Chemical Fingerprint and Metabolic Profile Analysis of Tianshu Tablets by Ultra-High Performance Liquid Chromatography/Quadrupole-Time of Flight Mass Spectrometry

Chen, Lin; Chen, Renhao; Ouyang, Hui; Wang, Qi; Li, Zhifeng; Feng, Yulin; Yang, Shilin

doi:https://doi.org/10.1155/2019/9158942

Journal of Analytical Methods in Chemistry

On this page

Abstract Introduction Experimental Results and Discussion Conclusion Data Availability Conflicts of Interest Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2019 | Article ID 9158942 | https://doi.org/10.1155/2019/9158942

Chemical Fingerprint and Metabolic Profile Analysis of Tianshu Tablets by Ultra-High Performance Liquid Chromatography/Quadrupole-Time of Flight Mass Spectrometry

Lin Chen,¹Renhao Chen,¹Hui Ouyang,²Qi Wang,¹Zhifeng Li,¹Yulin Feng,²and Shilin Yang²

Academic Editor: Eliseo Herrero-Hernández

Received29 Jun 2019

Accepted03 Oct 2019

Published23 Oct 2019

Abstract

In recent years, the chemical fingerprinting of traditional Chinese medicines and the metabolites in these compounds has been a hot topic. In the present study, the chemical fingerprint of Tianshu tablets (TST) and the metabolic characteristics of compounds in rats after intragastric administration were studied by ultra-high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UPLC/Q-TOF MS). In a preliminary study, 77 chemical components in TST were determined by comparison with retention times, accurate molecular mass, and characteristic fragment ions of the known compounds in the literature and some well-known compounds were analyzed in detail, and the fragmentation pathways for parishins B, gastrodin A, and cnidilide or neocnilide were specifically analyzed. After intragastric administration of TST (4 g/kg) to rats, a total of 61 compounds were detected in plasma samples, including 7 prototypes and 54 metabolites. After further analysis, it was found that these metabolites were subjected to glucuronidation, sulfation, methylation, hydroxylation, dehydrogenation, or mixed metabolic processes. Hydroxylation and glucuronidation were finally confirmed as the main metabolic pathways. This is the first research on the chemical fingerprint and metabolites of TST, which lays a foundation for further investigation of TST.

1. Introduction

In recent years, traditional Chinese medicine (TCM) has attracted increasing attention worldwide by virtue of its applications. Da Chuanxiong Formula (DCXF) is a well-known and extensively used traditional Chinese medicine (TCM) decoction for the treatment of migraine caused by blood stasis and wind-heat syndrome. It is composed of two herbs, namely, Chuanxiong (Chuanxiong rhizoma) and Tianma (Gastrodiae rhizoma), with a crude weight ratio of 4 : 1. Tianshu tablets (TST) are a representative DCXF preparation that is widely used in clinics for treating the blood stasis type of headache and migraine [1–3].

Phytochemical and pharmacological investigations of DXCF have shown that phenols, organic acids, phthalides, and nitrogen-containing compounds are the major active ingredients [4]. At present, several qualitative studies on the main components of DCXF have been performed [5–8]. One study used LC-Q-TOF/MS to identify 17 different components in a 50% ethanol extract of DCXF [5]. In one study, three compounds of Chuanxiong and eight components of Tianma were identified by HPLC-DAD-MSⁿ [6]. Two continuous studies showed that 10 different compounds were detected in rat plasma after intragastric administration of DCXF active components, including 6 compounds from Chuanxiong and 4 compounds from Tianma [7, 8]. These four studies were based on samples of a 50% ethanol extract from a 4 : 1 mixture of the two herbs or active ingredients from a single crude herb. In one study, 38 components were identified or preliminarily identified from a Tianshu capsule by means of HPLC, LC-DAD-MSN, and LC-DAD-ESI IT-TOF/MS analysis, although Tianshu tablets and Tianshu capsules are two different dosage forms [9]. This research is still very important because of its different applicability. This study enriches our understanding of the components of DCXF and studies the metabolites of TST for the first time.

