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Rapid discovery of chemical constituents and absorbed components in rat serum after oral administration of Fuzi-Lizhong pill based on high-throughput HPLC-Q-TOF/MS analysis

Contributed equally
Chinese Medicine201914:6

https://doi.org/10.1186/s13020-019-0227-z

  • Received: 7 November 2018
  • Accepted: 1 February 2019
  • Published:

Abstract

Background

Fuzi-Lizhong pill (FZLZP), which was first recorded in the Classic–“Taiping Huimin Heji Ju Fang” of the Song Dynasty, has been widely used to treat gastrointestinal disease in clinic for thousands of years in China. However, an in-depth understanding of the chemical constituents of FZLZP and its potential bioactive constituents is lacking.

Methods

A simple, sensitive and selective method of high-performance liquid chromatography coupled with quadrupole-time-of-flight high-definition mass spectrometry (HPLC-Q-TOF/MS) and automated data analysis (Agilent MassHunter Qualitative Analysis B.06.00 Workstation Software) was developed to simultaneously identify the chemical constituents of FZLZP and the absorbed prototypes as well as the metabolites in rat serum after the oral administration of FZLZP.

Results

Sixty-seven compounds, including alkaloids, flavonoids, triterpenes, gingerols, phenylpropanoids and volatile oil, in the FZLZP extract were tentatively characterized by comparing the retention time and mass spectrometry data and retrieving the reference literatures. Additionally, 23 prototype compounds and 3 metabolites in the rat serum samples were identified after oral administration of FZLZP, which might be the potential active components in vivo. In addition, the absorption of alkaloids decreased when Aconitum carmichaeli Debx. was in the form of combined application as a prescription compared to when it was in the form of herb powder.

Conclusions

Herein, the chemical constituent in vitro and the absorbed compounds in the serum of a traditional Chinese formula, Fuzi-Lizhong pill, were fully characterized using a rapid and comprehensive analysis approach based on high-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry coupled to MassHunter Qualitative Analysis software data processing approach. The results provide helpful chemical information on FZLZP for further pharmacology and active mechanism research. In view of the bioactive constitutes that basically were derived from these absorbed compounds in vivo, this work could provide a useful strategy to explore the bioactive substances of traditional Chinese medicine.

Keywords

  • Fuzi-Lizhong pill
  • Chemical constituents
  • Bioactive compounds
  • Metabolites
  • Traditional Chinese herbal medicine
  • High-performance liquid chromatography–electrospray ionization/quadrupole-time-of-flight high definition mass spectrometry

Background

Fuzi-Lizhong pill (FZLZP) is a popular Traditional Chinese medicine pill that was originally described in the Classic “Taiping Huimin Heji Ju F/ang” of the Song Dynasty (year 1102 by the Western calendar). It is composed of five herbal medicines, including Aconitum carmichaeli Debx. (Fuzi), Codonopsis pilosula (Franch.) Nannf. (Dangshen), Atractylodes macrocephala Koidz. (Baizhu), Glycyrrhiza uralensis Fisch. (Gancao) and Zingiber officinale Rosc. (Ganjiang). FZLZP is famous for warming the middle-jiao and tonifying the spleen and is used to treat spleen yang deficiency syndrome including enteritis, chronic diarrhoea, irritable bowel syndrome, abdominal pain, vomiting and spasm, peripheral chill, etc. [17]. Modern pharmacological research shows that FZLZP possesses a variety of pharmacological activities, including an increase in adaptive thermogenesis, pain relief, anti-inflammation, and spasmolytic benefits [815]. Although pharmacological activities of FZLZP have been extensively studied, very little is known about its systematic chemical constituents, and the bioactive compounds that account for its therapeutic effects remain unclear.

In our previous research, we focused on the dissolution behaviour of FZLZP in vitro and the results showed that some constituents in Aconitum carmichaeli Debx. and Glycyrrhiza uralensis Fisch., such as benzoylaconine, liquiritin and glycyrrhizic acid, were dissolved well in vitro [1618]. While FZLZP has the so-called active ingredients, there are no empirical data to prove their effectiveness as bioactive compounds. According to the theory of serum pharmacochemistry, while there are multiple components in herbs, only compounds that are absorbed into the blood have the possibility of showing pharmacological bioactivities [1924]. Therefore, simultaneous identification of systematic chemical constituents in vitro and potential active components in the blood of FZLZP are indispensable.

It was reported that the main components in Aconitum carmichaeli Debx. are monoester diterpenoid alkaloids (MDAs) and diester diterpenoid alkaloids (DDAs), which are toxicity and efficacy compounds and should be highly concerned [21]. Due to the toxicity, Fuzi is usually used in combination with other herbs as a prescription. Some researcher considered the combination to cause the reduction of the absorption of toxic compounds [21, 25]. As a typical combination, however, there are no detailed studies of this mechanism and the compound variations of FZLZP. The strategy of serum thermochemistry can provide us the accurate qualitative and the preliminary quantitative information for exploring the quantitative change of alkaloids and toxicity reducing mechanism.

Currently, LC–MS is widely applied for the analysis of herbal constituents in vitro and in vivo because of its superior sensitivity, selectivity and ability to conclusively identify the compounds [2629]. In this study, an approach of high-performance liquid chromatography (HPLC) quadrupole time-of-flight mass spectrometry (QTOF-MS) based on serum pharmacochemistry was developed to identify the phytochemical constituents of FZLZP and multiple absorbed components in rat serum.

Methods

The Minimum Standards of Reporting Checklist contains details of the experimental design, and statistics, and resources used in this study (Additional file 1).

Chemicals and materials

Nine reference compounds were obtained from Sichuan Victor Biological Technology Co. Ltd. (Chengdu China). HPLC grade Ethanol, formic acid and methanol were obtained from Fisher (ThermoFisher Scientific Inc, Waltham, MA, USA). Deionised water (18 MΩ) was prepared by distilled water through a Milli-Q system (Millipore, Milford, MA, USA). Fuzi (No. 1703003), Dangshen (No. 1705003), Baizhu (No. 1704088), Ganjiang (No. 1703060) and Gancao (No. 1703034) were purchased from Sichuan Neautus Traditional Chinese Medicine Co., Ltd. (Chengdu China) and were authenticated by Prof. Jin Pei, Department of Pharmacognosy of Chengdu University of Chinese Medicine.

Preparation of FZLZP

Fuzi, Ganjiang, Dangshen, Baizhu and Gancao were ground into fine powers and weighed according to the instructions recorded in Chinese Pharmacopoeia (2015 edition) and mixed well. Honey was heated at 116–118 °C until bright yellow uniform bubbles appeared on the surface and the honey became sticky. Mixed power and thermal refined honey were mixed at a ratio of 1:0.8 and were made into FZLZP (there is 0.153 kg crude aconite for every 1 kg FZLZP).

