Microwave-assisted extraction of cyclotides from Viola ignobilis

Accepted Manuscript Microwave-Assisted Extraction Of Cyclotides From Viola Ignobilis Mohsen Farhadpour, Hossein Hashempour, Zahra Talebpour, Nazanin A-Bagheri, Mozhgan Sadat Shushtarian, Christian W. Gruber, Alireza Ghassempour PII:

S0003-2697(15)00545-X

DOI:

10.1016/j.ab.2015.12.001

Reference:

YABIO 12246

To appear in:

Analytical Biochemistry

Received Date: 12 August 2015 Revised Date:

10 November 2015

Accepted Date: 3 December 2015

Please cite this article as: M. Farhadpour, H. Hashempour, Z. Talebpour, N. A-Bagheri, M.S. Shushtarian, C.W. Gruber, A. Ghassempour, Microwave-Assisted Extraction Of Cyclotides From Viola Ignobilis, Analytical Biochemistry (2016), doi: 10.1016/j.ab.2015.12.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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MICROWAVE-ASSISTED EXTRACTION OF CYCLOTIDES FROM VIOLA

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IGNOBILIS Mohsen Farhadpour1, Hossein Hashempour2, Zahra Talebpour3, Nazanin A-Bagheri1,

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Mozhgan Sadat Shushtarian1, Christian W. Gruber4, Alireza Ghassempour*,1

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Tehran, Iran.

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Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Evin,

Department of Chemistry, Faculty of Basic sciences, Azarbaijan Shahid Madani

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University, Tabriz, Iran.

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Departmen of Chemistry, Alzahra University, Vanac, Tehran, Iran.

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Center for

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Schwarzspanierstrasse 17, 1090 Vienna, Austria.

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First author

Medical University of Vienna,

[email protected], [email protected]

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Pharmacology,

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Physiology and

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Second author

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[email protected]

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Third author

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[email protected]

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Fourth author 1

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[email protected]

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Fifth author

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[email protected]

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sixth author

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[email protected]

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*

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, [email protected] , [email protected]

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Tel: +98-21-22431598 and Fax: +98-21-22431598

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Correspondence and reprints

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Abstract

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Cyclotides are an interesting group of circular plant peptides. Their unique three dimensional

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structure, defended by head-to-tail circular backbone chain containing three disulfide bonds,

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confers them stability in thermally, chemically and enzymatic conditions. Their unique stability

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in violent conditions creates an idea about the possibility of using harsh extraction methods such

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as microwave assisted extraction (MAE) without any changes of structures. MAE has been

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introduced as a potent extraction method for extraction of natural compounds, but it seldom be

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used for peptide and protein extraction. In this work, microwave irradiation was applied to the

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extraction of cyclotides. Procedure was performed in various steps using microwave instrument

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under different conditions. High performance liquid chromatography (HPLC) and matrix assisted

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laser desorption ionization-time of flight (MALDI-TOF) results shows stability of cyclotide

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structures on microwave radiation. The influential parameters including time, temperature and

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the ratio of solvents, which are affecting the MAE potency, were optimized. Optimal conditions

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were obtained at 20 minutes of irradiation time, 1200 W system power in 60 °C and

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methanol/water at the ratio of 90:10 (v/v) as solvent. The comparison of MAE results with

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maceration extraction are shown that there are similarities between cyclotides sequences and

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extraction yields.

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Keywords

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Viola ignobilis, Violaceae, microwave-assisted extraction, vigno cyclotides, cyclopeptides

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1 Introduction

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Cyclotides are a large family of mini-proteins that have been isolated from plants of the

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Rubiaceae, Violaceae, Cucurbitaceae, Fabaceae, Solanaceae and Poaceae families [1-7].

