198.1281, 105.0339 14-Benzoylhypaconine +++
28. 618.29210[M-H]− C32H45NO11 21.21 0.31 384.9167, 351.8983, 270.7405, 190.9267 14-Benzoyl-10-OH-aconine ++
29. 648.30023 C33H45NO12 21.93 −1.98 588.2775, 556.2513, 455.3509, 370.1645, 105.0340 10-OH-mesaconitine ++
30. 558.30530 C31H43NO8 22.07 −1.52 526.2800, 508.2674, 232.0710182.0626, 105.0341 14-Benzoyl-doxyhypaconine ++
31. 588.31561 C32H45NO9 22.32 −1.24 556.2905, 524.2639, 506.2443, 346.4250, 253.7027, 154.1226, 105.0341 14-Benzoyldeoxyaconine +
32. 542.31061 C31H43NO7 23.18 −1.15 510.2846, 492.2735, 482.2483, 460.2504, 154.1231 14-Benzoylneoline +
33. 632.30591 C33H45NO11 23.18 −0.63 572.2844, 540.2551, 522.2487, 508.2299, 354.1694, 105.0341 Mesaconitine * +++
34. 662.31683 C34H47NO12 23.39 −0.27 - Aconifine ++
35. 614.29553 C33H43NO10 24.17 −0.72 554.2743, 494.2534, 372.2162, 344.21622, 203.5583, 105.0341 2,3-didehydrohypaconitine +
36. 646.32135 C34H47NO11 24.58 −0.84 586.3002, 554.2727, 526.2797, 494.2520, 368.1843, 105.0340 Aconitine * ++
37. 616.31079 C33H45NO10 24.61 −0.83 556.2903, 524.2634, 496.2750, 464.2434, 338.1741, 310.1812, 105.0341 Hypaconitine * ++++
38. 600.31592 C33H45NO9 24.96 −1.32 540.2948, 508.2683, 480.2747, 476.2424, 448.2475, 354.2031, 254.4337, 105.0339 Secoyunaconitine +
39. 572.32117 C32H45NO8 24.95 −1.10 484.2688, 456.2745, 382.2002, 322.1798, 294.1857, 158.0964 14-O-Anisoylneoline +
40. 630.32635 C34H47NO10 26.07 −1.36 570.3046, 538.2788, 510.2882, 506.2528, 478.2571, 352.1898, 314.5361, 105.0341 3-Deoxyaconitine +++
41. 614.33173 C34H47NO9 27.60 −0.53 - Chasmaconitine ++
Phenolic compounds
42. 209.04474[M-H]− C10H10O5 8.56 −3.85 165.0545, 121.0281, 103.9187, 87.9238, 59.0123 Hydroxyferulic acid +++ ++
43. 433.13394[M-H]− C18H24O12 9.88 0.08 161.0442, 125.0230, 99.0436 Asperulosidic acid ++
44. 433.11407[M-H]− C21H22O10 13.87 −0.19 271.0615, 151.0024 5-Hydroxyliquiritin ++
45. 593.15137[M-H]− C27H30O15 15.51 0.29 473.1098, 383.9785, 353.0774 Vitexin II +++
46. 563.14055[M-H]− C26H28O14 15.83 −0.51 473.1089, 443.0985, 383.0769, 253.0502, 146.9367 Vitexin I +
47. 417.11890[M-H]− C21H22O9 16.74 −0.32 255.0662, 153.0182, 135.0074, 119.0488 Liquiritin * ++++
48. 505.13339 C24H24O12 18.72 −0.89 257.0809, 137.0234 Malonyl liquiritin +
49. 505.13358 C24H24O12 18.99 −0.07 257.0810, 137.0234 Malonyl liquiritin +
50. 431.13280 C22H22O9 20.26 −1.97 269.0809 Ononin ++++
51. 417.11908[M-H]− C21H22O9 20.39 −1.01 255.0662, 153.0180, 135.0072, 119.0481 Neoliquiritin 0.75 +++
52. 417.11900[M-H]− C21H22O9 20.74 −2.47 255.0662, 153.01816, 135.0074, 119.0488 Isoliquiritin 1.28 ++
53. 285.07670[M-H]− C16H14O5 21.23 −0.31 270.0536, 253.0505, 177.0182, 150.0310, 108.0203 Licochalcone B ++
54. 255.06560 C15H10O4 21.30 0.10 227.0704, 199.0754, 145.0286, 137.0234 Dihydroxyflavone ++++
55. 255.06580[M-H]− C15H12O4 21.70 −0.36 153.0180, 135.0073, 119.0487, 91.0173 Liquiritigenin * ++++ +++
56. 295.19040 C17H26O4 23.34 −0.22 177.0914, 163.0755, 137.0598, 131.0493, 99.0809 6-Gingerol ++++ ++
57. 269.04530[M-H]− C15H10O5 24.34 0.38 233.1537, 181.0644 Genistein +++
58. 255.06586[M-H]− C15H12O4 26.29 −0.49 153.0179, 135.0073, 119.0487, 91.0174 Isoliquiritigenin * ++++ +++
59. 269.08170 C16H12O4 26.58 1.04 253.0497, 237.0554, 213.0911, 118.0418, 107.0497 Formononetin * ++++ +++