Preparation of FZLZP extract samples for LC/MS analysis

FZLZP (1.5 g) was weighed and reflux-extracted with 50 mL 70% ethanol for 1 h. Then, the filtered supernatant sample was rotary evaporated at 40 °C to a concentration of 15 mL, and was centrifuged at 5000 revolutions/min (rpm) for 5 min. The solution was filtered through a 0.22-μm membrane for further analysis.

Animal handling and serum sample preparation

Eighteen male Sprague–Dawley rats (200 ± 20 g) were obtained from the Sichuan Dashuo Biotechnology Co., Ltd. and were randomly divided into three groups of 6 rats each (group A, FZLZP group for dosed rat serum; group B, Fuzi powder (FZP) group for dosed rat serum; group C, control group for blank rat serum). The animal facilities and protocols conformed to the Care and Use of Laboratory Animals published by the National Institutes of Health. The experiment was approved by the ethical committee of Chengdu University of TCM (No.20161105). The rats were housed in an animal room with a controlled environment (20–25 °C, 65–69% relative humidity, 12 h dark–light cycle), and were given water and fed normal food for 1 week before the experiment. All animals were fasted overnight before the experiments and had free access to water.

The FZLZP was dissolved in 0.5% CMC-Na and were grinded to prepare the FZLZP suspension (150 mg crude drug/mL). Fuzi powder was dissolved in 0.5% CMC-Na to prepare the FZPsuspension (23 mg crude drug/mL, the concentration of FuZi was calculated by the ratio in FZLZP). Group A was intragastric administration 1.5 g/kg body weight of FZLZP suspension for 3 days. Group B was intragastric administration 0.23 g/kg body weight of FZP suspension for 3 days. Group C was intragastric administration with an equivalent volume of 0.5% CMC-Na. Blood samples were collected from the abdominal aorta 45 min after oral administration on the 3rd day and were placed at room temperature for 1 h until solidification. Then, samples were centrifuged at 3000 rpm for 10 min at 4 °C. All samples were stored at − 80 °C until analysis. Three times methanol was added to the 2 mL serum samples, vortexed and then, centrifuged at 12,000 rpm for 20 min. The supernatant was dried with nitrogen gas. The residue was redissolved in 50 μL methanol, vortexed and then, centrifuged at 12,000 rpm for 20 min, and the filtrate was used as the LC/MS sample. 10 µL aliquot was injected for HPLC/MS analysis.

HPLC-QTOF-MS analysis condition

Chromatographic analysis was performed in an Agilent 1290 HPLC system controlled with MassHunter Workstation Software (V B.05.00, Agilent Technologies Inc, Santa Clara, CA, USA). Samples were separated on an Agilent HC-C18 column (4.6 × 250 mm, 5.0 μm, Agilent Technologies Inc.) held at 35 °C and the flow rate was 1.0 mL/min with the injection volume of 10 μL. The mobile phase consisted of 0.1% formic acid–water (v/v, A) and methanol (B). The optimal gradient elution programme was as follows: 0–15 min, 95–70% A; 15–30 min, 70–48% A; 30–45 min, 48–25% A; 45–48 min, 25–15% A; 48–55 min, 15–2% A; and 55–65 min, 2–2% A.

Mass spectrometry conditions

Mass spectrometry was performed using an Agilent 6540 QTOF–MS (Agilent Corp., USA) equipped with a Dual AJS electrospray ionization (ESI) source, and the following operating parameters were used: positive mode, drying gas (nitrogen, N2); flow rate, 8.0 L/min; gas temperature, 325 °C; nebulizer, 40 psig; sheath gas temperature, 350 °C; sheath gas flow, 11 L/min; capillary voltage, 4000 V; skimmer, 65 V; OCT 1 RF Vpp, 750 V; fragmentor, 110 V. The sample collision energy was set at 10, 20, 30 and 40 V. All the operations, acquisition, and analyses of data were controlled by Agilent LCMS-QTOF Mass Hunter Acquisition Software Ver. B.06.00 (Agilent Technologies Inc.) and operated under Mass Hunter Workstation Software Version B.06.00 (Agilent Technologies Inc.).

Establishment of FZLZP database

By searching databases, such as PubMed of the US National Library Medicine and the National Institutes of Health, SciFinder Scholar of American Chemical Society and the Chinese National Knowledge Infrastructure (CNKI) of Tsinghua University, all components reported in the literature on Aconitum carmichaeli Debx., Codonopsis pilosula (Franch.) Nannf., Atractylodes macrocephala Koidz., Glycyrrhiza uralensis Fisch. and Zingiber officinale Rosc. were summarized in an Agilent PCDL software Ver. B.06.00 (Agilent Technologies Inc.) to establish a database, which includes the name, molecular formula, chemical structure and literatures of each published known compound.

Results

Characterization of chemical constituents from FZLZP

Using the optimal conditions described above, all information on the MS data that was obtained from the robust HPLC-TOF-MS analysis, indicated the retention time and precise molecular mass and provided the MS/MS data. The protonated molecular weights of all target compounds were calculated within an error of 5 ppm. The base peak chromatogram (BPC) of the FZLZP extract sample in positive and negative ion modes are shown in Fig. 1A, and the data were processed by the Agilent MassHunter Qualitative Analysis B.06.00 Workstation Software with the “find compounds by molecular formula” tool. A total of 73 peaks were obtained, and 67 compounds were identified or tentatively characterized by comparing the tR values and the MS fragment characteristics of the compounds.
Fig. 1
Fig. 1

The HPLC-ESI/QTOF/MS BPC chromatograms (A FZLZP extract samples: a in positive mode, b in negative mode; B Serum samples: c controlled serum in positive mode, d dosed FZLZP serum in positive mode, e dosed FZP serum in positive mode.)

The reference standards are summarized in Table 1 and their fragmentation mechanism are proposed in Fig. 2. The compounds in FZLZP which are identified by the reference standards are summarized and marked in Table 2. For example, reference standards (RS) 1 liquiritigenin in Table 1 were detected in the positive ion mode at the Rt in 24.843 min with the m/z of 257.0809 (C15H13O4). Its MS/MS data were shown as m/z of 239.0698[M + H–H2O]+, 137.0234 [C7H4O3 + H]+, 121.0293[C8H8O + H]+ and 120.0721 [C7H4O3 + H–OH]+. And the compound 29 in Table 2 were detected in the positive ion mode at the Rt in 24.785 min with the m/z of 257.0819 (C15H13O4), 239.0707[M + H–H2O]+ and 137.0235 [C7H4O3 + H]+. Then compound 29 were characterized as liquiritigenin. Similar to the identification process above, among 67 compounds, 9 compounds were identified as benzoylaconine, benzoylmesaconine, benzoylhypaconine, mesaconitine, liquiritigenin, isoliquiritigenin, glycyrrhizic acid, glycyrrhetinic acid and atractylenolide II. The MS data of the (+) ESI–MS spectra are shown in Table 2.
Table 1

Retention time, m/z values of ions of reference standards

Peak no.

