South African Journal of Botany 77 (2011) 175–183 www.elsevier.com/locate/sajb

Phylogenetic selection of target in tribe Haemantheae for acetylcholinesterase inhibition and affinity to the serotonin reuptake transport protein

M.G.K. Bay-Smidt a, A.K. Jäger a, K. Krydsfeldt a, A.W. Meerow b,c, G.I. Stafford a,d, ⁎ J. Van Staden d, N. Rønsted a,

a Department of Medicinal Chemistry, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark b USDA-ARS-SHRS, National Germplasm Repository, 13601 Old Cutler Road, Miami, Florida, USA c Fairchild Tropical Garden, Miami, Florida, USA d Research Centre for Growth and Development, School of Biological and Conservation Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3201, South

Received 25 April 2010; received in revised form 14 July 2010; accepted 28 July 2010

Abstract

We present phylogenetic analyses of 37 taxa of Amaryllidaceae, tribe Haemantheae and belladonna L. as an outgroup, in order to provide a phylogenetic framework for the selection of candidate for lead discoveries in relation to Alzheimer's disease and depression. DNA sequences from the nuclear ribosomal internal transcribed spacer (ITS) and the trnL-F regions were used. Maximum parsimony analyses provide increased support for the sister relationship of and . Within Haemanthus, a well supported clade (89% BS) corresponds to a summer rainfall group (mainly Eastern Cape) with white-pale pink flowers. A second strongly supported clade (100% BS) corresponds to a winter rainfall group (mainly Western Cape) with red-pale pink flowers. Haemanthus montanus, which is from the summer rainfall region, is sister to the winter rainfall group. Alkaloid profiles and bioactivity profiles were investigated for 16 taxa using gas chromatography-mass spectrometry (GC–MS) and assays measuring acetylcholinesterase (AChE) inhibition and affinity to the serotonin reuptake transport protein (SERT). No alkaloids were detected by GC–MS in extracts of the two species of included in the present study suggesting that Gethyllis (and possibly ) species may not produce the alkaloids characteristic for the family. AChE inhibitory activity was found in all investigated clades except the Apodolirion–Gethyllis clade, which can be explained by the apparent lack of alkaloids in this clade. In spite of infra-specific variability of alkaloid profiles observed, dose-dependent SERT activity appears to be pronounced and restricted to the Haemanthus within tribe Haemantheae. Three of eight Haemanthus species tested had IC50 b10 μg/ml. Two of the most active extracts in the present study contained primarily montanine type alkaloids which have not been tested for SERT affinity previously. Simultaneous evaluation of bioactivity and alkaloid profiles in a phylogenetic framework can potentially be used to select candidate species for phytotherapy and drug discovery. © 2010 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Acetylcholinesterase; Amaryllidaceae; Drug discovery; Internal transcribed spacer; Phylogeny; Serotonin-reuptake-transport-protein; trnL-F

1. Introduction significant part of health care problems everywhere and are increasing as human life-expectancy increases in many Mental health problems such as epilepsy, convulsions, de- countries (WHO, 2010a). Plants provide a large number of pression, dementia and age-related diseases constitute a herbal remedies to treat mental illnesses. Many people are largely dependent on plants as the only readily available medicine (WHO, 2010b). In southern Africa well over 300 ⁎ Corresponding author. Tel.: +45 35 336504; fax: +45 35336001. species are used by traditional healers to treat mental illnesses E-mail address: [email protected] (N. Rønsted). (Sobiecki, 2002; Stafford et al., 2008).

0254-6299/$ - see front matter © 2010 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2010.07.016 176 M.G.K. Bay-Smidt et al. / South African Journal of Botany 77 (2011) 175–183

