1) University of Tübingen, Institute of Archaeological Sciences, Workgroup for Archaeobotany, Rümelinstrasse 23, Tübingen, Germany. contacting author: oezguer.cizer@uni-tuebingen.de Erysimum crassipes as neglected medicinal plant: Storage at Early Bronze Age Küllüoba/Turkey 2) Synovo Biotechnologies GmbH, Paul Ehrlich Strasse 19, Tübingen, Germany. Özgür Çizer1, Jan Guse2 and Birol Mutlu3 3) Inönü University, Faculty of Science and Letter, Department of Biology, Botany Section, Campus, Malatya, Turkey INTRODUCTION B L A C K İSTA M e- NFikirtepe M a r m a r a S e a Pendik Ilıpınar İznik L. S E A Sa ka r ya Yarımbur- Manyas L. Kumtepe TROY A E G E A N DemircihöOrman FidanYortan Susu rluk ESKİŞEHİR Porsuk R. KÜLLÜOSakarya R. Keçiça- Liman- Gediz R. İZMİR Settlement of Küllüoba is located on the fertile alluvial fan of Upper-Sakarya (Sangarius River), southern of modern province Eskişehir. KÜLLÜOBA Karamık S. 5 km According to archaeological evidence, Küllüoba was settled from Late Chalcolithic (ca 4.200 B.C.) until the beginning of Middle Bronze Age (ca. 1800 B.C.) with a main occupation phase in Early Bronze Age II. 10 km Building in square AG 22 gives evidence for an extended brand in almost catastrophic dimension. Reconstruction of the building is suggested as two-storey facility. Wooden piles, wood charcoals and seed remains are preserved in excellent condition. In the same building, Erysimum crassipes storage pot has been recovered in situ. Phytogeography: Iranio-Turanian vegetation, Archaeological records of Erysimum: ca. 80 species of genus Erysimum are distributed in Anatolia In Neolithic Hacılar in Central Anatolia, Erysimum inds are identiied on the species level as Erysimum sisymbrioides, mentioned as “consumed for its fat content” Climate: Hot-dry summers and cold winters., classiied as semi-arid climate. Main precipitation falls during spring and autumn, average precipitation is ca. 350 mm. Excavation trenches for analysed botanical samples Modern use of Erysimum: Use of numerous Erysimum species for medicinal purposes is recorded. Ethno-medicinal use of Erysimum crassipes is recorded solely in the Highlands of Northern Jordan Archaeologically, 2,5 million seed counts of Erysimum crassipes constitutes the irst recorded ind in the World. Efort was required to collect approximately 2.5 million seeds needs to be considered for the importance of the plant. Well preserved textile remains may have been a plug in the neck of the pot. METHODS Cardenolids in genus Erysimum: overview Modern accessions of Erysimum crassipes from Turkey Samples from each accesions are prepared separetely with 30 mg Acetonitril and Methanol Extracts are cerifuged with corund-sand in a fastprep ca. 45 sec. and stored by -20 for a week. For the HPLC/MS/MS the extractions are diluted with water (1/10). From hundreds of well-known cardiac glycosids, about 15 are used for the therapy Erysimum crassipes as modern specimen from the comparative collection (left) and carbonised specimen from Küllüoba (right). Testa pattern with reticules and papillae specific for the species E. crassipes can be observed. - Steroid structure - Lactone ring (functional group) - Sugar site chain (auxiliary group) Erysimum crassipes as medicinal plant The genus Erysimum includes poisonous chemical compounds like glycosides and Erysimoside which are speciically used for medical treatments (Bauer et al 1960). Digoxin (e.g., from foxglove plant, Digitalis lanata) Aims of the Analyses with spectometry methods First step: Targeting essential oil and glycoside of modern E. crassipes seeds to obtain a chemical proile using GS/MS (essential oils, analyses in progress and HPLC/MS/MS (glycosides). Second step: Thermal experiments on the modern seeds will be applied to detect the efects of charring on the chemical compounds. O H O OH HO O [M/z]+ = 697 O O HO O OH OH HO O OH O H O OH HO O [M/z]+ = 163 O O HO O k-Strophanthidin (e.g., Strophanthus kombé, Used in Africa also as arrow poison) Third step: Comparison of the chemical proiles of archaeological seeds and those of the modern seeds may allow reconstructing degraded chemical compounds in archaeological seeds. In modern medicine, combined therapy with the other medicaments is possible and in comparison to the other medications., less or no side-efects are expected. Glycosides are suitable for the treatment of the “chronic heartinsuiciency” but not suitable for the “acute” diseases . Cardiac glycosides do not have strong contraindications, if they are given in the right doses. In the modern medicine, the cardiac glycosides constitute the most cost-efective method for the therapy of chronic heartinsuiciency. In order to dedect the molecules like glycosides with low molecular weight, HPLC/MS-MS was applied fragmentation Some members of the genus Erysimum like E. oicinale, known as hedge mustard, was used by Ancient Greeks as expectorant, laxative and diuretic (Howard 1987). Indications of cardiac glycosides to heart diseases Enhancement of inotrophy (muscular contractions) of