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Fast determination of flavonoids in Glycyrrhizae radix by capillary zone electrophoresis
Analytica Chimica Acta 458 (2002) 345–354
Fast determination of flavonoids in Glycyrrhizae radix by capillary zone electrophoresis Tao Bo, Ke An Li, Huwei Liu∗ The College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China Received 6 November 2001; received in revised form 9 January 2002; accepted 29 January 2002
Abstract A fast capillary zone electrophoresis (CZE) method has been developed for the determination of four flavonoids (liquiritin, licoisoflavone A, licochalconel A and calycosin) in Glycyrrhizae radix. After a series of optimization experiments, 100 mM borate buffer (pH 10.5), 30 kV applied voltage and 35 ◦ C temperature were selected. The contents of four flavonoids in cultivated and wild crude drugs of Glycyrrhizae radix with different growth periods from one to four years, collected from different areas were successfully determined within 8 min, with satisfactory repeatability and recovery. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Capillary zone electrophoresis; Fast determination; Flavonoids; Glycyrrhizae radix
1. Introduction Glycyrrhizae radix is a Chinese herbal drug commonly used as an expectorant, detoxicant and spleen tonic and to restore vitality, reduce fever, arrest coughing, comfort the stomach, alleviate urgency and potentiate the effect of various other herbs. It has been known to contain mainly glycyrrhizin, glycyrrhetinic acid, coumarins and flavonoids in Glycyrrhizae radix [1,2]. This drug is widely found in Chinese medicinal preparations such as tonic, surdorfic, coordinative, vitality-regulating, blood-regulating, chill-dispelling and moistening formulas [3]. It has been found that glycyrrhizin and glycyrrhetinic acid showed anti-inflammatory, antitussive and antiallergic activity [4], liquiritin and isoliquiritin and their aglycones antiulcergenic, spasmolytic activities [4,5], ∗ Corresponding author. Tel.: +86-10-6275-4976; fax: +86-10-6275-1708. E-mail address: [email protected] (H. Liu).
glycycoumarin antibacterial activity and licochalcone A anti-HIV activity [6]. Methods for the determination of glycyrrhizin and glycyrrhetinic acid have been reported, including precipitation [7], thin-layer chromatography [8], gas–liquid chromatography [9], near infrared spectroscopy [10], high performance liquid chromatography (HPLC) [11–16] and capillary electrophoresis (CE) [17,18]. HPLC method for the determination of flavonoids in Glycyrrhizae radix has been also reported [19]. However, these methods suffer from materials and time consuming since large amounts of organic reagent and many operation steps are often required. The development of capillary electrophoresis (CE) has been comprehensively reviewed [20–23], and it is continuously a very active research area in separation science since this technique often provides higher resolving power, shorter analysis time and lower operation cost than conventional methods. Nowadays, some research groups are devoting themselves to the study of traditional medicine by CE
0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 0 0 7 5 - 2
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[24–28]. Among the analysis of herb medicine by CE, much work focus on the flavonoids due to their strong pharmacological effects [29–34], but the determination of the flavonoids in Glycyrrhizae radix by CE has not been reported. Owing to the complicated components in Chinese herbal medicines, as a commomly used chromatographic method for the analysis of flavonoids in Glycyrrhizae radix, HPLC requires gradient elution that tends to be inconvenient, time consuming (30 min) and often results in interference with other unknown constituents [19]. In this work, we describe a rapid capillary zone electrophoresis (CZE) methods for the determination of four flavonoids (liquiritin, licoisoflavone A, licochalconel A and calycosin) in Glycyrrhizae radix, and their chemical structures are shown in Fig. 1. The flavonoids contents of three crude drug and two extracts of Glycyrrhizae radix collected from different areas were determined and compared. The CE analysis described in this work has much shorter analytical time (8 min), higher separation ability, and more simple operation steps, compared with HPLC. With satisfactory repeatability, recovery and lower detection limit, this CE method is especially effective for bulky samples.
