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Kai-Yue Cao Chun-Feng Qiao Jing Zhao Jing Xie Shao-Ping Li State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China Received February 24, 2015 Revised March 29, 2015 Accepted April 2, 2015
Research Article
Quantitative analysis of acankoreoside A and acankoreagenin in the leaves of Schefflera octophylla and Schefflera actinophylla using pressurized liquid extraction and high-performance liquid chromatography coupled with evaporative light scattering detection A rapid method based on pressurized liquid extraction followed by high-performance liquid chromatography coupled with evaporative light scattering detection was firstly developed for the quantitative analysis of two bioactive triterpenoids (acankoreoside A and acankoreagenin) in the leaves of Schefflera octophylla and Schefflera actinophylla. The analysis was performed on an Agilent Zorbax SB-Aq column (4.6 × 50 mm, 3.5 m) with gradient elution of 0.1% formic acid and acetonitrile. Calibration curves of two analytes showed good linearity (R2 > 0.9990) within the tested ranges. This novel method is simple, rapid and accurate, and the results of quantification showed that contents of each investigated compound is significant high in natural S. octophylla (6.36–14.83%), which indicated that natural S. octophylla as potential medicinal resource. Furthermore, hierarchical clustering analysis based on the typical peaks of acankoreoside A and acankoreagenin from the 17 tested samples showed that natural and cultured Schefflera species were in different clusters, which could provide a means of discriminating between Schefflera species from different origins. Thus, acankoreoside A and acankoreagnin could be selected markers for quality control of S. octophylla and S. actinophylla. Keywords: Acankoreagenin / Acankoreoside A / High-performance liquid chromatography / Pressurized liquid extraction / Schefflera DOI 10.1002/jssc.201500223
1 Introduction Schefflera octophylla (Lour.) Harms, or Schefflera heptaphylla (Linn.) Frodin, and Schefflera actinophylla (Endl.) Harms (Fig. 1) are ornamental plants widely cultivated in Southeast Asia. They are two species of genus Schefflera, which belongs to the family of Araliaceae [1]. S. octophylla, commonly known as “E-zhang-chai” in Chinese, is a medicinal plant with compound leaves in Southeast Asia [2]. As a traditionally used folk medicine, leaves, and bark roots of natural S. Correspondence: Dr. Jing Zhao and Professor Shao-Ping Li, State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China E-mail: [email protected], [email protected] Fax: +853-28841358
Abbreviations: AA, acankoreoside A; AN, acankoreagenin; ELSD, evaporative light scattering detection; HCA, hierarchical clustering analysis; PLE, pressurized liquid extraction
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
octophylla have been revealed to have anti-pyretic, antiinflammatory, and anti-rheumatic effects [3–5]. Nowadays, as a principle ingredient of an herbal tea formulation, it is widely used to treat common cold in Hong Kong [4]. Modern pharmacological study showed that the aqueous extract of S. octophylla possesses the most potent antiviral activity against respiratory syncytial virus among 21 medicinal herbs traditionally used in southern China [6]. Moreover, triterpenoid compounds were isolated from an antiviral-guided fractionation of aqueous extract of S. octophylla [7]. Actually, phytochemical investigations also have demonstrated that triterpenoids and their glycosides (saponins) are the major chemical constituents in the leaves of S. octophylla and S. actinophylla [8–16]. Among them, acankoreoside A (3␣-hydroxylup-20(29)-ene-23,28-dioic acid (1→4)--D-glucopyranosyl28-O-␣-L-rhamnopyranosyl (1→6)--D-glucopyranoside, AA) and acankoreoagenin (3␣hydroxylup-20(29)-ene-23,28-dioic acid, AN) are a pair of lupane triterpenoid saponin and sapogenin isolated from the two species [8, 13, 16]. In previous bioactivity studies,
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Figure 1. Leaf of (A) natural S. octophylla, (B) cultured S. octophylla and (C) cultured S. actinophylla.
Figure 2. Chemical structures of investigated compounds.
the two compounds have been revealed to have various bioactivities [8, 13, 16–23]. AA has significant protective effect on cerebral infarction in rats [24], and it has not only showed moderate cytotoxicity against four kinds of human cancer cell lines [25], but also could significantly increase IFN-c and IL-2 release in spleen cells [26]. On the other hand, AN has been proved to possess broader antiviral activity against respiratory syncytial virus (RSV) [7]. It could inhibit the systemic release of pro-inflammatory cytokine HMGB1, which suggests it as a potential drug for the treatment of fulminant hepatitis [27]. Also, AN has been proved with the antitumor and anti-angiogenic effect, which has synergic action with chemical drugs [28]. Furthermore, within tested on various cancer cells, the result showed that AN possess selectivity for anti-cancer activity [29]. Therefore, leaves of the two Schefflera species are possibly potential medicinal part, and quantitative analysis of AA and AN is crucial for ensuing efficacy and quality of S. octophylla and S. actinophylla. Unfortunately, there was no report in this field. Complex components are big challenge for QC of natural herbs, especially for traditional Chinese medicines (TCMs). In recent years, rapid separation and analysis of polar components have become a hot research area [30, 31], and SB-Aq has been developed as ideal reverse phase column. Today, it has been successfully applied for separation and analysis of polar components from some well-known TCMs by our
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
group, such as Cordyceps, Ganoderma, Panax notoginseng, and Polygonum multiflorum [32–35]. In this present study, PLE combined with HPLC coupled to evaporative light scattering detection (ELSD) was firstly developed as a rapid and reliable method for simultaneous determination of AA and AN in S. octophylla and S. actinophylla. Besides, their contents in different samples were compared using hierarchical clustering analysis.
