Centrifugal partition chromatography in the isolation of minor ecdysteroids from Cyanotis arachnoidea

Accepted Manuscript Title: Centrifugal partition chromatography in the isolation of minor ecdysteroids from Cyanotis arachnoidea Authors: Halima Meriem Issaadi, Yu-Chi Tsai, Fang-Rong Chang, Attila Hunyadi PII: DOI: Reference:

S1570-0232(16)31277-6 http://dx.doi.org/doi:10.1016/j.jchromb.2017.03.043 CHROMB 20549

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

18-11-2016 24-3-2017 29-3-2017

Please cite this article as: Halima Meriem Issaadi, Yu-Chi Tsai, Fang-Rong Chang, Attila Hunyadi, Centrifugal partition chromatography in the isolation of minor ecdysteroids from Cyanotis arachnoidea, Journal of Chromatography Bhttp://dx.doi.org/10.1016/j.jchromb.2017.03.043 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.

Centrifugal partition chromatography in the isolation of minor ecdysteroids from Cyanotis arachnoidea

Halima Meriem Issaadia, Yu-Chi Tsaia,b, Fang-Rong Changb,c, Attila Hunyadia,d,*

a

Institute of Pharmacognosy, University of Szeged, Eötvös str. 6, 6726 Szeged, Hungary

b

Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Shih-Chuan 1st Rd. 100, Kaohsiung 80708, Taiwan c

Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 80708, Taiwan d

Interdisciplinary Centre for Natural Products, University of Szeged, Eötvös str. 6, 6720 Szeged, Hungary

* Corresponding Author: Tel: +3662546456. Fax: +3662545704. E-mail: [email protected].

1

Highlights:  Cyanotis arachnoidea extracts are worldwide commercialized as food supplements.  Minor phytoecdysteroids’ efficacy and safety is practically non-investigated.  CPC method was developed for ecdysteroid isolation from a commercial extract.  The separation was fast, efficient and reproducible.  The yield and purity allows biological studies on the isolated compounds. Abstract Phytoecdysteroids are known for their various beneficial bioactivities in mammals including a non-hormonal anabolic and adaptogenic effect. Cyanotis arachnoidea extracts are extensively utilized worldwide as ecdysteroid-rich materials for various purposes, e.g. food supplementation, use in agriculture and aquaculture, etc. Preparative chromatography of ecdysteroids requires extensive use of methods of different selectivity, and only a very limited number of papers are available on related application of modern liquid-liquid chromatographic techniques. In this work, a centrifugal partition chromatography (CPC) method was developed for the isolation of two minor ecdysteroids, dacryhainansterone and calonysterone, from a pre-purified commercial extract of Cyanotis arachnoidea. The biphasic solvent system was optimized by HPLC, and it was composed of n-hexane – ethyl acetate – methanol – water (1:5:1:5, v/v/v/v) in ascending mode. The isolated dacryhainansterone and calonysterone represented 99.1% and 99.7% purity, respectively. Calonysterone exerts a stronger effect on the protein kinase B (Akt) phosphorylation in mammalian skeletal muscle cells than the abundant 20-hydroxyecdysone, while no related data are available on dacryhainansterone. Despite their presence in food supplements, neither compound has appropriately been assessed for safety and efficacy. The reported method allows the gram scale isolation of these compounds, opening ways to their in-depth pharmacological investigation. Keywords: preparative liquid-liquid chromatography, Cyanotis arachnoidea extract, anabolic food supplement, ecdysterone, dacryhainansterone, calonysterone. 1. Introduction Ecdysteroids are the steroid hormones of all classes of arthropods; they are present at all stages of insect development, regulating many biochemical and physiological processes [1]. Phytoecdysteroids, their herbal analogues, are found in both gymnosperms and angiosperms and it appears that at least 5-6% of terrestrial plant species contain significant levels of these compounds [2] that can reach as much as 2-3% of dry weight (e.g., seeds of Rhaponticum carthamoides and stalks of Diploclisia glaucescens, inflorescences of Serrulata inermis and roots of Cyanotis arachnoidea) [3]. These compounds play a highly complex ecological role in the chemical communication between the Kingdoms of Nature through the food chain: as an amazing example, phytoecdysteroids can apparently make their way from plants through caterpillars into the bloodstream of songbirds, where these compounds can accumulate and significantly affect the apolysis of ticks feeding on the birds [4]. Phytoecdysteroids have long been studied for their non-hormonal metabolic effects on mammals, altogether accounting for a complex bioactivity that could be referred to a broadspectrum, general strengthening effect. Unfortunately, no well-designed, high quality clinical trials are available, nevertheless, a number of in vitro and in vivo studies confirm a significant anabolic activity [5]. For example, phytoecdysteroids enhanced protein synthesis by up to 20% in mouse and human myotubules after 4h of treatment [6], and a muscle specific fiber size 2

