Chemical changes of Angelicae Sinensis Radix and Chuanxiong Rhizoma by wine treatment: chemical profiling and marker selection by gas chromatography coupled with triple quadrupole mass spectrometry
© Zhan et al.; licensee BioMed Central Ltd. 2013
Received: 17 August 2012
Accepted: 26 May 2013
Published: 6 June 2013
Angelicae Sinensis Radix (ASR) and Chuanxiong Rhizoma (CR) can be treated with wine to promote their biological functions in Chinese medicine. Both ASR and CR contain similar volatile chemicals that could be altered after wine treatment. This study aims to identify the differential chemical profiles and to select marker chemicals of ASR and CR before and after wine treatment.
Chemical analyses were carried out by gas chromatography-triple quadrupole mass spectrometry (GC-QQQ-MS/MS) coupled with multivariate statistical analysis. Characterization of the compositions of essential oils was performed by automated matching to the MS library and comparisons of their mass spectra (NIST08 database). For ferulic acid, butylphthalide, Z-butylidenephthalide, senkyunolide A and Z-ligustilide, the mass spectrometer was operated in electron ionization mode, the selection reaction monitoring mode was used and an evaluation of the stability and sensitivity of the chromatographic system was performed for the tested extraction.
Principal component analysis (PCA) simultaneously distinguished ASR and CR from different forms. Ferulic acid, Z-butylidenephthalide, Z-ligustilide, butylphthalide and senkyunolide A were screened by PCA loading plots and can be used as chemical markers for discrimination among different groups of samples.
Different chemical profiles of ASR and CR after wine treatment could be identified by GC-QQQ-MS/MS. The five marker chemicals selected by PCA, namely ferulic acid, butylphthalide, Z-butylidenephthalide, senkyunolide A and Z-ligustilide, were sufficient to distinguish between the crude and corresponding wine-treated forms of ASR and CR.
Angelicae Sinensis Radix (ASR; roots of Angelica sinensis (Oliv.) Diels; family Apiaceae; Danggui in Chinese) and Chuanxiong Rhizoma (CR; rhizomes of Ligusticum chuanxiong Hort.; family Apiaceae; Chuanxiong in Chinese) are adopted as drugs or dietary supplements in Asian countries. ASR can tonify blood and relieve amenorrhea, and can thus be used as a health food supplement for women [1–3]. CR is used to relieve headache, rheumatic arthralgia and coronary heart diseases [4–6].
Herbal processing is a crucial procedure for improving herbal functions [7–9]. Treatment with wine is one of the widely adopted methods, after which the treated herbs show enhanced blood circulation and accelerated drug delivery . Wine treatment of ASR and CR alters the amounts of ferulic acid and total volatile oils [11–13], which are the pharmacologically active ingredients of both ASR and CR [14, 15]. However, the specific changes of the volatile oils in ASR and CR after wine treatment, especially the biologically active chemicals such as butylphthalide, Z-butylidenephthalide, senkyunolide A and Z-ligustilidehave not been delineated. Since the crude and wine-treated forms of ASR and CR may show significant differences in their chemical compositions and clinical efficacy [9, 16], identification of the different forms by chemical profiling methods would be a prerequisite for good quality control.
Some forms of ASR and CR are indistinguishable because of the similarities in their chemical compositions. Since their decoction or freeze-dried powder forms would cause technical problems if used in conventional chemical methods, sophisticated methods such as gas chromatography-triple quadrupole mass spectrometry (GC-QQQ-MS/MS) with high sensitivity and accuracy were adopted in this study to identify the chemical profiles of herbal extracts from ASR and CR. To our knowledge, this is the first study to analyze the changes in the chemical profiles of ASR and CR before and after wine treatment by GC-QQQ-MS/MS.
