A Validated LC Method for Simultaneous Determination of Phenolic, Coumarin and Phthalide Compounds in the Ethanolic Extract of Angelica tenuissima 2009, 70, 1079–1085
M. Nurul Islam1,2, Joo-Won Nam3, Jun Lee3, Eun-Kyoung Seo3, Sang Beom Han4, Dong-Hyun Kim1, Changbae Jin1, Hye Hyun Yoo1,& 1
2 3 4
Doping Control Center, Korea Institute of Science and Technology, P.O. BOX 131, Chungryang, Seoul 130-650, Republic of Korea; E-Mail:
[email protected];
[email protected] School of Biology and Environmental Science, University College Dublin, UCD Science Center West, Belfield, Dublin 4, Ireland College of Pharmacy, Ewha Women’s University, Seoul 120-750, Republic of Korea College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea
Received: 24 March 2009 / Revised: 23 June 2009 / Accepted: 25 June 2009 Online publication: 30 July 2009
Abstract In this study, an LC method for the simultaneous determination of six bioactive compounds from Angelica tenuissima, namely chlorogenic acid, ferulic acid, Z-ligustilide, nodakenin, decursin and decursinol angelate was developed and validated. Chromatographic analysis was carried out on a C18 column with a mobile phase consisting of 0.1% formic acid, methanol and acetonitrile at a flow rate of 0.8 mL min-1 and the effluent from the column was monitored by UV detector at 325 nm. The excellent linear behavior was observed over the investigated concentration range for reported compounds. The intra- and inter-day precision over the concentration range of compounds were lower than 1.7% (as relative standard deviation), and accuracy was between 97.2 and 106.0%. These results showed that the developed method is accurate, reproducible, and consequently applicable for the quantitation of bioactive components from the ethanolic extract of Angelica tenuissima Nakai.
Keywords Column liquid chromatography Phenolic acids Coumarins and phthalides Angelica tenuissima
Introduction The roots of Angelica tenuissima (Umbelliferae), known as Gubon in Original DOI: 10.1365/s10337-009-1263-0 0009-5893/09/10
Korea, is one of the extensively-used Korean traditional medicines. It has been used for the treatment of neuralgia and used to stop pain, cough, and
headache, to invigorate blood circulation, to disperse blood stasis and to provide relief from female diseases [1, 2]. The extensive phytochemical and pharmacological investigation on Angelica genus revealed the presence of several classes of bioactive compounds such as phenolic acids, coumarins, and phthalides and plenty of essential oils [1–3]. In our preliminary study for the phytochemical analysis of A. tenuissima, chlorogenic acid, ferulic acid, Z-ligustilide, nodakenin, decursin and decursinol angelate were found to be major constituents in the ethanolic extract of A. tenuissima. Chlorogenic acid is known to participate in glucose metabolism regulation and to be involved in the enhancement of fat metabolism in the liver [4, 5]. Ferulic acid is a ubiquitous plant constituent exhibiting natural antioxidant potential and strong antiinflammatory property [6]. Nodakenin, a coumarin has been reported to improve memory dysfunction by enhancing the cholinergic nervous system [7], and to have the neuroprotective effect against glutamate-induced toxicity [8]. Z-Ligustilide, a main component of Angelica genus, exhibited antioxidant and anti-apoptotic effects on transient
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forebrain ischemic mice [9] and demonstrated the therapeutic potential in treating vascular dementia and cerebrovascular insufficiency on experimental model [10]. Decursin, a novel class of pyranocoumarin, exhibited anti-tumor activities through activated protein kinase C and inhibiting androgen receptor signaling [11]. Decursinol angelate, a structural isomer of decursin, demonstrated the cytotoxic activities similar to decursin [11, 12]. Therefore, identification and determination of these bioactive compounds should be necessary in evaluation of the efficacy, safety and therapeutic consistency of A. tenuissima extract and its medical preparation. Several analytical tools such as LC, LC-MS, and GC-MS have been developed for the analysis of coumarins content of Angelica genus [13–17]. And analytical methods using electrophoresis, TLC and LC have been published for the quantitation of phenolic acids such as chlorogenic acid, ferulic acid and caffeic acid from natural products [18–21]. For phthalide compounds, GC-MS has been frequently used due to their volatile property. There are some reports describing the analysis of volatile compounds in Angelica tenuissima extract by GC-MS [1, 2]. However, most of the simultaneous analytical methods ever reported have dealt with limited compounds with similar polarity or chemical structure. The present study was designed to simultaneously determine the bioactive constituents of different chemical classes from A. tenuissima extracts using a popular analytical instrument, LC. Herein, an LC method for the determination of chlorogenic acid, ferulic acid, nodakenin, Z-ligustilide, decursin and decursinol angelate (Fig. 1) was developed and validated for the quality evaluation of A. tenuissima extract and its medicinal preparations.
