Journal of Analytical Methods in Chemistry

Journal of Analytical Methods in Chemistry / 2016 / Article

Research Article | Open Access

Volume 2016 |Article ID 1206391 | 8 pages | https://doi.org/10.1155/2016/1206391

Qualitative and Quantitative Analysis of Volatile Components of Zhengtian Pills Using Gas Chromatography Mass Spectrometry and Ultra-High Performance Liquid Chromatography

Academic Editor: Mohamed Abdel-Rehim
Received10 Oct 2015
Revised23 Dec 2015
Accepted24 Dec 2015
Published20 Jan 2016

Abstract

Zhengtian pills (ZTPs) are traditional Chinese medicine (TCM) which have been commonly used to treat headaches. Volatile components of ZTPs extracted by ethyl acetate with an ultrasonic method were analyzed by gas chromatography mass spectrometry (GC-MS). Twenty-two components were identified, accounting for 78.884% of the total components of volatile oil. The three main volatile components including protocatechuic acid, ferulic acid, and ligustilide were simultaneously determined using ultra-high performance liquid chromatography coupled with diode array detection (UHPLC-DAD). Baseline separation was achieved on an XB-C18 column with linear gradient elution of methanol-0.2% acetic acid aqueous solution. The UHPLC-DAD method provided good linearity (), precision (RSD < 3%), accuracy (100.68–102.69%), and robustness. The UHPLC-DAD/GC-MS method was successfully utilized to analyze volatile components, protocatechuic acid, ferulic acid, and ligustilide, in 13 batches of ZTPs, which is suitable for discrimination and quality assessment of ZTPs.

1. Introduction

In comparison with conventional fully porous particle columns, core-shell particle columns have higher column efficiency and sensitivity, achieve better separation, and remain constant over a wider linear range. In addition, the columns improve the speed of the mobile phase, reduce analysis time, and improve throughput. Moreover, the backpressure produced by the columns at optimum linear velocity is very low (<400 bar). So we take advantage of this new column to develop an efficient ultra-high performance liquid chromatography (UHPLC) method [14]. Gas chromatography mass spectrometry (GC-MS) has been employed in a broad range of analytical applications due to its high sensitivity and capacity to separate compounds effectively. GC-MS is commonly used to characterize and identify volatile organic compounds in complex mixtures [510]. GC-MS has become a popular and useful analytical tool in research on herbal medicines, especially in establishing chromatographic fingerprints for quality control of traditional Chinese medicines [11, 12], such as Fructus Xanthii [13].

Zhengtian pills (ZTPs) are Chinese patent medicine comprised of 15 medicinal herbs: Caulis Spatholobi, Radix Angelicae Sinensis, Rhizoma Chuanxiong, Asari Radix et Rhizoma, Uncariae Ramulus Cum Uncis, Paeoniae Radix Alba, Radix Rehmanniae, Radix Angelicae Dahuricae, Radix Saposhnikoviae, Notopterygii Rhizoma et Radix, Persicae Semen, Carthami Flos, Radix Angelicae Pubescentis, Ephedrae Herba, and Aconiti Lateralis Radix Praeparata [14]. ZTPs are used to treat tension headaches [15], headaches associated with spinal conditions, and premenstrual headaches. ZTPs are widely used in China to treat migraine headaches, a type of vascular headache [16, 17]. According to the Chinese Pharmacopoeia, paeoniflorin is the marker compound used for ZTP quality control [14].

In a previous study, HPLC was used to determine the major active components of ZTP, which included paeoniflorin, ferulic acid, prim-O-glucosylcimifugin, and 4′-O-beta-glucopyranosyl-5-O-methylvisamminol [18]; however, determination of these compounds is not sufficient for comprehensive quality control of ZTPs, which contain complex bioactive constituents, including volatile components of essential oils, alkaloids, flavonoids, and coumarin compounds. Several compounds from ZTPs ingredients, including protocatechuic acid from Spatholobi Caulis, and ferulic acid and ligustilide from Rhizoma Chuanxiong and Radix Angelicae Sinensis, have pharmacological activities, including antibacterial, anti-inflammatory, and analgesic effects, as well as protective effects on the cardiovascular system [1921]. Therefore, an effective and reliable method capable of qualitative and quantitative analysis of the diverse bioactive constituents of ZTPs is required to ensure their safety and efficacy. While significant research has been conducted on the volatile components of Rhizoma Chuanxiong and Radix Angelicae Sinensis, such studies have mainly focused on their cardiovascular, cerebrovascular, neuroprotective, antinociceptive, and anti-inflammatory effects [22, 23], and the volatile components of the other ZTPs constituents have not been reported.

