Chinese Journal of Natural Medicines 2013, 11(5): 0546−0552
Chinese Journal of Natural Medicines
The Spectrum-Effect integrated fingerprint of Polygonum cuspidatum based on HPLC-diode array detection-flow injection-chemiluminescence DING Xiao-Ping 1, ZHANG Cui-Ling 1, 3, QI Jin 1, 2, SUN Li-Qiong 1, QIN Min-Jian 4, YU Bo-Yang 1, 2* 1
Department of Complex Prescription of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing 211198, China; State Key Laboratory of Natural Medicines, Department of Complex Prescription of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing 210009, China; 3 Department of Traditional Chinese Medicine, Beng Bu Medical College, Bengbu 233030, China; 4 Department of Resources Science of Traditional Chinese Medicines, China Pharmaceutical University, Nanjing 211198, China 2
Available online 20 Sept. 2013 [ABSTRACT] AIM: To establish the Spectrum-Effect integrated fingerprint of Polygonum cuspidatum to evaluate the quality of
P. cuspidatum. METHODS: An on-line HPLC–DAD–flow injection chemiluminescence (FICL) method was developed to investigate the quality of P. cuspidatum from different habitats based on the established Spectrum-Effect integrated fingerprint. RESULTS: Nineteen batches of samples of P. cuspidatum were evaluated for the similarity of their chromatographic and free radical scavenging fingerprints, and the results compared. Main antioxidants were estimated by regression analysis between peak areas of thirteen compounds and their activities. Some active compounds were identified by HPLC-ESI-MS. CONSULSIONS: The results indicated that main antioxidants in P. cuspidatum could be rapidly screened by the established Spectrum-Effect integrated fingerprint based on on-line HPLC-DAD-FICL, and would be more efficient and objective method to evaluate the quality of P. cuspidatum. [KEY WORDS] Effect-Spectrum integrated fingerprint; HPLC-DAD-FICL; Polygonum cuspidatum; HPLC-ESI-MS
[CLC Number] R917
1
[Document code] A
[Article ID] 1672-3651(2013)05-0546-07
Introduction
Polygonum cuspidatum Siebold & Zucc. (Huzhang in Chinese), a well-known plant-based medicine, is dried root and rhizome and widely distributed in China. P. cuspidatum, which possesses antiviral [1], antioxidant [2] and estrogenic activities [3], has been used in China and Japan as a traditional medicine for the treatment of hypertension, tumors, atherosis [Received on] 28-Mar.-2012 [Research funding] This project was supported by the National Natural Science Foundation of China (Grant No. 30973965), 46th, 47th China Postdoctoral Fund (Nos. 20090461139, 20100471480), a grant from the Specialized Research Fund for the Doctoral Program of Higher Education of China (for the youth scholars) (No. 20090096120008), 2011’ Program for Excellent Scientific and Technological Innovation Team of Jiangsu Higher Education and the Priority Academic Program Development of Jiangsu Higher Education Institutions, Postdoctoral Fund of Jiangsu Province (No. 1001077C). [*Corresponding author] YU Bo-Yang: Prof., Tel/Fax: 86-2586185158, E-mail: [email protected] These authors have no any conflict of interest to declare. Published by Elsevier B.V. All rights reserved
and various inflammatory diseases [4–8]. Anthraquinones, stilbenes, flavonoids and phenols are considered as main active ingredients in P. cuspidatum [9]. At the present, some analytical methods, including HPLC, capillary electrophoresis and HPLC-ESI/MS [10–12], have been reported for the qualitative and quantitative analysis of single or several components in P. cuspidatum. However, the methods can not reveal the bioactivities of the compounds. Previous research has indicated that P. cuspidatum displayed superior antioxidant activity. Stilbenes, a class of biologically active components including resveratrol and piceid, were found to show very good antioxidant capacity [2, 7]. According to literature reports, although the activities of a few components or the extracts were studied, the integrated evaluation of the multiple active ingredients was still necessary. Previously, a concept of the “Spectrum-Effect Integrated Fingerprint” was proposed by this laboratory and some related research work was reported [13–18]. In this study, the comprehensive quality evaluation and the screening of active compounds was determined for P. cuspidatum through the combination of regression analysis, similarity, and the total activity of chromatographic and active fingerprints. The Spectrum-Effect
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integrated fingerprint can link the chromatographic fingerprint of some TCMs with different related spectra and bioactivity information to comprehensively evaluate the quality of a TCM online or offline. In the human body, oxygen-centered free radicals and reactive oxygen species may be produced in many physiological and biochemical processes, and excessive free radicals can cause pathological effects and diseases, such as cancer, ageing and cardiovascular diseases [19]. Therefore, much attention has been paid to the antioxidants, which are expected to prevent living systems from peroxidative damage. Plants can provide a large source of natural antioxidants that might serve as leads for the development of novel drugs. The investigation of natural antioxidants and bioactive compounds from traditional medicines or the treatment of certain human diseases is now receiving special attention. Typically, determination of the antioxidant activity of plants mainly focuses on a single compound or a total extract. In order to avoid the complex separation process and the possible loss of active components, several on-line methods, including HPLC-DAD-DPPH, HPLC-DAD-ABTS and HPLCDAD-MS for the analysis of DPPH• and ABTS+• scavenging activities and HPLC-DAD-chemiluminescence (CL) for the assay of H2O2 and O2– • scavenging activities [20-21], have been developed for the rapid screening of radical scavengers in complex samples. Although the on-line HPLC-DAD-CL method has been used to evaluate the antioxidant activity of some plant-based medicines [13-14], its effectiveness and extensive application need to be further verified. Traditional Chinese medicine has been used for pharmaceutical and dietary therapy for several millennia. Stable and rapid screening methods should be developed to search for natural antioxidants to replace synthetic antioxidants. In this study, the integrated Spectrum-Effect fingerprint of P. cuspidatum which was established based on the combined use of HPLC-DAD-FICL and HPLC-ESI-MS, was applied to screen the main antioxidants in P. cuspidatum. The comprehensive qualities of P. cuspidatum samples from nineteen habitats were evaluated by chemical and activity-based fingerprints. Similarity and correlation analyses were performed to evaluate the data from these fingerprints.