60. 367.11790[M-H]− C21H20O6 27.75 −1.99 352.0944, 309.0400, 298.0476, 283.0247 Glycycoum-arim/Licocoumarione +++
61. 271.09565 C16H14O4 28.56 1.19 254.2579, 161.0599, 137.0598, 123.04440, 100.0763 Echinatin ++
62. 355.11835[M-H]− C20H20O6 28.60 −1.07 328.1265, 269.11820, 269.11820, 178.9975, 125.0230 8-Dimethylallyleriodictyol/6-Dimethylallyleriodictyol ++
63. 277.18008 C17H24O3 28.84 2.28 177.0912, 145.0649, 137.0598 6-Shogaol +++ ++
64. 355.15480[M-H]− C21H24O5 29.81 −1.06 323.1284, 233.1176, 207.1017, 135.0438, 125.0230, 109.0280 Isopentadienyl glycyrrhizoflavone ++
65. 367.11790[M-H]− C21H20O6 29.53 −2.00 309.0400, 297.0400, 284.0325, 203.0702 Glycycoum-arim/Licocoumarione +++
66. 321.11262[M-H]− C20H18O4 30.07 −1.93 306.0892, 174.9549 Licoflavone A +
67. 353.10290[M-H]− C20H18O6 30.17 −1.33 339.1187, 321.1126, 295.0613, 283.0614, 270.0535 Isolicoflanonol +++ ++
68. 353.13782 C21H20O5 30.22 −1.59 299.0906, 297.0857, 267.0653, 199.0758, 147.0441, 135.0441 Gancaonin M ++
69. 383.11273[M-H]− C21H20O7 30.39 −2.33 338.2439, 247.1310, 227.0704, 207.1015, 155.0337, 140.0101 Licopyranocoumarin +
70. 383.14828 C22H22O6 30.66 −1.50 327.0859, 299.0913, 191.0704 Glycyrin ++ ++
71. 355.15320 C21H22O5 30.94 −2.01 289.0549, 287.0553, 191.1067, 153.0548, 69.0708 Licobenzofuran/liconeolignan +++
72. 337.10780[M-H]− C20H18O5 31.02 −1.07 314.0428, 282.0531 Licoflavone C ++
73. 365.10239[M-H]− C21H18O6 30.17 −1.63 307.0244, 295.0245, 282.0169 Isoglycyrol 4.84 ++
74. 365.10236[M-H]− C21H18O6 31.12 −1.99 307.0242, 295.0243, 282.0167 Glycyrol 5.04 +++
75. 333.24170 C21H32O3 34.17 −1.99 177.0911, 145.0649, 137.0598 10-Shogaol ++
76. 279.23264[M-H]− C18H32O2 38.27 1.13 261.2219, 199.8500 Linoleic acid ++
Saponins
77. 879.40173[M-H]− 881.41516
705.38361[M+H-glcA]+
511.34122[agl+H-H2O]+ 		C44H64O18 24.011 −0.67
−1.38		 (−) 351.0557, 193.0342, 113.0229
(+) 511.3408, 493.3279, 451.3188, 141.0183		 Uralsaponin M ++
78. 837.39105[M-H]−
839.40466
469.33072[gal+H-H2O]+ 		C42H62O17 25.49 −0.79
−1.32		 (−) 351.05603, 289.05652, 193.03430, 175.02340, 113.02294
(+) 469.3304, 487.3415, 451.3209, 141.0184		 Yunganoside K2 ++
79. 837.39178[M-H]−
839.40491
469.33084[agl+H-H2O]+ 		C42H62O17 26.07 −0.84
−1.07		 (−) 351.05557, 289.05621, 193.03413, 175.02360, 113.02285
(+) 469.3304, 487.3413, 451.3198, 141.0183		 Licoricesaponin G2 +
80. 471.34613 C30H46O4 26.62 −1.41 453.33508, 425.34262, 317.21100, 235.16887, 189.16374 Glycyrrhetinic acid (enoxolone) * +
81. 821.39630[M-H]−
823.40936
647.37744[M+H-glcA]+
453.33554[agl+H-H2O]+ 		C42H62O16 26.64 1.08
−1.70		 (−) 351.05573, 193.03406, 175.02338, 113.02288
(+) 453.3354, 471.3451, 435.3259		 Glycyrrhizic acid * ++++
82. 821.39612[M-H]−
823.40936
647.37787[M+H-glcA]+
453.33585[agl+H-H2O]+ 		C42H62O16 27.65 0.86
−0.47		 (−) 351.05640, 193.03404, 175.02319, 113.02289
(+) 453.3354, 435.3257		 Uralsaponin B or Licoricesaponine K2/H2 ++
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Note: * Compounds identified by comparing with reference standards; glcA: β-D-glucuronopyronosyl; agl: aglycone; +, response area below 106; ++, response area between 106 and 107; +++, response area between 107 and 108; ++++, response area above 108.