Chemicals, reagents and herbal materials
Ethyl acetate for GC analysis was purchased from Merck (Darmstadt, Germany). The chemical standards for butylphthalide, Z-butylidenephthalide and senkyunolide A were purchased from Weiqike Biotechnology Co. (Sichuan, China). Ferulic acid was obtained from Sigma (St. Louis, MO, USA). Z-Ligustilide was kindly provided by Prof. Pengfei Tu, Medical College of Peking University (Beijing, China). All chemical standards were tested and confirmed to show >98% purity based on their GC profiles and MS data. Fresh roots of ASR and fresh rhizomes of CR were collected in Gansu and Sichuan, China, respectively, as areas in which the best quality of the herbs can be guaranteed . The identity of the herbs was confirmed by morphological authentication, and their voucher specimens (#09-11-02 for ASR and #09-09-20 for CR) were deposited in the Center for Chinese Medicine R&D, Hong Kong University of Science & Technology (Clear Water Bay, Kowloon, Hong Kong). All chemical reagents used in the study were of analytical grade. Individual samples were prepared from 1 kg of powdered herbal materials. The dried roots (50 g) were sliced, sprayed with 5 mL of yellow wine (Best Spirits, Zhejiang, China; 15–16% alcohol) and placed in an oven at 80°C for 90 min with flipping twice per hour to ensure standardized processing of ASR and CR. The protocol was conducted in accordance with our previous study .
Ethyl acetate solution (25 mL) was added to 0.5 g of ground powders of ASR, CR, wine-treated ASR and wine-treated CR, respectively. Each mixture was then sonicated in an ultrasonic bath (240 W) for 20 min. In the second extraction, the residues from the first extraction were filtered, and the same extraction procedures were repeated. After centrifugation (Heraeus Megafue 1.0R; Waltham, MA, USA) at 2,500 × g and 4°C for 5 min, the supernatants were collected for GC-MS/MS analysis.
A 7000 GC/MS/MS series system (Agilent Technologies, Waldbronn, Germany) equipped with a 7890A gas chromatograph and GC-QQQ MassHunter workstation software (B.05.00.412; Agilent Technologies, Waldbronn, Germany) was used. Each extract was separated in an HP-5MS capillary column (250 μm × 30 m × 0.25 μm; Agilent Technologies, Waldbronn, Germany) with controlled temperature at 70°C in the initial stage, followed by elevation of the temperature to 280°C at a rate of 10°C/min. A pulsed splitless injection was conducted by injecting 1 μL of the sample extract. Helium was used as the carrier gas at a flow rate of 2.25 mL/min, and nitrogen was used as the collision gas at a flow rate of 1.5 mL/min. The spectrometer was operated in a full-scan electron-impact mode and the ionization energy was 70 eV. The inlet and ionization source temperatures were 250°C and 230°C, respectively. The solvent delay time was 3.5 min. The retention indices of all compounds were determined according to the Kovats method using n-alkanes as standards. The volatile flavor compounds were authenticated by comparing the mass spectra with the Kovats retention indices, NIST standard reference database (NIST 08) and other published databases of mass spectra. For the MS/MS analysis, suitable precursor ions and two product ions were selected for acquisition in the MRM mode of ferulic acid, butylphthalide, Z-butylidenephthalide, senkyunolide A and Z-ligustilide. The fragmentor voltage and collision energy values were optimized to achieve the greatest abundance of the detected compounds. The GC-QQQ MassHunter workstation software was applied for data acquisition and processing.
The relative standard deviation (RSD) was used to describe the precision, repeatability and recovery.
The peaks beyond the S/N ratios were labeled and manually integrated into chromatograms using the 7890A gas chromatograph and Chemstation software (B.03.01; Agilent Technologies, Waldbronn, Germany) to determine the differences in the volatile chemicals in the herbal materials after wine treatment. Principal component analysis (PCA) of the relative peak areas was performed using SPSS for Windows 16.0 software (SPSS Corporation, Armonk, NY, USA) to evaluate the discriminability of the peaks. Data were expressed as the mean ± standard deviation (SD) for n=3–8. Statistical tests were performed by one-way ANOVA. In the statistical analyses, differences were classed as significant for values of P < 0.05, and highly significant for values of P < 0.01 and P < 0.001.
