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Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 7  |  Issue : 1  |  Page : 33-46

Eleven absorbed constituents and 91 metabolites of chuanxiong rhizoma decoction in rats


Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University, Beijing, China

Date of Submission09-Sep-2020
Date of Acceptance09-Oct-2020
Date of Web Publication8-Mar-2021

Correspondence Address:
Prof. Dong-Hui Yang
School of Pharmaceutical Sciences, Peking University, No. 38, Xueyuan Road, Haidian District, Beijing 100191
China
Prof. Shao-Qing Cai
School of Pharmaceutical Sciences, Peking University, No. 38, Xueyuan Road, Haidian District, Beijing 100191
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/wjtcm.wjtcm_7_21

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  Abstract 


Objective: Chuanxiong Rhizoma (CR, the dried rhizomes of Ligusticum chuanxiong) is a well-known traditional Chinese medicine that promotes qi to activate blood, dispels wind, and relieves pain. To date, more than 118 constituents of CR have been isolated and identified. However, the in vivo mechanism of CR decoction is unclear and needs further investigation. In addition, to clarify the effective forms of CR, it is essential to reveal the absorbed constituents and metabolites of CR. Materials and Methods: The absorbed constituents and metabolites in urine and plasma samples of rats orally administered with CR decoction were screened and characterized using a high-performance liquid chromatography with diode array detector and combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry technique. Results: In total, 102 compounds, including 11 absorbed constituents (eight phthalides and three phthalic acids) and 91 metabolites (71 phthalide-related and 20 phenolic acid-related), were detected in drug-containing rat urine and plasma samples, among which 33 were new metabolites of either CR or its constituents. Based on the structures of these metabolites, six phthalides (ligustilide, senkyunolide I/H, senkyunolide J/N, and butylidenephthalide) and three phenolic acids (ferulic acid, isoferulic acid, and caffeic acid) were proposed as their precursors. They were also deduced to be the main absorbed constituents of CR decoction, which should have closer relationships with its pharmacological effects than other constituents. Phthalide-related metabolites were formed through the metabolic reactions of hydration, hydroxylation, cysteine conjugation, acetylcysteine conjugation, methanethiol conjugation, mercaptomethanol conjugation, glucuronidation, and sulfation, whereas the phenolic acid-related metabolites were mainly formed by glucuronidation and sulfation. Conclusions: Six phthalides and three phenolic acids were shown to be the main precursors of the metabolites of CR, and 33 compounds were new metabolites of either CR or its constituents. These results are helpful for further understanding of the in vivo mechanism and effective forms of CR.

Keywords: Chuanxiong Rhizoma, effective forms, metabolites, phenolic acids, phthalides


How to cite this article:
Xu F, Zhang L, Zhao X, Zhou QL, Liu GX, Yang XW, Yang DH, Cai SQ. Eleven absorbed constituents and 91 metabolites of chuanxiong rhizoma decoction in rats. World J Tradit Chin Med 2021;7:33-46

How to cite this URL:
Xu F, Zhang L, Zhao X, Zhou QL, Liu GX, Yang XW, Yang DH, Cai SQ. Eleven absorbed constituents and 91 metabolites of chuanxiong rhizoma decoction in rats. World J Tradit Chin Med [serial online] 2021 [cited 2021 Apr 21];7:33-46. Available from: https://www.wjtcm.net/text.asp?2021/7/1/33/310928




  Introduction Top


Chuanxiong Rhizoma (CR), the dried rhizome of Ligusticum chuanxiong Hort., is a well-known traditional Chinese medicine (TCM). The medicinal history of CR can be traced back to the “Shengnong Bencao Jing” (Divine Husbandman's Classic of the Materia Medica), which was written over 2000 years ago. CR promotes qi to activate blood, dispels wind, and relieves pain according to the China Pharmacopoeia 2020 edition. Modern pharmacological studies have indicated that CR has the activities of sedation and abirritation and shows significant bioactivities in the cardiovascular and cerebrovascular systems.[1],[2],[3],[4] In the clinic, CR has been used for the treatment of coronary heart disease,[5] stroke,[6] migraine,[7] various types of pain,[8] atherosclerosis,[9] angina pectoris,[9] hypertension,[9] thrombus formation,[9] sepsis,[10] and gynecological diseases.[11]

