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. 2022 Sep 20;15(1):139–150. doi: 10.1016/j.chmed.2022.08.002

Identification of prototype compounds and their metabolites in rats’ serum from Xuefu Zhuyu Decoction by UPLC-Q-TOF/MS

Xiaoyu Zhang a,1, Zhenzuo Jiang a,1, Lei Zhang a,1, Cheng Xue a, Xiafei Feng b, Xin Chai a, Yuefei Wang a,⁎
PMCID: PMC9975640  PMID: 36875444

Abstract

Objective

As a classic prescription in traditional Chinese medicine, Xuefu Zhuyu Decoction (XFZYD) has been widely used in the clinical treatment of cardiovascular and cerebrovascular diseases. In order to unveil the potentially effective compounds, a rapid ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) method was established to identify prototype compounds and their metabolites from XFZYD in rats’ serum.

Methods

The serum from rats after intragastric administration of XFZYD aqueous extract was analyzed by UPLC-Q-TOF/MS method. The prototype compounds and their metabolites were identified by comparison with the reference standards and tentatively characterized by comprehensively analyzing the retention time, MS data, characteristic MS fragmentation pattern and retrieving literatures.

Results

A total of 175 compounds (24 prototype compounds and 151 metabolites) were identified and tentatively characterized. The metabolic pathways of prototype compounds in vivo were also summarized, including glucuronidation, hydrolyzation, sulfation, demethylation, and hydroxylation, and so on.

Conclusion

In this study, a UPLC-Q-TOF/MS technique was developed to analyze prototype compounds and their metabolites from XFZYD in serum, which would provide the evidence for further studying the effective compounds of XFZYD.

Keywords: metabolites, prototype compounds, serum, UPLC-Q-TOF/MS, Xuefu Zhuyu Decoction

1. Introduction

Xuefu Zhuyu Decoction (XFZYD), developed by Qingren Wang (1768–1831) in Yilin Gaicuo (Correction of Medical Errors, 1850), is a well-known traditional Chinese medicine formula. XFZYD consists of eleven herbs, including Persicae Semen (Taoren), Angelicae Sinensis Radix (Danggui), Chuanxiong Rhizoma (Chuanxiong), Carthami Flos (Honghua), Paeoniae Radix Rubra (Chishao), Rehmanniae Radix (Dihuang), Aurantii Fructus (Zhiqiao), Bupleuri Radix (Chaihu), Platycodonis Radix (Jiegeng), Achyranthis Bidentatae Radix (Niuxi), and Glycyrrhizae Radix et Rhizoma (Gancao), which is widely used to treat diseases caused by qi stagnation and blood stasis with its effects of blood-activating, stasis-resolving and regulating qi-flowing (Wang et al., 2022, Wang, 2005). It was reported that XFZYD has the significant clinical effects on dysmenorrhoea, traumatic brain injury, and endometriosis (Jo et al., 2017, Zhang et al., 2018, Fu et al., 2019, Su et al., 2020). Especially, XFZYD is widely employed to treat cardiovascular diseases, including chest pain, headache, angina pectoris, heart failure, atherosclerosis, hypertension, and hyperlipidemia as the basic prescription (Yang et al., 2019, Lin et al., 2018, Jiang and Jiang, 2016, Wang et al., 2015, Wang and Qiu, 2019, Meng et al., 2018). However, its underlying action mechanism is still uncovered. Importantly, the clarification of chemical compounds in vivo is the prerequisite question which must be answered and worthy of in-depth study.

In recent years, researchers have made continuous efforts to develop various analytical methods to clarify the chemical compounds of XFZYD. Previously, our group employed ultra performance liquid chromatography with diode array detector tandem mass spectrometry (UPLC-DAD-MS/MS) method to identify 28 compounds and quantitatively analyze 12 compounds in XFZYD related products (Zhang et al., 2012). Also, we made use of ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) to systematically illuminate the chemical compounds of XFZYD in vitro, by which 103 compounds were identified, mainly including phenolic acids, flavonoids, saponins, terpenes, and other compounds (Zhang et al., 2015). Besides this, Fu et al (2016) qualitatively analyzed 34 major constituents, including organic acids, lactones, alkaloids, amino acids and cyanogenic glycosides in XFZYD by using ultra high performance liquid chromatography with hybrid ion trap time-of-flight mass spectrometry (UHPLC-ESI-IT-TOF-MS). However, there are few reports on clarification of the prototype compounds and their metabolites from XFZYD exposed in vivo, which will be important for illuminating the potentially active compounds. The metabolites always act as the active compounds in vivo. For instance, dihydroberberine, jatrorrhizine, columbamine, berberrubine, and demethyleneberberine, the metabolites of berberine produced by intestinal microbiota, have the significant effect of lipid-lowering (Feng et al., 2018, Zuo et al., 2006, Zhou et al., 2014). Therefore, it is essential to study the exposed compounds from XFZYD in vivo.

In this study, we employed UPLC-Q-TOF/MS for identification of chemical compounds and their metabolites in rats’ serum after oral administration of XFZYD aqueous extract at a dose of 16.38 g/kg body weight, which was expressed as the weight of decoction pieces. A total of 175 compounds were characterized and tentatively identified, including 24 prototype compounds and 151 metabolites. Also, the major metabolic pathways of prototype compounds from XFZYD in serum have been preliminarily proposed. Meaningfully, we established an analytical method with high sensitivity, rapid analysis, low consumption of samples, and abundant yield of structural information. Our study not only provides the information about the chemical substances of XFZYD in vivo, but also highly contributes to further investigation of the pharmacology and mechanism of XFZYD.