2 Materials and methods 2.1 Materials and chemicals Standards of AA and AN were separated and purified in our lab. Their purities were >98%, which were determined by HPLC. The structures (Fig. 2) were elucidated by their UV spectrophotometry, MS, and 1 H and 13 C NMR spectroscopy compared with literature data. Acetonitrile for HPLC was purchased from Merck (Darmstadt, Germany). Deionized water was prepared using a Millipore Milli Q-Plus system (Millipore, Bedford, MA, USA). Samples of raw leaves of cultured S. actinophylla, natural, and cultured S. octophylla were collected from Zhuhai, Guangdong Province, China, and Macao SAR, China. The species were authenticated by one of the authors, Dr. ChunFeng Qiao. The voucher specimens were deposited at the
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Figure 3. Effects of solvent, temperature and static extraction time on PLE extraction of (A) acankoreoside A and (B) acankoreagenin in natural S. octophylla (SOYL-3).
Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China.
the two analytes. The drift tube temperature for ELSD was set at 60⬚C, inlet gas pressure was 350 KPa and gain value was 8.
2.3 Sample preparation 2.2 HPLC analysis All separations were performed on an HPLC Agilent 1100 system (Agilent Technologies, Santa Clara, CA, USA), equipped with a vacuum degasser, a quaternary pump, an autosampler, and a Shimadzu LT-II ELSD (Shimadzu Corp, Kyoto, Japan), controlled by Agilent Chemstation LC Software. Separation was performed on an Agilent Zorbax SB-Aq column (4.6× 50 mm, 3.5 m) at 30⬚C. The flow rate was set at 1.0 mL/min and sample injection volume was 10 L. Gradient elution with (A) 0.1% formic acid and (B) acetonitrile was 0–4 min, 30% B; 4–5 min, 30–55% B; 5–7 min, 55–65% B; 7–9 min, 65–100% B; 9–10 min, 100% B. ELSD was used to determine C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Sample preparation was performed using a PLE on a Dionex ASE 350 system (Dionex, Sunnyvale, CA, USA) under optimized conditions. Dried powders (40⬚C, 6 h) of sample (0.1 g, 80 mesh) were mixed with diatomaceous earth in a proportion of 1:1 w/w and placed into a 10 mL stainless-steel extraction cell, respectively. The extraction was performed under the optimized conditions: solvent, 100% methanol; temperature, 100⬚C; Pressure, 1500 psi; static extraction time, 5 min; flush volume, 60%; one cycle and one of extraction time. Then the extract was transferred into a 25 mL volumetric flask, which was made up to its volume with methanol, and filtered through a 0.45 m Nylon membrane www.jss-journal.com
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Table 1. Summary for the test samples and their contents (mg/g) of investigated components
No.
Code
Cultivated S. octophylla 1 SOZL-1 2 SOZL-2 3 SOZL-3 4 SOZL-4 5 SOZL-5 Natural S. octophylla 6 SOYL-1 7 SOYL-2 8 SOYL-3 9 SOYL-4 10 SOYL-5 11 SOYL-6 12 SOYL-7 13 SOYL-8 14 SOYL-9 15 SOYL-10 Cultivated S. actinophylla 16 SAL-1 17 SAL-2
Collection information
Locations
Acankoreoside A
Acankoreagenin
Total a)
Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014
Macau, China Macau, China Macau, China Macau, China Zhuhai, China
NDb) ND ND ND ND
ND ND ND ND ND
/ / / / /
Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014
Macau, China Macau, China Macau, China Macau, China Macau, China Macau, China Macau, China Macau, China Macau, China Macau, China
53.7c 40.5 112.8 112.7 116 129 140.9 85.2 110.6 116.2
9.9 56.2 33.2 12.3 23 16.2 7.4 7.3 ±d) 5.7
63.6 96.7 146 125 139 145.2 148.3 92.5 110.6 121.9
Raw leaves, 2014 Raw leaves, 2014
Macau, China Macau, China
10.4 ±
± ND
10.4 /
a) Total amount of acankoreoside A and acankoreagenin. b) ND, not detected. c) The data were the average of two measurements with coefficient of variation less than 3%. d) ±, under limit of quantification.
in duplicates. For the two analytes, calibration curves were constructed by plotting the logarithmic value of the peak area (ELSD signal) versus logarithmic value of the concentration. The stock solution with the two reference compounds was diluted to a series of appropriate concentration with methanol, and an aliquot of the diluted solution were injected into HPLC for analysis. The LOD and LOQ were determined based on the S/N of about 3 and 10, respectively.
2.5 Precision, repeatability and recovery Figure 4. Typical HPLC–ELSD chromatograms of (A) mixed standards, (B) cultured S. octophylla, (C) cultured S. actinophylla, and (D) natural S. octophylla. 1, acankoreoside A; 2, acankoreagenin.
filter (Whatman, UK) prior for the injection into the HPLC system.
2.4 Calibration curves, LOD, and LOQ Stock solution was prepared by weighing the two reference compounds accurately and dissolving them in methanol. Then the stock solution was diluted to appropriate concentrations for construction of calibration curves. For each component, at least six concentrations of the solution were analyzed C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Intra- and inter-day variations were applied to measure the precision of the developed method. The known concentrations of two standard solutions were tested. For intra-day variability tests, the mixed standard solution was analyzed six times within one day, while for inter-day variability tests, the samples were examined in duplicate on three consecutive days. Variations were expressed as the RSDs. To confirm the repeatability, powders of natural S. octophylla (SOYL-3) in three levels (0.08, 0.1, and 0.12 g) were extracted by PLE in triplicate for each level, respectively, and analyzed by HPLC– ELSD mentioned above. The RSD was used as the measurement of repeatability. The recovery test was used to evaluate the accuracy of the method. A known amount of individual standards were added into certain amount (0.08 g) of sample SOYL-3. The www.jss-journal.com
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Figure 5. Dendrograms of hierarchical clustering analysis for 17 tested samples of Schefflera materials based on the contents of acankoreoside A and acankoreagenin. The sample codes were the same as in Table 1.
mixtures were extracted and analyzed using the developed method mentioned above. Three replicates were prepared for the test. The extract was transferred into a 25 mL volumetric flask that was made up to its volume with extraction solvent and filtered with a 0.45 m filter before analysis. The quantity of each analyte was subsequently obtained from the corresponding calibration curve. The recovery was calculated as follows: Recovery (%) = 100 × (amount found–original amount) / amount spiked.