increase was also observed in rats when treated with 20-hydroxyecdysone (20E), the most abundant phytoecdysteroid [7]. 20E was also reported as an antidiabetic agent whose daily administration can prevent obesity and insulin resistance in vivo, and which can reduce the glucose production in the hepatic cell culture predominantly through its influence on PI3Kdependent signaling pathways [8]. As for the mechanism of action of ecdysteroids, even though it is only partially resolved, it seems clear that the activation of Akt plays an important role in it [9]. The above-mentioned beneficial effects on mammals, supposedly including humans, led to the production and worldwide marketing of plant extracts with high ecdysteroid content. In this market, the roots of Cyanotis arachnoidea (Commelinaceae) appear to serve as by far the most typical raw material for manufacturing such preparations, in fact, our group has most recently revealed a case where food supplements were claimed to have been prepared from spinach but contained Cyanotis extract instead [10]. Since the isolation and identification of the first phytoecdysteroids in the mid-1960s, 487 natural analogues have become listed in the online database of herbal ecdysteroids (last update: August, 2015) [11], and the range of biochemical permutations detected so far suggests that probably > 1,000 analogues could exist in the Plant Kingdom [12]. Plants usually produce a rather complex ecdysteroid “cocktail,” and, due to the many possible minor differences between related analogues, the successful isolation of minor compounds usually require an extensive use of consecutively applied chromatographic steps of different selectivity [13-14]. Among these, counter-current chromatography (CCC), an all-liquid method utilizing the partition of a sample between two immiscible solvents to achieve separation [15-16], deserves particular attention. Counter-current chromatography is gaining popularity as a viable separation technique in natural products chemistry. In particular, high-speed counter-current chromatography (HSCCC) and centrifugal partition chromatography (CPC) have the following advantages: (i) no irreversible adsorption; (ii) full recovery of the injected sample; (iii) minimal tailing; (iv) low risk of sample denaturation; (v) economic maintenance (no expensive columns are required and only common solvents are consumed); (vi) low solvent consumption; (vii) crude plant extracts and semi-pure fractions can be chromatographed with sample loads ranging from milligrams to grams, (viii) aqueous and non-aqueous solvent systems are used and separation of compounds with a wide range of polarities is possible and (ix) a wide range of pH is tolerated in CCC, with implications in the separation of acidic and basic samples [16]. CPC is a hydrostatic CCC based on the use of a constant-gravity field produced by a rotation mechanism, which allows maintaining the liquid phase stationary while the mobile phase is pumped through it [17]. Known phytoecdysteroids have been previously isolated by the use of liquid-liquid partition chromatography. Droplet counter current chromatography (DCCC) has been widely used over the preceding years with solvent systems generally composed of chloroform, methanol and water at different ratios [18-19]. Just to mention two examples, 20E and Ajugasterone C were isolated by DCCC from Vitex madiensis in ascending mode (i.e. reverse-phase separation) using chloroform – methanol – water (13:7:4, v/v/v) solvent system [20], and 20E, 2-deoxy-20E and 20E 22-benzoate were isolated through a multistep procedure including the use of DCCC with the solvent system chloroform – methanol – water, (65:20:20, v/v/v) [21]. In contrast with DCCC, however, the use of modern liquid-liquid chromatographic techniques for the isolation of phytoecdysteroids is much less widespread than it would deserve to be. To the best of our knowledge, only a few reports are available on related applications. 20E was successfully isolated by means of HSCCC with a two-phase solvent system composed of ethyl acetate – nbutanol – water (4:1:5, v/v/v) from the roots of Serratula chinensis in 80 min [22]. As for CPC, our group has reported its use in multistep purifications of sensitive oxidized derivatives of 20E by using ethyl acetate – methanol – water (20:1:20, v/v/v) in ascending mode [23] or 3