Rt (min)

Systematic name

Molecular formula

[M + H]+

[M + Na]+

Fragmentations (m/z)

Measured mass (m/z)

Error (ppm)

Measured mass (m/z)

Error (ppm)

1

24.843

Liquiritigenin

C15H12O4

257.0809

0.3890

  

257.0809[M + H]+, 239.0698[M + H–H2O]+, 137.0234 [C7H4O3 + H]+, 121.0293 [C8H8O + H]+, 120.0721 [C7H4O3 + H–OH]+

2

27.507

Benzoylmesaconine

C31H43NO10

590.2952

− 1.3553

590.2952[M + H]+, 572.2832[M + H–H2O]+, 558.2683[M + H-CH3OH]+, 540.2580[M + H–CH3OH–H2O]+

3

28.228

Benzoylaconine

C32H45NO10

604.3130

2.3167

604.3130[M + H]+, 586.2995[M + H–H2O]+, 572.2852[M + H–CH3OH]+

554.2735[M + H–2H2O]+, 540.2577[M + H–CH3OH]+, 522.2475[M + H–2CH3OH–H2O]+

4

29.152

Benzoylhypaconine

C31H43NO9

574.3003

− 1.3930

574.3003[M + H] + , 542.2741[M + H–CH3OH]+, 524.2615[M + H–CH3OH–H2O]+, 510.2477[M + H–2CH3OH]+

5

31.663

Mesaconitine

C33H45NO11

632.3064

− 0.1582

632.3064[M + H]+, 600.2787[M + H–CH3OH]+, 572.2853[M + H–AcOH]+, 540.2594[M + H–AcOH–CH3OH]+, 512.2637[M + H–AcOH–CH3OH–CO]+,

6

39.648

Isoliquiritigenin

C15H12O4

257.0809

0.3890

  

257.0809[M + H]+, 239.0692[M + H–H2O]+, 137.0235[C7H4O3 + H]+, 121.0287 [C8H8O + H]+, 120.0514 [C7H4O3 + H–OH]+

7

48.854

Atractylenolide II

C15H20O2

233.1538

0.8578

  

233.1538[M + Na]+, 215.1440[M + Na–H2O]+, 187.1484[M + Na–CH2O2]+, 159.1165[M + Na–CH2O2–C2H4]+, 145.101 [M + Na–CH2O2–C3H6]+

131.0856[M + Na–CH2O2–C4H8]+, 105.0702[M + Na–CH2O2–C4H8–C2H2]+,

8

49.134

Glycyrrhizic acid

C42H62O16

  

845.3947

2.0109

845.3947[M + Na]+, 669.3614[M + Na–(GluA–H2O)]+

9

55.125

Glycyrrhetinic acid

C30H46O4

471.3458

− 2.3337

  

471.3458[M + H]+, 453.3349[M + H–H2O]+, 435.3244[M + H-2H2O]+

Fig. 2
Fig. 2

The mass fragment and fragmentation pathway of a Liquiritigenin, b Benzoylmesaconine, c Benzoylaconine, d Benzoylhypaconine, e mesaconitine, f Isoliquiritigenin, g Atractylenolide II, h Glycyrrhizic acid, i Glycyrrhetinic acid

Table 2

Identification information of constituents in vitro of FZLZP by HPLC-ESI/QTOF/MS

Peak no.

Rt (min)

Systematic name

Molecular formula

Molecular weight

[M + H]+

[M + Na]+

Fragmentations (m/z)

Source

Measured mass (m/z)

Error (ppm)

Measured mass (m/z)

Error (ppm)

1

5.091

l-Pyroglutamic acid

C5H7NO3

129.0426

130.0505

4.6136

  

130.0505[M + H]+, 112.0123[M + H–H2O]+, 84.0449[M + H–HCOOH]+

Dangshen

2

8.051

Codonopsine

C14H21NO4

267.1471

268.1543

0

  

268.1543[M + H]+, 250.1451[M + H–H2O]+,

205.0863[M + H–2CH3OH]+

Dangshen

3

9.229

5-hydroxymethyfurfural

C6H6O3

126.0317

127.0394

3.1486

  

127.0394[M + H]+, 109.0291[M + H–H2O]+

Dangshen

4

9.398

Karakolidine

C22H35NO5

393.2515

394.2590

0.5072

  

394.2590[M + H]+, 376.2489[M + H–H2O]+, 358.2371[M + H–2H2O]+

Fuzi

5

10.142

Phenylalanine

C9H11NO2

165.0790

166.0872

5.4188

  

166.0872[M + H]+, 120.0817[M + H–HCOOH]+

Dangshen

6

11.288

Senbusine A

C23H37NO6

423.2621

424.2696

0.4713

  

424.2696[M + H]+, 406.2579 [M + H–H2O]+

Fuzi

7

11.407

9-OH-senbusine A

C23H37NO7

439.2570

440.2635

− 1.8170

  

440.2635[M + H]+, 422.2531[M + H–H2O]+, 408.2318[M + H–CH3OH]+

Fuzi

8

12.042

16-β-hydroxycardiopetaline

C21H33NO4

363.2410

364.2480

− 0.5490

  

364.2480[M + H]+, 346.2372[M + H–H2O]+, 328.2273[M + H–2H2O]+

Fuzi

9

12.389

Mesaconine

C24H39NO9

485.2625

486.2697

− 0.2056

  

486.2697 M + H]+, 468.2573[M + H–H2O]+, 436.2323[M + H–H2O–CH3OH]

Fuzi

10

12.578

Songorine

C22H31NO3

357.2304

358.2382

1.3957

  

358.2382[M + H]+, 340.2267[M + H–H2O]+

Fuzi

11

12.908

Karakoline

C22H35NO4

377.2566

378.2639

0

  

378.2639[M + H]+, 360.2533[M + H–H2O]+

Fuzi

12

13.081

Isotalatizidine

C23H37NO5

407.2672

408.2743

− 0.2449

  

408.2743[M + H]+, 390.2630[M + H–H2O]+, 372.2517[M + H–2H2O]+, 358.2374[M + H–H2O–CH3OH]+

Fuzi

13

13.109

Senbusine B

C23H37NO6

423.2621

424.2707

3.0640

  

424.2707[M + H]+, 406.2584 [M + H–H2O]+

Fuzi

14

13.937

14-Acetylkarakoline

C24H37NO5

419.2672

420.2750

1.4276

  