In this study, a combined UHPLC coupled with diode array detection (UHPLC-DAD)/GC-MS method was developed to simultaneously identify volatile organic compounds in ZTPs and qualitatively and quantitatively analyze 3 main bioactive constituents present in various ZTPs ingredients. The combined UHPLC-DAD/GC-MS method can be utilized to evaluate the quality of ZTPs.

2. Experimental

2.1. Reagents and Chemicals

HPLC grade anhydrous ether and methanol were purchased from Merck (Darmstadt, Germany). Deionized water for the samples and mobile phase were prepared using a Milli-Q50 SP Reagent Water System (Millipore, France). Reference standards for protocatechuic acid, ferulic acid, and ligustilide were obtained from the National Institute for Food and Drug Control (Beijing, China). The purity of all standards was at least 98%. Thirteen batches of ZTP were purchased from China Resources Sanjiu Medical & Pharmaceutical Co. Ltd. (Shenzhen, China). Methanol, acetic acid, petroleum ether, ethyl ether, and ethyl acetate were purchased from Baishi Chemical Industry Co. Ltd. (Tianjin, China) and of analytical grade. All solvents were filtered through 0.22 μm membrane filters before analysis.

2.2. Sample Preparation

ZTPs were smashed into powder (40 mesh). For HPLC analysis, 3.0 g of pulverized ZTP was ultrasonically extracted at room temperature with 50 mL methanol for 0.5 h in a 250 mL triangular flask and dried. The ZTP residue was dissolved with 20 mL water and extracted 3 times with ethyl acetate (30, 30, and 20 mL). The ethyl acetate extracts were dried using an electrothermostatic water bath. Finally, the extract was reconstituted in 5 mL methanol. For GC-MS analysis, 3.0 g of pulverized ZTP was reflux-extracted twice for 1 h at 50°C with 20 mL ethyl acetate in a 250-mL triangular flask. The ethyl acetate extracts were dried and reconstituted in 5 mL ethyl acetate. All ZTP solutions were filtered through a 0.22 μm nylon membrane filter.

2.3. GC-MS Analysis

GC-MS analysis was performed on an Agilent 7890/5975C-GC/MSD instrument (Agilent Technologies, USA) coupled with a HP-5MS fused silica capillary column (30 m × 0.25 mm × 0.25 μm; Agilent Technologies, Santa Clara, CA, USA). The GC oven temperature was initially increased from 60°C to 120°C at a rate of 10°C/min, then elevated at a rate of 3°C/min up to 175°C, then increased at a rate of 5°C/min up to 205°C, then went up at a rate of 0.8°C/min up to 210°C, and then raised to 280°C at a rate of 5°C/min, held for 5 min, giving a total runtime of 55.583 min. 1 μL volume of ethyl acetate extracts was injected into the GC. Helium carrier gas at a constant flow rate of 1.0 mL/min and a 30 : 1 split ratio were used simultaneously. Mass spectrometer was operated in full scan with an electron energy of 70 eV; interface temperature: 280°C; MS source temperature: 230°C; MS quadrupole temperature: 150°C. The scan range was from m/z 30 to 550.