2. Experimental 2.1. Apparatus and conditions All separations were performed on an Agilent 3D CE system with air-cooling and a diode array detector (Agilent Technologies, Waldbronn, Germany). A 58.5 cm × 50 m i.d. fused silica capillary (Ruifeng Inc., Hebei, China) was utilized with an effective length of 50 cm, and its temperature was maintained at 35 ◦ C. The other conditions are as follows: applied voltage 30 kV, UV detection at 210 nm, and samples injection at 50 mbar for 10 s. The electrolyte solution was 100 mM borate buffer (pH 10.5), which was filtered through a 0.45 m membrane filter and degassed by ultrosonication for approximately 10 min before use. The capillary was conditioned daily by washing first with 0.5 M sodium hydroxide (10 min), then with water (10 min) and finally with the running buffer (15 min). Between consecutive analysis, the capillary was flushed with 0.5 M sodium hydroxide (1 min), then with water (2 min) and finally with the running buffer (3 min) in order to improve the migration time and peak area repeatability. 2.2. Chemicals The standards of liquiritin, licoisoflavone A, licochalconel A, calycosin and Glycyrrhizae radix crude drugs were provided by Institute of Medicine Plant Development (Beijing, China). All chemicals were of analytical-reagent grade: boric acid, hydroxide sodium, and methanol from Beijing Chemical Factory (Beijing, China); pure water prepared by Milli-Q system (Millipore, Bedford, MA, USA) was used for all buffer solutions. 2.3. Sample preparation
Fig. 1. The chemical structure of four studied flavonoids.
Pulverized dried crude drug of Cortex fraxini (0.35 g) was extracted with methanol (7 ml) by ultrasonication at room temperature for 30 min, then centrifuged at 1500 rpm for 10 min. The extraction was repeated three times. Extracts were combined and diluted to 25 ml with methanol, as the sample solution, which was then passed through a 0.45 m membrane filter.
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2.4. Solutions for construction of calibration curve
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Cortex fraxini from Gansu region. The mixtures were extracted and analyzed by the procedures described earlier.
Five calibration solutions containing liquiritin, licoisoflavone A, licochalconel A, and calycosin were prepared in methanol with different concentrations.
3. Results and discussions
2.5. Solution for recovery testing
3.1. Optimization of analytical conditions
Known amounts of liquiritin, licochalconel A, calycosin were added to the sample of the crude drug of
To verify the effect of buffer pH on migration behavior, experiments were performed by using the
Fig. 2. Migration times of the flavonoids as a function of buffer pH (A), and concentration (B). Conditions: (A) boric acid, 100 mM; applied voltage, 20 kV; temperature, 25 ◦ C; UV detection wavelength, 210 nm. (B) pH, 10.5; applied voltage, 20 kV; temperature, 25 ◦ C; UV detection wavelength, 210 nm.
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running buffers containing 100 mM borate with different pH under applied voltage 20 kV and 25 ◦ C temperature. The result showed that the migration times of four flavonoids increased with the increase of pH from 8–10.5 (see Fig. 2) due to greater ionization of the phenolic hydroxyl groups at higher pH resulting in greater mobilities of the flavonoids in the opposite direction to the electroosmotic flow (EOF), although rapid increase in EOF with the pH increase. However, the resolution between locochalconel A (peak 3) and calycosin (peak 4) was poor in the range of pH 8.5–10.0 compared with that at pH 10.5, because the ionization was less at the lower pH value, which could
not result in enough difference in the negative charge on the four analytes. The four flavonoids can be completely separated with moderate analysis time at pH 10.5, that was then selected as the preference pH for further optimization. The effect of borate concentration ranging from 50 to 200 mM on migration time under pH 10.5, 20 kV applied voltage and 25 ◦ C temperature indicated that the migration times of the flavonoids increased (see Fig. 2) because the increase in ionic strength reduced the EOF. As the resolution, peak shape, analytical time and repeatability were concerned, 100 mM borate was selected as the optium concentration.
Fig. 3. The effects of applied voltage (A), and temperture (B), on migration times of the flavonoids. Conditions: boric acid, 100 mM; pH, 10.5; applied voltage, 15–30 kV; 20 kV for (B); temperature, 20–35 ◦ C and 25 ◦ C for (A); UV detection, 210 nm.
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Finally, the effects of applied voltage (15–30 kV) and temperature (25–40 ◦ C) on the separation were also studied with 100 mM borate buffer at pH 10.5, as shown in Fig. 3. The optimum voltage and temperature were found to be 30 kV and 35 ◦ C, respectively, which combined sufficient resolution with a moderate analysis time. Therefore, the 100 mM borate buffer (pH 10.5), 30 kV applied voltage and 35 ◦ C column temperature were proved to be the optimized condition for the separation. Fig. 4 shows a typical electro-
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pherogram of a mixture of four flavonoid standards together with their on-line UV spectra obtained by DAD. Due to lower electric conductivity, boric acid cannot produce higher current at the higher concentration, which does not result in higher Joule heat and reduce the separation efficiency. Under the optimal CE conditions, the current in the capillary was 53 A. The theoretical plate numbers for liquiritin, licoisoflavone A, licochalconel A and calycosin were 179398, 143360, 172822 and 248950 m−1 , respectively.