photochemically modified ones by using chloroform – methanol –water in descending mode (10:7:3, v/v/v) [24]. In the course of investigating the chemical composition of commercially available Cyanotis extracts in connection with related bioactivity aspects, we have identified significant amounts of two minor ecdysteroids in such an extract during a preliminary study. Dacryhainansterone (1) was previously detected in the liquid waste left from the extraction of 20E from Cyanotis arachnoidea [25], whereas calonysterone (2), representing an unusual 6-hydroxy-5,8-dien-7one structure in its B ring, is reported here as a new ecdysteroid from this species. Structures of these compounds are presented in Fig. 1. Compound 2 has most recently been identified as a much stronger Akt activator in murine skeletal muscle cells than 20E itself [23], making this ecdysteroid particularly interesting for related bioactivity studies. In this work, our aim was to develop a simple and efficient preparative method for the isolation of this compound in order to allow its in-depth pharmacological investigation. 2. Materials and methods 2.1. General information Organic solvents used for TLC and CPC (analytical grade) and acetonitrile used for HPLC analysis (chromatographic grade) were purchased from Sigma-Aldrich (Budapest, Hungary). Calonysterone and dacryhainansterone, prepared during our previous related studies [23-26] and both possessing a purity of ≥ 98% by means of HPLC, were used as reference substances for qualitative analysis. Normal-phase (NP) TLC was performed on silica plates (Silica gel 60F254, E. Merck, Germany), by using the solvent system ethyl acetate ‒ ethanol ‒ water (4:0.25:0.25, v/v/v). Visualizations was performed under UV light λ1 = 254 nm and after spraying with vanillin/sulfuric acid reagent under λ2 = 365 nm. 2.2. Raw material and preliminary purification A commercial extract prepared from the roots of Cyanotis arachnoidea was purchased from Xi’an Olin Biological Technology Co., Ltd. (Xi’an, China). This extract (5.46 kg) was percolated with methanol (15.5 L) at room temperature. After evaporation to dryness, the dry residue (700 g) was fractionated through a silica gel column (2.1 kg) using a stepwise gradient of dichloromethane ‒ methanol, increasing the methanol content from 3% to 15% with a final wash with 100% methanol resulting in 6 fractions. Fraction 4 (244.8 g), eluted with dichloromethane – methanol (93:7, v/v), was subjected to further separation through silica using gradient of n-hexane ‒ ethyl acetate ‒ ethanol (60:40:0, v/v/v to 60:50:30, v/v/v) to obtain 13.16 g of a mixture containing mainly calonysterone and dacryhainansterone. 2.3. Reverse-phase HPLC Reverse-phase (RP) HPLC analyses were performed on a gradient system consisting of two Jasco PU-2080 pumps, a Jasco AS-2055Plus intelligent sampler connected to a JASCO LC-Net II/ADC equipped with a Jasco MD-2010 Plus PDA detector (Jasco Co., Tokyo, Japan). A Kinetex XB C-18 column (5 μm, 250x4.6 mm) was used with an isocratic solvent system of 30% aqueous acetonitrile at a flow rate of 1 mL/min. 2.4. CPC apparatus An Armen Spot CPC 250 mL (Armen Instrument, Saint Ave, France) was used, composed of an Armen Spot Prep II equipment (a 2-head pump with a flow rate range of 5–250 ml/min at a maximum pressure of 230 bar, a UV/Vis Diode Array Detector, a manual 10 mL sample loop injection valve and a fraction collector) and an Armen Spot CPC multilayer coil separation 4