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Cyclotides are in size between 27-37 amino acids. They are head-to-tail macrocyclic peptides

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containing six conserved cysteine residues that form three disulfide bounds in a cystine-knotted

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arrangement [8, 9]. This interesting three-dimensional structure confers them exceptional

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stability against chemical, enzymatic and thermal treatment, which would typically degrade

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linear proteins of similar size. Due to their stability cyclotides have been considered as promising

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template for peptide engineering and pharmaceutical applications [10]. Besides they exhibit a

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intrinsic biologically activities such as uterotonic [4, 5, 11], anti-HIV [12-14], antitumor [15],

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anthelmintic [16], insecticidal [17, 18], cytotoxicity [19, 20], and immunosuppressive properties

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[21-23]. Cyclotides are as interesting compounds for using in drug design. Pharmaceutical

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application of them is related to their uses in drug design as a template for grafting unstable

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peptide’s sequences to give them exceptional stability [24, 25].

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Similar to all plant-derived natural compounds, extraction is the critical step in the isolation of

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cyclotides from plant tissue. In previous studies, methanol and dichloromethane [26, 27], or

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combination of water and alcohols [28] have been used most commonly as solvents for

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extraction of cyclotides using maceration. This method usually endures over 24 hours to obtain

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efficient extraction yields. Hence other methods that could reduce extraction time are needed.

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Recently, the technique microwave-assisted extraction (MAE) has become attractive for the

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extraction of thermo labile components because of its properties, such as little solvent

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consumption and fast extraction efficiency with high yield [29, 30]. Microwave radiation has

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been shown to increase the temperature of aqueous solutions by dielectric heating [31]. In the

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case of proteins, prolonged irradiation using microwaves has also been proposed to cause protein

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unfolding and disruption of tertiary structure and enzyme activity [32]. For instance, Jun-Jun

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Guan et al. reported on the effect of microwave on isolation of soy protein, revealed that under

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the a long time exposure to high-power microwave irradiation, non-covalent bonds and the

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disulfide bonds in soybean protein were broken. But protein was disaggregated and unfolded,

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suggesting that forces handle protein structures, such as hydrophobic forces, can be destroyed by

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microwave to a certain extent as a result, but the less time will be spent on the microwave-

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assisted soy protein–saccharide graft reactions, and therefore the higher yields and the fewer by-

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products would be obtained [33]. However, Shin et al. observed no significant structural changes

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of human hair proteins using microwaves (600 W) for a specified irradiation period (5–120 min)

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due to the high cysteine content and hence great stability of hair proteins [34]. In many cases,

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direct interaction of protein or enzyme structure with the electromagnetic field of the microwave

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reactor were attributed to non-thermal effects [35-37] and evaluation of tertiary structure of

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trypsin and BSA by molecular mechanics calculations revealed that the employed

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electromagnetic field strength under laboratory microwave conditions without any significant

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heating, is too low to induce conformational changes in proteins or enzymes [38, 39].

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Since cyclotides are ultrastable mini-proteins, the aim of this work was to evaluate the

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effectiveness of cyclotide extraction from Viola ignobilis using microwave irradiation, and to

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determine overall yields of extracted cyclotides due to electromagnetic field effects.

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2 Materials and Methods

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2.1 Plant collection and extraction

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At first the dried plant material of Viola ignobilis (100 g) that was collected from the region of

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East Azarbaijan (Iran), was powdered. Then the dichloromethane extraction at room-temperature

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for 24 h as an appropriate extraction process was used to remove the non-polar components.

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After filtering through a cotton wool plug, the pulp plant was collected, dried and prepared for

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cyclotides extraction by maceration and microwave (MW) extraction methods using polar

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solvents.