The chemical constituents of CR can be divided into three main types: phthalides (e.g., ligustilide, senkyunolide A-S, butylphthalide, butylidenephthalide, neocnidilide, and phthalide dimers), phenolic acids (e.g., ferulic acid, isoferulic acid, and caffeic acid), and alkaloids (e.g., tetramethylpyrazine and pelolyrine).[1],[12],[13],[14],[15],[16],[17],[18] To date, more than fifty metabolites related to Z-ligustilide, butylphthalide, butylidenephthalide, ferulic acid, caffeic acid, and tetramethylpyrazine have been identified.[19],[20],[21],[22],[23],[24],[25],[26],[27],[28],[29],[30],[31],[32],[33],[34] However, there have been only two studies on the metabolism of CR decoction in vivo; 17 metabolites were isolated and identified from the methanol extract of urine samples of WZS-miniature pigs after the oral administration of CR decoction by our research group[35] and 12 conjugated metabolites related to senkyunolide I/J and butylidenephthalide were identified from rat plasma after the oral administration of CR (extracted with 70% ethanol) using high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS).[36]

To better understand the effective forms and action mechanism of CR, the absorption and in vivo metabolism of CR must be clarified. Therefore, in this study, the absorbed constituents of CR and their metabolites in the drug-containing urine and plasma samples of rats orally administered CR decoction were screened and characterized using an HPLC with diode array detector and combined with ESI ion trap time-of-flight multistage mass spectrometry (HPLC-DAD-ESI-IT-TOF-MSn) technique.


  Materials and Methods Top


Materials and reagents

CR was purchased from Yaoxing Medicinal Materials Company (Anguo, China) and identified as the dried rhizomes of L. chuanxiong Hort. by Dr. Dong-Hui Yang. A voucher specimen (No. 6332) was deposited in the Herbarium of Pharmacognosy, School of Pharmaceutical Sciences of Peking University.

Acetonitrile and methanol (HPLC grade) were purchased from Fisher Scientific (Fairlawn, NJ, USA), formic acid (AR grade) was purchased from Mreda Technology Inc.(Beijing, China), and deionized water was prepared using a Milli-Q system (Millipore, Billerica, MA, USA).

Preparation of CR decoction

The dried powders of CR (100 g) were immersed in water (1 L) for 1 h and then decocted for 1.5 h. After filtration, the residue was decocted again with 0.6 L of water for 1 h. The two filtered extracts were combined and concentrated to 140 mL (0.7 g crude drug/mL).

Animal protocols

Fourteen male Sprague-Dawley rats (250–300 g), purchased from the Department of Laboratory Animal Science of Peking University (Beijing, China), were randomly divided into two groups: drug-containing group (Group A, n = 7) and control group (Group B, n = 7). They were maintained in an environmentally controlled breeding room for 3 days. They were fasted for 12 h before the experiment but still had free access to water. The rats in Group A were orally administered the CR decoction at a dose of 5 g crude drug/kg three times (8:00 a.m., 8:00 p.m., and 8:00 a.m. of the next day), and an equal dose of distilled water was administered orally to the rats in Group B with the same administration schedule.

Urine samples were collected after the first drug administration and blood samples were taken from the heart, after the rats were anesthetized, 90 min after the third administration. The blood samples were collected into a sodium citrate tube and centrifuged (956 g, 15 min) to obtain plasma samples. All animal experiments were performed in accordance with the guidelines for Animal Experimentation of Peking University (Beijing, China), and the protocols were approved by the Animal Biomedical Ethical Committee of Peking University (Approval No. LA 2011-059).