2. Materials and methods

2.1. Chemicals and materials

Acetonitrile and methanol (HPLC grade) were purchased from Fisher Scientific (Fisher, USA). Formic acid and dimethyl sulfoxide were purchased from Meridian Medical Technologies (MREDA, USA). Water for UPLC analysis was purified by a Milli-Q water purification system (Millipore, USA). Reference compounds, including p-hydroxybenzoic acid (PHBA), hydroxysafflor yellow A (HSYA), amygdalin, p-hydroxycinnamic acid (PHCA), ferulic acid, albiflorin, paeoniflorin, liquiritin, isoquercitrin, narirutin, β-ecdysterone, naringin, rhoifolin, hesperidin, neohesperidin, liquiritigenin, naringenin, platycodin D, isoliquiritigenin, formononetin, ginsenoside-Ro, 18β-glycyrrhizic acid, nobiletin, and saikosaponin A were obtained from the National Institutes for Food and Drug Control (Beijing, China), Tianjin ZhongXin Pharmaceutical Group Co., Ltd. (Tianjin, China), and Top High Bio Technology Co., Ltd. (Nanjing, China). The purity of standards was above 98%. All samples were stored at 4 °C before analysis.

2.2. Preparation of standard solution

As individual standard stock solution, reference compounds were accurately weighed and directly prepared in methanol by using dimethyl sulfoxide as a cosolvent. Then, a mixed standard solution was prepared at about 5 μg/mL. All solution of reference standards was stored at 4 °C.

2.3. UPLC-Q-TOF/MS analytical conditions

2.3.1. Chromatographic conditions

Chromatographic analysis was performed on an ACQUITY™ UPLC system (Waters, Milford, USA) equipped with a binary solvent manager, sample manager and column oven, which was controlled by Masslynx V4.1 software. Chromatographic separation was carried out on an ACQUITY™ UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) held at 50 °C. The flow rate was set at 0.3 mL/min. The mobile phase consisted of 0.1% formic acid aqueous solution (A) and acetonitrile (B) in a gradient elution. The program applied was as follows: 0–6.5 min, 3%−11% B; 6.5–15 min, 11%−20% B; 15–20 min, 20%−36% B; 20–27 min, 36%−48% B; 27–30 min, 48%−55% B; 30–33 min, 55%−74% B, and 33–35 min, 74%−90%. The injection volume of the sample solution was 5 μL.

2.3.2. MS spectrometry conditions

The MS analysis was performed by employing a Waters ACQUITY SYNAPTTM G2-S high definition mass spectrometer system (Waters, Milford, USA) equipped with an electrospray ion (ESI) source. The optimal conditions were as follows: capillary voltage at 3.0 and −2.5 kV in positive and negative ion mode, respectively; sampling cone voltage at 40 V; source temperature at 120 °C; desolvation temperature at 400 °C; the flow rate of cone gas and desolvation gas (N2) at 50 L/h and 700 L/h, respectively; the flow rate of collision gas (Ar) at 0.20 mL/min. Data were acquired in centroid mode from m/z 100 to 1500 Da. For accurate mass to charge ratio acquisition, the MS was corrected during data acquisition using a lock mass of leucine-enkephalin (LE) at a concentration of 200 pg/mL via a LockSprayTM interface at a flow rate of 10 μL/min, monitoring the reference ions in the positive ion mode ([M+H]+ = 556.2771) and the negative ion mode ([M−H]– = 554.2615) during MS analysis.

2.4. Preparation of XFZYD

The decoction pieces (Taoren, Danggui, Chuanxiong, Honghua, Chishao, Dihuang, Zhiqiao, Chaihu, Jiegeng, Niuxi, and Gancao) were purchased from Anguo Oriental Medical Town (Hebei, China) and identified by Professor Tianxiang Li, which were deposited in Tianjin Key Laboratory of TCM Chemistry and Analysis (Tianjin, China).

According to the regulation of XFZYD, the decoction pieces (total weight of 78 g) were mixed and immersed in 600 mL deionized water for 1 h at room temperature, and then refluxed twice for two hours per time. After filtration and concentration, aqueous extract was dried at 45 °C in an oven under vacuum to give 30 g extract powder, the yield of which was 38.5%. The extract powder was stored at 4 °C before use.

2.5. Animals and drug administration

Male Sprague-Dawley (SD) rats (200 ± 10 g) were supplied by Beijing HFK Bioscience Co., Ltd. (SCXK2009-0004, Beijing, China). All animals were acclimated in a room (22–25 °C with 50% ± 10% humidity) for one week, which had free access to water and standard laboratory food, and gradually adapted to the facilities. Then, the rats were fasted with free access to water for 12 h prior to the experiment. For seven consecutive days, the aqueous extract of XFZYD was administrated to the rats intragastrically at a dose of 16.38 g/kg body weight, which was expressed as the weight of decoction pieces (Zhang et al., 2018, Fan et al., 2020). After the last administration, the blood samples were respectively collected from the retroorbital venous plexuses at 0.5, 1 and 2 h, which were placed at room temperature for 1 h and centrifuged at 8000 rpm for 10 min. Then, the serum samples were obtained from the different blood sampling time in one rat and equally mixed to acquire the blended serum samples, which were frozen immediately and stored at −80 °C until analysis. Blank serum samples were collected in the same way. Animal studies were conducted according to protocols approved by the Review Committee of Animal Care and Use.

2.6. Sample preparation

The serum samples were thawed and homogenized at room temperature in advance. After addition of 10% formic acid (10 μL) in serum (1 mL), the sample was vortex-mixed well and loaded on Cleanert S C18-SPE column (500 mg/3 mL, Bonna-Agela, China), which was respectively pretreated with 2 mL methanol and 2 mL water, and equilibrated with 3 mL 1% formic acid in sequence. The loaded sample was sequently eluted with 3 mL 1% formic acid and 2 mL methanol, then methanol eluant was collected and evaporated to dryness by a vacuum centrifugal concentrator (Eppendorf, Hamburg, Germany) at 40 °C. Finally, the residue was dissolved in 50% methanol (100 μL) for analysis.

2.7. Statistical analysis

All the MS data were acquired and processed by Masslynx V4.1 software (Waters Corporation, Milford, MA, USA). The structures of prototype compounds were drawn using ChemBioDraw 14 software (CambridgeSoft Corporation, USA). The network was portrayed by Cytoscape V3.8.0 software (National Resource for Network Biology, USA).