The extraction efficiency of PLE was determined by performing consecutive PLEs on the same sample under optimized PLE conditions, until no investigated components were detected. The extraction efficiency was calculated based on the total amount of individual investigated components, which was more than 95% for the first-time extraction. Taking into account the results of optimization and extraction efficiency experiments, the conditions of PLE method proposed were solvent, 100% methanol; temperature, 100⬚C; static extraction time, 5 min; pressure, 10.342 MPa (1500 psi); 60% of flush volume for one cycle and one of extraction time.
3 Results and discussion 3.1 Optimization of PLE
3.2 Method validation of HPLC–ELSD
The optimization of PLE was performed using the leaves of natural S. octophylla (SOYL-3) as sample that contained all of analytes. The pressure that has been reported with no significant effect on extraction efficiency [36, 37], was set at 10.342 MPa (1500 psi, system default value). Other parameters, including organic solvent ratio (100, 80, 60, 40% methanol), temperature (80, 100, and 120⬚C), and static time (5, 10, and 15 min) were optimized using univariate approach. Peak area of each analyte was used as the marker for evaluation of extraction efficiency. The results are shown in Fig. 3.
The calibration, LOD, LOQ, precision, repeatability, and recovery of the two analytes were performed using the developed HPLC–ELSD method. Good linearity was achieved in the range of 20.80–750.00 g/mL (AA) and 10.40– 166.67 g/mL (AN). The linear regression between logarithmic value of the peak area (y-axis) and logarithmic value of the concentration (x-axis) were y = 1.616x – 0.386 (AA, R2 = 0.9996) and y = 1.589x – 0.149 (AN, R2 = 0.9990). The LOD and LOQ were 8.75 and 16.39 g/mL (AA), 4.65 and 8.72 g/mL (AN). The intra- and inter-day variations (RSD)
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were 1.2 and 2.4% (AA), 2.4 and 3.9% (AN). The repeatability (RSD, n = 3) of three levels were 0.5, 2.5, and 0.9% (AA), 0.7, 1.0, and 2.2% (AN). The average recovery (n = 3) of was 102.9% (AA) and 94.5% (AN). Therefore, the developed HPLC–ELSD method could be considered as accurate for quantitative analysis of AA and AN.
simple, rapid and accurate, which could be used for discrimination and QC of S. octophylla and S. actinophylla. K.-Y. C. and C.-F. Q. contributed equally to this work. The research was partially supported by a grant from University of Macau to J. Zhao (MYRG085). The authors have declared no conflict of interest.
3.3 Quantification of acankoreoside A and acankoreagenin in Schefflera materials Typical chromatograms of PLE extracts for Schefflera materials are shown in Fig. 4. Identification of the investigated components was carried out by comparing their retention times and UV spectra with those obtained by injecting standards under the same conditions or spiking Schefflera samples with stock standard solutions. By using the calibration curve of each investigated compound, the contents of investigated compounds in two Schefflera species were determined. Table 1 summarized the result. In brief, natural S. octophylla was significant rich in AA and AN and their contents were in a big range from 6.36 to 14.83%, respectively. Although materials of natural S. octophylla were all collected from same origins, different environment and growth period of botanical could be key factors. However, using this method, AA and AN could not be detected in cultured S. octophylla. Generally, the contents of investigated compounds were: natural S. octophylla >> cultured S. actinophylla. The results indicated that AA and AN in high content contribute to the significant pharmacological activity of natural S. octophylla, and leaves of natural S. octophylla were proved to be potential medicinal resource.
3.4 Hierarchical clustering analysis (HCA) Although total content of AA and AN in the two species of Schefflera is obviously different, the morphological appearance of them is similar. Thus, hierarchical clustering analysis of 17 samples were performed on the basis of typical peaks of AA and AN. A Ward’s method was applied, and Euclidean distance was selected as measure. Fig. 5 shows the results for the tested 17 samples of Schefflera materials, which were divided into three main clusters. These are cultured S. actinophylla, natural and cultured S. octophylla, respectively. Hence, the results showed that AA and AN could be selected as marker compounds for discrimination and QC of S. octophylla and S. actinophylla.