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2.2 Microwave extraction

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MAE was performed using a Multiwave 3000 (Anton Paar Co., Graz, Austria) equipped with 16

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closed vessels; the maximum operating temperature and pressure were 190˚C and 20 bars. For

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this method, 1 g of the powdered plant material was weighed directly into each pre-cleaned MW

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vessel and 30 ml of different ratios of methanol/water were added to each vessel. Following

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incubating periods of 4, 8, 12, 16, 20 and 24 min with different conditions (Table 1), the mixture

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was cooled and solvent was separated from the solid material using a separating funnel. The

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resulting solutions were concentrated on a rotary evaporator and freeze dried; the crude extract

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was dissolved in 0.1 M NH4HCO3 buffer (pH = 8.1) and used for solid-phase extraction (SPE) of

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cyclotides. For comparison of yields of this method with maceration, conventional maceration

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was performed under optimum conditions, i.e. 30 mL of different ratios of methanol: were used

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as extraction solvents for 1 g of plant material over 24 h similar to the method described by

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Yeshak et al. for extraction of cyclotides from Viola odorata [28].

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2.3 Solid phase extraction and Chromatography analysis

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C18 SPE cartridges (Macherey-Nagel, Chromabond; 0.5 g; 3 mL) were activated with 3 mL of

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MeOH and equilibrated with 3 mL of water. The dried crude extract - dissolved in 0.1 M

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NH4HCO3 buffer - was loaded onto the C18 cartridge and washed with 10 mL of 0.1 M

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NH4HCO3 buffer, followed by 10 mL of 20% Ethanol. The cyclotide-containing fractions were

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collected by elution with 50 and 80 % Ethanol, respectively, and freeze dried. After dissolving in

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5% acetonitrile, they were analyzed using RP-C8 HPLC (Knauer, Eurospher I 5 µm;

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250 × 4.6 mm; 100 Å) using a Knauer 1200 series unit, with the following mobile phases;

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solvent A: 100% water containing 0.05% TFA; and solvent B: water/acetonitrile (10/90, v/v)

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containing 0.05% TFA. A flow rate of 1 mL/min was used for the analysis. The gradient

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employed started at 95% A and remained at this point for 5 min before changing to 85% over 10

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min. A linear gradient starting from 85 to 0% A was then employed during a 25-min period,

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remaining at 100% B for a further 5 min. Afterwards, the column was re-equilibrated to 95% A

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in 10 min. Peptide elution was monitored by UV-VIS detection at 218 nm.

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2.4 Mass Spectrometry analysis

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To investigate effects of microwave irradiation on the structure of the cyclotides extracted

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samples were analyzed by ThermoFisher Scientific ESI-MS system equipped with ion trap mass

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spectrometer (model LCQ, mass range m/z 10–2000) and a nanospray ionization interface

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(Bremen, Germany). Instrument control, data acquisition and processing were conducted by the

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Xcalibur software. Typical positive ESI-MS conditions were: capillary voltage 4.0 kV and

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skimmer cone voltage 20 V and MALDI-TOF/TOF using a 4800 Analyzer (AB Sciex, Canada)

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operated in reflector positive ion mode, acquiring 2000–3600 total shots per spectrum with a

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laser intensity set between 3200 and 3800. MS experiments were carried out using α-cyano-

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hydroxyl-cinnamic acid matrix at a concentration of 5 mg mL−1 dissolved in 50 % (v/v)

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acetonitrile. An aliquot of 0.5 µL of each sample was mixed with 3 µL of matrix solution and the

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mixture was spotted onto the target plate [40, 41].

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3 Results and Discussion

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3.1 Optimization of microwave extraction

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The MAE was carried in a four-step procedure to obtain the optimum condition for extraction of

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cyclotides. Cyclotides are characterized both by hydrophilic and hydrophobic surface patches.

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Due to this amphiphilic property, the selection of an appropriate extraction solvent is crucial. In

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2012, Yeshak et al. reported a protocol using a mixture of aqueous and organic solvents for

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efficient cyclotide extraction. They could show that a mixture of water: methanol (50:50) solvent

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would be the optimum condition for extraction of cyclotides from Viola odorata by maceration

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for 6 hours without application of re-maceration [28]. Application of a combination of aqueous

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and alcoholic solutions would be considered as the solvent of choice for extraction of cyclotides.