In our study, 77 chemical components of TST were preliminarily determined by a comparison with retention time, accurate molecular mass, and characteristic fragment ions of known compounds in the literature. Furthermore, UPLC/Q-TOF MS was used to analyze the plasma of rats after oral administration of TST. A total of 61 compounds were identified or preliminarily identified, including 7 prototypes and 54 metabolites.

2. Experimental

2.1. Chemicals and Materials

Some reference standards (pyroglutamic acid, 5-(hydroxymethyl)furoic acid, and parishin B) for Gastrodia rhizoma were isolated and purified in our laboratory, and other standards (uridine, gastrodin A, and neocnilide) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Purities of the standards were above 98% by HPLC analysis. HPLC-grade acetonitrile, methanol, and formic acid were purchased from Fisher Scientific (MA, USA). Deionized water was prepared by a Milli-Q Water purification system (Millipore, MA, USA). High-purity nitrogen (99.99%) and helium (99.999%) were purchased from Gas Supplies Center of Peking University Health Science Center (Beijing, China).

Gastrodia rhizoma and Chuanxiong rhizoma were purchased from Tian Heng pharmacy (Beijing, China). All herbal materials were authenticated by Professor Bei Wu (Nanchang Institute for Food and Drug Control). TianShu tablets were prepared according to Chinese Pharmacopoeia 2015 Edition [1].

2.2. Animals and Drug

Sprague–Dawley rats (male, 12–14 weeks; 200–240 g) were provided by Hunan SJA Laboratory Animal Co., Ltd. Protocols for all animal experiments were approved. Animals were kept in a controlled environment for 3 days and fasted for 12 h before experiments. TST were dissolved in a 9 g/L NaCl solution (NS) (250 mg/ml) and administered by oral gavage at a dose of 1000 mg/kg (equivalent to 4 g of crude drug per kg) body weight.

2.3. Sample Collection and Pretreatment

After oral administration of TST, blood samples were collected at 30, 60, and 120 min (n = 5) in an Eppendorf tube with heparin sodium and then centrifuged (16000 rpm) at 4°C for 10 min. The supernatant was then separated, and all samples were stored at −80°C immediately until analysis. The protocol for sample preparation is described below: 1 mL plasma was mixed with 5 mL methanol, vortexed for 5 min, and centrifuged at 16000 rpm at 4°C for 20 min. The supernatant was dried with nitrogen gas at 4°C, and 400 μl of 50% methanol was added to reconstitute the residue, which was then centrifuged at 16000 rpm for 10 min at 4°C. The supernatant was transferred to a vial, and 10 μl was injected for LC-MS analysis. All samples were filtered through a membrane (0.22 μm pore size). At the same time, in order to eliminate the influence of matrix, blank plasma was added to participate in the analysis.

2.4. UPLC/QTOF-MS Conditions

UPLC/QTOF-MS analysis was performed on a Shimadzu LC-30 AD system (Kyoto, Japan) coupled with an AB SCIEX Triple-TOF 5600 mass spectrometer (Foster City, CA, USA). All samples were separated on an Acquity UPLC C18 column (100 mm × 2.1 mm, 1.7 μm, Waters, USA) with a flow rate of 0.3 mL/min at 40°C. The mobile phase consisted of aqueous 0.1% formic acid (A) and 0.1% formic acid in acetonitrile (B). The gradient elution program for TST was set at 0–3.0 min, 5–8% B; 3.0–10.0 min, 8–15% B; 10.0–18 min, 15–20% B; 18–22 min, 20–35% B; 22–37 min, 35–45% B; 37–43 min, 45–95% B; and 43–48 min, 95–95% B. The gradient elution program for the plasma samples was set at 0–2.0 min, 5% B; 2.0–25.0 min, 5–95% B; and 25.0–30.0 min, 95–95%. The equilibration time was 5 min. The conditions for the ion source were as follows: compounds in TST were measured using the total ion chromatograms in negative and positive ion ESI-MS mode in the mass range m/z 50–1250, but plasma samples were analyzed only in positive ion ESI-MS mode. The other operating parameters were optimized as follows: source temperature, 500°C; ion spray voltage, 4500 V; gas 1, 50 psi; gas 2, 50 psi; curtain gas, 45 psi; decluttering potential, 100 V; and collision energy was set to 40 (15) eV.