Fig. 4. Electropherogram of a mixture of four flavonoids with their UV spectra obtained by DAD. Conditions: boric acid, 100 mM; pH, 10.5; applied voltage, 30 kV; temperature, 35 ◦ C; UV detection wavelength, 210 nm. See UV spectra for peak identification.
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Table 1 The R.S.D. of peak area and migration time, recoveries, and detection limits for the studied flavonoids Component
Liquiritin Licoisoflavone A Licochalconel A Calycosin a
Repeatability (R.S.D., n = 5) (%)
Recovery (n = 5)
Peak area
Migration time
Recovery (%)
R.S.D. (%)
2.14 2.47 1.12 1.67
1.78 2.06 2.08 2.48
99.7 –a 96.1 94.1
2.63 – 2.60 2.32
Detection limit (g ml−1 ) 2.4 1.2 1.4 1.3
Not tested.
3.2. Construction of calibration curves Calibration curves were constructed in the concentration range of 9.5–378.4 g ml−1 for liquiritin, 6.4–128.0 g ml−1 for licoisoflavone A, 15.4–154.0 g ml−1 for licochalconel A and 4.0–80.0 g ml−1 for calycosin. The linear regression equations and correlation coefficients were:
and for migration time from 1.78 to 2.48%, respectively. The detection limit (3 S/N) is varied from 1.2 to 2.4 g ml−1 . The recoveries of liquiritin, licochalconel A and calycosin from Glycyrrhizae radix were determined by the method of standard addition (the recovery of licoisoflavone A was not tested because it was not found in all studied samples). 3.4. Determination of the flavonoids in Cortex fraxini
liquiritin Y = 2.3430X − 7.3636(r = 0.9984) licoisoflavone A Y = 1.3861X − 3.5252(r = 0.9947) licochalconel A Y = 1.4196X + 4.0889(r = 0.9949) calycosin Y = 0.89881X + 3.7551(r = 0.9925) where, X is the peak area (in integration unit), Y the concentration (g ml−1 ) of the individual flavonoid, and r the correlation coefficient. 3.3. System suitability test The repeatability (relative standard deviation, R.S.D.), detection limit and recovery of the method for these analytes are listed in Table 1. The R.S.D. (n = 5) for peak area ranges from 1.12 to 2.47%,
The amounts of liquiritin, licoisoflavone A, licochalconel A and calycosin in Glycyrrhizae radix from three different areas and in two extracts of the drug have been quantified by using above linear regression equations and the results are listed in Table 2 (expressed as the percentage of crude drug). Fig. 5 shows typical electropherograms obtained from the crude drugs and extracts of Glycyrrhizae radix. The results indicated that the amounts of the four flavonoids in these samples were obviously different, and their fingerprint electropherograms greatly varied. In all studied samples, liquiritin has the highest content, so it can be regarded as a characteristic compound for the quality control of Glycyrrhizae radix. In most samples, due to their extremely low contents,
Table 2 The contents (%, w/w) of the four flavonoids in crude drugs and extracts of Glycyrrhizae radix from different areas (n = 5) Growth area
Liquiritin (%)
Licoisoflavone A (%)
Licochalconel A (%)
Calycosin (%)
Gansu Inner Mongolia Xinjiang Extract 1 Extract 2
2.95 (2.16%)a 0.60 (3.05%) Trace Trace 7.48 (1.93%)
Not Not Not Not Not
0.21 (2.61%) Not found Not found Not found Not found
0.083 (3.07%) 0.26 (2.86%) 0.19 (4.13%) 2.93 (3.27%) 0.27 (3.01%)
a
The data in the parentheses denote R.S.D.
found found found found found
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Fig. 5. Electropherograms obtained from the crude drugs and extract of Glycyrrhizae radix. (A) Gansu, (B) Inner Mongolia, (C) Xinjiang, (D) Extract 2. See Fig. 4 for separation conditions and peak identification.
licoisoflavone A, licochalconel A were not quantified. The reported HPLC method [19] also gave similar results. What is more, for investigating the effects of planting conditions (e.g. wild and cultivated as well
as different planting years) on the contents of the flavonoids in Glycyrrhizae radix, the amounts of the four flavonoids in the samples collected within different growth periods were determined as demonstrated
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Fig. 6. Electropherograms obtained from the crude drug of cultivated Glycyrrhizae radix. (A) 1 year, (B) 2 years, (C) 3 years, (D) 4 years. See Fig. 4 for separation conditions and peak identification.
in Table 3 and Fig. 6. The results are very important for cultivating Glycyrrhizae radix and reducing the destruction and waste of Glycyrrhizae radix resource. Furthermore, the contents of the four flavonoids in the stem of Glycyrrhizae radix were also determined,
indicating much lower contents of these flavonoids (0.044% for liquiritin and not found for licoisoflavone A, licochalconel A and calycosin). As a result, the stem cannot serve as an alternative resource in place of Glycyrrhizae radix.
