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ISSN 1615-9306 · JSSCCJ 38 (11) 1813–2006 (2015) · Vol. 38 · No. 11 · June 2015 · D 10609
JOURNAL OF
SEPARATION SCIENCE
Methods Chromatography · Electroseparation Applications Biomedicine · Foods · Environment
11 15
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J. Sep. Sci. 2015, 38, 1822–1827
Weifeng Yao1,2 Jin Dai2 Chuanzhu Zheng1 Beihua Bao1,4 Haibo Cheng3 Li Zhang1,4 Anwei Ding1,4 Wei Li1
Research Article
1 School
A comprehensive strategy was designed for the quality assessment of Fructus Ligustri Lucidi, a well-known and commonly used herbal medicine in clinical practice in China. First, a simple and stable method of high-performance liquid chromatography was developed for the simultaneous quantitative analysis of six compounds, namely, salidroside, nuzhenide, specnuezhenide, oleanic acid, ursolic acid, and acetyl oleanic acid in Fructus Ligustri Lucidi. The separation of analytes was conducted on a C18 column (200 mm × 4.6 mm, 5 m) at 30⬚C, and the wavelength of UV detector was set at 210 nm. In quantitative analysis, all of the calibration curves showed good linear regression (R2 > 0.9994) within the tested ranges, and the mean recoveries of three different concentrations ranged from 95.21–102.34%. The described method was applied to determine 11 batches of samples collected from different stores in China. Then multiple chemometrics analysis including hierarchical cluster analysis and principal component analysis were performed to classify samples and search significant compounds. Three notable compounds, specnuezhenide, oleanic acid, and acetyl oleanic acid, were discovered for better quality control compared with those stated in the China pharmacopeia. The results demonstrated that this strategy could be readily utilized for the comprehensive quality control of Fructus Ligustri Lucidi.
of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China 2 Northwest Metabolomics Research Center, Department of Anesthesiology and Pain Medicine, University of Washington, Seattle, WA, USA 3 Jiangsu Engineering Laboratory for Research and Industrialization of Empirical Formulae, Nanjing University of Chinese Medicine, Nanjing, China 4 Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, and National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Nanjing University of Chinese Medicine, Nanjing, China Received January 23, 2015 Revised March 2, 2015 Accepted March 4, 2015
Quality assessment of Fructus Ligustri Lucidi by the simultaneous determination of six compounds and chemometric analysis
Keywords: Chemometrics / Fructus Ligustri Lucidi / High-performance liquid chromatography / Quality assessment / Traditional Chinese medicine DOI 10.1002/jssc.201500094
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Fructus Ligustri Lucidi (FLL), the fruit of Ligustrum lucidum Ait, is well known and commonly used as herbal medicine in clinical practice of China. In the theory of traditional Chinese medicine, it has been known to ‘‘support the healthy energy, nourish the liver and kidneys’’ and used to refresh the liver and kidneys, improve eyesight and promote the growth of black hair [1]. It has also been reported to possess immunomodulatory, anti-inflammatory, hepatoprotective, anti-tumor and anti-aging activities [2–5]. Recently, the effect to prevent the osteoporosis has also been studied [6]. Several types of chemical constituents considered as the characteristic and active constituents from FLL were isolated including 40 triterpenoids, 48 iridoids, ten flavones, ten phenylethanoid Correspondence: College of Pharmacy, Nanjing University of Chinese Medicine, 003(Mail-box), 138 Xianlin Road, Nanjing, 210023, Jiangsu, China E-mail: [email protected]; [email protected] Fax: +86-25-85811524 E-mail: [email protected]
Abbreviations: FLL, Fructus Ligustri Lucidi; HCA, hierarchical cluster analysis; PCA, principal component analysis C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
glycosides, and others [7]. So, separation and analysis of the desired chemical components in herbal medicine are very important subjects for modernization research of traditional Chinese medicine [8]. Some compounds have been chosen as index for QC of FLL. Oleanolic acid and ursolic acid were separated and determined in FLL by MEKC [9] and HPLC [10, 11]. Salidroside and specnuezhenide were also quantitatively analyzed in FLL for QC by HPLC [12]. However, to our knowledge, the method for simultaneous separation and determination of six characteristic and active components in FLL by HPLC has not been found. In this work, three triterpenoids, oleanic acid, ursolic acid and acetyl oleanic acid, and three iridoids, salidroside, nuzhenide and specnuezhenide were determined by a simple and stable HPLC method in FLL samples from different drug stores in China. Both oleanolic acid and ursolic acid are effective in protecting against chemically induced liver injury in laboratory animals [13]. Oleanolic acid has been marketed in China as an oral drug for human liver disorders. And acetyl oleanic acid is the derivative of oleanic acid in FLL. Salidroside, nuzhenide, and specnuezhenide were recommended as antiosteoporotic compounds in FLL [14]. The alteration of the compound content were further explored using chemometric methods including hierarchical cluster analysis www.jss-journal.com
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(HCA) and principal component analysis (PCA). The main purpose of this study is to develop a comprehensive QC method for herbal medicine based on instrumental determination and chemometric analysis.
