ANALYTICAL SCIENCES JUNE 2007, VOL. 23 705 2007 © The Japan Society for Analytical Chemistry

Identification of Herb Acanthopanax senticosus (Rupr. Et Maxim.) Harms by Capillary Electrophoresis with Electrochemical Detection

Xiaoguang ZHOU,* Chunying ZHENG,** Jianshe HUANG,* and Tianyan YOU*†

*State Key Laboratory of Electroanalytical Chemistry, Institute of Applied Chemistry, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Changchun 130022, P. R. **Life Science College, University, 150080, P. R. China

In this paper, a rapid, high efficient, sensitive and inexpensive approach based on a combination of simple ultrasonic extract and capillary electrophoresis (CE) separation with electrochemical detection (ED), is described to identify herbs by comparing their CE-ED profiles (namely, CE-ED electropherograms). The proposed method takes advantage of ultra- small sample volume, low consumption of organic solvent, simple sample pretreatment and easy cleanup procedure. It was applied to analyze the CE-ED profiles of stems of herb Acanthopanax senticosus (Rupr. Et Maxim.) Harms from different sources and different parts (roots, rhizomes, stems and leaves) of this herb. By comparing peak number, peak height and peak height ratio, we found that the CE-ED profiles showed big differences for the herbs from the different sources and the different parts of this herb. In addition, the distribution of bioactive compounds (isofraxidin, rutin and chlorogenic acid) in the different parts of this herb and their content variations affected by the source were studied with the CE-ED method. Based on their own unique CE-ED profiles, these herbs from the different sources and the different parts of this herb could be easily distinguished. Therefore, the proposed approach could be used as a rapid, high efficient and sensitive method for the identification of herbal medicines.

(Received October 5, 2006; Accepted December 14, 2006; Published June 10, 2007)

Traditional Chinese medicines (TCMs) have been used to treat versatility but suffers from lack of high sensitivity because of various diseases for over a thousand years in oriental countries.1 the short capillary light path. In comparison, electrochemical The components in the TCMs are very complicated, containing detection (ED) provides high sensitivity with simple equipment. hundreds of different compounds.1 These compounds in the The identification analysis of the TCMs by the highly efficient TCMs are extremely influenced by some external factors such CE with the sensitive ED could ensure better authenticity, as soil, climate, and growth stage.2 Moreover, false and inferior quality, safety and efficacy of the raw material prior to use. herbal medicines sometimes appear on the market. Therefore, In our previous work, the CE-ED identification analyses of fast and sensitive quantitative and qualitative analysis of the herbs from different species with similar appearance and the TCMs is very essential to ensure the authenticity, quality, safety identical Chinese name of Mutong, namely, confusable Caulis and efficacy of the TCMs. aristolochiae manshuriensis, Caulis clematidis armandii and Recently, identification analysis, which is based on the overall Caulis akebiae, were studied and satisfactory results were characteristics of an herb, has been accepted as a common obtained.10 The identification of herbs from different sources, method to control herbal quality. Many methods have been which very much affect the components and the contents of developed to identify the TCMs, such as high performance active constituents in the herbs, is very important to ensure the liquid chromatography (HPLC),3 gas chromatography (GC),4 quality and the medicinal effect of this herb. Here, we discuss infrared spectrum (IR)5 and capillary electrophoresis (CE).6 The in detail the identification analysis of A. senticosus (Rupr. Et CE method has many advantages over other methods for the Maxim.) Harms from four different sources by the CE-ED analysis of the TCMs:7 1) easily cleaned capillary column; 2) method. simple sample pretreatment; 3) short analysis time; 4) A. senticosus (Rupr. Et Maxim.) Harms is a herb widely simultaneous separation of multi-component from the cultivated in Hebei, Jilin, Liaoning and the Northeast China.11 complicated TCMs samples by the CE with high separation Indeed, this herb from Heilongjiang province of China is efficiency; 5) ultra-small sample volume and low cost; 6) various considered the original and genuine herbal medicine.12 In the CE separation modes for the analysis of the complicated TCMs Chinese pharmacopoeia, the drug: Radix et Caulis constituents. The most commonly used detection method acanthopanacis senticosi (Ciwujia in Chinese) is the dried roots combined with the CE to identify the TCMs is ultraviolet and the rhizomes or the stems of this herb.13 In addition, the spectrophotometry (UV).6,8,9 The UV method possesses good leaves of this herb are also used as herbal medicine.14 This herb is regarded to have a similar pharmacological action to that of † To whom correspondence should be addressed. Chinese ginseng, and can be used as a tonic and adaptogen.15 E-mail: [email protected] Therefore, the study on A. senticosus Harms arises researchers’ 706 ANALYTICAL SCIENCES JUNE 2007, VOL. 23

Fig. 2 CVs of isofraxidin, rutin and chlorogenic acid at the CFE in 20 mM PBS (pH 6.0). (A) Blank solution, (B) isofraxidin, (C) rutin, (D) chlorogenic acid. The concentration of each compound was 0.1 mM. Scan rate, 50 mV s–1. Fig. 1 Structures of isofraxidin, rutin and chlorogenic acid.