2
Experimental
2.1 Materials and reagents P. cuspidatum samples were collected in different natural growth sites in China. All of them were identified by Professor Bo-yang Yu Voucher specimens (No. 100901-19) deposited in the Herbarium of China Pharmaceutical University. Their habitats and codes were listed as follows: Sichuan (S01), Guangzhou (S02), Anhui(S03), Fujian(S04), Hunan(S05), Hunan (S06), Hunan(S07), Hubei (S08), Hubei (S09), Zhejiang (S10), Guangxi (S11), Jiangsu (S12), Jiangsu (S13), Chengdu (S14), Sichuan (S15), Shanxi (S16), Jiangxi (S17), Hubei (S18), and Hunan (S19). A reference sample of
resveratrol was purchased from Jiangsu Provincial Institute of Material Medical. Epicatechin and emodin were purchased from Zelang Technical Co., Nanjing. HPLC-grade acetonitrile was acquired from Tedia (Fairfield, USA) and analytical grade phosphoric acid ((Nanjing Chemical Plant, Jiangsu, China) was used in HPLC analysis. Luminol (Sigma Co., USA), hydrogen peroxide (30% H2O2 in water), Na2CO3, and NaHCO3 (Nanjing Chemical Reagent Co., Jiangsu, China) and EDTA (Shanghai Chemical Reagent Co., Shanghai, China), were applied for CL detection. Deionized water used for HPLC analysis was purified using a Millipore water purification system (Millipore, MA, USA). 2.2 Sample preparation The samples from different habitats were ground to a powder and passed through 60 mesh sieve, and then dried at 60°C for 6 h. An accurately weighed sample (2.0 g) of powder was refluxed twice with 60 mL ethanol−water (95:5, V/V) for one and half an hour, respectively. The extract solutions were filtered, and the residues rinsed twice with ethanol−water (20 mL, 95:5, V/V). The filtrates and washings were combined and evaporated under vacuum. The dry residue was dissolved and diluted with ethanol−water (95:5, V/V) into a volumetric flask. The extracted solution (1mL) was diluted with ethanol-water (95:5, V/V) into a volumetric flask. After filtration through a 0.45 µm filter, an aliquot of 5 µL solution was injected onto the HPLC. 2.3 Preparation of reagent solutions for the determination of H2O2 scavenging activity Carbonate buffer (pH 10.5) was prepared by mixing of appropriate volumes of 0.1 mol·L−1 Na2CO3 and 0.1 mol·L−1 NaHCO3. A 1.83 × 10−2 mol·L−1 stock solution of luminol was prepared in 0.1 mol·L−1 Na2CO3 solution. The 1.83 × 10−5 mol·L−1 luminol solution including 6.3 × 10−3 mol·L−1 EDTA was prepared in carbonate buffers (pH 10.5) and the 8.8 × 10−4 mol·L−1 H2O2 solution was prepared from 30% H2O2 diluted in water. 2.4 HPLC-DAD-FICL analysis An Agilent 1100 series HPLC system (Agilent Technologies, MA, USA), consisting of a binary pump, an autosampler, a thermostated column compartment, and a photodiode array detector, was used for the chromatographic analysis. The UV detector was set at 280 nm. A LiChrospher C18 column (250 mm × 4.6 mm i.d., 5 µm) (Hanbon Sci & Tech, Jiangsu, China) was used for all chromatographic separations. The mobile phase consisted of 0.1% aqueous H3PO4 and acetonitrile (B) using a gradient program of 15%-20% B at 0-20 min, 20%-30% B at 20-35 min, 30%-50% B at 35-55 min, 50%-60% B at 55-80 min, and 60%-100% B at 80-85 min. The mobile phase flow rate was 1 mL·min−1, and the column temperature was set at 30°C. The CL emission was detected by BPCL system (Academia Sinica Biophysics Institute, Beijing, China). On-line post-column additions of CL solutions were delivered with a BT-200 peristaltic pump (Huxi Analysis Instrument Factory,
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Shanghai, China) at the flow rate of 1.1 mL·min−1 for luminol and H2O2 solutions. The CL detector was equipped with a flat glass coil as detection cell of 80 µL and a photomultiplier operated at −800 V. All other parts of the HPLC–DAD–FICL detection system were interconnected with PEEK tubes [16]. 2.5 HPLC-ESI-MS analysis An Agilent 1100 LC/MSD Trap XCT ESI system (Agilent Technologies, MA, USA) was used for mass spectrometric determination. The HPLC-MS analysis was performed under the same gradient program with HPLC-DADFICL using the 0.1% (V/V) aqueous formic acid (A) and acetonitrile (B). The ESI-MS spectra were acquired in the negative ionization modes. The conditions of MS analysis were as follows: drying gas (air) flow rate, 9.0 L·min−1; drying gas temperature, 350 °C; scan range, 200-800 amu; nebulizing pressure, 40 psi, capillary voltage, 3,300 V. 2.6 Data analysis Data analysis of chemcal fingerprint was performed by professional software named Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2004 A), which was edited by the Chinese Pharmacopoeia Committee. The similarities of different chromatographic patterns were compared with the mean chromatogram between the samples tested. Similarity of bioactivity fingerprint was evaluated by the vector angle [22]. It was assumed that vector X ( x1 , x2 , x3 , …, xn) (| X | =
x12 + x2 2 + x32 +⋅⋅⋅⋅⋅⋅ xn 2 ) represents the active
fingerprint and the other vector Y ( y1 , y 2 , y 3 , …, y n) (| Y | = y12 + y2 2 + y32 +⋅⋅⋅⋅⋅⋅ yn 2 ) represents the reference finger-
prints, thus the vector angle of X and Y is calculated by the formula. The two vectors are more similar when the cosine values are near 1. X ·Y cos θ = X ×Y
Where xi denotes absolute scavenging rates (%) of the peak in bioactivity fingerprints and yi denotes mean scavenging rate (%) of the peak. Correlation analysis was performed to analyze the relationships between peak area in chemical fingerprints and the scavenging rates (%) by SPSS software (SPSS for Windows 12.0, SPSS Inc., USA).