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In addition, the combination of aqueous and alcoholic solutions can enhance the extraction yield,

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because of their enhanced microwave absorbing capacity due to their dielectric properties [42].

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For these reasons, different ratios of methanol: water mixtures were chosen for extraction of

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cyclotides from Viola ignobilis. As initial step, different ratios of methanol/water were used as

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solvent at 500 W microwave irradiation intensity during 4 min at 50 ºC.

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HPLC and LC-MS analyses of these samples (Suppl. Fig. S1, A, B) indicated that in comparison

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with the results of maceration extraction (Fig. 1 A, B), no cyclotides were extracted from MAE

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using the initial protocol. However, as it is illustrated in chromatogram c in Fig. S1A, the solvent

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ratio of 90:10 (v/v) of methanol/water resulted in the appearance of more abundant peaks (as

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determined by the increased area under curve) in MAE extraction. Likely the small amount of

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water in solvent facilitates better heating and increased mass transfer of cyclotides into the

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solvent by penetrating the plant cell wall. In addition HPLC analysis of extracts in different

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ratios of methanol: water during maceration method indicated that the overall extraction

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efficiency was improved by raising the amount of methanol from 70 to 90% (Fig. 2).

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Accordingly, this optimized solvent ratio was chosen to continue with the next steps. In the

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closed vessel system, possibly elevated temperature above the boiling point of solvent, resulted

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in the improved extraction efficiencies, since desorption of analyte from the plant tissue

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increased. Additionally, the capacity of solvents to solubilize analytes will increase at higher

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temperature by decreasing the surface tension and solvent viscosity [42]. However, an increasing

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temperature is associated with increased in pressure that could raise safety concern. Therefore in

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the second step of our protocol, the experiment was performed at 60 ºC to analyze the effect of

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temperature on the extraction procedure without destroying of analytes. Similarly, HPLC

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chromatograms of these samples (Suppl. Fig. S2) indicated no cyclotides appeared in the extract.

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Nevertheless, the intensity (area under curve) of peaks, in comparison to the first protocol, has

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been increased.

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In the third protocol, the analysis of the HPLC chromatograms (Suppl. Fig. S3) indicated that

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better extraction efficiency was obtained with a microwave power of 1200 W, applied for 4 min

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irradiation time at 60ºC with the solvent 90:10 (v/v) methanol/water. Using this method, similar

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to maceration, four HPLC peaks that appear to be cyclotides were observed at the same retention

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times, but with less intensity. This indicated that MAE under optimum solvent ratio, temperature

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and irradiation power, is capable of extracting cyclotides without any changes to their

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hydrophobicity and hence to their structure. In addition, all molecular weights of cyclotides that

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had been analyzed with MALDI-TOF indicated no breakdown of covalent bonds such as

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disulfide bonds using high-power microwave irradiation (Fig. 3).

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Microwave power and irradiation time are two factors which influence each other to a great

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extent. A combination of moderate power with longer exposure may be a good approach. In

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general, reports showed that the optimum power and irradiation time depend on the type of

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analytes, and maximum extraction was improved by raising microwave power during short time

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exposure [42]. To enhance the yield of extraction, different irradiation time periods were applied

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in next protocol.

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HPLC chromatograms indicated that increasing irradiation time to 20 min at 1200 W raised

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extraction yield of cyclotides (Fig. 4). Irradiation time greater 20 min led to a decreased

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extraction yield and this it could be related to the decrease in cyclotides solubility resulting from

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microwave irradiation. This phenomenon provides evidence of changing the hydrophobic and

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hydrophilic patches of cyclotide structure compared to the study performed on soybean protein

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due to increasing of irradiation time (heating time) by Sadeghi et al. [43]. Hence the 20 min

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irradiation time was selected as the optimum time for cyclotide extraction with MAE (Fig. 4).