Table 3.

Metabolites of major alkaloids found in the urine and plasma samples.

ID [M+H]+
(m/z)		 Formula tR
(min)		 Error
(ppm)		 ms/ms Composition Change Identification ClogP Area
Urine Plasma
M1. 410.25302 C22H35NO6 3.72 −1.69 392.2425, 374.2317, 360.2165, 342.2054 +O Hydroxy karakolidine ++
M2. 374.23212 C22H31NO4 7.55 −1.25 356.2212, 338.2106, 198.1122 +O Hydroxy songorine ++
M3. 394.25812 C22H35NO5 7.76 −1.85 376.2476, 358.2362, 98.0971, 58.0611 +O Hydroxy karakoline ++
M4. 438.28433 C24H39NO6 10.76 0.67 406.2588, 388.2476, 374.230, 356.2226 +O 10-Hydroxy talatizamine ++
M5. 632.30560 C33H45NO11 22.61 −1.50 572.2853, 540.2590, 512.2641, 508.2310, 480.2390, 358.2004, 354.1703, 105.0341 +O Hydroxy hypaconitine ++
M6. 392.24268 C22H33NO5 8.28 −1.21 374.2315, 344.2221, 312.1962, 114.0916 -H2 Dehydrogenated karakolidine +++
M7. 392.24249 C22H33NO5 8.95 −2.15 374.2325, 344.2240 -H2 Dehydrogenated karakolidine +++
M8. 452.26361 C24H37NO7 9.85 −1.48 434.2529, 416.2419, 204.2270, 384.2155 -H2 Dehydrogenated fuziline +++
M9. 376.24780 C22H33NO4 10.38 −1.82 358.2373, 98.0969 -H2 Dehydrogenated karakoline ++
M10. 376.24756 C22H33NO4 10.96 −1.15 358.2375, 234.0137, 98.0970 -H2 Dehydrogenated karakoline +++
M11. 436.26895 C24H37NO6 10.24 −0.95 418.2581, 400.2475, 386.2315, 358.2355, 340.2265 -H2 Dehydrogenated neoline +++ +
M12. 436.26907 C24H37NO6 10.64 −0.67 418.2585, 400.2473, 386.2303, 358.2383 -H2 Dehydrogenated neoline +++
M13. 420.27393 C24H37NO5 13.60 −1.33 388.2477, 370.2375, 98.0972 -H2 14-Dehydrogenated talatizamine +++
M14. 364.24744 C21H33NO4 7.39 −2.20 346.2374, 328.2268 -CH2 Demethyl karakoline +++ +
M15. 364.24740 C21H33NO4 7.86 −2.20 346.2370, 328.2266 -CH2 Demethyl karakoline ++++ +
M16. 424.26892 C23H37NO6 10.68 −1.05 406.2583, 374.2327, 356.2211, 342.2069, 154.1226 -CH2 Demethyl neoline +++ +
M17. 424.26890 C23H37NO6 11.08 −0.98 406.2581, 374.2317, 356.2222, 342.2076, 154.1228 -CH2 Demethyl neoline ++++
M18. 408.27386 C23H37NO5 9.87 −1.44 376.2475, 358.2365, 326.2136 -CH2 18-o-Demethyl talatizamine −0.78 ++++ ++
M19. 408.27393 C23H37NO5 11.17 −1.29 376.2478, 358.2373, 326.2129 -CH2 16-o-Demethyl talatizamine −0.73 ++++ ++
M20. 602.29486 C32H43NO10 21.31 −1.70 542.2742, 510.2477, 482.2540, 478.2212, 324.1592, 105.0339 -CH2 Demethyl hypaconitine ++++ +
M21. 618.28992 C32H43NO11 22.17 −1.22 558.2684, 526.2423, 508.2394, 354.1695, 105.0341 -CH2 Demethyl mesaconitine ++
M22. 330.20581 C20H27NO3 8.19 −1.70 312.1954 -C2H4 Deethyl songorine +++
M23. 350.23181 C20H31NO4 9.05 −2.21 332.2215, 314.2106, 300.1958, 234.9901, 158.9743 -C2H4 Deethyl karakoline +++
M24. 410.25314 C22H35NO6 10.97 −1.40 392.2423, 378.2271, 360.2163, 328.1906 -C2H4 Deethyl neoline ++
M25. 408.27374 C23H37NO5 8.96 −1.74 390.2631, 372.2537, 358.2369 -CH2O Demethyl-deoxy neoline ++++ ++
M26. 392.27896 C23H37NO4 13.38 −1.46 360.2527, 342.2436, 328.2265 -CH2O 16-O-Demethyl-14-deoxy Talatizamine +++
M27. 602.29529 C32H43NO10 23.87 −1.20 542.2773, 510.2486, 478.2222, 324.1592, 105.0341 -CH2O Demethyl-deoxy mesaconitine +++
M28. 360.25272 C22H33NO3 9.38 −1.68 342.2422, 324.2325, 121.0651 -H2O Dehydrated karakoline +++ +
M29. 360.25250 C22H33NO3 9.92 −2.11 342.2422, 324.2307 -H2O Dehydrated karakoline ++++ +
M30. 614.2517 C33H43NO10 24.17 −1.57 544.2743, 522.2518, 494.2534, 372.2162, 344.2215 -H2O Dehydrated mesaconitine ++
M31. 380.24277 C22H33NO5 8.81 −1.01 362.2316, 344.2046, 330.2065 -CH2+O Demethyl-hydroxy karakoline ++
M32. 438.24811 C23H35NO7 12.09 −1.06 420.2374, 402.2265, 392.2440, 374.2317 -CH2-H2 Dehydrogenated-demethyl fuziline ++ +
M33. 438.24890 C23H35NO7 12.21 0.49 420.2367, 402.2283, 392.2442, 374.2323 -CH2-H2 Dehydrogenated-demethyl fuziline ++ +
M34. 362.23172 C21H31NO4 8.15 −0.45 344.2223, 185.0710 -CH2-H2 Dehydrogenated-demethyl karakoline +++
M35. 422.25327 C23H35NO6 10.99 −1.07 390.2268, 406.2597, 390.2268, 374.2324 -CH2-H2 Dehydrogenated-demethyl neoline +++ +
M36. 406.25839 C23H35NO5 10.70 −1.01 388.2477, 370.2368, 328.2266 -CH2-H2 14-Dehydrogenated-16-O-demethyl talatizamine +++
M37. 346.20090 C20H27NO4 7.79 −1.12 328.1904, 296.1645, 268.1701, 251.1437 -C2H4+O N-Deethyl-hydroxy songorine ++ +
M38. 346.20071 C20H27NO4 8.49 −1.65 328.1903, 296.1650, 268.1699, 251.1429 -C2H4+O N-Deethyl-hydroxy songorine +++
M39. 378.26337 C22H35NO4 10.98 −1.43 346.2371, 328.2279 -C2H4-O N-Deethyl-14-deoxy talatizamine 0.88 +++ +
M40. 378.26334 C22H35NO4 11.23 −1.45 346.2371, 328.2267 -C2H4-O N-Deethyl-8-deoxy talatizamine 1.15 +++
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Note: +, response area below 106; ++, response area between 106 and 107; +++, response area between 107 and 108; ++++, response area above 108.