Several Amaryllidaceae are used in traditional medicine for first isolated from the Caucasian snowdrop Galanthus wor- various illnesses (Neuwinger, 2000) including mental illnesses onowii Losinsk. and since from a range of other Amaryllidaceae (Sobiecki, 2002; Stafford et al., 2008). African Amaryllidaceae species, is approved as an AChE inhibitor in a number of constitutes 280–300 species in 20 genera, which are divided countries (Heinrich and Teoh, 2004). Galanthamine is a into three major clades corresponding to the tribes Amarylli- selective, reversible and competitive inhibitor of AChE, as deae, Cyrtantheae and Haemantheae (Meerow and Clayton, well as an allosteric modulator of the neural nicotinic receptor 2004; Meerow and Snijman, 1998, 2001, 2006; Meerow et al., for acetylcholine (Heinrich and Teoh, 2004). Crude extracts of a 1999). Most species used in traditional medicine belong to the number of Amaryllidaceae species have shown AChE inhibi- Amaryllidaceae tribe . For example, Boophone tion, but most tested species belong to Crinum L. (Houghton et disticha (L.f.) Herb., is widely used to induce hallucinations, to al., 2004; Jäger et al., 2004) and Narcissus (López et al., 2002). treat headaches and anxiety, and to sedate hysterical, violent The most active alkaloids reported belong to the lycorine- and and psychotic patients (Neergaard et al., 2009). However, galanthamine types (Elgorashi et al., 2004, 2006a; López et al., Aiton (tribe Cyrtantheae) and Scadoxus Raf. (tribe 2002). Haementheae) species are also used in traditional medicine to Depression is associated with reduction of neurotransmitters treat mental illnesses (Stafford et al., 2008). in the brain. Several antidepressants exert their effect by (L.) Friis & Nordal is known to cause dizziness, visual selective inhibition of serotonin reuptake and affinity to the disturbances, CNS excitation or depression (Veale et al., serotonin reuptake inhibitor (SERT) is associated with possible 1992). In Eastern , a bath with root infusion of antidepressant effect (SSRIs; Stahl, 1998). Crude extracts and (Martyn) Raf. is used to treat mental isolated alkaloids of Boophone disticha have shown affinity to illness (Chhabra et al., 1987). Activities of plants in SERT (Nielsen et al., 2004; Sandager et al., 2005) as have a Amaryllidaceae related to the central nervous system are number of the tested alkaloids from other Amaryllidaceae ascribed to norbelladine-derived alkaloids restricted to the species, primarily of the crinine-type (Elgorashi et al., 2006b). Amaryllidaceae subfamily Amarylloideae (AGP III, 2009). The purpose of the present study is to use a phylogenetic Over 500 alkaloids have been described from various species hypothesis for Amaryllidaceae tribe Haemantheae to evaluate and have been subdivided into 18 major types based on their the distribution of (a) alkaloids, (b) acetylcholinesterase hypothetical biosynthetic pathways (Jin, 2009). inhibitory activity, and (c) affinity to the serotonin reuptake Efforts are continuously being made to identify and develop transport protein. new and improved herbal remedies and lead compounds for drug discovery. However, identification and isolation of 2. Materials and methods bioactive compounds is both time-consuming and costly and only a few of the Amaryllidaceae species and their alkaloids 2.1. Materials have been evaluated for their CNS-effects. New rational approaches are needed to enable the more effective selection Taxon sampling included representatives of all genera of of the most promising organisms for herbal remedies and drug tribe Haemantheae. Apodolirion was included in the phyloge- discovery programmes (Bohlin et al., 2007). netic analyses, but due to lack of material available for this In a study of Narcissus L. (Amaryllidaceae), we have shown study, no chemical or bioactivity data was obtained. For the that the distribution of alkaloids with AChE inhibitory activity phylogenetic hypothesis, we expanded the matrix of Meerow is significantly restricted within the phylogeny of Narcissus and and Clayton (2004) with 17 samples also used for chemical and can be used to predict the occurrence of active alkaloids in bioactivity evaluation in the present study. Of these, Gethyllis uninvestigated species (Rønsted et al., 2008). In the present afra L., G. grandiflora L.Bolus, Haemanthus amarylloides study we take a phylogenetic approach to explore and select Jacq., H. coccineus L., H. deformis Hook.f., H. hirsutus Baker, potential new target species of tribe Haemantheae for lead H. montanus Baker, H. sanguineus Jacq., and Scadoxus discovery of acetylcholinesterase and serotonin reuptake multiflorus, were not included in the earlier study by Meerow transport protein inhibitors. In the most recent formal and Clayton (2004). We did not include a set of sequences classification of Amaryllidaceae by Meerow and Snijman available in Genbank from Spies and Strydom as these were (1998), tribe Haemantheae included only the genera unpublished and we did therefore not feel we could trust their Lindl., Welw. ex Baker, Haemanthus L. and identity. Amaryllis belladonna L. was used as an outgroup Scadoxus. However, phylogenetic analyses have supported an following Meerow and Clayton (2004). We followed circum- expanded tribe Haemantheae including also the genera scriptions and species concepts of Meerow and Snijman (1998), Apodolirion Baker and Gethyllis Plum. ex L. (tribe Gethylli- Snijman (1984) and Govaerts et al. (2010). Total genomic DNA deae; Meerow and Clayton, 2004; Meerow and Snijman, 1998). and alkaloids were extracted from 17 species. In total, 28 In western healthcare, Alzheimer's disease (AD) is one of the sequences were produced for this study and 59 sequences were most prevailing diseases of cognitive dysfunction in the elderly. downloaded from Genbank/EMBL from previous studies Acetylcholine deficiency in the brain is a major feature seen in (Meerow and Clayton, 2004; Meerow and Snijman, 2001; patients suffering from AD, and an important approach to Meerow et al., 1999). Specimen determinations, voucher manage AD is inhibition of the enzyme acetylcholinesterase information, localities, and EMBL/Genbank accession numbers (AChE) (Heinrich and Teoh, 2004). Galanthamine, an alkaloid (HM140798–HM140825) are listed in Table 1. M.G.K. Bay-Smidt et al. / South African Journal of Botany 77 (2011) 175–183 177

