Simultaneous determination of three sesquiterpene lactones in Aucklandia lappa Decne by high performance liquid chromatography
Chang-Seob Seo, Hyeun-Kyoo Shin
K-herb Research Center, Korea Institute of Oriental Medicine, 1672 Yuseongdae ro, Yuseong gu, Daejeon 305 811, Republic of Korea
|Date of Submission||07-Nov-2014|
|Date of Acceptance||04-Jan-2014|
|Date of Web Publication||10-Jul-2015|
K-herb Research Center, Korea Institute of Oriental Medicine, 1672 Yuseongdae ro, Yuseong gu, Daejeon 305 811
Republic of Korea
Source of Support: This research was supported by a grant (no. K14030) from the Korea Institute of Oriental Medicine, Conflict of Interest: None declared.
| Abstract|| |
Background: Aucklandia lappa Decne, a well known traditional herbal medicine, is used for the treatment of asthma, rheumatism, coughs, tuberculosis, and many other diseases. Objective: We performed simultaneous analysis of three sesquiterpene lactones, costunolide (1), dehydrocostus lactone (2), and alantolactone (3), obtained from a 70% methanol extract of A. lappa using high performance liquid chromatography–photodiode array (HPLC–PDA) techniques. Materials and Methods: The compounds 1–3 were separated using a reversed phase SunFire™ C18 analytical column kept at 35°C by the isocratic elution with distilled water and acetonitrile as mobile phase. The flow rate was 1.0 mL/min, and the injection volume was 10 μL. Results: The established analytical method showed high linearity, with a correlation coefficient ≥0.9999. The limit of detection and the limit of quantification of compounds 1–3 were 0.06–0.13 μg/mL and 0.21–0.42 μg/mL, respectively. The recovery of the compounds 1–3 was 97.27–103.00%. The intra and inter day relative standard deviations were 0.09–0.97% and 0.09–1.06%, respectively. The amounts of the compounds 1–3 were 17.32, 28.26, and 0.01 mg/g, respectively. Conclusion: The established and validated HPLC–PDA method may be help for the quality control of herbal medicine, A. lappa.
Keywords: Aucklandia lappa Decne, high performance liquid chromatography–photodiode array, simultaneous determination
|How to cite this article:|
Seo CS, Shin HK. Simultaneous determination of three sesquiterpene lactones in Aucklandia lappa Decne by high performance liquid chromatography. Phcog Mag 2015;11:562-6
|How to cite this URL:|
Seo CS, Shin HK. Simultaneous determination of three sesquiterpene lactones in Aucklandia lappa Decne by high performance liquid chromatography. Phcog Mag [serial online] 2015 [cited 2021 Oct 27];11:562-6. Available from: http://www.phcog.com/text.asp?2015/11/43/562/160471
| Introduction|| |
Aucklandiae Radix is derived from the root of Aucklandia lappa Decne (A. lappa, Asteraceae), syn Saussurea lappa, which has been officially recorded as “Mokhyang” in the Korean herbal pharmacopoeia. It has mainly been used as a traditional medicine for treating vomiting, gastric pain, abdominal pain, anorexia, distension, and nausea. Phytochemical studies with A. lappa have identified sesquiterpenes, sesquiterpene lactones, alkaloids, lignans, and tannins. Among these compounds, sesquiterpene lactones such as costunolide, dihydrocostunolide, isodihydrocostunolide, dehydrocostus lactone, alantolactone, isoalantolactone, and mokko lactone have been reported as the main constituents. Isolated sesquiterpene lactones from A. lappa have been shown to have various pharmacological activities including antiulcer,, anticancer,, anti inflammatory, antimycobacterial, and protein tyrosine phosphatase 1B inhibitory activities. Several analytical techniques have been reported the simultaneous determination and separation of various constituents in A. lappa.,, Kumar et al. performed the simultaneous analysis and method validation of isoalantolactone and alantolactone using ultra performance liquid chromatography with mass detection (mass spectrometry [MS]), and Li et al. developed a high speed counter current chromatography method for the separation and purification of costunolide and dehydrocostus lactone. In addition, Shum et al. carried out the chemical profile of the chemical components by gas chromatography with mass detection (MS).