Results and discussion
Validation of GC-QQQ-MS/MS methodology
Mass spectra properties of the marker chemicals
Retention time (min)3
Regarding the calibration of the marker chemicals, the regression equations of the five chemicals together with their LOD and LOQ values are shown in Additional file 2: Table S1. A mixed solution of the five analytes at various known concentrations was injected in triplicate, and the calibration curve of each analyte was plotted as the peak area against the concentration. The calibration curves demonstrated good linear regression with correlation coefficients of >0.990 in the test ranges. Using MS/MS analyses, the LOD and LOQ values were given for the ranges of 0.001–0.020 and 0.005–0.080 μg/mL, respectively. The overall intra-day and inter-day precisions were determined by analyzing the known concentrations of the five analytes. Solutions prepared from the herbal extracts of either ASR or CR were analyzed to test the repeatability. Variations were expressed as the RSD. The values of all intra-day and inter-day variations were found to be <5.0% (Additional file 3: Table S2). In addition, the validation studies showed that the assay had high reproducibility with an RSD of <5.0% (n=5). The average recoveries of the marker chemicals ranged from 95.58% to 98.75%, and their RSD values were all <5.0% (Additional file 3: Table S2). Therefore, the application of the GC-QQQ-MS/MS method in this study was precise, accurate and sensitive for simultaneous and quantitative evaluation of the marker chemicals in ASR and CR.
Chemical variations of ASR and CR after wine treatment
Quantitative determination based on marker chemicals of Angelicae Sinensis Radix and Chuanxiong Rhizoma
Angelicae Sinensis Radix (mg/100 g)
Chuanxiong Rhizoma (mg/100 g)
64.12 ± 8.03 1
86.35 ± 5.12*
135.01 ± 7.70
161.47 ± 12.64*
2.21 ± 0.10
2.34 ± 0.21
7.66 ± 0.27
4.54 ± 0.16*
21.73 ± 3.10
13.13 ± 1.35*
34.36 ± 0.17
33.90 ± 0.22
23.53 ± 5.34
21.91 ± 2.04
140.55 ± 5.44
101.21 ± 3.18**
452.32 ± 16.38
326.06 ± 11.67*
839.53 ± 10.47
825.58 ± 9.82
Components of the volatile chemicals in Angelicae Sinensis Radix and Chuanxiong Rhizoma
Senkyunolide A 4
Ferulic acid 4
ASR and CR both belong to the Apiaceae family, and are quite similar in their chemical constituents, including phthalides (e.g., Z-ligustilide, senkyunolide A, butylphthalide and Z-butylidenephthalide) and phenolic compounds (e.g., ferulic acid and coniferyl ferulate) . Wine treatment of ASR and CR is a commonly adopted procedure. In our findings, the changes of volatile chemicals in ASR and CR were distinctive after wine treatment. The amounts of β-pinene, β-myrcene and caryophyllene were reduced in wine-treated ASR, while these amounts were increased in wine-treated CR. Meanwhile, the amounts of Z-butylidenephthalide and Z-ligustilide were reduced in wine-treated ASR, but remained unchanged in wine-treated CR. Since wine treatment can reduce the amounts of volatile chemicals in ASR and CR, the chemical changes of these herbs would exhibit very different chemical profiles. We speculate that these differences may arise through differences in the cellular structures, tissue organizations and chemical compositions of the herbs.
PCA of the GC-MS/MS fingerprints and chemical markers
Different chemical profiles of ASR and CR after wine treatment could be identified by GC-QQQ-MS/MS. Five marker chemicals analyzed by PCA, namely ferulic acid, butylphthalide, Z-butylidenephthalide, senkyunolide A and Z-ligustilide, were sufficient to distinguish the crude and corresponding wine-treated forms of ASR and CR.
Angelicae Sinensis Radix
Principal component analysis
Gas chromatography coupled with tandem mass spectrometry
Gas chromatography-triple quadrupole mass spectrometry.
This research was supported by grants from the Research Grants Council of Hong Kong (6419/06 M, N_HKUST629/07, 662608 and 662911 to KWKT; 661110 to DTWL) and the Croucher Foundation (CAS-CF07/08.SC03 to KWKT).
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