420.2750[M + H]+, 402.1695[M + H–H2O]+,

356.1122[M + H–H2O–2CH3OH]+,

Fuzi

15

14.091

Aconine

C25H41NO9

499.2781

500.2850

− 0.7995

  

500.2850[M + H]+, 482.2741[M + H–H2O]+, 468.2564[M + H–CH3OH]+, 450.2478[M + H–H2O–CH3OH]+, 436.2309[M + H–2CH3OH]+, 418.2209[M + H–H2O–2CH3OH]+

Fuzi

16

14.380

Hetisine

C20H27NO3

329.1991

330.2064

0

  

330.2064[M + H]+, 312.1951[M + H–H2O]+

Fuzi

17

15.319

Hypaconine

C24H39NO8

469.2676

470.2744

− 0.8506

  

470.2744[M + H]+, 453.2301[M + H–OH]+, 438.2474[M + H–CH3OH]+, 406.2212[M + H–2CH3OH]+, 374.1941[M + H–3CH3OH]+

Fuzi

18

15.810

Fuzitine

C20H23NO4

341.1627

342.1697

− 0.8767

  

342.1697[M + H]+, 324.1026[M + H–H2O]+

Fuzi

19

16.070

Fuziline

C24H39NO7

453.2727

454.2800

0.2201

  

454.2800[M + H]+, 436.2677[M + H–H2O]+, 418.2583[M + H–2H2O]+, 404.2443[M + H–H2O–CH3OH]+, 386.2295[M + H–2H2O–CH3OH]+, 354.2069[M + H–2H2O–2CH3OH]+

Fuzi

20

16.248

Tau-cadinol

C15H26O

222.1984

245.1852

− 9.7884

  

245.1852[M + H]+, 213.0195[M + H–CH3OH]+, 199.1252[M + H–CH3OH–CH3]+, 184.9885[M + H–CH3OH–2CH3]+,

169.0055[M + H–CH3OH–3CH3]+,

Ganjiang

21

16.573

Neoline

C24H39NO6

437.2777

438.2848

− 0.4563

  

438.2848 M + H]+, 420.2756[M + H–H2O]+, 388.2478[M + H–H2O–CH3OH]+, 370.2365[M + H–2H2O–CH3OH]+, 356.2213[M + H–H2O–2CH3OH]+

Fuzi

22

16.743

Talatisamine

C24H39NO5

421.2828

422.2899

0.4736

  

422.2899[M + H]+, 390.2621[M + H–CH3OH]+, 358.2349[M + H–2CH3OH]+

Fuzi

23

18.651

Chasmanine

C25H41NO6

451.2934

452.3008

02210

  

452.3008[M + H]+, 420.2737[M + H–CH3OH]+

Fuzi

24

19.739

Geranial

C10H16O

152.1201

153.1275

0.6530

  

153.1275[M + H]+, 135.1162[M + H–H2O]+, 125.0940[M + H–CO]+

Ganjiang

25

20.390

14-Acetyltalatizamine

C26H41NO6

463.2934

464.3014

1.5076

  

464.3014[M + H]+, 432.2753[M + H–CH3OH]+, 414.2645[M + H–CH3OH–H2O]+, 400.2486[M + H–2CH3OH]+, 372.2522[M + H–CH3OH–AcOH]+

Fuzi

26

21.828

7-hydroxycoumarin

C9H6O3

162.0317

163.0395

3.0667

  

163.0395[M + H]+, 145.0627[M + H–H2O]+

Baizhu

27

23.891

Schaftoside

C26H28O14

564.1479

565.1542

− 1.7694

  

565.1542[M + H]+, 547.1434[M + H–H2O]+, 529.1303[M + H–2H2O]+, 511.1220[M + H–3H2O]+

Gancao

28

24.041

Scopoletin

C10H8O4

192.0423

193.0500

2.5900

  

193.0500[M + H]+, 161.0603[M + H–CH3OH]+

Baizhu

29#

24.785

Liquiritigenin

C15H12O4

256.0736

257.0819

4.2788

  

257.0819[M + H]+, 239.0707[M + H–H2O]+, 137.0235[C7H4O3 + H]+, 121.0280[C8H8O + H]+, 120.0525 [C7H4O3 + H–OH]+

Gancao

30#

27.065

Benzoylmesaconine

C31H43NO10

589.2887

590.2959

− 0.1694

590.2959[M + H]+, 572.2826[M + H–H2O]+, 558.2663[M + H–CH3OH]+ 540.2573[M + H–CH3OH–H2O]+

Fuzi

31

27.325

Isoviolanthin

C27H30O14

578.1636

579.1700

− 1.3812

  

579.1700[M + H]+, 561.1588[M + H–H2O]+, 543.1485[M + H–2H2O]+, 525.1382[M + H–3H2O]+

Gancao

32#

27.614

Benzoylaconine

C32H45NO10

603.3043

604.3114

− 0.3309

  

604.3114[M + H]+, 587.2801[M + H–OH]+, 554.2711[M + H–2CH3OH]+

Fuzi

33#

28.595

Benzoylhypaconine

C31H43NO9

573.2938

574.3011

0

  

574.3011[M + H]+, 542.2745[M + H–CH3OH]+,,510.2457[M + H–2CH3OH]+

Fuzi

34

28.748

Lobetyolinin

C26H38O13

558.2312

  

581.2203

− 0.3441

581.2203[M + Na]+, 419.1709[M + Na–C6H10O5]+

Dangshen

35

31.019

Liquiritin apioside or Isoliquiritin apioside

C26H30O13

550.1686

551.1751

− 1.4514

  

551.1751[M + H]+, 419.1333[M + H–(Apiose–H2O)]+, 257.0830[M + H–(Apiose–H2O)–(Glc–H2O)]+

Gancao

36#

31.163

Mesaconitine

C33H45NO11

631.2993

632.3067

0.3163

632.3067[M + H]+, 614.1110[M + H–H2O]+,

600.2748[M + H–CH3OH]+, 572.2834[M + H–AcOH]+

Fuzi

37

31.423

7-methoxy-liquiritin

C22H22O9

430.1264

431.1332

− 1.1597

  

431.1332[M + H]+, 269.0811[M + H–(Glc–H2O)]+

Gancao

38

31.646

14-Benzoylneoline

C31H43NO7

541.3040

542.3135

4.2411

  

542.3135[M + H]+, 524.3010[M + H–H2O]+,

510.2731[M + H–CH3OH]+, 492.2733[M + H–H2O–CH3OH]+

Fuzi

39

31.659

Dehydrated benzoylhypaconine

C31H41NO8

555.2832

556.2906

0.1798

  

