J. Sep. Sci. 2006, 29, J. Zhao et al. 2609

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1 J. Sep. Sci. 2006, 29, J. Zhao et al Jing Zhao 1 Xiao-Qi Zhang 2 Shao-Ping Li 1 Feng-Qing Yang 1 Yi-Tao Wang 1 Wen-Cai Ye 2 1 Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau, P. R. China 2 Institute of TCM & Natural Medicine, Jinan University, Guangzhou, P. R. China Original Paper Quality evaluation of Ganoderma through simultaneous determination of nine triterpenes and sterols using pressurized liquid extraction and high performance liquid chromatography A method combining HPLC and pressurized liquid extraction was developed for simultaneous quantification of nine components, including eight triterpenes (ganoderic acid A, ganoderic acid Y, ganoderic acid DM, ganoderol A, ganoderol B, ganoderal A, methyl ganoderate D and ganoderate G) and a sterol (ergosterol), in Ganoderma. The determination was achieved by using a Zorbax ODS C 18 analytical column ( mm id, 5 lm) and gradient elution with diode-array detection. All calibration curves showed good linearity (r 2 A0.9997) within the test ranges. The developed method showed good repeatability for the quantification of the nine investigated components in Ganoderma with intra- and inter-day variations of less than 2.4% and 4.1%, respectively. The validated method was successfully applied to quantify the nine components in two species of Ganoderma, i.e. G. lucidum and G. sinense, used as Lingzhi in China. Furthermore, hierarchical clustering analysis based on the nine components in HPLC profiles from the tested 11 samples showed that chemical characteristics were significantly different between G. lucidum and G. sinense, which suggested that clinical investigation should be performed so as to ensure the safety and efficacy of medication. Keywords: Ganoderma / HPLC / Pressurized liquid extraction / Sterols / Triterpenes / Received: May 2, 2006; revised: June 29, 2006; accepted: June 30, 2006 DOI /jssc Introduction Lingzhi (in Chinese), one of the well-known traditional Chinese medicines, has been deemed an elixir of life for thousands of years. About 98 species belonging to the family Ganodermataceae could be identified in China, a few of which have long been used as traditional Chinese medicine [1]. It is recorded that the fruiting bodies from two species of Ganoderma, i. e. Ganoderma lucidum and Ganoderma sinense, are used as Linzhi for replenishing Qi to calm the mind, and to relieve coughing and dyspnea [2]. Its beneficial clinical effects in patients with hepatitis, hyperglycemia, chronic bronchitis, cancer, muscular dystrophy, arteriosclerosis, hypertension, hypercholesterolemia, and leukopenia have been confirmed in pharmacological studies in recent years [3 8]. Modern Correspondence: Dr. Shao-Ping Li, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau SAR, P. R. China. spli@umac.mo Fax: Abbreviations: DAD, diode array detector; PLE, pressurized liquid extraction research has revealed that Lingzhi contains a variety of chemical ingredients, including carbohydrates, organic germanium, triterpenes, adenosine, alkaloids, numerous mineral elements, and amino acids [9 12]. Among these various phytochemicals, triterpenes, and related compounds have multiple pharmacological activities [13 21]. However, chemical components are quite varied among different species of Ganoderma [22, 23]. Therefore, quantitative analysis of triterpenes is very important for ensuring the efficacy and quality of Ganoderma. HPLC has been used for quantitative determination of triterpenes in Ganoderma [19, 24 26]. However, because of the structural similarity of triterpenes, few methods have been reported for their simultaneous analysis in Ganoderma. The present paper describes the development of a method combining HPLC and pressurized liquid extraction (PLE) for simultaneous determination of nine components, including eight triterpenes (ganoderic acid A, ganoderic acid Y, ganoderic acid DM, ganoderol A, ganoderol B, ganoderal A, methyl ganoderate D, and ganoderate G) and a sterol (ergosterol) in Ganoderma. The chromatographic characteristics of two species of Ganoderma,