2.4. HPLC Analysis

HPLC analysis was performed on a Thermo Ultimate-3000 system (Thermo Scientific, Waltham, MA, USA) coupled with a DAD. A Phenomenex Kinetex XB-C18 column (100 mm × 4.6 mm; 2.6 μm i.d.; 25°C column temperature) with a guard column (2.1 mm × 4.6 mm, 2.6 μm i.d.) was used (Phenomenex, Torrance, CA, USA). The mobile phase was 0.2% (v/v) aqueous acetic acid solution (A) and methanol (B). The linear gradient was as follows: 0–7 min, 10% B; 7–15 min, 10–15% B; 15–35 min, 15–40% B; 35–65 min, 40–50% B. The flow rate was 1.5 mL/min and the injection volume was 2 μL.

2.5. HPLC Method Validation

All components were quantified using chromatograms obtained at 260 nm. The quantification was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision.

The stock solution containing the 3 markers was prepared and diluted to appropriate concentration ranges for the establishment of calibration curves. The calibration graphs were plotted after linear regression of the peak areas versus the corresponding concentrations. Good linear behavior was observed, with correlation coefficients () between 0.9992 and 0.9994. LOD and LOQ were determined at signal-to-noise () ratios of approximately 3 and 10, respectively. Recovery experiments were performed at medium levels. The concentrations of protocatechuic acid, ferulic acid, and ligustilide were 0.0204 mg/mL, 0.1027 mg/mL, and 0.22 mg/mL. This procedure was accordingly repeated for six replicates. The spiked samples were extracted, processed, and quantified in accordance with the methods described above. Recoveries varied from 100.18 to 103.39%, with RSDs from 0.50 to 2.36% (Table 1).


AnalytesRegression equation Linearity range/μg mL−1LOD/μgLOQ/μgRecovery %RSD %

Protocatechuic acid0.99944.1–28.60.00190.0037100.68 0.35
Ferulic acid 0.999280.2–561.40.0250.074100.98 0.56
Ligustilide0.9994110–7700.0440.084102.69 0.28

Precision was evaluated with the solution of sample 1209051H under the selected optimal conditions 6 times in 1 day to measure intraday variation. The RSDs of the precision results were in the range of 0.54–0.89%. Repeatability was confirmed with 6 different working solutions prepared from sample 1209051H. The RSDs of the repeatability results were in the range of 1.88–2.31%. The stability of the solutions was tested by injecting them into the apparatus at 0, 2, 4, 6, 8, 12, and 24 h. The RSDs of the stability results were in the range of 1.80–2.53%.

Moreover, specificity was investigated by comparing the chromatograms of mixed standards and the ZTP extract (Figure 1). According to the three-dimensional plot of the absorbance as a function of retention time and wavelength in the HPLC-DAD data for sample number 1209051H, no evidence of peak of impurities overlapping the markers was found.

This is the first simultaneous analysis of protocatechuic acid, ferulic acid, and ligustilide with acceptable linearity, precision, repeatability, and accuracy.

3. Results and Discussion

3.1. Optimization of the GC Method and Extraction

In this study, the extraction efficiencies of petroleum ether, ethyl ether, and ethyl acetate were compared. Total ion current (TIC) was greatest when ethyl acetate was used. Investigation of the extraction efficiencies of the reflux and ultrasonic methods indicated that total TIC was higher when the reflux method was used. The GC results for the different solvent extracts and methods are described in Table 2 and Figure 2. In the ethyl acetate solvent extracts, 22 components were identified by artificial analysis and computer retrieval, while relative content was determined by the area normalization method. The main components of the ethyl acetate solvent extracts were ligustilide (19.381%), oleic acid (10.012%), 9,12-octadecadienoic acid (9.346%), butylidene phthalide (5.055%), dibutyl phthalate (5.891%), xanthyletin (3.813%), methyl eugenol (3.833%), n-docosane (3.545%), asarinin (2.085%), and safrole (1.555%). In comparison to the ethyl acetate solvent extract samples, the ethyl ether extract samples contained a greater amount of 2,6-di-tert-butyl-p-cresol (2.667%), while the petroleum ether extract samples contained a greater amount of sulfur (3.993%).