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Table 3 Contents (%, w/w) of the four flavonoids in the cultivated and wild Glycyrrhizae radix (n = 5) Liquiritin (%)
Licoisoflavone A (%)
Licochalconel A (%)
Calycosin(%)
Growth period 1 year 2 years 3 years 4 years
Cultivated Glycyrrhizae radix 1.28 (2.55%)a Not 1.86 (1.04%) Not 2.39 (0.96%) Not 1.36 (3.15%) Not
found found found found
Not found Not found Trace Not found
0.16 (4.19%) Not found Not found 0.13 (3.50%)
Growth period 1 year 2 years 3 years 4 years
Wild Glycyrrhizae radix 1.76 (2.04%) 2.28 (1.18%) 1.97 (2.79%) 1.51 (3.07%)
found found found found
Not found Not found Trace Not found
Not found Not found 0.15 (4.15%) Not found
a
Not Not Not Not
The data in the parentheses denote R.S.D.
4. Conclusions The contents of liquiritin, licoisoflavone A, licochalconel A and calycosin in Glycyrrhizae radix were determined by the proposed CZE method within 8 min under the optimized conditions, with satisfactory repeatability and recovery. This method could be effective for quality control of Glycyrrhizae radix. The flavonoids content in three crude drugs and two extracts of Glycyrrhizae radix from different areas were obviously different. Based on the comparison of flavonoids content in wild and cultivated Glycyrrhizae radix and the stem, we could make good use of Glycyrrhizae radix resource.
Acknowledgements The authors would like to acknowledge Agilent Technologies for providing the capillary electrophoresis system. The study is supported by NSFC, Grant no. 29875001. References [1] H.Y. Hsu, Y.P. Chen, S.J. Sheu, C.H. Hsu, C.C. Chen, H.C. Chang, Chinese Materia—A Concise Guide, Modern Drug Press, Taipei, 1984, p. 95. [2] G.J. Xiu, Sheng Yao Xue, Medicinal Materials, in Chinese, People’s Medical Publication House, Beijing, China, 1995, p. 172. [3] S.Y. Huang, A Collective Commentary to Herbal Formulas, Ba-Teh Education and Culture Press, Taipei, 1987, p. 1.
[4] W. Watanabe, Taisha 10 (1973) 688. [5] Drugs in Japan, Japan Pharmaceutical Information Centre, 9th Edition, Tokyo, Japan, 1985, p. 12. [6] T. Hatano, H. Kagawa, T. Yashuhara, T. Okuda, Chem. Pharm. Bull. 36 (1988) 2090. [7] P.A.H. Houseman, J. Assoc. Off. Anal. Chem. 36 (1922) 191. [8] Y. Tokino, M. Koshioka, M. Shiokawa, Y. Ishii, S.M. Higashino, T. Hayashi, Planta Med. 36 (1979) 74. [9] T. Vandenhaf, K.W. Glombitza, M. Steiner, Sci. Pharm. 41 (1973) 155. [10] Y.X. Chen, L.K. Sorensen, Fresenins J. Anal. Chem. 367 (2000) 491. [11] S. Ogawa, A. Yoshida, Y. Mitani, Yakugaku Zasshi 96 (1976) 1488. [12] Y. Akada, Y. Tanase, Yakugaku Zasshi 96 (1976) 1035. [13] J. Killacky, M.S.F. Ross, T.D. Turner, Planta Med. 30 (1976) 310. [14] K. Okada, J. Tanaka, A. Miyashita, K. Iwoto, Yakugaku Zasshi 101 (1981) 822. [15] N. Okamura, T. Maki, H. Miyauchi, Biol. Pharm. Bull. 24 (10) (2001) 1161. [16] R. Matissek, P. Sproer, Dtsch. Lebensm.-Rundsch. 92 (12) (1996) 381. [17] H.R. Chen, S.J. Sheu, J. Chromatogr. A 653 (1993) 184. [18] G.Q. Zhang, S.G. Ji, Y.F. Chai, Biomed. Chromatogr. 13 (1999) 407. [19] L. Zheng, R.Y. Zhang, T. Ming, Z.C. Lou, J. Chromatogr. 513 (1990) 247. [20] L.S.C. Robert III, Anal. Chem. 68 (1996) 569R. [21] R.K. Gilpin, L.A. Pachla, Anal. Chem. 73 (2001) 2805. [22] Stephen C. Beale, Anal. Chem. 70 (1998) 279. [23] Sergey N. Krylov, Norman J. Dovichi, Anal. Chem. 72 (2000) 111R. [24] H. Zhang, Z. Hu, G. Yang, Chromatographia 44 (1997) 162. [25] Y.R. Ku, Y.T. Lin, J.H. Lin, J. Chromatogr. A 805 (1998) 301. [26] B.L. Lee, C.N. Ong, J. Chromatogr. A 881 (1/2) (2000) 439. [27] H.K. Wu, W.C. Chuang, S.J. Sheu, J. Chromatogr. A 803 (1998) 179.
