Turk J Pharm Sci 11(1), 107-112, 2014

Short communication

Quantitative Determination of Galanthamine and Lycorine in elwesii by HPLC-DAD Gulen Irem KAYA*, Derya CICEK POLAT, Ahmet EMIR, Buket BOZKURT SARIKAYA, Mustafa Ali ONUR, Nehir UNVER SOMER Ege University, Faculty of Pharmacy, Department of Pharmacognosy, 35100 Bornova-İzmir, TURKEY

In this study, aerial parts and of Galanthus elwesii Hook. (), collected from three different localities in Southern Turkey, were quantitatively analyzed for their content of galanthamine and lycorine, by using High Performance Liquid Chromatography (HPLC). The chromatographic separation was performed using an isocratic system with a mobile phase of trifuoroacetic acid-water-acetonitrile (0.01:90:10) applied at a fow rate 1 mL/min using diode array detector. The content of galanthamine in the aerial parts and bulbs of G. elwesii collected from Cimi village (Antalya) was determined as 0.346 and 0.042 %, respectively. The aerial parts of G. elwesii collected from Ibradi (Antalya) was found to contain 0.287 % galanthamine, whereas the bulbs contained 0.095 % of this . Galanthamine was not detected in the samples of G. elwesii growing in Kayrak village (Mersin). Among the tested specimens, lycorine was only found in the bulbs of G. elwesii collected from Ibradi (Antalya) and Kayrak village (Mersin) as 0.005 and 0.015 %, respectively.

Key words: Galanthus elwesii, Amaryllidaceae, Galanthamine, Lycorine, HPLC-DAD.

Galanthus elwesii Üzerinde YBSK-DAD Yöntemi ile Galantamin Likorin Miktar Tayini

Bu gali§mada, Türkiye’nin gtineyinde tig farkh lokaliteden toplanan Galanthus elwesii Hook. (Amaryllidaceae) bitkisinin toprak üstii kısımlan ve soğanlarındaki galantamin ve likorin igeriği Yiiksek Basınglı Sıvı Kromatografisi (YBSK) kullanılarak, kantitatif olarak analiz edilmi§tir. Kromatografik ayinm, triforoasetik asit-su-asetonitril mobil fazimn (0.01:90:10) aki§ hızı 1 mL/dk olacak §ekilde uygulandigi izokratik bir sistemden yararlanılarak ve DAD detektörii kullamlmak suretiyle gergekle§tirilmi§tir. Cimi köytinden (Antalya) toplanan G. elwesii bitkisinin toprak üstii kısımlan ve soğanlarındaki galantamin igeriği sırasıyla % 0.346 ve 0.042 olarak tayin edilmi§tir. ibradi (Antalya)’den toplanan G. elwesii bitkisinin toprak iistii kısımlannin % 0.287 galantamin igerdiği tespit edilirken, soğanlann bu alkaloidi % 0.095 orarunda igerdiği saptanrm§tır. Kayrak köytinden (Mersin) toplanan G. elwesii örneklerinde ise galantamin tespit edilmemi§tir. Çali§ilan örnekler arasında likorin, sadece Ibradi (Antalya) ve Kayrak köytinden (Mersin) toplanan G. elwesii soğanlannda sırasıyla % 0.005 ve 0.015 oranlannda bulunmu§tur

Anahtar kelimeler: Galanthus elwesii, Amaryllidaceae, Galantamin, Likorin, YBSK-DAD.