2 Materials and methods 2.1 Materials and chemicals The samples of FLL were purchased from 11 drug stores in China and identified by Professor Qinan Wu of Traditional Chinese Medicine Identification at Nanjing University of Chinese Medicine. The specimens (S1–S11) were deposited at the authors’ laboratory. The reference standards of oleanic acid, ursolic acid, salidroside, nuzhenide, and specnuezhenide were purchased from National Institute for Food and Drug Control of China. Acetyl oleanic acid was separated and purified in our lab (98.86%). Acetonitrile and formic acid were obtained from Merck (Darmstadt, German). Deionized water was from a Milli-Q system (Millipore, Bedford, MA, USA).
2.2 Apparatus Quantitative HPLC was performed on a Waters 2695 with 2996 photo diode array detector HPLC system (Waters, USA), and a PC with Empower work station for data processing. Ultrasound-assisted extraction (UAE) was performed using an ultrasonic cleaning bath (KQ-250V; Kun-Shan Ultrasonic Instruments, Kun-Shan, China).
2.3 Preparation of stock solution The standard stock solutions of salidroside (0.34 mg/mL), nuzhenide (0.31 mg/mL), specnuezhenide (0.28 mg/mL), oleanic acid (0.18 mg/mL), ursolic acid (0.5 mg/mL), and acetyl oleanic acid (2.01 mg/mL) were prepared in methanol and stored away from light at 4⬚C. Working solutions were prepared by appropriate dilution of the stock solution.
2.4 Preparation of sample Sample preparation was slightly modified from Guo’s method [15]. The air-dried sample of Fructus Ligustrum lucidum was ground to powder and passed through a 60 mesh sieve. The powder (1.0 g) was mixed with 25 ml of 80% methanol in a 50 mL conical flask with cover. The mixture was extracted ultrasonically for 60 min. Ultrasound equipment operated at a frequency of 40 kHz, power of 100 W and temperature of 25⬚C. After cooling, the solution was then made up to the required volume. Then the mixture was centrifuged for 15 min at 4000 rpm for deposit suspension particle. Before use, all samples were filtered through a 0.45 m membrane filter. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Chromatograms of reference substances (A) and sample (B). 1: Salidroside; 2: Nuzhenide; 3: Specnuezhenide; 4: Oleanic acid; 5: Ursolic acid; 6: Acetyl oleanic acid.
2.5 Chromatographic conditions and system suitability tests For the quantitation of salidroside, nuzhenide, specnuezhenide, oleanic acid, ursolic acid and acetyl oleanic acid, HPLC was conducted using a Thermo C18 column (200 mm × 4.6 mm i.d., 5 m particle size); the mobile phase consisted of acetonitrile (solution A) and 0.1% Formic acid in water (solution B). The flow rate, column temperature and injection volume were set to 1 mL/min, 30⬚C and 10 L, respectively. The gradient program was set as follows: 0–5min with 5– 11% A, 5–13 min with 11–15% A, 13–14 min with 15–22% A, 14–25 min with 22–25% A, 25–35 min with 25–95% A, and 35–55 min with 95% A. The wavelength of UV detector was fixed at 210 nm for determination. Under the above chromatographic conditions, the chromatographic peak symmetry factors of salidroside, nuzhenide, specnuezhenide, oleanic acid, ursolic acid and acetyl oleanic acid were between 0.95 and 1.05. The resolution to the adjacent peaks was greater than 1.5, and the numbers of theoretical plates were more than 3000. The chromatograms of mixed reference standards and a real sample are shown in Fig. 1.
2.6 Chemometric analysis PCA and HCA were performed on the SIMCA-P 11.0 (Umetrics AB) and Matlab 7.0 (MathWork) software, www.jss-journal.com
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Table 1. Linear regression data and validation for six compounds (n = 3)
Compounds
Regression equation
R2
Linear range (g/mL)
LOD (g/mL)
LOQ (g/mL)
Salidroside Nuzhenide Specnuezhenide Oleanic acid Ursolic acid Acetyl oleanic acid
Y = 1354315X – 51864 Y = 2055274X – 64637 Y = 862015186X + 111057 Y = 568 196X – 105 661 Y = 403 968X – 71 520 Y = 124 904X – 15 260
0.999 4 0.999 8 0.999 9 0.999 6 0.999 4 0.999 7
3.42–136.80 3.12–124.80 28.40–1136.00 18.02–720.80 5.04–201.60 20.08–803.20
0.43 0.36 1.54 1.05 0.46 1.42
1.24 1.02 5.58 3.48 1.38 4.45
Y: peak area; X: concentration (mg/mL).
respectively, to demonstrate the variability of the six compounds among 11 batches of FLL samples. The dataset was organized in a matrix with 11 rows corresponding to the samples and six columns corresponding to the peak areas of the common peaks.
Table 1. The S/N is defined as the power ratio of a signal (meaningful information) to the background noise (unwanted signal). The LOD is considered to be the concentration when the S/N is equal to 3:1. The LOQ was the injection concentration corresponding to S/N ratio of 10.
