Optimization of cyclotide extraction parameters

Optimization of cyclotide extraction parameters

Phytochemistry Letters 5 (2012) 776–781 Contents lists available at SciVerse ScienceDirect Phytochemistry Letters journal homepage: www.elsevier.com...

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Phytochemistry Letters 5 (2012) 776–781

Contents lists available at SciVerse ScienceDirect

Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol

Optimization of cyclotide extraction parameters Mariamawit Y. Yeshak a,b, Robert Burman a, Camilla Eriksson a, Ulf Go¨ransson a,* a b

Department of Medicinal Chemistry, Division of Pharmacognosy, Uppsala University, Box 574, SE 751 23 Uppsala, Sweden Department of Pharmacognosy, School of Pharmacy, Addis Ababa University, Box 1176, Addis Ababa, Ethiopia



Article history: Received 25 April 2012 Received in revised form 31 August 2012 Accepted 6 September 2012 Available online 18 September 2012

Cyclotides are gene-encoded plant mini-proteins that contain a unique circular and cystine knotted amide backbone. Because of that ultra stable scaffold and the ability to harness a wide variety of sequences and biological activities within the scaffold, cyclotides find interesting potential applications for drug discovery and in agriculture. However, some fundamental knowledge is still missing to exploit these plant compounds, including finding the optimal process of their extraction from plant material. In the current work, the extraction parameters solvent type, time of extraction, number of re-macerations and the plant material to solvent ratio have been compared using the sweet violet (Viola odorata L.) as a model plant. That species is a well-characterized and rich source of cyclotides that contains prototypic cyclotides with different chemical and physical properties. We found that hydroalcoholic solutions of medium polarity give good yield of the cyclotide cocktail. In conclusion, single maceration with 50% MeOH for 6 h at a plant material to solvent ratio of 0.5:10 (g/mL) represents an optimum extraction method. ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Keywords: Plant peptide Cyclotide Extraction Optimization Cystine knot Solvent

1. Introduction Natural products have attracted researchers since ancient times until present day; the main idea is to make use of the structural diversity and biological activity of natural products in drug discovery and design (Clardy and Walsh, 2004; Harvey, 2007; Sasidharan et al., 2011). One class of natural products with a unique structure and a wide range of activities is the family of cyclotides. Cyclotides are gene encoded, mini-proteins derived from plants. They have a unique topology in their head-to-tail cyclized backbone and their core made of three disulfide bonds in a cystine knot motif (Craik, 1999). The motif is characterized by two disulfide bonds that together with the peptide backbone form an embedded ring that is penetrated by a third disulfide bond (Go¨ransson and Craik, 2003; Rosengren et al., 2003). The cyclized backbone in combination with this cystine knot motif is referred to as the cyclic cystine knot (CCK) (Fig. 1) (Craik, 1999). Apart from being a defining feature of the cyclotide family, the CCK also endows these proteins with a well defined 3D structure and an ultra-stable framework which resists treatment with heat, proteases and chemicals (Colgrave and Craik, 2004; Yeshak et al., in manuscript). The discovery of cyclotides dates back to the 1970s when during a Red Cross Relief mission, the Norwegian physician Lorens Gran

* Corresponding author. E-mail address: [email protected] (U. Go¨ransson).