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The fraction that was eluted with 50:50 ethanol: water from SPE, was injected in to preparative

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C8 column and the second peak was collected. Its purity was confirmed with analytical HPLC

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and MALDI-TOF analysis identified its molecular weight as varv A cyclotide (Suppl. Fig. S4).

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For quantification of extraction yields we used this model peptide exemplarily.

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To quantify the amount of extracted cyclotides by the two methods, a stock solution (0.5 mg/mL)

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was prepared by dissolving varv A in Milli Q water and accurately determining the concentration

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using the UV absorbance of the solution at 280 nm (ε = 6410 M

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solutions ranging in concentration from 1 to 300 µg /mL of varv A was prepared by dilution of

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the stock solution in 5% Acetonitrile and then a calibration curve was obtained by plotting their

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peak areas against concentration of each solution (Suppl. Fig. S5). The peak areas and amount

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of extracted cyclotides from both methods were calculated by use of this calibration curve and

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were summarized in Table 2 according to the results from the HPLC analysis. The obtained

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results surprisingly indicated that in microwave extraction method, three peaks were extracted to

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cm

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a lesser amount than the corresponding peaks obtained from maceration, but the other one was

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extracted at an amount of about three times more than during the conventional maceration

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method.

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3.2 HPLC result

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The comparison of peak area for extracted vigno cyclotides in each extraction method (Fig. 2)

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showed that the extraction of vigno 3 and vigno 4 (peak 4) was increased considerably by

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microwave irradiation. The biggest differences are between first and fourth peaks of cyclotides

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related to vigno 1-2 and vigno 3-4, respectively. Hence investigation of sequence differences in

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their structure can be useful for the explanation of observed differences in MAE. The sequence

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diversity of vigno cyclotides are shown in Table 3. Vigno 1 and vigno 2 have alanine and glycine

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in loop 2 and 3, respectively, and this alanine in vigno 3 and 4 is replaced by valine. This

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difference caused to increase the dipole moment of vigno 3 and 4 in loop 2 because of changing

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methyl to isopropyl group. In addition, replacing of alanine for glycine in vigno 4, caused to

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increase its dipole moment, slightly in loop 4 because of the changing hydrogen atom to methyl

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group. The other difference is in loop 6, which valine residue in vigno 1 and 2 replaced by

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threonine to make vigno 3 and 4. The dipole moment of threonine residue is greater than valine.

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So, vigno 3 and 4 have a greater dipole moment in loop 6 in comparison to vigno 1 and 2. All of

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these differences make dipole moments of vigno 3 and 4 greater than vigno 1 and 2. Hence,

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vigno 3 and 4 in fourth peak have more interaction with microwave irradiation, so, the yield of

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extraction by microwave is more than a maceration method. Other differences in vigno 2 can be

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effective on yield of extraction very slightly. Vigno 5 from a third HPLC peak, compared to

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vigno 1 and 2, have a valine in loop 2 and a glycine in loop 3, and the average of reducing and

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increasing of dipole moments could not alter its yield of extraction by microwave. Varv A from

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the second HPLC peak is similar to vigno 3 exception of replacing glycine for serine in loop 3.

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This changing maybe reduces interaction of varv A with microwave irradiation. Therefore the

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yield of extraction by MAE does not change considerably.

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3.3 MALDI-TOF analysis

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The MALDI-TOF spectra for the 50% eluted fractions from SPE with maceration and MAE have

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been shown in Fig. 4. According to the spectra, mass weight patterns are identical in two

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different extraction methods. This observation confirms the lack of significant changing in the

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primary structures of extracted peptides. All vigno cyclotides were extracted without

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considerable oxidation in their sequences. So, based on this study, microwave irradiation can be

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used for the extraction of cyclotides by retaining their intact structures.

11 4 Conclusions

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The novelty of this study is applying microwave assisted extraction for the analysis of cyclotides

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for the first time. The obtained result confirmed that MAE can be useful for discovering of new

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cyclotides with new sequences. For example, the maceration method could not get enough yields

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of extraction for identification of an unknown cyclotide in Viola ignobilis. Finally, this work

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proposed the using of microwave irradiation for extraction in cyclotides discovery parallel to the

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maceration method to keep chance for obtaining new sequences.