column (capacity: 250 ml; maximum rotation speed: 3000 rpm). The CPC equipment was controlled by the Armen Glider CPC software. 2.5. Selection of the two-phase solvent system The selection of the optimal solvent system to separate the two phytoecdysteroids was achieved by the evaluation of the partition coefficients (K(U/L)i) and the separation factors (α). The partition coefficients weredefined as the concentration of a solute in the Upper phase (CUi) divided by that in the Lower phase (CLi): K(U/L)i = CUi ⁄CLi. (1) The separation factors representing the ratios of partition coefficients between two solutes were determined by calculating: 𝛼 = K(U/L)i ⁄K(U/L)(i-1) where K(U/L)i > K(U/L)(i-1) (2) Aliquots of the upper and the lower phases of each solvent system were analysed by HPLC in order to compare the area under the curve of each corresponding peak. To do so, the same volume of each phase was introduced in test tubes (15x150 mm) and approximately 0.5 mg of the fraction to purify was added. The tubes were sonicated and subsequently shaken with a vortex mixer (VWR, Lab dancer S40) in order to guarantee a good distribution of the sample between the two phases and to equilibrate these later. Subsequently, 200 μL of each phase were pipetted and introduced into HPLC vials where they were evaporated to dryness under nitrogen. The dry residues were dissolved in 900 μL of methanol (HPLC grade) and analysed by HPLC. 2.6. Selection of the mobile phase In CPC, either the upper or the lower phase can be selected as the mobile phase. After the selection of the correct biphasic solvent system, the choice of the mobile phase was based on the K(U/L)i values. For an effective separation, K(U/L)i values need to fall within the range of 0.5-2. In our case, the K(U/L)i values also served as the basis for deciding which phase should serve as the mobile phase according to the following principle: the mobile phase should be the upper, organic phase if 1 ≤ K(U/L)i < 2, while it should be the lower, aqueous phase if 0.5 ≤ K(U/L)i < 1 [27]. 2.7. CPC procedure for separation 2.7.1. Preparation of the selected CPC biphasic solvent system The optimal solvent system, selected as described above, was composed of n-hexane – ethyl acetate – methanol – water (1:5:1:5, v/v/v/v). A total volume of 4800 mL of this mixture was prepared (2x2400 mL) in a 5L separation funnel and then shaken vigorously at room temperature. The two phases were left to equilibrate and then each one was collected before use for separation. Based on the above-mentioned principle, the upper organic phase was selected as the mobile phase and the lower aqueous phase was used as the stationary phase in ascending mode (tail-to-head way; representing a normal-phase separation mode). 2.7.2. Equilibration and separation procedures The CPC column was first filled in descending mode with the stationary phase with a high flow rate of 50 mL/min at a rotation speed of 500 rpm and pressure of 17 bar. Then the mobile phase was introduced in ascending mode into the column with a flow rate of 10 mL/min at a pressure of 86 bar and a rotation speed of 1600 rpm gradually adjusted to 2400 rpm in order to generate a centrifugal acceleration. A total weight of 1 g of the fraction to purify was dissolved in a 54 mL of one-to-one volume ratio mixture of the two solvent phases and then filtrated through a glass filter. The separation was performed through six consecutive injections. The elution was continuously monitored at λ=243 and 298 nm, and 20 mL fractions were collected. The combined fractions were 5

evaporated under vacuum to dryness and then dissolved in methanol for later analysis by TLC and HPLC. 2.8. Calculation of separation characteristics During the “filling step” of the CPC column, before reaching the hydrodynamic equilibrium, the expelled stationary phase volume from the column was collected in a volumetric flask for calculation of the stationary phase retention volume ratio (Sf): 𝑉c−𝑉e Sf = 𝑉c (4) where VC is the total volume of the column (250 mL) and Ve the expelled volume of the stationary phase. In CPC, the expected retention volume (VR) is linked to the partition coefficient Ki expressed as the amount of solute in the stationary phase divided by that of the mobile phase, accordingly, the volume of the mobile phase (VM) and the volume of the stationary phase remaining inside the column (VS): 𝑉 R = 𝑉 M + Ki ∗ 𝑉 S (5) The expected retention time (tR) was obtained by dividing VR by the flow rate. After each separation, the efficiency of the column was estimated by the calculation of the theoretical plate number (N) for compounds 1 and 2 and taking the average efficiency N = (N1 + N2)/2, as [28]: tR 2

N = 16 ∗ (w) (6) where tR is the retention time of the eluting component and w is the peak width at the baseline. The resolution (Rs) between compounds 1 and 2 defined according to the classical equation as: 2∗(tR2−tR1) Rs = w +w (7) 2 1 The CPC Rs was evaluated by combining the previous equations (4), (5), (6) and (7) as follows: Rs = Sf

K2−K1 √N [ ] 4 1−Sf[1−K1+K2]

(K2>K1)

(8)