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[28] H. Long, J.J. Yang, H.W. Liu, T.S. Wang, A.J. Huang, Y.L. Sun, Chin. Chem. Lett. 9 (10) (1998) 941. [29] X.M. Wu, Z.B. Yin, YaoWu FenXi ZaZhi 21 (2001) 138. [30] G. Chen, H.W. Zhang, J.N. Ye, Anal. Chim. Acta 423 (2000) 69.
[31] X.L. Gu, Q.Y. Chu, Mary Odwyer, M. Zeece, J. Chromatogr. A 881 (2000) 471. [32] G. Chen, H.W. Zhang, J.N. Ye, Talanta 53 (2000) 471. [33] H.J. Issaq, Electrophoresis 18 (1997) 2438. [34] P.J. Larger, A.D. Jones, C. Dacombe, J. Chromatogr. A 799 (1998) 309.
Fast determination of flavonoids in Glycyrrhizae radix by capillary zone electrophoresis Tao Bo, Ke An Li, Huwei Liu∗ The College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China Received 6 November 2001; received in revised form 9 January 2002; accepted 29 January 2002
Abstract A fast capillary zone electrophoresis (CZE) method has been developed for the determination of four flavonoids (liquiritin, licoisoflavone A, licochalconel A and calycosin) in Glycyrrhizae radix. After a series of optimization experiments, 100 mM borate buffer (pH 10.5), 30 kV applied voltage and 35 ◦ C temperature were selected. The contents of four flavonoids in cultivated and wild crude drugs of Glycyrrhizae radix with different growth periods from one to four years, collected from different areas were successfully determined within 8 min, with satisfactory repeatability and recovery. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Capillary zone electrophoresis; Fast determination; Flavonoids; Glycyrrhizae radix
1. Introduction Glycyrrhizae radix is a Chinese herbal drug commonly used as an expectorant, detoxicant and spleen tonic and to restore vitality, reduce fever, arrest coughing, comfort the stomach, alleviate urgency and potentiate the effect of various other herbs. It has been known to contain mainly glycyrrhizin, glycyrrhetinic acid, coumarins and flavonoids in Glycyrrhizae radix [1,2]. This drug is widely found in Chinese medicinal preparations such as tonic, surdorfic, coordinative, vitality-regulating, blood-regulating, chill-dispelling and moistening formulas [3]. It has been found that glycyrrhizin and glycyrrhetinic acid showed anti-inflammatory, antitussive and antiallergic activity [4], liquiritin and isoliquiritin and their aglycones antiulcergenic, spasmolytic activities [4,5], ∗ Corresponding author. Tel.: +86-10-6275-4976; fax: +86-10-6275-1708. E-mail address: [email protected] (H. Liu).