3.2. Identification of Prototype Components

An in-house database has been established for each compound involved in RALP, RGP, and RZ based on our previous experimental data and the related literature for the investigation of their chemical constituents. The database consisted of the compound name, molecular formula, accurate molecular mass, chemical structure, MS2 mass spectra, and related product ion information. The total ion chromatograms (BPIs) of TSD and the urine and plasma samples after oral administration by UHPLC-Q-Exactive-MS/MS in positive and negative ion modes are presented in Figure 2. It is found that the majority of alkaloids responded well in the positive mode, and the majority of phenolic compounds and saponins responded well in the negative mode. A total of 82 prototype compounds were identified or tentatively characterized, including 41 alkaloids, 35 phenolic compounds, and 6 saponins (shown in Table 2) by comparing the EICs among TSD, drugged, and blank samples and by comparison with reference standards, internal database, and the literature. Figure S3 (Supplementary Materials) displayed MS/MS spectra of major prototype compounds in the urine samples.

Figure 2.

Figure 2

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Total ion chromatograms (TIC) of TSD and the urine and plasma samples after oral administration by UHPLC-Q-Exactive-MS/MS ((A): Tongmai Sini decoction; (B): blank urine samples; (C): urine samples; (D): blank plasma samples; (E): plasma samples.).

3.2.1. Identification of Alkaloid Components

Metabolites for alkaloids obtained in this study could be classified into three subtypes, namely, diester-diterpenoid alkaloids (DDAs), monoester-diterpenoid alkaloids (MDAs), and amine-diterpenoid alkaloids (ADAs) [30]. We conducted an in-depth study of the chemical constituents of alkaloids of Aconitum carmichaeli in previous research [24,29], in which we carried out detailed mass fragmentation analysis of DDAs, MDAs, and ADAs, and a total of 42 DDAs and 120 diterpenoid alkaloids were identified, respectively.

In the MS2 spectra of DDAs, the most abundant ion yielded from the loss of a molecule of AcOH at the C8 site, which could be a diagnostic neutral loss for the differentiation of DDAs from MDAs and ADAs [29]. Thus, Compounds 29, 33, 35–38, and 40–41 were extracted by NLF for 60 Da in MS spectra for the urine sample, showing their molecular weight between 600 and 650 Da. Among them, Compounds 33, 36, and 37 were unambiguously identified as mesaconitine (MA), aconitine (AC), and hypaconitine (HA), respectively, by comparing their tR values and mass spectra data with those of reference compounds. Apart from the ion of [M+H-60(AcOH)]+ (m/z 572.2844, 586.3002, 556.2903), the ions of [M+H-60-32(MeOH)]+ (m/z 554.2727, 524.2634, 540.2551) and [M+H-60-32(MeOH)-28(carbonyl group)]+ (m/z 526.2797, 496.2750, 522.2487) of the three compounds, respectively, suggested the active elimination of MeOH occurred at C16 site and a neutral molecule of CO, which could also be regarded as characteristic fragments for identification of the DDAs. Compounds 29, 35, 38, and 40–41 were tentatively identified as 10-OH-mesaconitine, dehydrohypaconitine, secoyunaconitine, 3-deoxyaconitine, and chasmaconitine by comparing their acquired accurate mass data, characteristic fragment ions with those of compounds in our previous research [29].

In the MS spectra for the urine sample, by extraction of NLF for both 32 Da and 18 Da with limitation of molecular weight ranging from 500 to 620 Da, ten peaks were found. Neutral losses of 32, 18, and 122 Da, corresponding to the elimination of acetic acid, methanol, and benzoic acid, or combinations of these, could be considered diagnostic fragment ions for MDAs [31]. However, fragment peaks formed by the loss of the typical substituent group as BzOH (122 Da) were hardly detected for MDAs in this study. Thus, Compounds 22–28 and 30–32 were identified as MDAs accordingly by comparing the accurate mass data and diagnostic fragment ions with those of the compounds in our previous research [24].