3
Results and Discussion
3.1 On-line analysis for chemical and activity-based fingerprints of P. cuspidatum by HPLC-DAD-FICL The CL reaction of luminol and H2O2 is usually applied to study radical scavenging activity of plant extracts. With the development of flow injection chemiluminescence (FICL), HPLC-FICL has been developed to search for natural antioxidants in plants extract, and can rapidly screen the antioxidants in complex matrixes [13-17]. However, baseline drift was a common problem in HPLC-DAD-CL detection when the
gradient elution was carried out [23]. So the conditions of chromatographic separation and CL reaction should be optimized for a stable CL baseline and sensitive CL detection. The mobile phase for the chromatographic separation as well as the pH values and concentrations of CL solutions were investigated in previous studies. In this study, the factors were estimated, and the results suggested that 0.1 mol·L−1 carbonate buffers (pH 10.5), the proposed gradient elution program and the concentrations of CL solutions should be adopted for further studies. Chemical and activity-based fingerprints of P. cuspidatum were simultaneously obtained by HPLC-DAD-FLCL (Fig. 1). In Fig. 1, peaks 3, 4, 5, 7, 8, 10, and 12, showing strong CL inhibition, suggested their superiority in H2O2 scavenging activities, while the predominant peaks 6, 9, 11, and 13 displayed minor activities. In addition, it was apparent that some minor peaks also showed strong CL inhibition. The scavenging rate (%) was applied to reveal the capacity of each compound for scavenging H2O2, and was calculated by equation CL − CL1 ×100% Scavenging rate (%) = 0 CL0 Where CL0 was the blank CL intensity (without sample) and CL1 was the inhibited CL intensity of every compound in the samples. 3.2 Identification of bioactive compounds in P. cuspidatum Peak areas and scavenging rates (%) of various compounds in the nineteen batches of samples are displayed in Fig. 2. As shown in Fig. 2A, peaks 3, 7, 9, and 13 in some samples showed higher areas than other peaks, while the scavenging rates (%) of peaks 9 and 13 were hardly observed in Fig. 2B. Some minor peaks showing high scavenging rates (%), such as 1, 2, 4, 5, 8, 10, and 12, implied their dominance in H2O2 scavenging activity. According to the results of activity-based fingerprint of the nineteen batches of samples, it was apparent that compounds 1, 2, 3, 4, 5, 7, 8, 10, and 12 were the main active ingredients in P. cuspidatum. Negative ion mode was used to identify thirteen peaks in the chromatographic fingerprint by HPLC-ESI/MS, and the MS data are shown in Table 1. Peaks 2, 7 and 13 were identified as epicatchin, resveratrol, and emodin, respectively by the comparison with pure standards. Peaks 3 and 5 showed the same [M – H]– m/z at 389, [M – H – Glu]– at m/z 227 and similar MS3 fragments. This indicated that they were isomers, and were tentatively identified as resveratrol-4′-O-glucoside (resveratroloside) and resveratrol-3-O-glucoside (piceid) [24, 26] . Isomers were also found for peaks 6 and 9 [M – H]– m/z at 431, [M – H – Glu]– at m/z 269 and having similar MS3 fragments. They were identified according to their spectral data as summarized in Table 1 [27]. Peak 10 ([M – H]– m/z at 517, [M – H – Glu] – at m/z 473 and [M – H – Glu-6′-O-malonyl] –) at m/z 311) displayed the same spectral
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Fig. 1 Chemical and activity-based fingerprints of P. cuspidatum from the on-line HPLC-DAD-FICL detection Table 1 Identification of bioactive compounds in P. cuspidatum Peak No.
tR (min)
1 2
9.7 11.7
[M − H]−/ [M + HCOOH − H]− 415 289
3
21.5
389
4
22.3
441
5
30.8
389
6
34.2
431
7
36.9
227
8
40.7
407
9
42.0
431
10
45.0
517
11
46.8
283
12
50.2
283
13
70.8
269
HPLC-ESI-MSn m/z – – MS2[389]: 227. MS3[227]: 209, 184, 157, 143, 107. – MS2[389]: 227. MS3[227]: 185, 183, 159, 143. MS2[431]: 269. MS3[269]: 241, 225. MS2[227]: 185, 159, 143. MS2[407]: 245. MS3[245]: 230. MS2[431]: 269. MS3[269]: 241, 225, 197. MS2[517]: 473. MS3[473]: 311, 269, 255. MS2[283]: 268, 240. MS3[240]: 197. MS2[283]: 268, 240, 151. MS2[269]: 225, 153. MS3[225]: 210, 157.
Compounds Unknown compound Catechin Resveratroloside Flavanol gallate Piceid Unknown compound Resveratrol Torachrysone-8-O-glucoside Emodin-8-O-glucoside Unknown compound Questin/Physcion Questin/Physcion Emodin
Fig. 2 Peak areas and scavenging rates (%) of thirteen peaks in the nineteen batches of samples. A Peak areas, B scavenging rates (%)
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data as peak 9 and was assigned tentatively as emodin 8-O-(6′-O-malonyl)-glucoside [26]. Peak 8 ([M – H]– m/z at 407, [M – H – Glu]– at m/z 245) was tentatively identified as torachrysone-8-O-glucoside [12, 24]. 3.3 Similarity evaluation of P. cuspidatum from different habitats The consistency of plant-based medicines can be tested through comparing the similarity between the chemical fingerprints of samples and the reference/standard fingerprints. The software “Similarity Evaluation System for Chromatographic Fingerprint of TCM” was used to evaluate these chromatograms. The chromatograms were introduced in the form of AIA (*.cdf), which includes the information of peak areas and retention times. The reference chromatogram was produced, and the similarity values of all of the introduced
chromatograms could be calculated by comparison with the reference chromatogram. The qualities of samples were relatively consistent when the similarity values were higher than 0.900. In terms of chemical fingerprints, the “common peaks” 3, 5, 6, 7, 9, 10, 11, and 13 were chosen as markers for peak matching. Then, the data of the fingerprints of the nineteen batches of P. cuspidatum samples were used to analyze the similarity among these samples by comparing each chromatogram with the reference chromatogram. The similarity results of the samples are listed in Table 2. The similarity values of the nineteen samples were more than 0.93, expect for S19, which indicated that the activity-based fingerprints of the samples from the different locations were stable, and were generally consistent.