Experimental Materials The Angelica tenuissima samples of Korean origin were purchased from the oriental herb store in Korea. Reference standards (chlorogenic acid, ferulic acid,
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nodakenin, Z-ligustilide and decursin) were provided by Ewha Women’s University (Seoul, Korea). Purity of standard compounds estimated by LC were higher than 95%. Internal standard chrysin, a flavonoid was purchased from Sigma Chemicals (St. Louis, MO, USA). Methanol and acetonitrile of LC grade were purchased from JT Baker (NJ, USA). All other chemicals used were of analytical grade unless otherwise specified. Distilled water was prepared using Milli-Q purification system (Millipore, Bedford, MA, USA).
Standard Solutions Standard stock solution of chlorogenic acid, ferulic acid, nodakenin, Z-ligustilide and decursin were made up at concentration of 1 mg mL-1 in 80% methanol. The standard solutions were serially diluted with 80% methanol to obtain working standard solutions at several concentration levels. 10 lL of internal standard chrysin (200 lg mL-1 in 80% methanol) were added to 100 lL of standard solution of each concentration. The calibration curves were constructed using five calibration standard mixture solutions with a concentration range for chlorogenic acid of 0.8–40 lg mL-1, ferulic acid 0.36–18 lg mL-1, nodakenin 0.60–30 lg mL-1, Z-ligustilide 2.4–120 lg mL-1, and decursin 0.5–50 lg mL-1. The injection volume into the LC system was 15 lL.
Sample Preparation An aliquot (100 g) of each A. tenuissima sample (as powder) was extracted with 70% ethanol (700 mL) by refluxing for 3 h at 80 °C. After cooling, the extract solution was filtered. The supernatant was collected, concentrated and freezedried to produce dried powder of the ethanolic extract. The sample solution for LC analysis was prepared by dissolving the extract in 80% methanol at a concentration of 2.5 mg mL-1. An aliquot of the sample (100 lL) was spiked with 10 lL of internal standard chrysin (200 lg mL-1) and filtered through a 0.22 lm membrane filter to remove the undissolved particles before analysis.
15 lL of samples were subjected to injection into the LC system.
LC Condition Chromatographic procedure was performed with an LC system consisting of Nanospace SI-1 binary pump, SI-1 autosampler, SI-1 oven, and SI-1 UVVIS detector (Shiseido, Tokyo, Japan). The chromatographic separation of compounds was achieved using the analytical column Capcell Pak C18 (4.6 mm I.D. 9 150 mm, particle size 3 lm, Shiseido, Tokyo, Japan) and column oven temperature was maintained at 35 °C. The mobile phase consisted of 0.1% aqueous formic acid with 10% methanol (solvent A) and 90% acetonitrile containing 0.1% formic acid (solvent B). Elution was performed at a flow rate of 0.8 mL min-1 in a binary gradient mode. The initial composition of solvent B was 10%. The composition of solvent B was linearly increased to 20% at 7 min, to 50% at 10 min, and to 58% at 14 min, maintained at this condition up to 24 min, and returned to initial condition at 25 min, which was followed by 7 min-column re-equilibration. Chromatograms were acquired at 325 nm in the UV-Vis detector. The signals from the detector were collected and analyzed with a computer equipped with SMC21 system software.