Sample preparation

Urine samples (550 mL) were evaporated to approximately 5 mL by rotary evaporation under a vacuum at 50°C and extracted with 30 mL of methanol twice. After centrifugation (2,656 g, 30 min), the supernatants were combined, concentrated to approximately 5 mL and centrifuged for 30 min (12,000 g, 10°C). An 8-μL aliquot of the supernatant was injected into the HPLC-DAD-ESI-IT-TOF-MSn system for analysis.

Plasma samples (25 mL) were extracted with 25 mL of methanol twice using ultrasonication. After centrifugation (2,656 g, 20 min), the supernatants were combined, evaporated to dryness under nitrogen gas, dissolved in 1 mL of methanol-water (4:1, v/v) solution, and centrifuged for 30 min (12,000 g, 10°C). A 25-μL aliquot of the supernatant was injected into the HPLC-DAD-ESI-IT-TOF-MSn system for analysis.

Instruments and conditions

Chromatographic analyses were performed on a Shimadzu LC-20A (Shimadzu, Japan) chromatographic system equipped with two LC-20AD pumps, a micro degasser, a SIL-20AC autosampler, a CTO-20A thermostatically controlled column oven, an SPD-M20A PDA detector, and a CBM-20A system controller. A Phenomenex Gemini-C18 column (4.6 mm × 250 mm, 5 μm) was used for the analysis. The mobile phase consisted of 0.1% (v/v) formic acid-water (A) and acetonitrile (B) with the following gradient elution: 0–5 min, 3% B; 5–15 min, 3%–5% B; 15–25 min, 5%–8% B; 25–40 min, 8%–12% B; 40–70 min, 12%–18% B; 70–80 min, 18% B; 80–90 min, 18%–35% B; 90–95 min, 35%–60% B; 95–100 min, and 60%–100% B. The flow rate was 1.0 mL/min and 0.2 mL/min was split into the MS. The UV spectra were recorded from 190 to 700 nm.

The MS analysis was performed on a hybrid ion trap-time of flight mass spectrometer (Shimadzu LCMS-IT-TOF, Shimadzu, Japan) with an ESI source. The conditions were as follows: nebulizing nitrogen gas flow, 1.5 L/min; curved desolvation line and heat block temperature, 200°C; interface voltage, 4.5 kV (+)/3.5 kV (−); detector voltage, 1.70 kV; and ion accumulation time, 20 ms. All operations were performed using Shimadzu LCMSsolution (version 3.60) and Formula Predictor software (version 1.01) (Shimadzu Technologies, Japan). The mass, ranging from 50 to 1000 Da, was calibrated with trifluoroacetic acid sodium solution (2.5 mM). The data were collected in full scan mode over m/z 100–700 for MS1, and m/z 50–700 for MS2 and MS3 in positive ion (PI) and negative ion (NI) detection modes.

The peak area of each metabolite detected was calculated based on its extracted ion chromatogram (EIC). The relative content of each metabolite was calculated as the percentage of its peak area versus the total peak area of all identified compounds (except U56 in urine, which is too high). For the metabolites detected only in PI or NI mode, their relative contents were calculated based on their corresponding PI EICs or NI EICs; for those detected under both PI and NI modes, their content was calculated based on their EICs with higher responses.


  Results and Discussion Top


Identification of the characteristic compounds in drug-containing urine samples

The molecular weights of the characteristic compounds were determined based on pseudomolecular ions [M+H]+/[M+NH4]+ in positive-ion mode and [M−H] in negative-ion mode. The molecular formulae of the metabolites were predicted using Formula Predictor (ver. 1.01),[37] which included mass accuracy (<5 ppm), nitrogen rule, double-bond equivalent index, and isotopic pattern.

