GC-MS Investigation and Acetylcholinesterase Inhibitory Activity of Galanthus rizehensis

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Introduction

Plants of the Amaryllidaceae family are well known for their content of alkaloids with diverse chemical structures and interesting biological activities such as antitumour, antiviral, and acetylcholinesterase (AChE) inhibitory activity (Bastida et al., 2006). Besides this, several Ama- ryllidaceae species are used in many countries in gardening and also for decorative purposes.

Alzheimer’s disease (AD) is the most prevalent reason for dementia in elderly people, affecting more than 30 million people worldwide (Selt- zer, 2010; Williams et al., 2011). AChE inhibitors are presently major drugs for the symptomatic treatment of AD. The most prominent Amaryl- lidaceae alkaloid, galanthamine, is a long acting, selective, reversible, and competitive AChE inhibitor which is extensively used for the treatment of AD under the brand names Reminyl^® andRazadyne^®( Howland, 2010; Ago et al., 2011;

Williams et al., 2011). There is a strong relation- ship between cholinergic activity and learning ability and memory. Acetylcholine, an important neurotransmitter in the central nervous system, is hydrolyzed by AChE to yield choline. An in- adequate acetylcholine level in AD patients is the main element of the cholinergic hypothesis

(Bartus et al., 1985; Williams et al., 2011). Explor- ing new natural sources to ﬁ nd preferable AChE inhibitors is one of the major reasons of the present study.

Galanthus L. (Amaryllidaceae) is a genus of bulbous monocotyledons, which grow naturally in Europe, Anatolia, and the Middle East. It is represented by fourteen taxa and one hybrid in Turkey. Galanthus rizehensis Stern is a small plant occurring in northeastern Turkey (Davis, 1999, 2006).

In recent years, GC-MS (gas chromatography- mass spectrometry) has been proven a fast and reliable method for the investigation of the components in complex alkaloid mixtures (Kreh et al., 1995; Berkov et al., 2004a). This method provides identiﬁ cation of a great deal of compounds in small quantities of plant materials. Previously, the alkaloidal content of several Galanthus species has been investigated by GC-MS (Berkov et al., 2004a, 2008a). However, there is no report in the literature concerning the alkaloid proﬁ ling of G.

rizehensis by this technique. Therefore, in the cur- rent study, we performed GC-MS analyses on the alkaloidal extracts prepared from the aerial parts and bulbs of G. rizehensis collected during ﬂ owering and fruiting periods. In addition, AChE inhibitor potentials of the prepared extracts were de-

Inhibitory Activity of Galanthus rizehensis

Buket Bozkurt Sarikayaâ,*, Nehir Unver Somerâ, Gulen Irem Kayaâ, Mustafa Ali Onurâ, Jaume Bastida^b, and Strahil Berkov^c

a Department of Pharmacognosy, Faculty of Pharmacy, Ege University, 35100, Bornova, Izmir, Turkey. Fax: +90 232 3885258. E-mail: buket.bozkurt@ege.edu.tr

b Departament de Productes Naturals, Biologia Vegetal I Edafologia, Facultat de Farmàcia, Universitat de Barcelona, 08028, Barcelona, Spain

c AgroBio Institute, 8 Dragan Tzankov Blvd., 1164-Soﬁ a, Bulgaria

* Author for correspondence and reprint requests

Z. Naturforsch. 68 c, 118 − 124 (2013); received May 17, 2012/January 15, 2013

GC-MS (gas chromatography-mass spectrometry) analyses of alkaloids in the aerial parts and bulbs of Galanthus rizehensis Stern (Amaryllidaceae), collected during two different vegetation periods, was performed. Twenty three alkaloids were identiﬁ ed in four different alkaloid extracts. Acetylcholinesterase (AChE) inhibitory activities of the alkaloid extracts were tested. Both the highest alkaloid diversity and the most potent inhibitory activity (IC50 12.94 μg/ml) were obtained in extracts from the bulbs of G. rizehensis collected during the fruiting period.

Key words: Galanthus rizehensis, Alkaloids, GC-MS, Acetylcholinesterase Inhibition

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termined spectrophotometrically by a microplate assay modiﬁ ed from the method of Ellman et al.

(1961) using a 96-well microplate reader (López et al., 2002).

Material and Methods Plant material

G. rizehensis was collected from Maçka, Trab- zon on March 11, 2007 and May 2, 2007 during the ﬂ owering and fruiting periods, respectively. The plant was identiﬁ ed by Prof. M. Ali Onur from the Department of Pharmacognosy, Faculty of Pharmacy, Ege University, Izmir, Turkey. Voucher specimens have been deposited (Nos. 1371, 1376) in the herbarium of this department.