Table 2 Similarity values of the nineteen batches of P. cuspidatum Method
Sample number S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11 S12 S13 S14 S15 S16 S17 S18
S19
Bioactive finger0.855 0.887 0.883 0.989 0.855 0.918 0.977 0.974 0.978 0.956 0.953 0.961 0.927 0.864 0.989 0.943 0.977 0.975 0.858 print Chemical finger0.981 0.980 0.970 0.993 0.990 0.988 0.989 0.979 0.998 0.966 0.968 0.966 0.938 0.986 0.994 0.958 0.987 0.985 0.899 print
As to the bioactivity fingerprints, the vector angles of scavenging rates (%) of thirteen peaks and their average values were used to estimate the similarity of the nineteen batches of samples, and the results were listed in Table 2. It was apparent that the similarity values of the samples from thirteen habitats were higher than 0.900 except for S01, S02, S03, S05, S14, and S19. Compared with the results of the chemical fingerprints, the bioactivity fingerprints might decrease the differences among chromatographic peak areas of some “Common peaks” in various samples, such as peak 6, 9, 11 and 13. Furthermore, minor peaks showing good bioactivities might be ignored in chromatographic fingerprint analysis. According to the bioactivity information, the evaluation results of chemical fingerprints showed a limitation for the quality control of P. cuspidatum. Thus, the integrated evaluation method of chemical and activity-based fingerprints could become an efficient strategy for the quality control of P. cuspidatum. 3.4 Correlation analysis of chemical and activity-based fingerprints of P. cuspidatum Correlation analysis for chromatographic peak areas and radical scavenging rates (%) of the thirteen compounds in the nineteen batches of samples were performed by SPSS statistics software, and the results are shown in Fig. 3. The relationships of only ten compounds were revealed because compounds 9, 11, and 13 hardly exhibited any H2O2 scavenging activities. In addition, the correlation coefficients of compounds 5, 6, 10, and 12 were low because obvious differences between their peak areas and scavenging rates (%) were found. As shown in Fig. 2, the activities of compounds 5, 10, and 12 showing small peak areas were stronger, except for
compound 6, and their peak areas and activities in the nineteen batches of samples were significantly different. The results of the on-line screening of active components in the samples indicated that compounds 1, 2, 3, 4, 5, 7, 8, 10, and 12 were the main bioactive ingredients (Fig. 2), while the correlation
Fig. 3 Correlation value results of peak areas and scavenging rates (%) of ten peaks in the nineteen batches of P. cuspidatum samples
analysis results of the chemical and activity-based fingerprints revealed low correlation coefficients for the main bioactive compounds 5, 10, and 12. It was apparent that the correlation coefficient was not efficient enough for distinguishing between the main active ingredients in the samples, which showed the limitation of using the correlation coefficient for the quality evaluation of P. cuspidatum. In view of the comprehensive information from two perspectives, compounds 1, 2, 3, 4, 5, 7, 8, 10, and 12 were responsible for the antioxidant activity of P. cuspidatum. The integrated evaluation of the on-line screening method and the correlation analysis is therefore a powerful tool in the search for bioactive compounds in complex samples.
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The samples containing multiple, bioactive compounds might correlate with their fine quality. Thus, the total scavenging rates (%) of various compounds in the nineteen batches of samples were displayed in Fig. 4. It was apparent that most of the samples possessed similar total activities except for S02, S03, S08, S09, S13, S14, and S19, and that the total activity of S19 was the lowest. The samples S01, S04, S06, S07, S10, and S12 showed higher total activities which would imply their better quality. The evaluation method based on chemical and activity-based fingerprints could be an efficient strategy to reveal the comprehensive quality of Polygonum cuspidatum derived from different habitats.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9] [10]
Fig. 4 The total activities of the nineteen batches of P. cuspidatum samples
Conclusion. In this study, the Spectrum-Effect integrated fingerprint of P. cuspidatum was established based on the on-line HPLC-DAD-FICL method. Furthermore, the comprehensive quality evaluation of nineteen batches of Polygonum cuspidatum samples from various habitats were investigated by the combination of chemical and activity-based fingerprints derived from the Spectrum-Effect integrated fingerprint of Polygonum cuspidatum. Similarity evaluation results of the chemical fingerprints indicated that the quality of the samples from different locations were stable and consistent, while the results of the bioactivity fingerprints were different. The bioactivity fingerprints revealed the differences between the various samples. Moreover, correlation analysis was used to estimate the main bioactive compounds in P. cuspidatum samples by the relationships of peak areas and inhibition ratios of thirteen peaks. Some main active ingredients could not be sufficiently distinguished, while on-line HPLC-DAD-FICL could rapidly screen main antioxidants in P. cuspidatum. The “Integrated Spectrum-Effect Fingerprint”, an integrated analysis of chemical and bioactivity fingerprints method, offers a rational approach for the quality evaluation of plant-based medicines.
[11]
[12]
[13]
[14]
[15]
[16]
[17]
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基于 HPLC-DAD-FICL 法建立虎杖的谱-效整合指纹图谱 丁晓萍 1，张翠玲 1, 3，戚
进 1, 2，孙丽琼 1，秦民坚 4，余伯阳 1, 2*
1
中国药科大学中药复方研究室，南京 211198;
2
天然药物活性物质与功能国家重点实验室，南京 210009;
3
蚌埠医学院中药研究室，蚌埠 233030;
4
中国药科大学中药资源研究室，南京 211198
【摘 要】 目的：建立虎杖药材的谱-效整合指纹图谱，为科学评价其质量提供可靠方法。方法：采用高效液相色谱-二极 管阵列-流动注射化学发光法得到不同来源虎杖药材的谱-效整合指纹图谱，从而综合评价虎杖药材的质量。结果：对 19 批药材 的化学指纹图谱和清除自由基的活性指纹图谱分别进行了相似度评价，并对其结果进行了对比分析。对 13 个主要抗氧化成分的 色谱峰面积和活性进行了回归分析，并利用 HPLC-ESI-MS 鉴定了部分成分的化学结构。结论：基于高效液相色谱-二极管阵列流动注射化学发光法所建立的虎杖的谱-效整合指纹图谱能快速筛选虎杖药材中的主要抗氧化活性成分，这种综合评价方法能更 客观、有效地反映虎杖药材的质量。 【关键词】 谱-效整合指纹图谱；高效液相色谱-二极管阵列-流动注射化学发光法；虎杖；高效液相-电喷雾-质谱 【基金项目】 国家自然基金资助项目（No. 30973965）；第 46，47 批中国博士后基金（Nos. 20090461139，20100471480）；2010 年江苏省博士后基金（No. 1001077C） ；教育部新教师基金（No. 20090096120008）；2011 年度江苏省高等学校优秀科技创新团 队；江苏高校优势学科建设工程资助项目
Chinese Journal of Natural Medicines
The Spectrum-Effect integrated fingerprint of Polygonum cuspidatum based on HPLC-diode array detection-flow injection-chemiluminescence DING Xiao-Ping 1, ZHANG Cui-Ling 1, 3, QI Jin 1, 2, SUN Li-Qiong 1, QIN Min-Jian 4, YU Bo-Yang 1, 2* 1