4 Concluding remarks A rapid method based on PLE followed by HPLC–ELSD analysis was firstly developed to quantify AA and AN in S. octophylla and S. actinophylla. The results indicated leaves of natural S. octophylla as potential medicinal part. This method is C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
5 References [1] Flora of Commission of PRC (Eds.), Flora of the People’s Republic of China. Science Press, Peking 1978, pp. 50– 51. [2] Perry, L. M., Medicinal plants of east and south-east Asia. MIT Press, Cambridge 1980, pp. 44. [3] Kitajima, J., Shindo, M., Tanaka, Y., Chem. Pharm. Bull. 1990, 38, 714–716. [4] Kong, Y. C. (Eds.), Herbal medicines and teas of Hong Kong, Commerical Press, Hong Kong 2000, pp. 90– 91. [5] Chen, L., Zhu, Q. H., Lin, R. C., J. Chin. Med. Mater. 2002, 25, 353–366. [6] Li, Y. L., Ooi, L. S. M., Wang, H., But, P. P. H., Ooi, V. E. C., Phytother. Res. 2004, 18, 718–722. [7] Li, Y. L., Jiang, R. W., Ooi, L. S. M., But, P. P. H., Ooi, V. E. C., Phytother. Res. 2007, 21, 466–470. [8] Adam, G., Lischewski, M., Phiet, H. V., Preiss, A., Schmidt, J., Sung, T. V., Phytochemistry 1982, 21, 1385– 1387. [9] Lischewski, M., Ty, P. D., Schmidt, J., Preiss, A., Phiet, H. V., Adam, G., Phytochemistry 1984, 23, 1695–1697. [10] Schmidt, J., Nam, V. V., Lischewski, M., Hoang, V. P., Kuhnt, C., Adam, G., Phytochemistry 1984, 23, 2081– 2082. [11] Sung, T. V., Peter-Katalinic, J., Adam, G., Phytochemistry 1991, 30, 3717–3720. [12] Sung, T. V., Lavaud, C., Porzel, A., Steglich, W., Adam, G., Phytochemistry 1991, 31, 227–231. [13] Sung, T. V., Steglich, W., Adam, G., Phytochemistry. 1991, 30, 2349–2356. [14] Sung, T. V., Adam, G., Phytochemistry. 1991, 30, 2717– 2720. [15] Maeda, C., Ohtani, K., Kasai, R., Yamasaki, K., Duc, N. M., Nham, N. T., Cu, G. K. Q., Phytochemistry. 1994, 37, 1131–1137. [16] Wanas, A. S., Matsunami, K., Orsuka, H., Desoukey, S. Y., Fouad, M. A., Kamel, M. S., Chem. Pharm. Bull. 2010, 58, 1596–1601. [17] Van Kiem, P., Dat, N. T., Van Minh, C., Lee, J. J., Kim, Y. H., Arch. Pharm. Res. 2003, 26, 594–596. [18] Chang, S. Y., Yook, C. S., Nohara, T., Chem. Pharm. Bull. 1998, 46, 163–165. [19] Kim, J. A., Yang, S. Y., Koo, J. E., Koh, Y. S., Kim, Y. H., Bioorg. Med. Chem. Lett. 2010, 20, 6703– 6707.
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[20] Liu, X. Q., Chang, S. Y., Park, S. Y., Nohara, T., Yook, C. S., Arch. Pharm. Res. 2002, 25, 831–836.
[28] Qian, S. H., Yuan, L. H., Cao, P., An, S. Y., Cai, X. T., J. Chin. Med. Mater. 2009, 32, 1889–1891.
[21] Liu, X. Q., Chang, S. Y., Yook, C. S., J. Lanzhou Uni. 2006, 42, 86–91.
[29] Liu, P. P., Qu, Y., Dou, D. Q., Kang, T. G., Smith, D., J. Chem. Soc. Pakistan. 2012, 34, 419–423.
[22] Hirai, Y., Murayama, T., Miyakoshi, M., Hirono, S., Isoda, S., Ideura, N., Ida, Y., Shoji, J., Wang, G. S., Xu, J. D., J. Nat. Med. 1995, 49, 462–467.
[30] Toyo’oka, T., J. Chromatogr. Sci. 2008, 46, 233–247.
[23] Liu, P. P., Li, M., Kang, T. G., Dou, D. Q., Smith, D. C., Nat. Prod. Commun. 2010, 5, 1019–1022. [24] Liu, X. Q., Zou, Q. P., Feng, S., Zheng, L. S., Yook, C. S., Drug Evaluation Res. 2010, 33, 95. [25] Nhiem, N. X., Tung, N. H., Van Kiem, P., Van Minh, C., Ding, Y., Hyun, J. H., Kang, H. K., Kim, Y. H., Chem. Pharm. Bull. 2009, 57, 986–989. [26] Nhiem, N. X., Kiem, P. V., Minh, C. V., Tai, B. H., Tung, N. H., Ha do, T., Soung, K. S., Kim, J. H., Ahn, J. Y., Lee, Y. M., Kim, Y. H., Bioorg. Med. Chem. Lett. 2010, 20, 4927–4931. [27] Zhang, B. X., Li, N., Zhang, Z. P., Liu, H. B., Zhou, R. R., Zhou, B. Y., Zou, M. X., Dai, X. H., Xiao, M. F., Liu, X. Q., Fan, X. G., Int. Immunopharmacol. 2011, 11, 1018– 1023.
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[31] Liang, X. M., Jin, Y., Wang, Y. P., Jin, G. W., Fu, Q., Xiao, Y. S., J. Chromatogr. A. 2009, 1216, 2033– 2044. [32] Yang, F. Q., Li, D. Q., Feng, K., Hu, D. J., Li, S. P., J. Chromatogr. A. 2010, 1217, 5501–5510. [33] Qian, Z. M., Zhao, J., Li, D. Q., Hu, D. J., Li, S. P., J. Sep. Sci. 2012, 35, 2725–2734. [34] Han, D. Q., Zhao, J., Xu, J., Peng, H. S., Chen, X. J., Li, S. P., J. Pharmaceut. Biomed. 2013, 72, 223–230. [35] Qian, Z. M., Wan, J. B., Zhang, Q. W., Li, S. P., J. Pharmaceut. Biomed. 2008, 48, 1361–1367. [36] Bjorklund, E., Nilsson, T., Bowadt, S., TrAC, Trends Anal. Chem. 2000, 19, 434–445. [37] Ong, E. S., Woo, S. O., Yong, Y. L., J. Chromatogr. A. 2000, 904, 57–64.