NaH2PO4 and borax solution was adjusted with either 0.20 M interests.16–18 Isofraxidin, chlorogenic acid and rutin are the HCl or 0.20 M NaOH, respectively. All solutions were filtered main active compounds in this herb,11,15,19 and their structures through a 0.22-μm membrane before use. are illustrated in Fig. 1. Among these active compounds, the content of isofraxidin is an important indicator for the quality Apparatus and conditions valuation of the TCMs made of this herb.20 Many methods have A laboratory-built end-column CE-ED detection system has been used to analyze the constituents of A. senticosus Harms, been described previously.23 Separation voltage applied on two such as HPLC with UV/Vis, mass spectrometry (MS), nuclear ends of the capillary was provided by a high-voltage (±30 kV) magnetic resonance (NMR) or fluorescence (FL) DC power supply ( Institute of Applied Physics, detections.15,19,21 The active compounds in the roots and the China). A 40-cm length of 25 μm i.d., 360 μm o.d., fused silica leaves of this herb have been analyzed in detail.19,22 But, the uncoated capillary (Yongnian Optical Fiber Factory, Hebei, identification of this herb from the different sources and the China) was used for the separation. Before the first use, the different parts of this herb, and the distribution of these active capillary was flushed with 0.1 M NaOH aqueous solution constituents in the different parts have not been studied so far. overnight. In order to guarantee good reproducibility, we rinsed In this paper, the contents of the three active compounds in the capillary with doubly distilled water and the running buffer these herbs, the CE-ED profiles (namely, CE-ED for 2 min and 4 – 6 min between consecutive runs, respectively. electropherograms) of the stems of A. senticosus Harms from A traditional three electrodes electrochemical configuration, the different sources and the different parts of this herb were consisting of a 33 μm carbon fiber microdisk electrode (CFE) analyzed by the CE-ED method. By comparison of the CE-ED working electrode, a Pt wire auxiliary electrode, and an profiles obtained, the herbs from the different sources could be Ag/AgCl (saturated with KCl) reference electrode, was used in easily distinguished, as could the different parts of this herb. this experiment. The preparation of the CFE was reported Besides, the distributions of these active constituents in the before.23 Before use, the CFE was ground on a piece of fine different parts of this herb were also investigated. The results sandpaper and ultrasonicated in the doubly distilled water, and indicated that the contents of the three active compounds were finally carefully aligned at the outlet of capillary in order to clearly different in the different parts, and were influenced by arrange in a wall-jet configuration of end column mode. the source. Thus, the CE-ED method is proved to be a potential Electrochemical current was monitored by an electrochemical method for the TCMs identification. workstation of CH Instruments Model 800 voltammetric analyzer (CH Instruments, Austin, TX, USA). The CE separation was performed with a running buffer as a 7.0 mM Experimental NaH2PO4–7.5 mM borax (pH 7.0) solution at a separation voltage of 15 kV. The detection potential applied on the Reagents and solutions working electrode was +1.2 V (vs. Ag/AgCl). All samples were Isofraxidin, rutin and chlorogenic acid were from National injected at a height of 17 cm for 25 s. Institute for the Control of Pharmaceutical and Biological Products. All herb samples were kindly provided by Life Sample preparation Science College of Heilongjiang University. Other chemicals All herbs of A. senticosus collected from four different were of analytical grade and were used as received without sources and different parts of this herb were designed for the further purification. All aqueous solutions were prepared with study of effect of the different sources and the different parts on doubly distilled water. their active components. Stock solutions with concentrations of 5.0 mM for all Concerning the herbs from the different sources, only the standards were prepared in methanol and stored in a refrigerator sources are different, while the other conditions were ensured to (4˚C). A desired concentration was diluted with the doubly be the same, such as the same growing periods (3 years) and the distilled water prior to use. The pH value for a mixture of same harvest season (June, 2005). The herb sample from the ANALYTICAL SCIENCES JUNE 2007, VOL. 23 707