Department of Complex Prescription of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing 211198, China; State Key Laboratory of Natural Medicines, Department of Complex Prescription of Traditional Chinese Medicine, China Pharmaceutical University, Nanjing 210009, China; 3 Department of Traditional Chinese Medicine, Beng Bu Medical College, Bengbu 233030, China; 4 Department of Resources Science of Traditional Chinese Medicines, China Pharmaceutical University, Nanjing 211198, China 2
Available online 20 Sept. 2013 [ABSTRACT] AIM: To establish the Spectrum-Effect integrated fingerprint of Polygonum cuspidatum to evaluate the quality of
P. cuspidatum. METHODS: An on-line HPLC–DAD–flow injection chemiluminescence (FICL) method was developed to investigate the quality of P. cuspidatum from different habitats based on the established Spectrum-Effect integrated fingerprint. RESULTS: Nineteen batches of samples of P. cuspidatum were evaluated for the similarity of their chromatographic and free radical scavenging fingerprints, and the results compared. Main antioxidants were estimated by regression analysis between peak areas of thirteen compounds and their activities. Some active compounds were identified by HPLC-ESI-MS. CONSULSIONS: The results indicated that main antioxidants in P. cuspidatum could be rapidly screened by the established Spectrum-Effect integrated fingerprint based on on-line HPLC-DAD-FICL, and would be more efficient and objective method to evaluate the quality of P. cuspidatum. [KEY WORDS] Effect-Spectrum integrated fingerprint; HPLC-DAD-FICL; Polygonum cuspidatum; HPLC-ESI-MS
[CLC Number] R917
1
[Document code] A
[Article ID] 1672-3651(2013)05-0546-07
Introduction
Polygonum cuspidatum Siebold & Zucc. (Huzhang in Chinese), a well-known plant-based medicine, is dried root and rhizome and widely distributed in China. P. cuspidatum, which possesses antiviral [1], antioxidant [2] and estrogenic activities [3], has been used in China and Japan as a traditional medicine for the treatment of hypertension, tumors, atherosis [Received on] 28-Mar.-2012 [Research funding] This project was supported by the National Natural Science Foundation of China (Grant No. 30973965), 46th, 47th China Postdoctoral Fund (Nos. 20090461139, 20100471480), a grant from the Specialized Research Fund for the Doctoral Program of Higher Education of China (for the youth scholars) (No. 20090096120008), 2011’ Program for Excellent Scientific and Technological Innovation Team of Jiangsu Higher Education and the Priority Academic Program Development of Jiangsu Higher Education Institutions, Postdoctoral Fund of Jiangsu Province (No. 1001077C). [*Corresponding author] YU Bo-Yang: Prof., Tel/Fax: 86-2586185158, E-mail: [email protected] These authors have no any conflict of interest to declare. Published by Elsevier B.V. All rights reserved
and various inflammatory diseases [4–8]. Anthraquinones, stilbenes, flavonoids and phenols are considered as main active ingredients in P. cuspidatum [9]. At the present, some analytical methods, including HPLC, capillary electrophoresis and HPLC-ESI/MS [10–12], have been reported for the qualitative and quantitative analysis of single or several components in P. cuspidatum. However, the methods can not reveal the bioactivities of the compounds. Previous research has indicated that P. cuspidatum displayed superior antioxidant activity. Stilbenes, a class of biologically active components including resveratrol and piceid, were found to show very good antioxidant capacity [2, 7]. According to literature reports, although the activities of a few components or the extracts were studied, the integrated evaluation of the multiple active ingredients was still necessary. Previously, a concept of the “Spectrum-Effect Integrated Fingerprint” was proposed by this laboratory and some related research work was reported [13–18]. In this study, the comprehensive quality evaluation and the screening of active compounds was determined for P. cuspidatum through the combination of regression analysis, similarity, and the total activity of chromatographic and active fingerprints. The Spectrum-Effect
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integrated fingerprint can link the chromatographic fingerprint of some TCMs with different related spectra and bioactivity information to comprehensively evaluate the quality of a TCM online or offline. In the human body, oxygen-centered free radicals and reactive oxygen species may be produced in many physiological and biochemical processes, and excessive free radicals can cause pathological effects and diseases, such as cancer, ageing and cardiovascular diseases [19]. Therefore, much attention has been paid to the antioxidants, which are expected to prevent living systems from peroxidative damage. Plants can provide a large source of natural antioxidants that might serve as leads for the development of novel drugs. The investigation of natural antioxidants and bioactive compounds from traditional medicines or the treatment of certain human diseases is now receiving special attention. Typically, determination of the antioxidant activity of plants mainly focuses on a single compound or a total extract. In order to avoid the complex separation process and the possible loss of active components, several on-line methods, including HPLC-DAD-DPPH, HPLC-DAD-ABTS and HPLCDAD-MS for the analysis of DPPH• and ABTS+• scavenging activities and HPLC-DAD-chemiluminescence (CL) for the assay of H2O2 and O2– • scavenging activities [20-21], have been developed for the rapid screening of radical scavengers in complex samples. Although the on-line HPLC-DAD-CL method has been used to evaluate the antioxidant activity of some plant-based medicines [13-14], its effectiveness and extensive application need to be further verified. Traditional Chinese medicine has been used for pharmaceutical and dietary therapy for several millennia. Stable and rapid screening methods should be developed to search for natural antioxidants to replace synthetic antioxidants. In this study, the integrated Spectrum-Effect fingerprint of P. cuspidatum which was established based on the combined use of HPLC-DAD-FICL and HPLC-ESI-MS, was applied to screen the main antioxidants in P. cuspidatum. The comprehensive qualities of P. cuspidatum samples from nineteen habitats were evaluated by chemical and activity-based fingerprints. Similarity and correlation analyses were performed to evaluate the data from these fingerprints.
