Original Papers
237
Authors
Xue Qiao*, Chun-Fang Liu*, Shuai Ji, Xiong-Hao Lin, De-An Guo, Min Ye
Affiliation
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China
Key words " Glycyrrhiza uralensis l " Leguminosae l " licorice l " SPE‑HPLC‑DAD l " coumarins l " flavonoids l
Abstract
received revised accepted
July 16, 2013 October 21, 2013 Dec. 8, 2013
Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1360272 Published online February 4, 2014 Planta Med 2014; 80: 237–242 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Min Ye State Key Laboratory of Natural and Biomimetic Drugs School of Pharmaceutical Sciences Peking University 38 Xueyuan Road Beijing 100191 China Phone: + 86 10 82 80 20 24 Fax: + 86 10 82 80 20 24 [email protected]
!
Minor phenolic compounds in licorice (Glycyrrhiza uralensis) have recently been proved for diverse bioactivities and favorable bioavailability, indicating that they may play an important role in the therapeutic effects or herb-drug interactions of licorice. However, so far, their abundance in licorice remains unknown. In this study, a reliable solid-phase extraction coupled with a highperformance liquid chromatography and diode array detection method was established to determine the minor phenolic compounds in licorice. The analytes were enriched by a three-step solidphase extraction method, and then separated on a YMC ODS‑A column by gradient elution. Five coumarins and flavonoids were identified by electrospray ionization tandem mass spectrometry, and then quantified using high-performance liquid chromatography and diode array detection. The amounts of glycycoumarin, dehydroglyasperin C, glycyrol, licoflavonol, and glycyrin in G. uralensis were 0.81 ± 0.28, 1.25 ± 0.59, 0.20 ± 0.08,
Introduction !
Licorice is derived from the roots and rhizomes of Glycyrrhiza uralensis Fisch., G. inflata Bat., and G. glabra L. (Leguminosae family) [1]. It is one of the most frequently used herbal medicines, and is mainly used for the treatment of peptic ulcers, inflammation, coughs, hepatic disease, viral infections, cancer, and other ailments [2–5]. These biological activities are generally attributed to major chemical constituents such as flavanones, chalcones, and triterpenoid saponins [6, 7]. Recently, minor phenolic compounds in licorice (especially coumarins, free flavones, and isoflavones) have
* The first two authors contributed equally to this paper.
0.12 ± 0.04, and 0.17 ± 0.08 mg/g, respectively. Abundances of these compounds in other Glycyrrhiza species (G. glabra, G. inflata, and G. yunnanesis) were remarkably lower than G. uralensis.
Abbreviations !
ESI‑MSn:
electrospray ionization tandem mass spectrometry LOD: limit of detection LOQ: limit of quantification SPE-HPLC‑DAD: solid-phase extraction coupled with high-performance liquid chromatography and diode array detection RSD: relative standard deviation IS: internal standard S/N: signal-to-noise Supporting information available online at http://www.thieme-connect.de/ejournals/toc/ plantamedica
received more and more attention due to their significant antioxidant, antimicrobial, and anticarcinogenic activities [8, 9]. For instance, glycycoumarin (1), a representative coumarin in G. uralensis, showed antithrombotic [10] and antispasmodic [11] activities. Glycyrol (3) showed an antiinflammatory effect [12], and could inhibit neuraminidase [13] and aldose reductase [14]. Glycyrin (5) was reported to have antihypertension activity (which was related to PPAR-γ) [15] and antivirus activity [16]. Furthermore, the isoflavone compound dehydroglyasperin C (2) exhibited neuroprotective effects by inducing phase II enzymes [17], while the minor flavone licoflavonol (4) was reported as an anti-rotavirus agent, especially to G5P [7] and G8P [7, 16]. The structures of " Fig. 1. these compounds are shown in l
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Simultaneous Determination of Five Minor Coumarins and Flavonoids in Glycyrrhiza uralensis by Solid-Phase Extraction and High-Performance Liquid Chromatography/Electrospray Ionization Tandem Mass Spectrometry
Original Papers
Fig. 1 Structures of five minor phenolic compounds in Glycyrrhiza uralensis and the internal standard.
In addition to various bioactivities, some of these minor phenols were also proved for favorable bioavailability in vivo, according to our recent studies [18, 19]. Glycycoumarin (1) could be rapidly absorbed and distributed together with its phase II metabolites [19]. Glycyrol (3) and licoflavonol (4) could also be detected in rat plasma after administration of the licorice extract. These minor compounds may contribute to the therapeutic effects or herb-drug interactions of licorice. However, though the contents of major components have been clearly revealed [20, 21], little is known about the abundance of these minor compounds in licorice, except that the content of glycycoumarin has only been addressed once, so far [22]. SPE is an effective and reproducible approach to enrich minor compounds in complicated herbal extracts [23]. In this study, a reliable SPE-HPLC‑DAD method was established to determine five minor phenolic compounds in 20 batches of Glycyrrhiza uralensis. These compounds included three coumarins (glycycoumarin, glycyrol, and glycyrin) and two flavonoids (dehydroglyasperin C and licoflavonol).
Fig. 2 Optimization of the elution volume in solid-phase extraction using 100 % methanol. (Color figure available online only.)
Results and Discussion !