2

For the calculation of the theoretical plate number and the resolution, the CPC peaks obtained under 298 nm were fitted by non-linear multi-peaks gaussian regression model using OriginPro 9.1 software (OriginLab Corporation, Northampton, Massachusetts). 3. Results and Discussion The partially purified commercial extract of Cyanotis arachnoidea was analysed by TLC and HPLC-PDA in the range of 200 to 650 nm. The HPLC chromatogram indicated the presence of 4 peaks corresponding to the two targeted phytoecdysteroids dacryhainansterone (1) and calonysterone (2) detected at 5 and 9.86 min, respectively, along with two minor other components hereinafter referred to as impurity 3 and 4 (Fig. 2). The identification of the two main components was performed by comparison with reference substances based on the retention factors, the retention times and the UV spectra, and their structural identities were also confirmed by means of 1H NMR spectroscopy. The measured 1H NMR chemical shifts (in methanol-d4) were identical with NMR data reported previously for dacryhainansterone (1) [29] and calonysterone (2) [23]. 1H NMR spectra of compounds 1 (Fig. S1) and 2 (Fig. S2) are available as supplementary information. In order to isolate the targeted phytoecdysteroids, a CPC separation method was developed. The key to a successful CPC separation is finding a biphasic solvent system that provides a suitable partitioning of the solutes between the two phases. The selection of the suitable solvent system for a CPC separation must satisfy the following requirements [30]: 6

1. The settling time of the solvent system should be shorter than 30 s. 2. The partition coefficient of each target compound (K(U/L)i) should fall in the range of 0.5-2, and the separation factor (α) between any two consecutively eluting components should be greater than or equal to 1.5. 3. It is desirable that the solvent system provides nearly equal volumes of each phase to avoid excessive waste of the solvent. The optimal solvent system was estimated by means of HPLC analysis by computing the ratio of the areas under the curves of each peak. The partition coefficients and the separation factors are summarized in Table 1. In order to ensure the separation of the impurities 3 and 4 from the two targeted phytoecdysteroids, their partition coefficients and separation factors were also taken into account. The HEMWat (Hexane – Ethyl acetate – Methanol – Water) method covers a wide range of hydrophobicity by a stepwise and simultaneous increase of the applied ratio of n-hexane and methanol [31]. Considering this, the biphasic HEMWat system (0:5:0:5, v/v/v/v) was selected as starting point to our optimization procedure. In the n-hexane – ethyl acetate – methanol – water (0:1:0:1, v/v/v/v) solvent system, the K(U/L)i values of compounds 1 and 2 and impurities 3 and 4 were significantly greater than 2 indicating the high affinity of all the analytes to the upper organic phase, signifying that they would elute closer to the solvent front. Although the solvent system n-hexane – ethyl acetate – methanol – water (1:10:1:10, v/v/v/v) improved the K(U/L)i values for compound 2 and impurity 3, the ones corresponding to the other solutes remained quite high (greater than 2). For the solvent systems n-hexane – ethyl acetate – methanol – water (5:20:5:20, v/v/v/v) and (3:10:3:10, v/v/v/v), moderate low values of the partition coefficients of the compound 2 and the impurities 3 and 4 were obtained indicating that by using these solvent systems the compounds would elute after a too long running time. Finally, the results showed that the two-phase solvent system composed of n-hexane – ethyl acetate – methanol – water (1:5:1:5, v/v/v/v) provided the suitable K(U/L)i values ranging between 0.5 and 2 and gave acceptable calculated α values. The settling time of this solvent system was also short (27 s), and the volume ratio of the upper and lower phases was 0.90. Therefore the solvent system n-hexane – ethyl acetate – methanol – water (1:5:1:5, v/v/v/v) was selected to make the CPC separation. For the selection of the mobile phase, based on the values of K(U/L)i the upper organic phase was selected as the mobile phase. Under the optimized parameters comprising biphasic system n-hexane – ethyl acetate – methanol – water (1:5:1:5, v/v/v/v), constant pressure of 86 bars, flow rate of 10 mL/min and rotation speed of 2400 rpm, six consecutive injections of the crude extract (1 g) were performed (Fig. 3). Compounds 1 and 2 were separated in less than 30 min for each injection. The 20 fractions collected automatically after each injection were all combined into four pooled fractions (fraction I (0-14 min), II (14-18min), III (18-22min), IV (22-30min). After the CPC separation, combined fractions (I, II, III and IV) were submitted to TLC and HPLC-PDA measurements. The chromatograms of the combined fractions II, III and IV are presented in Fig. 4. A total weight of 89.54 mg of fraction I was obtained. The amount of fraction III, composed of the two phytoecdysteroids of interest and the impurities 3 and 4 was only 38.71 mg (i.e. 4.06% of the recovered weight). As fraction II and IV, 357.33 mg of dacryhainansterone (1) and 299.22 mg of calonysterone (2) was obtained, respectively, and the evaporation of the stationary phase yielded 168 mg of dry residue. Accordingly, an altogether 95.28% of the initial weight was recovered after the separation. 7