LC-MS Condition for Identification of Decursinol Angelate Identification of decursinol angelate was by an Agilent 1100 series, LC-MSD system (USA) equipped with an autosampler, binary pump, diode array detector (DAD) and coupled to an electrospray ion trap mass spectrometer. The analysis was performed using a Hypersil Gold column (150 9 2.1 mm, 3 lm, Thermo Electron Corporation, USA) with column temperature kept at 35 °C and the injection volume was 15 lL. The mobile phase consisted of 0.1% formic acid (solvent A) and 90% acetonitrile in 0.1% formic acid (solvent B) at a flow rate of 0.25 mL min-1 in a
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Fig. 1. Chemical structure along with UV spectrum of chlorogenic acid (a), ferulic acid (b), nodakenin (c), Z-ligustilide (d), decursin (e) and decursinol angelate (f)
gradient mode. The solvent gradient was changed according to the following schedule; solvent B (10%) was increased to 50% at 12 min, maintained at this condition up to 30 min, and returned to the initial condition in 2 min, followed by 8 min-column re-equilibration. The LC chromatogram was monitored at 325 nm. The electrospray capillary potential was set to 3,500 V. Nitrogen was used as a drying gas for solvent evaporation and temperature was kept at 350 °C. The MS and MS-MS spectra over the mass range of m/z 100–400 were obtained in the positive ionization mode.
Results and Discussion Chromatography In this paper, our goal was to develop a simple procedure with the best chroOriginal
matographic peak resolution, reduced run time and low cost of analysis for use in routine quality control of marker compounds in A. tenuissima. The rapid and simultaneous determination of chlorogenic acid, ferulic acid, nodakenin, Z-ligustilide, decursin and decursinol angelate in a single LC run in the isocratic mode has considerable difficulty due to variation of physicochemical properties between analytes. Thus, gradient elution method was developed as to ensure that all the constituents could be well separated and determined in one analysis run. In the course of experiments, various mixtures of water and methanol were tried as a mobile phase, but separation of decursin and its isomeric compound decursinol angelate was not satisfactory. Accordingly, a mixture of acetonitrile and formic acid was used as an organic modifier, which showed a significant improvement
in separation and good resolution (RS > 1.8), as well as satisfactory peak symmetry and shape were achieved. Subsequently a gradient condition was optimized. The gradient program initiated with solvent A (0.1% formic acid) and solvent B (90% acetonitrile containing 0.1% formic acid) in the ratio of 85:15 resulted in good peak shape but early elution of chlorogenic acid was observed. To overcome the early elution of chlorogenic acid, the gradient program was set in the ratio of 90:10 (solvent A: solvent B) but this broadened the peak shape of chlorogenic acid. Finally, solvent A was replaced with 10% methanol in 0.1% formic acid, which produced an excellent peak shape and optimum retention time for chlorogenic acid. The typical chromatograms of samples and standard mixture are shown in Fig. 2, from which one can observe that all compounds and internal
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Time (min) Fig. 2. Typical chromatograms of chlorogenic acid (1), ferulic acid (2), nodakenin (3), Z-ligustilide (4), decursin (5) and decursinol angelate (6) from a standard mixture (a) and extract samples (b)
standard are completely separated within 21 min. Notable, decursin and decursinol angelate, isomeric compounds were well separated for a relatively short run time in this study. A previously published report described the separation of decursin and dercursinol angelate within 40 min using a gradient elution system [16]. Another literature reported the separation of these isomers from Angelica species within 22 min, which is comparable to our analytical condition but the expensive reagent alcohol was used for this LC system [15]. However, our method achieved the analysis separating all major constituents within a shorter time using a conventional column and mobile phase, which may be a convenient method for the routine quality control assessment of A. tenuissima as well as other Angelica species. Full UV spectra of standard compounds were obtained by DAD detection in the range of 200–400 nm to optimize the wavelength for UV detection. It was observed that 325 nm was the most appropriate wavelength for all the target compounds. Typical UV spectrum along with the chemical structures of the standard compounds is shown in Fig. 1.