To efficiently extract minor coumarins and flavonoids from licorice, the experimental conditions were optimized, including solvent (70% and 100 % methanol), solvent volume (6-, 10-, 20-, and 30-fold), extraction method (reflux, ultrasonication, and maceration), and extraction time (15, 30, 60, and 120 min). The chromatograms from different conditions are given in Fig. 1S. The optimized method was to extract 0.25 g of the powder with 6 mL of methanol (containing 0.037 mg/mL of bergapten) in an ultrasonic water bath for 30 min. HPLC conditions were also optimized (Fig. 2S), including column type (Zorbax SB‑C18/Aglient, Symmetry C18/Waters, YMC ODS‑A/YMC, and Atlantis T3/Waters), column temperature (30 °C, 35 °C), and detection wavelength (230 nm, 254 nm, 280 nm, 306 nm, 365 nm). With the optimized conditions, all analytes could be well separated within 40 min. Due to the low polarity of the target compounds, solid-phase extraction was used to remove the polar major interfering components (flavonoid glycosides and saponins) of licorice. The SPE method was optimized to improve its efficiency and reproducibility. The Oasis HLB cartridge (packed with N-vinylpyrrolidone and divinylbenzene) was selected to trap the analytes. The licorice extract (see “Sample preparation” section) was reconstituted in 600 µL of methanol and then loaded onto the SPE column. A three-step elution program was developed: 1) elute with 6 mL of water to remove hydrophilic constituents (Fig. 3S); 2) elute with 8 mL of 65 % methanol to remove unwanted compounds, while
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effectively retaining the target analytes; and 3) elute with 8 mL of 100 % methanol to obtain the enriched analytes. Our results suggested that analytes were retained on the column at below 60 % and 65 % methanol (5 mL), and started to be eluted when 70 % methanol (5 mL) was used (Fig. 4S). In addition, 65 % methanol was more effective to remove the unwanted compounds than 60 % methanol (Fig. 5S), and 8 mL of 65 % methanol could remove the majority of the unwanted major compounds (Fig. 6S). The elution of 8 mL of 100% methanol could yield 99.82% (1), 99.74 % (2), 99.60 % (3), 100.00 % (4), and 100.00 % (5) of the analytes " Fig. 2). To validate the recovery of this SPE method, an internal (l standard (bergapten) was spiked into a licorice extract and pretreated with the same procedure. The compound had a similar UV absorption and similar chromatographic behavior as the analytes, and was absent in the licorice samples (Fig. 7S). Recovery of the internal standard (calculated by the peak area ratio of SPEtreated solution/original solution) was above 93 %. Therefore, the optimal SPE pretreatment procedure was developed as described in the “Sample preparation” section. In the chromatogram of SPE-treated licorice samples, five analytes were identified by high-performance liquid chromatography coupled with HPLC‑DAD‑MSn. Both negative and positive ESI ion modes were tested, and (−)-ESI was chosen for its higher sensitivity of coumarins and flavonoids. The retention times of the five " Fig. 3 B) were consistent peaks in the licorice sample GC-18 (l
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" Fig. 3 A). Tandem mass spectra of with the pure standards 1–5 (l " Fig. 4. Three coumarins (1, 3, the five analytes are provided in l and 5) showed maximum UV absorptions at 254 nm and 352 nm. Take compound 1 for example, (−)-ESI‑MS showed an intense [M – H]− ion at m/z 367. The MS/MS spectra were predominated by the elimination of CH3· (from the methoxyl group), C3H7·, and C5H8· (from the isoprenyl chain). The m/z 309 ion was generated by the simultaneous cleavage of CH3· and C3H7·. It yielded daughter ions at m/z 265 and 281 due to the loss of CO2 and CO, respectively. All three coumarins showed similar path" Fig. 4 A, C, and E). On the other hand, the two flavonoids ways (l had the same [M – H]− ion, but they showed different maximum UV absorptions at 330 nm (2) and 340 nm (4). Predominant cleavage occurred on the isoprenyl group to produce m/z 298 " Fig. 4 B and (-C4H7), which further fragmented into m/z 269 (l D). Tandem mass spectra and UV spectra of the analytes in real samples matched with those of authentic standards, indicating " Fig. 3 was acceptable to furthat the resolution of peaks 1–5 in l ther conduct the quantitative analysis. A validated HPLC-UV method was then established to determine the contents of these minor phenolic compounds in licorice. For quantitative analysis, the working solution samples were analyzed in duplicate to establish the calibration curves. The curves for compounds 1–5 were y = 6.3237 x + 0.1832 (linear range from 15.65 to 1370 µg/mL), y = 1.8786 x + 0.0854 (19.54 to 2736 µg/ mL), y = 11.2660 x + 0.0187 (2.057 to 360 µg/mL), y = 10.0555 x − 0.019 (3.171 to 222 µg/mL), and y = 8.8812 x + 0.022 (7.057 to 494 µg/mL), respectively. The calibration curves were constructed by plotting the ratio of mean peak areas of the samples and the IS against the concentration of each compound. All calibration curves showed good linearity with correlation coefficients (r2) no less than 0.999, in relatively wide dynamic ranges (70- to 175-fold). The LOD and LOQ of five compounds were determined by injecting a series of standard solutions until the S/N " Table 1). ratio was three for LOD and 10 for LOQ, respectively (l