reported that the medicinal plant called ‘‘Kalata-Kalata’’ (Oldenlandia affinis, Rubiaceae) was used during labor to accelerate uterine contractions by the Lulua tribe in Congo (Gran, 1973). A handful of the powdered, dried plant material is boiled with approximately 1 L of water, the decoction is then taken orally or applied directly to the birth canal after contractions have started (Gran et al., 2000). The uteroactive substance in the decoction was found to be a peptide, given the name kalata B1, and its amino acid content was determined (Sletten and Gran, 1973). However, it took more than two decades until the unique structure of kalata B1 was fully understood (Saether et al., 1995). During the early 90s reports about cyclic peptides from plants with the same unique structure as kalata B1’s have started to appear (Gustafson et al., 1994; Scho¨pke et al., 1993; Witherup et al., 1994). The macrocyclic peptides were eventually given the name ‘cyclotides’ (Craik et al., 1999). As can be seen in the Cybase (data base of circular proteins, www.cybase.org.au) the cyclotides are today the largest existing family of circular proteins (Mulvenna et al., 2006; Wang et al., 2008a,b). The potential applications of cyclotides in the discovery include the employment of the ultra-stable framework as a scaffold to unstable drugs, and also the use of cyclotides for the inherent biological/pharmacological properties they exhibit across a wide range of biological systems. Their effects include anti-HIV (Gustafson et al., 1994, 2004; Wang et al., 2008a,b), anthelmintic (Colgrave et al., 2009), insecticidal (Jennings et al., 2001; Pinto et al., 2012) uterotonic (Gran, 1973), neurotensin inhibitory (Witherup et al., 1994), hemolytic (Scho¨pke et al., 1993), trypsin

1874-3900/$ – see front matter ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.phytol.2012.09.001

M.Y. Yeshak et al. / Phytochemistry Letters 5 (2012) 776–781


2. Results and discussion

Fig. 1. Topology of cyclotides illustrating the cyclic cystine knot (CCK). Note that the cyclotides further fall into two main sub-families based on a conceptual topological difference. The first subfamily contains a cis-Pro amide bond that creates a twist to the circular peptide backbone, a graphic representation of the backbone resembling a Mo¨bius strip, consequently it is termed as the Mo¨bius sub-family. On the other hand, cyclotides that lack the cis-Pro do not have the conceptual twist in their circular backbone and are classified under the bracelet subfamily (Craik et al., 1999). Here, cycloviolacin O2 represents the bracelets whereas varv A represents the Mo¨bius. Structures were built from PDB files: cycloviolacin O2 (PDB file: 2KNM) (Go¨ransson et al., 2009) and varv A based on PDB file of kalata B1 (1NB1) (Rosengren et al., 2003). The sequence between the conserved cysteines (marked I–VI) is referred to as a loop.

inhibitory (Hernandez et al., 2000), molluscicidal (Plan et al., 2008), antifouling (Go¨ransson et al., 2004) cytotoxic (Lindholm et al., 2002; Burman et al., 2011), and antimicrobial (Pra¨nting et al., 2010; Tam et al., 1999) activities. To date, the biological sources of cyclotides are plants belonging to Rubiaceae, Violaceae, Cucurbitaceae and Fabaceae (Craik and Conibear, 2011). However, out of these, Violaceae stands out in that all of the so far investigated members have been found to contain cyclotides; moreover, the large share of the cyclotides known today are isolated from Violaceae species. Different laboratories have been using different separation techniques over the past years to isolate cyclotides from plant biomass, yet there is still a need to develop an optimized separation method in order to achieve an optimum yield of the cocktail of cyclotides from a given plant (Colgrave et al., 2010). Similar to all plant-derived natural products, extraction is the first critical step in the isolation of cyclotides from their sources. The aim of this work is hence tuning conditions for extraction of cyclotides so that the extraction process is made efficient in terms of solvent, time and plant material. We have chosen Viola odorata L. (sweet violet as a model plant), a species in which the cyclotide content is well characterized by our group and others (Craik et al., 1999; Trabi et al., 2004; Svanga˚ rd et al., 2003). It is known to express up to 30 different cyclotides (Colgrave et al., 2010). Ninety-one different extracts were prepared by changing parameters around type of solvent, plant material to solvent ratio, time of extraction and number of re-macerations. Yields were analyzed by LC–MS using four cyclotides, namely cycloviolacin O2 (cyO2), cycloviolacin O18 (cyO18), cycloviolacin O19 (cyO19) and varv A as marker compounds.