2.5. Data Process

TST compounds from the extracts and metabolites data were acquired by full scan, which rely on dynamic background subtraction (DBS) and multiple mass defect filtering (MMDF) and includes some compounds with very low concentrations (MDF window was set to ±50 mDa around the mass defects of the templates and over a mass range of ±50 Da around the filter template masses).

Analysis of data on TST compounds in extracts and metabolites was performed using a variety of data mining tools, including extract ion chromatograms (XIC) of PeakView®1.6 (AB SCIEX, CA, USA), MMDF, and NLF&DPLs of Metabolitepilot™ 1.5 (AB SCIEX, Foster City, CA, USA). All compounds were analyzed after removal of the matrix effects.

3. Results and Discussion

3.1. Optimization of LC/MS Conditions

In order to obtain the best analytical data, our analysis builds upon another recent study [10]. The separation conditions, supplements, and chromatographic columns were optimized at the beginning of the experiment. Firstly, in order to obtain sharp peaks and reduce the pressure on the UPLC column, methanol was used as the mobile phase instead of acetonitrile. At the same time, 0.1% formic acid was added to improve peak shape and ionization of the analytes. The gradient was improved, and it was shown that the compounds in TST could be separated within 48 minutes, while plasma samples could be separated within 30 minutes (the specific methods can be found in Section 2.4). In addition, in order to obtain the most abundant mass spectrometry information, the collision energy was optimized. The results showed that when the collision energy rose to 40 eV, the main fragments were seen, but when the energy reached 55 eV, the second order fragments were too fragmented to be easily analyzed. Therefore, a collision energy of 40 eV was selected. As for UPLC/Q-TOF MS, mass spectra were recorded in both positive and negative detection modes.

3.2. UPLC/Q-TOF MS Analysis of TST Extracts

To characterize the chemical constituents of TST, a fast, efficient, and reliable UPLC/Q-TOF MS method was established. By virtue of the high resolution and speed of UPLC and the accurate mass measurement of the TOF MS, a total of 77 compounds were identified. The mass spectra of these components were examined in negative ion mode and positive ion mode. The total ion chromatogram (TIC) of TST in positive and negative ion modes are shown in Figures 1 and 2. Details of the identified components are summarized in Table 1.

Through analysis, it was found that the 77 compounds contained 19 organic acids, 9 nitrogen-containing compounds, 11 glucosides, 8 phenols, 24 phthalides, and 6 other compounds. The numbering information of these compounds is shown in Figure 3.

3.2.1. Chemical Fingerprint of TST in Negative Ion Modes

According to the literature, the main components of Tianma are phenols and organic acids [9]. However, there are also glycosides in the components of Tianma [12]. Many characteristic components of Tianma were analyzed and identified in the negative ion mode. Because the structures of organic acids and phenols are relatively simple, the characteristic glycoside compounds X18 and X23 were identified here and the chromatographic and spectral data for compounds X18 and X23 were preliminarily characterized by referring to the literature and reference materials.

Peak X18 gave an [M−H]⁻ ion at m/z 447.1508. Peak X18 produced MS² base peaks at m/z 269.1028 and 161.0449 corresponding to [M-H-179 Da]⁻. This suggests that Peak X18 may contain a glucose group and a fructose group. Therefore, we deduced that the molecular structure likely contains sucrose. According to literature reports [12] and reference standards, we identified Peak X18 as gastrodin A. Peak X23 gave a [M-H]⁻ ion at m/z 727.2091 and had characteristic fragment ions at m/z 459.1156, 441.1045, 423.0937, 397.1142, 369.1188, and 217.0496. Based on previous studies [12] and a reference standard, Peak X23 was identified as parishin B. The characteristic fragmentation patterns of gastrodin A and parishin B are described in Figures 4(a) and 4(b).

