Elsevier

Journal of Chromatography B

Volume 965, 15 August 2014, Pages 231-237
Journal of Chromatography B

Simulated moving bed purification of flaxseed oil orbitides: Unprecedented separation of cyclolinopeptides C and E

https://doi.org/10.1016/j.jchromb.2014.06.037Get rights and content

Highlights

  • Enrichment of orbitides from a flaxseed oil extract using a three-zone simulated moving bed.

  • Investigation of flow rate effect (feed, desorbent, and extract) on orbitide separation.

  • The use of a single low toxicity solvent that can be easily recovered and recycled.

  • The use of rapid chromatography to monitor SMB purification offline.

Abstract

The purification and enrichment of most natural products with potential pharmaceutical applications has been performed mainly employing conventional batch-mode chromatographic processes. There is a growing interest in use of simulated moving bed (SMB) chromatography for natural product enrichment as this method enables conservation of mobile phase, while increasing productivity of chromatography medium. SMB increases yield while decreasing cost. Cyclolinopeptides C ([1-9-NaC],[1-MetO]-CLB, 3) and E ([1-8-NaC],[1-MetO]-CLE, 8) were extracted as a mixture from flaxseed oil and then enriched using a three-zone simulated moving bed. The current research extends the SMB technology to enrichment of cyclolinopeptides (CLs), a group of biologically active hydrophobic cyclic peptides that occur in flaxseed oil. Of interest are [1-9-NaC],[1-MetO]-CLB (3) and [1-8-NaC],[1-MetO]-CLE (8) that provide synthetic scaffolds for modified CLs. The influence of flow rate (feed, desorbent, and extract) on the separation of [1-9-NaC],[1-MetO]-CLB (3) and [1-8-NaC],[1-MetO]-CLE (8) was investigated.

Introduction

Orbitides are cyclic oligopeptide natural products that occur in plants [1]. Cyclolinopeptides (CLs) are a group of biologically active orbitides that are found in flaxseed, flax roots, and flaxseed products (Fig. 1). To date, twenty CLs, each possessing eight or nine amino acid residues, have been reported [2], [3], [4], [5], [6], [7], [8], [9], [10]. The biological activity of some CLs and their analogs is summarized in a recent review [11]. Specifically, CLs 1, 2, 4, 8 and 9 suppress immune responses in several assays [4], [11], [12], [13], [14]. Interestingly, other than 1, the rest of the aforementioned CLs (2, 4 and 9) are structurally related to 3 and 8, only differing in the oxidation state of the methionyl residue that plays a role in their chromatographic separation. The biological activity of 4 and 9 is intriguing when compared to that of their precursors 3 and 8, respectively [6]. This biological activity may lead to the use of these compounds as pharmaceuticals.

The commercial pharmaceutical potential of biologically active orbitides requires either economical synthesis or purification procedures. A synthetic approach could be very costly due to complexity of reactions, number of reaction steps required, the cost of reagents, and the efficiency of each reaction step. For example, the synthesis of hydrophobic peptides that contain a high portion of Ala, Val and Ile on solid phase media is associated with aggregation during peptide strand elongation. Aggregation may cause termination or deletion of the peptide sequence. Solid phase synthesis of plant orbitides would face this difficulty. Isolation of orbitides from flaxseed (Linum usitatissimum) [14] is difficult as these compounds occur as complex mixtures and in low concentrations of just 0.2% in flaxseed oil [15]. Reaney et al. [16] developed methods for concentrating flax orbitides. Although the orbitide fraction was amenable to batch chromatography, the yield was low. Therefore, we explored the use of simulated moving bed (SMB) technology for high throughput separation of a binary mixture of 3 and 8.

SMB technology, developed in the 1960s, utilizes a multi-column chromatographic countercurrent separation process that allows for continuous injection of sample, with continuous separation and simultaneous recovery of enriched products through two separate streams [17]. Solid (stationary phase) movement is simulated by periodic switching of inlet (feed and desorbent) and outlet (raffinate and extract) ports of the unit, both in the direction of solvent flow as illustrated in Fig. 2A and B [18]. Complete separation of a binary mixture is achieved upon establishment of equilibrium in flow rates between mobile and stationary phases such that the more retained analyte (A) is carried by the solid phase to the extract port, whereas the less retained analyte (B) is carried by the mobile phase to the raffinate outlet (Fig. 2A and B). Hence, SMB chromatography enables enhanced throughput with lower solvent consumption over a shorter period of time compared to batch chromatography [19], [20]. This chromatographic technique has been successfully used in separation of hydrocarbons and fine chemicals (particularly enantiomers) in scale-up operations that results in high productivity and low solvent consumption [21]. The technology has also found use in the purification of proteins [22] and sugar alcohols [23]. The major disadvantage of SMB technology is its limitation in purification of multi-component mixtures, especially natural products with pharmaceutical potential. The technology efficiently separates binary mixtures under conditions of constant temperature, pressure, and mobile phase composition. More elaborate separations have been designed including ternary [24] and gradient separations [25]. Nonetheless, the limitations of SMB are compensated by higher efficiency in terms of separation of binary mixtures at higher concentrations at which they would normally not be resolved, and achieving such difficult separations with lower solvent consumption than batch chromatography [19], [20].