Correspondence: E-mail: [email protected]; Tel: +90 232 311 4079; Fax:+90 232 388 5258

107 Gulen Irem KAYA, Derya CICEK POLAT, Ahmet EMIR, Buket BOZKURT SARIKAYA, Mustafa Ali ONUR, Nehir Unver SOMER

INTRODUCTION XPERIMENTAL

The family Amaryllidaceae, consisting of material The family Amaryllidaceae, consisting of Plan about 1100 in 85 genera (1), is we l t material about 1100 species in 85 genera (1), is well G. G. elwesii was collected from Cimi village known for its with diverse chemical elwesii was collected from Cimi village n for its alkaloids with diverse chemical and and Ibradi (both in Antalya) and from Kayrak structures and bio ogical activities (2). bradi (both in Antalya) and from Kayrak ures and biological activities (2). villa village (Mersin). The were identifed G lanthamine (Figure 1), important alkaloid ge (Mersin). The plants were identified thamine (Figure 1), an important by P by Prof. M. Ali Onur from the Department of rof. M. Ali Onur from the Department of founid found din inAmaryllidacea Amaryllidaceae especies species, , ii s a lonPhga Pharmacognosy, Faculty of Pharmacy, Ege acting, selective, reversibl and competitive macognosy, Faculty of Pharmacy, Ege acting, selective, reversible and Uni ersityUniversity, Izmi,r Izmi(Turkey)r (Turkey). Vouche. Voucher sampler samples s etitivacetylcholinesterae acetylcholinesterass inhibitore inhibitor. It is . useIt d ofof r .o felwesii G. elwesii (No’ s (No’1404s ,1404 1405, ,1405 1408, )1408 are ) are is used th foe rtreat the mtreatmen n of tmil ofd mil andd anmoderatd moderate casee s deofp sited posited in thde i Herbariun the Herbarium of thme o Departmenf the Department t cases Aolfz hAeilmzheeri’ms edri’ssea dsies e(a3s)e. L(y3c).orLinyec o(Friingeure o2f),P armacognosyof Pharmacognosy, Facult, Faculty of Pharmacyy of Pharmacy, Ege , Ege (Figu r ae com2)m,o n aalkaclomidm inonAmarlyklalliodiadceaeinfamiUl yneU, ir snitiyv.ersity. Amar yhlalsid abceeeane pfaromvielyn, thoashabveena pwroivden sptoectrum Chemicals have aofw ibdieo lsopgeictar lu ma cotfiv bi tiioelso gincacl uadci tnivgi tiaenstivi Crahle icals The standard galanthamine and lycorine were (i4n)g, aannttitiuvimr aol r (54), aannttiimt uaml aoriral (5(6),) anTd standarh d galanthamine and lycorine previously isolated from several Galanthus a lnatriianlfam(m6)atoryan adctiviatnietisin (f7la).m mDuateor tyo t hwe ier r previously isolated from several tiime sp (o7r)ta.n D t u em teod tihceinirali mpprorptearnttiems,e diitc ihnasl beGennthusspecieal sspecie in ous r inla bou ratorr laboratory and authenticatey and d rotfie sg,reiat t hinatserbesetento o df e tgerrematinein t ehr e s ct ontotent aouft h nticateby sp dctra byl analysespectrasl (UVanalyse, IRs , NM(UVR, IRan,d MS). mthineese t haelk caolonitdesn ti no fthteh epsleanatlska olfo Ai dms ainry tlhl iedaceNaHPL anMe d CMS) grad. HPLe acetonitrilC grade eacetonitril (Lab Scaen (LaAnalyticab l faomfi Al ym. Uarpyl tloid daacteea, e vafarimo iul sy .m Ue tphotodsd haatev,e b eSeSciencna Acneas)ly, tTi cFaAl (TrifSucoiernocaeces)t,ic acidT) F (AMerck) udsescrmibeethdod csonchearnvien g btehe n q udaenstcifricbaetdion (Tofr i luaonrdo achetriocmatogacraidp)hic g( rMadeerckd)oublea-nddistilled rgnailnagntthhaem qiunaen atnifdi c laytcionrinoef (g8a-l1a1n)t.h amine chrmwao t aotegr awpheirce gurasedde dforubtlhee-d ipsrteilpleadratwi oant e ro f the and l corine (8-11). wer mu soebdilefo rphtahsee.prOepthaer rat i ocnh eomfic thaels muosbedil ein the Am Among the Amaryllidaceae genera in Turkey, ea. sOsatyh ewrere c ohfemanicalaylsticuasl egdradine.the assay onGalanthusg the LAmaryllidacea. is epresenteed bygener 14 t a xa iann d onphae s Turk y, Galanthus L. is represented by 14 wer of analytical grade. hybrid (12). Of these taxa, Galanthus elwesii HPLC instrument taxa and one hybrid (12). Of these taxa, Hook. has a wide natural distribution and can Galanthus elwesii Hook. has a wide natural HPLC instrumentHPLC a alysi s was carried out using a liquid be found in Bulgaria, northeastern Greece, the distribution and can be found in Bulgaria, HPL chroC analysim tographis wacs systecarriem d(Agilen out usint 110g0 aseries) , eastern Aeg an Islands, southern Ukr ine and northeastern Greece, the eastern Aegean liqu dequippe chromatographid with ac DAsysteD m(Agilen (Agilent 120t 1100 series)0 , Turkey. Within Turkey, this species has the s)a, equippequaternard ywit pumh a pDA systeD (Agilenm (Agilent 120t0 1100 Islands, southern Ukraine and Turkey. Within seri Turkeywides, thit distributios speciens amonhasg oth er s anwidesd naturallt sery i s)s,eraiesquGa1te3r1n1aAry), paumvapcu suyms t e dmega(Assgeirle (nAt gilent distributiogrowns amonin orthg otherw stes anr ,d westernaturalln yan growd southers 11n0 1s1e0r0ies erGie1s3 G11A32),2Aa), va a tchueurm osdteagttaesds ecrolumn Anatolia (13). in northwestern, western and southern (Ag ecnotmpar1t1m0e0n t (Asegriilesnt 1 1G01032se2rAie)s, G1a316A), Anatolia (13). ther aos mtatatneudalc oinlujemcntorc owmi tpha r2tm0 eμnLt (lAoogpile(nAt gilent

