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Application of high-speed counter-current chromatography for preparative separation of cyclic peptides from Vaccaria segetalis

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Abstract

Following an initial clean-up step on silica gel, high-speed counter-current chromatography (HSCCC) was used to separate cyclic peptides from an extract of the seeds of Vaccaria segetalis. The two-phase solvent system used for HSCCC separation was composed of petroleum ether–ethyl acetate–methanol–water at an optimized volume ratio of 0.5:3.5:1:5. From 190 mg of crude extract, 38.0 mg of segetalin B and 28.5 mg of segetalin A were obtained with purities of 98.1% and 95.6% as determined by HPLC, respectively. The chemical structures of the target compounds were confirmed by high resolution electrospray ionization time of flight MS (HRESI-TOF-MS) and 1H NMR analyses.

Introduction

Vaccaria segetalis (Neck.) Garcke (Caryophyllaceae) is an annual herb widely distributed in Asia, Europe and North America [1]. Wang-Bu-Liu-Xing, the dried seeds of V. segetalis, is a well-known traditional Chinese medicine and officially listed in the Chinese Pharmacopoeia, which has been widely used to promote diuresis, facilitate milk secretion, activate blood circulation and relieve carbuncles in Chinese medical practice [2], [3]. In phytochemical studies, varieties of compounds were isolated including cyclic peptides, triterpene saponins, alkaloids, phenolic acid, flavonoids and steroids from the seeds of this herb [1], [4]. Cyclic peptides comprise a class of naturally occurring molecules, which exhibit a range of biological activities [5]. Cyclic peptides obtained from the seeds of V. segetalis have become a subject of interest because of bioactivities including oestrogen-like activity [6], [7], vasorelaxant activity [8]. Although these cyclic peptides can be chemically synthesized [9], [10], a large amount of organic solvents were used and also toxic waste in organic synthesis was produced. Therefore, development of an efficient method for the preparation of these compounds from the natural sources is warranted.

High-speed counter-current chromatography (HSCCC), as a support-free liquid–liquid partition chromatography, eliminates the irreversible adsorptive loss of the samples onto solid support and has an excellent sample recovery [11]. Compared to other liquid–liquid techniques, HSCCC has its own advantages such as shorter separation time, wider range of selection of solvent systems and quantitative material recovery. This method has been successfully applied to separate and purify various natural products [12], [13], [14]. In this paper, an efficient new method for separation and purification of cyclic peptides segetalin B and segetalin A (Fig. 1) from the Chinese medicinal plant V. segetalis is reported.

Section snippets

Reagents and materials

Acetonitrile used for HPLC analyses was of chromatographic grade (Yucheng Chemical Factory, Yucheng, China). The water used in mobile phase mixtures was treated with a Milli-Q water purification system (Millipore, USA). Petroleum ether (60–90 °C), ethyl acetate, CHCl3 and methanol for preparation of crude samples and HSCCC separation were all of analytical grades (Juye Chemical Factory, Jinan, China).

The dried seeds of V. segetalis were purchased from a local drug store. The botanical

Optimization of HPLC conditions

The optimum HPLC condition aimed at chromatogram with good resolution of adjacent peaks within a short analysis time, especially when numerous samples were to be analyzed. Several elution systems were tested in HPLC separation of crude sample, such as isocratic elution of methanol–0.1%HCOOH, acetonitrile–0.1%HCOOH, methanol–acetonitrile–0.1%HCOOH, etc. The results indicated that when acetonitrile–0.1%HCOOH (30:70, v/v) was used as mobile phase in isocratic elution mode, the major peaks were

Conclusion

An efficient HSCCC method for separation and purification of two cyclic peptides, segetalin B and segetalin A, from the seeds of V. segetalis was developed by using petroleum ether–ethyl acetate–methanol–water (0.5:3.5:1:5, v/v) as the two-phase solvent system. Segetalin B (38.0 mg) at purity of 98.1% and segetalin A (28.5 mg) at purity of 95.6% could be obtained from 190 mg crude extract in a single run. The compounds obtained can be used as reference substances for chromatographic purposes as

Acknowledgements

Financial supports from the Natural Science Foundation of China (20872083), the Major National S&T Program (no. 2009ZX09308-005), the Key Science and Technology Program of Shandong Province and the Key Science and Technology Program of Institute of Chinese Material Medical, CACMS (ZZ20090107) are gratefully acknowledged.

References (23)

  • H. Morita et al.

    Tetrahedron Lett.

    (1995)
  • H. Itokawa et al.

    Alkaloid: Chem. Pharmacol.

    (1997)
  • H. Morita et al.

    Tetrahedron Lett.

    (1994)
  • H. Morita et al.

    Bioorg. Med. Chem. Lett.

    (2006)
  • P. Sonnet et al.

    Tetrahedron Lett.

    (2003)
  • P. Sonnet et al.

    Tetrahedron Lett.

    (2001)
  • Y. Ito

    J. Chromatogr. A

    (2005)
  • X. Wang et al.

    J. Chromatogr. A

    (2004)
  • S. Yao et al.

    J. Chromatogr. A

    (2007)
  • Q. Tang et al.

    J. Chromatogr. B

    (2008)
  • H. Oka et al.

    J. Chromatogr. A

    (1998)
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