V, respectively, while an irreversible oxidation peak for chlorogenic acid was observed at about +0.60 V. The current of their second irreversible oxidation peak was higher than the first for both isofraxidin and rutin. The two oxidation peaks were probably caused by the oxidation of methoxyl and hydroxyl group for isofraxidin, and several electron transfer steps proceeded while hydroxyl groups were oxidated for rutin.24,25

Selection of running buffer The nature of running buffer extremely influences CE

separation efficiency (Rs). Because Rs values of chlorogenic acid and rutin were much larger than 5, while the migration time of rutin was similar to that of isofraxidin, we investigated the

effects of different buffers on Rs of isofraxidin and rutin (Fig. 3). We firstly used a phosphate buffer solution (PBS) as the running buffer for the CE separation of isofraxidin and rutin. The resolution of isofraxidin and rutin was poor when 20 mM PBS (pH 6.0) was used (Fig. 3A). For the good resolution of isofraxidin and rutin, we changed the pH value of PBS to 8.8. Nevertheless, the rutin was not detected within 13 min and the peak current of isofraxidin decreased (Fig. 3B). Then, a 0.10 M borate buffer solution (pH 9.1) was used as the running buffer (Fig. 3C). The good resolution of isofraxidin and rutin could finally be achieved. However, lower peak currents of isofraxidin and rutin were obtained.

Finally, we tried a mixture of 7.5 mM NaH2PO4 and 7.5 mM borax (pH 6.0) for the separation. Under this condition, the excellent resolution and the higher peak currents for isofraxidin and rutin could be obtained (Fig. 3D) by comparison with the results using other buffers. Thus, the mixture of borax and Fig. 3 Effects of the running buffer nature on the CE separation. NaH2PO4 solutions was selected as the running buffer, which (A) 20 mM PBS (pH 6.0), (B) 20 mM PBS (pH 8.8), (C) 0.10 M was accordant with previous reports.26 The reason of borate (pH 9.1), (D) 7.5 mM NaH PO –7.5 mM borax (pH 6.0). 2 4 improvement of the resolution for isofraxidin and rutin was that the borate can chelate with the analytes to form more soluble complex anions.27 same source was not from one individual plant, but from a mixture of many plants collected together. Optimization of analytical procedure For all the herb samples from the different parts, they had the The selection of the detection potential applied on the working same source (Wuchang), the same growing periods (3 years) electrode is of vital importance in order to obtain a high and the same harvest season (June, 2005), only the parts were sensitivity. Figure 4 depicts the hydrodynamic voltammograms different. In addition, the herb sample of each part was from a (HDVs) of isofraxidin, rutin and chlorogenic acid at the 33 μm mixture of this part of many plants collected together. CFE. As for isofraxidin, the peak current was very low from Samples of all herbs were ground into powder with an herbal 0.45 to 0.70 V, then increased dramatically and reached a crusher through a 40-mesh sieve (FZ 102 Minisize Crusher for plateau at 0.90 V. After 1.15 V, the peak current again Herbal Sample, Yongguangming Medical Instrument Factory, increased and reached another plateau at 1.2 V. Finally, the , China). Then, 0.5 g powder samples for each herb peak current began to decrease with the higher detection were extracted with 5 mL of methanol separately for 20 min in potential. Rutin exhibited a variation trend similar to that of an ultrasonic bath. The supernatant was moved out. The isofraxidin. But the two plateaus stood between 0.80 – 1.1 V, extraction procedure was repeated three times. Next, each of and 1.2 – 1.35 V, respectively. These HDVs results of the total extracted solutions was concentrated nearly to dryness. isofraxidin and rutin were consistent with their CVs, in which Methanol was added to dissolve the residue to a final volume of there were two irreversible oxidation peaks and their second 5.0 mL. Before analysis, all sample solutions were filtered irreversible oxidation peak was higher than the first. As for through a 0.22-μm membrane and diluted in the doubly distilled chlorogenic acid, the peak current increased with increasing the water to a required concentration (50% methanol), which could detection potential and reached the highest value at 1.2 V, and be directly injected for analysis. then decreased after that. During the experiments, the background noise increased very slowly with the detection potential from 0.45 to 1.35 V. Therefore, the 1.2 V was Results and Discussion selected as the optimized applied potential for the sensitive and the stable detection of isofraxidin, rutin and chlorogenic acid. Cyclic voltammetry Subsequently, the effects of injection time (from 8 to 35 s),