556.2906[M + H]+, 524.2647[M + H–CH3OH]+, 492.2381[M + H–2CH3OH]+

Fuzi

40

31.683

Liquiritin or Isoliquiritin

C21H22O9

418.1264

419.1335

0.4771

  

419.1335[M + H]+, 257.0811[M + H–(Glc–H2O)]+

Gancao

41

31.921

Aconifine

C34H47NO12

661.3098

662.3172

0.1509

  

662.3172[M + H]+, 644.3095[M + H–H2O]+,

626.1346 [M + H–2H2O]+

Fuzi

42

32.100

Hypaconitine

C33H45NO10

615.3043

616.3116

0

  

616.3116[M + H]+, 584.2843[M + H–CH3OH]+

556.2899[M + H–C2H4O2]+, 524.2533[M + H–C2H4O2–CH3OH]+, 496.2678[M + H–C2H4O2–CH3OH–CO]+

Fuzi

43

32.245

Formononetin

C16H12O4

268.0736

269.0814

2.2298

  

269.0814[M + H]+, 254.0580[M + H–CH3]+, 237.0536[M + H–CH3OH]+, 225.0554[M + H–CH3–CO]+, 213.0908[M + H–C2O2]+, 181.0666[M + H–C2O2–CH3OH]+

Gancao

44

32.528

Aconitine

C34H47NO11

645.3149

646.3216

− 0.9283

  

646.3216[M + H]+, 628.3140[M + H–H2O]+,

596.2849[M + H–H2O–CH3OH]+

Fuzi

45

33.241

Deoxyaconitine

C34H47NO10

629.3200

630.3273

0

  

630.3273[M + H]+, 598.3070[M + H–CH3OH]+

Fuzi

46

36.853

Echinatin

C16H14O4

270.0892

271.0963

− 0.7377

  

271.0963[M + H]+, 253.0850[M + H–H2O]+

Gancao

47

38.085

Benzoic acid

C7H6O2

122.0368

123.0447

4.8763

  

123.0447[M + H]+, 77.0379[M + H–HCOOH]+

Baizhu

48#

39.763

Isoliquiritigenin

C15H12O4

256.0736

257.0814

2.334

  

257.0814[M + H]+, 239.0704[M + H–H2O]+, 137.0235[C7H4O3 + H]+, 121.0277[C8H8O + H]+, 120.0527 [C7H4O3 + H–OH]+

Gancao

49

40.720

Glycycoumarin

C21H20O6

368.1260

369.1345

3.2508

  

369.1345[M + H]+, 333.2235[M + H–2H2O]+,

313.1057 [M + H–C4H8]+,

Gancao

50

41.513

6-gingerdione

C17H24O4

292.1675

293.1736

− 2.7520

  

293.1736[M + H]+, 275.1650[M + H–H2O]+

257.1517[M + H–2H2O]+

Ganjiang

51

42.593

Kumatakenin

C17H14O6

314.0790

315.0859

− 1.2694

  

315.0859[M + H]+, 298.2146[M + H–OH]+,

279.0782[M + H–2H2O]+

Ganjiang

52

43.486

6-gingerol

C17H26O4

294.1831

  

317.1737

4.4140

317.1771[M + Na]+, 299.2546[M + Na-H2O]+

Ganjiang

53

43.507

Gingerenone-A

C21H24O5

356.1624

357.1710

3.6397

  

357.1710[M + H]+, 339.2718[M + H–H2O]+, 321.2612[M + H–2H2O]+

Ganjiang

54

43.544

6-shogaol

C17H24O3

276.1725

277.1795

1.0823

  

277.1795[M + H]+, 259.1694[M + Na–H2O]+,

Ganjiang

55

45.779

lupiwighteone

C20H18O5

338.1154

339.1239

3.5385

  

339.1239[M + H]+, 321.2818[M + H–H2O]+

Gancao

56

46.339

Atractylenolide III

C15H20O3

248.1412

245.1485

0

  

249.1485[M + H]+, 231.1389[M + H–H2O]+, 175.0751[M + H–H2O–2CO]+, 163.0756[M + H–H2O–C5H8]+

Baizhu

57

48.364

Gancaonin L

C20H18O6

354.1103

355.1189

3.6607

  

355.1189[M + H]+, 337.2536[M + H–H2O]+

Gancao

58

48.398

Licoricesaponin G2

C42H62O17

838.3987

839.4076

1.9061

  

839.4076[M + H]+, 663.3722[M + H–(GluA–H2O)]+, 469.3308[M + H–2 (GluA–H2O)–H2O]+

Gancao

59#

48.887

Atractylenolide II

C15H20O2

232.1463

233.1541

2.1445

  

233.1541[M + Na]+, 187.1485[M + Na–CH2O2]+,

159.0806[M + Na–CH2O2–C2H4]+, 145.1013 [M + Na–CH2O2–C3H6]+, 131.0857[M + Na–CH2O2–C4H8]+, 105.0703[M + Na–CH2O2–C4H8–C2H2]+

Baizhu

60#

49.296

Glycyrrhizic acid

C42H62O16

822.4038

823.4130

2.3075

  

823.4130 [M + H]+, 647.3793[M + H–(GluA–H2O)]+

Gancao

61

49.667

Farnesal

C15H24O

220.1827

221.1907

3.1647

  

221.1907 M + H]+, 192.9740[M + H–CO]+

Ganjiang

62#

49.841

Glycyrrhetinic acid

C30H46O4

470.3396

471.3488

4.031

  

471.3488[M + H]+, 453.3354[M + H–H2O]+, 435.3224[M + H–2H2O]+, 425.3378[M + H–HCOOH]+

Gancao

63

50.671

Licorice saponin B2

C42H64O15

808.4245

  

831.4151

1.6838

831.4151 [M + Na]+, 655.3825[M + Na–(GluA–H2O)]+,

479.3547[M + Na–2 (GluA–H2O)]+

Gancao

64

51.232

Licoricone

C22H22O6

382.1416

383.1502

3.3929

  

383.1502[M + H]+, 355.1587[M + H–C2H4]+

Gancao

65

51.390

Atractylenolide I

C15H18O2

230.1307

231.1383

1.2979

  

231.1383[M + H]+, 185.1326[M + H–HCOOH]+,

157.1012[M + H–HCOOH–C2H4]+, 105.0701 [M + H–HCOOH–2C2H4–2C]+

Baizhu

66

52.950

Neoglycyrol

C21H18O6

366.1103

367.1165

− 0.5447

  

367.1165[M + H]+, 349.2239[M + H–H2O]+,

335.2389[M + H–CH3OH]+,

317.2283[M + H–2H2O–CH3OH]+

Gancao

67

54.310

Licorice-saponin J2

C42H64O16

824.4194

825.4286

2.3018

  