2 2610 J. Zhao et al. J. Sep. Sci. 2006, 29, Ganoderma lucidum and Ganoderma sinense, were also compared. 2 Experimental 2.1 Materials and chemicals Acetonitrile for LC was purchased from Merck (Darmstadt, Germany). Deionized water was prepared using a Millipore Milli Q-Plus system (Millipore, Bedford, MA). Ergosterol was purchased from Sigma (St. Louis, USA). Ganoderic acid A, ganoderic acid Y, ganoderic acid DM, ganoderol A, ganoderol B, ganoderal A, methyl ganoderate D, and ganoderate G were separated and purified by ourselves. The purity of all compounds was A98%, which was tested by HPLC. The structures (Fig.1) were confirmed by their UV, IR, MS, and NMR data. Six batches of Ganoderma lucidum (Leyss. ex Fr.) Karst. and five batches of Ganoderma sinense Zhao, Xu et Zhang were obtained from different locations in China (Table 1). Voucher specimens of Ganoderma were deposited at the Institute of Chinese Medical Sciences, University of Macau, Macau, China. Table 1. Summary for the tested samples of Ganoderma. No. Code Samples Sources 1 GL-1 Ganoderma lucidum Yunnan Province 2 GL-2 Ganoderma lucidum Local market 3 GL-3 Ganoderma lucidum Sichuan Province 4 GL-4 Ganoderma lucidum Local market 5 GL-5 Ganoderma lucidum Anhui Province 6 GL-6 Ganoderma lucidum Guangdong Province 7 GS-1 Ganoderma sinense Yunnan Province 8 GS-2 Ganoderma sinense Local market 9 GS-3 Ganoderma sinense Local market 10 GS-4 Ganoderma sinense Sichuan Province 11 GS-5 Ganoderma sinense Local market 2.2 Pressurized liquid extraction Sample preparation was performed by PLE in a Dionex ASE 200 system (Dionex Corp., Sunnyvale, CA, USA). Powdered (ca. 2 mm) Ganoderma (1.0 g) was placed in an 11-mL stainless steel extraction cell, and extracted under optimized extraction conditions (see Section 3.1). Then the extract was transferred to a 50-mL volumetric flask, made up to volume with methanol, and filtered through a 0.45-lm Econofilter prior to injection into the HPLC system. The parameters affecting the extraction efficiency in PLE include the solvent type, static extraction time, number of extraction cycles, and temperature and were optimized using univariate design. The pressure applied did not have a significant effect on the extraction efficiency as it was used to keep the solvent in the liquid state [27 29]. Thus MPa (1500 psi, system default value) was set as the extraction pressure. The best PLE conditions for preparing ergosterol, ganoderma alcohols, and ganoderma acids were obtained. Figure 1. Structures of the investigated compounds in Ganoderma. 2.3 HPLC analysis Analyses were performed on an Agilent Series 1100 liquid chromatograph, equipped with a vacuum degasser, a quaternary pump, an autosampler, and a diode array detector (DAD), connected to an Agilent ChemStation running ChemStation software. A Zorbax ODS C 18 column ( mm id, 5 lm) and a Zorbax ODS C 18 guard column ( mm id, 5 lm) were used. Solvents constituting the mobile phase were water (A) and methanol (B). The elution conditions applied were: 0 10 min, 52% B isocratic; min, linear gradient 52 53% B; min, linear gradient 53 85% B; min, linear gradient % B; min, 100% B isocratic; and, finally, the reconditioning step of the column was 52% B isocratic for 15 min. The flow rate was 1.0 ml/min and the injection volume was 10 ll. The system was operated at 258C, and monitored at 275 nm for ergosterol and at 254 nm for the other analytes.

3 J. Sep. Sci. 2006, 29, PLE followed by HPLC to determine triterpenes and sterols in Ganoderma Calibration curves The methanol stock solution of standards was prepared and diluted with methanol to appropriate concentrations for the establishment of calibration curves. At least six concentrations of the eight analytes (ganoderic acid A, ganoderic acid Y, ganoderic acid DM, ganoderol A, ganoderal A, methyl ganoderate D, ganoderate G, and ergosterol) mixture and ganoderol B solution were injected in triplicate, respectively, and then the calibration curves were constructed by plotting the peak areas versus the concentration of each analyte. 