The purity of the two obtained phytoecdysteroids was calculated based on the maximum absorbance chromatograms provided by the HPLC-PDA data, and it was 93% for dacryhainansterone (1) and 96% for calonysterone (2). After recrystallizing them from ethyl acetate − methanol (2:1, v/v), the purity reached 99.1% and 99.7% respectively. Prior to the separation, the retention volume ratio Sf and the expected retention volume VR for each targeted compound were evaluated in order to predict the performance of the developed process. According to the high value of the stationary phase retention 0.7, an excellent resolution between was expected with well gaussian shaped peaks at 22.9 min and 16 min and thus a high number of theoretical plates (Table 2). Although the CPC chromatograms of the 6 consecutive injections showed a good reproducibility in separation (with a constant back pressure at 86 bar so a constant Sf value) leading to compounds with very high purity, a slight decrease of the resolution with the increasing number of injections and thus a gradually decreasing theoretical plate number could be observed. Nevertheless, our method could clearly overcome the instrumental limitations in separating the two compounds of interest, and, as such, it can be considered as a good starting point for further method development initiatives aiming the isolation of phytoecdysteroids by using CPC. 4. Conclusions In the present study, a CPC method was successfully developed for the isolation and purification of two phytoecdysteroids: dacryhainansterone and calonysterone from a partially purified extract of Cyanotis arachnoidea using a solvent system composed of n-hexane – ethyl acetate – methanol – water (1:5:1:5, v/v/v/v). A good repeatability was found in six consecutive sample injections. The method allows obtaining these bioactive compounds, present in significant amounts in commercial extracts extensively utilized for food supplementation, in high purity and yield necessary to further studies on their efficacy and safety in the near future. Funding: This work was funded by the National Research, Development and Innovation Office, Hungary (NKFIH; K119770). Acknowledgements Networking contribution from COST Actions CM1106, “Chemical Approaches to Targeting Drug Resistance in Cancer Stem Cells”, and CM1407, “Challenging Organic Syntheses Inspired by Nature - from Natural Products Chemistry to Drug Discovery” are acknowledged. A.H. acknowledges the János Bolyai fellowship of the Hungarian Academy of Sciences and the Kálmán Szász Prize. The authors wish to thank Dr. Norbert Kúsz (Institute of Pharmacognosy, University of Szeged) for taking the 1H NMR spectra of compounds 1 and 2.