2
Experimental
2.1 Materials and reagents P. cuspidatum samples were collected in different natural growth sites in China. All of them were identified by Professor Bo-yang Yu Voucher specimens (No. 100901-19) deposited in the Herbarium of China Pharmaceutical University. Their habitats and codes were listed as follows: Sichuan (S01), Guangzhou (S02), Anhui(S03), Fujian(S04), Hunan(S05), Hunan (S06), Hunan(S07), Hubei (S08), Hubei (S09), Zhejiang (S10), Guangxi (S11), Jiangsu (S12), Jiangsu (S13), Chengdu (S14), Sichuan (S15), Shanxi (S16), Jiangxi (S17), Hubei (S18), and Hunan (S19). A reference sample of
resveratrol was purchased from Jiangsu Provincial Institute of Material Medical. Epicatechin and emodin were purchased from Zelang Technical Co., Nanjing. HPLC-grade acetonitrile was acquired from Tedia (Fairfield, USA) and analytical grade phosphoric acid ((Nanjing Chemical Plant, Jiangsu, China) was used in HPLC analysis. Luminol (Sigma Co., USA), hydrogen peroxide (30% H2O2 in water), Na2CO3, and NaHCO3 (Nanjing Chemical Reagent Co., Jiangsu, China) and EDTA (Shanghai Chemical Reagent Co., Shanghai, China), were applied for CL detection. Deionized water used for HPLC analysis was purified using a Millipore water purification system (Millipore, MA, USA). 2.2 Sample preparation The samples from different habitats were ground to a powder and passed through 60 mesh sieve, and then dried at 60°C for 6 h. An accurately weighed sample (2.0 g) of powder was refluxed twice with 60 mL ethanol−water (95:5, V/V) for one and half an hour, respectively. The extract solutions were filtered, and the residues rinsed twice with ethanol−water (20 mL, 95:5, V/V). The filtrates and washings were combined and evaporated under vacuum. The dry residue was dissolved and diluted with ethanol−water (95:5, V/V) into a volumetric flask. The extracted solution (1mL) was diluted with ethanol-water (95:5, V/V) into a volumetric flask. After filtration through a 0.45 µm filter, an aliquot of 5 µL solution was injected onto the HPLC. 2.3 Preparation of reagent solutions for the determination of H2O2 scavenging activity Carbonate buffer (pH 10.5) was prepared by mixing of appropriate volumes of 0.1 mol·L−1 Na2CO3 and 0.1 mol·L−1 NaHCO3. A 1.83 × 10−2 mol·L−1 stock solution of luminol was prepared in 0.1 mol·L−1 Na2CO3 solution. The 1.83 × 10−5 mol·L−1 luminol solution including 6.3 × 10−3 mol·L−1 EDTA was prepared in carbonate buffers (pH 10.5) and the 8.8 × 10−4 mol·L−1 H2O2 solution was prepared from 30% H2O2 diluted in water. 2.4 HPLC-DAD-FICL analysis An Agilent 1100 series HPLC system (Agilent Technologies, MA, USA), consisting of a binary pump, an autosampler, a thermostated column compartment, and a photodiode array detector, was used for the chromatographic analysis. The UV detector was set at 280 nm. A LiChrospher C18 column (250 mm × 4.6 mm i.d., 5 µm) (Hanbon Sci & Tech, Jiangsu, China) was used for all chromatographic separations. The mobile phase consisted of 0.1% aqueous H3PO4 and acetonitrile (B) using a gradient program of 15%-20% B at 0-20 min, 20%-30% B at 20-35 min, 30%-50% B at 35-55 min, 50%-60% B at 55-80 min, and 60%-100% B at 80-85 min. The mobile phase flow rate was 1 mL·min−1, and the column temperature was set at 30°C. The CL emission was detected by BPCL system (Academia Sinica Biophysics Institute, Beijing, China). On-line post-column additions of CL solutions were delivered with a BT-200 peristaltic pump (Huxi Analysis Instrument Factory,
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Shanghai, China) at the flow rate of 1.1 mL·min−1 for luminol and H2O2 solutions. The CL detector was equipped with a flat glass coil as detection cell of 80 µL and a photomultiplier operated at −800 V. All other parts of the HPLC–DAD–FICL detection system were interconnected with PEEK tubes [16]. 2.5 HPLC-ESI-MS analysis An Agilent 1100 LC/MSD Trap XCT ESI system (Agilent Technologies, MA, USA) was used for mass spectrometric determination. The HPLC-MS analysis was performed under the same gradient program with HPLC-DADFICL using the 0.1% (V/V) aqueous formic acid (A) and acetonitrile (B). The ESI-MS spectra were acquired in the negative ionization modes. The conditions of MS analysis were as follows: drying gas (air) flow rate, 9.0 L·min−1; drying gas temperature, 350 °C; scan range, 200-800 amu; nebulizing pressure, 40 psi, capillary voltage, 3,300 V. 2.6 Data analysis Data analysis of chemcal fingerprint was performed by professional software named Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2004 A), which was edited by the Chinese Pharmacopoeia Committee. The similarities of different chromatographic patterns were compared with the mean chromatogram between the samples tested. Similarity of bioactivity fingerprint was evaluated by the vector angle [22]. It was assumed that vector X ( x1 , x2 , x3 , …, xn) (| X | =
x12 + x2 2 + x32 +⋅⋅⋅⋅⋅⋅ xn 2 ) represents the active
fingerprint and the other vector Y ( y1 , y 2 , y 3 , …, y n) (| Y | = y12 + y2 2 + y32 +⋅⋅⋅⋅⋅⋅ yn 2 ) represents the reference finger-
prints, thus the vector angle of X and Y is calculated by the formula. The two vectors are more similar when the cosine values are near 1. X ·Y cos θ = X ×Y
Where xi denotes absolute scavenging rates (%) of the peak in bioactivity fingerprints and yi denotes mean scavenging rate (%) of the peak. Correlation analysis was performed to analyze the relationships between peak area in chemical fingerprints and the scavenging rates (%) by SPSS software (SPSS for Windows 12.0, SPSS Inc., USA).