In this study, we developed a method for the simultaneous determination of three sesquiterpene lactones, costunolide (1), dehydrocostus lactone (2), and alantolactone (3), the chemical structures of which are shown in [Figure 1], for quality control of A. lappa using high performance liquid chromatography (HPLC) coupled with a photodiode array (PDA) detector.
|Figure 1: Chemical structures of the three biomarker compounds in Aucklandia lappa|
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| Materials and methods|| |
The roots of A. lappa Decne used in this study was purchased from HMAX (Jecheon, Korea) in July 2009. The botanical origin of this sample was confirmed taxonomically by Prof. Je Hyun Lee, Dongguk University, Gyeongju, Republic of Korea. A voucher specimen (2009 KIOM62) has been deposited at the K-herb Research Center Korea Institute of Oriental Medicine.
Chemicals and materials
Reference compounds 1–3 were purchased from ChemFaces (Wuhan, China). The purities of the three sesquiterpene lactones were >98.0% by HPLC analysis. HPLC grade solvents, methanol, acetonitrile, and water were obtained from JT Baker (Phillipsburg, NJ, USA). Glacial acetic acid (analytical grade) was purchased from Merck KGaA (Darmstadt, Germany).
Preparations of 70% methanol extract and sample solution
Dried sample powder of A. lappa (100 g) was extracted 3 times with 70% methanol (1 L) by heating to reflux for 90 min. The extracted solution was filtered through filter paper, evaporated at 40°C using a Büchi R 210 rotary evaporator (Flawil, Switzerland) under vacuum to dryness and freeze dried. The yield of the freeze dried 70% methanol extract obtained was 28.57% (28.57 g). For the HPLC analysis of the compounds 1–3, the 70% methanol extract (20 mg) was dissolved in 10 mL of 70% methanol and extracted by sonication for 30 min. The solution was filtered through a 0.2 μm membrane filter (Woongki Science, Seoul, Korea) before injection into the HPLC instrument.
Apparatus and conditions
The simultaneous determination was performed with a Shimadzu Prominence LC 20A series HPLC (Shimadzu Co., Kyoto, Japan) consisting of a solvent delivery unit (LC 20AT), on line degasser (DGU 20A3), column oven (CTO 20A), auto sample injector (SIL 20AC), and PDA detector (SPD M20A). Data were collected and processed by LCsolution software (Version 1.24, Shimadzu, Kyoto, Japan). Separation of compounds 1–3 was carried out on a reversed phase SunFire™ C18 analytical column (Waters, Milford, MA, USA; 150 mm × 4.6 mm and 5 μm particle size). The mobile phase for chromatographic separation of the three analytes was distilled water (A) and acetonitrile (B) with isocratic elution (i.e. 40% A and 60% B). The flow rate was 1.0 mL/min, the column temperature was maintained at 35°C, and the detection wavelength of quantification was set at 225 nm. The injection volume was 10 μL.
Calibration curves, limits of detection, and quantification
To prepare the stock solutions, reference compounds 1–3 were accurately weighed and dissolved in methanol to a concentration of 1.0 mg/mL; the samples were stored below 4°C. The calibration curves for 1–3 were calculated by plotting the peak areas (y) versus the concentrations (x, μg/mL) using the standard solutions. The tested concentration ranges for calibration curves were 1.56–200.00, 2.34–300.00, and 0.23–30.00 μg/mL for compounds 1, 2, and 3, respectively. The limits of detection (LOD) and limits of quantification (LOQ) for the three sesquiterpene lactones were calculated at signal to noise ratios of 3 and 10, respectively.