Chemicals

Authentic compounds as standards for GC-MS analyses and galanthamine for AChE inhibitory activity determination had previously been isolated from several Galanthus or other Amarylli- daceae species in our laboratory as indicated in Table I and authenticated by means of spectral analyses (UV, IR, 1D NMR, 2D NMR, and MS).

Other chemicals were of analytical grade.

Extraction

Four different alkaloidal extracts were prepared from the specimens of G. rizehensis (aerial parts – fl owering period, bulbs – fl owering period, aerial parts – fruiting period, and bulbs – fruiting period) to be used in GC-MS analyses and in the AChE assay. Briefl y, air-dried, powdered plant material (500 mg) was extracted consecutively three times with methanol (5 ml) in an ultrasonic bath for 30 min each at room temperature. The solvent was evaporated from the combined extracts, the residue was dissolved in 10 ml 2% sul- furic acid, and neutral compounds were removed with diethyl ether (3 x 10 ml). The acidic aque- ous phase was basifi ed with 25% ammonia to pH 9 – 10 and extracted with chloroform (3 x 10 ml).

The combined chloroform extracts were then dried over anhydrous sodium sulfate, ﬁ ltered, and the organic solvent was evaporated in vacuo to afford the alkaloidal extract. For the GC-MS analyses, the extracts were dissolved in methanol (5 mg extract in 250 μl methanol).

GC-MS analysis

The GC-MS analyses were recorded on a Hewlett-Packard 6890+MSD 5975 instrument (Hewlett-Packard, Palo Alto, CA, USA) operat- ing in the electron impact mode (EI) at 70 eV. A HP-5 MS column (30 m x 0.25 mm x 0.25 μm) was used. The temperature conditions followed the program: 100 – 180 °C at 15 °C/min, 180 – 300 °C at 5 °C/min, and a 10-min hold at 300 °C. The in- jector temperature was 250 °C. The ﬂ ow rate of the carrier gas (helium) was 0.8 ml/min. The split ratio was 1:20.

The spectra of co-eluting chromatographic peaks were investigated and deconvoluted by the use of AMDIS 2.64 (NIST, National Institute of Standardization and Technology, Gaithers- burg, MD, USA). The alkaloids were identiﬁ ed by comparing their mass spectral fragmentation with standard reference spectra from the NIST 05 database (NIST Mass Spectral Database, PC- Version 5.0, 2005) or by GC-MS co-chromatography with previously isolated authentic standards. Moreover, data obtained from the literature were used for the identiﬁ cation of the alkaloids.

The percentage of the total ion current (TIC) for each compound is given in Table I. The area of the GC-MS peaks depends not only on the concentration of the related compounds, but also on the intensity of their mass spectral fragmentation.

Thus, the data in Table I do not stand for absolute quantiﬁ cation but allow the comparison of alkaloid proﬁ les in different samples.

AChE inhibitory activity

AChE inhibitory activity was determined spectrophotometrically by using a microplate assay modiﬁ ed from the colorimetric method of Ellman et al. (1961) with a 96-well microplate reader as previously described (López et al., 2002). Sam- ples with concentrations of 600, 400, 200, 100, 50, 10, 1 μg/ml in phosphate buffer (8 mM K2HPO4, 2.3 mM NaH2PO4, 0.15 M NaCl, 0.05% Tween 20, pH 7.5 – 7.6) were prepared and ﬁ nal concentrations of 150, 100, 50, 25, 12.5, 2.5, 0.25 μg/ml were tested. The enzyme inhibitory activity was calculat- ed as the percentage compared to the blank. Gal- anthamine was used as a positive control. The inhibitory concentration of 50% (IC50) was obtained using the software package GraphPad Prism V3.0 (GraphPad Software, San Diego, CA, USA).

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Results and Discussion GC-MS analysis

In the present work, the alkaloid proﬁ le of G.

rizehensis is reported for the ﬁ rst time using GC- MS analysis. Twenty-three alkaloids were detected in the four different extracts prepared from G.

rizehensis (Table I, Figs. 1 – 3). The extracts ex- hibited remarkable differences in their alkaloid profi les. Six alkaloids were detected in the aerial parts of the fl owering period, twelve in the bulbs of the fl owering period, six in the aerial parts of the fruiting period, and as many as 22 in the bulbs of the fruiting period (see Table I). Alka- loids belonging to major subgroups of the Ama- ryllidaceae alkaloids, including lycorine, tazettine, and galanthamine, were identifi ed in the extracts of G. rizehensis (Table I). Hordenine, a common base reported previously from Amaryllidaceae and other plant families (Pellati and Benvenuti, 2007; Berkov et al., 2009), was found in the bulbs of the plant. Incartine was the major alkaloid in the bulbs of the fl owering period (45%) followed by lycorine (18.6%). Similarly, lycorine-type alkaloids were the main constituents of the bulbs collected in the fruiting period. Trisphaeridine and 11,12-didehydroanhydrolycorine were the major components of the aerial parts collected in both vegetation periods. One β-carboline alkaloid, i.e.

