YHUBEPCHTET NO XPAHUTEIHU TEXHOJOFHH - TLI0B AUB UNIVERSITY OF FOOD TECHNOLOGIES - PLOVDIV SCIENTIFIC WORKS Volume LVII, Issue 1 Plovdiv, October 15-16, 2010 HAYUHA KOH®EPEHIMA C MEXKYHAPOJHO YYACTHE “XPAHUTEJIHA HAYKA, TEXHHKA TEXHOJIOI'HH 2010” ‘FOOD SCIENCE, ENGINEERING AND TECHNOLOGIES 2010’ HAY4UHM TPYOBE Tom LVI, Caurex 1 TInosans, 15 - 16 oxromepn 2010TEXHONOTMN — 2010" TECHNOLOGIES ‘2010 15-16 oxromapu, Mnopnus = S15 ~ 16 October, Plovdi See “XPAHTESTHA HAYKA, TEXHUKA M1 G FOOD SCIENCE, ENGINEERING AND Auetunxonuuectepasa uHxnOupauio gelicTBMe Ha ankanonaHn ekcTpaktu or in vitro cuctemu Ha Galanthus elwesii Hook. fil. Bacun Teoprmes, Vsax Visaton u AtaHac Nasnos MonyyeHu ca dee shoot (H2L8 u LEV) u edva Kanycua (H1) Kynmypu om Eneesueso koxuye (Galanthus elwesii Hook. fil), pacmawo @ pationa Ha ceeepousmouna Basieapun. MnyseHume kynmypu ca avanusupanu 3a cbd»pxaHuemo Ha ankanoudu, npumexaeaLyu auemunxonunecmepasa —ubxuumopHa akmueHocm u @ ycmanoeeHo, 46 Subepeyupanume shoot Kynmypu npodyyupam ankanoudu @ no-2onemu Konuyecmea 6 cpasHenue ¢ KanycHama Kynmypa. AuemunxonuHecmepasa unxu6upawyama akmueHoom Ha mexdume ekcmpakmu @ sHayumento no-sucoKa om masu Ha KanycHama Kynmypa (76.3% u 82.1% unxu6upane 3a nunuu H2L8 u LEV, CbhomeemuHo, 6 cpaeHeHue Cc 13.3% 3a nuts H1). YomaHoseno e, 4e ayemunxonunecmepasa UHXU6upauyama akmueHocm ce Obimku Ha ankarioud c Rf cmotinocm 0.17. Acetylcholinesterase inhibitory action of alkaloids extracts from Galanthus elwesii Hook. fil. in vitro systems. Vasil Georgiev, Ivan Ivanov and Atanas Pavlov One callus (H1) and two shoot (H2L8 and LEV) cultures of snow drop (Galanthus elwesii Hook. fi.) from the north-east Bulgaria region were obtained and analyzed for the production of alkaloids with acetylcholinesterase inhibitory action. Differentiated shoot lines produced higher amount of alkaloids and demonstrated significantly higher activity to inhibit acetylcholinesterase enzyme compared to the callus culture (76.3% and 82.1% for lines H2L8 and LEV compared to 13.3 for H1). The alkaloid with Rf value of 0.17 was found to be responsible for the acetylcholinesterase inhibitory action. Keywords: Amaryllidaceae alkaloids, Acetylcholinesterase inhibitors, Galanthus elwesii Hook. fil., TLC Introduction Galanthus elwesii Hook. fil. (snow drop) is a small bulbous plant distributed throughout South-Eastern European countries and Euro-Asia [1]. This plant produced a large variety of Amaryllidaceae alkaloids, many of which have been found to exhibit strong acetylcholinesterase inhibitory, cytotoxic and antiviral activities [1]. A lot of the scientific attention in the last few years was focused to the screening of the new natural compounds with higher acetylcholinesterase inhibitory action for the symptomatic treatment of Alzheimer's disease [2]. In the present study we report the potentials of two different type G. elwesii Hook. fil. in vitro systems to produce Amaryllidaceae alkaloids with acetylcholinesterase inhibitory action. 415Materials and methods Plant material G. elwesii Hook. fil. plants were collected from their natural habitat located near to the town Karnobat, Markela locality and from vilage Obrochishte, located in north-east Bulgaria. For the experiments stems, ovaries and bulbs were cut and surface sterilized with 70% ethanol for 20 sec followed by sterilization in 20% Domestos bleach solution (www.unilever.com) for 20 min. The sterilized explants were used for the next initiation of in vitro cultures after removing of necrotic parts. Obtaining of in vitro cultures The calli and the shoots formation was initiated by transferring of the sterile explants on MS media, supplied with 3% sucrose (Duchefa, The Netherlands), 5.5 g/L “Plant agar” (Duchefa, The Netherlands) and different combinations of auxin and cytokinin, presented in Table 1. The cultivation was carried out at 26°C, in darkness or under illumination (16 h light and 8 h darkness). The subcultivation period of the explants, as well as of the obtained in vitro cultures was 28 days at the same conditions. Extraction of alkaloids Lyophilized biomass for shoots and callus culture (200 mg and 500 mg, respectively) were homogenised and the alkaloids, were extracted according to Georgiev et al. [3]. TLC alkaloids assay Dried alkaloid mixtures were dissolved in 100 uL methanol and 5 UL or 20 UL of extracts (for shoots or callus, respectively) were dropped on Silicagel G60 TLC plate (ALUGRAM® SIL G Silica gel 60, Macherey-Nagel, Germany). The galanthamine standard (Galantamine hydrobromide, Sigma-Aldrich, Germany) was spotted in concentrations of 2, 5, 10 and 20 ug on the same plate as well. The mobile phase was Chloroform : Methanol 26% Ammonia (12 : 1 : 0.1). The spots were visualized by Dragendorff reagent. The alkaloid contents were quantified densitometry using QuantiScan® software (BioSoft, Cambridge, UK). The amounts of the detected alkaloids were expressed as GAL equivalents. TLC acetylcholine esterase inhibitory actions assay The assay was performed according to the method discribed by Georgiev et al. [3], modified as follow: 2 UL of alkaloid mixtures were spotted on previously washed with acetone and dried Silicagel G60 TLC plate (ALUGRAM® SIL G Silica gel 60, Macherey- Nagel, Germany). The galanthamine standard (Galantamine hydrobromide, Sigma-Aldrich, Germany) was spotted in concentrations of 2, 4 and 5 ig on the same plate as well. The plate was developed as described above. After drying, the plate was immediately sprayed with enzyme solution (Acetylcholinesterase EC. 3.1.1.7, Sigma-Aldrich, Germany — 500U in 150 ml of 0.05 M Tris-HCI buffer with pH=7.8, supplied with 150 mg bovine serum albumin - Sigma-Aldrich, Germany) and dried on could air.