Cloud-Point Extraction Combined with Liquid Chromatography for the Determination of Ergosterol, a Natural Product with Diuretic Activity, in Rat Plasma, Urine, and Faeces
Ergosterol from many medicinal fungi has been demonstrated to possess a variety of pharmacological activities in vivo and in vitro. A new method based on cloud-point extraction has been developed, optimized and validated for the determination of ergosterol in rat plasma, urine and faeces by liquid chromatography. The non-ionic surfactant Triton X-114 was chosen as the extract solvent. The chromatographic separation was performed on an Inertsil ODS-3 analytical column with a mobile phase consisting of methanol and water (98？:？2, v/v) at a flow rate of 1？mL/min. The methodology was validated completely. The results indicated good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. The method was successfully applied to the pharmacokinetic studies of ergosterol in rats. The results indicate that the ergosterol levels in feces are much higher than those in plasma and urine of the rat. 1. Introduction Ergosterol is one of the best-known steroids, which exists widely in many medicinal fungi such as Polyporus umbellatus, Cordyceps sinensis, and Hypsizigus marmoreus [1–3]. We have recently reported the ergosterol has diuretic activity from Polyporus umbellatus [4]. Ergosterol has also been reported to possess cytotoxic activity [5] and anti-inflammatory activity [3]. Despite the fact that ergosterol showed multiple pharmacological activities, several pharmacokinetic and biochemical aspects of this compound remain unclear. However, the final effect of the drug in vivo might be influenced by many factors, such as body-and/or cell-compartment distribution, drug metabolism, lipophilicity, membrane permeability, and protein binding. So, these multiple pharmacological activities of ergosterol make it worth carrying out further study on pharmacokinetic properties and elimination pathway of ergosterol. A number of methods have been reported for the quantification of ergosterol in raw materials, such as high performance liquid chromatography-ultraviolet detection (HPLC-UV) [1, 6–11], high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) [12, 13], and gas chromatography-mass spectrometry (GC-MS) [14–16]. To the best of our knowledge, there is no information describing the quantification of ergosterol in biological samples such as rat or human plasma, urine, and faeces. In addition, the effects on the elimination pathway of ergosterol have not been reported. In general, preclinical research including metabolism and pharmacokinetics of herbal medicine components are of great importance in
References
[1]
Y. Y. Zhao, X. L. Cheng, Y. Zhang, Y. Zhao, R. C. Lin, and W. J. Sun, “Simultaneous determination of eight major steroids from Polyporus umbellatus by high-performance liquid chromatography coupled with mass spectrometry detections,” Biomedical Chromatography, vol. 24, no. 2, pp. 222–230, 2010.
[2]
J. W. Bok, L. Lermer, J. Chilton, H. G. Klingeman, and G. H. N. Towers, “Antitumor sterols from the mycelia of Cordyceps sinensis,” Phytochemistry, vol. 51, no. 7, pp. 891–898, 1999.
[3]
K. Yasukawa, T. Aoki, M. Takido, T. Ikekawa, H. Saito, and T. Matsuzawa, “Inhibitory effects of ergosterol isolated from the edible mushroom Hypsizigus marmoreus on TPA-induced inflammatory ear oedema and tumour promotion in mice,” Phytotherapy Research, vol. 8, no. 1, pp. 10–13, 1994.
[4]
Y. Y. Zhao, R. M. Xie, X. Chao, Y. Zhang, R. C. Lin, and W. J. Sun, “Bioactivity-directed isolation, identification of diuretic compounds from Polyporus umbellatus,” Journal of Ethnopharmacology, vol. 126, no. 1, pp. 184–187, 2009.
[5]
H. Matsuda, J. Akaki, S. Nakamura et al., “Apoptosis-inducing effects of sterols from the dried powder of cultured mycelium of Cordyceps sinensis,” Chemical and Pharmaceutical Bulletin, vol. 57, no. 4, pp. 411–414, 2009.
[6]
Y. Y. Zhao, Y. Zhao, R. C. Lin, and W. J. Sun, “HPLC determination of ergosta-4, 6, 8(14), 22-tetraen-3-one in Polyporus umbellatus,” Chinese Journal of Pharmaceutical Analysis, vol. 29, no. 9, pp. 1579–1581, 2009.
[7]
D. Abramson and D. M. Smith, “Determination of ergosterol in canola (Brassica napus L.) by liquid chromatography,” Journal of Stored Products Research, vol. 39, no. 2, pp. 185–191, 2002.
[8]
J. P. Yuan, J. H. Wang, X. Liu, H. C. Kuang, and S. Y. Zhao, “Simultaneous determination of free ergosterol and ergosteryl esters in Cordyceps sinensis by HPLC,” Food Chemistry, vol. 105, no. 4, pp. 1755–1759, 2007.