A total of 21 prototype compounds were identified as ADAs, most of which possessed molecular weight between 390 and 500 Da and were eluted within the initial 16 min. The substitutions of C1 and C3 sites of ADAs were relatively active sites and could be easily cleaved, yielding major peaks [M+H-H2O]+ or [M+H-CH3OH]+ in MS2 spectra as the diagnostic ion accordingly. Fragmentation pathways of differently substituted ADAs included different diagnostic ions. Compounds 1, 4, 6, 7, 8, 13, 14, as ADAs with C1-OH substitution, firstly fragmented into [M+H-H2O]+ as diagnostic fragment ions and followed by losses of typical substituent groups (CH3OH and H2O) in their MS2 spectra. By comparing their accurate mass data with our chemical database and the literature [24,32], they were identified as karakolidine, senbusine A, senbusine B, karakoline, isotalatizidine, fuziline, and neoline, respectively. For Compounds 18 (talatizamine), the most prominent fragmentation ions were designated as 390.2696 ([M+H-CH3OH]+), suggesting its C1 site with -OCH3 substitutions. It also yielded 372.2517 ([M+H-CH3OH-H2O]+), 358.2379 ([M+H-CH3OH-CH3OH]+), and 340.2238 ([M+H-CH3OH-CH3OH-H2O]+), and its characteristic fragmentation patterns are shown in Figure 3.

Figure 3.

Figure 3

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Probable fragmentation pathways of talatizamine, isotalatizidine, and 16-O-demethyl talatizamine.

3.2.2. Identification of Phenolic Compounds

In addition to alkaloids from RALP, the main prototype compounds identified in vivo included flavonoids, isoflavonoids, coumarins, and saponins from RGP, and volatile oils from RZ, as shown in Table 2. The MS data of these compounds were compared with those of reference standards, internal databases, and the literature, while isomers could be initially identified by comparing their ClogP.

Flavonoids are important active components of RGP, among which four components, namely liquiritigenin, isoliquiritigenin, iquiritin, and isoliquiritin, have the highest content and are regarded as the indicator components of RGP, which were identified by comparing mass data with those of the reference standards. Compound 47, as reference compound liquiritin, formed the [M-H]−-based peak at m/z 417.11890 (C21H21O9−), for which furtherly formed fragmentation ion m/z 255.0662 [M-H-glu]− of the aglycone element in the MS/MS spectrum, accompanied by three characteristic fragments at m/z 135.0074 (C7H3O3−), 119.0488 (C8H7O−), and 91.0173 (C6H3O−), which can be used for the identification of the same type of licorice flavonoids.

Compound 50 formed the [M+H]+ molecular ion peak at m/z 431.13280 and further removed one molecule of glucose residues to form the aglycone at m/z 269.08121, which was identified as ononin, the main isoflavone of RGP. Its aglycone formed the same ion at m/z 269.08170 at the retention time of 26.58 min and was fragmented into the fragments of m/z 253.0497, 237.0554, and 213.0911, which is identified as formononetin, and the two prototypes are the most important isoflavonoid components in RGP.

The elemental compositions of other types of licorice flavonoid constituents determined by LC-MS were compared with the data of existing database compounds. Compounds 44, 53, 54, 67, and 68 were preliminarily identified as 5-hydroxyliquiritin, licochalcone B, dihydroxyflavone, licoflavone A, and isolicoflanonol. Similarly, other types of phenolic compounds, such as coumarins, were identified or preliminarily identified, including Compounds 60, 66, 64, 70, 73, and 74, which were identified as glycycoum-arim, licocoumarione, licopyranocoumarin, glycyrin isoglycyrol, and glycyrol, correspondingly. A few other phenolic components observed in vivo of TSD were derived from RZ, while compounds 56, 63, and 75 were tentatively identified as 6-gingerol, 6-shogaol, and 10-shogaol, respectively, with fragment ions m/z 177.09 and 137.06 as their characteristic fragment ions in PI mode, which is consistent with the literature [33].

3.2.3. Identification of Saponins

From the LC-MS/MS profiles, six saponin components were found as absorbed prototype components, all of which were derived from RGP. The saponins (Compounds 77, 78, 79, 81, and 82) were within the retention time of 14–21 min and had both mass spectral response in NI and in PI mode.

As a general rule for triterpenoid saponins in MS/MS spectra, the fragmentation reactions undergone by activated saponin ions almost occur within the glycan part of the saponin ions, and the sugar chains can be eliminated successively from end to inner and finally to obtain an aglycone ion [34]. Through glycosidic cleavages or cross-ring cleavages, the parent ion obtained a series of ions retaining the charge at the reducing terminus were termed Y and Z (glycosidic cleavages) and X (cross-ring cleavages), whereas those ions retaining the charge at the non-reducing terminus are termed B, C (glycoside cleavages), and A (cross-ring cleavages) [35].

The MS cleavage pathways of saponins from RGP, however, were incompletely abided by this rule. Take glycyrrhizic acid as an example; in MS spectra of PI mode, the ions of [M-H]− were obtained, accompanied by the fragment ions of m/z 647.37744 [M+H-β-D-glucuronopyronosyl (glcA)]+ and m/z 453.33554 [aglycottne (agl)+H-H2O]+, which were similarly for the other detected saponins and has not been reported up to present. More interestingly, in the MS/MS spectra of the detected saponins, the ions of [agl+H-H2O]+ rather than [agl+H]+ were observed as the base peaks, namely, m/z 453.34 (C30H45O3+), 469.33 (C30H45O4+), and 511.34 (C30H45O4+), corresponding to the aglycone of enoxolone, hydroxyenoxolone, and acetoxyenoxolone, respectively.

The produced ions obtained in NI mode were quite different from those in PI mode. The fragment ions of glycosidic cleavages or cross-ring cleavages, as well as the aglycone, were hardly detected in NI mode. The ions of m/z 351.05 (C12H15O12−), 193.03 (C6H9O7−), 175.02 (C6H7O6−), and 113.02 (C5H5O3−) were observed, corresponding to the successive loss of two glucuronopyranosyls. Thus, the identification information for aglycone s and sugar chains of licorice saponins can be obtained from PI and NI ion modes, respectively.