2.5 Limits of detection and quantification The stock solutions containing reference compounds were diluted with methanol to appropriate concentrations, and an aliquot of the diluted solutions was injected into HPLC for analysis. The limits of detection (LOD) and quantification (LOQ) for each analyte were determined at a signal-to-noise ratio (S/N) of about 3 and 10, respectively. Figure 2. Influence of selected factors including solvent type, temperature, static extraction time, and number of cycles on the PLE extraction of nine investigated compounds in Ganoderma. Conditions: The investigated levels of selected factors, methanol, ethanol, and ethyl acetate as solvent type, 808C, 1008C, and 1208C as temperature, 5 min, 10 min, and 15 min as static extraction time, as well as 1, 2, and 3 as number of cycles, were defined as L1, L2, and L3, respectively. To determine one of the parameters, the others were set at the system default value (temperature, 1008C; pressure, MPa (1500 psi); static extraction time, 5 min; flush volume, 60%; and extraction cycle, 1). The sample was Ganoderma lucidum from Anhui. 2.6 Precision and accuracy Intra- and inter-day variations were chosen to determine the precision of the method. The known concentrations of nine standard solutions were tested. For intra-day variability tests, the standard solutions were 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 relative standard deviations. For every calibration curve, the calibration concentrations were back-calculated from the peak area of the analytes. The deviation from the nominal concentration was defined as accuracy. The recovery was determined by adding known amount of individual standards to 50 mg of GL-5. The mixture was extracted and analyzed using the method mentioned above. The extract was dried under vacuum and the residue was transferred to a 5 ml volumetric flask which was made up to its volume with extraction solvent and filtered through a 0.45-lm filter before analysis. The quantity of each analyte was subsequently obtained from the corresponding calibration curve. 2.7 Data analysis Hierarchical clustering analysis was performed by SPSS 11.5 for Windows (SPSS Inc, Chicago, IL, USA), which comprises a number of procedures graphical, statistical, reporting, processing, and tabulating procedures that enable simple and rapid data evaluation. A method called between groups linkage was applied, and Cosine, which is a pattern similarity measure, was selected as measure for hierarchical clustering analysis. 3.2 Validation of the developed method The linearity, regression, and linear ranges of the nine analytes were determined using the developed HPLC method. The correlation coefficient (r 2 A0.9997) values indicated appropriate correlations between the investigated compound concentrations and their peak areas within the test ranges. The limits of detection and quani 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 3 Results and discussion 3.1 Optimization of PLE procedure The optimization of the PLE procedure was performed using GL-5 which contained most of the analytes. The parameters, including the type of solvent (methanol, ethanol, and ethyl acetate), temperature ( C), number of extraction cycles (1 3), and static extraction time (5 15 min), were studied by using the univariate approach (Fig. 2). The total peak area of the nine analytes was used as the marker for evaluation of extraction efficiency. The recovery efficiency for the PLE procedure was determined by performing consecutive pressurized liquid extractions on the same sample under optimized PLE conditions, until no investigated compounds were detected by the analysis. The recovery was calculated on the basis of the total amount of individual investigated components, which was more than 98% for the first-time extraction. Taking into account the results of optimization and recovery experiments, the conditions of the PLE method proposed were: solvent, methanol; temperature, 1008C; static extraction time, 5 min; pressure, MPa (1500 psi); 60% of the flush volume for one cycle and one extraction time.

4 2612 J. Zhao et al. J. Sep. Sci. 2006, 29, Table 2. Linear regression data of investigated compounds from Ganoderma. Analytes LOD LOQ Linear range Linear regression data Slope Intercept r 2 Ganoderic acid A Ganoderic acid Y Ganoderic acid DM Ganoderol A Ganoderol B Ganoderal A Methyl ganoderate D Ganoderate G Ergosterol Squares of correlation coefficients for the standard curves. Mean and percentage of relative standard deviation (RSD) for three replicates. Table 3. Intra- and inter-day variability for the assay of ganoderma alcohols, ganoderma acids, and ergosterol in Ganoderma. Analytes Conc. Found RSD Intra-day Accuracy b) Found RSD Inter-day Accuracy Ganoderic acid A Ganoderic acid Y Ganoderic acid DM Ganoderol A Ganoderol B Ganoderal A Methyl ganoderate D Ganoderate G Ergosterol Mean and relative standard deviation (RSDs) for three replicates. b) Accuracy = 1006mean of measured concentration/nominal concentration. tification were less than 1.72 lg/ml and 5.33 lg/ml, respectively (Table 2), and the overall intra- and inter-day variations (RSD) of the nine analytes were less than 2.4% and 4.1%, respectively (Table 3). The developed method had good accuracy with overall recovery of 98.1% 100.8% for the analytes (Table 4). The results indicated that this HPLC method was precise, accurate, and sensitive for quantitative determination of nine components including ganoderma acids, ganoderma alcohols, and ergosterol in Ganoderma. 