Cyclic voltammograms (CVs) of isofraxidin, rutin and buffer concentration (from 5.0 to 8.5 mM for NaH2PO4, and chlorogenic acid at the CFE are shown in Fig. 2. From the CVs, from 5.0 to 9.0 mM for borax), pH value of buffer (from 5.0 to it could be seen that isofraxidin yielded two irreversible 9.0) and separation voltage (from 5.0 to 17.5 kV) on the peak oxidation peaks at about +0.60 and +0.90 V, respectively; rutin current, the migration time and the separation efficiency gave two irreversible oxidation peaks at about +0.40 and +1.0 (namely, theoretic plate numbers (N)) were each studied. 708 ANALYTICAL SCIENCES JUNE 2007, VOL. 23

Fig. 4 HDVs of 1.0 × 10–5 M isofraxidin, 2.0 × 10–5 M rutin and 5.0 Fig. 5 Electropherogram of a standard solution containing 1.0 × –5 –5 –5 –5 × 10 M chlorogenic acid. Running buffer, 7.5 mM NaH2PO4–7.5 10 M isofraxidin, 2.0 × 10 M rutin and 2.0 × 10 M chlorogenic mM borax (pH 6.0); sampling, injection 20 s at a height of 17 cm; acid under the optimum conditions. Running buffer, 7.5 mM separation voltage, 15 kV. borax–7.0 mM NaH2PO4 (pH 7.0); working potential, 1.2 V (vs. Ag/AgCl); separation voltage, 15 kV; sampling, injection 25 s at a height of 17 cm.

In order to obtain a sharper peak and a higher detection signal, we selected 25 s as the injection time. When the pH value of the buffer was 7.0, the highest peak currents and shorter Method evaluation migration time for the analytes were obtained. Besides, we Under the optimized conditions described previously, a series found that the migration time of isofraxidin did not change over of the standard mixture solutions with concentration ranging –7 –4 the whole buffer concentration range of NaH2PO4 and borax. from 1.0 × 10 to 1.0 × 10 M for isofraxidin and rutin, and This result is accordant with previous reports.26 High detection from 1.0 × 10–6 to 5.0 × 10–5 M for chlorogenic acid were each sensitivity was thus achieved when the mixture of 7.0 mM studied. Detection limits actually detected and linear ranges of

NaH2PO4 and 7.5 mM borax solution was used. Therefore, we these analytes obtained by the CE-ED method are listed in chose it as the running buffer. Table 1. The detection limit and the linear range of rutin The high separation efficiency is extremely significant in obtained in this experiment was comparable to that reported order to avoid the interference from complicated samples such previously with the CE-ED method.28,29 As for isofraxidin, the as the TCMs. Therefore, the effects of separation voltage on the detection limit was 1 order of magnitude lower and the linear separation efficiency and the peak current were investigated. It range was 1 order of magnitude wider than that with CE-UV was found that the highest peak currents for isofraxidin and method.26 But for chlorogenic acid, the detection limit was 1 rutin were achieved when the separation voltage was 15 kV. At order of magnitude higher and the linear range was 1 order of the same time, the N value was the largest for rutin, but for magnitude narrower than that reported before with the CE-ED isofraxidin increased slowly from 15 to 17.5 kV. Considering method.30 In this research, the running buffer of 30 mM borate the peak current and the N together, we selected 15 kV as the solution (pH 9.5) used was different from ours, which affected optimum separation voltage. Under this condition, the N values the peak response. Such results indicated that the optimum for isofraxidin and rutin were of 2.8 × 105 and 1.9 × 105 (40 cm condition of the separation and the detection for isofraxidin and separation capillary), respectively. rutin was not the optimum for the analysis of chlorogenic acid. Based on the experiments, we determined the optimized The intra-day reproducibility of the method was carried out by separation and detection conditions: detection potential at 1.2 V, consecutive detections of 6 injections of a standard mixture –5 –5 7.0 mM NaH2PO4–7.5 mM borax (pH 7.0) buffer, injection time solution containing 1.0 × 10 M isofraxidin, 2.0 × 10 M rutin 25 s at a height of 17 cm and separation voltage at 15 kV to and 2.0 × 10–5 M chlorogenic acid under the optimum accomplish the highly sensitive and the highly efficient conditions. The RSDs (n = 6) for the peak current (< 2.4%) and separation. the migration time (< 0.70%) are shown in Table 2. The low Under such optimized conditions, baseline separation of RSD values obtained indicated that the reproducibility of this isofraxidin, rutin and chlorogenic acid with the high sensitivity method for the analysis of the standard samples was was accomplished within 6 min. A typical electropherogram for satisfactory. the standard mixture solution was shown in Fig. 5. The peak identification was performed with spiking method. The Analysis of herb extract unknown peak was an electroactive foreign substance in the Detection contents of analytes in herbs. As mentioned above, rutin standard sample, which was confirmed by comparing the contents of the active compounds in the herb are influenced electropherograms of rutin and blank. by the source. Here, the stems of A. senticosus (Rupr. Et In the CE-ED experiment, the conditions of the electrode and Maxim.) Harms from the four different sources were analyzed. the capillary greatly influence stability and reproducibility of All the sources are located in Heilongjiang province; they are the detection results. In this experiment, the treatment methods Maoershan, Wudalianchi, and Wuchang. In of the CFE and the capillary were the same as before.10 The addition, the contents of the analytes in the different parts of this capillary was only treated with the doubly distilled water and herb were also detected. The peak identifications of isofraxidin, the running buffer for 2 and 4 min. The CFE was treated in an rutin and chlorogenic acid in the real sample solutions were ultrasonic bath for 1 min before experiment everyday. assured by spiking methods. The contents of isofraxidin, rutin ANALYTICAL SCIENCES JUNE 2007, VOL. 23 709