Intraday variation, interday variation, interbatch variation, stability, and recovery of the method were validated for each analyte. The analysis was repeated using the same sample six times in the same day, and additionally on three consecutive days to determine intra- and interday precision, respectively. Intra- and interday variations of the signals were less than 3.00 % and 0.97 % (rep" Table 1). The interbatch variation resented by RSD values, see l was evaluated by preparing and analyzing six samples of the same batch of licorice with the same procedure. Variation among " Table 1). The same licorice these analyses was less than 2.43 % (l sample was stored at 25 °C, and analyzed at 0, 2, 4, 8, 12, and 24 h " Tafor the stability test. The variation was no more than 2.26 % (l ble 1). Recovery tests were performed by adding a known amount of each pure analyte into 0.125 g of licorice crude drug powder (n = 3). The mixture was pretreated following the “Sample preparation” section, and then analyzed following the “HPLCUV quantitation” section. The developed analytical method showed acceptable recoveries between 85.30 % and 105.22 % (RSD < 7.84 %, Table 1S). Using the validated SPE-HPLC‑DAD method, the contents of five phenolic compounds were determined in 20 batches of G. uralen" Table sis, as well as in samples from other Glycyrrhiza species (l 2). Among the analytes, the contents of glycycoumarin (1) and dehydroglyasperin C (2) were 0.40–1.28 mg/g and 0.40–2.28 mg/ g, respectively, in G. uralensis. Glycyrol (3), licoflavonol (4), and glycyrin (5) had relatively lower abundances between 0.08– 0.32 mg/g, 0.06–0.21 mg/g, and 0.07–0.44 mg/g, respectively. The contents of the five phenols were remarkably lower in other Glycyrrhiza species. G. yunnanesis is mainly distributed in Yunnan Province in China, and is used as licorice by local people. However, it contained none of the five phenolic compounds. For G. inflata and G. glabra, compound 5 was missing in their samples, and the content of compounds 1, 2, and 3 was lower than those of G. uralensis. Due to the limited sample volume, this requires further investigation with more samples. It is also noteworthy that G. uralensis samples collected from Xinjiang and In-
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Planta Med 2014; 80: 237–242
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Fig. 3 HPLC‑UV chromatograms of different Glycyrrhiza species after SPE treatment (365 nm). A Mixed standards, B G. uralensis, C G. inflata, D G. glabra, and E G. yunnanensis. 1 Glycycoumarin, 2 dehydroglyasperin C, 3 glycyrol, 4 licoflavonol, 5 glycyrin, IS internal standard (bergapten). (Color figure available online only.)
Original Papers
Fig. 4 (−)-ESI‑MSn spectra of the five analytes. A Glycycoumarin (1), B dehydroglyasperin C (2), C glycyrol (3), D licoflavonol (4), and E glycyrin (5). (Color figure available online only.)
Table 1 Variation, stability, and detection limits of the HPLC-UV quantitation method. Analyte 1 2 3 4 5
LOD
LOQ
Intraday variation
Interday variation
Interbatch variation
Stability
(µg/mL)
(µg/mL)
(n = 5)
(n = 3)
(n = 6)
(n = 6)
0.157 0.326 0.069 0.106 0.141
0.261 3.257 0.686 1.057 0.235
0.09 0.11 3.00 0.36 0.98
0.63 0.17 0.47 0.97 0.48
0.20 0.36 2.43 0.18 0.75
0.28 0.98 2.26 0.54 0.46
Note: 1 glycycoumarin, 2 dehydroglyasperin C, 3 glycyrol, 4 licoflavonol, 5 glycyrin, LOD limit of detection (S/N = 3), LOQ limit of quantification (S/N = 10). Intraday variation, interday variation, interbatch variation, and stability are shown in RSD (%)
ner Mongolia contained lower amounts of the phenolic compounds, although these places are considered authentic production areas of licorice in China. Therefore, further studies are necessary for comprehensive quality control of licorice, and the quality of licorice crude drugs needs to be standardized to guarantee its therapeutic efficacy.
Materials and Methods !
Chemicals, reagents, and plant materials HPLC grade acetonitrile and formic acid were purchased from Mallinckrodt Baker Technology, Inc. Deionized water was purified by a Milli-Q system (Millipore). Other reagents were purchased from Beijing Chemical Corporation. Oasis HLB cartridges (600 cc, 200 mg) were purchased from Waters. Five pure reference standards, glycycoumarin (1), dehydroglyasperin C (2), glycyrol (3), licoflavonol (4), and glycyrin (5) were isolated from Glycyrrhiza uralensis by the authors. Their structures were identified by NMR and MS spectroscopy (Fig. 8S), by comparison to previous publications (listed in the note to Fig. 8S). The IS, bergapten, was purchased from ZeLang Medical Technology Co., Ltd. All the reference compounds showed purities above 98 % by HPLC analy" Fig. 1. Twentysis. Structures of these compounds are shown in l three batches of crude drugs were collected in different provinces " Table 2) and were authenticated by the authors as of China (l G. uralensis (20 batches), G. inflata (1 batch), G. glabra (1 batch), and G. yunnanensis (1 batch). Among them, G. yunnanensis is used as folk medicine in Yunnan Province. Voucher specimens (LIC12GC-1 to LIC12GC-20, LC11GI-1, LC11GG-1, LC11GY-1)
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were deposited at the School of Pharmaceutical Sciences, Peking University.
Sample preparation For HPLC/MSn and HPLC-UV analyses, raw materials of licorice were pulverized into fine powder, accurately weighed, and then methanol was added (containing 0.037 mg/mL of bergapten) to make a concentration of 250 mg per 6 mL. The licorice was then extracted in an ultrasonic water bath for 30 min at 30 °C. Following centrifugation (10 min, 7000 rpm), a 5-mL aliquot of the supernatant was transferred into a clean tube, and dried under a gentle nitrogen flow. The residue was reconstituted in 600 µL of methanol and was then loaded onto an SPE column (preconditioned with 5 mL of methanol, followed by 5 mL of water). The column was sequentially eluted with 6 mL of water, 8 mL of 65 % methanol, and 8 mL of 100 % methanol. The flow rate was maintained at approximately 0.5 mL/min using a 12-position vacuum extraction manifold. The 100% methanol fraction was collected, evaporated to dryness under a gentle flow of nitrogen, and reconstituted in 200 µL of methanol for analysis.