In e Ipresenn th present studyt study, aeria, aerial partls p an rtds anbulbd bulbs of s of11 G.0 1100 series G1328 A Rheodyne 7725i). Data elwesiiwesii, , collectec ll cted frofromm threthree differene different localitiet 20s analysis was carried out with "Agilent Chem tiein s southerin nsouther Turkeyn , Turkeywer , quantitativelwere Rhye Station Softw are". The chromatographic assay itatively analyzed for their content of out analyzed for their content of galanthamine and was performed on a Hichrom C column (5(j,, hamine and lycorine, by using High The lycorine, by using High Performance Liquid 250 m m, 4.6 mm) at 290.4 nm. The mobile rmance Liquid Chromatography coupled a H Chromatography coupled with diode array phase consisted of TF A:wa ter:aceto nitrile with iode array detector (HPLC-DAD). at 2 detector (HPLC-DAD). (0.01:90:10, v/v/v) a pplied at a flow rate of

108 Turk J Pharm Sci 11(1), 107-112, 2014

1mL/min (11,14). The analysis was performed Preparation of standard solutions at 25°C. The quantitative determination of For the preparation of the calibration curves galanthamine and lycorine was carried out by of galanthamine and lycorine, 2 mg of each the external standard method based on peak alkaloid was dissolved in 5 mL 0.1 % TFA and areas. filtered (Sem Concept Syringe 100 x 13 mm, 0.45 μm). By diluting the stock solutions, nine