Table 1 Detection limits and regression equations of isofraxidin, rutin and chlorogenic acida Analyte Detection limitb/M Linear range/M Regression equationc R2 Isofraxidin 1.0 × 10–7 1.0 × 10–7 – 1.0 × 10–4 y = 5.89677x + 0.19198 1 Rutin 2.0 × 10–7 2.0 × 10–7 – 1.0 × 10–4 y = 1.56552x – 0.70353 0.9998 Chlorogenic acid 1.5 × 10–6 1.5 × 10–6 – 5.0 × 10–5 y = 1.949x – 2.7043 0.9988 a. CE-ED conditions were the same as in Fig. 5. b. Detection limit actually detected was estimated to be three times the signal-to-nose. c. In the regression equation, the x-value was the concentration of analytes (10–6 M), the y-value was the peak current (10–12 A).

and chlorogenic acid in the real samples were determined by Table 2 Precision (RSD) of the present methoda comparison of each one’s peak heights in the standard solution b c and that in the real samples. Detection results are summarized Standard sample Real sample (n = 6) (n = 3) in Table 3. Analyte In all the stems of A. senticosus Harms from the four different Peak Migration Peak Migration sources, no rutin was detected. The contents of isofraxidin and current time current time chlorogenic acid were different for the different sources. The Isofraxidin 1.5 0.63 3.4 1.6 concentration of isofraxidin in the stems from Wuchang (0.021 –1 Rutin 2.4 0.69 6.0 1.8 mg g ) was comparable with that from Mudanjiang (0.023 mg Chlorogenic acid 1.2 0.70 5.7 0.70 g–1), and was the highest. In contrast, the content of isofraxidin in the stems from Wudalianchi (0.0021 mg g–1) was the lowest, a. CE-ED conditions were the same as in Fig. 5. and was only a tenth of that from Wuchang, while the content of b. The concentrations of analytes were 1.0 × 10–5 M for isofraxidin, isofraxidin from Maoershan (0.0065 mg g–1) was three-tenths of 2.0 × 10–5 M for rutin and 2.0 × 10–5 M for chlorogenic acid in the that from Wuchang. The content of chlorogenic acid was the standard sample. –1 c. The stems from Wudalianchi, Heilongjiang and the leaves of A. highest for Mudanjiang (1.1 mg g ) and was the lowest for –1 –1 senticosus (Rupr. Et Maxim.) Harms with concentration 0.1 g mL Wudalianchi (0.23 mg g ). were examined for the precision analysis of isofraxidin and From Table 3 it could be found that the concentration chlorogenic acid, and rutin, respectively. difference of isofraxidin for the different districts in Heilongjiang province was very clear. Therefore, we suggest that the source of the herb A. senticosus Harms should be definitely demanded because the content of isofraxidin is an 3.3% for isofraxidin, and 102 and 4.8% for chlorogenic acid, important indicator for quality valuation of the TCMs made of respectively (n = 3). Because no rutin was found in the stems of the herb A. senticosus Harms. this herb, the extract of the leaves of this herb was studied. The In addition, as for the different parts of the herb A. senticosus average recovery and the RSD for rutin were 98 and 4.6%, Harms, the contents of the analytes were also obviously respectively. different. The rutin was only found in the leaves of A. senticosus Harms, which was accordant with a previous report.19 Identification analysis of herbs The content of isofraxidin in the stems was comparable with Because the contents and the components of the active that in the rhizomes, and was 10 times that in the leaves, which compounds in the herbs are influenced by the sources,2 the was the same as that in the roots. The content of