3 Results and discussion
3.3 Precision
3.1 The selection of UV wavelength for determination
The repeatability test was conducted by repeatedly measuring a standard solution. The intra-day precision was determined by performing six aliquots at each sample level during a single day, and the inter-day precision was determined by performing six aliquots at each sample level on different days. The RSD, also termed coefficient of variation (CV), was used as a statistical parameter. The intra- and inter-day precision RSD were less than 2.42%, indicating that the proposed method was highly precise (Table S1).
To better select the UV wavelength in HPLC, the UV spectra of salidroside, nuzhenide, specnuezhenide, oleanic acid, ursolic acid, and acetyl oleanic acid were extracted from photodiode array detection (Fig. S1). HPLC–UV has been already employed for the separation and determination of some of the six compounds in FLL. Oleanolic acid and ursolic were detected at the wavelength of 210 nm by HPLC [10]. Since the spectrum of acetyl oleanic acid is similar to those of oleanic acid and ursolic acid, the detection wavelength could be set at 210 nm (Fig. S1D–F). Shi determined salidroside in FLL by an accurate HPLC method with the UV detection wavelength at 230 nm [12]. The spectrum of salidroside has a peak at 221.5 nm, so the selection of 210 nm at the symmetric position is also suitable for its quantitation. An HPLC method was also established for simultaneous determination of nuzhenide, specnuezhenide, and oleanic acid in the prescription of FLL with the detection wavelength at 215 nm [16]. Therefore, the detection wavelength of UV detector was set at 210 nm for the determination of all six compounds.
3.2 Linearity, range, and LOD To prepare the standard solutions, the standard stock solutions were diluted with methanol in a 10 mL volumetric flask, respectively. Then, the standard solutions at six different concentrations were analyzed in triplicate per level. Table 1 shows the linear calibration regression equations and correlation coefficients of salidroside, nuzhenide, specnuezhenide, oleanic acid, ursolic acid, and acetyl oleanic acid. The LOD and LOQ of each compound using the abovementioned chromatographic conditions are also shown in C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.4 Recovery To verify the accuracy of the method, different amounts of known standards were added to the sample solution. Accurate amounts of each standard solution at three different concentrations (low, medium, and high) were added to 1.0 g of S10. The samples were prepared in triplicate at each level and the accuracy was expressed as recovery percentage. Triplicate experiments were performed at each level. The mixture was extracted and analyzed as described in Sections 2.4 and 2.5. The extraction recovery of each analyte was calculated using the following equation: Recovery (%) = (found amount–original amount)/spiked amount × 100 (1) Good percentage recoveries were obtained, and the results are shown in Table S2. The mean recoveries of three different concentrations ranged from 95.21 to 102.34%.
3.5. Assay of 11 samples from different drug stores and chemometric analysis The HPLC assay and chemometric analysis were subsequently applied to the comprehensive quality evaluation of www.jss-journal.com
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Table 2. The content of six compounds from different sources (mg/g) (n = 3)
Batch No.
Salidroside
Nuzhenide
Specnuezhenide
Oleanic acid
Ursolic acid
Acetyl oleanic acid
S1(Bozhou, Anhui) S2(Siping, Jilin) S3(Chengdu, Sichuan) S4(Haerbin, Heilongjiang) S5(Ningbo, Zhejiang) S6(Hangzhou, Zhejiang) S7(Wuhan, Hubei) S8(Zhengzhou, Henan) S9(Nanning, Guangxi) S10(Jiaxing, Zhejiang) S11(Nanjing, Jiangsu)
2.936 1.850 2.006 0.385 0.469 4.128 3.531 2.807 0.530 1.673 1.781
1.490 1.337 1.566 0.908 0.719 1.281 0.819 3.002 1.550 1.632 1.653
16.939 8.217 9.737 2.740 1.469 16.165 9.458 19.319 5.281 16.806 16.256
10.268 7.367 9.766 12.482 9.552 8.867 9.429 13.262 9.105 9.330 12.535
1.400 1.053 1.457 1.786 0.958 1.042 1.132 3.158 0.913 1.333 2.366
6.576 8.483 7.779 10.230 10.721 6.205 6.532 8.073 6.881 6.021 8.200
Note: The content of compound represents the weight (mg) in 1 g of FLL power.