3.3 Identification and quantitation of investigated compounds Chromatograms of a PLE extract from two species of Ganoderma are shown in Fig. 3. Identification of the investigated compounds was carried out by comparison of their retention time and UV spectra with those obtained injecting standards under the same conditions or by spiking Cordyceps samples with stock standard solutions. By using the calibration curve of each investigated compound, the contents of the nine compounds in the two species of Ganoderma were determined. Table 5 summarizes the results. In general, both G. lucidum and G. sinense contain a much higher amount of ergosterol, which is one of the chemical components from mycelium cells and is commonly used as a mould growth indicator [30]. However, the content of triterpenes was obviously different between the two species of Ganoderma. Under these analytical conditions, the amounts of the eight triterpenes in G. sinense were not above their LOD (Fig. 3C), which was significantly different from the levels in G. lucidum. Therefore, the clinic efficacy of the two species of Ganoderma should be carefully investigated because it is considered that triterpenes and related compounds are their bioactive components. Although it was reported that the amounts of chemical components vary considerably among different species of Ganoderma [22, 23], no data were presented for G. sinense. Recently, six ganoderic acids in G. sinense were determined by RP-HPLC

5 J. Sep. Sci. 2006, 29, PLE followed by HPLC to determine triterpenes and sterols in Ganoderma 2613 Table 4. Recoveries for the assay of ganoderma alcohols, ganoderma acids, and ergosterol in Ganoderma. Analytes Added (lg) Found (lg) Recovery Mean l SD RSD Ganoderic acid A Ganoderic acid Y Ganoderic acid DM Ganoderol A Ganoderol B Ganoderal A Methyl ganoderate D Ganoderate G Ergosterol The data were presented as average of three determinations. Recovery = 1006(amount found original amount)/amount spiked. RSD = 1006SD/mean. Table 5. Contents (mg/g) of ganoderma alcohols, ganoderma acids, and ergosterol in Ganoderma. Analytes Ganoderma lucidum Ganoderma sinense GL-1 GL-2 GL-3 GL-4 GL-5 GL-6 GS-1 GS-2 GS-3 GS-4 GS-5 Ganoderic acid A Ganoderic acid Y + Ganoderol B + b) 0.17 c) Ganoderic acid DM Ganoderol A Ganoderal A Methyl ganoderate D Ganoderate G Ergosterol Undetectable. b) Beyond lower limit of linear range. c) Calculated as ganoderic acid Y. [26], but the analytes are different from ours. Using the eight peaks' area of nine analytes in the HPLC chromatograms, hierarchical cluster analysis of 11 samples was performed. A method known as between groups linkage was applied, and Cosine was selected as measure. Figure 4 shows 11 samples of Ganoderma, which are divided into two main clusters, G. lucidum and G. sinense, respectively. The result demonstrate that the two species of Ganoderma are significantly different. It is thought that interaction of multiple chemical compounds contributes to the therapeutic effects of Chinese medicines. However, the overall clinical efficacy of the two species of Ganoderma used as Linzhi has not been determined. Therefore, comparison of chemical components and pharmacologii 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