chlorogenic quantitative analysis and the qualitative analysis of the acid in the roots was the highest, and such content in the leaves compounds in the TCMs are very important to ensure was the lowest. authenticity, quality, safety and efficacy. We applied the CE- Reproducibility of CE-ED method and recoveries of analytes for ED method for the identification analysis of the herb A. analysis of herb. Owing to the very complicated components in senticosus Harms from the different sources and the different the herbs, which extremely influence the stability of the parts of this herb, and investigated the distribution of the separation capillary and the activity of the working electrode, bioactive ingredients (isofraxidin, chlorogenic acid and rutin) in the capillary and the electrode should be treated between the different parts (roots, rhizomes, stems and leaves) of this continuous determinations of the TCMs extract solution. herb. Between runs, the capillary was washed with water for 2 min The distinguishability was investigated based on peak and with the running buffer for 6 min; the CFE electrode should number, peak height and peak height ratio of the electroactive be activated by scanning between –0.2 and 1.2 V for 20 cycles compounds in the context of the same diluted mass besides ultrasonic treatment for 1 min prior to experiment concentration for each herb. To explain it more clearly, we everyday. Otherwise, the peak current decreased 24% and the divided the electropherograms into several electropherogram migration time prolonged 45 s for isofraxidin. Thus, after the segments for the identification analyses of these herbs. The simple treatment, the satisfactory reproducibility was achieved isofraxidin, rutin and chlorogenic acid are the important (Table 2). The precisions of the peak current and the migration bioactive ingredients in the herb A. senticosus Harms. time (as RSD) were between 3.4 and 6.0%, and 0.7 and 1.8%, Therefore, these three compounds were commended as markers. respectively. These results proved that the CE-ED method Identification of herbs from different sources. Because the provided the good stability and the good reproducibility for the sources are different, the conditions of soil and climate are also analysis of the real samples. different. Thus, not only the contents of the active compounds The recoveries were determined by spiking method under the are different, but also are the kinds of the compounds in the optimum conditions. The standard-spiked extract of the stems herb. Therefore, the electropherograms obtained by the CE-ED of A. senticosus Harms (Wudalianchi, Heilongjiang) was method for the stems of A. senticosus (Rupr. Et Maxim.) Harms examined. The average recoveries and the RSDs were 107 and from Wuchang, Maoershan, Mudanjiang and Wudalianchi in 710 ANALYTICAL SCIENCES JUNE 2007, VOL. 23

Table 3 Detection results of isofraxidin, rutin and chlorogenic acid in the herbal medicines with the CE-ED method (n = 3, mg g–1)a

Sample Cultivation Isofraxidin Rutin Chlorogenic acid A. senticosus Harms region Mean content RSD Mean content RSD Mean content RSD

Stem Maoershan 0.0065 0.34 NFb 0.40 5.4 Wudalianchi 0.0021 2.1 NF 0.23 2.6 Mudanjiang 0.023 1.9 NF 1.1 4.8 Wuchang 0.021 0.75 NF 0.80 5.0 Rhizome Wuchang 0.020 2.6 NF 1.7 3.6 Leaf Wuchang 0.0012 0.28 0.013 0.15 5.6 Root Wuchang 0.0012 1.6 NF 4.1 2.4 5.9

a. CE-ED conditions were the same as in Fig. 5. b. NF, not found.