Using our previous strategy,[38] ninety characteristic compounds were detected in the drug-containing urine sample [Figure 1] and [Table 1], and the structures of 86 compounds, including 8 absorbed constituents and 78 metabolites, were tentatively identified. Among them, 32 metabolites are new metabolites of CR.
Figure 1: The typical BPCs (Base peak chromatograms) of the samples analyzed by high performance liquid chromatography with diode array detector and combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry under negative ion detection mode (a, drug-containing urine; b, blank urine; c, drug-containing plasma; d, blank plasma; e, Chuanxiong Rhizoma decoction). The 90 peaks of 1–90 indicated the peaks detected in the drug-containing urine; eight of them (18, 21–24, 29, 57, 69) were prototype constituents from the Chuanxiong Rhizoma decoction, and twenty of them (23–25, 29, 34–35, 79, etc.,) also detected in the drug-containing plasma. The 16 peaks of P1–P2, P6–P7, P9, P32–P33, etc., indicated the peaks only detected in the drug-containing plasma

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Table 1: Liquid chromatography - mass spectrometry data of 90 absorbed constituents and metabolites in drug-containing urine after oral administration of Chuanxiong Rhizoma decoction

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The eight prototype constituents were deduced as senkyunolide J/N or its isomers (U21, U22, U23, and U29), senkyunolide D/4,7-dihydroxy-3-butylphthalide or its isomers (U18, U57, and U69), and phthalic acid or its isomers (U24) [Table 1]. The metabolites could be categorized into two groups: 67 phthalide-related metabolites and 11 phenolic acid-related metabolites.

Identification of phthalide-related metabolites

In total, 67 compounds were detected as phthalide-related metabolites including 41 senkyunolide-like metabolites [Figure 2]a], 2 ligustilide-like metabolites [Figure 2]b, and 24 butylidenephthalide-like metabolites [Figure 2]c based on their mass data and UV spectra information.[31],[35],[39],[40],[41] The senkyunolide-like metabolites showed UV λmax at 277 nm [Figure 2]a, whereas the ligustilide-like metabolites exhibited UV λmax at 230 and 287 nm [Figure 2]b, and the butylidenephthalide-like metabolites exhibited UV λmax at 229 and 280 nm, with a shoulder peak at 320 nm [Figure 2]c.
Figure 2: Typical structures and ultraviolet spectra of metabolites (a, senkyunolide-like metabolites; b, ligustilide-like metabolites; c, butylidenephthalide-like metabolites; d, phenolic acid-like metabolites) and positive ion MS2 spectrum of U1 (e)

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The senkyunolide-like metabolites had similar chemical structures to senkyunolide I/H ( m/z 225.11, C12H17O4) and senkyunolide J/N ( m/z 227.12, C12H19O4). Senkyunolide I/H-like metabolites showed the characteristic fragment ions of senkyunolide I/H at m/z 225.11, 207.10 [C12H17O4−H2O]+, and 189.09 [C12H17O4−2H2O]+ in positive-ion mode, whereas the characteristic fragment ions of senkyunolide J/N-like metabolites were 2 Da higher than those of senkyunolide I/H, that is, at m/z 227.12, 209.11 [C12H19O4−H2O]+, 191.10 [C12H19O4−2H2O]+, and 163.11 [C12H19O4−H2O−CO]+ [Figure 2]e. Their specific structural types were inferred by their characteristic neutral losses. For example, glucuronide conjugates have a neutral loss of 176.03 Da (C6H8O6), and sulfate conjugates have a neural loss of 79.96 Da (SO3). The neutral losses of 163.03 Da (C5H9NO3S), 121.02 Da (C3H7NO2S), 48.00 Da (+HSCH3), and 64.00 Da (+HSCH2OH) indicated acetylcysteine, cysteine, methanethiol, and mercaptomethanol, respectively.[21] Accordingly, the structures of 41 senkyunolide-like metabolites were tentatively identified including 24 new metabolites that were reported as the metabolites of CR for the first time [Table 1] and [Figure 3]. The senkyunolide-like metabolites (accounting for 33% of the total content) were thought to originate from senkyunolides I/H, senkyunolides J/N, and their isomers in CR.
Figure 3: Senkyunolide-like metabolites identified in the drug-containing urine and the proposed metabolic pathways (*New metabolites)