8

References 1. L. Dinan, Phytoecdysteroids: biological aspects, Phytochemistry. 57 (2001) 325-339. 2. L. Dinan, J. Harmatha, V. Volodin, R. Lafont, Phytoecdysteroids: diversity, biosynthesis and distribution, in: Smagghe G (ed.) Ecdysone: structures and functions. Springer, Berlin, 2009, pp. 3-45. 3. G. Laekeman, A. Vlietinck, Phytoecdysteroids: phytochemistry and pharmacological activity, in: K.G. Ramawat, J.M Merillon (eds.), Natural products: phytochemistry, botany and metabolism of alkaloids, phenolics and terpenes, Springer, Berlin, 2013, pp. 3827-3850. 4. S. Hornok, D. Kováts, B. Flaisz, T. Csörgő, Á. Könczöl, G. Tibor Balogh, A. Csorba, A.Hunyadi, An unexpected advantage of insectivorism: insect moulting hormones ingested by song birds affect their ticks, Sci. Rep. 6 (2016) 23390. 5. M. Báthori, N. Tóth, A. Hunyadi, A. Márki, E. Zádor, Phytoecdysteroids and anabolicandrogenic steroids-structure and effects on humans, Curr. Med. Chem. 15 (2008) 7591. 6. J. Gorelick-Feldman, D. Maclean, N. Ilic, A. Poulev, M.A. Lila, D. Cheng, I. Raskin, Phytoecdysteroids increase protein synthesis in skeletal muscle cells, J. Agric. Food. Chem. 56 (2008) 3532-3537. 7. N. Tóth, A. Szabó, P. Kacsala, J. Héger, E. Zádor, 20-Hydroxyecdysone increases fiber size in a muscle-specific fashion in rat, Phytomedicine 15 (2008) 691-8. 8. P. Kizelsztein, D. Govorko, S. Komarnytsky, A. Evans, Z. Wang, W.T. Cefalu, I. Raskin, 20-Hydroxyecdysone decreases weight and hyperglycemia in a diet-induced obesity mice model, Am. J. Physiol. Endocrinol. Metab. 296 (2009) 433-9. 9. J. Gorelick-Feldman, W. Cohick, I. Raskin, Ecdysteroids elicit a rapid Ca2+ flux leading to Akt activation and increased protein synthesis in skeletal muscle cells, Steroids 75 (2010) 632-637. 10. A. Hunyadi, I. Herke, K. Lengyel, M. Báthori, Z. Kele, A. Simon, G. Tóth, K. Szendrei, Ecdysteroid-containing food supplements from Cyanotis arachnoidea on the European market: evidence for spinach product counterfeiting. Sci. Rep. 6 (2016) 37322. 11. R. Lafont, J. Harmatha, F. Marion-Poll, L. Dinan, I.D. Wilson, Ecdybase: a free ecdysteroid database). http://ecdybase.org/, 2002 → continuously updated (accessed 04.11.2016). 12. L. Dinan, The Karlson Lecture. Phytoecdysteroids: what use are they?, Arch. Insect Biochem. Physiol. 72 (2009) 126-41. 13. A. Ványolós, M. Báthori, New perspectives in the analysis of ecdysteroids: a promising group of biologically active compounds, Curr. Pharm. Anal. 4 (2008) 162-175. 14. R. Lafont, C. Blais, J. Harmatha, I.D. Wilson, Ecdysteroids: Chromatography, in: , Reference Module in Chemistry, Molecular Sciences and Chemical Engineering, EPublishing Elsevier Inc., 2013, pp. 1-15 ISBN: 978-0-12-409547-2. 15. A.P. Foucault, L. Chevolot, Counter-current chromatography: instrumentation, solvent selection and some recent applications to natural product purification, J. Chromatogr. A 808 (1998) 3-22. 16. A. Marston, K. Hostettmann, Developments in the application of counter-current chromatography to plant analysis, J. Chromatogr. A 1112 (2006) 181-194. 17. E. Delannay, A. Toribio, L. Boudesocque, J.M. Nuzillard, M. Zèches-Hanrot, E. Dardennes, G. Le Dour, J. Sapi, J.H. Renault, Multiple dual-mode centrifugal partition chromatography, a semi-continuous development mode for routine laboratory-scale purifications, J. Chromatogr. A 1127 (2006) 45-51. 18. D. Rudel, M. Bathori, J. Gharbi, J.P. Girault, I. Racz, K. Melis, K. Szendrei, R. Lafont, New ecdysteroids from Serratula tinctoria, Planta Med. 58 (1992) 358-364. 9

19. J.P. Girault, M. Bathori, E. Varga, K. Szendrei, R. Lafont, Isolation and identification of new ecdysteroids from the Caryophyllaceae, J. Nat. Prod. 53 (1990) 279-293. 20. I. Kubo, A. Matsumoto, J.F. Ayafor, Efficient isolation of a large amount of 20hydroxyecdysone from Vitex madiensis (Verbenaceae) by droplet counter-current chromatography, Agric. Biol. Chem. 48 (1984) 1683-1684. 21. M. Báthori, I. Máthé, Droplet countercurrent chromatography to isolate ecdysteroids from the herb Silene tatarica (L.), Acta Pharm. Hung. 66 (1996) 125-131. 22. H. Jieyun, X. Xinjun, X. Zhiyong, X. Zhisheng, Y. Mei, Y. Depo, Isolation and purification of 20-hydroxyecdysone from Radix Serratulae Chinensis by high-speed counter-current chromatography, J. Liq. Chromatogr. Relat. Technol. 37 (2014) 19091916. 23. J. Csábi, T.J. Hsieh, F. Hasanpour, A. Martins, Z. Kele, T. Gáti, A. Simon, G. Tóth, A. Hunyadi, Oxidized metabolites of 20-hydroxyecdysone and their activity on skeletal muscle cells: preparation of a pair of desmotropes with opposite bioactivities, J. Nat. Prod. 78 (2015) 2339-2345. 24. W.C. Lai, B. Dankó, J. Csábi, Z. Kele, F.R. Chang, M.L. Pascu, T. Gáti, A. Simon, L. Amaral, G. Tóth, A. Hunyadi, Rapid, laser-induced conversion of 20-hydroxyecdysone – A follow-up study on the products obtained, Steroids 89 (2014) 56-62. 25. N. Ruilin; Q. Minhua, Phytoecdysones in liquid waste during molting hormone production with Cyanotis arachnoidea, Yunnan Zhiwu Yanjiu 9 (1987) 253-6. 26. A. Hunyadi, A. Gergely, A. Simon, G. Tóth, G. Veress, M. Báthori, Preparative-scale chromatography of ecdysteroids of Serratula wolffii Andrae, J. Chromatogr. Sci. 45 (2007) 76-86. 27. Y. Ito, Golden rules and pitfalls in selecting optimum conditions for high-speed countercurrent chromatography, J. Chromatogr. A 1065 (2005) 145-168. 28. A. Berthod, K. Faure, Revisiting resolution in hydrodynamic countercurrent chromatography: tubing bore effect, J. Chromatogr. A 1390 (2015) 71-77. 29. P.C. Bourne, P. Whiting, T.S. Dhadialla, R.E. Hormann, J-P. Girault, J. Harmatha, R. Lafont,L. Dinan, Ecdysteroid 7,9(11)-dien-6-ones as potential photoaffinity labels for ecdysteroid binding proteins, J. Insect. Sci. 2 (2002) 11. 30. J.Cazes, Encyclopedia of Chromatography 2004 Update Supplement Marcel Dekker, Inc. New York, 2003. 31. JB. Friesen, JB. McAlpine, SN. Chen, GF. Pauli, Countercurrent separation of natural products: an update, J. Nat. Prod. 78 (2015) 1765-1796.