3. Results and discussion

3.1. Optimization of pretreatment method and analytical condition

In order to remove interfering substances in serum samples and enrich the targeted compounds for more sensitive detection, serum samples were generally pretreated by the methods of protein precipitation, liquid–liquid extraction and solid-phase extraction, respectively (Feng et al., 2020, Yan et al., 2019, Zhang et al., 2019). Moreover, addition of a certain amount of acid, the performance of pretreatment can be improved for the acidic compounds. By our study, Cleanert S C18-SPE column (500 mg/3 mL, Bonna-Agela, China) was proved to be suitable as pretreatment method for the serum sample, resulting in the excellent enrichment of the interesting compounds and the satisfactory elimination of interfering signal in MS analysis. The structures of the detected prototype compounds are displayed in Fig. 1.

Fig. 1.

Fig. 1

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Chemical structures of prototype compounds.

Furthermore, chromatographic separation and MS detection were accomplished to excellently detect the interesting compounds. Acetonitrile – water as the mobile phase system was optimized through adding 0.1% formic acid in water by taking the characteristics of acidic and alkaline analytes into account. Aiming at obtaining comprehensive MS information of analytes as much as possible, UPLC-Q-TOF/MS was performed in positive and negative ion modes. Because of the existence of phenolic hydroxyl and carboxyl groups in the analytes, it is found that the negative ion mode has good MS response.

3.2. Identification of prototype compounds from XFZYD in serum

Illumination of prototype compounds absorbed into blood is the prerequisite issue to clarify the exposure of chemical compounds in vivo from Chinese materia medica (CMM), which will pave the way for identification of metabolites derived from these prototype compounds. Through our previous study, flavanones, saponins and phenolic acids were proven to be the representative compounds in XFZYD (Zhang et al., 2012, Zhang et al., 2015). In our study, characterization of prototype compounds was performed from the dosed serum by analyzing the blank serum, dosed serum, and reference standards. The extracted ion chromatograms of prototype compounds from the serum samples and mixed standards are shown in Fig. 2. Compared the MS data of the serum samples with those of authentic compounds (Table 1 and Supplementary Table S1), 24 prototype compounds were characterized, including 13 flavanones (HSYA, liquiritin, isoquercitrin, narirutin, naringin, rhoifolin, hesperidin, neohesperidin, liquiritigenin, naringenin, isoliquiritigenin, formononetin and nobiletin), four saponins (platycodin D, ginsenoside-Ro, 18β-glycyrrhizic acid and saikosaponin A), three phenolic acids (PHBA, PHCA, and ferulic acid), and four others (β-ecdysterone, amygdalin, albiflorin and paeoniflorin).

Fig. 2.

Fig. 2

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Extracted ion chromatograms of prototype compounds from serum sample solution and mixed standards solution (Blank serum sample (A), dosed serum sample (B), and mixed standards sample (C) in negative mode; Blank serum sample (D), dosed serum sample (E), and mixed standards sample (F) in positive mode.

Table 1.

MS data of prototype compounds from XFZYD in serum by UPLC-Q-TOF/MS.

No. Compounds tR (min) Formula Negative ion mode (−)
 Positive ion mode (+)
[M−H]−, (m/z) [M+H]+, (m/z)
P1 PHBA 4.080 C7H6O3 137.0239 –
P2 HSYA 5.512 C27H32O16 611.1612 613.1769
P3 Amygdalin 6.200 C20H27NO11 456.1506 458.1662
P4 PHCA 7.532 C9H8O3 163.0395 –
P5 Albiflorin 7.922 C23H28O11 525.1608 * 481.1710
P6 Paeoniflorin 8.739 C23H28O11 525.1608 * 498.1985 #
P7 Ferulic acid 9.182 C10H10O4 193.0501 –
P8 Liquiritin 10.355 C21H22O9 417.1186 419.1342
P9 Isoquercitrin 10.698 C21H20O12 463.0877 465.1033
P10 Narirutin 12.73 C27H32O14 579.1714 581.1870
P11 β-Ecdysterone 12.814 C27H44O7 479.3009 481.3165
P12 Naringin 13.474 C27H32O14 579.1714 581.1870
P13 Rhoifolin 13.711 C27H30O14 577.1557 579.1714
P14 Hesperidin 14.372 C28H34O15 609.1819 611.1976
P15 Neohesperidin 15.112 C28H34O15 609.1819 611.1976
P16 Liquiritigenin 15.521 C15H12O4 255.0657 257.0814
P17 Naringenin 18.424 C15H12O5 271.0606 273.0763
P18 Platycodin D 19.254 C57H92O28 1223.5697 1225.5853
P19 Isoliquiritigenin 20.214 C15H12O4 255.0657 257.0814
P20 Formononetin 20.776 C16H12O4 267.0657 269.0814
P21 Ginsenoside-Ro 21.398 C48H76O19 955.4903 957.5059
P22 18β-Glycyrrhizic acid 22.356 C42H62O16 821.3960 823.4116
P23 Nobiletin 23.130 C21H22O8 – 403.1393
P24 Saikosaponin A 24.174 C42H68O13 779.4582 781.4738
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P = prototype compound, “−” undetected, “*” [M−H+HCOOH]−, “#” [M+NH4]+.