glycycoumarin antibacterial activity and licochalcone A anti-HIV activity [6]. Methods for the determination of glycyrrhizin and glycyrrhetinic acid have been reported, including precipitation [7], thin-layer chromatography [8], gas–liquid chromatography [9], near infrared spectroscopy [10], high performance liquid chromatography (HPLC) [11–16] and capillary electrophoresis (CE) [17,18]. HPLC method for the determination of flavonoids in Glycyrrhizae radix has been also reported [19]. However, these methods suffer from materials and time consuming since large amounts of organic reagent and many operation steps are often required. The development of capillary electrophoresis (CE) has been comprehensively reviewed [20–23], and it is continuously a very active research area in separation science since this technique often provides higher resolving power, shorter analysis time and lower operation cost than conventional methods. Nowadays, some research groups are devoting themselves to the study of traditional medicine by CE
0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 0 0 7 5 - 2
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[24–28]. Among the analysis of herb medicine by CE, much work focus on the flavonoids due to their strong pharmacological effects [29–34], but the determination of the flavonoids in Glycyrrhizae radix by CE has not been reported. Owing to the complicated components in Chinese herbal medicines, as a commomly used chromatographic method for the analysis of flavonoids in Glycyrrhizae radix, HPLC requires gradient elution that tends to be inconvenient, time consuming (30 min) and often results in interference with other unknown constituents [19]. In this work, we describe a rapid capillary zone electrophoresis (CZE) methods for the determination of four flavonoids (liquiritin, licoisoflavone A, licochalconel A and calycosin) in Glycyrrhizae radix, and their chemical structures are shown in Fig. 1. The flavonoids contents of three crude drug and two extracts of Glycyrrhizae radix collected from different areas were determined and compared. The CE analysis described in this work has much shorter analytical time (8 min), higher separation ability, and more simple operation steps, compared with HPLC. With satisfactory repeatability, recovery and lower detection limit, this CE method is especially effective for bulky samples.
2. Experimental 2.1. Apparatus and conditions All separations were performed on an Agilent 3D CE system with air-cooling and a diode array detector (Agilent Technologies, Waldbronn, Germany). A 58.5 cm × 50 m i.d. fused silica capillary (Ruifeng Inc., Hebei, China) was utilized with an effective length of 50 cm, and its temperature was maintained at 35 ◦ C. The other conditions are as follows: applied voltage 30 kV, UV detection at 210 nm, and samples injection at 50 mbar for 10 s. The electrolyte solution was 100 mM borate buffer (pH 10.5), which was filtered through a 0.45 m membrane filter and degassed by ultrosonication for approximately 10 min before use. The capillary was conditioned daily by washing first with 0.5 M sodium hydroxide (10 min), then with water (10 min) and finally with the running buffer (15 min). Between consecutive analysis, the capillary was flushed with 0.5 M sodium hydroxide (1 min), then with water (2 min) and finally with the running buffer (3 min) in order to improve the migration time and peak area repeatability. 2.2. Chemicals The standards of liquiritin, licoisoflavone A, licochalconel A, calycosin and Glycyrrhizae radix crude drugs were provided by Institute of Medicine Plant Development (Beijing, China). All chemicals were of analytical-reagent grade: boric acid, hydroxide sodium, and methanol from Beijing Chemical Factory (Beijing, China); pure water prepared by Milli-Q system (Millipore, Bedford, MA, USA) was used for all buffer solutions. 2.3. Sample preparation
Fig. 1. The chemical structure of four studied flavonoids.
Pulverized dried crude drug of Cortex fraxini (0.35 g) was extracted with methanol (7 ml) by ultrasonication at room temperature for 30 min, then centrifuged at 1500 rpm for 10 min. The extraction was repeated three times. Extracts were combined and diluted to 25 ml with methanol, as the sample solution, which was then passed through a 0.45 m membrane filter.
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2.4. Solutions for construction of calibration curve
347
Cortex fraxini from Gansu region. The mixtures were extracted and analyzed by the procedures described earlier.
Five calibration solutions containing liquiritin, licoisoflavone A, licochalconel A, and calycosin were prepared in methanol with different concentrations.
3. Results and discussions
2.5. Solution for recovery testing
3.1. Optimization of analytical conditions
Known amounts of liquiritin, licochalconel A, calycosin were added to the sample of the crude drug of
To verify the effect of buffer pH on migration behavior, experiments were performed by using the
Fig. 2. Migration times of the flavonoids as a function of buffer pH (A), and concentration (B). Conditions: (A) boric acid, 100 mM; applied voltage, 20 kV; temperature, 25 ◦ C; UV detection wavelength, 210 nm. (B) pH, 10.5; applied voltage, 20 kV; temperature, 25 ◦ C; UV detection wavelength, 210 nm.