Extraction parameters that influence the yield of cyclotides have been evaluated and optimized using V. odorata as a model plant. For any given plant, extractive yield is a factor of the extraction solvent, chemical nature of the sample, time and temperature of extraction. Other factors kept constant, the extraction solvent plays a key role in obtaining the target constituents in a desired quality and quantity (Samuelsson et al., 1985; Sasidharan et al., 2011). The choice of the solvent is mainly done based on the chemical properties, i.e. polarity or hydrophobicity of the target compounds. For samples selected on the basis of traditional use, the choice is many times done in a way to, as much as possible, mimic the traditional extraction method (Sasidharan et al., 2011). The traditional healers of the Lulua tribe use decoction with water to extract the cyclotide containing plant Kalata-Kalata (Gran et al., 2000). Although decoction is the method of extraction for most of the traditionally prepared remedies (Samuelsson et al., 1985), organic solvents with medium or high polarity have been most commonly used in laboratories dealing with natural products; and there are evidences where extracts of these solvents could show better activity as compared with the aqueous extracts (Samuelsson et al., 1985). The characterization of complex structures such as peptides

Fig. 2. Analysis of extracts of V. odorata. Ninety-one types of extracts were prepared by varying solvent type (0–100% (v/v), in steps of 10%, of either MeOH or EtOH in water), time of extraction, plant material to solvent ratio and number of remaceration times. Chromatograms shown are examples of LCMS analyses of the extracts, note that a higher yield of cyclotides was obtained with solvents of medium polarity (for e.g. 70% EtOH) and the yield was very low with a polar solvent i.e. water (V. odorata photo courtesy of Dr. Erika Svedlund).


M.Y. Yeshak et al. / Phytochemistry Letters 5 (2012) 776–781

and proteins from aqueous or organic solvent extracts, however, has been overlooked in the past. But during the last decades the interest in this class of natural products has increased with the development of automated and affordable techniques for their isolation (Franco, 2011; O‘Keefe, 2001). Due to the reasons above and the overall amphiphilic nature of cyclotides, that is the combination of hydrophobic patches with charged residues, aqueous and alcoholic solutions of MeOH and EtOH (0–100% (v/v) and in steps of 10%) were chosen as extraction solvents in the current work. Relative yields were compared using LC–MS and the peak integrals of four cyclotides. Yields were normalized towards the highest yield, which was set to one. Selected cyclotides (cyO18, varv A, cyO19 and cyO2) differ in hydrophobicity, demonstrated by the their different retention times (Fig. 2); their content of charged residues. Moreover they represent minor (cyO18 and cyO19) as well as major (varv A and cyO2) cyclotides in V. odorata. The latter are also prototypes for the two main cyclotide subfamilies. Analyses of the LC–MS chromatograms show that hydroalcoholic solutions of medium polarity—between the range of 30% and 60% of MeOH—give comparatively high yields. That was also the case for 20% EtOH: one of the cyclotides i.e. cyO2 actually had a maximum yield at that EtOH concentration (Fig. 3). Solvents of extreme polarity/hydrophobicity in this study, i.e. water and the pure alcohols, resulted in low yields of all of the four cyclotides. For instance, when comparing the relative yield of cyO2 at 20% EtOH (where maximum yield was obtained), extraction with pure EtOH gave only 5% as much while water and pure MeOH gave only 15% and 22%, respectively. The same also holds true for varv A where maximum yield was obtained at 20% and 40% EtOH and the other solvents, i.e. water and either of pure EtOH and MeOH yielded only 19%, 3%, and 11% as much, respectively. High extractive yield of the cocktail, was obtained at 20% EtOH and 50% MeOH.