(a)

(b)

(c)

3.2.2. Chemical Fingerprint of TST in Positive Ion Modes

The analysis of the positive ion mode results showed that the characteristic components of Chuanxiong, including phthalides, were present. Here, compound Y27 was selected for analysis, and the chromatographic and spectral data of this compound were analyzed by comparison with the literature and reference materials. The cleavage pathway of phenyl peptides in Chuanxiong was also analyzed.

Peak Y27 gave a [M+H]⁺ ion at m/z 195.1378 and fragment ions at m/z 177.1344, 149.1309, and 107.0550. According to previous literature reports [9] and a reference standard, we identified peak Y27 as cnidilide or neocnilide. The characteristic fragmentation pattern of Y27 is shown in Figure 4(c)

According to our analysis, the main components of Tianma in negative ion mode were organic acids, phenols, and glycosides, with mainly phthalides detected in positive ion mode. The specific pyrolysis fragments were similar to the standards.

3.3. Detection and Identification of the Metabolites of TST in Rat Plasma

In order to identify as many potential pharmacologically active compounds as possible in TST, metabolic profiling of TST in rat plasma was performed. Compounds absorbed in vivo can be further metabolized by a variety of enzymes through oxidation, hydrolyzation, methylation, glucuronidation, and sulfation. Only peaks that were detected in the dosed plasma samples but not in blank samples were considered as probable metabolites. The mass spectra of the metabolites were examined in positive ion mode. These were further analyzed by using Peakview 1.2 to identify expected and unexpected metabolites from different metabolic pathways, and their structures were identified by tandem MS. We selected senkyunolide D or 4,7-dihydroxy-3-butylphthalide and senkyunolide A as examples of the structural identification process. The metabolites of these compounds and others are summarized in Table 2, and their TIC and extract ion chromatogram (EICs) are shown in Figures 5–8.

Metabolite M1, which eluted at 9.75 min, formed a molecular ion of [M+H]⁺ at m/z 223.0963 corresponding to C₁₂H₁₄O₄. M1 was found to have major fragment ions in common with senkyunolide D, so M1 is most likely senkyunolide D or 4,7-dihydroxy-3-butylphthalide. Metabolite M2, which eluted at 10.78 min, formed a molecular ion of [M+H]⁺ at m/z 253.1071 corresponding to C₁₃H₁₆O₅. The characteristic production of m/z 235.0963 and 221.0839 was generated by loss of 18 Da and 18 + 14 Da, which implied loss of a H₂O group and methyl group. Other product ions were identical to that of M1. Therefore, M2 may be a metabolite of senkyunolide D after hydroxyl and methyl conjugation. Metabolite M3, which eluted at 9.2 min, formed a molecular ion of [M+H]⁺ at m/z 303.0535 corresponding to C₁₂H₁₄SO₇. Its major fragment ions at m/z 285.0430 and 205.0858 were generated by the loss of 18 Da and 18 + 80 Da, which implied a H₂O group and sulfate group. Other product ions were identical to that of M1. Therefore, M3 may be a metabolite of senkyunolide D following sulfation. Metabolite M4, which eluted at 10.6 min, formed a molecular ion of [M+H]⁺ at m/z 399.1285 corresponding to C₁₈H₂₂O₁₀. Its major fragment ion (m/z 223.0969) was generated by a loss of 176 Da, which implied a glucuronide group. Other product ions were identical to that of M1. Therefore, M4 might be a metabolite of senkyunolide D after glucuronidation. Metabolites M5 and M6 appear to correspond to M4 plus 2 Da or 16 Da, respectively. The product ions m/z 225.4427 and m/z 227.0584 have both lost 176 Da. Therefore, M5 might be a metabolite of senkyunolide D after hydrogenation and glucuronidation, while M6 might be a metabolite of senkyunolide D after hydroxylation and glucuronidation (metabolites of M1 and extract ion chromatograms (EICs) are shown in Figure 6).