Peak numberRetention time (min) Compound nameMolecular formulaRelative content (%)
Ethyl acetate refluxingEthyl acetate ultrasonicEther ultrasonicPetroleum ether ultrasonic

12.808 3,5-Dimethoxytoluene C9H12O2152.190.6371.7181.3721.703
24.312SafroleC10H10O2162.191.555
310.0592-Methoxy-4-vinylphenol phenol C9H10O2150.171.6911.3730.861
412.2411,2-Dimethoxy-4-(2-propenyl)benzene C12H18O3210.273.8332.8393.2380.768
512.4423,4,5-TrimethoxytolueneC10H14O3182.220.7550.5710.689
614.901Pentadecane C15H32212.411.3191.2690.795
715.3842,6-Di-tert-butyl-4-methylphenolC15H24O220.352.667
820.422Butylidene phthalideC12H12O2188.225.0554.8714.0863.426
922.455LigustilideC12H14O2190.2419.38116.10917.65415.063
1024.366trans-LigustilideC12H14O2190.241.0071.5661.060.79
1126.5021,2-Benzenedicarboxylic acid, bis(2-methylpropyl) esterC16H22O4278.342.7240.7191.881
1228.946Dibutyl phthalateC16H22O4278.345.8913.1725.8915.193
1330.251SulfurS832.063.993
1433.188(Z,Z)-9,12-Octadecadienoic acid C18H32O2280.449.34610.60511.4845.865
1533.327Oleic acidC18H34O2282.4610.01212.33412.34716.905
1633.892(Z,Z)-9,12-Octadecadienoic acid ethyl esterC20H36O2383.51.1810.711.9361.268
1738.18TricosaneC23H48324.630.7060.6780.7890.888
1844.619(S)-8,8-Dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano(3,2-g)chromen-7-yl 3-methylbut-2-enoateC19H20O5328.361.4641.2092.3181.962
1945.038,8-Dimethyl-2H,8H-benzo[1,2-:5,4-]dipyran-2-oneC14H12O3228.243.8134.6294.1667.711
2046.766EicosaneC20H42282.552.0052.392.9462.839
2149.893n-DocosaneC22H46310.63.5453.944.2725.138
2253.837AsarininC20H18O6354.352.0851.91.6712.01

3.2. Optimization of the HPLC Method and Extraction

Due to the existence of acidic components in the ZTP extraction, a small amount of acid was added to the mobile phase to ease ionization of these components, with the goal of improving peak shape and restraining peak tailing. Different concentrations of acetic acid, phosphoric acid, and formic acid were compared for this purpose. The results showed that all compounds could be baseline-separated when 0.2% aqueous acetic acid solution was added.

DAD detection was performed within a wavelength range of 190–400 nm. When the chromatograms and characteristic UV spectra of the 3 reference compounds were compared, it was found that the 3 active compounds had higher absorbance, better separation, and a steady baseline at 260 nm in comparison with the other tested wavelengths.

The extraction procedure was optimized prior to sample analysis. The samples (3.0 g each) were extracted with different volumes and percentages of methanol, as well as different volumes and percentages of ethanol, respectively. The optimum results were obtained using 50 mL methanol. Investigation of the dependence of the yield on the duration of the extraction (15, 30, and 60 min) showed that all of the investigated compounds were almost completely extracted when 30 min extraction was used.

3.3. Sample Analysis

The UHPLC-DAD/GC-MS method was applied to analyze 13 batches of ZTP (Tables 3 and 4). Protocatechuic acid content ranged from 6.07 to 17.30 μg/g, ferulic acid content ranged from 79.59 to 115.90 μg/g, and ligustilide content ranged from 79.59 to 388.69 μg/g. The GC-MS results showed that the average relative contents of ligustilide, oleic acid, asarinin, safrole, 2-methoxy-4-vinylphenol, and 1,2-dimethoxy-4-(2-propenyl)benzene were 6.991%, 23.275%, 1.631%, 0.586%, 0.575%, and 0.574%, respectively. 3,5-Dimethoxytoluene was not detected. These results suggested that the contents of each component varied greatly among batches of ZTPs. The largest observed difference in content was nearly 5-fold, indicating large variations in the ZTP production process and/or incorrectly identified herbal sources.