The major factor affecting the quantitative yield is the ability of the solvent to solubilize the compound of interest from the plant material. The amphiphilic surface of cyclotides is a property of both their hydrophobic regions and their content of charged residues (Fig. 4), hence it is not surprising that higher amount of cyclotides was extracted by solvents of medium polarity and very little/none with highly polar or hydrophobic ones. Once the solvents for the highest possible yield of the cocktail were established, five solvents (water alone, and 30% and 60% of MeOH or EtOH in water) were chosen to optimize the other extraction parameters. Those concentrations were chosen because the total yield of cyclotides was then the highest (30% and 60% of both MeOH and EtOH, Fig. 3). Results from the effect of time of extraction have shown that both the major and minor cyclotides were readily extracted after maceration for 0.5 h, but the yield increased with time of maceration and reached its optimum level at 6 h (Fig. 5). Re-maceration of a marc with fresh menstruum is the common procedure of extraction in order to dissolve out as much of the active ingredients as possible from a given plant material. The number of re-macerations should be optimized, however, since it may not be economical material and time wise to repeatedly extract the same marc if the repetitive extraction does not add sound difference in yield. In this study, extractive yields from V. odorata were compared up to four times of re-maceration. As depicted in Fig. 5, the amount of the extractive yield decreased with each re-maceration. This is expected, as cyclotides would be dissolved out each time a fresh solvent is added, leaving lesser amount of cyclotides within the marc. Moreover, we looked into cyclotide yield after the first maceration in comparison with that of the total yield after the four re-macerations. The water extracts for all cyclotides had the maximum difference in yield between single and four time macerations, for instance the yield of varv A after four macerations was 92% higher than that of a single maceration. On the other

Fig. 3. Extractive yields from V. odorata by eleven different hydroalcoholic solvents as compared by taking four cyclotides as marker compounds. W: water; M1–M10: 10% MeOH–100% MeOH; E1–E10: 10–100% EtOH.

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Fig. 4. Surface structures of cycloviolacin O2, O18, O19 and varv A. The color scheme of the models is: yellow for hydrophobic residues (Ala, Leu, Ile, Pro, Trp and Val), blue for cationic residues (Arg and Lys), red for anionic residues (Glu) and white for the rest of the amino acids. 3D structure of cycloviolacin O2 was built from the PDB file (2KNM) (Go¨ransson et al., 2009). SWISS-MODEL (Guex and Peitsch, 1997; Arnold et al., 2006; Schwede et al., 2003) was used to build the 3D structures of the other three cyclotides based on PDB files of cycloviolacin O2 (for cycloviolacin O18 and cycloviolacin O19) and kalata B1 (1NB1) (Rosengren et al., 2003) for varv A. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

hand, re-maceration is of less importance for the 30% MeOH extract. The yield of four time macerates for cyO2 and cyO18 added only 9% to the yield of the single maceration. According to the current results, repetitive extraction is beneficiary when using water as an extraction solvent while a single maceration can suffice when hydroalcohols are used for extraction. Then, the plant material to solvent ratio was evaluated for extraction. The cocktail of cyclotides were extracted at an optimum level when the ratio was 0.5 g/10 mL solvent (Fig. 5). The yield of cyO2 and varv A decreased at the ratio of 2 g/10 mL solvent whereas that of cyO18 and cyO19 remained similar between 0.5 and 2 g/10 mL. This phenomenon may be exploited in cases of targeting selected cyclotides from the plant. The mixtures of water and alcohol that have been shown to give the best yields in the current work represent low cost,

Fig. 5. Amount of cyclotide extracted under various conditions of extraction, i.e. at different extraction times (A), different number of macerations (B) and at different plant material to solvent ratio (C). W: water; M3, M6: 30 respective 60% MeOH in water; E3, E6: 30 respective 60% MeOH in water; R1–R4: repetition (re-maceration) 1–4.

environmentally friendly solvents. Moreover, these aqueous based extracts are also amenable for loading on e.g. ion exchange or reverse phase resins (adjustment of pH or dilution with water may be required). Other solvents have been used for cyclotide extraction, for example CH2Cl2:MeOH (1:1 (v/v)) (Craik et al., 1999; Pinto et al., 2012) or aqueous buffers (Poth et al., 2011).