Studies have shown that the prototype compounds identified in XFZYD play an important role in the prevention and treatment of cardiovascular and cerebrovascular diseases. For instance, flavonoids can exert vasodilatory effects in vitro by regulating eNOS or Ca2+ channels (Tang et al., 2021). Phenolic acids protect the blood brain barrier through MMP-9 inhibition and anti-inflammation (Zhang & Song et al., 2018). Saponins have the pharmacological effects of anti-atherosclerosis, myocardial protection, and anti-thrombosis (Li et al., 2015). For example, HSYA, as the marker compound of safflower, has been widely used for the treatment of cerebrovascular and cardiovascular diseases due to its property of promoting blood circulation and removing blood stasis (Yang et al., 2020). Formononetin could alleviate the development of atherosclerosis by regulating the interaction between KLF4 and SRA (Ma et al., 2020). Amygdalin was reported to have the effect of reducing the development of atherosclerosis by inhibiting inflammatory reaction and promoting the immune regulation function of T cells (Deng et al., 2011). Naringin, a major compound of flavanone, which can be extracted from many CMM herbs, has the effects of anti-atherosclerosis, anti-hypertension, and myocardial protection (Hsueh et al., 2016, Sun et al., 2019). Isoliquiritigenin is a flavonoid compound from Glycyrrhiza glabra that has been proven to attenuate atherosclerosis lesion and decrease blood lipid level by inhibiting TRPC5 channel (Qi et al., 2020). Also, it has been reported that hesperidin prevents the redox imbalance induced by hyperlipidemia (Kumar, Akhtar, & Rizvi, 2020). In vitro and in vivo experiments showed that ferulic acid has antithrombotic, hypolipidemic activities, and so on (Choi et al., 2018, Kamal-Eldin et al., 2000, Zhu and Zhang, 2014).

3.3. Identification of metabolites from XFZYD in serum

Enzymes play an important role in the metabolism of CMM compounds. It can metabolize prototype compounds derived from the studied formula into active metabolites, contributing to the treatment of diseases. β-Glucosidase is abundant enzyme in intestinal, which can easily transform glycoside into aglycone by deglycosylation reaction (Yan et al., 2018). In liver, liver microsomal enzymes can metabolize prodrug into metabolites through Ⅰ and Ⅱ phase metabolism (Zeng et al., 2018). By analyzing the chromatograms of the blank serum and dosed serum (Fig. 3), a total of 151 metabolites were tentatively identified by the Metabolyxn (Masslynx V4.1 software) and detailed MS data (Table 2 and Supplementary Table S2). There were 71, 53, 14, and 13 metabolites transformed from flavanones, saponins, phenolic acids and other compounds, respectively. The network was adopted to display the relationship between prototype compounds and metabolites, and 24 prototype compounds (P1–P24) identified from XFZYD have been modified into 151 metabolites, which was shown in Fig. 4. Interestingly, it can be drawn that one prototype compound can be metabolized into the different metabolites, the different prototype compounds can be metabolized into the same metabolite. M10-8 taken as a typical example was simultaneously produced by narirutin, naringin, and neohesperidin in vivo. Meanwhile, narirutin, naringin, and neohesperidin can be metabolized into the different metabolites besides M10-8, respectively.

Fig. 3.

Fig. 3

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Extracted ion chromatograms of metabolites of serum samples. Blank serum sample (A) and dosed serum sample (B) in negative mode; Blank serum sample (C) and dosed serum sample (D) in positive mode.

Table 2.

MS data and metabolic pathways of metabolites after oral administration of XFZYD by UPLC-Q-TOF/MS.

Prototypes Metabolic pathways No. tR (min) Peak No. Negative ion mode (−)
 Positive ion mode (+)
 Formula
[M−H]−, (m/z) [M+H]+, (m/z)
PHBA Parent P1 3.99 – 137.0242 – C7H6O3
Hydroxylation + Methylation M1-1 5.74 11 167.0350 – C8H8O4
Hydroxylation M1-2 6.04 14 153.0195 – C7H6O4



HSYA Parent P2 5.51 – 611.1616 – C27H32O16
Deoxidation M2-1 11.43 42 595.1682 – C27H32O15



Amygdalin Parent P3 6.13 – 456.1506 480.1478 # C20H27NO11
Deglycosylation (glucose) M3-1 7.42 20 294.0985 318.0961 # C14H17NO6
M3-2 7.58 21 294.0984 318.0962 # C14H17NO6
Deglycosylation (2 × glucose) + Sulfate conjugation M3-3 8.90 23 212.0025 – C8H7NO4S



PHCA Parent P4 7.51 – 163.0402 – C9H8O3
Hydroxylation + Sulfation conjugation M4-1 4.90 9 258.9922 – C9H8O7S
Sulfate conjugation M4-2 2.65 2 242.9968 – C9H8O6S
M4-3 4.77 8 242.9970 – C9H8O6S
M4-4 5.84 13 242.9969 – C9H8O6S
M4-5 6.07 15 242.9969 – C9H8O6S



Albiflorin Parent P5 7.91 – 525.1614 * 503.1537 # C23H28O11
De-benzoic acid M5-1 5.74 12 359.1338 383.1314 # C16H24O9
De-benzoic acid + Dehydration M5-2 10.65 38 341.1243 365.1212 # C16H22O8
De-benzoic acid + Acetylation M5-3 10.80 40 401.1451 425.1432 # C18H26O10



Paeoniflorin Parent P6 8.72 – 525.1609 * 503.1525 # C23H28O11
De-debenzoylpaeoniflorin + Glucuronide conjugation M6-1 2.38 1 343.0662 * – C13H14O8
De-debenzoylpaeoniflorin + Hydroxylation + sulfate conjugation M6-2 2.73 3 262.9869 * – C7H6O6S
De-debenzoylpaeoniflorin + Sulfate conjugation M6-3 3.06 4 246.9924 * – C7H6O5S



Ferulic acid Parent P7 9.18 – 193.0507 – C10H10O4
Glucuronide conjugation M7-1 4.34 6 369.0823 – C16H18O10
M7-2 6.57 18 369.0834 – C16H18O10
Sulfate conjugation M7-3 3.13 5 273.0078 – C10H10O7S
M7-4 5.38 10 273.0081 – C10H10O7S
M7-5 6.41 16 273.0080 – C10H10O7S
M7-6 6.56 17 273.0080 – C10H10O7S
Demethylation + Sulfate conjugation M7-7 4.90 9 258.9918 – C9H8O7S
Demethylation + Deoxidation + Sulfate conjugation M7-8 2.65 2 242.9968 – C9H8O6S
M7-9 4.66 7 242.9973 – C9H8O6S
M7-10 4.77 8 242.9970 – C9H8O6S
M7-11 6.07 15 242.9968 – C9H8O6S