Table 1 Details of material included in this study. Taxa Voucher information Origin ITS trnL-F Apodolirion Baker A. cedarbergense D.Müll.-Doblies Dulse s.n. (NBG) AY280344 a AY278957/AY278971 a A. lanceolatum (Thunb.) Benth. & NBG 714/88 (NBG) AY280345 a AY278958/AY2789721 a Hook.f. ex B.D.Jacks. Clivia Lindl. C. caulescens R.A.Dyer Rourke 2167 (NBG) AY280346 a AY278959/AY278973 a C. gardenii Hook. Rourke 2160 (NBG) AY280347 a AY278960/AY278974 a C. miniata (Lindl.) Bosse Rourke 2143 (NBG) AY280348 a AY278961/AY278975 a C. miniata (Lindl.) Bosse Rønsted 344 (C) (C) E1291−0002 HM140798 HM140815 C. nobilis Lindl. MW Chase 3080 (K) AY280349 a AF104776/AF104763 b C. nobilis Lindl. Rønsted 325 (C) (C) E1291−0001 HM140799 – Cryptostephanus Welw. ex Baker C. haemanthoides Pax Koopowitz 11040 (NBG) AY280350 a AY278962/AY278976 a C. vansonii Verd. Meerow 2310 (FTG) AY280351 a AF104804/AF104743 b C. vansonii Verd. Rønsted 332 (C) (C) S2002−0104, HM140800 – Gethyllis Plum. ex L. G. afra L. Rønsted 355 (C) Telos Rare , USA HM140801 – G. britteniana Baker Van Jaarsveld 5618 (NBG) AY280352 a AY278963/AY278977 a G. ciliaris (Thunb.) Thunb. Duncan 1123 (NBG) AY280353 a AF104816/AF104745 b G. grandiflora L.Bolus Rønsted 470 (C) Rare Plants, UK HM140805 – G. lanuginosa Marloth Van Jaarsfeld 4377 (NBG) AY280354 a AY278964/AY278978 a G. verticillata R.Br. Meerow 2350 (FTG) AY280355 a AY278965/AY278979 a Haemanthus L. H. albiflos Jacq. Meerow 2351 (FTG) AY280356 a AY278966/AY278980 a H. albiflos Jacq. Rønsted 314 (NU) University of KZN, Nursery HM140803 – H. amarylloides Jacq. Rønsted 443 (C) Telos Rare Bulbs, USA HM140804 HM140816 H. coccineus L. Rønsted 471 (C) Rare Plants, UK HM140805 – H. deformis Hook.f. Rønsted 373 (C) (C) HM140806 HM140817 H. deformis Hook.f. Rønsted 315 (NU) Midmar Dam, KZN, SA HM140807 HM140818 H. graniticus Snijman Snijman 308 (NBG) AY280357 a AY278967/AY278981 a H. hirsutus Baker Rønsted 468 (C) Rare Plants, UK HM140808 HM140819 H. humilis Jacq. MW Chase 2025 (K) – AF104781/AF104721 b H. montanus Baker Rønsted 365 (C) Rare Plants, UK HM140809 HM140820 H. pumilio Jacq. Snijman 668 (NBG) AY280358 a AY278968/AY278982 a H. sanguineus Jacq. Rønsted 326 (C) (C) P1999–5533, HM140810 HM140821 H. sanguineus Jacq. Rønsted 469 (C) Rare Plants, UK HM140811 HM140822 Scadoxus Raf. S. cinnabarinus (Decne.) Friis & Nordal MW Chase 549 (K) AY280359 a AF104783/AF104754 b S. membranaceus (Baker) Friis & Nordal NBG 708/88 (NBG) AY280360 a AY278969/AY278983 a S. multiflorus (Martyn) Raf. Rønsted 317 (NU) Andrew James Nursery, KZN HM140812 HM140823 S. puniceus (L.) Friis & Nordal NBG 43/72 (NBG) AY280361 a AY278970/AY278984 a S. puniceus (L.) Friis & Nordal Rønsted 321 (NU) Val-Lea Vista Nursery, KZN HM140813 HM140824 S. puniceus (L.) Friis & Nordal Rønsted 313 (NU) Ngoye forest, KZN HM140814 HM140825 Outgroup Amaryllis belladonna L. MW Chase 612 (K) AF373084 c AF104795/AF104744 b Official herbaria: (C): Herbarium, Botanical Garden and Museum, University of Copenhagen, Denmark; (FTG): Fairchild Tropical Botanic Garden, Miami, USA; (K): Royal Botanic Gardens, Kew, UK; (NBG): South African National Biodiversity Institute, Cape Town, SA; (NU): Bews Herbarium, School of Biological& Conservation Sciences, University of KwaZulu-Natal, Pietermaritzburg, SA. Abbreviations: KZN: KwaZulu-Natal; SA: South Africa; UK: United Kingdom. a Meerow and Clayton (2004). b Meerow et al. (1999). c Meerow and Snijman (2001).