stems of A. senticosus Harms from Wuchang and Mudanjiang could be easily distinguished by comparing their unique electropherograms obtained by the CE-ED method. Secondly, except for segment 1, the identification of the electropherograms a and b was also conducted by comparing segments 2′ and 3′, respectively. In segment 2′, there were 3 peaks for the electropherogram b, but none for the electropherogram a. In segment 3′, 4 peaks were found for the electropherogram a, and only 2 peaks for the electropherogram b. Therefore, the electropherograms a and b could be easily distinguished based on comparison of the 3 segments. From Fig. 6 one can see that the CE-ED profiles of the stems of A. senticosus Harms from the different sources had clear differences and could be easily identified. This indicated that the proposed CE-ED method could be used to identify not only the herbs from the different species,10 but also those from the different sources, which is very important for the identification analysis of the TCMs. Identification of different parts of herb. The distribution of the compounds in the different parts of an herb is different. Here, we used the CE-ED method to distinguish the different parts of the herb A. senticosus (Rupr. Et Maxim.) Harms (namely, roots, stems, rhizomes and leaves). The electropherograms of the Fig. 6 Electropherograms of the stems of A. senticosus (Rupr. Et different parts of this herb obtained with the CE-ED method are Maxim.) Harms from the different sources. (a) Maoershan, (b) shown in Fig. 7. Wudalianchi, (c) Wuchang, (d) Mudanjiang. Peaks (1) isofraxidin, In segment 1, the electropherograms could be divided into two (3) chlorogenic acid. Conditions were the same as in Fig. 5. groups: the stems and the rhizomes, and the leaves and the roots, based on peak height of peak 1. The peak heights of peak 1 in the roots and the leaves were much lower than that in the stems and the rhizomes. And the further identification within Heilongjiang province were different (Fig. 6). the two groups was conducted as follows. From segment 1, the four electropherograms could be divided Firstly, because in segment 1 the peak heights of the peak 1 into two groups: electropherograms c and d, and were similar, we could distinguish the electropherograms of the electropherograms a and b, based on the peak height difference roots and the leaves from segments 2 and 3. In segment 2, there between peaks 1 and 1a (1b, 1c, 1d). In the electropherograms c were higher and more peaks for the leaves (8 peaks) than for the and d, the peak heights of the peak 1 were higher than that of roots (3 peaks). Moreover, the marker rutin (peak 2) was found c d c the peaks 1 and 1 , and their peak height ratios (i.e., ip1/ip1 , only in the leaves. In segment 3, the higher and more peaks d ip1/ip1 ) were similar. But, the peak height of peak 1 was less were obtained for the roots (5 peaks), but not for the leaves (3 than 1b for the electropherogram b and similar to that of peak 1a peaks). for the electropherogram a. The further identification within the Secondly, the rhizomes and the stems could be identified from two groups was conducted as follows. the segments 1 and 3′. In segment 1, the peak currents of the Firstly, because the electropherograms c and d could not be peak 1 were similar for the stems and the rhizomes, but peak identified in the segment 1, the identification of the height ratios of the peak 1 and 1′ (1″) (i.e., ip1/ip1′ and ip1/ip1″) electropherograms c and d was carried out by comparing were somewhat different. In the electropherogram of the stems, segments 2 and 3. In segment 2, there were much higher peak the peak height ratio (ip1/ip1′) was larger than 1. But the peak currents for 5 peaks in the electropherogram d, compared with height ratio (ip1/ip1″) was less than 1 for the rhizomes. In that in the electropherogram c. In segment 3, the peak height of segment 3′, 4 peaks were found for the rhizomes, and only 2 peak 3 was higher than that of peaks 3d for the electropherogram peaks for the stems. d, but just the reverse for the electropherogram c. Thus, the Thus, the different parts of the herb A. senticosus Harms were ANALYTICAL SCIENCES JUNE 2007, VOL. 23 711