Liquiritin Parent P8 10.32 – 417.1195 419.1353 C21H22O9
Glucuronide conjugation M8-1 6.61 19 593.1511 – C27H30O15
M8-2 10.19 31 593.1508 595.1645 C27H30O15
Hydroxylation + Dehydrogenation M8-3 10.04 25 431.0979 433.1131 C21H20O10
M8-4 10.36 35 431.0979 433.1134 C21H20O10
Deglycosylation (glucose) + Hydroxylation + Glucuronide conjugation M8-5 13.11 54 447.0931 449.1082 C21H20O11
Hydroxylation + Methylation M8-6 17.54 80 447.1289 449.1444 C22H24O10
Hydroxylation + Sulfation conjugation M8-7 14.31 63 513.0705 – C21H22O13S
Deglycosylation (glucose) + Methylation M8-8 18.62 88 269.0817 271.0978 C16H14O4
Deglycosylation (glucose) + Sulfate conjugation M8-9 12.41 49 335.0229 337.0376 C15H12O7S
M8-10 18.14 85 335.0230 337.0384 C15H12O7S



Isoquercitrin Parent P9 10.69 – 463.0880 465.102 C21H20O12
Methylation + Deoxidation M9-1 13.98 56 461.1090 – C22H22O11
M9-2 14.15 60 461.1086 – C22H22O11
M9-3 18.98 93 461.1088 – C22H22O11
Deoxidation M9-4 15.33 66 447.0936 – C21H20O11
Deoxidation + Sulfate conjugation M9-5 10.11 30 527.0497 – C21H20O14S
M9-6 10.26 33 527.0498 – C21H20O14S



Narirutin Parent P10 12.72 – 579.1718 581.1868 C27H32O14
Deglycosylation (rhamnose) + Dehydrogenation M10-1 10.04 26 431.0979 433.1131 C21H20O10
M10-2 10.36 36 431.0979 433.1134 C21H20O10
Deglycosylation (rutinose) + Glucuronide conjugation M10-3 13.11 54 447.0931 449.1082 C21H20O11
Hydroxylation M10-4 10.72 39 595.1658 597.181 C27H32O15
M10-5 11.43 43 595.1677 – C27H32O15
Hydroxylation + Dehydrogenation M10-6 18.11 82 593.1506 595.1644 C27H30O15
Hydroxylation + Methylation M10-7 17.23 79 609.1833 611.1979 C28H34O15
Deglycosylation (rutinose) + Hydroxylation + Methylation M10-8 13.04 52 301.0722 303.0875 C16H14O6
M10-9 13.23 55 301.0717 303.0872 C16H14O6
M10-10 14.23 62 301.0722 303.0883 C16H14O6
Methylation M10-11 14.01 58 593.1874 – C28H34O14



β-Ecdysterone Parent P11 12.82 – 525.3080 * – C27H44O7
Methylation M11-1 22.44 100 539.3206 * – C28H46O7
Reduction + Deoxidation M11-2 22.48 101 511.3267 * – C27H46O6
Demethylation + Deoxidation M11-3 22.63 102 495.2962 * – C26H42O6
Acetylation M11-4 23.97 111 567.3167 * – C29H46O8



Naringin Parent P12 13.47 – 579.1719 581.1863 C27H32O14
Deglycosylation (rhamnose) + Dehydrogenation M12-1 10.04 26 431.0979 433.1131 C21H20O10
M12-2 10.36 36 431.0979 433.1134 C21H20O10
Deglycosylation (neohesperidose) + Glucuronide conjugation M12-3 13.11 54 447.0931 449.1082 C21H20O11
Hydroxylation + Dehydrogenation M12-4 18.11 83 593.1506 595.1644 C27H30O15
Deglycosylation (neohesperidose) + Hydroxylation + Methylation M12-5 13.04 52 301.0722 303.0875 C16H14O6
Methylation M12-6 14.01 59 593.1874 – C28H34O14



Rhoifolin Parent P13 13.71 – 577.1590 579.1697 C27H30O14
Deglycosylation (neohesperidose) + Hydroxylation + Glucuronide conjugation M13-1 12.33 48 461.0726 463.0879 C21H18O12
Deglycosylation (rhamnose) + Hydroxylation + Methylation M13-2 14.15 61 461.1086 – C22H22O11
M13-3 13.98 57 461.1090 – C22H22O11
M13-4 18.98 94 461.1088 – C22H22O11
M13-5 19.10 95 461.1084 – C22H22O11
Deglycosylation (neohesperidose) + Hydroxylation + Sulfation conjugation M13-6 9.86 24 364.9967 – C15H10O9S
Deglycosylation (rhamnose) + Hydroxylation + Sulfation conjugation M13-7 10.09 28 527.0500 – C21H20O14S
M13-8 10.25 32 527.0500 – C21H20O14S
Deglycosylation (rhamnose) + Hydroxylation M13-9 15.33 67 447.0936 – C21H20O11
Deglycosylation (rhamnose) + Methylation M13-10 18.64 91 445.1133 447.1294 C22H22O10
Deglycosylation (rhamnose) M13-11 10.04 26 431.0979 433.113 C21H20O10
M13-12 10.36 36 431.0979 – C21H20O10
M13-13 15.62 72 – 433.1136 C21H20O10
M13-14 16.30 77 – 433.113 C21H20O10
Deglycosylation (rhamnose) + Reduction M13-15 11.57 45 433.1135 – C21H22O10
M13-16 17.09 78 – 435.129 C21H22O10
Deglycosylation (rhamnose) + Sulfate conjugation M13-17 12.54 51 511.0547 – C21H20O13S
M13-18 8.22 22 511.0553 – C21H20O13S