In the current chromatographic separation, an 8-column 3-Zone SMB system in a 3-2-3 configuration comprising 3, 2 and 3 columns in Zones 1, 2 and 3, respectively, was used (Fig. 2A and B). Each of the three Zones plays a significant role in the purification of a given binary mixture. This configuration utilizes three pumps, that is, a pump for each stream of feed, desorbent, and extract. The separation of the mixture occurs between Zones 2 and 3 comprising 2 and 3 columns, respectively. Zone 2 is located between the feed inlet and extract outlet (between Zones 1 and 3). In this Zone, the less retained adsorbate B must be desorbed and carried by the mobile phase through to Zone 3 where it exits the system through the raffinate stream. On the other hand, the more retained adsorbate A is adsorbed and enriched in Zone 2 and is carried by the solid phase to Zone 1 (comprising 3 columns and is located between the desorbent inlet and extract outlet) where it exits the system through the extract stream with the aid of the extract pump [26].

In this configuration, a shutoff valve between Zones 3 and 1 is closed to ensure that all adsorbate B exits the system after Zone 3, thus preventing adsorbate B from entering Zone 1 [26]. Regeneration of the solid phase with fresh stream of solvent occurs in Zone 1 where adsorbate A is washed out through the extract stream. The enhancement of 3 and 8 was conducted using a semi-industrial SMB-Unit with 8-columns divided in 3 Zones with a 3-2-3 configuration as shown in Fig. 2.

Section snippets

Analytical methods

Crude CL extract (Fig. 3), enriched peptide mixtures from flash column chromatography, and SMB enriched fractions from extract and raffinate streams (Fig. 4) were analyzed by an Agilent 1200 series HPLC system (Agilent Technologies Canada, Mississauga, ON) equipped with a quaternary pump, a degasser, an autosampler, a thermostated column compartment and diode array detector (wavelength range 190–300 nm). Chromatographic separation was performed on a Chromolith® SpeedROD RP-18e column (50 mm × 4.6 mm

Results and discussion

Preparative SMB units are composed of multi-chromatographic columns ranging between six and sixteen, and may use three, four or five pumps. The columns are arranged in series and are connected through a valve block that enables different inlet (feed and desorbent) and outlet (extract and raffinate) ports to allow continuous flow of sample and mobile phase, and withdrawal of the enriched products [29]. Given that the solid bed is fixed, movement is simulated by periodic shifts of inlet and

Conclusion

We have developed a method using SMB technology for enrichment of CLs 3 and 8. This is the first demonstration of SMB use in the separation of large quantities of CLs. Cyclolinopeptides 3 and 8 are valuable orbitides that occur in flaxseed and are readily oxidized or reduced to other known peptides (2, 4, 7, and 9). The peptides may also be used as scaffolds for synthesis of methionine modified peptides [28]. Although orbitides display remarkable biological activities [11], their potential

Acknowledgements

Financial support for the authors’ work was obtained from the Strategic Research Program and Agricultural Development Fund (Research Grant to Martin Reaney) of the Saskatchewan Ministry of Agriculture. The Canada Foundation for Innovation and Agriculture and AgriFood Canada provided funding for purchase of the SMB system and associated equipment making up the University of Saskatchewan Bioprocessing Pilot Plant. Additional funds for operation were realized through Genome Canada support of

References (31)

  • H. Morita et al.

    Bioorg. Med. Chem. Lett.

    (1997)
  • H. Morita et al.

    Tetrahedron

    (1999)
  • T. Matsumoto et al.

    Phytochemistry

    (2001)
  • A. Gorski et al.

    Transplant Proc.

    (2001)
  • P.D. Jadhav et al.

    Food Chem.

    (2013)
  • E.R. Francotte et al.

    J. Chromatogr. A

    (1997)
  • J. Andersson et al.

    J. Chromatogr. A

    (2006)
  • S. Abel et al.

    J. Chromatogr. A

    (2002)
  • T.B. Jensen et al.

    J. Chromatogr. A

    (2000)
  • C.M. Olivia et al.

    J. Chromatogr. B

    (2012)
  • M. Juza

    J. Chromatogr. A

    (1999)
  • C. Langel et al.

    J. Chromatogr. A

    (2009)
  • P.G. Arnison et al.

    Nat. Prod. Rep.

    (2013)
  • H.P. Kaufmann et al.

    Chem. Ber.

    (1959)
  • T. Matsumoto et al.

    Tennen Yuki Kagobutsu Toronkai Koen Yoshishu

    (2001)
  • Cited by (9)

    View all citing articles on Scopus
    View full text