FLL samples. The quantitative determination results are summarized in Table 2 and demonstrate that there are distinct differences in concentration of analytes from different store. Therefore, further HCA and PCA were used to demonstrate the difference of FLL samples from 11 drug stores based on the HPLC quantitation results (Fig. 2). HCA, one of the most commonly used unsupervised pattern recognition methods, was a useful multivariate statistic technique to assign samples into groups by creating a cluster tree or dendrogram, according to similarity [17]. To assess the resemblance and differences of these samples, HCA of FLL samples was performed based on the content of all the six characteristic compounds by Matlab 7.0. The Ward method was applied as the amalgamation rule and the Euclidean distance was selected to measure the resemblance and classify the 11 samples. The results obtained following HCA are shown in a dendrogram of Fig. 2A in which three welldefined clusters were visible. S4 and S5 were categorized into cluster I; S2, S3, S7, and S9 were categorized into cluster II, while the rest were put into cluster III. As a multivariate analysis technique, PCA could visualize similarities or differences within multivariate data [18]. The content of six compounds were set as variables, while 11 batches of samples were set as observations. The variables were centered and scaled to “Unit Variance” before PCA in SIMCA-P. The first principal component (t1) explained 92.48% of the total variance in the data set while the second principal component (t2) explained 6.96% (Fig. 2B). The first two principal components reduced the multidimensional dataset to a 2D data set which explained more than 99% of the total variance. Thus, it was notable that all samples were classified into three groups, including group I (S4 and S5), group II (S2, S3, S7, and S9), and group III (S1, S6, S8, S10, and S11). This was quite consistent with the results of HCA. In PCA, the loading coefficients represent the importance of each individual feature in a reduced dimension [19]. In general, 2D loading plots (e.g., p1/p2 loading plot) provide useful information to identify important features in the first and second PC dimensions. Here, the results from Fig. 2C show that the compounds, C3 (specnuezhenide), C4 (oleanic acid), and C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
C6 (acetyl oleanic acid), have the largest loading coefficients, indicating the degree of importance of the original feature in the p1/p2 dimension. Combined with the information from Table 2, the content of specnuezhenide in FLL samples of group I were much lower than those in other groups, and the content of acetyl oleanic acid were higher. Specnuezhenide in FLL of group III had relatively higher content. Specnuezhenide is a major active compound in FLL, and it is the only compound which should be quantified according to Edition 2010 of China Pharmacopeia [1]. In former edition of China Pharmacopeia, oleanic acid was used as the quantitative index of FLL [20]. However, the assay of oleanic acid, a common compound in several herbal medicines, is lack of specificity for QC of FLL, especially for the QC of FLL in Chinese medicinal formulae. So a particular compound in FLL, specnuezhenide, was selected to replace oleanic acid in Edition 2010 of China Pharmacopeia. In this work, we found that, besides specnuezhenide, oleanic acid, and acetyl oleanic acid could also influence the classification in FLL samples and should be taken into account for QC of FLL. Although the definitive action of acetyl oleanic acid in FLL has not been reported as we know, some studies have demonstrated its potential abilities of exhibiting anti-angiogenic effects in human umbilical vein endothelial cells [21] and inducing apoptosis in human colon carcinoma HCT-116 cells [22].
4 Conclusion In this study, a comprehensive strategy using HPLC–UV coupled with chemometrics was proposed for QC of FLL samples. The developed quantitative method is promising to be the routing analysis for six compounds (salidroside, nuzhenide, specnuezhenide, oleanic acid, ursolic acid, and acetyl oleanic acid) in FFL samples. Furthermore, HCA and PCA were applied to the classification and evaluation of FLL samples and the significant variables were selected based on PCA loading efficiencies. This strategy is highly recommendable for quality study of other herbal medicine. www.jss-journal.com
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Figure 2. A: The cluster analysis of 11 samples (the distance was calculated by Euclidean distance); B: The score plot of PCA (t1/t2); C: The loading plot of PCA (p1/p2). C1: Salidroside; C2: Nuzhenide; C3: Specnuezhenide; C4: Oleanic acid; C5: Ursolic acid; C6: Acetyl oleanic acid.
The authors are grateful for the financial support by National Natural Science Foundation of China (Grant No. 81001599, 81173547 and 81373972) and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
5 References [1] National Pharmacopoeia Committee, Pharmacopoeia of the People’s Republic of China Part 1, Chinese Medical Science and Technology Press, Beijing 2010, 43. [2] Yim, T. K., Wu, W. K., Pak, W. F., Phytother. Res. 2001, 15, 589–592.
The authors have declared no conflict of interest. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[3] Yim, T. K., Ko, K. M., Pharmaceut. Biol. 2001, 39, 146–151.
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[5] Tsai, S. J., Yin, M. C., J. Food. Sci. 2008, 73, H174–H178.
[15] Guo, N., Yu, Y., Ablajan, K., Li, L., Fan, B., Peng, J., Yan, H., Ma, F., Nie, Y., Rapid Commun. Mass Spectrom. 2011, 25, 1701–1714.
[6] Dong, X. L., Zhao, M., Wong, K. K., Br. J. Nutr. 2012, 108, 92–101.
[16] He, M., Yao, W., Zhang, L., Ding, A., Zhongguo Zhong Yao Za Zhi 2012, 37, 594–596.
[7] Gao, L., Li, C., Wang, Z., Liu, X., You, Y., Wei, H., Guo, T., Nat. Prod. Res. 2014, 23, 1–18.
[17] Liao, H. J., Lai, Z. Q., Su, J. Y., Yi, Y. Y., Li, Y. C., Lai, X. P., Su, Z. R., Lin, Z. X., J. Sep. Sci. 2012, 35, 2193–2202.
[8] Liu, S., Yi, L. Z, Liang, Y. Z., J. Sep. Sci. 2008, 31, 2113– 2137.
[18] Yao, W., Yin, X., Hu, Y., J. Chromatogr. A 2007, 1160, 254–262.
[9] Liu, H., Shi, Y., Wang, D., Yang, G., Yu, A., Zhang, H., J. Pharm. Biomed. Anal. 2003, 32, 479–485.
[19] Cho, H. W., Kim, S. B., Jeong, M. K., Park, Y., Miller, N. G., Ziegler, T. R., Jones, D. P., Int. J. Data Min. Bioinform. 2008, 2, 176–192.