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J. Sep. Sci. 2015, 38, 2201–2207
Kai-Yue Cao Chun-Feng Qiao Jing Zhao Jing Xie Shao-Ping Li State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China Received February 24, 2015 Revised March 29, 2015 Accepted April 2, 2015
Research Article
Quantitative analysis of acankoreoside A and acankoreagenin in the leaves of Schefflera octophylla and Schefflera actinophylla using pressurized liquid extraction and high-performance liquid chromatography coupled with evaporative light scattering detection A rapid method based on pressurized liquid extraction followed by high-performance liquid chromatography coupled with evaporative light scattering detection was firstly developed for the quantitative analysis of two bioactive triterpenoids (acankoreoside A and acankoreagenin) in the leaves of Schefflera octophylla and Schefflera actinophylla. The analysis was performed on an Agilent Zorbax SB-Aq column (4.6 × 50 mm, 3.5 m) with gradient elution of 0.1% formic acid and acetonitrile. Calibration curves of two analytes showed good linearity (R2 > 0.9990) within the tested ranges. This novel method is simple, rapid and accurate, and the results of quantification showed that contents of each investigated compound is significant high in natural S. octophylla (6.36–14.83%), which indicated that natural S. octophylla as potential medicinal resource. Furthermore, hierarchical clustering analysis based on the typical peaks of acankoreoside A and acankoreagenin from the 17 tested samples showed that natural and cultured Schefflera species were in different clusters, which could provide a means of discriminating between Schefflera species from different origins. Thus, acankoreoside A and acankoreagnin could be selected markers for quality control of S. octophylla and S. actinophylla. Keywords: Acankoreagenin / Acankoreoside A / High-performance liquid chromatography / Pressurized liquid extraction / Schefflera DOI 10.1002/jssc.201500223
1 Introduction Schefflera octophylla (Lour.) Harms, or Schefflera heptaphylla (Linn.) Frodin, and Schefflera actinophylla (Endl.) Harms (Fig. 1) are ornamental plants widely cultivated in Southeast Asia. They are two species of genus Schefflera, which belongs to the family of Araliaceae [1]. S. octophylla, commonly known as “E-zhang-chai” in Chinese, is a medicinal plant with compound leaves in Southeast Asia [2]. As a traditionally used folk medicine, leaves, and bark roots of natural S. Correspondence: Dr. Jing Zhao and Professor Shao-Ping Li, State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China E-mail: [email protected], [email protected] Fax: +853-28841358
Abbreviations: AA, acankoreoside A; AN, acankoreagenin; ELSD, evaporative light scattering detection; HCA, hierarchical clustering analysis; PLE, pressurized liquid extraction
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
octophylla have been revealed to have anti-pyretic, antiinflammatory, and anti-rheumatic effects [3–5]. Nowadays, as a principle ingredient of an herbal tea formulation, it is widely used to treat common cold in Hong Kong [4]. Modern pharmacological study showed that the aqueous extract of S. octophylla possesses the most potent antiviral activity against respiratory syncytial virus among 21 medicinal herbs traditionally used in southern China [6]. Moreover, triterpenoid compounds were isolated from an antiviral-guided fractionation of aqueous extract of S. octophylla [7]. Actually, phytochemical investigations also have demonstrated that triterpenoids and their glycosides (saponins) are the major chemical constituents in the leaves of S. octophylla and S. actinophylla [8–16]. Among them, acankoreoside A (3␣-hydroxylup-20(29)-ene-23,28-dioic acid (1→4)--D-glucopyranosyl28-O-␣-L-rhamnopyranosyl (1→6)--D-glucopyranoside, AA) and acankoreoagenin (3␣hydroxylup-20(29)-ene-23,28-dioic acid, AN) are a pair of lupane triterpenoid saponin and sapogenin isolated from the two species [8, 13, 16]. In previous bioactivity studies,
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Figure 1. Leaf of (A) natural S. octophylla, (B) cultured S. octophylla and (C) cultured S. actinophylla.
Figure 2. Chemical structures of investigated compounds.
the two compounds have been revealed to have various bioactivities [8, 13, 16–23]. AA has significant protective effect on cerebral infarction in rats [24], and it has not only showed moderate cytotoxicity against four kinds of human cancer cell lines [25], but also could significantly increase IFN-c and IL-2 release in spleen cells [26]. On the other hand, AN has been proved to possess broader antiviral activity against respiratory syncytial virus (RSV) [7]. It could inhibit the systemic release of pro-inflammatory cytokine HMGB1, which suggests it as a potential drug for the treatment of fulminant hepatitis [27]. Also, AN has been proved with the antitumor and anti-angiogenic effect, which has synergic action with chemical drugs [28]. Furthermore, within tested on various cancer cells, the result showed that AN possess selectivity for anti-cancer activity [29]. Therefore, leaves of the two Schefflera species are possibly potential medicinal part, and quantitative analysis of AA and AN is crucial for ensuing efficacy and quality of S. octophylla and S. actinophylla. Unfortunately, there was no report in this field. Complex components are big challenge for QC of natural herbs, especially for traditional Chinese medicines (TCMs). In recent years, rapid separation and analysis of polar components have become a hot research area [30, 31], and SB-Aq has been developed as ideal reverse phase column. Today, it has been successfully applied for separation and analysis of polar components from some well-known TCMs by our
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
group, such as Cordyceps, Ganoderma, Panax notoginseng, and Polygonum multiflorum [32–35]. In this present study, PLE combined with HPLC coupled to evaporative light scattering detection (ELSD) was firstly developed as a rapid and reliable method for simultaneous determination of AA and AN in S. octophylla and S. actinophylla. Besides, their contents in different samples were compared using hierarchical clustering analysis.