Metabolite M27, which eluted at 14.81 min, formed a molecular ion of [M+H]⁺ at m/z 193.1222 corresponding to C₁₂H₁₆O₂. A major fragment ion was shared with senkyunolide A, suggesting that M27 is prototype of senkyunolide A. Metabolite M28, which eluted at 14.81 min, formed a molecular ion of [M+H]⁺ at m/z 223.1328 corresponding to C₁₃H₁₈O₃. Its major fragment ions m/z 205.1224 and 191.1060 were generated by loss of 18 Da and 18 + 14 Da, which implied loss of a H₂O group and methyl group. Other product ions were identical to that of M27. Based on the possible metabolic reactions, M28 might be a metabolite of senkyunolide A after hydroxylation and methylation. Metabolite M29, which eluted at 10.35 min, formed a molecular ion of [M+H]⁺ at m/z 372.1475 corresponding to C₁₇H₂₅O₆NS. Its major fragment ions m/z 209.1269 and 191.1074 were generated by loss of 163 Da and 163 + 18 Da, which implied loss of an acetylcysteine group and H₂O group. Other product ions were identical to that of M27. Therefore, M29 may be a metabolite of senkyunolide A after hydroxyl and acetylcysteine conjugation. Metabolite M30, which eluted at 10.60 min, formed a molecular ion of [M+H]⁺ at m/z 399.1285 corresponding to C₁₈H₂₂O₁₀. Its major fragment ions m/z 223.0969, 205.0847, and 177.0883 were generated by the loss of 176 Da, 176 + 18 Da, and 176 + 18 + 28 Da, which implied loss of a glucuronide group, H₂O group, and CO group. Other product ions were identical to that of M27. Therefore, M30 may be a metabolite of senkyunolide A following carboxylation and glucuronidation. Metabolite M31, which eluted at 8.67 min, formed a molecular ion of [M+H]⁺ at m/z 401.1444 corresponding to C₁₈H₂₄O₁₀. Its major fragment ions m/z 225.1127, 207.1017, and 189.0924 were generated by loss of 176 Da, 176 + 18 Da, and 176 + 18 + 18 Da, which implied loss of a glucuronide group and two H₂O groups. Other product ions were identical to that of M27. Therefore, M31 might be a metabolite of senkyunolide A after 2 hydroxylation events and glucuronidation. Metabolite M32, which eluted at 10.54 min, formed a molecular ion of [M+H]⁺ at m/z 211.1327 corresponding to C₁₂H₁₈O₃. Its major fragment ions were m/z 193.1225 and 175.1096. An m/z of 193.1225 (loss of 18 Da) corresponds to senkyunolide A, suggesting that M32 might be a metabolite of senkyunolide A after H₂O conjugation. Metabolite M33, which eluted at 9.56 min, formed a molecular ion of [M+H]⁺ at m/z 225.1119 corresponding to C₁₂H₁₆O₄. Its major fragment ions m/z 207.1013 and 189.0914 were generated by loss of 18 Da and 18 + 18 Da, which implied loss of one or two H₂O groups. Other product ions were identical to that of M27. Therefore, M33 might be a metabolite of senkyunolide A after 2 hydroxylation events (metabolites of M27 and extracted ion chromatograms (EICs) are shown in Figure 7).

Sixty-one metabolites were identified in rat plasma. Through the analysis of these 61 metabolites, it was found that hydroxylation and glucuronidation were the main metabolic ways following oral administration of TST. From the identified metabolites, it can be speculated that after absorption of TST by human blood, most of the compounds undergo hydroxylation and glucuronidation, which allow TST to play a positive role in the treatment of migraine and blood stasis headaches. This provides a basis for follow-up research on the medical uses of TST. At the same time, from the information obtained on the metabolites, it can be seen that the main metabolites in positive ion mode of TST are concentrated as chuanxiong lactones, but there are no effective metabolites from Tianma. It is possible that Tianma metabolites are mainly present in the negative ion mode of plasma or in feces, urine, and bile, which requires further study.