[4] Gao, D. W., Li, Q. W., Li, Y., Can. J. Physiol. Pharmacol. 2007, 85, 1076–1083.
[10] Xia, E. Q., Yu, Y. Y., Xu, X. R., Deng, G. F., Guo, Y. J., Li, H. B., Ultrason. Sonochem. 2012, 19, 772–776. [11] Wang, C., Liu, H., Zhang, B., Guo, H., J Sep Sci. 2011, 34, 3023–3028. [12] Shi, L., Ma, Y., Cai, Z., Biomed. Chromatogr. 1998, 12, 27–30. [13] Liu, J., J. Ethnopharmacol. 1995, 49, 57–68. [14] Chen, Q., Yang, L., Zhang, G., Wang, F., Phytother. Res. 2013, 27, 973–979.
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[20] National Pharmacopoeia Committee, Pharmacopoeia of the People’s Republic of China Part 1, Chemical Industry Press, Beijing 2005, pp. 31. [21] Cui, E. J., Hwang-Bo, J., Park, J. H., Baek, N. I., Kim, J., Hong, S. G., Chung, I. S., Biotechnol. Lett. 2013, 35, 1807–1815. [22] Yoo, K. H., Park, J. H., Cui, E. J., Kim, K. I., Kim, J. Y., Kim, J., Hong, S. G., Baek, N. I., Chung, I. S., Phytother. Res. 2012, 26, 1541–1546.
www.jss-journal.com
ISSN 1615-9306 · JSSCCJ 38 (11) 1813–2006 (2015) · Vol. 38 · No. 11 · June 2015 · D 10609
JOURNAL OF
SEPARATION SCIENCE
Methods Chromatography · Electroseparation Applications Biomedicine · Foods · Environment
11 15
www.jss-journal.com
1822
J. Sep. Sci. 2015, 38, 1822–1827
Weifeng Yao1,2 Jin Dai2 Chuanzhu Zheng1 Beihua Bao1,4 Haibo Cheng3 Li Zhang1,4 Anwei Ding1,4 Wei Li1
Research Article
1 School
A comprehensive strategy was designed for the quality assessment of Fructus Ligustri Lucidi, a well-known and commonly used herbal medicine in clinical practice in China. First, a simple and stable method of high-performance liquid chromatography was developed for the simultaneous quantitative analysis of six compounds, namely, salidroside, nuzhenide, specnuezhenide, oleanic acid, ursolic acid, and acetyl oleanic acid in Fructus Ligustri Lucidi. The separation of analytes was conducted on a C18 column (200 mm × 4.6 mm, 5 m) at 30⬚C, and the wavelength of UV detector was set at 210 nm. In quantitative analysis, all of the calibration curves showed good linear regression (R2 > 0.9994) within the tested ranges, and the mean recoveries of three different concentrations ranged from 95.21–102.34%. The described method was applied to determine 11 batches of samples collected from different stores in China. Then multiple chemometrics analysis including hierarchical cluster analysis and principal component analysis were performed to classify samples and search significant compounds. Three notable compounds, specnuezhenide, oleanic acid, and acetyl oleanic acid, were discovered for better quality control compared with those stated in the China pharmacopeia. The results demonstrated that this strategy could be readily utilized for the comprehensive quality control of Fructus Ligustri Lucidi.
of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China 2 Northwest Metabolomics Research Center, Department of Anesthesiology and Pain Medicine, University of Washington, Seattle, WA, USA 3 Jiangsu Engineering Laboratory for Research and Industrialization of Empirical Formulae, Nanjing University of Chinese Medicine, Nanjing, China 4 Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, and National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Nanjing University of Chinese Medicine, Nanjing, China Received January 23, 2015 Revised March 2, 2015 Accepted March 4, 2015
Quality assessment of Fructus Ligustri Lucidi by the simultaneous determination of six compounds and chemometric analysis
Keywords: Chemometrics / Fructus Ligustri Lucidi / High-performance liquid chromatography / Quality assessment / Traditional Chinese medicine DOI 10.1002/jssc.201500094
Additional supporting information may be found in the online version of this article at the publisher’s web-site
1 Introduction Fructus Ligustri Lucidi (FLL), the fruit of Ligustrum lucidum Ait, is well known and commonly used as herbal medicine in clinical practice of China. In the theory of traditional Chinese medicine, it has been known to ‘‘support the healthy energy, nourish the liver and kidneys’’ and used to refresh the liver and kidneys, improve eyesight and promote the growth of black hair [1]. It has also been reported to possess immunomodulatory, anti-inflammatory, hepatoprotective, anti-tumor and anti-aging activities [2–5]. Recently, the effect to prevent the osteoporosis has also been studied [6]. Several types of chemical constituents considered as the characteristic and active constituents from FLL were isolated including 40 triterpenoids, 48 iridoids, ten flavones, ten phenylethanoid Correspondence: College of Pharmacy, Nanjing University of Chinese Medicine, 003(Mail-box), 138 Xianlin Road, Nanjing, 210023, Jiangsu, China E-mail: [email protected]; [email protected] Fax: +86-25-85811524 E-mail: [email protected]
Abbreviations: FLL, Fructus Ligustri Lucidi; HCA, hierarchical cluster analysis; PCA, principal component analysis C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