2 Materials and methods 2.1 Materials and chemicals Standards of AA and AN were separated and purified in our lab. Their purities were >98%, which were determined by HPLC. The structures (Fig. 2) were elucidated by their UV spectrophotometry, MS, and 1 H and 13 C NMR spectroscopy compared with literature data. Acetonitrile for HPLC was purchased from Merck (Darmstadt, Germany). Deionized water was prepared using a Millipore Milli Q-Plus system (Millipore, Bedford, MA, USA). Samples of raw leaves of cultured S. actinophylla, natural, and cultured S. octophylla were collected from Zhuhai, Guangdong Province, China, and Macao SAR, China. The species were authenticated by one of the authors, Dr. ChunFeng Qiao. The voucher specimens were deposited at the
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Figure 3. Effects of solvent, temperature and static extraction time on PLE extraction of (A) acankoreoside A and (B) acankoreagenin in natural S. octophylla (SOYL-3).
Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China.
the two analytes. The drift tube temperature for ELSD was set at 60⬚C, inlet gas pressure was 350 KPa and gain value was 8.
2.3 Sample preparation 2.2 HPLC analysis All separations were performed on an HPLC Agilent 1100 system (Agilent Technologies, Santa Clara, CA, USA), equipped with a vacuum degasser, a quaternary pump, an autosampler, and a Shimadzu LT-II ELSD (Shimadzu Corp, Kyoto, Japan), controlled by Agilent Chemstation LC Software. Separation was performed on an Agilent Zorbax SB-Aq column (4.6× 50 mm, 3.5 m) at 30⬚C. The flow rate was set at 1.0 mL/min and sample injection volume was 10 L. Gradient elution with (A) 0.1% formic acid and (B) acetonitrile was 0–4 min, 30% B; 4–5 min, 30–55% B; 5–7 min, 55–65% B; 7–9 min, 65–100% B; 9–10 min, 100% B. ELSD was used to determine C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Sample preparation was performed using a PLE on a Dionex ASE 350 system (Dionex, Sunnyvale, CA, USA) under optimized conditions. Dried powders (40⬚C, 6 h) of sample (0.1 g, 80 mesh) were mixed with diatomaceous earth in a proportion of 1:1 w/w and placed into a 10 mL stainless-steel extraction cell, respectively. The extraction was performed under the optimized conditions: solvent, 100% methanol; temperature, 100⬚C; Pressure, 1500 psi; static extraction time, 5 min; flush volume, 60%; one cycle and one of extraction time. Then the extract was transferred into a 25 mL volumetric flask, which was made up to its volume with methanol, and filtered through a 0.45 m Nylon membrane www.jss-journal.com
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Table 1. Summary for the test samples and their contents (mg/g) of investigated components
No.
Code
Cultivated S. octophylla 1 SOZL-1 2 SOZL-2 3 SOZL-3 4 SOZL-4 5 SOZL-5 Natural S. octophylla 6 SOYL-1 7 SOYL-2 8 SOYL-3 9 SOYL-4 10 SOYL-5 11 SOYL-6 12 SOYL-7 13 SOYL-8 14 SOYL-9 15 SOYL-10 Cultivated S. actinophylla 16 SAL-1 17 SAL-2
Collection information
Locations
Acankoreoside A
Acankoreagenin
Total a)
Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014
Macau, China Macau, China Macau, China Macau, China Zhuhai, China
NDb) ND ND ND ND
ND ND ND ND ND
/ / / / /
Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014 Raw leaves, 2014
Macau, China Macau, China Macau, China Macau, China Macau, China Macau, China Macau, China Macau, China Macau, China Macau, China
53.7c 40.5 112.8 112.7 116 129 140.9 85.2 110.6 116.2
9.9 56.2 33.2 12.3 23 16.2 7.4 7.3 ±d) 5.7
63.6 96.7 146 125 139 145.2 148.3 92.5 110.6 121.9
Raw leaves, 2014 Raw leaves, 2014
Macau, China Macau, China
10.4 ±
± ND
10.4 /
a) Total amount of acankoreoside A and acankoreagenin. b) ND, not detected. c) The data were the average of two measurements with coefficient of variation less than 3%. d) ±, under limit of quantification.
in duplicates. For the two analytes, calibration curves were constructed by plotting the logarithmic value of the peak area (ELSD signal) versus logarithmic value of the concentration. The stock solution with the two reference compounds was diluted to a series of appropriate concentration with methanol, and an aliquot of the diluted solution were injected into HPLC for analysis. The LOD and LOQ were determined based on the S/N of about 3 and 10, respectively.
2.5 Precision, repeatability and recovery Figure 4. Typical HPLC–ELSD chromatograms of (A) mixed standards, (B) cultured S. octophylla, (C) cultured S. actinophylla, and (D) natural S. octophylla. 1, acankoreoside A; 2, acankoreagenin.
filter (Whatman, UK) prior for the injection into the HPLC system.
2.4 Calibration curves, LOD, and LOQ Stock solution was prepared by weighing the two reference compounds accurately and dissolving them in methanol. Then the stock solution was diluted to appropriate concentrations for construction of calibration curves. For each component, at least six concentrations of the solution were analyzed C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Intra- and inter-day variations were applied to measure the precision of the developed method. The known concentrations of two standard solutions were tested. For intra-day variability tests, the mixed standard solution was analyzed six times within one day, while for inter-day variability tests, the samples were examined in duplicate on three consecutive days. Variations were expressed as the RSDs. To confirm the repeatability, powders of natural S. octophylla (SOYL-3) in three levels (0.08, 0.1, and 0.12 g) were extracted by PLE in triplicate for each level, respectively, and analyzed by HPLC– ELSD mentioned above. The RSD was used as the measurement of repeatability. The recovery test was used to evaluate the accuracy of the method. A known amount of individual standards were added into certain amount (0.08 g) of sample SOYL-3. The www.jss-journal.com
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Figure 5. Dendrograms of hierarchical clustering analysis for 17 tested samples of Schefflera materials based on the contents of acankoreoside A and acankoreagenin. The sample codes were the same as in Table 1.
mixtures were extracted and analyzed using the developed method mentioned above. Three replicates were prepared for the test. The extract was transferred into a 25 mL volumetric flask that was made up to its volume with extraction solvent and filtered with a 0.45 m filter before analysis. The quantity of each analyte was subsequently obtained from the corresponding calibration curve. The recovery was calculated as follows: Recovery (%) = 100 × (amount found–original amount) / amount spiked.