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running buffers containing 100 mM borate with different pH under applied voltage 20 kV and 25 ◦ C temperature. The result showed that the migration times of four flavonoids increased with the increase of pH from 8–10.5 (see Fig. 2) due to greater ionization of the phenolic hydroxyl groups at higher pH resulting in greater mobilities of the flavonoids in the opposite direction to the electroosmotic flow (EOF), although rapid increase in EOF with the pH increase. However, the resolution between locochalconel A (peak 3) and calycosin (peak 4) was poor in the range of pH 8.5–10.0 compared with that at pH 10.5, because the ionization was less at the lower pH value, which could
not result in enough difference in the negative charge on the four analytes. The four flavonoids can be completely separated with moderate analysis time at pH 10.5, that was then selected as the preference pH for further optimization. The effect of borate concentration ranging from 50 to 200 mM on migration time under pH 10.5, 20 kV applied voltage and 25 ◦ C temperature indicated that the migration times of the flavonoids increased (see Fig. 2) because the increase in ionic strength reduced the EOF. As the resolution, peak shape, analytical time and repeatability were concerned, 100 mM borate was selected as the optium concentration.
Fig. 3. The effects of applied voltage (A), and temperture (B), on migration times of the flavonoids. Conditions: boric acid, 100 mM; pH, 10.5; applied voltage, 15–30 kV; 20 kV for (B); temperature, 20–35 ◦ C and 25 ◦ C for (A); UV detection, 210 nm.
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Finally, the effects of applied voltage (15–30 kV) and temperature (25–40 ◦ C) on the separation were also studied with 100 mM borate buffer at pH 10.5, as shown in Fig. 3. The optimum voltage and temperature were found to be 30 kV and 35 ◦ C, respectively, which combined sufficient resolution with a moderate analysis time. Therefore, the 100 mM borate buffer (pH 10.5), 30 kV applied voltage and 35 ◦ C column temperature were proved to be the optimized condition for the separation. Fig. 4 shows a typical electro-
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pherogram of a mixture of four flavonoid standards together with their on-line UV spectra obtained by DAD. Due to lower electric conductivity, boric acid cannot produce higher current at the higher concentration, which does not result in higher Joule heat and reduce the separation efficiency. Under the optimal CE conditions, the current in the capillary was 53 A. The theoretical plate numbers for liquiritin, licoisoflavone A, licochalconel A and calycosin were 179398, 143360, 172822 and 248950 m−1 , respectively.
Fig. 4. Electropherogram of a mixture of four flavonoids with their UV spectra obtained by DAD. Conditions: boric acid, 100 mM; pH, 10.5; applied voltage, 30 kV; temperature, 35 ◦ C; UV detection wavelength, 210 nm. See UV spectra for peak identification.
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Table 1 The R.S.D. of peak area and migration time, recoveries, and detection limits for the studied flavonoids Component
Liquiritin Licoisoflavone A Licochalconel A Calycosin a
Repeatability (R.S.D., n = 5) (%)
Recovery (n = 5)
Peak area
Migration time
Recovery (%)
R.S.D. (%)
2.14 2.47 1.12 1.67
1.78 2.06 2.08 2.48
99.7 –a 96.1 94.1
2.63 – 2.60 2.32
Detection limit (g ml−1 ) 2.4 1.2 1.4 1.3
Not tested.
3.2. Construction of calibration curves Calibration curves were constructed in the concentration range of 9.5–378.4 g ml−1 for liquiritin, 6.4–128.0 g ml−1 for licoisoflavone A, 15.4–154.0 g ml−1 for licochalconel A and 4.0–80.0 g ml−1 for calycosin. The linear regression equations and correlation coefficients were:
and for migration time from 1.78 to 2.48%, respectively. The detection limit (3 S/N) is varied from 1.2 to 2.4 g ml−1 . The recoveries of liquiritin, licochalconel A and calycosin from Glycyrrhizae radix were determined by the method of standard addition (the recovery of licoisoflavone A was not tested because it was not found in all studied samples). 3.4. Determination of the flavonoids in Cortex fraxini
liquiritin Y = 2.3430X − 7.3636(r = 0.9984) licoisoflavone A Y = 1.3861X − 3.5252(r = 0.9947) licochalconel A Y = 1.4196X + 4.0889(r = 0.9949) calycosin Y = 0.89881X + 3.7551(r = 0.9925) where, X is the peak area (in integration unit), Y the concentration (g ml−1 ) of the individual flavonoid, and r the correlation coefficient. 3.3. System suitability test The repeatability (relative standard deviation, R.S.D.), detection limit and recovery of the method for these analytes are listed in Table 1. The R.S.D. (n = 5) for peak area ranges from 1.12 to 2.47%,
The amounts of liquiritin, licoisoflavone A, licochalconel A and calycosin in Glycyrrhizae radix from three different areas and in two extracts of the drug have been quantified by using above linear regression equations and the results are listed in Table 2 (expressed as the percentage of crude drug). Fig. 5 shows typical electropherograms obtained from the crude drugs and extracts of Glycyrrhizae radix. The results indicated that the amounts of the four flavonoids in these samples were obviously different, and their fingerprint electropherograms greatly varied. In all studied samples, liquiritin has the highest content, so it can be regarded as a characteristic compound for the quality control of Glycyrrhizae radix. In most samples, due to their extremely low contents,
Table 2 The contents (%, w/w) of the four flavonoids in crude drugs and extracts of Glycyrrhizae radix from different areas (n = 5) Growth area
Liquiritin (%)
Licoisoflavone A (%)
Licochalconel A (%)
Calycosin (%)
Gansu Inner Mongolia Xinjiang Extract 1 Extract 2
2.95 (2.16%)a 0.60 (3.05%) Trace Trace 7.48 (1.93%)
Not Not Not Not Not
0.21 (2.61%) Not found Not found Not found Not found
0.083 (3.07%) 0.26 (2.86%) 0.19 (4.13%) 2.93 (3.27%) 0.27 (3.01%)
a
The data in the parentheses denote R.S.D.