2.2. DNA extraction, amplification and sequencing which only the forward strand of the trnL-F region was sequenced. The trnL-F region could not be readily amplified DNA was extracted using the Qiagen DNeasy plant from a number of taxa due to the presence of microsattelite extraction kit (Qiagen, Copenhagen, Denmark) from 20 mg of regions (poly-A and poly-T) towards the middle and the end of dried leaf-fragments. The internal transcribed spacer region the trnL-F region. (ITS) and the trnL-F region were amplified and sequenced using standard protocols (e.g. Meerow and Clayton, 2004) and 2.3. Phylogenetic reconstructions primers ITS4 and ITS5 (White et al., 1990) and c and f (Taberlet et al., 1991) respectively. Both strands were sequenced for each Sequences were edited and assembled using Sequencer region for all taxa, except for NR321 Scadoxus puniceus, for 4.8.TM software (Gene Codes, Ann Arbor, MI, USA). Sequences 178 M.G.K. Bay-Smidt et al. / South African Journal of Botany 77 (2011) 175–183 were aligned using default options in Clustal X v. 2.0.11 between 50 and 74 bootstrap percentages (BS) as weak support, software (Larkin et al., 2007). We analyzed the two regions scores between 75 and 89% BS as moderate support, and separately to identify phylogenetic conflicts among the regions scores of greater than 90% BS as strong support. We consider prior to performing a combined analysis. For the separate percentages b50% to be unsupported because such clades analyses we show only the bootstrap tree (Fig. 1) to establish that are not present in the majority of trees for the resampled there were no cases of strongly supported conflict, namely clades datasets. that are highly supported by bootstrap analysis in a single-loci analysis that are incongruent with highly supported clades in 2.4. Alkaloid diversity another single-loci analysis. All data were therefore combined into one analysis. Alkaloids were extracted from 300 mg dried scales. The Phylogenetic analyses were conducted using PAUP v. pulverized plant material was macerated in 400 μl MeOH for 4.0b10 (Swofford, 2002). All changes were assessed as 5 min. Three ml 0.1% H2SO4 was added and the material was unordered and equally weighted. Indels were coded as missing extracted for 45 min under ultrasonification followed by data and not included in the analyses because all groups marked shaking for 2 h. The sample was centrifuged for 10 min at by indel patterns were already well supported by the bootstrap 4000 rpm and the supernatant taken off. Extraction was procedure. Most parsimonious trees (MP) were obtained using repeated twice more, but the sample was left on the shaker 1,000 replicates of random taxon addition sequence and TBR overnight during the second extraction. The three extracts were branch swapping saving multiple trees (MULTREES). Relative pooled, centrifuged for 10 min at 4000 rpm, and retained on an levels of homoplasy were assessed using the consistency index ion-exchange SPE (solid phase extraction) column (Isolute (CI) and the retention index (RI). SCX, 500 mg, 3 ml, Mikrolab, Denmark). The column was Clade support was assessed using non-parametric bootstrap preconditioned with 2 ml MeOH and 2 ml 0.1% H2SO4. After re-sampling analysis (Felsenstein, 1985). Bootstrap analyses of addition of the alkaloid extract, the column was washed with the three individual datasets and the combined dataset were 2 ml 0.1% H2SO4 and 4 ml MeOH. The alkaloid extracts were carried out using 1000 replicates each consisting of 10 simple then eluted three times with 2 ml 25% NH4OH in MeOH, pH addition sequence replicates, with TBR swapping, and a limit 11–12, concentrated under vacuum until dryness and re- of 10 trees retained for each replicate. We defined scores dissolved to a concentration of 5 mg/ml in MeOH.