109 Gulen Irem KAYA, Derya CICEK POLAT, Ahmet EMIR, Buket BOZKURT SARIKAYA, Mustafa Ali ONUR, Nehir Unver SOMER different concentrations of galanthamine and found in the bulbs of the specimens of G. elwesii lycorine were prepared within the ranges of 1.0- growing in Kayrak village (Mersin) and Ibradi 400 μg/mL and 1.0-300 μg/mL respectively. (Antalya). Of these specimens, the bulbs of G. Each standard solution was injected into the elwesii collected from Kayrak village (Mersin), column in triplicate with a volume of 20 μL was found to contain the highest content of and the regression equation of the calibration lycorine (Figure 5) (Table 1). curve was obtained as y=11.451763 x - Although, G. elwesii has been a subject of 2.40657 (r2= 0.99993) for galanthamine and as several phytochemical studies (16-18), there is y= 15.8423032 x-1.339608 (r2=0.99985) for scanty data on the quantification of alkaloids lycorine. of this species. To the best of our knowledge, quantitative analysis of the alkaloids has been Extract preparation carried out only in Galanthus elwesii of Turkish The extraction procedure was carried out origin. The results of previous investigations as previously, described. Prepacked columns revealed that G. elwesii specimens collected based on diatomaceous earth for liquid-liquid from Karaburun, Izmir contained both sample purification has been used for the rapid galanthamine (0.007-0.026 %) and lycorine preparation of the extracts (14,15). Briefy, (0.004-0.013 %) (19). In another study, lycorine about 200 mg of powdered plant material was content was reported as 0.011 % in G. elwesii macerated with 5 mL of 2 % hydrochloric acid specimens collected from Antalya, Akseki for 5 hours in an ultrasonic bath at 40oC and then (20). Moreover, in a previous study, G. elwesii the extract was made alkaline with 1 mL of 26 growing in Yamanlar, Izmir was not found to % ammonium hydroxide and the volume was contain galanthamine and lycorine (10,21). adjusted to 10 mL in a volumetric fask with distilled water. The basic solution centrifuged ACKNOWLEDGEMENTS at 5000 rpm for 10 min and aliquots of 3 mL were applied on the Extrelut® (Merck) columns. This study was financially supported by Ege The alkaloids were eluted with chloroform University Research Fund (09/ECZ/037) (3 x 5 mL) for 10 min. The organic solvent and partially funded by TUBITAK (TBAG- was distilled in vacuo to afford the alkaloidal 104T272) and EBILTEM (2007/BIL/007). extract. The extract was dissolved in 1 mL 0.1 % TFA and filtered. 20 μL was injected into the REFERENCES HPLC column. Each analysis was carried out 1. Davis AP, The Genus Galanthus, pp 15-170, in triplicate. Timber Press Inc, Oregon, 1999. 2. Hoshino O, Amaryllidaceae alkaloids, In: The RESULTS AND DISCUSSION Alkaloids Chemistry and Pharmacology, Ed: Cordell GA, pp 323-424, Academic Press Inc, The identification and quantitative New York, 1998. determination of galanthamine and lycorine in 3. Heinrich M, Teoh HL, Galanthamine from G. elwesii collected from different localities in snowdrop-the development of a modern southern Turkey, was established by comparison drug against Alzheimer’s disease from local of the retention times and peak areas with those Caucasian knowledge, J Ethnopharmacol 92(2- of standards. The HPLC chromatogram of the 3), 147-162, 2004. standards is given in Figure 3. Detection by DAD 4. Szlavik L,Gyuris A, Minarovits J, Forgo P, Molnar J, Hohmann J, Alkaloids from increased the sensitivity of the HPLC method. Leucojum vernum and Antiretroviral Activity Galanthamine was found in the specimens of of Amaryllidaceaea Alkaloids, Planta Med 70, G. elwesii growing in Cimi village and Ibradi 871-873, 2004. (both in Antalya). Of these specimens, the aerial 5. Lamoral-Theys D, Andolfi A, Goietsenoven GV, parts of G. elwesii collected from Cimi village, Cimmino A, Le Calve B, Wauthoz N, Megalizzi was found to contain the highest content of V, Gras T, Bruyere C, Dubois J, Mathieu V, this alkaloid (Figure 4). Galanthamine was not Kornienko A, Kiss R, Evidente A, Lycorine, the detected in the samples of G. elwesii growing main phenanthridine Amaryllidaceae alkaloid, in Kayrak village (Mersin). Lycorine, was only exhibits significant antitumor activity in cancer