found found found found found
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Fig. 5. Electropherograms obtained from the crude drugs and extract of Glycyrrhizae radix. (A) Gansu, (B) Inner Mongolia, (C) Xinjiang, (D) Extract 2. See Fig. 4 for separation conditions and peak identification.
licoisoflavone A, licochalconel A were not quantified. The reported HPLC method [19] also gave similar results. What is more, for investigating the effects of planting conditions (e.g. wild and cultivated as well
as different planting years) on the contents of the flavonoids in Glycyrrhizae radix, the amounts of the four flavonoids in the samples collected within different growth periods were determined as demonstrated
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Fig. 6. Electropherograms obtained from the crude drug of cultivated Glycyrrhizae radix. (A) 1 year, (B) 2 years, (C) 3 years, (D) 4 years. See Fig. 4 for separation conditions and peak identification.
in Table 3 and Fig. 6. The results are very important for cultivating Glycyrrhizae radix and reducing the destruction and waste of Glycyrrhizae radix resource. Furthermore, the contents of the four flavonoids in the stem of Glycyrrhizae radix were also determined,
indicating much lower contents of these flavonoids (0.044% for liquiritin and not found for licoisoflavone A, licochalconel A and calycosin). As a result, the stem cannot serve as an alternative resource in place of Glycyrrhizae radix.
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Table 3 Contents (%, w/w) of the four flavonoids in the cultivated and wild Glycyrrhizae radix (n = 5) Liquiritin (%)
Licoisoflavone A (%)
Licochalconel A (%)
Calycosin(%)
Growth period 1 year 2 years 3 years 4 years
Cultivated Glycyrrhizae radix 1.28 (2.55%)a Not 1.86 (1.04%) Not 2.39 (0.96%) Not 1.36 (3.15%) Not
found found found found
Not found Not found Trace Not found
0.16 (4.19%) Not found Not found 0.13 (3.50%)
Growth period 1 year 2 years 3 years 4 years
Wild Glycyrrhizae radix 1.76 (2.04%) 2.28 (1.18%) 1.97 (2.79%) 1.51 (3.07%)
found found found found
Not found Not found Trace Not found
Not found Not found 0.15 (4.15%) Not found
a
Not Not Not Not
The data in the parentheses denote R.S.D.
4. Conclusions The contents of liquiritin, licoisoflavone A, licochalconel A and calycosin in Glycyrrhizae radix were determined by the proposed CZE method within 8 min under the optimized conditions, with satisfactory repeatability and recovery. This method could be effective for quality control of Glycyrrhizae radix. The flavonoids content in three crude drugs and two extracts of Glycyrrhizae radix from different areas were obviously different. Based on the comparison of flavonoids content in wild and cultivated Glycyrrhizae radix and the stem, we could make good use of Glycyrrhizae radix resource.
Acknowledgements The authors would like to acknowledge Agilent Technologies for providing the capillary electrophoresis system. The study is supported by NSFC, Grant no. 29875001. References [1] H.Y. Hsu, Y.P. Chen, S.J. Sheu, C.H. Hsu, C.C. Chen, H.C. Chang, Chinese Materia—A Concise Guide, Modern Drug Press, Taipei, 1984, p. 95. [2] G.J. Xiu, Sheng Yao Xue, Medicinal Materials, in Chinese, People’s Medical Publication House, Beijing, China, 1995, p. 172. [3] S.Y. Huang, A Collective Commentary to Herbal Formulas, Ba-Teh Education and Culture Press, Taipei, 1987, p. 1.
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