Hesperidin Parent P14 14.37 – 609.1814 611.1979 C28H34O15
Deglycosylation (rutinose) + 2 × Glucuronide conjugation M14-1 11.23 41 653.1345 – C28H30O18
Glucuronide conjugation M14-2 11.60 46 785.2145 787.2285 C34H42O21
Deglycosylation (rhamnose) + Dehydrogenation M14-3 14.15 61 461.1087 463.1242 C22H22O11
Deglycosylation (rutinose) + Glucuronide conjugation M14-4 15.03 65 477.1036 479.1185 C22H22O12
Dehydrogenation + Demethylation M14-5 18.11 82 593.1506 595.1644 C27H30O15



Neohesperidin Parent P15 15.09 – 609.1842 611.1967 C28H34O15
Deglycosylation (neohesperidose) + 2 × Glucuronide conjugation M15-1 11.23 41 653.1345 – C28H30O18
Demethylation M15-2 11.43 43 595.1677 597.1825 C27H32O15
Deglycosylation (rhamnose) + Dehydrogenation M15-3 14.15 61 461.1087 463.1241 C22H22O11
Glucuronide conjugation M15-4 11.60 47 785.2145 787.2269 C34H42O21
Deglycosylation (neohesperidose) + Glucuronide conjugation M15-5 15.03 65 477.1036 479.1185 C22H22O12
M15-6 15.47 70 477.1034 – C22H22O12
Deglycosylation (neohesperidose) M15-7 13.04 52 301.0722 303.0876 C16H14O6
M15-8 13.23 55 301.0717 303.087 C16H14O6
M15-9 14.23 62 301.0722 303.0882 C16H14O6
Deoxidation M15-10 14.01 59 593.1874 – C28H34O14
Deglycosylation (rhamnose) + Sulfate conjugation M15-11 14.58 64 543.0828 – C22H24O14S



Liquiritigenin Parent P16 15.51 – 255.0659 257.0822 C15H12O4
Glucuronide conjugation M16-1 10.04 27 431.0979 433.1131 C21H20O10
M16-2 10.36 37 431.0979 433.1134 C21H20O10
Hydroxylation M16-3 15.38 68 271.0621 – C15H12O5
Hydroxylation + Glucuronide conjugation M16-4 10.09 29 447.0932 – C21H20O11
M16-5 13.11 54 447.0931 449.1082 C21H20O11
Methylation M16-6 18.62 88 269.0817 271.0978 C16H14O4
Sulfate conjugation M16-7 11.55 44 335.0225 337.0385 C15H12O7S
M16-8 12.47 50 335.0229 337.0376 C15H12O7S



Naringenin Parent P17 18.42 – 271.0602 273.0771 C15H12O5
Glucuronide conjugation M17-1 13.13 54 447.0930 449.1082 C21H20O11
Sulfate conjugation M17-2 15.40 69 351.0183 353.0337 C15H12O8S
Deoxidation + Sulfate conjugation M17-3 18.15 85 335.0230 337.0384 C15H12O7S
Methylation + Deoxidation M17-4 18.62 88 269.0817 271.0978 C16H14O4



Platycodin D Parent P18 19.25 – 1223.5681 1225.5792 C57H92O28
Deglycosylation (apiose + xylose + rhamnose + arabinose) M18-1 21.40 98 681.3850 – C36H58O12
Deglycosylation (apiose) + Methylation + Deoxidation M18-2 30.90 142 1089.5479 – C53H86O23



Isoliquiritigenin Parent P19 20.21 – 255.0669 257.0823 C15H12O4
Hydroxylation + Sulfation conjugation M19-1 10.27 34 351.0177 353.0334 C15H12O8S
Glucuronide conjugation M19-2 10.36 37 431.0979 433.1134 C21H20O10
Sulfate conjugation M19-3 12.47 50 335.0229 337.0376 C15H12O7S
Hydroxylation + Glucuronide conjugation M19-4 13.11 54 447.0931 449.1082 C21H20O11
M19-5 15.51 71 447.0931 449.1086 C21H20O11
Glucuronide conjugation M19-6 15.62 73 431.0982 433.1137 C21H20O10
M19-7 16.23 76 431.0978 433.113 C21H20O10
Sulfate conjugation M19-8 18.16 86 335.0230 337.0384 C15H12O7S
Methylation M19-9 18.62 89 269.0817 271.0978 C16H14O4



Formononetin Parent P20 20.77 – 267.0666 269.0825 C16H12O4
Glucuronide conjugation M20-1 16.11 74 443.0978 445.1135 C22H20O10
Hydroxylation + Glucuronide conjugation M20-2 13.10 53 459.0933 461.108 C22H20O11
M20-3 17.88 81 459.0924 461.108 C22H20O11
Reduction M20-4 18.62 90 269.0817 271.0978 C16H14O4
Sulfate conjugation M20-5 18.68 92 347.0228 349.0386 C16H12O7S



Ginsenoside-Ro Parent P21 21.39 – 955.4894 – C48H76O19
Deglycosylation (glucose) + Deglucuronidation + 2 × Dehydrogenation M21-1 20.69 97 613.3737 615.3871 C36H54O8
Deglycosylation (glucose) + Hydroxylation + Dehydrogenation M21-2 25.43 115 807.4169 – C42H64O15
Deglycosylation (glucose) + Deglucuronidation + Hydroxylation + Dehydrogenation M21-3 30.22 139 631.3857 – C36H56O9