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The selectivity of the method was determined by comparing the chromatographic profile of the standards to that from A. tenuissima extract samples. Thus, retention time, maximum lambda and UV spectrum were compared to each other. In addition, the A. tenuissima extract sample was fortified with authentic standards and checked to guarantee the agreement of each peak from the A. tenuissima extract sample to the corresponding standard. As a result, a discrepancy between the standard mixture and the sample fortified with the standards was not observed. Moreover, no interference was observed in the eluting and sample dissolving solvents at the retention times of chlorogenic acid, ferulic acid, nodakenin, Z-ligustilide, decursin and decursinol angelate (chromatogram not shown).
Validation The calibration curve for cholorogenic acid, ferulic acid, nodakenin, Z-ligustilide and decursin was generated by plotting the peak area ratio for analytes to an internal standard versus the concentration of analytes by least-square regression analysis. The calibration
range for each analyte was described in Table 1 and each calibration curve was obtained using six-point concentrations. The range of the calibration curve was found to be adequate for the simultaneous analysis of the six bioactive compounds in A. tenuissima extract. The linear correlation co-efficient (r2) for all calibration curves were greater than 0.999, indicating a good linearity and intercept was close to zero (Table 1). The intra-assay precision and accuracy were performed by analyzing the five sets of quality control samples prepared at concentrations of low, middle, and high levels within a calibration range and the inter-assay precision and accuracy were examined over 5 days by the analysis of quality control samples prepared each day. The relative standard deviation (RSD) was considered as a measure of precision for data evaluation during the validation study. The RSD values of intra-assay and inter-assay precision were both below 1.7% and accuracy remained between 97.2 and 106.0%. These results of precision and accuracy were all satisfactory for the analysis of bioactive constituents from A. tenuissima extract samples (Table 2). The stock solution of standard compounds was diluted to a series of appropriate concentrations with 80% methanol, and an aliquot of diluted solutions were injected into LC for analysis. The limit of detection (LOD) was evaluated based on the lowest detectable signal in the chromatogram having a signal-to-noise (S/N) ratio of 3. Under our experimental conditions, listed in Table 1, the LODs were found to be 0.01, 0.01, 0.02, 0.01, 0.01 lg mL-1 for chlorogenic acid, ferulic acid, nodakenin, Z-ligustilide and decursin respectively. The limits of quantitation (LOQ) were assessed based on the lowest quantitative level having an S/N ratio 10. The LOQs of chlorogenic acid, ferulic acid, nodakenin, Z-ligustilide and decursin were found to be 0.05, 0.04, 0.06, 0.03 and 0.04 lg mL-1, respectively. Both LOD and LOQ for these five standards were low enough that the method would be capable of detecting traces of these compounds, either in crude extract or its medicinal dosage form.