110 Turk J Pharm Sci 11(1), 107-112, 2014

cells that display resistance to proapoptotic 15. Bozkurt Sarıkaya B, Unver Somer N, Onur stimuli: an investigation of structure-activity MA, Quantitative determinations on Galanthus relationship and mechanistic insight, J Med rizehensis, Turk J Pharm Sci 9(1), 61-66, 2012. Chem 52(20), 6244-6256, 2009. 16. Berkov S, Sidjimova B, Evstatieva L, Popov 6. Şener B, Orhan İ, Satayavivad J, Antimalarial S, Intraspecific variability in the alkaloid activity screening of some alkaloids and the of Galanthus elwesii, Phytochem plant extracts from Amaryllidaceae, Phytother 65(5), 579-86, 2004. Res 17(10), 1220-1223, 2003. 17. Berkov S, Bastida J, Sidjimova B, Viladomat 7. Çitoğlu GS, Acikara OB, Yilmaz BS, Ozbek F, Codina C, Phytochemical differentiation H, Evaluation of analgesic, anti-infammatory of and Galanthus elwesii and hepatoprotective effects of lycorine (Amaryllidaceae): A case study, Biochem Syst from Sternbergia fisheriana (Herbert) Rupr, Ecol 36(8), 638-645, 2008. Fitoterapia 83(1), 81-7, 2012. 18. Berkov S, Cuadrado M, Osorio E, Viladomat F, 8. Berkov S, Pavlov A, Ilieva M, Burrus M, Codina C, Bastida J, Three new alkaloids from Popov S, Stanilova M, GC-MS of alkaloids Galanthus nivalis and Galanthus elwesii, Planta in Leucojum aestivum plants and their in vitro Med 75(12), 1351-1355, 2009. cultures, Phytochem Anal 16, 98-103, 2005. 19. Kaya GI, Gözler T, Quantitative evaluation 9. Abou-Donia AH, Toaima SM, Hammoda of the specimens of Galanthus elwesii, J Fac HM, Sawky E, New rapid validated Pharm Gazi 20 (2), 71-78, 2003. HPTLC method for the determination of 20. Muhtar F, Şener B, Evaluation of Amaryllidaceae lycorine in Amaryllidaceae plants extracts plants exported from Turkey regarding lycorine, Chromatographia 65, 497-500, 2007. In The Proceedings of the XI.th Meeting on 10. Kaya GI, Fillik A, Hısıl Y, Unver N, High Plant Crude Drugs 1996, ed. Coşkun, Ankara pressure liquid chromatographic analysis of University Press, Ankara, pp. 384-388, 1997. lycorine in four Galanthus species growing in 21. Sarier Çelik D, Pharmacognostical studies Turkey, Turk J Pharm Sci 1,105-114, 2004. on Galanthus elwesii Hook. growing around 11. Mustafa NR, Rhee IK, Verpoorte R, Rapid Karagol, Yamanlar, İzmir, MSc Thesis, Ege method for determination of galanthamine University, İzmir 2002. in Amaryllidaceae plants using HPLC, J Liq Chromatogr Rel Technol 26, 3217-3233, 2003. 12. Davis AP, The Genus Galanthus-Snowdrops in the Wild. In Snowdrops, A Monograph of Received: 21.11.2012 Cultivated Galanthus, Bishop, M Davis, AP Grimshaw J (Eds.), Griffin Press Publishing Accepted: 13.02.2013 Ltd, Cheltenham, 9-63, 2006. 13. Yüzbaşıoğlu S, Morphological variations of Galanthus elwesii in Turkey and difficulties on identification, Bocconea 24,335-339, 2012. 14. Kaya GI, Cicek D, Sarikaya B, Onur MA, Unver-Somer N, HPLC-DAD analysis of lycorine in Amaryllidaceae species, Nat Prod Com 5(6), 873- 876, 2010.

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