825.4286[M + H]+, 649.3906 [M + H–(GluA–H2O)]+, 455.3537[M + H-2 (GluA–H2O)–H2O]+, 437.3435 [M + H-2 (GluA–H2O)–2H2O]+

Gancao

#Indicates compounds identified by comparing with the reference standards

The remaining 58 compounds were tentatively characterized based on their chromatographic and spectrometric data, referring to previous literature [25, 3033]. For example, MS2 spectra of compound 4 (molecular ion at m/z [M + H]+ 394.2590) in Table 2 gave characteristic fragment ions of [M + H–H2O]+ at m/z 376.2489 and [M + H–2H2O]+ at m/z 358.2371. Thus, it corresponded to Karakolidine by comparison with literature data [30]. Moreover, MS2 spectra of compound 12 (molecular ion at m/z [M + H]+ 408.2743) in Table 2 gave characteristic fragment ions of [M + H–H2O]+ at m/z 390.2630, 372.2517[M + H–2H2O]+ and [M + H–CH3OH]+ at m/z 358.2374. Then it was identified as Isotalatizidine. All the MS data of the (+) ESI–MS spectra are shown in Table 2. Besides, all the structures of the compounds identified are shown in Figs. 3 and 4. The deriving herb for each compound was also assigned. The majority of constituents are identified as alkaloids, flavonoids, triterpenes, gingerols, phenylpropanoids and volatile oil.
Fig. 3
Fig. 3

Structures of compounds identified in the extract of Fuzi Lizhong Pill

Fig. 4
Fig. 4

Structures of compounds identified in the extract of Fuzi Lizhong Pill

Characterization of the absorbed chemical constituents in rat serum

Identification of the bioactive chemical prototype constituents in rat serum

As the results of constituents in rat serum show in Table 3, by comparing the tR values and MS fragment characteristics between compounds in serum and compounds in FZLZP extract, 10 alkaloid components sourced from Aconitum carmichaeli Debx. were identified, including benzoylaconine, benzoylmesaconine, benzoylhypaconine, mesaconitine, Hypaconitine, fuziline, neoline, talatisamine, chasmanine, and 14-acetyltalatizamine. These constituents have been reported as parts of the main constituents with significant effects of analgesia, anti-inflammation, thermogenesis and increasing blood oxygen in Fuzi [34, 35]. The MS data of the (+) ESI–MS spectra are shown in Table 3. For example, MS2 spectra of compound 19 in Table 2 was detected at the Rt in 16.070 min with the molecular ion at m/z 454.2800[M + H]+ and gave characteristic fragment ions of [M + H–H2O]+ at m/z 436.2677. Similarly, MS2 spectra of compound 2 in Table 3 was detected at the Rt in 16.615 min with the molecular ion at m/z 454.2808[M + H]+ and gave characteristic fragment ions of [M + H–H2O]+ at m/z 436.0243. Thus, compound 2 in Table 3 was identified as the absorbed prototype of Fuziline in rat serum. The other alkaloid components were identified in a similar way.
Table 3

Characterization of chemical constituents in vivo and metabolites of FZLZP by HPLC-ESI/QTOF/MS

Peak no.

Rt (min)

Systematic name

Molecular formula

Molecular weight (Da)

[M + H]+

[M + Na]+

Fragmentations (m/z)

Source/prototype

Measured value (Da)

Error (ppm)

Measured value (Da)

Error (ppm)

1

4.841

l-Pyroglutamic acid

C5H7NO3

129.0426

130.0498

− 0.7689

  

130.0498[M + H]+, 112.9741[M + H–H2O]+

Dangshen

2

16.615

Fuziline

C24H39NO7

453.2727

454.2808

1.981

  

454.2808[M + H]+, 436.0243[M + H–H2O]+

Fuzi

3

17.021

Talatisamine

C24H39NO5

421.2828

422.2905

0.9472

  

422.2905[M + H]+, 390.2651[M + H–CH3OH]+, 359.3263[M + H–CH2OH–CH3OH]+

Fuzi

4*

24.357

Glucuronide conjugation metabolite

C21H20O10

432.1056

433.1132

0.6927

  

433.1132[M + H]+, 257.0843[M + H–(GluA–H2O)]+

Liquiritigenin

5

25.811

Liquiritigenin

C15H12O4

256.0736

257.0819

4.2788

  

257.0819 [M + H]+, 239.0713[M + H–H2O]+, 137.0237[C7H4O3 + H]+

Gancao

6

27.236

Benzoylmesaconine

C31H43NO10

589.2887

590.2948

− 2.033

  

590.2948[M + H]+, 558.2657[M + H–CH3OH]+ 540.2537[M + H–CH3OH–H2O]+, 508.2218[M + H-2CH3OH–H2O]+

Fuzi

7

27.520

Benzoylaconine

C32H45NO10

603.3043

604.3134

2.97

  

604.3134[M + H]+, 540.6158[M + H-2CH3OH]+, 508.8095[M + H-3CH3OH]+

Fuzi

8

28.379

Liquiritin or Isoliquiritin

C21H22O9

418.1264

  

441.1144

− 2.72

441.1144 [M + Na]+, 424.0979 [M + Na–OH]+, 350.8191[M + Na–C6H3O]+

Gancao

9

28.595

Benzoylhypaconine

C31H43NO9

573.2938

574.3025

0

574.3025[M + H]+, 443.8613[M + H-3CH3OH–H2O–HO]+

Fuzi

10

31.405

Mesaconitine

C33H45NO11

631.2993

632.3079

2.2141

  

632.3079[M + H]+, 599.9372[M + H–CH3OH]+, 540.2653[M + H–AcOH–CH3OH]+

Fuzi

11

32.453

Hypaconitine

C33H45NO10

615.3043

616.3089

− 4.381

  

616.3089[M + H]+, 597.8211 [M + H–H2O]+, 556.2792[M + H–C2H4O2]+

Fuzi

12*

33.299

Glucuronide conjugation metabolite

C30H47NO13

629.3047

630.3295

27.7640

  

630.3295 [M + H]+, 454.8397[M + H–(GluA–H2O)]+

Fuziline

13*

33.165

Glucuronide conjugation metabolite

C21H20O10

432.1056

433.1145

3.6942

  

433.1145[M + H]+, 257.0829[M + H–(GluA–H2O)]+

Isoliquiritigenin

14

40.710

Isoliquiritigenin

C15H12O4

256.0736

257.0807

− 0.3889

  

257.0807[M + H]+, 239.1624[M + H–H2O]+

Gancao

15

42.275

6-gingerdione

C17H24O4

292.1675

293.1734

− 4.4342

  

293.1734[M + H]+, 275.1586[M + H–H2O]+

Ganjiang

16

42.514

Formononetin

C16H12O4

268.0736

269.0799

− 3.3447

  

