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. 2019 Mar 1;14:6. doi: 10.1186/s13020-019-0227-z

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

Zhen Zhang 1,#, Maoyuan Jiang 1,#, Xinyi Wei 1, Jinfeng Shi 1, Zhao Geng 2, Shasha Yang 1, Chaomei Fu 1,✉, Li Guo 1,✉
PMCID: PMC6397459  PMID: 30867675

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.

Electronic supplementary material

The online version of this article (10.1186/s13020-019-0227-z) contains supplementary material, which is available to authorized users.

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. [1–7]. 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 [8–15]. 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 [16–18]. 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 [19–24]. 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 [26–29]. 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

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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]+
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Fig. 2.

Fig. 2

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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
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#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, 30–33]. 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

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Structures of compounds identified in the extract of Fuzi Lizhong Pill

Fig. 4.

Fig. 4

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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
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* 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 [36–38].

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 [39–42]. 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

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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.

Additional file

13020_2019_227_MOESM1_ESM.pdf (2.1MB, pdf)

Additional file 1. Minimum standards of reporting checklist.

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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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

Contributor Information

Zhen Zhang, Email: zhangzhendr@126.com.

Maoyuan Jiang, Email: 362265312@qq.com.

Xinyi Wei, Email: 2627019775@qq.com.

Jinfeng Shi, Email: 191006842@qq.com.

Zhao Geng, Email: gengzhao713@hotmail.com.

Shasha Yang, Email: 312891469@qq.com.

Chaomei Fu, Email: chaomeifu@126.com.

Li Guo, Email: gli64@sina.com.

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Associated Data

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

Supplementary Materials

13020_2019_227_MOESM1_ESM.pdf (2.1MB, pdf)

Additional file 1. Minimum standards of reporting checklist.

Data Availability Statement

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

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