[9]
L. M. Seitz and Y. Pomeranz, “Ergosterol, ergosta-4,6,8(14),22-tetraen-3-one, ergosterol peroxide, and chitin in ergoty barley, rye, and other grasses,” Journal of Agricultural and Food Chemistry, vol. 31, no. 5, pp. 1036–1038, 1983.
[10]
S. P. Li, P. Li, C. M. Lai et al., “Simultaneous determination of ergosterol, nucleosides and their bases from natural and cultured Cordyceps by pressurised liquid extraction and high-performance liquid chromatography,” Journal of Chromatography A, vol. 1036, no. 2, pp. 239–243, 2004.
[11]
C. Arnezeder, W. Koliander, and W. A. Hampel, “Rapid determination of ergosterol in yeast cells,” Analytica Chimica Acta, vol. 225, no. 1, pp. 129–136, 1989.
[12]
M. Varga, T. Bartók, and á. Mesterházy, “Determination of ergosterol in Fusarium-infected wheat by liquid chromatography-atmospheric pressure photoionization mass spectrometry,” Journal of Chromatography A, vol. 1103, no. 2, pp. 278–283, 2006.
[13]
J. V. Headley, K. M. Peru, B. Verma, and R. D. Robarts, “Mass spectrometric determination of ergosterol in a prairie natural wetland,” Journal of Chromatography A, vol. 958, no. 1-2, pp. 149–156, 2002.
[14]
Z. Parsi and T. Górecki, “Determination of ergosterol as an indicator of fungal biomass in various samples using non-discriminating flash pyrolysis,” Journal of Chromatography A, vol. 1130, no. 1, pp. 145–150, 2006.
[15]
B. O. Axelsson, A. Saraf, and L. Larsson, “Determination of ergosterol in organic dust by gas chromatography-mass spectrometry,” Journal of Chromatography B, vol. 666, no. 1, pp. 77–84, 1995.
[16]
F. Q. Yang, K. Feng, J. Zhao, and S. P. Li, “Analysis of sterols and fatty acids in natural and cultured Cordyceps by one-step derivatization followed with gas chromatography-mass spectrometry,” Journal of Pharmaceutical and Biomedical Analysis, vol. 49, no. 5, pp. 1172–1178, 2009.
[17]
K. Kobashi and T. Akao, “Relation of intestinal bacteria to pharmacological effects of glycoside,” Bioscience and Microflora, vol. 16, no. 1, pp. 1–7, 1997.
[18]
D. H. Kim, K. W. Yu, E. A. Bae, H. J. Park, and J. W. Choi, “Metabolism of kalopanaxsaponin B and H by human intestinal bacteria and antidiabetic activity of their metabolites,” Biological and Pharmaceutical Bulletin, vol. 21, no. 4, pp. 360–365, 1998.
[19]
J. Shen and X. Shao, “Cloud point extraction and its applications to the separation and analysis of organic components,” Progress in Chemistry, vol. 18, no. 4, pp. 482–487, 2006.
[20]
H. Ma, J. You, and Y. Liu, “Cloud-point extraction combined with HPLC for determination of larotaxel in rat plasma: a pharmacokinetic study of liposome formulation,” Journal of Separation Science, vol. 35, no. 12, pp. 1439–1446, 2012.
[21]
M. D. Rukhadze, S. K. Tsagareli, N. S. Sidamonidze, and V. R. Meyer, “Cloud-point extraction for the determination of the free fraction of antiepileptic drugs in blood plasma and saliva,” Analytical Biochemistry, vol. 287, no. 2, pp. 279–283, 2000.
[22]
Y. Y. Zhao, L. B. Yang, M. C. Wang et al., “1β-hydroxylfriedelin, a new natural pentacylic triterpene from the sclerotia of Polyporus umbellatus,” Journal of Chemical Research, no. 11, pp. 699–701, 2009.
[23]
J. X. Li, J. Li, L. Z. Xu, S. L. Yang, and Z. M. Zou, “Studies on chemical constituents of Cordyceps sinensis (Berk) Sacc,” Chinese Pharmaceutical Journal, vol. 38, no. 7, pp. 499–501, 2003.
[24]
U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), and Center for Veterinary Medicine (CVM), Guidance for Industry-Bioanalytical Method Validation, Center for Drug Evaluation and Research (CDER), Rockville, Md, USA, 2001.
[25]
Y. Y. Zhao, X. L. Cheng, Y. Zhang et al., “A fast and sensitive HPLC-MS/MS analysis and preliminary pharmacokinetic characterization of ergone in rats,” Journal of Chromatography B, vol. 878, no. 1, pp. 29–33, 2009.
[26]
Y. Y. Zhao, X. L. Cheng, F. Wei, et al., “Ultra performance liquid chromatography coupled with electrospray and atmospheric pressure chemical ionization-quadrupole time-of-flight mass spectrometry with novel mass data collection technique: determination and pharmacokinetics, tissue distribution and biliary excretion study of ergone in rat,” Journal of Separation Science, vol. 35, no. 13, pp. 1619–1629, 2012.