269.0799[M + H]+, 181.0511[M + H–C2O2–CH3OH]+

Gancao

17

44.584

14-Acetyltalatizamine

C26H41NO6

463.2934

464.3015

1.7230

  

464.3015[M + H]+, 446.2652[M + H– H2O]+, 432.6414[M + H–CH3OH]+

Fuzi

18

46.555

6-gingerol

C17H26O4

294.1831

295.1905

0.3388

  

295.1905[M + H]+, 263.1618[M + H–CH3OH]+, 179.1028[M + H–C7H15O]+

Ganjiang

19

46.980

6-shogaol

C17H24O3

276.1725

277.1781

− 6.1332

  

277.1794[M + H]+, 260.1816[M + Na–OH]+, 245.1533[M + H–CH3OH]+

Ganjiang

20

47.690

Atractylenolide II

C15H20O2

232.1463

233.1533

− 1.2867

  

233.1533[M + Na]+, 187.1487[M + Na–CH2O2]+,

159.1179[M + Na–CH2O2–C2H4]+, 145.1005[M + Na–CH2O2–C3H6]+

Baizhu

21

48.102

Chasmanine

C25H41NO6

451.2934

  

474.2841

3.1627

474.2841[M + H]+, 442.0836[M + H–CH3OH]+

Fuzi

22

49.895

Glycyrrhizic acid

C42H62O16

822.4038

823.4094

− 2.0646

  

823.4094[M + H]+, 647.3792[M + H–(GluA–H2O)]+

Gancao

23

50.826

Atractylenolide I

C15H18O2

230.1307

231.1382

0.8653

  

231.1382[M + H]+, 105.9823[M + H–HCOOH–2C2H4–2C]+

Baizhu

24

51.095

Neoline

C24H39NO6

437.2777

  

460.2669

− 0.2173

460.2669 [M + Na]+, 442.2666[M + Na–H2O]+

Fuzi

25

54.144

7-hydroxycoumarin

C9H6O3

162.0317

163.0396

3.6801

  

163.0396[M + H]+, 145.5012[M + H–H2O]+

Baizhu

26

56.004

Glycyrrhetinic acid

C30H46O4

470.3396

471.3479

− 2.122

  

471.3479[M + H]+, 453.4285[M + H–H2O]+

Gancao

* Indicates metabolites

Six compounds sourced from Glycyrrhiza uralensis Fisch. were identified, including 3 flavonoids, namely, liquiritigenin, isoliquiritigenin, and formononetin and 2 triterpenes, namely, glycyrrhetinic acid and glycyrrhizic acid. The MS data of the (+)ESI–MS spectra are shown in Table 3. For example, MS2 spectra of compound 48 in Table 2 was detected at the Rt in 39.763 min with the molecular ion at m/z 257.0814[M + H]+ and gave characteristic fragment ions of 239.0704[M + H–H2O]+, 137.0235[C7H4O3 + H]+, 121.0277[C8H8O + H]+, 120.0527 [C7H4O3 + H–OH]+. Similarly, MS2 spectra of compound 14 in Table 3 was detected at the Rt in 40.710 min with the molecular ion at m/z 257.0807[M + H]+ and gave characteristic fragment ions of [M + H–H2O]+ at m/z 239.1624. Thus, compound 14 in Table 3 was identified as the absorbed prototype of Isoliquiritigenin in rat serum. Furthermore, liquiritin or isoliquiritin may also have been found, but further comparison with reference compounds is needed to identify these isomers. The flavonoids and triterpenes in Glycyrrhiza uralensis Fisch. have been reported as having significant anti-inflammatory, abirritation and immunoregulation effects [3638].

7-Hydroxycoumarin, atractylenolide I and atractylenolide II have been identified as bioactive chemical constituents sourced form Atractylodes macrocephala Koidz. (Baizhu) and were found as the main institutes with the effect of anti-inflammatory, antitumor and gastrointestinal regulation in Baizhu [3942]. The MS data of the (+) ESI–MS spectra are shown in Table 3. For example, MS2 spectra of compound 26 in Table 2 was detected with the molecular ion at m/z 163.0395 [M + H]+ and gave characteristic fragment ions of 145.0627[M + H–H2O]+. Similarly, MS2 spectra of compound 25 in Table 3 was detected with the molecular ion at m/z 163.0396[M + H]+ and gave characteristic fragment ions of [M + H–H2O]+ at m/z 145.5012. Thus, compound 25 in Table 3 was identified as the absorbed prototype of 7-hydroxycoumarin in rat serum.

6-Gingerdione, 6-gingerol and 6-shogaol sourced from Zingiber officinale Rosc (Ganjiang) were identified and were reported as having obvious antioxidant, anti-inflammatory, gastrointestinal protective and antitumor effects [43, 44]. The MS data of the (+) ESI–MS spectra are shown in Table 3. For example, MS2 spectra of compound 50 in Table 2 was detected with the molecular ion at m/z 293.1736[M + H]+ and gave characteristic fragment ions of 275.1650[M + H–H2O]+, 257.1517[M + H–2H2O]+. Similarly, MS2 spectra of compound 15 in Table 3 was detected with the molecular ion at m/z 293.1734[M + H]+ and gave characteristic fragment ions of [M + H–H2O]+ at m/z 257.1586. Thus, compound 15 in Table 3 was identified as the absorbed prototype of 6-gingerdione in rat serum.

One compound was sourced from Codonopsis pilosula (Franch.) Nannf. (Dangshen) and was identified as l-pyroglutamic acid. MS2 spectra of compound 1 in Table 2 was detected with the molecular ion at m/z 130.0505[M + H]+ and gave characteristic fragment ions of 112.0123[M + H–H2O]+, 84.0449[M + H–HCOOH]+. Similarly, MS2 spectra of compound 1 in Table 3 was detected with the molecular ion at m/z 130.0498[M + H]+ and gave characteristic fragment ions of [M + H–H2O]+ at m/z 112.9741. Thus, compound 1 in Table 3 was identified as the absorbed prototype of l-pyroglutamic acid in rat serum.