1-acetyl-β-carboline, was detected in the aerial parts of G. rizehensis. This is an interesting ﬁ nd- ing, since β-carboline alkaloids are not common in the Amaryllidaceae family. Recently, together with this alkaloid, vittatine, 11-hydroxyvittatine, lycorine, and incartine have been isolated from G.

rizehensis by our research group (Sarikaya et al., 2012). Consequently, eighteen alkaloids are reported for the ﬁ rst time in G. rizehensis by this study.

Among the detected alkaloids, lycorine-type alkaloids dominated in the studied extracts (63.7%).

Galanthamine and tazettine-type alkaloids were present in very low amounts in the samples.

Mass spectral fragmentation of the compounds GR-1 and P-3 (Table I) display speciﬁ c features

of Amaryllidaceae alkaloids. Based on the avail- able literature, they could not be identiﬁ ed.

AChE inhibitory activity

The AChE inhibitory activities of the four different alkaloid extracts were tested at fi nal concentrations of 150 to 0.25 μg/ml. The most signifi - cant results were observed for the extracts of the bulbs (IC50 17.15 μg/ml, fl owering period, and IC50

12.94 μg/ml, fruiting period), while extracts of the aerial parts showed lesser activities (IC50 122.6 μg/

ml, ﬂ owering period, and IC50 45.78 μg/ml, fruit- ing period). Galanthamine was used as a positive control (IC50 0.043 μg/ml).

In conclusion, extracts of plant parts collected in the fruiting period showed more diverse alkaloid proﬁ les than those of plant parts collected in the ﬂ owering period. Furthermore, bulbs contained a higher number of alkaloids than the aerial parts.

The greatest alkaloid diversity (22 alkaloids) was recorded in bulbs collected in the fruiting period.

This extract also exhibited the most potent AChE inhibitory activity (IC50 12.94 μg/ml). Bulbs in the ﬂ owering period also exhibited signiﬁ cant activity (IC50 17.15 μg/ml), probably due to the abundance of lycorine-type compounds, since galanthamine and lycorine-type compounds are known to pos- sess higher anticholinesterase activity than compounds of other types of Amaryllidaceae alkaloid skeletons (López et al., 2002; Elgorashi et al., 2004).

Acknowledgements

This study was ﬁ nancially supported by TU- BITAK (TBAG-104T272), EBILTEM (2007/

BIL/007), Ege University Research Fund (09/

ECZ/014, 09/ECZ/021). B. B. S. thanks TUBI- TAK-BIDEB for a research fellowship. The au- thors also thank Dr. Asunción Marín (Serveis Cientiﬁ cotècnics, Universitat de Barcelona, Facul- tat de Farmàcia, Barcelona, Spain) for performing the GC-MS analyses.

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Table I. GC-MS data of the compounds found in the extracts of G. rizehensis. CompoundRI[M+]m/z (relative intensity, %)Content (percentage of TIC)References Flowering periodFruiting period Aerial partsBulbsAerial partsBulbs Hordenine a1469165(1)121(3), 107(5), 91(4), 77(7), 58 (100)–13.9–tr.Berkov et al. (2009); S 1-Acetyl-β-carbo- lineb1995210(86)182(51), 168(100), 140(35)4.4–4.6–NIST 05; Zhou et al. (1998); S P-32194253(51)252(100), 224(42), 181(13), 166(15), 152(12), 128(13)3.10.6–10.5Berkov et al. (2004b) Ismine c2280257(39)238(100), 211(5), 196(17), 168(12)1.1–1.40.9Berkov et al. (2009); S Trisphaeridine c2282223(100)222(39), 167(9), 165(11), 164(15), 138(22)46.2–40.35.8Berkov et al. (2009); S Galanthamine d2406287(86)286(100), 270(14), 244(24), 230(13), 216(34)–––0.1NIST 05; Berkov et al. (2008b); S Vittatine e 2472271(100)254(13), 228(24), 199(78), 187(66), 157(21), 128(28)–4.7–4.3Berkov et al. (2009); S 11-Deoxytazettine f2485315(26)300(49), 250(6), 231(100), 211(169), 169(9), 141(15)–––0.1NIST 05; Berkov et al. (2009) Anhydrolycorine g 2501251(42)250(100), 220(2), 192(11), 191(10), 165(3), 124(6) –2.5–17.9NIST 05; Berkov et al. (2009) 8-O-Demethylmari- tidinee2505273(100)256(22), 230(20), 201(82), 189(42), 174(23)–––0.4Berkov et al. (2009); S O-Acetylcaranine g2522313(76)312(8), 270(2), 252(98), 240(10), 226(100), 211(4), 194(8), 154(3)–1.2–0.2NIST 05; Berkov et al. (2005) 1-Acetyl pluvine g2547329(81)328(11), 286(3), 268(80), 254(20), 242(100), 228(13), 198(5), 182(7), 151(5)