Fig. 1. Bootstrap consensus trees from analysis of (A) ITS nrDNA sequences and (C) trnL-F plastid DNA sequences. M.G.K. Bay-Smidt et al. / South African Journal of Botany 77 (2011) 175–183 179

Alkaloid profiles were obtained by gas chromatography- with the same buffer 1:10 (w/v). The supernatant was discarded, mass spectrometry (GC–MS) using a method developed by the pellet was suspended in buffer (Tris base 5 mM; EDTA Berkov et al. (2008). The GC–MS spectra were recorded on an 5 mM; pH 7.5), left for 20 min at 0 °C and centrifuged at 6000 g Agilent GC–MS system (5973 N Mass Selective Detector, 6890 for 10 min. The supernatant was discarded and the pellet was Network GC-system, 7683 Series Injector and Autosampler, suspended in buffer (Tris base 50 mM; NaCl 120 mM; KCl Agilent Technologies, Santa Clara, USA) operating in EI mode 50 mM; pH 7.5) and centrifuged at 6000 g for 10 min. The at 70 ev. An HP-5MS column (30 m×0.25 mm×0.25 μm) was supernatant was discarded and the protein pellet finally used (Agilent Technologies, Denmark). The temperature suspended in 1:10 (w/v) buffer (Tris base 50 mM; NaCl programme was: 100–180 °C at 15 °C/min, 1 min hold at 120 mM; KCl 50 mM; pH7.5). The tissue homogenate was kept 180 °C and 180–300 °C at 5 °C/min and 1 min hold at 300 °C. at −80 °C until use. The extracts were tested in a minimum of The injector temperature was 280 °C. The flow-rate of carrier six concentrations from 0.05 μg/ml to 5 mg/ml (4.17 ng/ml– gas (Helium) was 0.8 mL/min. The split ratio was 1:20. Samples 417 μl/ml in the assay) of dried plant material. Twenty-five μl were filtered through a 0.45 μl Millipore filter before injection of of each dilution was mixed with 50 μlof[3H]-citalopram 1 μl into the GC–MS. Alkaloids were identified to type by (4 nM, 0.67 nM in the assay) and 225 μl of tissue suspension, comparison with the NIST 08 Mass Spectral Search Program, respectively. The total binding of [3H]-citalopram was deter- version 2.0 (NIST, Gaithersburg, Maryland) and with published mined with a solvent blank. Paroxetin (120 μM, 10 μM in the spectral data. Alkaloid structures were scored to one of the assay) was used for the determination of the non-specific eighteen types proposed by Jin (2009) based on hypothetical binding. All samples were incubated for 2 h at 25 °C and then biosynthetic pathways (Fig. 3). filtered under vacuum using Avantec GC50-25 mm glass fibre filters. The radioactivity on the filters was determined by liquid 2.5. Acetylcholinesterase inhibitory activity scintillation using Ultimo Gold XR as scintillation fluid. Specific binding was calculated as total binding minus Measuring of AChE activity was conducted using an assay unspecific binding. All experiments were done in triplicate. modified from the assay by Adsersen et al. (2006).25μl15mM Fluoxetine hydrochloride (Dr. Ehrenstorfer GmbH, Germany) acetylthiocholine iodide (ATCl), 125 μl 3 mM 5,5-dithiobis-(2- was used a positive standard (IC50 =0.062 μg/ml). SERT nitrobenzoic acid) (DTNB), 50 μl buffer B (50 mM Tris–HCl, inhibitory activity expressed as IC50 (μg dried plant material/ pH 8.0, 0.1% bovine serum albumin) and 25 μl of the sample ml) was mapped onto the MP consensus tree from the combined dissolved in 10% MeOH in buffer A (50 mM Tris–HCl, pH 8) analysis (Fig. 3). IC50 values were determined for samples with were added to the wells in a 96 well microplate. Each sample an initial IC50 b50 μg/ml. was tested over a range of minimum five concentrations. Absorbance corresponding to spontaneous hydrolysis was read 3. Results in a BIOTEK EL808 microplate reader (Holm and Halby, Biotek, Denmark) at 405 nm every min for 10 min. 25 μl 3.1. Phylogenetic analyses 0.22 U/ml acetylcholinesterase (Electrophorus electricus, Sigma, Germany) was added to each well and absorbance was The aligned ITS region included 36 taxa and 899 characters again read every min for 10 min. Enzyme activity was of which 527 were constant and 209 (23%) were potentially calculated as a percentage compared to an assay using buffer parsimony informative. Parsimony analysis of the ITS region only. The AChE inhibitory data were analyzed with the produced 2340 trees of 639 steps with CI=0.75 and RI=0.86. software package GraFit 5 (Erithacus Software Ltd., East The aligned trnL-F region included 31 taxa and 1050 characters Grinstead, UK). AChE inhibition values are means of three of which 861 were constant and 67 (6%) were potentially individual determinations each performed in triplicate. parsimony informative. Parsimony analysis of the trnL-F region Galanthamine hydrobromide (Sigma, Germany) was used as produced 6550 trees of 237 steps with CI=0.87 and RI=0.84. positive control (IC50 =2.9 μM±0.29≈ 0.84 μg/ml±0.84). The combined matrix included 37 taxa and 1949 characters of AChE inhibitory activity expressed as IC50 (mg dried plant which 1388 were constant and 276 (14%) were potentially material/ml) was mapped onto the MP consensus tree from the parsimony informative. Parsimony analysis of the combined combined analysis (Fig. 3). IC50 values were determined for matrix produced 50 trees of 890 steps with CI=0.77 and samples with an initial IC50 b50 μg/ml. RI=0.85, which suggests that multiple islands of equally parsimonious trees are unlikely (Maddison, 1991). 2.6. Affinity to the serotonin reuptake transport protein 3.2. Alkaloid profiles Affinity to SERT was tested using the method described by Plenge et al. (1990) and Nielsen et al. (2004) with modifications Extraction of alkaloids yielded from 0.9 to 5.6% of the dried (Neergaard et al., 2009). Briefly, whole rat brains, except bulb weight. Alkaloid types (Jin, 2009; Fig. 3) were mapped cerebellum, were homogenized with an Ultra Turax homoge- onto the MP consensus tree from the combined analysis (Fig. 2). nizer in 1:10 (w/v) buffer (Tris base 5 mM; NaCl 150 mM; Six of the eighteen alkaloid types proposed by Jin (2009) were EDTA 20 mM; pH 7.5). The homogenate was centrifuged at recorded by GC–MS in the present study (Fig. 3). Cherylline 6000 g for 10 min and the homogenized tissue pellet washed type alkaloids (Fig. 3G) were only found in Cryptostephanus 180 M.G.K. Bay-Smidt et al. / South African Journal of Botany 77 (2011) 175–183