Preparation of standard working solutions and internal standard solution For HPLC quantification, a stock solution was prepared by dissolving the five analytes in methanol. Concentrations of the analytes were 2.74 mg/mL (1), 3.42 mg/mL (2), 0.36 mg/mL (3), 1.11 mg/mL (4), and 2.47 mg/mL (5), respectively. The stock solution was diluted with methanol (1.25-, 2.00-, 2.50-, 3.33-, 4.00-, 4.67-, 5.60-, 7.00-, 9.33-, 14.00-, and 28.00-fold, respectively) to produce serial concentrations of working solutions. The IS solution was prepared by dissolving bergapten in methanol to make
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Table 2 Contents of the five analytes (1−5) in G. uralensis and other Glycyrrhiza species.
GC-1 GC-2 GC-3 GC-4 GC-5 GC-6 GC-7 GC-8 GC-9 GC-10 GC-11 GC-12 GC-13 GC-14 GC-15 GC-16 GC-17 GC-18 GC-19 GC-20 GC-21 GC-22 GC-23
Species G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. glabra G. inflata G. yunnanesis
Collected from
Content of analytes (mg/g)
Gansu Anhui Yulin, Shaanxi Pingdingshan, Heʼnan Huaiyuan, Anhui Haerbin Wenlin, Zhejiang Xinjiang Langfang, Hebei Baotou, Inner Mongolia Jinzhou, Liaoning Inner Mongolia Inner Mongolia Hangjinqi, Inner Mongolia Hangjinqi, Inner Mongolia Chifeng, Inner Mongolia Zhangjiakou, Hebei Xingtai, Hebei Huʼnan Gansu Xinjiang Xinjiang Yunnan
1
2
3
4
5
0.879 1.125 1.022 1.280 1.187 0.821 1.043 0.636 0.826 0.405 0.456 0.779 0.409 0.400 0.726 1.003 0.474 1.091 0.857 0.789 0.006 0.320 –
0.608 1.652 1.094 2.279 1.692 1.251 1.523 0.547 1.159 0.907 0.399 0.729 0.517 0.487 1.181 1.817 1.672 2.179 1.966 1.310 – 0.319 –
0.155 0.242 0.245 0.324 0.322 0.158 0.273 0.122 0.202 0.084 0.093 0.183 0.083 0.108 0.176 0.240 0.279 0.246 0.224 0.167 – 0.123 –
0.166 0.166 0.214 0.125 0.117 0.115 0.103 0.172 0.095 0.111 0.061 0.092 0.062 0.149 0.136 0.087 0.188 0.101 0.091 0.079 0.094 0.048 –
0.173 0.199 0.215 0.198 0.188 0.128 0.163 0.170 0.122 0.068 0.101 0.112 0.087 0.149 0.215 0.156 0.442 0.182 0.135 0.125 – – –
Note: 1 glycycoumarin, 2 dehydroglyasperin C, 3 glycyrol, 4 licoflavonol, 5 glycyrin, – below the limit of quantitation
a concentration of 0.037 mg/mL. To each 200 µL of working solution, 5 mL of IS solution was added, and the mixtures were dried under a nitrogen flow. The residue was reconstituted in 200 µL methanol to obtain a set of working solutions to establish the calibration curves. All solutions and samples were kept at 4 °C until use.
4.5 kV; sheath gas (N2) 40 arbitrary units; auxiliary gas (N2) 10 units; capillary temperature 320 °C; capillary voltage − 30 V; and tube lens offset voltage − 20 V. Full-scan spectra were recorded in the range of m/z 120–1000. Data were analyzed using Xcalibur™ 1.4 software (Thermo Fisher).
Supporting information HPLC‑UV quantitation The HPLC determination was performed on an Agilent series 1100 HPLC system including a quaternary pump, a diode array detector, an autosampler, and a column compartment. Samples were separated on a YMC-Pack ODS‑A column (250 mm × 4.6 mm I. D., 5 µm) equipped with an Agilent ZORBAX SB‑C18 guard column (20 mm × 3.9 mm I. D., 5 µm). The mobile phase consisted of acetonitrile (A) and water containing 0.1 % (v/v) formic acid (B). A gradient program was used as follows: 0–5 min 44–46 % A; 5–25 min 46–50% A; 25–30 min 50–60% A; 30– 32 min 60–65% A; 32–34 min 65–95% A; and 34–36 min 95 % A. A 15-min post-run time was set to fully equilibrate the column. The flow rate was 1.0 mL/min. The column temperature was 35 °C. The detection wavelength was set from 190 to 800 nm, and the samples were detected at 365 nm. The sample injection volume was 10 µL. All data were processed by Agilent LC B.02.01 ChemStation software.
Optimization of the extraction method, the HPLC condition and SPE elution, as well as recovery of the quantitation method are available as Supporting Information.
Acknowledgements !
This work was supported by the National Natural Science Foundation of China (No. 81173644, No. 81222054) and the Program for New Century Excellent Talents in University from the Chinese Ministry of Education (No. NCET-11–0019).
Conflict of Interest !
No conflict of interest exists in the submission of this manuscript and this manuscript has been approved by all authors for publication.