Sample number3,5-Dimethoxytoluene (%)Safrole (%)2-Methoxy-4-vinylphenol phenol (%)1,2-Dimethoxy-4-(2-propenyl)benzene (%)Ligustilide (%)Oleic acid (%)Asarinin (%)

1209051H0.7440.3210.2674.9726.1660.758
1303002H0.3930.5870.7537.10116.7691.806
1212023H0.3210.3930.4385.74732.8441.084
1210019H0.7360.5568.90520.5813.120
1212024H0.5560.4200.3095.90334.8152.163
1301023H0.4200.4004.28534.9891.119
1302026H0.4000.7537.55818.5681.334
1303036H0.7530.9735.91620.6912.074
1304001H0.9730.80310.84519.5931.560
1306019H0.8030.5887.39620.6272.164
1306020H0.5880.4585.72518.0161.344
1306027H0.4580.4750.8166.51723.2261.154
1309014H0.4750.7440.85810.01215.6911.523


Sample numberContent (μg/g)
⁢Protocatechuic acid⁢Ferulic acid⁢Ligustilide
MeanRSD (%)Mean RSD (%)Mean RSD (%)

1209051H14.011.9180.112.78174.131.65
1303002H14.431.2679.591.4279.592.52
1303002H17.301.3980.371.84178.852.65
1210019H8.360.8278.062.02363.420.39
1212024H10.642.7582.511.21348.782.48
1301023H12.211.2586.420.88252.372.57
1302026H10.081.67115.902.62323.112.47
1303036H6.072.5184.983.00388.691.05
1304001H10.021.61111.972.35268.612.42
1306019H9.621.6186.681.32303.620.81
1306020H8.131.2680.932.89355.711.72
1306027H9.710.3793.140.49260.750.52
1309014H9.642.8696.502.89301.900.59

The UHPLC-DAD/GC-MS method we established could detect as much volatile components as possible. Compared to previous study using a two-dimensional liquid chromatography coupled to mass spectrometry, there were more compounds identified by the GC-MS method, such as 1,2-dimethoxy-4-(2-propenyl)benzene, 2-methoxy-4-vinylphenol phenol, butylidene phthalide, oleic acid, and (Z,Z)-9,12-octadecadienoic acid ethyl ester, which were mainly from Rhizoma Chuanxiong [24]. It is very helpful for a comprehensive understanding of volatile components of ZTPs. At the same time, the established UHPLC-DAD method could determine protocatechuic acid, ferulic acid, and ligustilide simultaneously, which was very helpful and practicable for the quality control of ZTPs for companies. Since TCM is a complex system containing tens or even hundreds of different chemical constituents, the active compounds of most TCM still remain unknown. It is reported that molecular biochromatography was a novel strategy for the screening and analysis of biologically active compounds in TCM [25, 26]. Thus, in our further study, we will make an attempt to screen active compounds of ZTPs by using molecular biochromatography.

4. Conclusions

A UHPLC-DAD/GC-MS method was established for the comprehensive analysis of ZTPs that allowed separation of complex constituents in a short time. GC-MS provided accurate masses of protonated molecules, which were helpful for compound identification. The UHPLC-DAD/GC-MS method was successfully applied for simultaneous determination of 3 bioactive compounds in ZTP. The UHPLC-DAD/GC-MS method is readily available, rapid, and reliable. Therefore, the UHPLC-DAD/GC-MS method is suitable for routine analysis, original discrimination, and effective quality control of ZTPs. The amounts of protocatechuic acid, ferulic acid, and ligustilide in the ZTP batches varied considerably. Therefore, future studies should focus on evaluating the influence of bioactive constituent of ZTPs on their therapeutic effects using preclinical pharmacodynamic and clinical testing.

Conflict of Interests

The authors declare that there is no conflict of interests.

Acknowledgments

This work was supported by the Chinese Medicinal Materials Production and Construction Projects of the Ministry of Industry and Information Technology of China ([2015] no. 282) and the Collaborative Innovation Center Research Team Construction Project in Guangdong Province—the Innovation Research Team of Traditional Chinese Medicine Resources (A1-AFD01514A04).