Fig. 2. Strict consensus of 50 MP trees from combined analyses of ITS and trnL-F DNA sequences. Bootstrap support indicated above branches. Distribution of AChE and SERT inhibitory activity expressed as IC50 (μg dry weight of plant material/ml) and content of alkaloids types according to Jin (2009) of different tribe Haemantheae bulb extracts. Alkaloid types are: (A) lycorine, (B) homolycorine (C) galanthamine (D) montanine, (E) crinine, (F) tazettine, and (G) cherylline. Abbreviations of genera are: A=Apodolirion,Cl=Clivia,Cr=Cryptostephanus,G=Gethyllis,H=Haemanthus,S=Scadoxus. nt.=not tested.

and homolycorine and galanthamine type alkaloids (B and C may not produce alkaloids, which are otherwise found respectively) were not detected in Scadoxus. However, none of throughout the family. However, this would need to be verified the major alkaloid types appear to be characteristic for specific by testing additional Gethyllis and Apodolirion species. clades in Haemantheae. Previous work on Gethyllis ciliaris (not included in this study) has suggested that this species may not 3.3. AChE and SERT inhibition produce alkaloids (Elgorashi et al., 2007). No alkaloids were detected by GC–MS in extracts of the two species of Gethyllis AChE and SERT inhibition expressed as IC50 (μg dried plant included in the present study, indicating that not only G. ciliaris material/ml) was mapped onto the MP consensus tree from the but possibly the whole genus Gethyllis (including Apodolirion) combined analyses (Fig. 2). M.G.K. Bay-Smidt et al. / South African Journal of Botany 77 (2011) 175–183 181

3

3 3 3

3 (A) Lycorine (B) Homolycorine (C) Galanthamine (D) Montanine

3

3

3

3 (E) Crinine (F) Tazettine (G) Cherylline

Fig. 3. Alkaloid types according to Jin (2009) identified in tribe Haemantheae bulb extracts.