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Table 1. Calibration curve data for chlorogenic acid, ferulic acid, nodakenin, Z-ligustilide and decursin
a
Compound
Range (lg mL-1)
Slope Aa
y-Intercept Ba
Correlation coefficient (r2)
LOD (lg mL-1)
LOQ (lg mL-1)
Chlorogenic acid Ferulic acid Nodakenin Z-Ligustilide Decursin
0.80–40 0.36–18 0.60–30 2.40–120 0.50–50
0.049 0.074 0.066 0.050 0.057
-0.004 -0.005 0.006 -0.017 -0.006
0.9996 0.9999 0.9999 0.9999 0.9999
0.01 0.01 0.02 0.01 0.01
0.05 0.04 0.06 0.03 0.04
Values are mean of three calibration curves. The slope and y-intercept refer to the regression equation y = Ax + B
Table 2. Intra- and inter-day precisions and accuracies for the determination of chlorogenic acid, ferulic acid, nodakenin, Z-ligustilide and decursin Analyte
Chlorogenic acid Ferulic acid Nodakenin Z-Ligustilide Decursin
Nominal conc. (lg mL-1)
0.80 10 40 0.36 4.5 18 0.60 7.5 30 2.4 30 120 0.50 12.5 50
Intra-day (n = 5)
Inter-day (n = 5)
Observed conc. (n = 5) Mean ± SD
Precision (%)
Accuracy (%)
Observed conc. (n = 5) Mean ± SD
Precision (%)
Accuracy (%)
0.82 9.93 38.95 0.35 4.49 17.97 0.58 7.51 30.05 2.37 29.90 120.45 0.53 12.47 50.33
1.4 0.7 0.5 0.9 0.4 1.0 1.7 0.8 0.8 0.6 0.6 1.0 0.7 0.6 0.9
102.5 99.3 97.4 97.2 99.8 99.8 97.4 100.1 100.2 98.8 99.7 100.4 106.0 99.8 100.7
0.79 9.80 38.93 0.35 4.51 18.11 0.59 7.61 30.19 2.43 30.01 120.89 0.53 12.57 50.57
1.3 0.5 0.3 0.7 0.7 1.1 0.8 0.9 0.4 0.9 0.9 0.4 1.5 1.2 0.5
98.8 98.0 97.3 98.0 100.2 100.6 98.8 101.5 100.7 101.1 100.1 100.7 105.8 100.6 101.1
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.01 0.06 0.19 0.003 0.02 0.18 0.01 0.06 0.24 0.01 0.19 1.19 0.004 0.08 0.47
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.01 0.04 0.12 0.002 0.03 0.20 0.004 0.07 0.12 0.02 0.28 0.53 0.01 0.16 0.27
Table 3. Recovery of the five analytes from the Angelica tenuissima ethanolic extract Analyte
Spiked conc. (lg mL-1)
Observed conc. (lg mL-1) (n = 3)
Mean recovery (%)
CV (%)
Chlorogenic acid
2 5 20 0.9 4.5 9 1.5 7.5 15 6 30 60 2.5 12.5 25
1.94 5.2 19.45 0.95 4.60 8.98 1.58 7.93 15.80 5.66 30.09 60.10 2.37 12.48 25.00
97.0 104.0 97.3 105.4 102.3 99.8 105.0 105.8 105.4 94.3 100.3 100.2 94.6 99.8 100.0
3.6 1.7 1.1 4.4 1.3 0.5 5.3 1.6 1.0 1.2 0.4 0.5 3.6 0.5 0.7
Ferulic acid Nodakenin Z-Ligustilide Decursin
In the recovery test, the extract sample was spiked with the mixed standard solution at low, medium and high concentration levels and analyzed. The extract sample spiked with 80% methanol was prepared and used as a control samples. The percentage Original
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.07 0.09 0.21 0.04 0.06 0.05 0.08 0.13 0.16 0.07 0.13 0.30 0.09 0.06 0.18
of the quantified amount to the spiked amount was calculated and expressed as a recovery. The average recoveries were 94.3–105.8% with RSDs below 5.3% for all analytes investigated. The detailed results are shown in Table 3.
The stability test was performed with standard solutions containing chlorogenic acid, ferulic acid, nodakenin, Z-ligustilide and decursin in different conditions. As a result, after the standard solution was maintained at room temperature (22 ± 3 °C) for 2 days and
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Intensity 7 x10 229.1 1.5
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Fig. 3. MS-MS spectrum of decursinol angelate
Table 4. Content of the bioactive constituents in ethanolic extract of Angelica tenuissima (mg g-1 of root extract) Code no.