3.3. Identification of Metabolites

Prototypes and metabolites exist simultaneously in plasma and urine samples. Thirty-two major prototypes, including 11 alkaloids from RALP, as well as 21 phenolic and saponin compounds from RGP and RZ, were selected as MDF templates for metabolite screening. The 32 compounds contained a wide range of chemical structure types with relatively high content in TDS. A total of 40 alkaloids and 25 phenolic compounds were identified or tentatively characterized by comparing the mass data with those of prototype compounds and metabolic pathways reported by the literature [36,37,38,39,40].

After prototypes are absorbed into the body, some of them are excreted as prototypes, and some of them can be converted into other metabolites. DDAs were ester hydrolyzed to MDAs in rats; for example, MA, HA, and AC could be ester hydrolyzed to 14-Benzoylmesaconine (BM), 14-Benzoylhypaconine (BH), and 14-Benzoylaconitine (BA) during the process of metabolism in rat, while BM, BH, and BA themselves could be metabolized to mesaconine, hypaconine, and aconine [36]. Therefore, certain prototypes are themselves metabolites and metabolized from other prototypes in rats.

3.3.1. Identification of Alkaloid Metabolites

For diterpenoid alkaloids, most metabolites from hydroxylation, deoxylation, demethylation, deethylation, dehydrogenation, ester hydrolysis, and demethylation with deoxylation have been found in vivo. Metabolites of alkaloids were identified or tentatively identified based on their metabolic pathways, as reported in the literature [37].

The metabolites for major alkaloids were found in the urine and plasma samples, as displayed in Table 3. Most metabolites observed were mainly metabolized from karakolidine, songorine, karakoline, talatizamine, hypaconitine, mesaconitine, neoline, and fuziline. These results manifested that alkaloids mainly underwent oxidation, dehydrogenation, demethylation, N-deethylation, hydrolysis, demethylation with deoxidation, and dehydrogenation with demethylation, etc.

After oral administration of TSD, eight related metabolites of talatizamine (18) were identified in urine samples. Metabolite M18 and M19 showed [M+H]+ ion at m/z 408.27386 and 408.27393 (giving formula C23H37NO5), 14 Da (CH2) less than the parent compound. In the MS2 spectra, characteristic ions at m/z 376.25 ([M+H-CH3OH]+), 358.24 ([M+H-CH3OH-H2O]+), and 326.21 ([M+H-CH3OH-H2O-CH3OH]+), suggesting its C1 site with -OCH3 substitutions. Those characteristic ions were different from the characteristic ions of the prototype component, isotalatizidine (Compound 8), although they shared the same elemental composition (C23H37NO5). Isotalatizidine, with -OH substitutions at the C1 site, first yielded 390.2631 ([M+H-H2O]+) by loss of H2O at the C1 site. The fragmentation pathways of demethyl talatizamine and isotalatizidine can be compared in Figure 3. The methyl group of the C16 site or C18 site could easily be metabolized instead of that of the C1 site for M18 and M19. The Clog p values of 18-O-demethyl talatizamine and 16-O-demethyl talatizamine were −0.78 and −0.74, calculated by ChemDraw 14.0. Hence, M18 and M19 were tentatively determined as 18-O-demethyl talatizamine and 16-O-demethyl talatizamine.

M4 was confirmed as hydroxylated talatizamine for the [M+H]+ ion at m/z 438.28433 (formula C24H39NO6), 16 Da (O) more than talatizamine, and the fragment ions at m/z 406.2588, 388.2476, 374.230 and 356.2226 were all 16 Da less than those of talatizamine. Therefore, M4 was deduced as 10-Hydroxy Talatizamine, as for the C10 site in diterpenoid alkaloids prone to be hydroxylated by the literature [38].

Apart from these three metabolites, other metabolites (M13, M26, M36, M39, and M40) of talatizamine were produced through the reaction of dehydrogenation, demethylation, N-deethylation, and deoxidation. The proposed metabolic pathways of talatizamine are shown in Figure 4. The other metabolites of alkaloids were deduced accordingly by their acquired accurate mass data, retention time, and characteristic fragment ions, as well as the Clog p values, and biotransformation pathways information and composition change calculated by ChemDraw 14.0 and Compound Discoverer 3.2.

Figure 4.

Figure 4

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Proposed metabolic pathways of talatizamine in vivo.

3.3.2. Identification of Phenolic Compound Metabolites

Metabolites of phenolic compounds, mainly from hydroxylation, oxylation, methylation, dehydrogenation, hydration, methylation with oxylation, dehydrogenation with oxylation, and sulfation, have been observed in vivo. They were identified or tentatively identified by comparing their accurate mass data with prototypes and their metabolic pathways reported by the literature [39,40]. The metabolites for major phenolic compounds found in the urine and plasma samples were exhibited in Table 4.

Table 4.

Metabolites of phenolic compounds found in the urine and plasma samples.