4. Discussion humilis and H. hirsutus are sister taxa in the present analyses, but only with moderate support (66% BS). A second strongly 4.1. Phylogenetic inferences supported clade D (100% BS) corresponds to a winter rainfall group (mainly Western Cape) with red-pale pink flowers The ITS region provided more informative characters than (H. amarylloides, H. coccineus, H. crispus, H. graniticus, the trnL-F region and helped resolve most of the branches, but H. pumilio and H. sanguineus). Haemanthus montanus, which the trnL-F regions helped resolve the deeper branches. For is from the summer rainfall region, is sister to the winter rainfall example the ITS sequence of Cryptostephanus haemanthoides group, with which it shares leaf characteristics (Snijman, 1984). was so divergent from the ITS sequence of C. vansonii (19%) that the genus did not resolve as monophyletic in the ITS 4.2. Phylogenetic correlation of alkaloids with AChE inhibitory analyses, but the trnL-F sequences (1% divergence) provided activity 87% bootstrap support for the monophyly of Crypstostephanus, which was also reflected in the combined analysis although with AChE inhibitory activity was found in all investigated clades only weak support (55%). (Fig. 2) except the Apodolirion–Gethyllis clade, which can be The topology obtained in both the individual and the explained by the apparent lack of alkaloids in the investigated combined analyses is in agreement with Meerow and Clayton species of this clade. Lowest IC50 values were generally (2004) and with unpublished thesis work by Conrad (2008).In expressed by extracts containing crinine, galanthamine, and/ the combined analyses (Fig. 2) tribe Haemantheae is divided or lycorine type alkaloids (e.g. Clivia miniata NR344, into two main clades; A (66% BS) and B (88% BS). Clade A IC50 =18μg/ml of dried extract; Haemanthus sanguineus corresponds to genera Clivia (100% BS) and Cryptostephanus NR326, IC50 =8 μg/ml; and Scadoxus puniceus NR321, (53% BS). Clade B corresponds to genera Apodolirion– IC50 =18μg/ml) corresponding well with previous studies Gethyllis–Haemanthus–Scadoxus. Within clade B genera (Elgorashi et al., 2004, 2006a; López et al., 2002). In the Apodolirion and Gethyllis form a strongly supported clade present study, Haemanthus sanguineus NR469 also showed (100% BS). Gethyllis is paraphyletic to Apodolirion because G. weak activity (IC50 =49 μg/ml) despite presenting only mon- lanuginosa is more closely related to Apodolirion than to the tanine type alkaloids (Crouch et al., 2005; Wildman and remainder of Gethyllis species included in this study. Scadoxus Kaufman, 1955), which have to our knowledge not been tested (95% BS) and Haemanthus (89% BS) are sister clades with for AChE inhibitory activity before. Duplicate samples of H. moderate support (74%BS). sanguineus and S. puniceus did not show consistent results, Compared to the study by Meerow and Clayton (2004),we indicating that within species variations of chemistry may also included 9 additional samples of Haemanthus, improving the depend on other factors such as natural variation within and sampling to almost half of the genus (9–10 of 22 species). between populations (infraspecific variation), collected indivi- Within Haemanthus, a well supported clade C (89% BS) duals being exposed to different ecological conditions, and the corresponds to a summer rainfall group (mainly Eastern Cape) stage, age and maturity of the bulb, harvest time, drying with white-pale pink flowers (H. albiflos, H. deformis, H. conditions etc. reflecting seasonal and circadian fluctuations. hirsutus and H. humilis). Haemanthus hirsutus was reduced to a Infraspecific variability was for example reported in Crinum subspecies of H. humilis by Snijman (1984). Haemanthus macowanii Baker (Elgorashi et al., 2002), Galanthus elwesii 182 M.G.K. Bay-Smidt et al. / South African Journal of Botany 77 (2011) 175–183

Hook.f. by Berkov et al. (2004), and Leucojum aestivum L. pronounced and restricted to the genus Haemanthus within tribe (Georgieva et al., 2007). Haemantheae. Simultaneous evaluation of bioactivity and alkaloid profiles in a phylogenetic framework can potentially be used to 4.3. Phylogenetic correlation of alkaloids with SERT inhibitory select candidate species for phytotherapy and drug discovery as activity exemplified here by Amaryllidaceae tribe Haemantheae.