HPLC‑DAD‑MSn analysis HPLC‑DAD‑MSn analysis was performed on an Agilent series 1100 HPLC instrument coupled with an LCQ Advantage ion-trap mass spectrometer (Thermo Fisher) via an electrospray ionization interface. The HPLC conditions were the same as those for HPLC‑DAD analysis. The HPLC eluent was introduced into the electrospary ionization source in a post-column splitting ratio of 5 : 1. It was operated in the negative ion mode: source voltage
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237
Authors
Xue Qiao*, Chun-Fang Liu*, Shuai Ji, Xiong-Hao Lin, De-An Guo, Min Ye
Affiliation
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China
Key words " Glycyrrhiza uralensis l " Leguminosae l " licorice l " SPE‑HPLC‑DAD l " coumarins l " flavonoids l
Abstract
received revised accepted
July 16, 2013 October 21, 2013 Dec. 8, 2013
Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1360272 Published online February 4, 2014 Planta Med 2014; 80: 237–242 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Min Ye State Key Laboratory of Natural and Biomimetic Drugs School of Pharmaceutical Sciences Peking University 38 Xueyuan Road Beijing 100191 China Phone: + 86 10 82 80 20 24 Fax: + 86 10 82 80 20 24 [email protected]
!
Minor phenolic compounds in licorice (Glycyrrhiza uralensis) have recently been proved for diverse bioactivities and favorable bioavailability, indicating that they may play an important role in the therapeutic effects or herb-drug interactions of licorice. However, so far, their abundance in licorice remains unknown. In this study, a reliable solid-phase extraction coupled with a highperformance liquid chromatography and diode array detection method was established to determine the minor phenolic compounds in licorice. The analytes were enriched by a three-step solidphase extraction method, and then separated on a YMC ODS‑A column by gradient elution. Five coumarins and flavonoids were identified by electrospray ionization tandem mass spectrometry, and then quantified using high-performance liquid chromatography and diode array detection. The amounts of glycycoumarin, dehydroglyasperin C, glycyrol, licoflavonol, and glycyrin in G. uralensis were 0.81 ± 0.28, 1.25 ± 0.59, 0.20 ± 0.08,
Introduction !
Licorice is derived from the roots and rhizomes of Glycyrrhiza uralensis Fisch., G. inflata Bat., and G. glabra L. (Leguminosae family) [1]. It is one of the most frequently used herbal medicines, and is mainly used for the treatment of peptic ulcers, inflammation, coughs, hepatic disease, viral infections, cancer, and other ailments [2–5]. These biological activities are generally attributed to major chemical constituents such as flavanones, chalcones, and triterpenoid saponins [6, 7]. Recently, minor phenolic compounds in licorice (especially coumarins, free flavones, and isoflavones) have
* The first two authors contributed equally to this paper.
0.12 ± 0.04, and 0.17 ± 0.08 mg/g, respectively. Abundances of these compounds in other Glycyrrhiza species (G. glabra, G. inflata, and G. yunnanesis) were remarkably lower than G. uralensis.
Abbreviations !
ESI‑MSn:
electrospray ionization tandem mass spectrometry LOD: limit of detection LOQ: limit of quantification SPE-HPLC‑DAD: solid-phase extraction coupled with high-performance liquid chromatography and diode array detection RSD: relative standard deviation IS: internal standard S/N: signal-to-noise Supporting information available online at http://www.thieme-connect.de/ejournals/toc/ plantamedica
received more and more attention due to their significant antioxidant, antimicrobial, and anticarcinogenic activities [8, 9]. For instance, glycycoumarin (1), a representative coumarin in G. uralensis, showed antithrombotic [10] and antispasmodic [11] activities. Glycyrol (3) showed an antiinflammatory effect [12], and could inhibit neuraminidase [13] and aldose reductase [14]. Glycyrin (5) was reported to have antihypertension activity (which was related to PPAR-γ) [15] and antivirus activity [16]. Furthermore, the isoflavone compound dehydroglyasperin C (2) exhibited neuroprotective effects by inducing phase II enzymes [17], while the minor flavone licoflavonol (4) was reported as an anti-rotavirus agent, especially to G5P [7] and G8P [7, 16]. The structures of " Fig. 1. these compounds are shown in l
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Simultaneous Determination of Five Minor Coumarins and Flavonoids in Glycyrrhiza uralensis by Solid-Phase Extraction and High-Performance Liquid Chromatography/Electrospray Ionization Tandem Mass Spectrometry
Original Papers
Fig. 1 Structures of five minor phenolic compounds in Glycyrrhiza uralensis and the internal standard.
In addition to various bioactivities, some of these minor phenols were also proved for favorable bioavailability in vivo, according to our recent studies [18, 19]. Glycycoumarin (1) could be rapidly absorbed and distributed together with its phase II metabolites [19]. Glycyrol (3) and licoflavonol (4) could also be detected in rat plasma after administration of the licorice extract. These minor compounds may contribute to the therapeutic effects or herb-drug interactions of licorice. However, though the contents of major components have been clearly revealed [20, 21], little is known about the abundance of these minor compounds in licorice, except that the content of glycycoumarin has only been addressed once, so far [22]. SPE is an effective and reproducible approach to enrich minor compounds in complicated herbal extracts [23]. In this study, a reliable SPE-HPLC‑DAD method was established to determine five minor phenolic compounds in 20 batches of Glycyrrhiza uralensis. These compounds included three coumarins (glycycoumarin, glycyrol, and glycyrin) and two flavonoids (dehydroglyasperin C and licoflavonol).
Fig. 2 Optimization of the elution volume in solid-phase extraction using 100 % methanol. (Color figure available online only.)
Results and Discussion !