glycosides, and others [7]. So, separation and analysis of the desired chemical components in herbal medicine are very important subjects for modernization research of traditional Chinese medicine [8]. Some compounds have been chosen as index for QC of FLL. Oleanolic acid and ursolic acid were separated and determined in FLL by MEKC [9] and HPLC [10, 11]. Salidroside and specnuezhenide were also quantitatively analyzed in FLL for QC by HPLC [12]. However, to our knowledge, the method for simultaneous separation and determination of six characteristic and active components in FLL by HPLC has not been found. In this work, three triterpenoids, oleanic acid, ursolic acid and acetyl oleanic acid, and three iridoids, salidroside, nuzhenide and specnuezhenide were determined by a simple and stable HPLC method in FLL samples from different drug stores in China. Both oleanolic acid and ursolic acid are effective in protecting against chemically induced liver injury in laboratory animals [13]. Oleanolic acid has been marketed in China as an oral drug for human liver disorders. And acetyl oleanic acid is the derivative of oleanic acid in FLL. Salidroside, nuzhenide, and specnuezhenide were recommended as antiosteoporotic compounds in FLL [14]. The alteration of the compound content were further explored using chemometric methods including hierarchical cluster analysis www.jss-journal.com
J. Sep. Sci. 2015, 38, 1822–1827
Liquid Chromatography
1823
(HCA) and principal component analysis (PCA). The main purpose of this study is to develop a comprehensive QC method for herbal medicine based on instrumental determination and chemometric analysis.
2 Materials and methods 2.1 Materials and chemicals The samples of FLL were purchased from 11 drug stores in China and identified by Professor Qinan Wu of Traditional Chinese Medicine Identification at Nanjing University of Chinese Medicine. The specimens (S1–S11) were deposited at the authors’ laboratory. The reference standards of oleanic acid, ursolic acid, salidroside, nuzhenide, and specnuezhenide were purchased from National Institute for Food and Drug Control of China. Acetyl oleanic acid was separated and purified in our lab (98.86%). Acetonitrile and formic acid were obtained from Merck (Darmstadt, German). Deionized water was from a Milli-Q system (Millipore, Bedford, MA, USA).
2.2 Apparatus Quantitative HPLC was performed on a Waters 2695 with 2996 photo diode array detector HPLC system (Waters, USA), and a PC with Empower work station for data processing. Ultrasound-assisted extraction (UAE) was performed using an ultrasonic cleaning bath (KQ-250V; Kun-Shan Ultrasonic Instruments, Kun-Shan, China).
2.3 Preparation of stock solution The standard stock solutions of salidroside (0.34 mg/mL), nuzhenide (0.31 mg/mL), specnuezhenide (0.28 mg/mL), oleanic acid (0.18 mg/mL), ursolic acid (0.5 mg/mL), and acetyl oleanic acid (2.01 mg/mL) were prepared in methanol and stored away from light at 4⬚C. Working solutions were prepared by appropriate dilution of the stock solution.
2.4 Preparation of sample Sample preparation was slightly modified from Guo’s method [15]. The air-dried sample of Fructus Ligustrum lucidum was ground to powder and passed through a 60 mesh sieve. The powder (1.0 g) was mixed with 25 ml of 80% methanol in a 50 mL conical flask with cover. The mixture was extracted ultrasonically for 60 min. Ultrasound equipment operated at a frequency of 40 kHz, power of 100 W and temperature of 25⬚C. After cooling, the solution was then made up to the required volume. Then the mixture was centrifuged for 15 min at 4000 rpm for deposit suspension particle. Before use, all samples were filtered through a 0.45 m membrane filter. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Chromatograms of reference substances (A) and sample (B). 1: Salidroside; 2: Nuzhenide; 3: Specnuezhenide; 4: Oleanic acid; 5: Ursolic acid; 6: Acetyl oleanic acid.
2.5 Chromatographic conditions and system suitability tests For the quantitation of salidroside, nuzhenide, specnuezhenide, oleanic acid, ursolic acid and acetyl oleanic acid, HPLC was conducted using a Thermo C18 column (200 mm × 4.6 mm i.d., 5 m particle size); the mobile phase consisted of acetonitrile (solution A) and 0.1% Formic acid in water (solution B). The flow rate, column temperature and injection volume were set to 1 mL/min, 30⬚C and 10 L, respectively. The gradient program was set as follows: 0–5min with 5– 11% A, 5–13 min with 11–15% A, 13–14 min with 15–22% A, 14–25 min with 22–25% A, 25–35 min with 25–95% A, and 35–55 min with 95% A. The wavelength of UV detector was fixed at 210 nm for determination. Under the above chromatographic conditions, the chromatographic peak symmetry factors of salidroside, nuzhenide, specnuezhenide, oleanic acid, ursolic acid and acetyl oleanic acid were between 0.95 and 1.05. The resolution to the adjacent peaks was greater than 1.5, and the numbers of theoretical plates were more than 3000. The chromatograms of mixed reference standards and a real sample are shown in Fig. 1.