3
Results and Discussion
3.1 On-line analysis for chemical and activity-based fingerprints of P. cuspidatum by HPLC-DAD-FICL The CL reaction of luminol and H2O2 is usually applied to study radical scavenging activity of plant extracts. With the development of flow injection chemiluminescence (FICL), HPLC-FICL has been developed to search for natural antioxidants in plants extract, and can rapidly screen the antioxidants in complex matrixes [13-17]. However, baseline drift was a common problem in HPLC-DAD-CL detection when the
gradient elution was carried out [23]. So the conditions of chromatographic separation and CL reaction should be optimized for a stable CL baseline and sensitive CL detection. The mobile phase for the chromatographic separation as well as the pH values and concentrations of CL solutions were investigated in previous studies. In this study, the factors were estimated, and the results suggested that 0.1 mol·L−1 carbonate buffers (pH 10.5), the proposed gradient elution program and the concentrations of CL solutions should be adopted for further studies. Chemical and activity-based fingerprints of P. cuspidatum were simultaneously obtained by HPLC-DAD-FLCL (Fig. 1). In Fig. 1, peaks 3, 4, 5, 7, 8, 10, and 12, showing strong CL inhibition, suggested their superiority in H2O2 scavenging activities, while the predominant peaks 6, 9, 11, and 13 displayed minor activities. In addition, it was apparent that some minor peaks also showed strong CL inhibition. The scavenging rate (%) was applied to reveal the capacity of each compound for scavenging H2O2, and was calculated by equation CL − CL1 ×100% Scavenging rate (%) = 0 CL0 Where CL0 was the blank CL intensity (without sample) and CL1 was the inhibited CL intensity of every compound in the samples. 3.2 Identification of bioactive compounds in P. cuspidatum Peak areas and scavenging rates (%) of various compounds in the nineteen batches of samples are displayed in Fig. 2. As shown in Fig. 2A, peaks 3, 7, 9, and 13 in some samples showed higher areas than other peaks, while the scavenging rates (%) of peaks 9 and 13 were hardly observed in Fig. 2B. Some minor peaks showing high scavenging rates (%), such as 1, 2, 4, 5, 8, 10, and 12, implied their dominance in H2O2 scavenging activity. According to the results of activity-based fingerprint of the nineteen batches of samples, it was apparent that compounds 1, 2, 3, 4, 5, 7, 8, 10, and 12 were the main active ingredients in P. cuspidatum. Negative ion mode was used to identify thirteen peaks in the chromatographic fingerprint by HPLC-ESI/MS, and the MS data are shown in Table 1. Peaks 2, 7 and 13 were identified as epicatchin, resveratrol, and emodin, respectively by the comparison with pure standards. Peaks 3 and 5 showed the same [M – H]– m/z at 389, [M – H – Glu]– at m/z 227 and similar MS3 fragments. This indicated that they were isomers, and were tentatively identified as resveratrol-4′-O-glucoside (resveratroloside) and resveratrol-3-O-glucoside (piceid) [24, 26] . Isomers were also found for peaks 6 and 9 [M – H]– m/z at 431, [M – H – Glu]– at m/z 269 and having similar MS3 fragments. They were identified according to their spectral data as summarized in Table 1 [27]. Peak 10 ([M – H]– m/z at 517, [M – H – Glu] – at m/z 473 and [M – H – Glu-6′-O-malonyl] –) at m/z 311) displayed the same spectral
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Fig. 1 Chemical and activity-based fingerprints of P. cuspidatum from the on-line HPLC-DAD-FICL detection Table 1 Identification of bioactive compounds in P. cuspidatum Peak No.
tR (min)
1 2
9.7 11.7
[M − H]−/ [M + HCOOH − H]− 415 289
3
21.5
389
4
22.3
441
5
30.8
389
6
34.2
431
7
36.9
227
8
40.7
407
9
42.0
431
10
45.0
517
11
46.8
283
12
50.2
283
13
70.8
269
HPLC-ESI-MSn m/z – – MS2[389]: 227. MS3[227]: 209, 184, 157, 143, 107. – MS2[389]: 227. MS3[227]: 185, 183, 159, 143. MS2[431]: 269. MS3[269]: 241, 225. MS2[227]: 185, 159, 143. MS2[407]: 245. MS3[245]: 230. MS2[431]: 269. MS3[269]: 241, 225, 197. MS2[517]: 473. MS3[473]: 311, 269, 255. MS2[283]: 268, 240. MS3[240]: 197. MS2[283]: 268, 240, 151. MS2[269]: 225, 153. MS3[225]: 210, 157.
Compounds Unknown compound Catechin Resveratroloside Flavanol gallate Piceid Unknown compound Resveratrol Torachrysone-8-O-glucoside Emodin-8-O-glucoside Unknown compound Questin/Physcion Questin/Physcion Emodin
Fig. 2 Peak areas and scavenging rates (%) of thirteen peaks in the nineteen batches of samples. A Peak areas, B scavenging rates (%)
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data as peak 9 and was assigned tentatively as emodin 8-O-(6′-O-malonyl)-glucoside [26]. Peak 8 ([M – H]– m/z at 407, [M – H – Glu]– at m/z 245) was tentatively identified as torachrysone-8-O-glucoside [12, 24]. 3.3 Similarity evaluation of P. cuspidatum from different habitats The consistency of plant-based medicines can be tested through comparing the similarity between the chemical fingerprints of samples and the reference/standard fingerprints. The software “Similarity Evaluation System for Chromatographic Fingerprint of TCM” was used to evaluate these chromatograms. The chromatograms were introduced in the form of AIA (*.cdf), which includes the information of peak areas and retention times. The reference chromatogram was produced, and the similarity values of all of the introduced
chromatograms could be calculated by comparison with the reference chromatogram. The qualities of samples were relatively consistent when the similarity values were higher than 0.900. In terms of chemical fingerprints, the “common peaks” 3, 5, 6, 7, 9, 10, 11, and 13 were chosen as markers for peak matching. Then, the data of the fingerprints of the nineteen batches of P. cuspidatum samples were used to analyze the similarity among these samples by comparing each chromatogram with the reference chromatogram. The similarity results of the samples are listed in Table 2. The similarity values of the nineteen samples were more than 0.93, expect for S19, which indicated that the activity-based fingerprints of the samples from the different locations were stable, and were generally consistent.