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Two ligustilide-like glucuronides (U74 and U86), which exhibited the diagnostic ions of hydroxyligustilide at m/z 209.11 ([C12H15O2 + H2O]+), 191.10 (C12H15O2), and 163.11 [C12H15O2−CO]+ in the MS2 spectrum of [M+NH4]+ at m/z 402.17, as well as characteristic fragment ion of the glucuronide residue at m/z 175.02 (C6H8O6) in the MS2 spectrum of [M−H]+ at m/z 383.13, were detected. Because hydration is generally proposed to occur at C3–C8, they were tentatively identified as 3-hydroxysendanenolide A-3- O-glucuronide[39],[40] and its isomer. The concentration of ligustilide, which is the major constituent in the essential oil of CR,[42] was lower in the CR decoction; therefore, only two ligustilide-related metabolites (accounting for 5.4% of the total content) were detected in the drug-containing urine sample [Table 1] and [Figure 4]. According to previous studies,[43],[44] ligustilide is unstable at high temperature and under light exposure and rapidly degrades; thus, we deduced that the ligustilide in CR was most likely transformed into senkyunolides I/H or their isomers after being decocted.
Figure 4: Butylidenephthalide-like and ligustilide-like metabolites identified in the drug-containing urine and the proposed metabolic pathways (*New metabolites

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Butylidenephthalide-like metabolites yielded the fragment ions of butylidenephthalide at m/z 189.09 (C12H13O2) and hydrated butylidenephthalide at m/z 207.10 [C12H13O2+H2O]+. If the monohydroxylation of butylidenephthalide occurred at C-6 or C-7, the fragment ion at m/z 205.08, which was 16 Da (O) higher than that of butylidenephthalide, would be observed in the MS2 spectrum. Furthermore, if dihydroxylation occurred at C-6 and C-7, the fragment ion at m/z 221.08, which was 32 Da higher than that of butylidenephthalide, would be observed.[22] When the compounds were conjugated with mercaptomethanol, it yielded the diagnostic neutral loss of HSCH2OH (64 Da). Hence, 24 butylidenephthalide-like metabolites, which accounted for 18.7% of the total content, including 8 new metabolites, were identified [Table 1] and [Figure 4]. They were mainly formed through hydration, hydroxylation, acetylcysteine conjugation, glucuronidation, sulfation, and mercaptomethanol conjugation.

Isomers were common for most phthalides in CR owing to the chiral centers in their structures. Therefore, there were many isomers for phthalide-type metabolites, which were formed not only because of the multiple chiral carbons but also owing to the multiple substituent positions. For example, four prototype constituents were detected as senkyunolides J/N and their isomers from the CR decoction, and ten isomers of mercaptomethanol conjugates of 3-hydroxysenkyunolides J/N were detected in the drug-containing urine sample [Figure 3] and [Table 1]. Nevertheless, the exact structure identification of these isomers was beyond the capacity of the HPLC-DAD-ESI-IT-TOF-MSn technique.

Identification of phenolic acid-related metabolites

Fifteen phenolic acid-related metabolites were detected, which showed UV λmax at 236 nm and 329 nm, with a characteristic shoulder peak at 295 nm[45] [Figure 2]d. Among these, 11 were identified including six ferulic acid-related and five caffeic acid-related metabolites [Table 1] and [Figure 5].
Figure 5: Ferulic acid-like and caffeic acid-like metabolites identified in the drug-containing urine and the proposed metabolic pathways