In spite of infra-specific variability of alkaloid profiles Acknowledgements observed in both Haemanthus sanguineus and Scadoxus puniceus, dose-dependent SERT activity appears to be pro- NR thanks the Botanical Gardens of the University of nounced and restricted to the genus Haemanthus within tribe Copenhagen for plant material. This research was supported by μ Haemantheae. Haemanthus hirsutus (NR468; IC50 =1.7 g/ml; a Steno grant (No. 272-07-0281) to NR from the Danish 10% lycorine type, 60% homolycorine type and 20% montanine Council for Independent Research – Natural Sciences and a μ type alkaloids), H. sanguineus (NR469; IC50 =2.4 g/ml; Marie Curie International Incoming Fellowship within the 7th only montanine type alkaloids) and H. coccineus (NR471; European Community Framework Programme to GIS and NR μ IC50 =8.7 g/ml; 98% montanine type and 2% crinine type (No. 235167). alkaloids) showed the best activity (IC50 b50 μg/ml). Previous studies have shown SERT activity of several crinine type alkaloids (Elgorashi et al., 2006b; Sandager et al., References 2005). However, also cherylline (cherryline type), tazettine A.P.G. III, 2009. An update of the Angiosperm Phylogeny Group classification (tazettine type) and 1-O-acetyllycorine (lycorine type) have for the orders and families of flowering plants: APG III. Botanical Journal of showed affinity to SERT, but these were the only compounds of the Linnean Society 161, 105–121. the respective types with reported activity in a study by Adsersen, A., Gauguin, B., Gudiksen, L., Jäger, A.K., 2006. Screening of plants Elgorashi et al. (2006b). Two of the most active extracts in the used in Danish folk medicine to treat memory dysfunction for acetylcho- linesterase inhibitory activity. Journal of Ethnopharmacology 104, 418–422. present study (Haemanthus coccineus NR471, IC50 =8.7 μg/ml; μ Berkov, S., Sidjimova, B., Evstatieva, L., Popov, S., 2004. Intra-specific and Haemanthus sanguineus NR469, IC50 =2.4 g/ml), variability in the alkaloid metabolism of Galanthus elwesii. Phytochemistry contained primarily montanine type alkaloids, which have not 65, 579–586. been tested for SERT affinity previously. Montanine type Berkov, S., Bastida, J., Viladomat, F., Codina, C., 2008. Analysis of alkaloids were also detected in Scadoxus puniceus (NR321) and galanthamine-type alkaloids by capillary gas chromatography-mass spec- – Cryptostephanus vansonii (NR332), and showed no SERT trometry in plants. Phytochemical Analysis 19, 285 293. N μ – Bohlin, L., Göransson, U., Backlund, A., 2007. Modern pharmacognosy: activity (IC50 50 g/ml) but GC MS profiles suggests that the connecting biology and chemistry. Pure and Applied Chemistry 79, Haemanthus alkaloids are of a different montanine subtype. 763–774. Haemanthus hirsutus NR468 (IC50 =1.7 μg/ml) was the most Chhabra, S.C., Mahunnah, B.L.A., Mshiu, E.N., 1987. Plants used in traditional SERT active extract, which may be due to the presence of medicine in eastern Tanzania. I. Pteridophytes and angiosperms (Acantha- – homolycorine or lycorine type alkaloids in addition to ceae to Canellaceae). Journal of Ethnopharmacology 21, 253 277. Conrad, F., 2008. Molecular systematics, biogeography and dating of the tribe montanine type alkaloids. Homolycorine type alkaloids have Haemantheae (Amaryllidaceae) and the phylogeography of Clivia. PhD also not been tested for SERT activity before. Thesis, University of Cape Town. Haemanthus species have not been recorded in traditional Crouch, N.R., Pohl, T.L., Mulholland, D.A., Ndlovu, E., 2005. Alkaloids from use to treat mental illnesses (Sobiecki, 2002; Stafford et al., three ethnomedicinal Haemanthus species: H. albiflos, H. deformis and H. – 2008), but could potentially be interesting. However, it must be pauculifolius (Amaryllidaceae). South African Journal of Botany 71, 49 52. Elgorashi, E.E., Drewes, S.E., Van Staden, J., 2002. Organ-to-organ and emphasized that several species are rare or threatened in the seasonal variation in alkaloids from Crinum macowanii. Fitoterapia 73, wild (Raimondo et al., 2009). Simultaneous evaluation of 490–495. bioactivity and alkaloid profiles in a phylogenetic framework Elgorashi, E.E., Stafford, G.I., Van Staden, J., 2004. Acetylcholinesterase has successfully been used to select candidate species for enzyme inhibitory effects of Amaryllidaceae alkaloids. Planta Medica 70, – traditional medicine and drug discovery. Tribe Haementheae s.l. 260 262. Elgorashi, E.E., Malan, S.F., Stafford, G.I., Van Staden, J., 2006a. Quantitative includes about 76 species, but SERT activity appear restricted to structure-activity relationship studies of acetylcholinesterase enzyme the genus Haemanthus with only 22 species. Three of eight inhibitory effects of Amaryllidaceae alkaloids. South African Journal of Haemanthus species tested showed IC50 b10 μg/ml. However, Botany 72, 224–231. IC values of extracts always have to be interpreted with Elgorashi, E.E., Stafford, G.I., Jäger, A.K., Van Staden, J., 2006b. Inhibition of 50 3 caution as activity can be due to potent compounds in low [ H] citalopram binding to the rat brain serotonin transporter by Amaryllidaceae alkaloids. Planta Medica 72, 470–473. concentrations, less potent compounds in high concentration or Elgorashi, E.E., Coombes, P.H., Mulholland, D.A., Van Staden, J., 2007. a combination of active compounds. Several Haemanthus Isoeugenitol, a cyclooxygenase-1 inhibitor from Gethyllis ciliaris. South extracts have been selected and are currently undergoing further African Journal of Botany 73, 156–158. studies to isolate and identify the presumed SERT active Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the – alkaloids. bootstrap. Evolution 39, 783 791. 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Edited by JC Manning