References

  1. Kinetex core-shell particles column, G. F. S. I. C., http://www.gzflm.com/productinfo/detail_4_6_74.aspx.
  2. R. Hayes, A. Ahmed, T. Edge, and H. Zhang, “Core-shell particles: preparation, fundamentals and applications in high performance liquid chromatography,” Journal of Chromatography A, vol. 1357, pp. 36–52, 2014. View at: Publisher Site | Google Scholar
  3. A. Natale, D. Nardiello, C. Palermo, M. Muscarella, M. Quinto, and D. Centonze, “Development of an analytical method for the determination of polyphenolic compounds in vegetable origin samples by liquid chromatography and pulsed amperometric detection at a glassy carbon electrode,” Journal of Chromatography A, vol. 1420, pp. 66–73, 2015. View at: Publisher Site | Google Scholar
  4. R. Preti, M. L. Antonelli, R. Bernacchia, and G. Vinci, “Fast determination of biogenic amines in beverages by a core-shell particle column,” Food Chemistry, vol. 187, pp. 555–562, 2015. View at: Publisher Site | Google Scholar
  5. E. A. Nonato, F. Carazza, F. C. Silva, C. R. Carvalho, and Z. D. L. Cardeal, “A headspace solid-phase microextraction method for the determination of some secondary compounds of Brazilian sugar cane spirits by gas chromatography,” Journal of Agricultural and Food Chemistry, vol. 49, no. 8, pp. 3533–3539, 2001. View at: Publisher Site | Google Scholar
  6. T. Górecki, J. Harynuk, and O. Panić, “The evolution of comprehensive two-dimensional gas chromatography (GC × GC),” Journal of Separation Science, vol. 27, no. 5-6, pp. 359–379, 2004. View at: Publisher Site | Google Scholar
  7. F. Gong, Y.-Z. Liang, H. Cui, F.-T. Chau, and B. T.-P. Chan, “Determination of volatile components in peptic powder by gas chromatography-mass spectrometry and chemometric resolution,” Journal of Chromatography A, vol. 909, no. 2, pp. 237–247, 2001. View at: Publisher Site | Google Scholar
  8. S. Shen, Y. Sha, C. Deng, X. Zhang, D. Fu, and J. Chen, “Quality assessment of Flos Chrysanthemi Indici from different growing areas in China by solid-phase microextraction-gas chromatography-mass spectrometry,” Journal of Chromatography A, vol. 1047, no. 2, pp. 281–287, 2004. View at: Publisher Site | Google Scholar
  9. C. Deng, Y. Mao, N. Yao, and X. Zhang, “Development of microwave-assisted extraction followed by headspace solid-phase microextraction and gas chromatography-mass spectrometry for quantification of camphor and borneol in Flos Chrysanthemi Indici,” Analytica Chimica Acta, vol. 575, no. 1, pp. 120–125, 2006. View at: Publisher Site | Google Scholar
  10. C. Deng, N. Yao, B. Wang, and X. Zhang, “Development of microwave-assisted extraction followed by headspace single-drop microextraction for fast determination of paeonol in traditional Chinese medicines,” Journal of Chromatography A, vol. 1103, no. 1, pp. 15–21, 2006. View at: Publisher Site | Google Scholar
  11. H. Zhu, Y. Wang, H. Liang, Q. Chen, P. Zhao, and J. Tao, “Identification of Portulaca oleracea L. from different sources using GC-MS and FT-IR spectroscopy,” Talanta, vol. 81, no. 1-2, pp. 129–135, 2010. View at: Publisher Site | Google Scholar
  12. L. Tong, Y. Wang, J. Xiong, Y. Cui, YigangZhou, and L. Yi, “Selection and fingerprints of the control substances for plant drug Eucommia ulmodies Oliver by HPLC and LC–MS,” Talanta, vol. 76, no. 1, pp. 80–84, 2008. View at: Publisher Site | Google Scholar