The extraction efficiency of PLE was determined by performing consecutive PLEs on the same sample under optimized PLE conditions, until no investigated components were detected. The extraction efficiency was calculated based on the total amount of individual investigated components, which was more than 95% for the first-time extraction. Taking into account the results of optimization and extraction efficiency experiments, the conditions of PLE method proposed were solvent, 100% methanol; temperature, 100⬚C; static extraction time, 5 min; pressure, 10.342 MPa (1500 psi); 60% of flush volume for one cycle and one of extraction time.
3 Results and discussion 3.1 Optimization of PLE
3.2 Method validation of HPLC–ELSD
The optimization of PLE was performed using the leaves of natural S. octophylla (SOYL-3) as sample that contained all of analytes. The pressure that has been reported with no significant effect on extraction efficiency [36, 37], was set at 10.342 MPa (1500 psi, system default value). Other parameters, including organic solvent ratio (100, 80, 60, 40% methanol), temperature (80, 100, and 120⬚C), and static time (5, 10, and 15 min) were optimized using univariate approach. Peak area of each analyte was used as the marker for evaluation of extraction efficiency. The results are shown in Fig. 3.
The calibration, LOD, LOQ, precision, repeatability, and recovery of the two analytes were performed using the developed HPLC–ELSD method. Good linearity was achieved in the range of 20.80–750.00 g/mL (AA) and 10.40– 166.67 g/mL (AN). The linear regression between logarithmic value of the peak area (y-axis) and logarithmic value of the concentration (x-axis) were y = 1.616x – 0.386 (AA, R2 = 0.9996) and y = 1.589x – 0.149 (AN, R2 = 0.9990). The LOD and LOQ were 8.75 and 16.39 g/mL (AA), 4.65 and 8.72 g/mL (AN). The intra- and inter-day variations (RSD)
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were 1.2 and 2.4% (AA), 2.4 and 3.9% (AN). The repeatability (RSD, n = 3) of three levels were 0.5, 2.5, and 0.9% (AA), 0.7, 1.0, and 2.2% (AN). The average recovery (n = 3) of was 102.9% (AA) and 94.5% (AN). Therefore, the developed HPLC–ELSD method could be considered as accurate for quantitative analysis of AA and AN.
simple, rapid and accurate, which could be used for discrimination and QC of S. octophylla and S. actinophylla. K.-Y. C. and C.-F. Q. contributed equally to this work. The research was partially supported by a grant from University of Macau to J. Zhao (MYRG085). The authors have declared no conflict of interest.
3.3 Quantification of acankoreoside A and acankoreagenin in Schefflera materials Typical chromatograms of PLE extracts for Schefflera materials are shown in Fig. 4. Identification of the investigated components was carried out by comparing their retention times and UV spectra with those obtained by injecting standards under the same conditions or spiking Schefflera samples with stock standard solutions. By using the calibration curve of each investigated compound, the contents of investigated compounds in two Schefflera species were determined. Table 1 summarized the result. In brief, natural S. octophylla was significant rich in AA and AN and their contents were in a big range from 6.36 to 14.83%, respectively. Although materials of natural S. octophylla were all collected from same origins, different environment and growth period of botanical could be key factors. However, using this method, AA and AN could not be detected in cultured S. octophylla. Generally, the contents of investigated compounds were: natural S. octophylla >> cultured S. actinophylla. The results indicated that AA and AN in high content contribute to the significant pharmacological activity of natural S. octophylla, and leaves of natural S. octophylla were proved to be potential medicinal resource.
3.4 Hierarchical clustering analysis (HCA) Although total content of AA and AN in the two species of Schefflera is obviously different, the morphological appearance of them is similar. Thus, hierarchical clustering analysis of 17 samples were performed on the basis of typical peaks of AA and AN. A Ward’s method was applied, and Euclidean distance was selected as measure. Fig. 5 shows the results for the tested 17 samples of Schefflera materials, which were divided into three main clusters. These are cultured S. actinophylla, natural and cultured S. octophylla, respectively. Hence, the results showed that AA and AN could be selected as marker compounds for discrimination and QC of S. octophylla and S. actinophylla.