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Ferulic acid-like metabolites produced a series of fragment ions of ferulic acid at m/z 193.05 (C10H9O4), m/z 178.03 [C10H9O4−CH3], m/z 149.06 [C10H9O4−CO2], and m/z 134.04 [C10H9O4−CO2−CH3] in the MS2 spectrum. Caffeic acid-like metabolites yielded the diagnostic fragment ions of caffeic acid at m/z 179.03 (C9H7O4) and m/z 135.04 [C9H7O4−CO2].[27],[28],[29] Therefore, six ferulic acid-like metabolites and five caffeic acid-like metabolites were identified. The content of these compounds (especially for U56, sulfated ferulic acid) was generally higher than that of other metabolites, which indicated that ferulic acid was easily absorbed into the blood and that the sulfated form may greatly promote its excretion. The caffeic acid-like metabolites, accounting for 7% of the total content, might originate from dicaffeoylquinic acids or caffeoylquinic acids (easily hydrolyzed to caffeic acid and quinic acid by colonic microflora) in CR.[19],[46],[47] Glucuronidation and sulfation were the two major metabolic pathways for phenolic acid-related metabolites.

The structures of four phenolic acid-related metabolites (U7, U15, U44, and U58) are still unknown because of insufficient fragment ion information.

Identification of the characteristic compounds in the drug-containing plasma samples

Thirty-six compounds were tentatively identified from the drug-containing plasma samples using the same method and strategy including six prototype constituents (P1, P3, P4, P7, P8, and P21) that were present in CR and 30 metabolites (14 phthalide-related metabolites and 16 phenolic acid-related metabolites) [Table 2], among which three prototype constituents (P3, P4, and P8) and 17 metabolites (ten phthalide-related metabolites and seven phenolic acid-related metabolites) were also detected in the urine samples.
Table 2: Liquid chromatography-mass spectrometry data of 36 absorbed constituents and metabolites in drug-containing plasma after oral administration of Chuanxiong Rhizoma decoction

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Similar to the metabolites detected in the urine samples, phthalide-related and phenolic acid-related metabolites were the predominant metabolites in the plasma samples. The phthalide-related metabolites (accounting for 27% of the total content), mainly, in the form of glucuronidation, were deduced to be derived from the prototype constituents of senkyunolides J/N, senkyunolides I/H, and hydrated butylidenephthalide present in CR. The phenolic acid-related metabolites (accounting for 73% of the total content) included (iso) ferulic acid glucuronides/sulfates (63%) and caffeic acid glucuronides/sulfates (7%). Consistent with the urine samples, ferulic acid sulfates with a relative content of 53% were the major metabolites in the plasma samples.

Compared with the metabolites identified from the urine samples, 16 additional compounds were detected in the plasma samples [Table 2]. They were three prototype constituents (P21: senkyunolides J/N or isomer; P1 and P7: phthalic acid or isomer), four phthalide-related glucuronides (P20: senkyunolide J/N-6/7- O-glucuronide; P25: senkyunolide I/H-6/7- O-glucuronide; P27: 3-hydroxybutylphthalide-3- O-glucuronide; P32: 6,7-dihydro-6/7-hydroxybutylidenephthalide-6/7- O-glucuronide), and nine phenolic acid-relatedmetabolites (P15: caffeic acid-1- O-sulfate; P17 and P18: isocaffeic acid-3′/4′- O-sulphate; P33: ferulic acid-4′- O-sulfate, isoferulic acid-3′- O-sulfate, or 1-methoxyl-caffeic acid-3′/4′- O-sulfate; P2, P6, P9, P11, and P13: 1-methoxyl-caffeic acid-3′/4′- O-glucuronide, isoferulic acid-1- O-glucuronide, and 1-methoxyl-isocaffeic acid-3′/4′- O-glucuronide).