To efficiently extract minor coumarins and flavonoids from licorice, the experimental conditions were optimized, including solvent (70% and 100 % methanol), solvent volume (6-, 10-, 20-, and 30-fold), extraction method (reflux, ultrasonication, and maceration), and extraction time (15, 30, 60, and 120 min). The chromatograms from different conditions are given in Fig. 1S. The optimized method was to extract 0.25 g of the powder with 6 mL of methanol (containing 0.037 mg/mL of bergapten) in an ultrasonic water bath for 30 min. HPLC conditions were also optimized (Fig. 2S), including column type (Zorbax SB‑C18/Aglient, Symmetry C18/Waters, YMC ODS‑A/YMC, and Atlantis T3/Waters), column temperature (30 °C, 35 °C), and detection wavelength (230 nm, 254 nm, 280 nm, 306 nm, 365 nm). With the optimized conditions, all analytes could be well separated within 40 min. Due to the low polarity of the target compounds, solid-phase extraction was used to remove the polar major interfering components (flavonoid glycosides and saponins) of licorice. The SPE method was optimized to improve its efficiency and reproducibility. The Oasis HLB cartridge (packed with N-vinylpyrrolidone and divinylbenzene) was selected to trap the analytes. The licorice extract (see “Sample preparation” section) was reconstituted in 600 µL of methanol and then loaded onto the SPE column. A three-step elution program was developed: 1) elute with 6 mL of water to remove hydrophilic constituents (Fig. 3S); 2) elute with 8 mL of 65 % methanol to remove unwanted compounds, while
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effectively retaining the target analytes; and 3) elute with 8 mL of 100 % methanol to obtain the enriched analytes. Our results suggested that analytes were retained on the column at below 60 % and 65 % methanol (5 mL), and started to be eluted when 70 % methanol (5 mL) was used (Fig. 4S). In addition, 65 % methanol was more effective to remove the unwanted compounds than 60 % methanol (Fig. 5S), and 8 mL of 65 % methanol could remove the majority of the unwanted major compounds (Fig. 6S). The elution of 8 mL of 100% methanol could yield 99.82% (1), 99.74 % (2), 99.60 % (3), 100.00 % (4), and 100.00 % (5) of the analytes " Fig. 2). To validate the recovery of this SPE method, an internal (l standard (bergapten) was spiked into a licorice extract and pretreated with the same procedure. The compound had a similar UV absorption and similar chromatographic behavior as the analytes, and was absent in the licorice samples (Fig. 7S). Recovery of the internal standard (calculated by the peak area ratio of SPEtreated solution/original solution) was above 93 %. Therefore, the optimal SPE pretreatment procedure was developed as described in the “Sample preparation” section. In the chromatogram of SPE-treated licorice samples, five analytes were identified by high-performance liquid chromatography coupled with HPLC‑DAD‑MSn. Both negative and positive ESI ion modes were tested, and (−)-ESI was chosen for its higher sensitivity of coumarins and flavonoids. The retention times of the five " Fig. 3 B) were consistent peaks in the licorice sample GC-18 (l
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" Fig. 3 A). Tandem mass spectra of with the pure standards 1–5 (l " Fig. 4. Three coumarins (1, 3, the five analytes are provided in l and 5) showed maximum UV absorptions at 254 nm and 352 nm. Take compound 1 for example, (−)-ESI‑MS showed an intense [M – H]− ion at m/z 367. The MS/MS spectra were predominated by the elimination of CH3· (from the methoxyl group), C3H7·, and C5H8· (from the isoprenyl chain). The m/z 309 ion was generated by the simultaneous cleavage of CH3· and C3H7·. It yielded daughter ions at m/z 265 and 281 due to the loss of CO2 and CO, respectively. All three coumarins showed similar path" Fig. 4 A, C, and E). On the other hand, the two flavonoids ways (l had the same [M – H]− ion, but they showed different maximum UV absorptions at 330 nm (2) and 340 nm (4). Predominant cleavage occurred on the isoprenyl group to produce m/z 298 " Fig. 4 B and (-C4H7), which further fragmented into m/z 269 (l D). Tandem mass spectra and UV spectra of the analytes in real samples matched with those of authentic standards, indicating " Fig. 3 was acceptable to furthat the resolution of peaks 1–5 in l ther conduct the quantitative analysis. A validated HPLC-UV method was then established to determine the contents of these minor phenolic compounds in licorice. For quantitative analysis, the working solution samples were analyzed in duplicate to establish the calibration curves. The curves for compounds 1–5 were y = 6.3237 x + 0.1832 (linear range from 15.65 to 1370 µg/mL), y = 1.8786 x + 0.0854 (19.54 to 2736 µg/ mL), y = 11.2660 x + 0.0187 (2.057 to 360 µg/mL), y = 10.0555 x − 0.019 (3.171 to 222 µg/mL), and y = 8.8812 x + 0.022 (7.057 to 494 µg/mL), respectively. The calibration curves were constructed by plotting the ratio of mean peak areas of the samples and the IS against the concentration of each compound. All calibration curves showed good linearity with correlation coefficients (r2) no less than 0.999, in relatively wide dynamic ranges (70- to 175-fold). The LOD and LOQ of five compounds were determined by injecting a series of standard solutions until the S/N " Table 1). ratio was three for LOD and 10 for LOQ, respectively (l