Precision, reproducibility, and accuracy
Intra day and inter day variations, which were used to evaluate the precision of the established HPLC method, were determined using the standard addition method for samples spiked with low, medium, and high concentration levels of analyte. The relative standard deviation (RSD) was used as a measurement of precision. To confirm the reproducibility of the proposed method, we measured six replications using the mixed standard stock solutions. The RSD values of peak areas and retention times of the three compounds were evaluated to establish the reproducibility of the method, and recovery tests were performed to evaluate the accuracy of the method. The test for recovery was carried out by adding known amounts of low, medium, and high concentration levels of the three reference compounds to 20 mg of A. lappa extract. This test was evaluated using the calibration curves determined for compounds 1–3.
| Results and discussion|| |
Optimization of chromatographic conditions
High performance liquid chromatography conditions including column type, column temperature, and mobile phases were assessed to accomplish the simultaneous separation of the three analytes 1–3. For the optimized separation of the three components, columns including Phenomenex Gemini C18 (250 mm × 4.6 mm, 5 μm), Waters SunFire C18 (250 mm × 4.6 mm, 5 μm), Waters SunFire C18 (150 mm × 4.6 mm, 5 μm), and OptimaPak C18 (250 mm × 4.6 mm, 5 μm), various column temperatures (30, 35, and 40°C), and a range of mobile phases composed of acids such as acetic, formic, and phosphoric acid, and organic solvents methanol and acetonitrile, were examined. As a result, optimized simultaneous separation of the three analytes with respect to baseline, resolution, and peak shape could be achieved using a Waters SunFire™ C18 column (150 mm × 4.6 mm, 5 μm) with isocratic elution of distilled water (A) and acetonitrile (B) at 35°C. The optimized conditions enabled the three compounds to be eluted within 15 min with a resolution of better than 1.56. Detection wavelength for quantification of the three analytes was set at 225 nm, and each compound in the HPLC chromatogram was identified by comparing the retention times and ultraviolet spectra with those of standards and by co injection with an authentic sample. The retention times of compounds 1–3 under the optimized conditions were 7.81, 8.70, and 10.22 min, respectively. Representative HPLC chromatograms of standard solutions and the A. lappa extracts are shown in [Figure 2].
|Figure 2: High-performance liquid chromatography chromatogram of standard solution (a) and Aucklandia lappa extract (b). Costunolide (1); dehydrocostus lactone (2); and alantolactone (3)|
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The system suitability, that is., capacity factor (K′), relative retention (a), resolution (Rs), theoretical plate number (N) and tailing factor (TF) was measured in order to evaluate the reliability of optimized HPLC method. As shown in [Table 1], the capacity factor and relative retention were 2.88–5.82 and 1.14–1.78. The resolutions of the compounds 1–3 were >1.5, which suggested that the peaks of three components were not severely overlapped by adjacent peaks and interference from other components. The number of theoretical plate and TF were 1392–3340 and 1.12–1.20.
Linearity, range, limits of detection, and limits of quantification
Each regression equation (y = ax + b) was calculated based on the ratio of peak area (y), and concentration (x, μg/mL) of reference compounds 1–3. Standard curves plotted with compounds 1–3 showed high linearity, with r2 ≥0.9999 in the eight different concentration ranges tested. The LODs and LOQs compounds 1–3 were 0.06–0.13 and 0.21–0.42 μg/mL, respectively. The results are summarized in [Table 2].
|Table 2: Regression equation, linear range, correlation coefficient, LOD, and LOQ for marker compounds|
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Precision, reproducibility, and accuracy
As shown in [Table 3], the recoveries of the three analytes were 97.27–13.00% at low, medium, and high concentration levels, and the RSD values were <1.02%. The RSD for reproducibility of analysis of compounds 1–3 was 0.19–0.28% for peak area and 0.08–0.09% for retention times [Table 4]. The precisions (RSD) of intra and inter day variations for the three analytes were <0.97 and 1.06%, respectively. The results, which are summarized in [Table 4], therefore, suggest that the established method was suitable for quantitative analysis of the three investigated compounds.
The established HPLC analytical method was applied for the simultaneous quantification of the compounds 1–3 in A. lappa. The amounts of three sesquiterpene lactones, costunolide (1), dehydrocostus lactone (2), and alantolactone (3) were 17.32 mg/g, 28.26 mg/g, and 0.01 mg/g, respectively [Table 5].
|Table 5: The amounts of the three marker compounds in A. lappa determined by HPLC analysis|
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| Conclusion|| |
A convenient, simple, and accurate HPLC PDA method was established for the simultaneous determination of the three sesquiterpene lactones, costunolide (1), dehydrocostus lactone (2), and alantolactone (3), in A. lappa. The developed method has been successfully applied to the quantitative analysis for the purpose of quality control and showed good linearity, precision, and accuracy. The established HPLC PDA method was demonstrated to be a suitable method for quality control of the components of A. lappa.
| Acknowledgments|| |
This research was supported by a grant (no. K14030) from the Korea Institute of Oriental Medicine.
| References|| |
Kim H, Kang K, Choi G, Kim H, Jeong S, Ju Y. A study on external and internal morphology and pattern analysis in 4 kinds of Mok Hyaeng Radix. Korean J Orient Med 2006;12:117 30.