M.Y. Yeshak et al. / Phytochemistry Letters 5 (2012) 776–781

Judged by the current results, there is a risk of using low polarity solvents, as they do not extract the most hydrophilic cyclotides. Indeed, that extraction bias may lead to that more potent cyclotides escape discovery (Colgrave et al., 2008). Another point to take into account is that plants contain highly lipohilic constituents such as photosynthetic pigments, which would be obtained in higher amounts if one uses non-polar solvents for extraction. Cost of extraction solvent and environmental factors would also be questions when using solvents such as CH2Cl2, particularly in industrial scale extraction. In conclusion, hydroalcoholic solutions of medium polarity may be tuned to give good yields of cyclotides from V. odorata. A single 6-h maceration at a ratio of 0.5 g plant material/10 mL solvent was found to be an optimum method to extract V. odorata cyclotides if hydroalcoholic solvents are used. High yield of the cocktail was obtained at 20% EtOH and 50% MeOH, however, extraction with 50% MeOH may be preferred since it is a relatively safe solvent, not attractive for user abuse, and because it is widely employed in natural product laboratories.

3. Experimental 3.1. General experimental conditions LC–MS analyses were carried out on extracts of V. odorata, which were prepared under varying conditions of extraction, i.e. type of solvent, plant material: solvent ratio, time of extraction and number of re-maceration. A total number of 273 (91  3) samples were analyzed for the 91 different conditions of extraction. The LC– MS runs were performed on a nanoAcquity UPLC system coupled to a Q-Tof micro MS (Waters, Milford, MA). An aliquote of 0.5 ml was injected onto a C-18 nanoAcquity UPLC1 column (75 mm  150 mm i.d., 1.7 mm), operated with a linear gradient from 99.5% water in 0.5% MeCN (buffer A) to 99.5% MeCN in 0.5% water (buffer B) over 22 min at a flow rate of 0.3 mL/min. The capillary voltage on the mass spectrometer was maintained at 4200 V. Data were acquired between the mass range of 500– 2000 Da. Four cyclotides, which have been previously known to be contained in V. odorata were selected as marker peptides: relative yields at the different conditions were determined using the integral of each marker peptide as calculated in MassLynx (Waters). 3.2. Plant material Dried aerial parts of V. odorata L., were obtained from Alfred Galke GmbH (Gittelde, Germany) (Lot No. 6922). The plant material was harvested on the 5th November 2004. 3.3. Extraction conditions The dried aerial parts of V. odorata were finely ground; extracts in varying extraction solvents were prepared by macerating the plant material (0.5 g) in 10 mL of water, and at increasing concentrations of MeOH in steps of 10% (between 10% and 100%) for 24 h. The extractions were repeated in a similar manner for EtOH. Four hydroalcoholic solutions (30% and 60% of MeOH and EtOH each) were then chosen as solvents for the rest of the extraction procedures because of high yield of cyclotides observed at those concentrations. The procedures followed for preparing extracts with varying times of extraction, plant material to solvent ratio and re-maceration are summarized in Fig. 2. All extracts were dried in a SpeedVac (SpeedVac1 Plus, SC 100A, Savant) after filtration. The dry extracts were re-dissolved in 10% MeCN, centrifuged, and supernatants were taken for LC–MS analysis.

Acknowledgments The work was supported by a grant from the Swedish International Development Cooperation Agency/The Department for Research Cooperation (7500723501). Work in the UG laboratory is supported by the Swedish Research Council (621-2007-5167) and the Swedish Foundation for Strategic Research (F06-0058). References Arnold, K., Bordoli, L., Kopp, J., Schwede, T., 2006. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22, 195–201. Burman, R., Herrmann, A., Tran, R., Kivela¨, J.E., Lomize, A., Gullbo, J., Go¨ransson, U., 2011. Cytotoxic potency of small macrocyclic knot proteins: structure-activity and mechanistic studies of native and chemically modified cyclotides. Org. Biomol. Chem. 9, 4306–4314. Clardy, J., Walsh, C., 2004. Lessons from natural molecules. Nature 432, 829–837. Colgrave, M.L., Craik, D.J., 2004. 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