cardiac muscles (direct indication on myocardial system) through mobilisation of intracellular Ca+ in to the cells. Targetting the cardiac glycosides using HPLC/MS-MS: SK 6270 (Malatya) BM 11739 (Malatya) BM 11740 (Ankara) BM 11741 (Kayseri) Steroid part of the glycoside (agylcone) is biochemically active part of the glycosides. On the modern specimens (right) the reticules have regular shapes with five edges, the papillae have intact appearance. In case of the archaeological specimens (left), the reticules are irregular in shape with slightly pufed walls; papillae are more sunken, due to efects of charring. Sample preparation for HPLC/MS/MS Digitoxin longer-lasting efects, much longer elimination half-life in the human body, around seven days Erysimoside Molecular Formula C35H52O14 Average mass 696.779 OH OH HO O OH Precursor Scans: Scanning for all masses in the first Quadrupol (Q1) between a given range of 300-1200 U (Targetted molercular weight for glycone part of glycosid) Fragmentation in Q2 and isolation for the scanned molecule weight in Q3. A signal comes out as peak, if a fragment is broken from the targeted molecule in Q1. a peak occurs with molecule weight 697, when the mass in Q1 isolated, the same molecule lost in Q2 a glucose fragment als cation (positive) with a fragmentation of 163. RESULTS/ DISCUSSION References: Akhgar, M.R. and Roknabadi, S. (2015) Composition of the essential of Erysimum crassicaule. Chemistry of Natural Compounds 51 (3): 575-576. Bauer, S., Országh Š., Bauerová, O., Mokrý, J., Masler, L. Šikl, D. and Tomko, J. (1960) Isolierung von Erysimosid und Hevetikosid aus graublätrigem Hederich (Erysimum canascens Roth.) durch Gegenstromverteilung. Planta Med: 8(2): 145-151. Cizer, Ö. (2015) Archaeobotanical investigations of plant cultivation and husbandry practices at the Early Bronze Age settlement Küllüoba in West-Central Turkey: Considerations on environment, climate and economy. BAR International Series 2766, Oxford. Davis, P. H. (eds.) 1965-1988. Flora of Turkey and the east Aegean Islands. (Vol 1. Crucifereae) Edinburg University Press. Es-Sai N.-E., Kerhoas, L., Einhorn, J., Paul-Henri, D. (2005) Application of ESI/MS, CID/MS and tandem MS/MS to the fragmentation study of eriodictyol 7-O-glucosyl-( 2)-glucoside and luteolin 7-O-glucosyl-(1 2)-glucoside. International Journal of Mass Spectrometry 247: 93–100. Jawla, S., Mogla, O.P. and Kumar, Y. (2010) Herbal Remedies for Asthma: An Overview Journal of Chemical and Pharmaceutical Research 2(1): 267-272 Precursor scan (Q1, top: intensity/time), down: intensity/mass (m/z) Full Scan (top: intensity/time), base peak (bottom: intensity/mass (m/z) Preliminary results for Erysimum using HPLC/MS/MS Discussion/Future work Full scanns and base peak chromatogram (intensity of the highest peak) for dedection of the ionisated molecule. Further analyses using spectometry methods with modern seeds are required. Evidence for glucone in form of digi-glucose . No clear evidence for an ionised molecule weight for aglycone part (steroide) of the glycoside (e.g., Erysimoside). Signiicant peak might consist of a Glucose and Digitoxose (Digi-Glucoce), with a molecule weight 310 (the peak has a H as + ion). Problem: if this disaccharide dereives as fragmentation from an ‚ aglycone‘, the peak is not clearly visible. Thermal experiments will be conducted on modern seeds and their chemical proile will be provided. Chemical proiles of archaeological seeds will be obtained. Chemical proiles of both modern and thermally degredated seeds will be compared with the modern seeds in order to understand the degredation patterns of the chemical compounds. Understanding the chemical proile of the plant may constitute an additional tool to the taxonomic identiication. Lei, H-Z., Yahara, S., Nohara, T., Tai, B.-S., Xiong, J.-Z., and Ma, Y.-L. (2000) Cardiac Glycosides from Erysimum cheiranthoides. Chemical & pharmaceutical bulletin 48(2): 290-292. Makarevich, I. F., Zhernoklev, K. V., Slyusarskaya, T. V. and Yarmolenko, G. N. (1993) Cardiac Glycosides of Erysimum contractum HI. Glucocanescein Chemistry of Natural Compounds 29 (5): 488-489. March, R.E., Lewars, E.G., Stadey, C.J., Miao, X.-S., Zhao, X. and Metcalfe, C.D. (2006) A comparison of lavonoid glycosides by electrospray tandem mass spectrometry. International Journal of Mass Spectrometry 248 (61–85). Maslennikova, V. A., Genkina, G. L., Umarova, R. U., Navruzova, A. M. and Abubakirov, N. K. (1967) Glycosides of Erysimum VI. Cardenolides of E. altaicum, E. cuspidatum, E. difusum, E. marschallianum, E. nuratensae, E. violascens. Khimiya Prirodnykh Soedinenii (Chemistry of Natural Compounds) 3 (2): 173-178. Oran, S. and Al-Eisawi, D. (2014) Medicinal plants in the high mountains of northern Jordan. International Journal of Biodiversity and Conservation: 6(6):436-443. Umarova, R. U., Maslennikova, V. A. and Abubakirov, N. K. (1977) steroid compounds of Erysimum XI. sterols of Erysimum difusum and E. euspidatum Chemistry of Natural Compounds 13 ( 6): 692-693.