ID [M+H]+
(m/z)		 Formula tR
(min)		 Error
(ppm)		 ms/ms Composition Change Identification Area
Urine Plasma
M41. 259.09701 C15H14O4 17.57 2.53 153.0548, 135.0441, 107.0496 +H2 Hydrogenated liquiritigenin ++++ ++
M42. 259.09689 C15H14O4 20.78 1.83 153.0549, 107.0497 +H2 Hydrogenated isoliquiritigenin ++++ +++
M43. 297.20602 C17H28O4 22.11 −0.23 177.0912, 163.0755, 137.0598, 131.0494 +H2 Hydrogenated 6-gingerol ++++ ++
M44. 269.08170[M-H]− C16H14O4 26.82 −0.03 254.0582, 153.0178, 135.0073, 91.0173 +H2 Hydrogenated formononetin ++++ ++
M45. 367.11792[M-H]− C21H20O6 29.54 −2.01 352.0936, 309.0400, 310.0434, 284.0325 +H2 Hydrogenated glycyrol +++ +
M46. 355.15311 C21H22O5 30.71 −2.57 337.1065, 299.0912, 189.0911, 177.0546, 151.0393 +H2 Hydrogenated gancaonin M ++
M47. 309.20578 C18H28O4 26.17 −0.70 163.0756, 137.0599, 131.0494 +CH2 Methyl 6-gingerol ++
M48. 309.20572 C18H28O4 26.80 −0.92 179.0704, 150.068, 137.0598, 83.0864 +CH2 Methyl 6-gingerol ++
M49. 285.07587 C16H12O5 18.49 0.51 270.0525, 253.0499, 299.0866, 225.0546, 123.0443 +O Hydroxy formononetin +++
M50. 273.07593 C15H12O5 19.06 0.72 255.066, 179.0339, 153.0184, 147.0442, 123.044, 119.0496 +O Hydroxy liquiritigenin/isoliquiritigenin +++ ++
M51. 369.13266 C21H20O6 29.18 −1.71 351.1222, 229.0860, 193.0497, 165.0548, 151.0389 +O Hydroxy gancaonin M +++ +
M52. 313.20038 C17H28O5 15.07 −0.96 203.1066, 163.0754, 137.0598 +H2O Hydrated 6-gingerol +++
M53. 273.07629[M-H]− C15H14O5 20.08 −1.36 255.0661, 167.0337, 109.0279 +H2O Hydrated liquiritigenin +++
M54. 273.07660[M-H]− C15H14O5 23.05 −0.68 255.0655, 151.0387, 135.0072, 109.0280 +H2O Hydrated isoliquiritigenin +++
M55. 293.17447 C17H24O4 16.57 −0.91 163.0756, 137.0598, 99.0811 -H2 Dehydrogenated 6-gingerol ++ +
M56. 255.06552 C15H10O4 18.49 0.41 227.0703, 199.0756, 137.0234 -H2 Dehydrogenated liquiritigenin +++ +
M57. 255.06550 C15H10O4 21.29 −0.16 227.0699, 199.0755, 137.0234 -H2 Dehydrogenated isoliquiritigenin ++++ +
M58. 307.15466[M-H]− C17H24O5 26.17 −1.41 275.1288, 171.1014, 153.0907, 121.0280, 111.0799 -H2+O Dehydrogenated-hydroxy 6-gingerol ++
M59. 277.18039 C17H24O3 28.85 2.07 189.0914, 177.09123, 145.05493, 137.0597 -H2-O Dehydrated 6-gingerol ++++ ++
M60. 285.07648[M-H]− C16H14O5 22.03 −0.35 270.0533, 153.0180, 149.0594, 135.0073, 134.0358, 91.0174 +CH2+O Methyl-hydroxy liquiritigenin +++ +
M61. 285.07645[M-H]− C16H14O5 26.88 −1.06 270.0535, 153.0180, 149.0595, 135.0073, 91.0174 +CH2+O Methyl-hydroxy isoliquiritigenin ++
M62. 299.09170 C17H14O5 26.99 0.69 284.0680, 243.1061, 166.0268 +CH2+O Methyl-hydroxy formononetin +++ +
M63. 335.02261[M-H]− C15H12O7S 18.62 −1.33 255.0661, 199.0064, 135.0073, 119.0487 +SO3 Liquiritigenin sulfate ++++ +++
M64. 335.02271[M-H]− C15H12O7S 23.32 −0.94 255.0663, 199.0055, 135.0073, 119.0486 +SO3 Isoliquiritigenin sulfate ++++ ++++
M65. 347.02263[M-H]− C16H12O7S 23.56 −1.41 267.0664, 252.0427 +SO3 Formononetin sulfate ++++ +++
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Note: +, response area below 106; ++, response area between 106 and 107; +++, response area between 107 and 108; ++++, response area above 108.

Metabolites of phenolic compounds observed in vivo were mainly derived from the metabolism of liquiritigenin, isoliquiritigenin, and 6-gingerol, which were the most important aglycones from RGP and RZ in TSD. According to the MS data and the metabolic pathways reported in the literature, eleven related metabolites were identified in urine and plasma samples after the absorption of liquiritigenin and isoliquiritigenin. M60 and M61 showed [M-H]− ion at m/z 285.0765 (C16H13O5+), 30 Da (CH2+O) heavier than parent compounds. In the MS/MS spectra, characteristic ions at m/z 270.05 [M-H-CH2]− indicated the methyl substitution, and m/z 135.01 or 119.05 were used for the characterization of liquiritigenin or isoliquiritigenin derivatives. According to the polarity of liquiritigenin and isoliquiritigenin, M60 and M61 were confirmed as methyl-hydroxy liquiritigenin and methyl-hydroxy isoliquiritigenin. In vivo, liquiritigenin and isoliquiritigenin could be metabolized to a series of metabolites (M41, M42, M50, M53, M54, M56, M57, M60, M61, M63, and M64) by reaction of hydrogenation, dehydrogenation, hydroxylation, oxylation, hydration, methylation, and sulfation. In vivo, seven metabolites (M43, M47, M48, M52, M55, M58, and M59) of 6-gingerol were produced through the reaction of hydrogenation, methylation, hydration, hydroxylation, and dehydrogenation, with characteristic ions at m/z 163.08 or 137.06.