18β-Glycyrrhizic acid Parent P22 22.37 – 821.3950 823.4107 C42H62O16
2 × Deglucuronidation + 3 × Hydroxylation M22-1 23.34 107 517.3167 519.3307 C30H46O7
2 × Deglucuronidation + Demethylation + Hydroxylation M22-2 24.13 112 471.3106 473.3261 C29H44O5
M22-3 25.58 116 471.3114 473.3272 C29H44O5
M22-4 25.73 119 471.3110 473.3272 C29H44O5
M22-5 26.10 122 471.3113 473.327 C30H44O5
M22-6 26.65 125 471.3113 473.3267 C29H44O5
M22-7 30.59 141 471.3115 473.3269 C29H44O5
Deglucuronidation + Demethylation + Hydroxylation M22-8 20.62 96 647.3424 649.3581 C35H52O11
2 × Deglucuronidation + Demethylation M22-9 23.38 108 – 457.332 C29H44O4
M22-10 30.23 140 455.3157 457.3316 C29H44O4
M22-11 31.01 143 455.3166 457.3317 C29H44O4
M22-12 31.31 144 455.3159 457.332 C29H44O4
2 × Deglucuronidation + Dehydrogenation M22-13 28.34 134 – 469.3318 C30H44O4
Deglucuronidation + Deoxidation + Dehydrogenation M22-14 23.88 110 659.3431 661.3585 C36H52O11
Hydroxylation M22-15 18.31 87 837.3928 839.4043 C42H62O17
2 × Deglucuronidation + Hydroxylation + Dehydrogenation M22-16 25.83 121 483.3111 485.3264 C30H44O5
M22-17 27.13 128 483.3103 485.3266 C30H44O5
M22-18 27.99 133 483.3132 485.3272 C30H44O5
M22-19 29.86 136 483.3112 485.3268 C30H44O5
Deglucuronidation + Hydroxylation + Dehydrogenation M22-20 22.96 104 – 661.3583 C36H52O11
2 × Deglucuronidation + Hydroxylation + Sulfation conjugation M22-21 22.93 103 565.2838 567.2986 C30H46O8S
M22-22 23.16 106 565.2840 567.2985 C30H46O8S
2 × Deglucuronidation + Hydroxylation M22-23 23.85 109 485.3260 487.3422 C30H46O5
M22-24 24.98 114 485.3271 487.3424 C30H46O5
M22-25 25.64 117 485.3273 487.3425 C30H46O5
M22-26 26.12 123 485.3271 487.3426 C30H46O5
M22-27 27.17 129 485.3259 487.3423 C30H46O5
M22-28 27.85 131 485.3275 487.3426 C30H46O5
M22-29 29.87 138 485.3259 487.3424 C30H46O5
Deglucuronidation + Hydroxylation M22-30 21.51 99 661.3586 663.3733 C36H54O11
2 × Deglucuronidation M22-31 32.40 146 469.3322 471.3491 C30H46O4
M22-32 32.69 149 469.3313 471.3472 C30H46O4
Deglucuronidation M22-33 26.81 127 645.3626 647.3785 C36H54O10
Deglucuronidation + Sulfate conjugation M22-34 22.99 105 725.3210 727.3356 C36H54O13S



Nobiletin Parent P23 23.12 – – 403.1392 C21H22O8
Hydroxylation M23-1 16.22 75 – 419.1338 C21H22O9
Hydroxylation + Glucuronide conjugation M23-2 18.11 84 – 595.1641 C27H30O15



Saikosaponin A Parent P24 24.17 – 779.4595 – C42H68O13
Deglycosylation (glucose + rhamnose) + Dehydrogenation M24-1 32.40 147 469.3332 471.3491 C30H46O4
M24-2 32.47 148 469.3318 471.3492 C30H46O4
M24-3 32.69 150 469.3313 471.3472 C30H46O4
M24-4 33.15 151 469.3311 471.3474 C30H46O4
Deglycosylation (glucose + rhamnose) + Hydroxylation + Dehydrogenation M24-5 24.89 113 – 487.3425 C30H46O5
M24-6 25.64 118 485.3273 487.3425 C30H46O5
M24-7 26.12 124 485.3271 487.3426 C30H46O5
M24-8 26.70 126 – 487.3424 C30H46O5
M24-9 27.17 130 485.3262 487.3423 C30H46O5
M24-10 27.85 132 485.3257 487.3426 C30H46O5
M24-11 29.18 135 485.3266 487.341 C30H46O5
M24-12 29.86 137 485.3264 487.3424 C30H46O5
Deglycosylation (glucose + rhamnose) M24-13 25.75 120 471.3467 473.3642 C30H48O4
Deglycosylation (glucose + rhamnose) + 2 × Dehydrogenation M24-14 32.31 145 467.3164 469.3309 C30H44O4
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P = prototype compound, M = metabolite, “−” undetected, “*” [M−H+HCOOH]-, “#” [M+Na]+.

Fig. 4.

Fig. 4

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Metabolic network of prototype compounds in XFZYD.

As follows, we introduced the identified metabolites and summarized metabolic pathways of prototype compounds derived from the different characteristics of structures.

3.3.1. Identification of metabolites from flavanones

In this study, 13 prototype flavonoids were identified in rat' serum after oral administration of XFZYD, which were mainly originated from Honghua, Gancao, Zhiqiao, and Danggui. With the aid of Metabolyxn (Masslynx V4.1 software), 71 metabolites from flavonoids were identified (Table 2 and Supplementary Table S2), which were derived from P2 (one metabolite), P8 (10 metabolites), P9 (six metabolites), P10 (11 metabolites), P12 (six metabolites), P13 (18 metabolites), P14 (five metabolites), P15 (11 metabolites), P16 (eight metabolites), P17 (four metabolites), P19 (nine metabolites), P20 (five metabolites) and P23 (two metabolites), respectively. By studying the metabolic pathways in detail, we found that flavonoids mainly undergo glucuronidation, sulfation deglycosylation, demethylation, and deoxidation reactions. For example, neohesperidin (P15) was modified into eleven metabolites (M15-1–M15-11) through above-mentioned metabolic pathways (Fig. 5).

Fig. 5.

Fig. 5

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Metabolic pathways of neohesperidin.

Glucuronidation: M15-4 showed a quasi-molecular ion at m/z 785.2145 in the negative ion mode, 176 Da heavier than neohesperidin at m/z 609.1842, suggesting that glucuronidation reaction has happened to neohesperidin. Fragment ions of M15-4 at m/z 301.0714, 151.0059, 149.0621, and 175.0255 were detected, whose fragmentation characteristics were similar to neohesperidin. The characteristic fragment ion of glucuronic acid was monitored at m/z 175.0255. This result indicated that the occurrence of glucuronidation conjugation to neohesperidin.