Chlorogenic acid
Ferulic acid
Nodakenin
Z-Ligustilide
Decursin
Decursinol angelate
AT-1 AT-2 AT-3 AT-4 AT-5 AT-6 AT-7 AT-8 AT-9 AT-10
1.24 3.22 1.73 1.31 1.78 6.20 2.90 1.47 2.31 1.45
0.72 0.75 0.49 0.93 0.71 3.05 0.68 0.84 0.64 0.91
1.19 1.24 – – 0.85 5.66 0.48 – 1.27 –
33.44 22.62 9.44 58.78 41.77 4.98 21.66 19.66 13.86 30.39
5.90 12.37 0.74 1.30 13.21 22.55 2.46 0.51 4.13 1.32
3.86 7.46 0.51 0.93 9.77 14.06 1.95 0.25 2.41 0.82
± ± ± ± ± ± ± ± ± ±
0.001 0.123 0.016 0.025 0.012 0.100 0.106 0.010 0.020 0.005
± ± ± ± ± ± ± ± ± ±
0.002 0.002 0.001 0.004 0.004 0.008 0.007 0.003 0.004 0.006
± 0.003 ± 0.003 ± 0.006 ± 0.004 ± 0.011 ± 0.010
± ± ± ± ± ± ± ± ± ±
0.096 0.007 0.027 0.195 0.136 0.013 0.230 0.140 0.100 0.212
± ± ± ± ± ± ± ± ± ±
0.026 0.011 0.001 0.051 0.066 0.074 0.027 0.002 0.029 0.012
± ± ± ± ± ± ± ± ± ±
0.005 0.016 0.004 0.016 0.042 0.044 0.025 0.008 0.019 0.005
Data are presented as the mean ± SD (n = 3)
in the autosampler (4 °C) for a day, the content of its components decreased by 0–2.2%. The standard solution was found to be stable for 1 month when stored at -20 °C.
Identification and Quantitation of Decursinol Angelate The peak observed behind the peak of decursinol was expected to be decursinol angelate by the literature describing phytochemical analysis of Angelica species. Subsequently, the peak was characterized by LC-MS-MS analysis due to the lack of the authentic standard. The MS-MS spectrum of the unknown peak was presented along with its possible fragmentation behavior in Fig. 3. The [M+H]+ ion at m/z 329 yielded major fragment ions at m/z 247 and m/z 229 which suggested successive loss of an angeloyl moiety
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(82 Da) and a hydroxyl group as a water molecule (18 Da). The UV and MS-MS spectra are similar to the previously published data [15, 16] which confirm the identity of decursinol angelate. Lack of availability of the authentic standard of decursinol angelate, its quantitation was carried out using the equation derived from the calibration curve of decursin, its isomeric compound.
Sample Analysis The developed analytical method was subsequently applied to the simultaneous determination of six bioactive components in ethanolic extracts of A. tenuissima. A representative chromatogram of the extract was shown in Fig. 2 along with the chromatogram of the standard compounds. The amount of each compound present in the extract
samples was determined and the results are summarized in Table 4. Generally, the contents of Z-ligustilide, decursin and decursinol angelate were shown to be high. The highest amounts of nodakenin, chlorogenic and ferulic acid were found in sample AT-6 whereas nodakenin was not detected in sample AT-3, AT-4, AT-8 and AT-10. As shown in Table 4, the content of these major ingredients vary significantly in different samples. This variation may influence the pharmacological activities of the extracts. Therefore, in order to ensure the consistency of therapeutic benefits, it is necessary to quantify each of the major bioactive components in the extract.
Conclusion The results demonstrate that the developed LC method is a useful, simple and
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rapid technique for identification, separation and quantitation of chlorogenic acid, ferulic acid, nodakenin, Z-ligustilide, decursin and decursinol angelate in A. tenuissima. The developed method could be readily utilized as a quality control method for the quantification of major bioactive ingredients in A. tenuissima and its related medicinal products.
Acknowledgment This work was supported in part by grants from Korea Food and Drug Administration and in part by Seoul Research and Business Development Program.
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