Intraday variation, interday variation, interbatch variation, stability, and recovery of the method were validated for each analyte. The analysis was repeated using the same sample six times in the same day, and additionally on three consecutive days to determine intra- and interday precision, respectively. Intra- and interday variations of the signals were less than 3.00 % and 0.97 % (rep" Table 1). The interbatch variation resented by RSD values, see l was evaluated by preparing and analyzing six samples of the same batch of licorice with the same procedure. Variation among " Table 1). The same licorice these analyses was less than 2.43 % (l sample was stored at 25 °C, and analyzed at 0, 2, 4, 8, 12, and 24 h " Tafor the stability test. The variation was no more than 2.26 % (l ble 1). Recovery tests were performed by adding a known amount of each pure analyte into 0.125 g of licorice crude drug powder (n = 3). The mixture was pretreated following the “Sample preparation” section, and then analyzed following the “HPLCUV quantitation” section. The developed analytical method showed acceptable recoveries between 85.30 % and 105.22 % (RSD < 7.84 %, Table 1S). Using the validated SPE-HPLC‑DAD method, the contents of five phenolic compounds were determined in 20 batches of G. uralen" Table sis, as well as in samples from other Glycyrrhiza species (l 2). Among the analytes, the contents of glycycoumarin (1) and dehydroglyasperin C (2) were 0.40–1.28 mg/g and 0.40–2.28 mg/ g, respectively, in G. uralensis. Glycyrol (3), licoflavonol (4), and glycyrin (5) had relatively lower abundances between 0.08– 0.32 mg/g, 0.06–0.21 mg/g, and 0.07–0.44 mg/g, respectively. The contents of the five phenols were remarkably lower in other Glycyrrhiza species. G. yunnanesis is mainly distributed in Yunnan Province in China, and is used as licorice by local people. However, it contained none of the five phenolic compounds. For G. inflata and G. glabra, compound 5 was missing in their samples, and the content of compounds 1, 2, and 3 was lower than those of G. uralensis. Due to the limited sample volume, this requires further investigation with more samples. It is also noteworthy that G. uralensis samples collected from Xinjiang and In-
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Fig. 3 HPLC‑UV chromatograms of different Glycyrrhiza species after SPE treatment (365 nm). A Mixed standards, B G. uralensis, C G. inflata, D G. glabra, and E G. yunnanensis. 1 Glycycoumarin, 2 dehydroglyasperin C, 3 glycyrol, 4 licoflavonol, 5 glycyrin, IS internal standard (bergapten). (Color figure available online only.)
Original Papers
Fig. 4 (−)-ESI‑MSn spectra of the five analytes. A Glycycoumarin (1), B dehydroglyasperin C (2), C glycyrol (3), D licoflavonol (4), and E glycyrin (5). (Color figure available online only.)
Table 1 Variation, stability, and detection limits of the HPLC-UV quantitation method. Analyte 1 2 3 4 5
LOD
LOQ
Intraday variation
Interday variation
Interbatch variation
Stability
(µg/mL)
(µg/mL)
(n = 5)
(n = 3)
(n = 6)
(n = 6)
0.157 0.326 0.069 0.106 0.141
0.261 3.257 0.686 1.057 0.235
0.09 0.11 3.00 0.36 0.98
0.63 0.17 0.47 0.97 0.48
0.20 0.36 2.43 0.18 0.75
0.28 0.98 2.26 0.54 0.46
Note: 1 glycycoumarin, 2 dehydroglyasperin C, 3 glycyrol, 4 licoflavonol, 5 glycyrin, LOD limit of detection (S/N = 3), LOQ limit of quantification (S/N = 10). Intraday variation, interday variation, interbatch variation, and stability are shown in RSD (%)
ner Mongolia contained lower amounts of the phenolic compounds, although these places are considered authentic production areas of licorice in China. Therefore, further studies are necessary for comprehensive quality control of licorice, and the quality of licorice crude drugs needs to be standardized to guarantee its therapeutic efficacy.
Materials and Methods !
Chemicals, reagents, and plant materials HPLC grade acetonitrile and formic acid were purchased from Mallinckrodt Baker Technology, Inc. Deionized water was purified by a Milli-Q system (Millipore). Other reagents were purchased from Beijing Chemical Corporation. Oasis HLB cartridges (600 cc, 200 mg) were purchased from Waters. Five pure reference standards, glycycoumarin (1), dehydroglyasperin C (2), glycyrol (3), licoflavonol (4), and glycyrin (5) were isolated from Glycyrrhiza uralensis by the authors. Their structures were identified by NMR and MS spectroscopy (Fig. 8S), by comparison to previous publications (listed in the note to Fig. 8S). The IS, bergapten, was purchased from ZeLang Medical Technology Co., Ltd. All the reference compounds showed purities above 98 % by HPLC analy" Fig. 1. Twentysis. Structures of these compounds are shown in l three batches of crude drugs were collected in different provinces " Table 2) and were authenticated by the authors as of China (l G. uralensis (20 batches), G. inflata (1 batch), G. glabra (1 batch), and G. yunnanensis (1 batch). Among them, G. yunnanensis is used as folk medicine in Yunnan Province. Voucher specimens (LIC12GC-1 to LIC12GC-20, LC11GI-1, LC11GG-1, LC11GY-1)
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were deposited at the School of Pharmaceutical Sciences, Peking University.
Sample preparation For HPLC/MSn and HPLC-UV analyses, raw materials of licorice were pulverized into fine powder, accurately weighed, and then methanol was added (containing 0.037 mg/mL of bergapten) to make a concentration of 250 mg per 6 mL. The licorice was then extracted in an ultrasonic water bath for 30 min at 30 °C. Following centrifugation (10 min, 7000 rpm), a 5-mL aliquot of the supernatant was transferred into a clean tube, and dried under a gentle nitrogen flow. The residue was reconstituted in 600 µL of methanol and was then loaded onto an SPE column (preconditioned with 5 mL of methanol, followed by 5 mL of water). The column was sequentially eluted with 6 mL of water, 8 mL of 65 % methanol, and 8 mL of 100 % methanol. The flow rate was maintained at approximately 0.5 mL/min using a 12-position vacuum extraction manifold. The 100% methanol fraction was collected, evaporated to dryness under a gentle flow of nitrogen, and reconstituted in 200 µL of methanol for analysis.