2.6 Chemometric analysis PCA and HCA were performed on the SIMCA-P 11.0 (Umetrics AB) and Matlab 7.0 (MathWork) software, www.jss-journal.com
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Table 1. Linear regression data and validation for six compounds (n = 3)
Compounds
Regression equation
R2
Linear range (g/mL)
LOD (g/mL)
LOQ (g/mL)
Salidroside Nuzhenide Specnuezhenide Oleanic acid Ursolic acid Acetyl oleanic acid
Y = 1354315X – 51864 Y = 2055274X – 64637 Y = 862015186X + 111057 Y = 568 196X – 105 661 Y = 403 968X – 71 520 Y = 124 904X – 15 260
0.999 4 0.999 8 0.999 9 0.999 6 0.999 4 0.999 7
3.42–136.80 3.12–124.80 28.40–1136.00 18.02–720.80 5.04–201.60 20.08–803.20
0.43 0.36 1.54 1.05 0.46 1.42
1.24 1.02 5.58 3.48 1.38 4.45
Y: peak area; X: concentration (mg/mL).
respectively, to demonstrate the variability of the six compounds among 11 batches of FLL samples. The dataset was organized in a matrix with 11 rows corresponding to the samples and six columns corresponding to the peak areas of the common peaks.
Table 1. The S/N is defined as the power ratio of a signal (meaningful information) to the background noise (unwanted signal). The LOD is considered to be the concentration when the S/N is equal to 3:1. The LOQ was the injection concentration corresponding to S/N ratio of 10.
3 Results and discussion
3.3 Precision
3.1 The selection of UV wavelength for determination
The repeatability test was conducted by repeatedly measuring a standard solution. The intra-day precision was determined by performing six aliquots at each sample level during a single day, and the inter-day precision was determined by performing six aliquots at each sample level on different days. The RSD, also termed coefficient of variation (CV), was used as a statistical parameter. The intra- and inter-day precision RSD were less than 2.42%, indicating that the proposed method was highly precise (Table S1).
To better select the UV wavelength in HPLC, the UV spectra of salidroside, nuzhenide, specnuezhenide, oleanic acid, ursolic acid, and acetyl oleanic acid were extracted from photodiode array detection (Fig. S1). HPLC–UV has been already employed for the separation and determination of some of the six compounds in FLL. Oleanolic acid and ursolic were detected at the wavelength of 210 nm by HPLC [10]. Since the spectrum of acetyl oleanic acid is similar to those of oleanic acid and ursolic acid, the detection wavelength could be set at 210 nm (Fig. S1D–F). Shi determined salidroside in FLL by an accurate HPLC method with the UV detection wavelength at 230 nm [12]. The spectrum of salidroside has a peak at 221.5 nm, so the selection of 210 nm at the symmetric position is also suitable for its quantitation. An HPLC method was also established for simultaneous determination of nuzhenide, specnuezhenide, and oleanic acid in the prescription of FLL with the detection wavelength at 215 nm [16]. Therefore, the detection wavelength of UV detector was set at 210 nm for the determination of all six compounds.
3.2 Linearity, range, and LOD To prepare the standard solutions, the standard stock solutions were diluted with methanol in a 10 mL volumetric flask, respectively. Then, the standard solutions at six different concentrations were analyzed in triplicate per level. Table 1 shows the linear calibration regression equations and correlation coefficients of salidroside, nuzhenide, specnuezhenide, oleanic acid, ursolic acid, and acetyl oleanic acid. The LOD and LOQ of each compound using the abovementioned chromatographic conditions are also shown in C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.4 Recovery To verify the accuracy of the method, different amounts of known standards were added to the sample solution. Accurate amounts of each standard solution at three different concentrations (low, medium, and high) were added to 1.0 g of S10. The samples were prepared in triplicate at each level and the accuracy was expressed as recovery percentage. Triplicate experiments were performed at each level. The mixture was extracted and analyzed as described in Sections 2.4 and 2.5. The extraction recovery of each analyte was calculated using the following equation: Recovery (%) = (found amount–original amount)/spiked amount × 100 (1) Good percentage recoveries were obtained, and the results are shown in Table S2. The mean recoveries of three different concentrations ranged from 95.21 to 102.34%.
3.5. Assay of 11 samples from different drug stores and chemometric analysis The HPLC assay and chemometric analysis were subsequently applied to the comprehensive quality evaluation of www.jss-journal.com
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Table 2. The content of six compounds from different sources (mg/g) (n = 3)
Batch No.
Salidroside
Nuzhenide
Specnuezhenide
Oleanic acid
Ursolic acid
Acetyl oleanic acid
S1(Bozhou, Anhui) S2(Siping, Jilin) S3(Chengdu, Sichuan) S4(Haerbin, Heilongjiang) S5(Ningbo, Zhejiang) S6(Hangzhou, Zhejiang) S7(Wuhan, Hubei) S8(Zhengzhou, Henan) S9(Nanning, Guangxi) S10(Jiaxing, Zhejiang) S11(Nanjing, Jiangsu)
2.936 1.850 2.006 0.385 0.469 4.128 3.531 2.807 0.530 1.673 1.781
1.490 1.337 1.566 0.908 0.719 1.281 0.819 3.002 1.550 1.632 1.653
16.939 8.217 9.737 2.740 1.469 16.165 9.458 19.319 5.281 16.806 16.256
10.268 7.367 9.766 12.482 9.552 8.867 9.429 13.262 9.105 9.330 12.535
1.400 1.053 1.457 1.786 0.958 1.042 1.132 3.158 0.913 1.333 2.366
6.576 8.483 7.779 10.230 10.721 6.205 6.532 8.073 6.881 6.021 8.200
Note: The content of compound represents the weight (mg) in 1 g of FLL power.