6 2614 J. Zhao et al. J. Sep. Sci. 2006, 29, Figure 4. Dendrograms resulting from eight peaks' area of nine analytes on HPLC chromatograms of the tested 11 Ganoderma samples using average linkage between groups of hierarchical cluster analysis. GL1 GL6 and GS1 GS5 are G. lucidum and G. sinense derived from different locations, respectively. cal activities of the two species of Ganoderma will be helpful in elucidating the mechanism of therapeutic effects and active components of Linzhi. 4 Concluding remarks Figure 3. HPLC chromatograms of PLE extract of (A) mixed standards, (B) Ganoderma lucidum, and (C) Ganoderma sinense. Conditions: solvent, methanol; temperature, 1008C; static extraction time, 5 min; pressure, MPa (1500 psi); flush volume, 60%; cycle, 1; and extraction number, 1. Analyses were performed on an Agilent Series 1100 liquid chromatograph, equipped with a vacuum degasser, a quaternary pump, an autosampler, and a DAD detector, connected to Agilent ChemStation software. A Zorbax ODS C 18 column ( mm id, 5 lm) and a Zorbax ODS C 18 guard column ( mm id, 5 lm) were used. Solvents that constituted the mobile phase were A (water) and B (methanol). The elution conditions applied were: 0 10 min, 52% B isocratic; min, linear gradient 52 53% B; min, linear gradient 53 85% B; min, linear gradient % B; min, 100% B isocratic; and, finally, the reconditioning step of the column was 52% B isocratic for 15 min. The flow rate was 1.0 ml/min and the injection volume was 10 ll. The system was operated at 258C, and monitored at 254 nm. 1, ganoderic acid A; 2+3, ganoderic acid Y+ ganoderol B; 4, ganoderic acid DM; 5, ganoderol A; 6, methyl ganoderate D; 7, ganoderate G; 8, ganoderal A; 9, ergosterol. The method described provides efficient simultaneous determination of ganoderma acids, ganoderma alcohols, and ergosterol using pressurized liquid extraction and HPLC, which has good precision and recovery. Chemical variation is obvious among the two species of Ganoderma, which suggests that the pharmacological activities should be compared between the two species of Ganoderma in order to ensure their safety and efficacy in clinical use. We are grateful to Mr. Wan Jianbo of our institute for his expert technical assistance. 5 References [1] Zhao, J. D., Zhang, X. Q., Flora Fungorum Sinicorum, Vol. 18: Ganodermataceae, Scientific Publishing House, Beijing [2] Pharmacopoeia Commission of PRC, Eds., Pharmacopoeia of the People s Republic of China, Chemical Industry Press, Beijing [3] Miyazaki, T., Nishijima, M., Chem. Pharm. Bull. 1981, 29, [4] Franz, G., Planta Med. 1989, 55, [5] Wang, S. Y., Hsu, M. L., Hsu, H. H., Tzeng, C. H., et al., Int. J. Cancer 1997, 70, [6] Kim, D. H., Shim, S. B., Kim, N. J., Jang, I. S., Biol. Pharm. Bull. 1999, 22,

7 J. Sep. Sci. 2006, 29, PLE followed by HPLC to determine triterpenes and sterols in Ganoderma 2615 [7] Lin, Z. B., Recent Advances in Chinese Herbal Drugs Action and Uses, Scientific Publishing House, Beijing [8] Lin, Z. B., Modern Research on Ganoderma lucidum, 2nd Ed., Beijing Medical University Press, Beijing [9] Chiu, S. W., Wang, Z. W., Leung, T. M., Moore, D., Food Chem. Toxicol. 2000, 38, [10] Wu, T. S., Shi, L. S., Kuo, S. C., J. Nat. Prod. 2001, 64, [11] Cheung, H. Y., Ng, C. W., Hood, D. J., J. Chromatogr. A 2001, 911, [12] Mau, J. L., Lin, H. C., Chen, C. C., Food Res. Int. 2001, 34, [13] El-Mekkawy, S., Meselhy, M. R., Nakamura, N., Tezuka, Y., et al., Phytochemistry 1998, 49, [14] Sonoda, Y., Sekigawa, Y., Sato, Y., Chem. Pharm. Bull. 1988, 36, [15] Komoda, Y., Shimizu, M., Sonoda, Y., Sato, Y., Chem. Pharm. Bull. 1989, 37, [16] Koyama, K., Imaizumi, T., Akiba, M., Kinoshita, K., et al., Planta Med. 1997, 63, [17] Kohoda, H., Tokumoto, W., Sakamoto, K., Fuji, M., et al., Chem. Pharm. Bull. 1985, 33, [18] Min, B. S., Gao, J. J., Hattori, M., Lee, H. K., Kim, Y. H., Planta Med. 2001, 67, [19] Min, B. S., Nakamura, N., Miyashiro, H., Bae, K. H., Hattori, M., Chem. Pharm. Bull. 1998, 46, [20] Min, B. S., Gao, J. J., Nakamura, N., Hattori, M., Chem. Pharm. Bull. 2000, 48, [21] Gao, J. J., Min, B. S., Ahn, E. M., Nakamura, N., et al., Chem. Pharm. Bull. 2002, 50, [22] Nishitoba, T., Sato, H., Shirasu, S., Sakamura, S., Agric. Biol. Chem. 1986, 50, [23] Di, X., Chan, K. K. C., Leung, H. W., Huie, C. W., J. Chromatogr. A 2003, 1018, [24] Ma, L., Wu, F., Chen, R. Y., Acta Pharm. Sin. 2003, 38, [25] Gao, J. J., Nakamura, N., Min, B. S., Hirakawa, A., et al., Chem. Pharm. Bull. 2004, 52, [26] Wang, X. M., Yang, M., Guan, S. H., Liu, R. X., et al., J. Pharm. Biomed. Anal. 2006, 41, [27] Björklund, E., Bøwadt, S., Nilsson, T., Trends Anal. Chem. 2000, 19, [28] Ong, E. S., Woo, S. O., Yong, Y. L., J. Chromatogr. A 2000, 904, [29] Richter, B. E., Jones, B. A., Ezzell, J. L., Porter, N. L., et al., Anal. Chem. 1996, 68, [30] Gutarowska, B., Zakowska, Z., Int. Biodeterior. Biodegrad. 2002, 49,