Yoon TS, Sung YY, Jang JY, Yang WK, Ji Y, Kim HK. Anti obesity activity of extract from Saussurea lappa
. Korean J Med Crop Sci 2010;18:151 6.
Hasson SS, Al Balushi MS, Alharthy K, Al Busaidi JZ, Aldaihani MS, Othman MS, et al
. Evaluation of anti resistant activity of Auklandia (Saussurea lappa
) root against some human pathogens. Asian Pac J Trop Biomed 2013;3:557 62.
Korea Food and Drug Administration. The Korean Herbal Pharmacopoeia. Seoul: Dongwon Munhwasa; 2007. p. 132.
Choi JY, Na M, Hyun Hwang I, Ho Lee S, Young Bae E, Yeon Kim B, et al
. Isolation of betulinic acid, its methyl ester and guaiane sesquiterpenoids with protein tyrosine phosphatase 1B inhibitory activity from the roots of Saussurea lappa
C.B.Clarke. Molecules 2009;14:266 72.
Robinson A, Kumar TV, Sreedhar E, Naidu VG, Krishna SR, Babu KS, et al
. A new sesquiterpene lactone from the roots of Saussurea lappa
: Structure anticancer activity study. Bioorg Med Chem Lett 2008;18:4015 7.
Li A, Sun A, Liu R. Preparative isolation and purification of costunolide and dehydrocostuslactone from Aucklandia lappa
Decne by high speed counter current chromatography. J Chromatogr A 2005;1076:193 7.
Rasul A, Khan M, Ali M, Li J, Li X. Targeting apoptosis pathways in cancer with alantolactone and isoalantolactone. Scientific World Journal 2013;2013:248532.
Yoshikawa M, Hatakeyama S, Inoue Y, Yamahara J. Saussureamines A, B, C, D, and E, new anti ulcer principles from Chinese Saussureae
Radix. Chem Pharm Bull (Tokyo) 1993;41:214 6.
Matsuda H, Kageura T, Inoue Y, Morikawa T, Yoshikawa M. Absolute stereostructures and syntheses of saussureamines A, B, C, D and E, amino acid sesquiterpene conjugates with gastroprotective effect, from the roots of Saussurea lappa
. Tetrahedron 2000;56:7763 77.
Ohnishi M, Yoshimi N, Kawamori T, Ino N, Hirose Y, Tanaka T, et al
. Inhibitory effects of dietary protocatechuic acid and costunolide on 7,12 dimethylbenz[a] anthracene induced hamster cheek pouch carcinogenesis. Jpn J Cancer Res 1997;88:111 9.
Kassuya CA, Cremoneze A, Barros LF, Simas AS, Lapa Fda R, Mello Silva R, et al
. Antipyretic and anti inflammatory properties of the ethanolic extract, dichloromethane fraction and costunolide from Magnolia ovata (Magnoliaceae
). J Ethnopharmacol 2009;124:369 76.
Fischer NH, Lu T, Cantrell CL, Castañeda Acosta J, Quijano L, Franzblau SG. Antimycobacterial evaluation of germacranolides. Phytochemistry 1998;49:559 64.
Kumar A, Kumar S, Kumar D, Agnihotri VK. UPLC/MS/MS method for quantification and cytotoxic activity of sesquiterpene lactones isolated from Saussurea lappa
. J Ethnopharmacol 2014;155:1393 7.
Shum KC, Chen F, Li SL, Wang J, But PP, Shaw PC. Authentication of Radix Aucklandiae and its substitutes by GC MS and hierarchical clustering analysis. J Sep Sci 2007;30:3233 9.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]