Overall metabolite profile of Chuanxiong Rhizoma decoction in vivo

In total, 102 compounds, including 11 absorbed constituents and 91 metabolites, were detected in the rats. Seventy-eight metabolites were tentatively identified from the drug-containing urine samples including 67 phthalide-related metabolites (41 senkyunolide-like, 24 butylidenephthalide-like metabolites, and two ligustilide-like metabolites) and 11 phenolic acid-related metabolites (six ferulic acid-like and five caffeic acid-like metabolites) [Figure 6] and [Table 1]. Among these, 17 metabolites were also detected in the plasma samples (10 phthalide-related metabolites and seven phenolic acid-related metabolites). Furthermore, 13 additional metabolites were identified from the drug-containing plasma samples (four phthalide-related metabolites and nine phenolic acid-related metabolites) [Figure 6] and [Table 2]. Among the identified 91 metabolites, 33 were reported as the metabolites of either CR or its constituents for the first time [Table 1] and [Table 2].
Figure 6: The numbers and percentages of each type of metabolites identified in the drug-containing samples (a, urine; b, plasma)

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Phthalide-related metabolites were abundant in the urine (67/78) and plasma (14/30) samples; therefore, phthalides were the most important absorbed constituents in CR. These compounds underwent hydration, hydroxylation, cysteine conjugation, acetylcysteine conjugation, methanethiol conjugation, mercaptomethanol conjugation, glucuronidation, and sulfation. Phenolic acid-related metabolites, which were also major metabolites of CR in the urine (11/78) and plasma (16/30) samples, mainly, underwent glucuronidation and sulfation. Apart from ferulic acid-4′- O-sulfate (U56, the content of which was too high to determine), the relative content of phenolic acid-related metabolites versus that of all metabolites reached 37% in the urine sample and 73% in the plasma sample [Table 1] and [Table 2]. Nine constituents in CR were deduced to be the precursors of the identified metabolites, namely, senkyunolide I/H, senkyunolide J/N, butylidenephthalide, ligustilide, ferulic acid, isoferulic acid, and caffeic acid. They were considered as the most important constituents that contribute to the pharmacological effects of CR decoction. The vasodilator and antiproliferative effects of ligustilide,[48],[49] antianginal effect of butylidenephthalide,[50] and antioxidant effect of senkyunolide H/I[51] have been well documented. Ferulic acid and caffeic acid have also been reported to have various biological activities such as antioxidant activity[52] and anti-carcinogenicity.[53] Therefore, further studies on the pharmacokinetics and cotherapeutic effects of the relevant absorbed constituents are necessary to understand the effects of CR.

An interesting phenomenon observed in this study was that most metabolites were in the form of different isomers. For example, there were ten isomers of 3-hydroxy-senkyunolide J/N-6/7-mercaptomethanol, ten isomers of 6,7-dihydro-6/7-hydroxybutylidenephthalide-6/7- O-glucuronide, eight isomers of 3-hydroxy-senkyunolide J/N-6/7- S-acetylcysteine, and five isomers of senkyunolide J/N [Table 1] and [Table 2]. Because the multiconstituents in CR decoction could be metabolized into many metabolites (including their isomers), based on the results of the present study, we believe that many types of compound (including both prototype constituents and their metabolites) entered the body after the administration of CR decoction, and that their blood concentrations may be too low to reach an effective level. However, compounds with similar structures are likely to have the same pharmacological effects on the same target and when the concentrations of many of these compounds are superimposed to reach a higher and effective concentration, they can produce a synergistic healing effect.[54],[55] This mechanism is worth investigation in future research to provide a scientific explanation for the phenomenon that effective substances in TCMs produce curative effects in vivo, even at low blood concentrations.


  Conclusions Top


Eleven absorbed constituents and 91 metabolites of Chuanxiong Rhizoma were detected in rats, and six phthalides and three phenolic acids were the main precursors of these metabolites. Thirty-three compounds were new metabolites of either CR or its constituents. These findings provide a solid basis for discovering the effective forms of CR in future.

Financial support and sponsorship

This work was financially supported by the National Science and Technology Major Project for Major New Drugs Innovation and Development of China (No. 2009ZX09502-006, No. 2009ZX09301-010, No. 2019ZX09201004-001-023), and the National Natural Science Foundation of China (Grant No. 30830120; 81473321).

Conflicts of interest

There are no conflicts of interest.



 
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