4 Concluding remarks A rapid method based on PLE followed by HPLC–ELSD analysis was firstly developed to quantify AA and AN in S. octophylla and S. actinophylla. The results indicated leaves of natural S. octophylla as potential medicinal part. This method is C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
5 References [1] Flora of Commission of PRC (Eds.), Flora of the People’s Republic of China. Science Press, Peking 1978, pp. 50– 51. [2] Perry, L. M., Medicinal plants of east and south-east Asia. MIT Press, Cambridge 1980, pp. 44. [3] Kitajima, J., Shindo, M., Tanaka, Y., Chem. Pharm. Bull. 1990, 38, 714–716. [4] Kong, Y. C. (Eds.), Herbal medicines and teas of Hong Kong, Commerical Press, Hong Kong 2000, pp. 90– 91. [5] Chen, L., Zhu, Q. H., Lin, R. C., J. Chin. Med. Mater. 2002, 25, 353–366. [6] Li, Y. L., Ooi, L. S. M., Wang, H., But, P. P. H., Ooi, V. E. C., Phytother. Res. 2004, 18, 718–722. [7] Li, Y. L., Jiang, R. W., Ooi, L. S. M., But, P. P. H., Ooi, V. E. C., Phytother. Res. 2007, 21, 466–470. [8] Adam, G., Lischewski, M., Phiet, H. V., Preiss, A., Schmidt, J., Sung, T. V., Phytochemistry 1982, 21, 1385– 1387. [9] Lischewski, M., Ty, P. D., Schmidt, J., Preiss, A., Phiet, H. V., Adam, G., Phytochemistry 1984, 23, 1695–1697. [10] Schmidt, J., Nam, V. V., Lischewski, M., Hoang, V. P., Kuhnt, C., Adam, G., Phytochemistry 1984, 23, 2081– 2082. [11] Sung, T. V., Peter-Katalinic, J., Adam, G., Phytochemistry 1991, 30, 3717–3720. [12] Sung, T. V., Lavaud, C., Porzel, A., Steglich, W., Adam, G., Phytochemistry 1991, 31, 227–231. [13] Sung, T. V., Steglich, W., Adam, G., Phytochemistry. 1991, 30, 2349–2356. [14] Sung, T. V., Adam, G., Phytochemistry. 1991, 30, 2717– 2720. [15] Maeda, C., Ohtani, K., Kasai, R., Yamasaki, K., Duc, N. M., Nham, N. T., Cu, G. K. Q., Phytochemistry. 1994, 37, 1131–1137. [16] Wanas, A. S., Matsunami, K., Orsuka, H., Desoukey, S. Y., Fouad, M. A., Kamel, M. S., Chem. Pharm. Bull. 2010, 58, 1596–1601. [17] Van Kiem, P., Dat, N. T., Van Minh, C., Lee, J. J., Kim, Y. H., Arch. Pharm. Res. 2003, 26, 594–596. [18] Chang, S. Y., Yook, C. S., Nohara, T., Chem. Pharm. Bull. 1998, 46, 163–165. [19] Kim, J. A., Yang, S. Y., Koo, J. E., Koh, Y. S., Kim, Y. H., Bioorg. Med. Chem. Lett. 2010, 20, 6703– 6707.
www.jss-journal.com
J. Sep. Sci. 2015, 38, 2201–2207
Liquid Chromatography
2207
[20] Liu, X. Q., Chang, S. Y., Park, S. Y., Nohara, T., Yook, C. S., Arch. Pharm. Res. 2002, 25, 831–836.
[28] Qian, S. H., Yuan, L. H., Cao, P., An, S. Y., Cai, X. T., J. Chin. Med. Mater. 2009, 32, 1889–1891.
[21] Liu, X. Q., Chang, S. Y., Yook, C. S., J. Lanzhou Uni. 2006, 42, 86–91.
[29] Liu, P. P., Qu, Y., Dou, D. Q., Kang, T. G., Smith, D., J. Chem. Soc. Pakistan. 2012, 34, 419–423.
[22] Hirai, Y., Murayama, T., Miyakoshi, M., Hirono, S., Isoda, S., Ideura, N., Ida, Y., Shoji, J., Wang, G. S., Xu, J. D., J. Nat. Med. 1995, 49, 462–467.
[30] Toyo’oka, T., J. Chromatogr. Sci. 2008, 46, 233–247.
[23] Liu, P. P., Li, M., Kang, T. G., Dou, D. Q., Smith, D. C., Nat. Prod. Commun. 2010, 5, 1019–1022. [24] Liu, X. Q., Zou, Q. P., Feng, S., Zheng, L. S., Yook, C. S., Drug Evaluation Res. 2010, 33, 95. [25] Nhiem, N. X., Tung, N. H., Van Kiem, P., Van Minh, C., Ding, Y., Hyun, J. H., Kang, H. K., Kim, Y. H., Chem. Pharm. Bull. 2009, 57, 986–989. [26] Nhiem, N. X., Kiem, P. V., Minh, C. V., Tai, B. H., Tung, N. H., Ha do, T., Soung, K. S., Kim, J. H., Ahn, J. Y., Lee, Y. M., Kim, Y. H., Bioorg. Med. Chem. Lett. 2010, 20, 4927–4931. [27] Zhang, B. X., Li, N., Zhang, Z. P., Liu, H. B., Zhou, R. R., Zhou, B. Y., Zou, M. X., Dai, X. H., Xiao, M. F., Liu, X. Q., Fan, X. G., Int. Immunopharmacol. 2011, 11, 1018– 1023.
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[31] Liang, X. M., Jin, Y., Wang, Y. P., Jin, G. W., Fu, Q., Xiao, Y. S., J. Chromatogr. A. 2009, 1216, 2033– 2044. [32] Yang, F. Q., Li, D. Q., Feng, K., Hu, D. J., Li, S. P., J. Chromatogr. A. 2010, 1217, 5501–5510. [33] Qian, Z. M., Zhao, J., Li, D. Q., Hu, D. J., Li, S. P., J. Sep. Sci. 2012, 35, 2725–2734. [34] Han, D. Q., Zhao, J., Xu, J., Peng, H. S., Chen, X. J., Li, S. P., J. Pharmaceut. Biomed. 2013, 72, 223–230. [35] Qian, Z. M., Wan, J. B., Zhang, Q. W., Li, S. P., J. Pharmaceut. Biomed. 2008, 48, 1361–1367. [36] Bjorklund, E., Nilsson, T., Bowadt, S., TrAC, Trends Anal. Chem. 2000, 19, 434–445. [37] Ong, E. S., Woo, S. O., Yong, Y. L., J. Chromatogr. A. 2000, 904, 57–64.
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