Identification of the bioactive metabolites in rat serum

Based on a comparison of the information for ions, 8 peaks were detected only in dosed serum and were assigned to metabolites. Detailed information about the elemental compositions, retention times, and the characteristic fragment ions of metabolites are shown in Table 3. Alkaloid-, phenylpropanoids- and gingerols-related metabolites are the main metabolic constituents of FZPLP absorbed in vivo, and the main metabolic pathways in vivo were glucuronide conjugation and glucuronide. Identification of the corresponding fragment ions was obvious. For example, compound 4 (24.357 min) in Table 3 produced [M + H] + at m/z 433 and MS2 yielded a major ion at m/z 257 (− 176, Da with the loss of C6H8O6) in the positive ion mode, combined with the retention time of the reference standard 1 in Table 1 and compound 29 in Table 2. Therefore, the peak was identified tentatively as a glucuronide conjugation metabolite of liquiritigenin. Similarly, compound 13 (the tR 33.165 min) in Table 3 has the similar retention time compared with the reference standard 6 in Table 1 and compound 48 in Table 2. And it produced [M + H] + at m/z 433 and MS2 yielded a major ion at m/z 257 (− 176, Da with the loss of C6H8O6) in the positive ion mode. Therefore, the peak was identified tentatively as a glucuronide conjugation metabolite of isoliquiritigenin. The possible structures of metabolites were elucidated as described above. All of the structures of metabolites were identified, and the MS data of the (+) ESI–MS spectra are shown in Table 3. This article reports these metabolites of FZLZP for the first time. The bioactivities are the subject of ongoing research.

Alkaloids difference between Group A and Group B

As the result shows in Fig. 5a, 10 kinds of alkaloids were detected in Group A. Most of them were trace amounts in vivo, which indicated the alkaloids’ poor absorption in the prescription. Conversely, unlike Group A, the amount of the alkaloids in vivo increased obviously in Group B (Fig. 5b). The difference indicated that the absorption amount of alkaloids in the prescription can be decreased compared to the absorption amount of alkaloids in the herb powder.
Fig. 5
Fig. 5

The difference in the absorbed compounds in vitro and in vivo. (a The difference in the absorbed compounds in vitro and in vivo of Group A; b The difference in the absorbed alkaloids in vitro and in vivo of Group B.) (Columns: A, l-pyroglutamic acid; B, Fuziline; C, Talatisamine; D, Benzoylmesaconine; E, Benzoylaconine; F, Benzoylhypaconine; G, Mesaconitine; H, Hypaconitine; I, 14-Acetyltalatizamine; J, Chasmanine; K, Neoline; L, Liquiritigenin; M, Liquiritin or Isoliquiritin; N, Isoliquiritigenin; O, Formononetin; P, Glycyrrhizic acid; Q, Glycyrrhetinic acid; R, 6-gingerdione; S, 6-gingerol; T, 6-shogaol; U, Atractylenolide II; V, Atractylenolide I; W, 7-hydroxycoumarin)

Discussion

To obtain LC chromatograms of lower pressure, greater baseline stability, better resolution and higher ionization efficiency, methanol and acetonitrile and series of concentrations of aqueous formic acid solution were prepared for analysis. The best result was achieved when the mobile phase consisted of 0.1% formic acid aqueous solution and methanol. Both positive and negative modes were investigated, and the results showed that the positive ion mode was more sensitive and could provide more information for both extract samples and serum samples analyses.

FZLZP is a formula composed under the guidance of traditional Chinese medicine theory. According to TCM theory, Aconitum carmichaeli Debx. is the “monarch drug” and the main herb in FZLZP recipe to warm middle jiao and eliminate cold. This was confirmed in this research with 10 constituents among 23 prototype components sourced from Aconitum carmichaeli Debx., which maintains the maximum bioactive compounds. Glycyrrhiza uralensis Fisch. is frequently prescribed in combination with other herbs to decrease toxicity and to increase efficacy. In this recipe, it is the “envoy drug” and is considered to be the paramount assistant herb, which can detoxify the toxicity of aconitum. In this study, we found that Glycyrrhiza uralensis Fisch. was the second most-absorbed herb. The results that some compounds absorbed well in vivo derived from Aconitum carmichaeli Debx. and Glycyrrhiza uralensis Fisch. are consistent with our previous studies that they were dissolved very well in vitro [16].

Alkaloids in Fuzi herb are the toxicity as well as the efficacy compounds. The prescriptions which contains Fuzi herb should be highly concerned. In our study, the results on the differences in alkaloids between Group A and Group B show that the amount of absorption of bioactive constituents in Fuzi can be significantly reduced when this herb is used as part of a prescription rather than used alone. We think there are two reasons. Firstly, according to the TCM theory, the toxicity of Fuzi can be reduced in combination with Gancao [25]. This should be further confirmed by researching the relationship and differences in the chemistry constituents between Fuzi-Gancao herb pairs in FZLZP. Secondly, the pill form is the embryonic form of sustained-release preparations. As a TCM classic says: only pill among all dosage forms can reduce the toxicity of toxic drugs. The toxic herb was usually made into a pill form to reduce the toxicity in TCM [17]. And it can be further confirmed by researching differences in the chemistry constituents between FZLZP and the Fuzi pill that made from Aconitum carmichaeli Debx. powder.

Conclusions

This study describes a simple, sensitive and selective HPLC-QTOF-MS method for structural characterization of chemical constituents in FZLZP and bioactive components in rat serum following oral administration of FZLZP. As a result, in vitro, a total of 67 compounds were successfully identified, and 23 prototype compounds that were absorbed in vivo were identified for the first time. In addition, 3 metabolites of the bioactive compounds were tentatively identified. In this prescription, the majority of compounds absorbed in vivo derived from Fuzi and Gancao. The results provide helpful chemical information for FZLZP for further pharmacological and active mechanism research. In addition, it helped to classify the material basis responsible for the therapeutic effects of FZLZP. Furthermore, the HPLC-QTOF-MS was a potentially powerful strategy for simultaneously achieving screening and analysis of multiple bioactive compounds in FZLZP.

Notes

Abbreviations

HPLC-QTOF-MS: 

high-performance liquid chromatography–electrospray ionization/quadrupole-time-of-flight high-definition mass spectrometry

FZLZP: 

Fuzi Lizhong Pill

FZP: 

Fuzi powder

Group A: 

FZLZP group for dosed rat serum

Group B: 

Fuzi powder group for dosed rat serum

Group C: 

control group for blank rat serum

Declarations

Authors’ contributions

ZZ and MJ carried out the screening experiments, ZZ wrote the manuscript and analyzed the data, MJ, XW, SY, JS graphed the picture, GZ revised the manuscript, CF and LG conceived of the study, contributed to the design and interpretation of the research. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

The dataset supporting the conclusions of this article is included within the article.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

Funding

This work was supported by the National Natural Science Foundation of China (No. 81803742), Key Project of Natural Science Fund of Sichuan Province (No. 18ZA0187), Pre-research National Natural Science Foundation of Chengdu University of Traditional Chinese Medicine (No. ZRYY1718).

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Authors’ Affiliations

(1)
The Ministry of Education Key Laboratory of Standardization of Chinese Herbal Medicine, State Key Laboratory Breeding Base of Systematic Research, Development and Utilization of Chinese Medicine Resources, College of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, 611137, China
(2)
Sichuan Institute for Food and Drug Control, Chengdu, 611137, China

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