2. Y. X. Zhou, H. M. Lei, Y. H. Xu, L. X. Wei, and X. F. Xu, “Research Technology of Fingerprint of Chinese Traditional Medicine”, 2002, Press of Chemical Industry, Beijing, China. 3. S. K. Yan, W. F. Xin, G. A. Luo, Y. M. Wang, and Y. Y. Cheng, J. Chromatogr., A, 2005, 1090, 90. 4. J. Meng, K. S. Y. Leung, Z. H. Jiang, X. P. Dong, and Z. Z. Zhao, Chem. Pharm. Bull., 2005, 53, 1484. 5. Y. Q. Xu, S. Q. Sun, X. F. Feng, and S. L. Hu, Spectrosc. Spect. Anal., 2003, 23, 502. 6. J. Pazourek, D. Gajdotová, M. Spanilá, M. Farková, K. Novotná, and J. Havel, J. Chromatogr., A, 2005, 1081, 48. 7. R. Wang, S. W. Liang, J. B. Wei, and X. F. Liu, Chin. J. Experimental Traditional Medical Formulae, 2004, 10, 61. 8. M. Gu, S. F. Zhang, Z. G. Su, Y. Chen, and F. Ouyang, J. Chromatogr., A, 2004, 1057, 133. 9. Y. Sun, T. Guo, Y. Sui, and F. M. Li, J. Chromatogr., B, 2003, 792, 147. 10. X. G. Zhou, C. Y. Zheng, J. Y. Sun, and T. Y. You, J. Chromatogr., A, 2006, 1109, 152. 11. Y. L. Zhou, S. L. Dong, and S. Q. Nie, “Flora of Woods in Fig. 7 Electropherograms of the different parts of the herb A. Heilongjiang Province”, 1986, Heilongjiang Scientific and senticosus (Rupr. Et Maxim.) Harms. Peaks: (1) isofraxidin, (2) Technical Press, Harbin, China. rutin, (3) chlorogenic acid. Conditions were the same as in Fig. 5. 12. Heilongjiang Institute of Traditional Chinese Medicine, “Chinese Ciwujia Studies”, 1981, Heilongjiang Scientific and Technical Press, Harbin, China, 5. 13. The Pharmacopoeia Commission of PRC, “Chinese identified based on these different electropherogram segments. Pharmacopoeia”, 2005, Vol. 1, Chemical Industry Press, And the application scope of the CE-ED method in the Beijing, China, 143. identification analysis of the herbs could be further extended. 14. M. Zhao, Y. Wang, and L. Kang, Chin. J. Chin, Materia Medica, 2001, 26, 534. 15. G. Zgórka and S. Kawka, J. Pharm. Biomed. Anal., 2001, Conclusions 24, 1065. 16. T. Fujikawa, H. Soya, H. Hibasami, H. Kawashima, H. Because the constituents in the TCMs are complicated, the Takeda, S. Nishibe, and K. Nakashima, Phytother. Res., quantitative and the qualitative analyses of the TCMs are very 2002, 16, 474. important to assure the efficacy and the safety of the TCMs. In 17. Y. Kimura and M. Sumiyoshi, J. Ethnopharmacol., 2004, this paper, the CE-ED profiles of the stems of A. senticosus 95, 447. (Rupr. Et Maxim.) Harms from the different sources both in 18. T. Fujikawa, S. Miguchi, N. Kanada, N. Nakai, M. Ogata, Heilongjiang province (namely Wuchang, Maoershan, I. Suzuki, and K. Nakashima, J. Ethnopharmacol., 2005, Wudalianchi and Mudanjiang) and the different parts of this 97, 375. herb were analyzed. The experiment results indicated that the 19. M. L. Chen, F. R. Song, M. Q. Guo, Z. Q. Liu, and S. Y. herbs from the different sources could be easily identified based Liu, Rapid Commun. Mass Spectrom., 2002, 16, 264. on several different segments in their electropherograms 20. S. M. Zhang, H. W. Wang, and Y. Q. Liu, J. obtained by the CE-ED method. So could the different parts in Pharm. Univ., 1996, 13, 216. this herb. In addition, the identification of the herbs from the 21. A. Tolonen, T. Joutsamo, S. Mattlla, T. Kämäräinen, and J. different sources further affirmed that the CE-ED method could Jalonen, Phytochem. Anal., 2002, 13, 316. be considered as a potential technology for the identification 22. S. Apers, T. Naessens, S. V. Miert, L. Pieters, and A. analysis of the TCMs. Vlietinck, Phytochem. Anal., 2005, 16, 55. 23. J. Y. Sun, C. Y. Zheng, X. L. Xiao, L. Niu, T. Y. You, and E. K. Wang, Electroanalysis, 2005, 17, 1675. Acknowledgements 24. G. Chen, X. H. Ding, Z. G. Cao, and J. N. Ye, Anal. Chim. Acta, 2000, 408, 249. We gratefully acknowledge the financial supports of the 25. G. Chen, H. F. Luo, J. N. Ye, and C. Q. Hu, Tatanta, 2001, National Natural Science Foundation of China (20605020), 54, 1067. Foundation of National Excellent Ph. D. Thesis and 26. W. J. Zheng, S. F. Wang, X. G. Chen, and Z. D. Hu, Distinguished Young Scholars of Jilin Province (20060112). Talanta, 2003, 60, 955. The authors also thank the Excellent Youth Foundation of 27. S. Hoffstetter-Kuhn, A. Paulus, E. Gassmann, and H. M. Heilongjiang University and the Scientific Foundation of Widmer, Anal. Chem., 1991, 63, 1541. Heilongjiang University for Ph. Ds. for their supports. 28. G. Chen, H. W. Zhang, and J. N. Ye, Anal. Chim. Acta, 2000, 423, 69. 29. Q. C. Chu, T. Wu, L. Fu, and J. N. Ye, Microchim. Acta, References 2004, 148, 311. 30. A. F. Wang, Y. Zhou, F. Wu, P. G. He, and Y. Z. Fang, J. 1. P. Dratar and J. Moravcova, J. Chromatogr., B, 2004, 812, 3. Pharm. Biomed. Anal., 2004, 35, 959.