  13. G.-H. Ruan and G.-K. Li, “The study on the chromatographic fingerprint of Fructus xanthii by microwave assisted extraction coupled with GC-MS,” Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, vol. 850, no. 1-2, pp. 241–248, 2007. View at: Publisher Site | Google Scholar
  14. State Pharmacopoeia Commission of the People's Republic of China, Pharmacopoeia of People's Republic of China, Chemical Industry Press, Beijing, China, 2010.
  15. H. R. Qian, X. K. Qi, G. Huang, and D. G. Feng, “Comparison of effect of zhengtian pill and tongtian oral solution in treating tension-type headache,” Chinese Journal of New Drugs and Clinical Remedies, vol. 22, no. 5, pp. 279–282, 2003. View at: Google Scholar
  16. Z. Xie, W. L. Li, and G. J. Shi, “Randomized, double-blind, dontrolled clinical study of Zhengtianwan pill in treating 42 cases of migraine,” Journal of Mathematical Medicine, vol. 24, no. 1, pp. 70–73, 2011. View at: Google Scholar
  17. C. X. Y. Y. Wang, H. C. Shang, M. Ren, and J. Yuan, “double-blind and double-dummy, multi-center clinical study of Zhengtian Pill for patients with migraine,” Chinese Traditional Patent Medicine, vol. 34, no. 5, pp. 791–794, 2012. View at: Google Scholar
  18. L. Huang, H.-L. Chen, and L.-L. Li, “Simultaneous determination of paeoniflorin, ferulic acid, prim-O-glucosylcimifugin and 4′-O-beta-glucopyranosyl-5-O-methylvisamminol in Zhengtian pills by HPLC,” Zhongguo Zhongyao Zazhi, vol. 38, no. 13, pp. 2114–2117, 2013. View at: Publisher Site | Google Scholar
  19. A. G. Z. Q. Lin, J. N. Chen, X. P. Lai, S. H. Gui, and C. P. Fang, “Anti-inflammatory and analgesic effects of ligustilide,” Chinese Journal of Experimental Traditional Medical Formulae, vol. 17, no. 77, pp. 165–168, 2011. View at: Google Scholar
  20. J. M. L. M. Zhai, R. An, F. L. Wu, and H. H. Xu, “Content determination of protocatechuic acid in Spatholobus suberectus dumn,” Traditional Chinese Drug Research & Clinical Pharmacology, vol. 20, no. 5, pp. 462–465, 2009. View at: Google Scholar
  21. X. Y. X. Y. Y. Hu, “Chemical and pharmacological research progress of ferulic acid,” Chinese Traditional Patent Medicine, vol. 28, no. 2, pp. 253–255, 2006. View at: Google Scholar
  22. X.-P. Chen, W. Li, X.-F. Xiao, L.-L. Zhang, and C.-X. Liu, “Phytochemical and pharmacological studies on Radix Angelica sinensis,” Chinese Journal of Natural Medicines, vol. 11, no. 6, pp. 577–587, 2013. View at: Publisher Site | Google Scholar
  23. X. Ran, L. Ma, C. Peng, H. Zhang, and L.-P. Qin, “Ligusticum chuanxiong Hort: a review of chemistry and pharmacology,” Pharmaceutical Biology, vol. 49, no. 11, pp. 1180–1189, 2011. View at: Publisher Site | Google Scholar
  24. X. Chen, L. Kong, X. Su et al., “Separation and identification of compounds in Rhizoma chuanxiong by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry,” Journal of Chromatography A, vol. 1040, no. 2, pp. 169–178, 2004. View at: Publisher Site | Google Scholar
  25. H. Wang, L. Kong, H. Zou, J. Ni, and Y. Zhang, “Screening and analysis of biologically active compounds in Angelica sinensis by molecular biochromatography,” Chromatographia, vol. 50, no. 7-8, pp. 439–445, 1999. View at: Publisher Site | Google Scholar
  26. H. Wang, H. Zou, L. Kong, J. Ni, and Y. Zhang, “Screening and analysis of biologically active components in traditional Chinese medicine by molecular biochromatography,” Chinese Journal of Chromatography, vol. 17, no. 2, pp. 123–127, 1999. View at: Google Scholar

Copyright © 2016 Cui-ting Liu 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.

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