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Figure captions

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Figure 1: A) HPLC analysis of sample which has been eluted by 50% ethanol from SPE

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obtained by the maceration method using 90:10 methanol/water for 24 h, that peak1,2,3 and 4

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are vigno1& vigno 2, varv A , vigno 5, and vigno 3 & vigno 4 respectively. B) LC-MS analysis

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of extracted cyclotides,

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Figure 2: Extractive yields from V. ignobilis with maceration by three different hydroalcoholic

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solvents; 70, 80, 90% methanol.

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Figure 4: HPLC analysis of samples which have been eluted by 50% ethanol from SPE after

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extraction by microwave at conditions (step 4): 90:10 methanol/water, 1200 W power, 60 ˚C and

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A) 4 min B) 12 min C) 20 min D) 24 min as irradiation times

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Figure 3: MALDI-TOF spectrum of vigno cyclotides extracted by A) maceration and B)

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microwave-assisted extraction with optimum condition; 90:10 methanol/water, 1200 W power,

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60 ˚C and 20min as irradiation time

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[19] R. Burman, A. Herrmann, R. Tran, J.-E. Kivelä, A. Lomize, J. Gullbo, U. Göransson, Cytotoxic potency of small macrocyclic knot proteins: Structure–activity and mechanistic studies of native and chemically modified cyclotides, Org. Biomol. Chem. 9 (2011) 4306-4314. [20] P. Lindholm, U. Göransson, S. Johansson, P. Claeson, J. Gullbo, R. Larsson, L. Bohlin, A. Backlund, Cyclotides: A Novel Type of Cytotoxic Agents 1 PL and UG contributed equally to this manuscript, Mol. Cancer Therapeutics 1 (2002) 365-369. [21] C. Grundemann, J. Koehbach, R. Huber, C.W. Gruber, Do plant cyclotides have potential as immunosuppressant peptides?, J. Nat. Prod. 75 (2012) 167-174.

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[24] R. Clark, N. Daly, D. Craik, Structural plasticity of the cyclic-cystine-knot framework: implications for biological activity and drug design, J. Biochem. 394 (2006) 85-93. [25] S.T. Henriques, D.J. Craik, Cyclotides as templates in drug design, Drug Discov. Today 15 (2010) 57-64.

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[26] U. Goransson, E. Svangard, P. Claeson, L. Bohlin, Novel strategies for isolation and characterization of cyclotides: the discovery of bioactive macrocyclic plant polypeptides in the Violaceae, Curr. Protein Pep. Sci. 5 (2004) 317-329. [27] P. Claeson, U. Göransson, S. Johansson, T. Luijendijk, L. Bohlin, Fractionation protocol for the isolation of polypeptides from plant biomass, J. Nat. Prod. 61 (1998) 77-81. [28] M.Y. Yeshak, R. Burman, C. Eriksson, U. Göransson, Optimization of cyclotide extraction parameters, Phytochem. Lett. 5 (2012) 776-781. [29] B. Kaufmann, P. Christen, Recent extraction techniques for natural products: microwave‐assisted extraction and pressurised solvent extraction, Phytochem. Anal. 13 (2002) 105-113.

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[30] C. Sparr Eskilsson, E. Björklund, Chromatography A, 902 (2000) 227-250.

Analytical-scale

microwave-assisted

extraction,

J.

[31] B.W. Renoe, Microwave assisted extraction, Am. Lab. 26 (1994) 34-34.

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[34] S. Shin, A. Lee, S. Lee, K. Lee, J. Kwon, M.Y. Yoon, J. Hong, D. Lee, G.-H. Lee, J. Kim, Microwave-assisted extraction of human hair proteins, Anal. Biochem. 407 (2010) 281-283. [35] Y.S. Hafez, A.I. Mohamed, F.M. HEWEDY, G. Singh, Effects of microwave heating on solubility, digestibility and metabolism of soy protein, J. Food Sci. 50 (1985) 415-417.