4. Conclusion

In this study, UPLC/Q-TOF MS was used to comprehensively determine the chemical fingerprint and metabolic profile of TST after intragastric administration. In the analysis of the chemical constituents of TST, 77 compounds were identified, including 39 compounds identified in negative ion mode and 38 compounds identified in positive ion mode. In order to elucidate the mass spectrometric pyrolysis law of the main compounds in TST, gastrodin A, parishin B, and cnidilide or neocnilide were specifically analyzed, and the results were completely consistent with the results in reference standards and the reported literature. And 61 metabolites of TST in rat plasma were detected, which were mainly metabolites of 7 compounds. Two prototypes (senkyunolide D or 4,7-dihydroxy-3-butylphthalide and senkyunolide A) and their metabolites were analyzed in detail, which showed hydroxylation and glucuronidation were the main metabolic pathways following oral administration. This study expanded our understanding of the chemical constituents of TST, studied its metabolic spectrum for the first time, and clarified its main metabolic pathway in plasma, which will lay the foundation for follow-up studies of the pharmacological mechanism of TST.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare no conflicts of interest in publication of this study.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (no. 81560638), Young Scientists Training Project of Jiangxi Province (no. 20162BCB23003), Jiangxi Province 5511 R&D Projects (no. 20165BCB19009), Jiangxi Province Talent Project (no. [2016]332), Nanchang Innovative Talent Team (no. [2018]274), and Research Projects of Traditional Chinese Medicine of Jiangxi Provincial Health Department (no. 2016A018).