Table 2 Similarity values of the nineteen batches of P. cuspidatum Method
Sample number S01 S02 S03 S04 S05 S06 S07 S08 S09 S10 S11 S12 S13 S14 S15 S16 S17 S18
S19
Bioactive finger0.855 0.887 0.883 0.989 0.855 0.918 0.977 0.974 0.978 0.956 0.953 0.961 0.927 0.864 0.989 0.943 0.977 0.975 0.858 print Chemical finger0.981 0.980 0.970 0.993 0.990 0.988 0.989 0.979 0.998 0.966 0.968 0.966 0.938 0.986 0.994 0.958 0.987 0.985 0.899 print
As to the bioactivity fingerprints, the vector angles of scavenging rates (%) of thirteen peaks and their average values were used to estimate the similarity of the nineteen batches of samples, and the results were listed in Table 2. It was apparent that the similarity values of the samples from thirteen habitats were higher than 0.900 except for S01, S02, S03, S05, S14, and S19. Compared with the results of the chemical fingerprints, the bioactivity fingerprints might decrease the differences among chromatographic peak areas of some “Common peaks” in various samples, such as peak 6, 9, 11 and 13. Furthermore, minor peaks showing good bioactivities might be ignored in chromatographic fingerprint analysis. According to the bioactivity information, the evaluation results of chemical fingerprints showed a limitation for the quality control of P. cuspidatum. Thus, the integrated evaluation method of chemical and activity-based fingerprints could become an efficient strategy for the quality control of P. cuspidatum. 3.4 Correlation analysis of chemical and activity-based fingerprints of P. cuspidatum Correlation analysis for chromatographic peak areas and radical scavenging rates (%) of the thirteen compounds in the nineteen batches of samples were performed by SPSS statistics software, and the results are shown in Fig. 3. The relationships of only ten compounds were revealed because compounds 9, 11, and 13 hardly exhibited any H2O2 scavenging activities. In addition, the correlation coefficients of compounds 5, 6, 10, and 12 were low because obvious differences between their peak areas and scavenging rates (%) were found. As shown in Fig. 2, the activities of compounds 5, 10, and 12 showing small peak areas were stronger, except for
compound 6, and their peak areas and activities in the nineteen batches of samples were significantly different. The results of the on-line screening of active components in the samples indicated that compounds 1, 2, 3, 4, 5, 7, 8, 10, and 12 were the main bioactive ingredients (Fig. 2), while the correlation
Fig. 3 Correlation value results of peak areas and scavenging rates (%) of ten peaks in the nineteen batches of P. cuspidatum samples
analysis results of the chemical and activity-based fingerprints revealed low correlation coefficients for the main bioactive compounds 5, 10, and 12. It was apparent that the correlation coefficient was not efficient enough for distinguishing between the main active ingredients in the samples, which showed the limitation of using the correlation coefficient for the quality evaluation of P. cuspidatum. In view of the comprehensive information from two perspectives, compounds 1, 2, 3, 4, 5, 7, 8, 10, and 12 were responsible for the antioxidant activity of P. cuspidatum. The integrated evaluation of the on-line screening method and the correlation analysis is therefore a powerful tool in the search for bioactive compounds in complex samples.
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The samples containing multiple, bioactive compounds might correlate with their fine quality. Thus, the total scavenging rates (%) of various compounds in the nineteen batches of samples were displayed in Fig. 4. It was apparent that most of the samples possessed similar total activities except for S02, S03, S08, S09, S13, S14, and S19, and that the total activity of S19 was the lowest. The samples S01, S04, S06, S07, S10, and S12 showed higher total activities which would imply their better quality. The evaluation method based on chemical and activity-based fingerprints could be an efficient strategy to reveal the comprehensive quality of Polygonum cuspidatum derived from different habitats.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9] [10]
Fig. 4 The total activities of the nineteen batches of P. cuspidatum samples
Conclusion. In this study, the Spectrum-Effect integrated fingerprint of P. cuspidatum was established based on the on-line HPLC-DAD-FICL method. Furthermore, the comprehensive quality evaluation of nineteen batches of Polygonum cuspidatum samples from various habitats were investigated by the combination of chemical and activity-based fingerprints derived from the Spectrum-Effect integrated fingerprint of Polygonum cuspidatum. Similarity evaluation results of the chemical fingerprints indicated that the quality of the samples from different locations were stable and consistent, while the results of the bioactivity fingerprints were different. The bioactivity fingerprints revealed the differences between the various samples. Moreover, correlation analysis was used to estimate the main bioactive compounds in P. cuspidatum samples by the relationships of peak areas and inhibition ratios of thirteen peaks. Some main active ingredients could not be sufficiently distinguished, while on-line HPLC-DAD-FICL could rapidly screen main antioxidants in P. cuspidatum. The “Integrated Spectrum-Effect Fingerprint”, an integrated analysis of chemical and bioactivity fingerprints method, offers a rational approach for the quality evaluation of plant-based medicines.
[11]
[12]
[13]
[14]
[15]
[16]
[17]
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基于 HPLC-DAD-FICL 法建立虎杖的谱-效整合指纹图谱 丁晓萍 1，张翠玲 1, 3，戚
进 1, 2，孙丽琼 1，秦民坚 4，余伯阳 1, 2*
1
中国药科大学中药复方研究室，南京 211198;
2
天然药物活性物质与功能国家重点实验室，南京 210009;
3
蚌埠医学院中药研究室，蚌埠 233030;
4
中国药科大学中药资源研究室，南京 211198
【摘 要】 目的：建立虎杖药材的谱-效整合指纹图谱，为科学评价其质量提供可靠方法。方法：采用高效液相色谱-二极 管阵列-流动注射化学发光法得到不同来源虎杖药材的谱-效整合指纹图谱，从而综合评价虎杖药材的质量。结果：对 19 批药材 的化学指纹图谱和清除自由基的活性指纹图谱分别进行了相似度评价，并对其结果进行了对比分析。对 13 个主要抗氧化成分的 色谱峰面积和活性进行了回归分析，并利用 HPLC-ESI-MS 鉴定了部分成分的化学结构。结论：基于高效液相色谱-二极管阵列流动注射化学发光法所建立的虎杖的谱-效整合指纹图谱能快速筛选虎杖药材中的主要抗氧化活性成分，这种综合评价方法能更 客观、有效地反映虎杖药材的质量。 【关键词】 谱-效整合指纹图谱；高效液相色谱-二极管阵列-流动注射化学发光法；虎杖；高效液相-电喷雾-质谱 【基金项目】 国家自然基金资助项目（No. 30973965）；第 46，47 批中国博士后基金（Nos. 20090461139，20100471480）；2010 年江苏省博士后基金（No. 1001077C） ；教育部新教师基金（No. 20090096120008）；2011 年度江苏省高等学校优秀科技创新团 队；江苏高校优势学科建设工程资助项目