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Tables Table 1 : Microwave extraction process microwave extraction Temperature process (°C) Step1 50 Step 2 60 Step 3 60 Step 4 60

Solvent (% methanol ) 70 ,80 , 90 90 90 90

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2

3

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4

5

6

11

12

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10

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9

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7

8

Irradiation time (min) 4 4 4 8, 10 ,12 16, 20

13

14

17

Microwave power (W) 500 500 1200 1200

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1

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Table 2: Comparison of peak area and amount of extracted cyclotides by microwave and maceration protocols Peak area and cyclotide amount

Cyclotides which were eluted by 50% Ethanol from SPE Peak 1 (vigno 1 & 2)

Peak 2 (varv A)

Peak 3 (vigno 5)

Peak 4 (vigno 3 & 4)

µg/ga

Area

µg/g

Area

µg/g

Area

µg/g

1005346

101.84

630794

61.94

547071

54.18

2243650

233.76

Maceration 2407125 249.71 1128395 116.73 1306701 a micrograms of extracted cyclotide per grams of dried plant b 1200 W power for 20 min at 60 ˚C by use of 90:10 methanol/water as solvent

133.75

826692

83.86

Microwave b

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1

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Area

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Table 3: Sequence diversity of cyclotides from Viola ignobilis Peak number

amino acid sequence

loops

1

2

3

4

5

6

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Cyclotide

G-LPLCGETCAGGTCNTP--GCSCS-WPVCVRN

vigno 1 1

GSSPLCGETCAGGTCNTP--GCSCS-WPVCVRD

vigno 2 2

G-LPVCGETCVGGTCNTP--GCSCS-WPVCTRN

vigno 5

3

G-LPLCGETCVGGTCNTP--GCSCG-WPVCVRN

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varv A

4

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G-LPLCGETCVGGTCNTP--GCSCS-WPVCTRN

vigno 3

G-LPLCGETCVGGTCNTP--ACSCS-WPVCTRN

vigno 4 CYS

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1

I

19

II

III

IV V

VI

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Figure 1

50TOTAL; #1-8 RT: 0.00-0.16 AV: 8 NL: 1.81E4 T: + p ESI Full ms [700.00-2000.00] x4

B

220

180 160 140 120 960.87

100 80

972.87

60

1010.47

954.67

40

946.07

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Relative Abundance

x2

1440.13

200

20

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A

1061.27

887.00 0 1000

1100

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900

1165.80

1234.07

1200

1515.07 1306.60 1300

1379.87 1400

1537.00 1500

1636.80 1600

m/z

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Figure 1: A) HPLC analysis of sample which has been eluted by 50% ethanol from SPE obtained by the maceration method using 90:10 methanol/water for 24 h that peak1,2,3 and 4 are vigno1& vigno 2, varv A , vigno 5, and vigno 3 & vigno 4 respectively. B) LC-MS analysis of extracted cyclotides,

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70

80 %methanol

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400 350 300 250 200 150 100 50 0

90

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Peak area

Figure 2

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Figure 2: Extractive yields from V. ignobilis with maceration by three different hydroalcoholic solvents; 70, 80, 90% methanol.

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Figure 3

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Figure 3: MALDI-TOF spectrum of vigno cyclotides extracted by A) maceration and B) microwave-assisted extraction with optimum condition; 90:10 methanol- water, 1200 w power, 60 ˚C and 20min as irradiation time

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Figure 4

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Figure 4: HPLC analysis of samples which had been eluted by 50% Ethanol from SPE after extraction by microwave with step 4 condition: 90:10 methanol: water, 1200 w power, 60 ˚C and

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A) 4 min B) 12 min C) 20 min D) 24 min as irradiation times