References

Chinese Pharmacopoeia Commission, Chinese Pharmacopoeia, vol. 1, China Medical Science Press, Beijing, China, 2010.
W. Xia, M. Zhu, Z. Zhang et al., “Effect of Tianshu capsule in treatment of migrane: a meta-analysis of randomized control trials,” Journal of Traditional Chinese Medicine, vol. 33, no. 1, pp. 9–14, 2013.
View at: Publisher Site | Google Scholar
L. Zhao, H. Ouyang, and Q. Wang, “Chemical fingerprint analysis and metabolic profiling of 50% ethanol fraction of lomatogonium rotatum by ultra-performance liquid chromatography/quadrupole–time of flight mass spectrometry,” Biomedical Chromatography, vol. 33, 2019.
View at: Publisher Site | Google Scholar
L. Wang, J. Zhang, Y. Hong et al., “Phytochemical and pharmacological review of da chuanxiong formula: a famous herb pair composed of chuanxiong rhizoma and gastrodiae rhizoma for headache,” Evidence-Based Complementray and Alternative Medicine, vol. 2013, Article ID 425369, 16 pages, 2013.
View at: Publisher Site | Google Scholar
S. M. Ni, D. W. Qian, E. X. Shang, J. A. Duan, J. M. Guo, and Y. P. Tang, “Analysis of chemical composition of dachuanxiong fang by ultra performance liquid chromatography-electrospray ionization-quadrupole time of flight/mass spectrometry,” Chinese Journal of Experimental Traditional Medical Formulae, vol. 16, no. 1, pp. 40–45, 2010.
View at: Google Scholar
L. Shen, X. Lin, Y. Hong et al., “Study on HPLC characteristic fingerprint of active components of dachuanxiong fang in plasma and cerebrospinal fluid,” China Journal of Chinese Materia Medica, vol. 37, no. 13, pp. 2017–2021, 2012.
View at: Publisher Site | Google Scholar
Y. Wei, X. Lin, N. Zhang et al., “UPLC-MS analysis of constituents of dachuanxiong fang active parts absorbed into blood,” China Journal of Chinese Materia Medica, vol. 36, no. 9, p. 1245, 2011.
View at: Publisher Site | Google Scholar
W. Yuan-Feng, Z. Ning, L. Xiao et al., “Release characteristics in vitro and pharmacokinetics of Da Chuanxiong Fang multiunit drug delivery system in rats,” Acta Pharmaceutica Sinica, vol. 46, no. 9, pp. 1150–1155, 2011.
View at: Google Scholar
L. Juanjuan, G. Huimin, C. Liangmian et al., “Chemical profiling of an antimigraine herbal preparation, Tianshu capsule, based on the combination of HPLC, LC-DAD-MSⁿ, and LC-DAD-ESI-IT-TOF/MS analyses,” Evidence-Based Complementary and Alternative Medicine, vol. 2014, Article ID 580745, 11 pages, 2014.
View at: Publisher Site | Google Scholar
D. Gao, B. Wang, Z. Huo et al., “Analysis of chemical constituents in an herbal formula Jitong Ning Tablet,” Journal of Pharmaceutical and Biomedical Analysis, vol. 140, pp. 301–312, 2017.
View at: Publisher Site | Google Scholar
L. I. Wen-Lan, J. Bai, Z. Sun et al., “Material base of in vivo invigorating Qi and enriching blood of Bazhen decoction by HPLC-ESI-MS,” Chinese Herbal Medicines, vol. 4, no. 4, pp. 282–286, 2012.
View at: Publisher Site | Google Scholar
Z. Li, Y. Wang, H. Ouyang et al., “A novel dereplication strategy for the identification of two new trace compounds in the extract of Gastrodia elata using UHPLC/Q-TOF-MS/MS,” Journal of Chromatography B, vol. 988, pp. 45–52, 2015.
View at: Publisher Site | Google Scholar
D. Kammerer, A. Claus, R. Carle, and A. Schieber, “Polyphenol screening of pomace from red and white grape varieties (Vitis vinifera L.) by HPLC-DAD-MS/MS,” Journal of Agricultural and Food Chemistry, vol. 52, no. 14, pp. 4360–4367, 2004.
View at: Publisher Site | Google Scholar
C. Schieber, L. Wang, X. Liu, M. Cheng, and H. Xiao, “Chemical fingerprint and metabolic profile analysis of ethyl acetate fraction of Gastrodia elata by ultra performance liquid chromatography/quadrupole-time of flight mass spectrometry,” Journal of Chromatography B, vol. 1011, pp. 233–239, 2016.
View at: Publisher Site | Google Scholar
M. Keabetswe, K. Bradley, S. Paul et al., “A metabolomics-guided exploration of the phytochemical constituents of vernonia fastigiata with the aid of pressurized hot water extraction and liquid chromatography-mass spectrometry,” Molecules, vol. 22, no. 8, p. 1200, 2017.
View at: Publisher Site | Google Scholar
C. Miao, S. Wu, B. Luo, J. Wang, and Y. Chen, “A new sesquiterpenoid from Ligusticum chuanxiong Hort,” Fitoterapia, vol. 81, no. 8, pp. 1088–1090, 2010.
View at: Publisher Site | Google Scholar
X.-L. Chang, Z.-Y. Jiang, Y.-B. Ma, X.-M. Zhang, K. W. K. Tsim, and J.-J. Chen, “Two new compounds from the roots of Ligusticum chuanxiong,” Journal of Asian Natural Products Research, vol. 11, no. 9, pp. 805–810, 2009.
View at: Publisher Site | Google Scholar
M. Ruan, Y. Li, X. Li, J. Luo, and L. Kong, “Qualitative and quantitative analysis of the major constituents in Chinese medicinal preparation Guan-Xin-Ning injection by HPLC-DAD-ESI-MSn,” Journal of Pharmaceutical and Biomedical Analysis, vol. 59, pp. 184–189, 2012.
View at: Publisher Site | Google Scholar
Y. Li, S. Peng, Y. Zhou, K. B. Yu, and L. S. Ding, “Two new phthalides from Ligusticum chuanxiong,” Planta Medica, vol. 72, no. 7, pp. 652–656, 2006.
View at: Google Scholar
J. Huang, X.-Q. Lu, C. Zhang et al., “Anti-inflammatory ligustilides from Ligusticum chuanxiong hort,” Fitoterapia, vol. 91, pp. 21–27, 2013.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2019 Lin Chen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Views

784

Downloads

806

Citations