3.4. Difference between Urine and Plasma Samples

Xenobiotics usually vary at trace levels and are interfered with endogenous components. Comparative analysis of metabolites between plasma and urine samples was carried out by the same LC-MS/MS method. Most prototype components and metabolites possessed suitable signal responses in urine samples, mainly as metabolites from phase I metabolism referring to dehydrogenation, demethylation, hydroxylation, deoxylation, and deethylation. A few phase II metabolites were detected in the urine, including sulfate conjugates of liquiritigenin, isoliquiritigenin, and formononetin.

Metabolites of TSD detected in the plasma samples are fewer than those in the urine samples. As for plasma samples, 10 prototype components (eight phenolic compounds and two alkaloids) were detected and tentatively identified, most of which were flavonoid aglycones. Fifteen metabolites derived from neoline, talatizamine, karakoline songorine, and fuziline, as well as sixteen metabolites derived from liquiritigenin, isoliquiritigenin, formononetin, gancaonin M, and 6-gingerol, respectively, were found in plasma samples, which indicated there were fewer metabolites identified in plasma samples. These results are reasonable due to their relatively lower concentration and higher matrix interference in plasma than in urine samples.

In the present study, ADAs and their metabolites from RALP were mainly detected in rats after oral administration of TSD. DDAs are the most toxic but chemically unstable alkaloids in RALP, and the alkaloidal composition changed during concocting and decocting, with DDAs changing to MDAs, and both transformed further to ADAs while the toxicity gradually diminished. ADAs, such as fuziline and neoline, showed activity against pentobarbital sodium-induced cardiomyocyte damage by obviously recovering beating rhythm and increasing the cell viability [41]. Mesaconine and hypaconine showed strong cardiac actions on the isolated perfused bullfrog heart. Moreover, mesaconine has protective effects, including improved inotropic effect and left ventricular diastolic function, on myocardial ischemia-reperfusion injury in rats [42].

Metabolites of licorice flavonoids and 6-gingerol were also mainly detected. Liquiritigenin offers cytoprotective effects against various cardiac injuries, and it could protect against myocardial ischemic injury by antioxidation, antiapoptosis, counteraction mitochondrial dysfunction, and damping intracellular Ca2+ [43]. 6-Gingerol was identified as a novel angiotensin II type 1 receptor antagonist for cardiovascular disease by high-throughput screening, which partially clarified the mechanism of ginger regulating blood pressure and strengthening the heart [44]. 6-gingerol administration protected I/R-induced cardiomyocyte apoptosis via the JNK/NF-κB pathway in the regulation of HMGB2 [45].

The results of the in vivo metabolite study of TSD in this study suggested that in the following pharmacokinetic, pharmacological, and efficacy studies, attention should be paid primarily to the ADAs alkaloids, licorice flavonoids, gingerol-6, and their metabolites

4. Conclusions

A total of 82 compounds, including 41 alkaloids, 35 phenolic compounds, and 6 saponins, were identified or tentatively characterized in TSD by UHPLC-Q-Exactive-MS/MS. Among them, 32 representative compounds with relatively high mass spectral peak areas and different core structures were selected as parent compound templates for further investigation of their metabolic profiles in rats. In total, 65 metabolites were screened out and tentatively characterized in rats’ urine and plasma based on their MS characteristic fragmentation patterns and information. The main metabolic reactions involved hydrogenation, demethylation, hydroxylation, hydration, methylation, deoxylation, and sulfation. This is a systematic study of in vivo metabolism of TSD, and it will be beneficial for further understanding of the pharmacological and pharmacokinetic study of TSD.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/metabo14060333/s1, Figure S1: Workflow for the identification of prototype components, Figure S2: Workflow for the identification of metabolites, Figure S3: MS/MS spectra of major prototype compounds in the urine samples. Table S1: Compound information of in-house database, Table S2: Parameters of data processing by Compound Discoverer software.

metabolites-14-00333-s001.zip (691.8KB, zip)

Author Contributions

Conceptualization, G.D.; Methodology, W.X.; Formal analysis, X.Z., Y.Z. and M.P.; Data curation, X.Z. and Y.Z.; Writing—original draft, X.Z. and M.P.; Writing—review & editing, X.Z., W.X. and G.D.; Supervision, G.D.; Project administration, G.D.; Funding acquisition, G.D. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

All animal experiments were performed at the SPF animal laboratory [experimental animals license number SYXK (Guangdong, China) 2008–0094]. The Institutional Animal Ethics Committee of Guangdong Provincial Hospital of Chinese Medicine approved all experimental protocols (approval code: No. 2023131, approval date: 21 December 2023).

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to confidentiality.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research is supported by special funds for international cooperation from the Guangdong Provincial Hospital of Chinese Medicine (YN2024RD01) and the Scientific Research Project of Guangdong Provincial Bureau of Traditional Chinese Medicine (20231144).

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

metabolites-14-00333-s001.zip (691.8KB, zip)

Data Availability Statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to confidentiality.

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