10

Legends to the figures

Fig. 1. Structures of dacryhainansterone and calonysterone.

11

Fig. 2. HPLC chromatogram of the partially purified crude sample of Cyanotis arachnoidea. Column: Kinetex XB-C18 250x4.6 mm; mobile phase: acetonitrile aqueous solution (3:7, v/v); flow-rate: 1 mL/min ; λ = 243 nm. Since we did not aim to purify the minor constituents of the sample, two partially separated peaks were considered jointly as impurity 4.

12

Fig. 3. CPC separation of 1 g of crude commercial extract of Cyanotis arachnoidea using six consecutive injections of 8 mL sample volume; three were of a concentration of 14 mg/ml and three were of a concentration of 23 mg/ml. The sample was dissolved in a 1:1 ratio of the two solvent phases, slight shifting of retention times comes from unavoidable variations in the injected solvent composition of each consecutive run.

13

Fig. 4. CPC chromatogram and HPLC chromatograms of fraction II, III and IV at the maximum absorbance for the separation of a pre-purified commercial extract of Cyanotis arachnoidea. Fractions II and IV contain dacryhainansterone (1; 93% purity) and calonysterone (2; 96% purity), respectively. HPLC-PDA fingerprints are presented at maximum absorbance (λ=200650 nm), and UV spectra of both compounds of interest are shown above their corresponding chromatograms.

14

Table 1 Partition coefficients and separation factors for the selected biphasic systems in separation of compounds 1 and 2 from commercial extract of Cyanotis arachnoidea. Solvent Partition coefficients Separation factors system n-hexane – ethyl acetate – K K(U/L)2 K(U/L)3 K(U/L)4 (U/L)1 α(1/2) α(2/3) α(3/4) α(1/4) methanol – water (v/v/v/v) 0:1:0:1 10.916 5.260 6.343 7.880 2.075 1.206 1.242 1.385 1:10:1:10 2.699 1.542 1.798 2.283 1.750 1.166 1.270 1.182 1:5:1:5 2.042 1.136 1.730 1.307 1.800 1.523 1.323 1.562 5:20:5:20 1.341 0.702 0.395 0.887 1.910 1.777 2.246 1.512 3:10:3:10 0.965 0.529 0.375 0.566 1.824 1.411 1.509 1.705

15

Table 2 Predicted and Experimental CPC results separation for the six consecutive injections. VRi, N and Rs refer to the average retention volumes of the six injections, the number of theoretical plates and the resolution, respectively. Separation characteristics

Sf

VR (mL)

Calonysterone (2) 229 Dacryhainansterone 0.7 160 (1)

VRi Number of injection average N / Rs (mL) 1st 2nd 3rd 4th 5th 6th 236 250 / 238/ 256/ 219/ 210/ 203/ 1.383 1.350 1.400 1.294 1.268 1.262 169.1

16