Deglycosylation + Glucuronidation: In the negative ion mode, M15-7, M15-8, and M15-9 showed quasi-molecular ion at m/z 301.0722 and were identified as aglycone of neohesperidin, suggesting that deglycosylation reaction has occurred to neohesperidin by intestine microbiota (Lin et al., 2020). M15-5 and M15-6 showed quasi-molecular ion both at m/z 477.1034, indicating that glucuronidation has occurred at the different hydroxyl group of hesperetin. The fragment ions of M15-5 and M15-6 were detected at m/z 301.0721, 151.0040 and 149.0617, which were similar to that of neohesperidin. M15-1 was identified as a product derived from the successive glucuronidation reaction to hesperetin, whose quasi-molecular ion was found at m/z 653.1345.

Sulfation: As a sulfated product, M15-11 showed a quasi-molecular ion at m/z 543.0828 and fragment ions at m/z 463.1240, 301.0385, 151.0044, and 149.0606 in the negative ion mode, indicating that sulfation reaction has happened to the metabolite generated from neohesperidin by losing a rhamnose.

Demethylation: M15-2 was tentatively identified as a demethylated product by comparing the quasi-molecular ion between M15-2 and neohesperidin, whose gap was 14 Da in the negative ion mode.

In addition, M15-10 was metabolized by neohesperidin through deoxidation reaction by loss of oxygen (16 Da) and showed a quasi-molecular ion at m/z 593.1874 in the negative ion mode.

In general, illumination of the proposed metabolic pathway about neohesperidin paved the way for studying the metabolites of other flavonoids. The identified metabolites of flavonoids are displayed in Table 2 and Supplementary Table S2.

3.3.2. Identification of metabolites from saponins

Based on the information of chromatographic behavior and MS data, four prototype compounds from saponins primarily derived from Jiegeng, Chuanxiong, Gancao, and Chaihu, were identified by comparing with standards, which were transformed into 53 metabolites, including two metabolites from P18, three metabolites from P21, 34 metabolites from P22, and 14 metabolites from P24 (Table 2 and Supplementary Table S2). Hydrolyzation, dehydrogenation, and hydroxylation were found to happen in the process of saponins metabolism. Give an example about how to identify metabolites, saikosaponin A underwent the mentioned metabolic pathways to produce 14 metabolites (M24-1–M24-14) in negative ion mode.

Compared to the quasi-molecular ion of saikosaponin A at m/z 779.4595, that of M24-13 was found to lose 308 Da and detected at m/z 471.3467, indicating that glucose and rhamnose were sequentially hydrolyzed from saikosaponin A to produce aglycone. The fragment ion at m/z 453.3000 was generated from M24-13 by losing H2O. The quasi-molecular ion of M24-1–M24-4 at m/z 469.3318 and the fragment ion at m/z 451.3203 were observed, which was inferred that M24-1–M24-4 were produced by dehydrogenation of M24-13. The successive dehydrogenation to M24-13 produced M24-14, whose quasi-molecular ion was monitored at m/z 467.3164 and a fragment ion at m/z 449.3234. The hydroxylation reaction occurred to M24-1–M24-4, which generated M24-5-M24-12 with the quasi-molecular ion at m/z 485.3264.

3.3.3. Identification of metabolites from phenolic acids

As phenolic acids derived from Dihuang, Danggui, Chuanxiong, and Jiegeng, P1, P4, and P7 were metabolized into two, five, and 11 metabolites detected in the dosed serum, respectively (Table 2 and Supplementary Table S2). The prominent metabolic reactions were hydroxylation and methylation for the tested compounds. For example, M1-2 showed a quasi-molecular ion at m/z 153.0195, which was 16 Da more than that of PHBA at m/z 137.0242 and produced the main fragment ions at m/z 109.0293 in the MS spectra, indicating that hydroxylation reaction has happened to PHBA. M1-1 was metabolized from PHBA by the sequential reaction of hydroxylation (16 Da) and methylation (14 Da), whose quasi-molecular ion was detected at m/z 167.0350.

3.3.4. Identification of metabolites from other prototype compounds

Originated from Niuxi, Taoren, and Chishao, β-ecdysterone, amygdalin, albiflorin, and paeoniflorin were identified and modified into four, three, three, and three metabolites detected in the serum after oral administration of XFZYD (Table 2 and Supplementary Table S2). Methylation, demethylation, acetylation, reduction, glucuronide conjugation, and sulfate conjugation were summarized from the identified metabolites. Taking M11-1–M11-4 metabolized from P11 as an example, M11-1 provided the quasi-molecular ion at m/z 539.3206, 14 Da more than that of β-ecdysterone at m/z 525.3080, suggesting that methylation reaction has occured. M11-2 displayed a quasi-molecular ion at m/z 511.3267 and the fragment ions at m/z 351.2148, 333.2022, and 205.0889, showing that reduction and deoxidation reactions have happened to β-ecdysterone. M11-3 was presumed to be the product of β-ecdysterone through demethylation and deoxidation reactions, whose quasi-molecular ion was monitored at m/z 495.2962. The quasi-molecular ion of M11-4 was detected at m/z 567.3167, indicating that acetylation reaction has generated to β-ecdysterone.

4. Conclusion

In this study, we have established a rapid and effective UPLC-Q-TOF/MS method to identify the prototype compounds and their metabolites in rat' serum after intragastric administration of XFZYD, from which 175 compounds (24 prototype compounds and 151 metabolites) were identified and tentatively characterized. Metabolic rules of compounds with the different type of structures were summarized from the relationship between the prototype compounds and their metabolites. This study is meaningful for clarification of the absorbed compounds in vivo. Also, this study has demonstrated the feasibility of UPLC-Q-TOF/MS application in exploring the prototype compounds and metabolites originated from compound formula.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by National Natural Science Foundation of China (No. 81873192 and 81202877) and Postgraduate Research and Innovation Project of Tianjin (No. 2019YJSS197).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.chmed.2022.08.002.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary data 1
mmc1.doc (344.5KB, doc)

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