Preparation of standard working solutions and internal standard solution For HPLC quantification, a stock solution was prepared by dissolving the five analytes in methanol. Concentrations of the analytes were 2.74 mg/mL (1), 3.42 mg/mL (2), 0.36 mg/mL (3), 1.11 mg/mL (4), and 2.47 mg/mL (5), respectively. The stock solution was diluted with methanol (1.25-, 2.00-, 2.50-, 3.33-, 4.00-, 4.67-, 5.60-, 7.00-, 9.33-, 14.00-, and 28.00-fold, respectively) to produce serial concentrations of working solutions. The IS solution was prepared by dissolving bergapten in methanol to make
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Table 2 Contents of the five analytes (1−5) in G. uralensis and other Glycyrrhiza species.
GC-1 GC-2 GC-3 GC-4 GC-5 GC-6 GC-7 GC-8 GC-9 GC-10 GC-11 GC-12 GC-13 GC-14 GC-15 GC-16 GC-17 GC-18 GC-19 GC-20 GC-21 GC-22 GC-23
Species G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. uralensis G. glabra G. inflata G. yunnanesis
Collected from
Content of analytes (mg/g)
Gansu Anhui Yulin, Shaanxi Pingdingshan, Heʼnan Huaiyuan, Anhui Haerbin Wenlin, Zhejiang Xinjiang Langfang, Hebei Baotou, Inner Mongolia Jinzhou, Liaoning Inner Mongolia Inner Mongolia Hangjinqi, Inner Mongolia Hangjinqi, Inner Mongolia Chifeng, Inner Mongolia Zhangjiakou, Hebei Xingtai, Hebei Huʼnan Gansu Xinjiang Xinjiang Yunnan
1
2
3
4
5
0.879 1.125 1.022 1.280 1.187 0.821 1.043 0.636 0.826 0.405 0.456 0.779 0.409 0.400 0.726 1.003 0.474 1.091 0.857 0.789 0.006 0.320 –
0.608 1.652 1.094 2.279 1.692 1.251 1.523 0.547 1.159 0.907 0.399 0.729 0.517 0.487 1.181 1.817 1.672 2.179 1.966 1.310 – 0.319 –
0.155 0.242 0.245 0.324 0.322 0.158 0.273 0.122 0.202 0.084 0.093 0.183 0.083 0.108 0.176 0.240 0.279 0.246 0.224 0.167 – 0.123 –
0.166 0.166 0.214 0.125 0.117 0.115 0.103 0.172 0.095 0.111 0.061 0.092 0.062 0.149 0.136 0.087 0.188 0.101 0.091 0.079 0.094 0.048 –
0.173 0.199 0.215 0.198 0.188 0.128 0.163 0.170 0.122 0.068 0.101 0.112 0.087 0.149 0.215 0.156 0.442 0.182 0.135 0.125 – – –
Note: 1 glycycoumarin, 2 dehydroglyasperin C, 3 glycyrol, 4 licoflavonol, 5 glycyrin, – below the limit of quantitation
a concentration of 0.037 mg/mL. To each 200 µL of working solution, 5 mL of IS solution was added, and the mixtures were dried under a nitrogen flow. The residue was reconstituted in 200 µL methanol to obtain a set of working solutions to establish the calibration curves. All solutions and samples were kept at 4 °C until use.
4.5 kV; sheath gas (N2) 40 arbitrary units; auxiliary gas (N2) 10 units; capillary temperature 320 °C; capillary voltage − 30 V; and tube lens offset voltage − 20 V. Full-scan spectra were recorded in the range of m/z 120–1000. Data were analyzed using Xcalibur™ 1.4 software (Thermo Fisher).
Supporting information HPLC‑UV quantitation The HPLC determination was performed on an Agilent series 1100 HPLC system including a quaternary pump, a diode array detector, an autosampler, and a column compartment. Samples were separated on a YMC-Pack ODS‑A column (250 mm × 4.6 mm I. D., 5 µm) equipped with an Agilent ZORBAX SB‑C18 guard column (20 mm × 3.9 mm I. D., 5 µm). The mobile phase consisted of acetonitrile (A) and water containing 0.1 % (v/v) formic acid (B). A gradient program was used as follows: 0–5 min 44–46 % A; 5–25 min 46–50% A; 25–30 min 50–60% A; 30– 32 min 60–65% A; 32–34 min 65–95% A; and 34–36 min 95 % A. A 15-min post-run time was set to fully equilibrate the column. The flow rate was 1.0 mL/min. The column temperature was 35 °C. The detection wavelength was set from 190 to 800 nm, and the samples were detected at 365 nm. The sample injection volume was 10 µL. All data were processed by Agilent LC B.02.01 ChemStation software.
Optimization of the extraction method, the HPLC condition and SPE elution, as well as recovery of the quantitation method are available as Supporting Information.
Acknowledgements !
This work was supported by the National Natural Science Foundation of China (No. 81173644, No. 81222054) and the Program for New Century Excellent Talents in University from the Chinese Ministry of Education (No. NCET-11–0019).
Conflict of Interest !
No conflict of interest exists in the submission of this manuscript and this manuscript has been approved by all authors for publication.
HPLC‑DAD‑MSn analysis HPLC‑DAD‑MSn analysis was performed on an Agilent series 1100 HPLC instrument coupled with an LCQ Advantage ion-trap mass spectrometer (Thermo Fisher) via an electrospray ionization interface. The HPLC conditions were the same as those for HPLC‑DAD analysis. The HPLC eluent was introduced into the electrospary ionization source in a post-column splitting ratio of 5 : 1. It was operated in the negative ion mode: source voltage
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