FLL samples. The quantitative determination results are summarized in Table 2 and demonstrate that there are distinct differences in concentration of analytes from different store. Therefore, further HCA and PCA were used to demonstrate the difference of FLL samples from 11 drug stores based on the HPLC quantitation results (Fig. 2). HCA, one of the most commonly used unsupervised pattern recognition methods, was a useful multivariate statistic technique to assign samples into groups by creating a cluster tree or dendrogram, according to similarity [17]. To assess the resemblance and differences of these samples, HCA of FLL samples was performed based on the content of all the six characteristic compounds by Matlab 7.0. The Ward method was applied as the amalgamation rule and the Euclidean distance was selected to measure the resemblance and classify the 11 samples. The results obtained following HCA are shown in a dendrogram of Fig. 2A in which three welldefined clusters were visible. S4 and S5 were categorized into cluster I; S2, S3, S7, and S9 were categorized into cluster II, while the rest were put into cluster III. As a multivariate analysis technique, PCA could visualize similarities or differences within multivariate data [18]. The content of six compounds were set as variables, while 11 batches of samples were set as observations. The variables were centered and scaled to “Unit Variance” before PCA in SIMCA-P. The first principal component (t1) explained 92.48% of the total variance in the data set while the second principal component (t2) explained 6.96% (Fig. 2B). The first two principal components reduced the multidimensional dataset to a 2D data set which explained more than 99% of the total variance. Thus, it was notable that all samples were classified into three groups, including group I (S4 and S5), group II (S2, S3, S7, and S9), and group III (S1, S6, S8, S10, and S11). This was quite consistent with the results of HCA. In PCA, the loading coefficients represent the importance of each individual feature in a reduced dimension [19]. In general, 2D loading plots (e.g., p1/p2 loading plot) provide useful information to identify important features in the first and second PC dimensions. Here, the results from Fig. 2C show that the compounds, C3 (specnuezhenide), C4 (oleanic acid), and C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
C6 (acetyl oleanic acid), have the largest loading coefficients, indicating the degree of importance of the original feature in the p1/p2 dimension. Combined with the information from Table 2, the content of specnuezhenide in FLL samples of group I were much lower than those in other groups, and the content of acetyl oleanic acid were higher. Specnuezhenide in FLL of group III had relatively higher content. Specnuezhenide is a major active compound in FLL, and it is the only compound which should be quantified according to Edition 2010 of China Pharmacopeia [1]. In former edition of China Pharmacopeia, oleanic acid was used as the quantitative index of FLL [20]. However, the assay of oleanic acid, a common compound in several herbal medicines, is lack of specificity for QC of FLL, especially for the QC of FLL in Chinese medicinal formulae. So a particular compound in FLL, specnuezhenide, was selected to replace oleanic acid in Edition 2010 of China Pharmacopeia. In this work, we found that, besides specnuezhenide, oleanic acid, and acetyl oleanic acid could also influence the classification in FLL samples and should be taken into account for QC of FLL. Although the definitive action of acetyl oleanic acid in FLL has not been reported as we know, some studies have demonstrated its potential abilities of exhibiting anti-angiogenic effects in human umbilical vein endothelial cells [21] and inducing apoptosis in human colon carcinoma HCT-116 cells [22].
4 Conclusion In this study, a comprehensive strategy using HPLC–UV coupled with chemometrics was proposed for QC of FLL samples. The developed quantitative method is promising to be the routing analysis for six compounds (salidroside, nuzhenide, specnuezhenide, oleanic acid, ursolic acid, and acetyl oleanic acid) in FFL samples. Furthermore, HCA and PCA were applied to the classification and evaluation of FLL samples and the significant variables were selected based on PCA loading efficiencies. This strategy is highly recommendable for quality study of other herbal medicine. www.jss-journal.com
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Figure 2. A: The cluster analysis of 11 samples (the distance was calculated by Euclidean distance); B: The score plot of PCA (t1/t2); C: The loading plot of PCA (p1/p2). C1: Salidroside; C2: Nuzhenide; C3: Specnuezhenide; C4: Oleanic acid; C5: Ursolic acid; C6: Acetyl oleanic acid.
The authors are grateful for the financial support by National Natural Science Foundation of China (Grant No. 81001599, 81173547 and 81373972) and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
5 References [1] National Pharmacopoeia Committee, Pharmacopoeia of the People’s Republic of China Part 1, Chinese Medical Science and Technology Press, Beijing 2010, 43. [2] Yim, T. K., Wu, W. K., Pak, W. F., Phytother. Res. 2001, 15, 589–592.
The authors have declared no conflict of interest. C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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