–0.9–0.7Berkov et al. (2005) Assoanine g2579267(54)266(100), 250(20), 222(11), 205(5), 193(6), 180(8)–––4.3Bastida et al. (2006); S GR-12592315(100)315(100), 272(30), 256(85), 226(34), 218(56), 200(26)–––0.1– 11,12-Didehydroan- hydroly-corine g2605249(60)248(100), 190(23), 163(7), 123(6), 95(10)45.28.451.916.5Berkov et al. (2009) Tazettine f2655331(33)316(16), 298(20), 260(4), 247(100), 227(13), 211(10), 201(18), 181(7), 152(9), 141(10), 128(8), 115(15)

–––0.1Berkov et al. (2004b); S Hippamine g2677301(21)300(15), 250(16), 227(82), 226(100)–0.1–0.2Berkov et al. (2008c); S Galanthine g2704317(20)318(5), 316(17), 298(9), 284(14), 268(18), 266(14), 244(15), 243(98), 242(100) –3.8–3.2S Sternbergine g2713331(37)270(33), 229(70), 228(100)–0.3–0.5Evidente et al. (1984); S 11-Hydroxyvitta- tine e2719287(7)258(100), 242(19), 212(12), 186(14), 181(18), 152(13)–––10.7Bastida et al. (2006); Berkov et al. (2009); S

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Table I continued. Lycorine g 2746287(28)268(25), 250(28), 227(70), 226(100), 211(5), 147(11)–18.6–11.0Likhitwitayawuid et al. (1993); Berkov et al. (2004a); S Incartine g2761333(48)332(100), 296(16), 259(71), 258(81)–45.01.012.3Berkov et al. (2008c); S Epimacronine f2812329(25)314(20), 255(22), 245(100), 201(67)tr.–0.80.1Berkov et al. (2008c); S a Other type. bβ-Carboline. c Phenanthidirine. d Galanthamine. e Crinine. f Tazettine. g Lycorine. S, Authentic isolated standard compound; tr. (trace) < 0.1. Fig. 1. GC-MS chromatogram of an alkaloidal extract from bulbs, ﬂ owering period, of G. rizehensis.

Hordenine Vittatine

Anhydrolycorine

11,12-Didehydroanhydrolycorine GalanthineLycorine Incartine Fig. 2. GC-MS chromatogram of an alkaloidal extract from bulbs, fruiting period, of G. rizehensis.

P-3 Trisphaeridine Vittatine Anhydrolycorine Assoanine 11,12-Didehydroanhydrolycorine Galanthine

Incartine Lycorine

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Fig. 3. Chemical structures of alkaloids identiﬁ ed in G. rizehensis: hordenine (1), 1-acetyl-β-carboline (2), ismine (4), trisphaeridine (5), galanthamine (6), vittatine (7), 11-deoxytazettine (8), anhydrolycorine (9), 8-O-demethyl- maritidine (10), O-acetylcaranine (11), 1-acetylpluvine (12), assoanine (13), 11,12-didehydroanhydrolycorine (15), tazettine (16), hippamine (17), galanthine (18), sternbergine (19), 11-hydroxyvittatine (20), incartine (21), lycorine (22), epimacronine (23).

N N

H

H₃C O

O

O NHCH₃

OH

1 2 4

O

O N

O O

O

NCH3 OCH₃

R¹

H

R² R³

^N

O

O ^N

MeO

H H HO

OMe

O

15 21 N

H₃C CH₃ HO

N R¹O

R²O

R⁴O

R³O ^N

R²

R¹

H H R⁴O

R³O ^N

R²

R¹

H H

7 R¹+R²= CH_2,R³= H 10 R¹= CH3, R²= R³= H 20 R¹+R²= CH2, R³= OH 6

5

8 R¹= ĮH R²= R³= H 16 R¹= ĮOH R²= R³= H 23 R¹= ȕH R²+R³= O

9 R¹+R²= CH2 13R¹= R²= CH3

11 R¹= OAc, R²= H, R³+R⁴= CH2

12 R¹= OAc, R²= H, R³= R⁴= CH3

17 R¹= OH, R²= OCH3, R³+R⁴= CH2

18 R¹= OH, R²= OCH₃, R³= R⁴= CH₃ 19 R¹= OAc, R²= OH, R³= H, R⁴= CH3

22 R¹= R²= OH, R³+R⁴= CH2 O

N CH₃ H₃CO

HO

H

N

OH

R³ R¹O

R²O

N

OH

R³

N

OH

R³ R¹O

R²O

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